Patent Publication Number: US-2021163890-A1

Title: T lymphocyte production methods and t lymphocytes produced thereby

Description:
This application claims benefit of U.S. Provisional Patent Application No. 62/272,984, filed Dec. 30, 2015, the disclosure of which is incorporated by reference herein in its entirety. 
    
    
     1. FIELD 
     The disclosure herein relates to the field of immunology, and more specifically, to the generation of antigen-specific T lymphocytes and methods of use thereof. 
     2. BACKGROUND 
     Generation of antigen-specific T cells, e.g., cytotoxic T lymphocytes (CTLs) ex vivo involves T cell antigen-specific activation followed, e.g., by T cell proliferation. Full activation of such T cells involves ligation of T cell receptor (TCR) with cognate MHC-peptide complexes presented by antigen presenting cells (APC), including dendritic cells (DC), macrophages, and B cells, and ligations of co-stimulatory molecules between T cells and APC. Sometimes, cell-free MHC-peptide complexes are used in lieu of MHC-peptide complexes presented by APCs. If co-stimulation activation is deficient, T cells will become either partially activated, undergo apoptosis, or enter into anergy status. 
     Both B cells and monocytes typically express HLA molecules required for MHC-peptide complex formation, but lack high expression of co-stimulatory molecules, such as CD80 and CD86. Without further activation or maturation to obtain fully activated APC, a failed or suboptimal antigen-specific T cell priming and amplification may ensue. Thus, a separate APC induction and isolation step is usually necessary prior to antigen-specific T cell priming ex vivo, by producing either mature dendritic cells (mDC) from the precursor monocytes, or activated B cells. 
     3. SUMMARY 
     Provided herein are methods of generating antigen-specific T cells, e.g., cytotoxic T lymphocytes (T cells), starting from peripheral blood mononuclear cells without a separate step of generating and isolating antigen-presenting cells, such as dendritic cells, macrophages, and B cells, and with a single round of antigen stimulation. 
     In one aspect, provided herein are methods of producing a cell population comprising antigen-specific T cells, e.g., CTLs, comprising the steps of: (a) isolating blood mononuclear cells, e.g., peripheral blood mononuclear cells (PBMCs) or cord blood mononuclear cells, from a subject; (b) culturing said blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, in an antigen presenting cell (APC) induction medium comprising interleukin 4 (IL-4) and soluble CD40 ligand (sCD40L) and/or comprising granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon, e.g., interferon α (IFN-α), to produce a first population of cells; (c) culturing the first population of cells in the presence of one or more antigens, to produce a second population of cells; and (d) culturing the second population of cells in a T cell expansion medium comprising interleukin 7 (IL-7), interleukin 15 (IL-15), and, optionally, IL-4, to produce a third population of cells; wherein the third population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the method further comprises a step of culturing the third population of cells in a second T cell expansion medium comprising IL-7 and IL-15, but not comprising IL-4, to create a fourth population of cells; wherein the fourth population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the method further comprises a step of isolating T cells that are CD3+ from the third population of cells or the fourth population of cells. In certain aspects, step (c) is performed in APC induction medium. In certain aspects, the T cell expansion medium comprises IL-4. In certain aspects, the subject is a human. It certain aspects, the antigen is a human antigen. In certain aspects, the antigen is a full-length protein. In certain aspects, the antigen is a fragment of a protein. In certain aspects, the antigen is a peptide representing a part of the protein. In certain aspects, the APCs produce the antigen from exogenous genetic material. 
     In certain aspects, the third population of cells comprises T cells that are additionally CD4+. In certain aspects, the third population of cells comprises T cells that are additionally CD8+. In certain aspects, the third population of cells comprises T cells that are additional CD4+ and CD8+. 
     In certain aspects, the fourth population of cells comprises T cells that are additionally CD4+. In certain aspects, the fourth population of cells comprises T cells that are additionally CD8+. In certain aspects, the fourth population of cells comprises T cells that are additionally CD4+ and CD8+. 
     In certain aspects, the APC induction medium comprises IL-4 and sCD40L. In certain aspects, the APC induction medium comprises 1-50 ng/mL of IL-4 and 0.1-5 μg/mL of sCD40L. In certain aspects, the APC induction medium comprises 8-12 ng/mL of IL-4 and 0.8-1.2 μg/mL of sCD40L. In certain aspects, the APC induction medium comprises 10 ng/mL of IL-4 and 1 μg/mL of sCD40L. 
     In certain aspects, the APC induction medium further comprises synthetic oligonucleotides with one or more unmethylated CpG dinucleotide motifs. In certain aspects, the APC induction medium further comprises GM-CSF and IFN-α. In certain aspects, the APC induction medium consists essentially of GM-CSF and IFN-α. 
     In certain aspects, the one or more antigens are, or are contained within, a pool of peptides, e.g., lyophilized peptides. In specific aspects, the pool of lyophilized peptides cover the sequences of human HPV16E6, HPV16E7, HPV18E6, and/or HPV18E7 proteins. In specific aspects, the peptides are at a concentration of 1 μg/mL. 
     In certain aspects, the T cell expansion medium comprises 10-100 ng/mL of IL-7, 2-20 ng/mL of IL-15, and 10-100 ng/mL of IL-4. In certain aspects, the T cell expansion medium comprises 40-60 ng/mL of IL-7, 7-11 ng/mL of IL-15, and 45-65 ng/mL of IL-4. In specific aspects, the T cell expansion medium comprises 50 ng/mL of IL-7, 9 ng/mL of IL-15, and 55 ng/mL of IL-4. 
     In certain aspects, the second T cell expansion medium comprises 10-100 ng/mL of IL-7 and 2-20 ng/mL of IL-15. In certain aspects, the second T cell expansion medium comprises 40-60 ng/mL of IL-7 and 7-11 ng/mL of IL-15. In specific aspects, the second T cell expansion medium comprises 50 ng/mL of IL-7 and 9 ng/mL of IL-15. 
     In certain aspects, the duration of step (b) is 1-3 days. In specific aspects, the duration of step (b) is 1 day. In certain aspects, the duration of step (d) is 8-16 days. In specific aspects, the duration of step (d) is 12 days. 
     In certain aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are isolated from whole blood, buffy coat, or an enriched leukapheresis product. In certain aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded in the APC induction medium at a density of 4-6×10 6 /cm 2 . In specific aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded at a density of 5×10 6 /cm 2 . In specific aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded at a density greater than 5×10 6 /cm 2 . 
     In certain aspects, the antigen-specific CD3+ cells are identified by, or are identifiable by, intracellular cytokine staining (ICCS). In certain aspects, the culturing steps are performed in a gas-permeable enclosure, e.g., a G-REX® device. In certain aspects, one or more culturing steps are performed in T-flasks. In certain aspects, one or more culturing steps are performed in bags. In specific aspects, the bags are static bags. In certain aspects, the bags are gas-permeable. In certain aspects, one or more culturing steps are performed in a WAVE™ bioreactor (GE Healthcare Life Sciences). In certain aspects, one or more culturing steps are performed in spinner flasks. 
     In one aspect, provided herein are populations of antigen-specific T cells produced by a method comprising the steps of: (a) isolating blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, from a subject; (b) culturing said blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, in an antigen presenting cell (APC) induction medium comprising interleukin 4 (IL-4) and soluble CD40 ligand (sCD40L) and/or comprising granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon α (IFN-α), to produce a first population of cells; (c) culturing the first population of cells in the presence of one or more antigens, to produce a second population of cells; and (d) culturing the second population of cells in a T cell expansion medium comprising interleukin 7 (IL-7), interleukin 15 (IL-15), and IL-4, to produce a third population of cells; wherein the third population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the populations of cells are produced by a method which further comprises a step of culturing the third population of cells in a second T cell expansion medium comprising IL-7 and IL-15, but not comprising IL-4, to create a fourth population of cells; wherein the fourth population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the populations of cells are produced by a method which further comprises a step of isolating T cells that are CD3+ from the third population of cells or the fourth population of cells. In certain aspects, step (c) is performed in APC induction medium. 
     In one aspect, provided herein are compositions, e.g., pharmaceutical compositions, comprising the antigen-specific T cells described herein. In certain aspects, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% or more of the cells in the composition are antigen-specific T cells. In certain aspects, 50% of the cells in the composition are antigen-specific T cells. In certain aspects, the compositions comprise a pharmaceutically acceptable excipient. 
     In one aspect, provided herein are methods of treating a cancer or a viral infection comprising administering to a patient in need thereof a population of antigen-specific T cells produced by the methods described herein, wherein the isolated blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are autologous to the patient. In another aspect, provided herein are methods of treating a cancer or a viral infection comprising administering to a patient in need thereof a population of antigen-specific T cells produced by the methods described herein, wherein the isolated blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are not autologous to the patient. In certain aspects, the viral infection is a human papillomavirus (HPV) infection. In certain aspects, the viral infection is an HPV infection and the antigen is an HPV antigen. In certain other aspects, the viral infection is an Epstein-Barr virus (EBV) infection. In certain aspects, the viral infection is an EBV infection and the antigen is an EBV antigen. In a specific embodiment of the foregoing embodiments, the antigens are expressed in tumor cells comprising the viral infection. In certain aspects, provided herein are methods of administering a population of antigen-specific T cells to a patient in need thereof. In certain aspects, the method further comprises administering to said patient an immunomodulatory drug or an epigenetic modifier. In specific aspects, the method further comprises administering to said patient an immune checkpoint inhibitor. In more specific aspects, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD1 antibody, and an anti-PD-L1 antibody. 
     In one aspect, provided herein are methods of treating a cancer comprising administering to a patient in need thereof a population of antigen-specific T cells produced by the methods described herein, wherein the isolated blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are autologous to the patient. In certain aspects, the cancer is an HPV positive (HPV+) cancer. In certain aspects, the HPV+ cancer is head and neck cancer. In certain aspects, the head and neck cancer is squamous cell carcinoma of the head and neck. In certain aspects, the head and neck cancer is oropharyngeal cancer. In certain aspects, the HPV+ cancer is penile cancer. In certain aspects, the HPV+ cancer is cervical cancer. In certain aspects, the HPV+ cancer is anal cancer. In certain aspects, the HPV+ cancer is vulval cancer. In certain aspects, the HPV+ cancer is vaginal cancer. In certain aspects, the HPV+ cancer is lung cancer. In certain aspects, the HPV+ cancer is metastatic. In certain aspects, the HPV+ cancer is recurrent. In certain aspects, the HPV+ cancer is metastatic and recurrent. In certain aspects, the method further comprises administering to said patient an immunomodulatory drug or an epigenetic modifier. In certain aspects, the method further comprises administering to said patient an immune checkpoint inhibitor. In certain aspects, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD1 antibody, and an anti-PD-L1 antibody. 
    
    
     
       4. BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 : Expansion fold change of cells of all donors under various APC induction cytokine conditions described in Example 1. 
         FIG. 2 : HPV T cell frequency of all donors under various APC induction cytokine conditions described in Example 1. 
         FIG. 3 : HPV T cell yield in all donors under various APC induction cytokine conditions described in Example 1. 
         FIG. 4A-B : Total cell expansion folds at harvest under various T cell expansion cytokine conditions described in Example 2. (A) Summary plot of all 4 donors. (B) Individual plot for each donor. 
         FIG. 5A-B : T cell frequency at harvest under various T cell expansion cytokine conditions described in Example 2. (A) Summary plot for all 4 donors. (B) Individual plot for each donor. 
         FIG. 6A-B : T cell yields at harvest under various T cell expansion cytokine conditions described in Example 2. (A) Summary plot for all 4 donors. (B) Individual plot for each donor. 
         FIG. 7 : Tcm, Tem and Teff percentages within CD45RO+ T cell populations under various T cell expansion cytokine conditions described in Example 2 for two different donors. Runs C1-C17 for each population (Tcm, Tem, and Teff) are shown in numerical order from left to right. 
         FIG. 8 : CD45RO+CD62Lhi T cell populations under various T cell expansion cytokine conditions described in Example 2 for two different donors. 
         FIG. 9 : Expansion Fold change of cells of all donors under various APC induction duration conditions described in Example 3. 
         FIG. 10 : HPV-T cell frequency of all donors under various APC induction duration conditions described in Example 3. 
         FIG. 11 : HPV T cell yield from all donors under various APC induction duration conditions described in Example 3. 
         FIG. 12 : Cell growth curve and doubling time under the conditions described in Example 4. 
         FIG. 13A-B : Changes with cell viability (A) and cell size (B) with expansion extension under the conditions described in Example 4. 
         FIG. 14A-D : Total cell expansion (A), fold change (B), T cell frequency (C) and T cell yield (D) at harvest on day 12, 14 and 16 post antigen stimulation under the conditions described in Example 4. 
         FIG. 15 : T cell purity and CD4/CD8 cell percentage at harvest from day 12 to day 16 under the conditions described in Example 4. 
         FIG. 16 : Non-T cell population at harvest from day 12 to day 16 under the conditions described in Example 4. 
         FIG. 17 : Changes of Memory T cell and CD45RO+CD62Lhi percentage with culture extension under the conditions described in Example 4. Statistical significance was determined by one-way ANOVA analysis. 
         FIG. 18 : Changes of percentages of central memory, effect memory and effect T cell populations with culture extension from 12 days to 16 days under the conditions described in Example 4. Within the CD45RO+ population, reduction of central memory T cell population and raise of effect T cell population were seen from day 12 to day 16 cultures. Statistical significance was determined by one-way ANOVA analysis. 
         FIG. 19 : HPV T cell frequency of all donors under the various seeding density and APC cytokine cocktail concentration conditions described in Example 5. 
         FIG. 20 : HPV T cell yield of all donors under the various seeding density and APC cytokine cocktail concentration conditions described in Example 5. 
         FIG. 21 : Cell expansion fold change of all donors under the conditions described in Example 6. The results are grouped with APC induction media type at the x-axis. The legend of the graphs is shown at the bottom right corner. “IL-4 Disc.” means IL-4 discontinuation. 
         FIG. 22 : HPV T cell frequency of all donors under the conditions described in Example 6. The results are grouped with APC induction media type at the x-axis. The legend of the graphs is shown at the bottom right corner. “IL-4 Disc.” means IL-4 discontinuation. 
         FIG. 23 : HPV T cell yield of all donors under the conditions described in Example 6. The results are grouped with APC induction media type at the x-axis. The legend of the graphs is shown at the bottom right corner. “IL-4 Disc.” means IL-4 discontinuation. 
         FIG. 24 : IFN-γ-HPV T cell frequency of all donors under the conditions described in Example 6. The results are grouped with APC induction media type at the x-axis. The legend of the graphs is shown at the bottom right corner. “IL-4 Disc.” means IL-4 discontinuation. The results are obtained from cells that are gated within CD3+ population. 
         FIG. 25 : Th1/Th2 ratio based on cytokine secretion of all donors under the conditions described in Example 6. IFN-γ level, IL-5 level and IFN-γ/IL-5 ratio as the Th1/Th2 ratio are presented. The results are grouped with APC induction media type at the x-axis. The legend of the graphs is shown at the bottom. “IL-4 Disc.” means IL-4 discontinuation. 
         FIG. 26 : Th1, Th2, Treg subset analysis based on transcription factor expression of all donors under the conditions described in Example 6. The results are grouped with APC induction media type at the x-axis. The legend of the graphs is shown at right. D #1: Donor #1; D #2: Donor #2; D #3: Donor #3. “IL-4 Conc.” means IL-4 continuation. “IL-4 Disc.” means IL-4 discontinuation. 
         FIG. 27 : T cells were generated from five cervical cancer patients, one healthy donor positive for HPV 16 and HPV 18, and three healthy donors of unknown HPV status. The T cells were than activated with HPV 16 or HPV 18 E6 or E7 antigenic peptides, or E6 and E7 antigenic peptides from both HPV 16 and HPV 18, and the percentage of T cells was measured. 
         FIG. 28A-B : T cells were tested for cytotoxicity against autologous targets or HLA-A2 matched targets in vitro at effector to target ratios of 2.5:1, 5:1, 10:1, 20:1, and 40:1. 
         FIG. 29A-D : Cells from seven individual donors were tested before (PBMCs) and after (T cells) generation of T cells. HPV antigen specificity was tested by measuring surface expression of CD137 ( FIG. 29A ). Effector activity was measured in HPV-stimulated PBMCs and in HPV or non-HPV stimulated T cells by measuring secretion of IFN-γ ( FIG. 29B ), GM-CSF ( FIG. 29C ), and TNF-α, ( FIG. 29D ). 
     
    
    
     5. DETAILED DESCRIPTION 
     Provided herein are methods of generating T cells, e.g., cytotoxic T lymphocytes (T cells) starting from blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, without a separate step of generating and isolating antigen-presenting cells, such as dendritic cells, macrophages, and B cells, and with a single round of antigen stimulation. As used herein, the term “T cells” means T lymphocytes, and includes cytotoxic T lymphocytes (T cells). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of abbreviations for key terms. 
               
            
           
           
               
               
               
            
               
                   
                 Term 
                 Full name 
               
               
                   
                   
               
               
                   
                 AE 
                 adverse event 
               
               
                   
                 APC 
                 antigen presenting cells 
               
               
                   
                 CBA 
                 cytometric beads array 
               
               
                   
                 CTL 
                 cytotoxic T lymphocytes 
               
               
                   
                 Cy 
                 cyclophosphamide 
               
               
                   
                 DC 
                 dendritic cells 
               
               
                   
                 DLT 
                 dose-limiting toxicity 
               
               
                   
                 DMSO 
                 dimethyl sulfoxide 
               
               
                   
                 Flu 
                 fludarabine 
               
               
                   
                 GM-CSF 
                 granulocyte-macrophage 
               
               
                   
                 HPV 
                 Human papillomavirus 
               
               
                   
                 ICCS 
                 intracellular cytokine staining 
               
               
                   
                 iDC 
                 immature dendritic cells 
               
               
                   
                 IFN-α 
                 interferon alpha 
               
               
                   
                 IFN-γ 
                 interferon gamma 
               
               
                   
                 IL-10 
                 interleukin 10 
               
               
                   
                 IL-13 
                 interleukin 13 
               
               
                   
                 IL-15 
                 interleukin 15 
               
               
                   
                 IL-1β 
                 interleukin 1 beta 
               
               
                   
                 IL-2 
                 interleukin 2 
               
               
                   
                 IL-4 
                 interleukin 4 
               
               
                   
                 IL-5 
                 interleukin 5 
               
               
                   
                 IL-6 
                 interleukin 6 
               
               
                   
                 IL-7 
                 interleukin 7 
               
               
                   
                 IM 
                 intramuscular 
               
               
                   
                 IMDM 
                 Iscove&#39;s Modified Dulbecco&#39;s 
               
               
                   
                 IV 
                 intravenous 
               
               
                   
                 mDC 
                 mature dendritic cells 
               
               
                   
                 MTD 
                 maximum tolerated dose 
               
               
                   
                 OS 
                 overall survival 
               
               
                   
                 PBMC 
                 peripheral blood mononuclear 
               
               
                   
                 PBS 
                 Phosphate-buffered saline 
               
               
                   
                 pDC 
                 plasmacytoid DC 
               
               
                   
                 PHA 
                 phytohemagglutinin 
               
               
                   
                 PD 
                 progressive disease 
               
               
                   
                 PFS 
                 progression-free survival 
               
               
                   
                 PO 
                 orally 
               
               
                   
                 RPMI 
                 Roswell Park Memorial Institute 
               
               
                   
                 RT 
                 room temperature 
               
               
                   
                 SCCHN 
                 squamous cell carcinoma of the head 
               
               
                   
                   
                 and neck 
               
               
                   
                 sCD40L 
                 soluble CD40 ligand 
               
               
                   
                 TNF-α 
                 tumor necrosis factor alpha 
               
               
                   
                   
               
            
           
         
       
     
     5.1 Production of T Cells and T Cell Populations 
     In one aspect, provided herein are methods of producing a cell population comprising antigen-specific T cells, e.g., cytotoxic T lymphocytes (CTLs), comprising the steps of: (a) isolating blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, from a subject; (b) culturing said blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, in an antigen presenting cell (APC) induction medium comprising interleukin 4 (IL-4) and soluble CD40 ligand (sCD40L) and/or comprising granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon α (IFN-α), to produce a first population of cells; (c) culturing the first population of cells in the presence of one or more antigens, to produce a second population of cells; and (d) culturing the second population of cells in a T cell expansion medium comprising interleukin 7 (IL-7), interleukin 15 (IL-15), and, optionally, IL-4, to produce a third population of cells; wherein the third population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the method further comprises a step of culturing the third population of cells in a second T cell expansion medium comprising IL-7 and IL-15, but not comprising IL-4, to create a fourth population of cells; wherein the fourth population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the method further comprises a step of isolating T cells that are CD3+ from the third population of cells or the fourth population of cells. In certain aspects, step (c) is performed in APC induction medium. In certain aspects, the T cell expansion medium comprises IL-4. In certain aspects, the subject is a human. It certain aspects, the antigen is a human antigen. In certain aspects, the antigen is a full-length protein. In certain aspects, the antigen is a fragment of a protein. In certain aspects, the antigen is a peptide representing a part of a protein. In certain aspects, the APCs produce the antigen from exogenous genetic material. In certain aspects, one or more steps are repeated in order to increase the yield of the target population of cells generated by those one or more steps. In certain aspects, a step is repeated with cells that remain unmodified after said step. In certain aspects, one or more steps are repeated with cells that remain unmodified after said steps. In certain aspects, steps (b) to (d) are repeated one time. In certain aspects, steps (b) to (d) are repeated two times. In certain aspects, steps (b) to (d) are repeated three times. In certain aspects, steps (b) to (d) are repeated four times. In certain aspects, steps (b) to (d) are repeated five times. In certain aspects, steps (b) to (d) are repeated with blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, remaining after step (d). In certain aspects, one or more of steps (b), (c), and (d) are repeated one, two, three, four, or five times. 
     In certain aspects, the third population of cells comprises T cells that are additionally CD4+. In certain aspects, the third population of cells comprises T cells that are additionally CD8+. In certain aspects, the third population of cells comprises T cells that are additionally CD4+ and CD8+. 
     In certain aspects, the fourth population of cells comprises T cells that are additionally CD4+. In certain aspects, the fourth population of cells comprises T cells that are additionally CD8+. In certain aspects, the fourth population of cells comprises T cells that are additionally CD4+ and CD8+. 
     In certain aspects, the APC induction medium comprises IL-4 and sCD40L. In certain aspects, the APC induction medium comprises 1-50 ng/mL of IL-4 and 0.1-5 μg/mL of sCD40L. In certain aspects, the APC induction medium comprises 8-12 ng/mL of IL-4 and 0.8-1.2 μg/mL of sCD40L. In certain aspects, the APC induction medium comprises 10 ng/mL of IL-4 and 1 μg/mL of sCD40L. 
     In certain aspects, the APC induction medium further comprises synthetic oligonucleotides with one or more unmethylated CpG dinucleotide motifs. In certain aspects, the APC induction medium further comprises GM-CSF and IFN-α. In certain aspects, the APC induction medium comprises GM-CSF and IFN-α. In certain aspects, the APC induction medium consists essentially of GM-CSF and IFN-α. 
     In certain aspects, the APC induction medium further comprises IMDM, 2-mercaptoethanol, Pen Strep, and/or human serum. In certain aspects, the APC induction medium further comprises RPMI-1640, Click&#39;s media, human serum, L-GLUTAMAX™ (Life Technologies), Pen Strep, and/or 2-mercaptoethanol. In certain aspects, the APC induction medium further comprises RPMI-1640, Click&#39;s media, human serum, glutamine, Pen Strep, and/or 2-mercaptoethanol. 
     In certain aspects, the IL-4 in the APC induction medium is at a concentration of 40 ng/mL. In certain aspects, the IL-4 in the APC induction medium is at a concentration of 10 ng/mL. In certain aspects, the CD40L in the APC induction medium is at a concentration of 1 μg/mL. In certain aspects, the CpG dinucleotide motifs in the APC induction medium are at a concentration of 4 μg/mL. In certain aspects, the GM-CSF in the APC induction medium is at a concentration of 800 U/mL. In certain aspects, the IFN-α in the APC induction medium is at a concentration of 1000 U/mL. 
     In certain aspects, the one or more antigens are a pool of peptides, e.g., a pool of lyophilized peptides. In certain aspects, the one or more antigens are an HPV peptide mixture, e.g., PEPMIX™. In certain aspects, the one or more antigens are a pool of overlapping 15-mers spanning E6 and E7 proteins from HPV types 16 and 18. In specific aspects, the pool of peptides cover the sequences of one or more of, or all of, human HPV16E6, HPV16E7, HPV18E6, and/or HPV18E7 proteins. In certain aspects, the one or more antigens comprise one or more antigens from a single virus, e.g., only from HPV16 or only from HPV18. In certain aspects, the peptides are at a concentration of 0.5-1 μg/mL. In specific aspects, the peptides are at a concentration of 1 μg/mL. In specific aspects, the peptides are at a concentration of 0.5 μg/mL. 
     In certain aspects, the T cell expansion medium comprises 10-100 ng/mL of IL-7, 2-20 ng/mL of IL-15, and 10-100 ng/mL of IL-4. In certain aspects, the T cell expansion medium comprises 40-60 ng/mL of IL-7, 7-11 ng/mL of IL-15, and 45-65 ng/mL of IL-4. In specific aspects, the T cell expansion medium comprises 50 ng/mL of IL-7, 9 ng/mL of IL-15, and 55 ng/mL of IL-4. In certain aspects, addition of the cytokines from the T cell expansion medium is performed 2 hours, 24 hours, or 48 hours after addition of the one or more antigens. In specific aspects, addition of the cytokines from the T cell expansion medium is performed 2 hours after addition of the one or more antigens. In specific aspects, addition of the cytokines from the T cell expansion medium is performed 24 hours after addition of the one or more antigens. In specific aspects, addition of the cytokines from the T cell expansion medium is performed 48 hours after addition of the one or more antigens. 
     In certain aspects, the T cell expansion medium comprises 1 ng/mL IL-15, 15 ng/mL IL-4, and 10 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 1 ng/mL IL-15, 15 ng/mL IL-4, and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 1 ng/mL IL-15, 35 ng/mL IL-4, and 30 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 1 ng/mL IL-15, 55 ng/mL IL-4, and 10 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 1 ng/mL IL-15, 55 ng/mL IL-4, and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 5.5 ng/mL IL-15, 15 ng/mL IL-4, and 10 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 5.5 ng/mL IL-15, 15 ng/mL IL-4, and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 5.5 ng/mL IL-15, 35 ng/mL IL-4, and 30 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 5.5 ng/mL IL-15, 55 ng/mL IL-4, and 10 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 5.5 ng/mL IL-15, 55 ng/mL IL-4, and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 10 ng/mL IL-15, 15 ng/mL IL-4, and 10 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 10 ng/mL IL-15, 15 ng/mL IL-4, and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 10 ng/mL IL-15, 35 ng/mL IL-4, and 30 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 10 ng/mL IL-15, 55 ng/mL IL-4, and 10 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 10 ng/mL IL-15, 55 ng/mL IL-4, and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 55 ng/mL IL-4 and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium comprises 5.5 ng/mL IL-15 and 50 ng/mL IL-7. In certain aspects, the T cell expansion medium consists essentially of 5.5 ng/mL IL-15 and 50 ng/mL IL-7. 
     In certain aspects, the second T cell expansion medium comprises 10-100 ng/mL of IL-7 and 2-20 ng/mL of IL-15. In certain aspects, the second T cell expansion medium comprises 40-60 ng/mL of IL-7 and 7-11 ng/mL of IL-15. In specific aspects, the second T cell expansion medium comprises 50 ng/mL of IL-7 and 9 ng/mL of IL-15. 
     In certain aspects, the duration of step (b) is 1-3 days. In specific aspects, the duration of step (b) is 1 day. In certain aspects, the duration of step (d) is 8-16 days. In specific aspects, the duration of step (d) is 12 days. In certain aspects, IL-4 is removed from the T cell expansion medium prior to the end of T cell expansion. In certain aspects, IL-4 is not removed from the T cell expansion medium prior to the end of T cell expansion. 
     In certain aspects, the blood mononuclear cells, e.g., PBMCs, are isolated from whole blood, buffy coat, or an enriched leukapheresis product. In certain aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded into the APC induction medium at a density of 4-6×10 6 /cm 2 . In specific aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded at a density of 5×10 6 /cm 2 . In specific aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded at a density greater than 5×10 6 /cm 2 . 
     In certain aspects, the antigen-specific CD3+ cells are identified by intracellular cytokine staining (ICCS). In certain aspects, the culturing steps are performed in a gas-permeable container, e.g., plastic bag or a G-REX® device. In certain aspects, one or more culturing steps are performed in T-flasks. In certain aspects, one or more culturing steps are performed in bags. In specific aspects, the bags are static bags. In certain aspects, the bags are gas-permeable. In certain aspects, one or more culturing steps are performed in a WAVE™ bioreactor. In certain aspects, one or more culturing steps are performed in a spinner flasks. 
     5.2 Isolation and Characterization of T cells 
     Methods of isolating T cells are known in the art and can be used to isolate (e.g., purify) the T cells produced using the methods described herein. For example, T cells can be isolated or enriched by staining cells, in one embodiment, with antibodies to CD3, and selecting for CD3+ cells. T cells may also be isolated with antibodies to CD4 and/or CD8, and selecting for CD4+ and/or CD8+ cells. T cells, for example, T cells produced by the methods described herein can also be isolated or enriched by removal of cells other than T cells in a population of cells that comprise the T cells, e.g., T cells produced by the methods described herein. For example, T cells, e.g., cells produced using the methods described herein, may be isolated or enriched by depletion of cells displaying non-T cell markers. Cells isolated by these methods may be additionally sorted. 
     Antigen-specific T cells, e.g., antigen-specific CTLs, e.g. HPV-specific T cells and CTLs, may, for example, be identified using an ICCS assay, a flow cytometry based assay which detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation. ICCS can be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers to detect antigen reactivity of T cells. In specific embodiments, a ICCS assay is used to measure antigen-specific T cell, e.g., HPV-T cell, frequency at various time points of the culture process of the T cells. ICCS works via the following principles: 1) antigen specific T cells can be activated by the same specific antigen re-stimulation, 2) in response to the antigen specific re-stimulation, T cell can produce cytokines (such as IFN-γ, TNF-α, IL-2, et al) and express activation markers (such as CD154, CD137, et al), 3) an inhibitor of protein transport (e.g. brefeldin A) is added during the re-stimulation to retain the cytokines within the cell, 4) the cells are then fixed in paraformaldehyde and permeabilized, and 5) the anti-cytokine antibody is added and the cells can be analyzed by flow cytometer. 
     As T cells will activate in response to a wide variety of stimulatory signals in addition to the antigen specific stimulation, positive staining (especially low intensity staining) does not unequivocally demonstrate true antigen specificity when using ICCS. Thus, in one embodiment, a non-antigen stimulation control is set up along with the specific antigen stimulation conditions. The antigen-specific, e.g., HPV, T cell number is counted by measuring the T cells that have at least one of the cytokine production or up-regulated activation markers, and subtracting the number from the non-antigen stimulation control. In certain embodiments, the panel used for flow cytometry is a 6 marker panel, including CD3, CD4, CD8, CD154 (CD40L), IFN-γ and TNF-α. CD3, CD4 and CD8 are T cell lineage markers that can define a total T cell population, optionally including natural killer T (NKT) cells, and CD4/CD8 T cell ratio. IFN-γ and TNF-α are cytokines that T cells produce after overnight re-activation. CD154 is a T cell activation marker that is up-regulated in newly antigen-activated T cells. 
     Determination and/or isolation of T cells may be performed, for example, using a T cell purity panel comprising antibodies against CD19, CD56, CD3, CD11a, CD4, and CD8. Determination and/or isolation of T cells at a certain stage of differentiation may be performed using a T cell differentiation panel comprising antibodies against CD3, CD62L, CD4, CD27, CCR7, and CD45RO. To determine whether cells comprise T cells, secreted cytokines can also be measured, for example, by an 8-plex cytometrix beads array (CBA). The panel may include one or more sets of cytokines selected from the group consisting of Th1 cytokines (e.g., IL-2, IFN-γ and TNF-α), Th2 cytokines (e.g., IL-5, IL-13), Treg cytokines (e.g., IL-10), and T cell effector function related cytokines (e.g., Granzyme B and GM-CSF). To determine whether cells comprise T cells, transcription factors may be measured, for example, one or more of T-bet, GATA-3, and FoxP3. 
     Cell separation, e.g., separating T cells produced by the methods described herein from non-T cells, can be accomplished by, e.g., flow cytometry, fluorescence-activated cell sorting (FACS), or, in one embodiment, magnetic cell sorting using microbeads conjugated with specific antibodies. The cells may be isolated, e.g., using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (e.g., about 0.5-100 μm diameter) that comprise one or more specific antibodies, e.g., anti-CD3 antibodies. Magnetic cell separation can be performed and automated using, e.g., an AUTOMACS™ Separator (Miltenyi) or a CLINIMACS® System (Miltenyi). A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or hapten. The beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker. In one embodiment, these cells can then be isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation. 
     5.3 T Cells and Populations of T Cells 
     In one aspect, provided herein are populations of antigen-specific T cells that are specific for a specific antigen or a set of related antigens, e.g., specific to antigens from the same protein. In one aspect, provided herein are populations of antigen-specific T cells that are specific for a set of unrelated antigens, e.g., specific to antigens from different proteins. In certain embodiments, the T cells are produced by the method comprising the steps of: (a) isolating blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, from a subject; (b) culturing said blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, in an antigen presenting cell (APC) induction medium comprising interleukin 4 (IL-4) and soluble CD40 ligand (sCD40L) and/or comprising granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon α (IFN-α), to produce a first population of cells; (c) culturing the first population of cells in the presence of one or more antigens, to produce a second population of cells; and (d) culturing the second population of cells in a T cell expansion medium comprising interleukin 7 (IL-7), interleukin 15 (IL-15), and IL-4, to produce a third population of cells; wherein the third population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the populations of cells are produced by a method which further comprises a step of culturing the third population of cells in a second T cell expansion medium comprising IL-7 and IL-15, but not comprising IL-4, to create a fourth population of cells; wherein the fourth population of cells comprises T cells that are CD3+ and specific for an antigen added in step (c). In certain aspects, the populations of cells are produced by a method which further comprises a step of isolating T cells that are CD3+ from the third population of cells or the fourth population of cells. In certain aspects, the populations of cells are produced by a method wherein step (c) is performed in APC induction medium. 
     In certain aspects, the third population of cells comprises T cells that are additionally CD4+. In certain aspects, the third population of cells comprises T cells that are additionally CD8+. In certain aspects, the third population of cells comprises T cells that are additional CD4+ and CD8+. 
     In certain aspects, the fourth population of cells comprises T cells that are additionally CD4+. In certain aspects, the fourth population of cells comprises T cells that are additionally CD8+. In certain aspects, the fourth population of cells comprises T cells that are additionally CD4+ and CD8+. 
     In certain aspects, the APC induction medium comprises IL-4 and sCD40L. In certain aspects, the APC induction medium comprises 8-12 ng/mL of IL-4 and 0.8-1.2 μg/mL of sCD40L. In certain aspects, the APC induction medium comprises 10 ng/mL of IL-4 and 1 μg/mL of sCD40L. 
     In certain aspects, the one or more antigens are, or are comprised within, a pool of peptides, e.g., lyophilized peptides. Preferably, the peptides are each derived from, e.g., represent a portion of, a sequence of a protein against which the T cells are to be activated. In certain embodiments, the peptides are 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers or 20-mers. In certain embodiments, the pool of peptides are non-overlapping across a specific protein sequence from which they are derived. In certain other embodiments, the pool of peptides are overlapping across a specific protein sequence from which they are derived. In specific embodiments, such peptides may successively overlap by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the protein is a Tumor-Associated Antigen (TAA). In other embodiments, the protein is a Tumor-Specific Antigen (TSA). In specific aspects, the peptides cover the sequences of human HPV16E6, HPV16E7, HPV18E6, and/or HPV18E7 proteins. In certain aspects, such peptides comprise every amino acid residue present in human HPV16E6, HPV16E7, HPV18E6, and/or HPV18E7 proteins. In certain aspects, such peptides comprise a portion of the amino acid residues present in human HPV16E6, HPV16E7, HPV18E6, and/or HPV18E7 proteins. In a specific embodiment, the peptides cover the sequences of human HPV16E6, HPV16E7, HPV18E6, and HPV18E7 proteins. In a specific embodiment, the peptides cover the sequences of human HPV16E6 and/or HPV16E7 proteins and do not cover HPV18E6, and/or HPV18E7 proteins. In another specific embodiment, the peptides cover the sequences of human HPV18E6 and/or HPV18E7 proteins and do not cover HPV16E6, and/or HPV16E7 proteins. In specific aspects, the peptides are at a concentration of 1 μg/mL. In certain aspects, the one or more antigens are produced from exogenous genetic material by the APCs. 
     In certain embodiments, the antigen is selected from the group consisting of B-cell maturation antigen (BCMA), IL13Rα2, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, high molecular weight melanoma-associated antigen (HMW-MAA), protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal ras protein, an abnormal p53 protein, fuc-GM1, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EBNA, EF2, Epstein Barr virus antigens, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAA0205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, triosephosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Horn/Niel-40, PRAME, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD30, CD70, EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), integrin αvβ3 (CD61), galactin, and Ral-B. 
     In certain embodiments, the antigen is a tumor-associated antigen or a tumor-specific antigen. In various specific embodiments, without limitation, the tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, high molecular weight melanoma-associated antigen (HMW-MAA), protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein. 
     In certain embodiments, the TAA or TSA is a cancer/testis (CT) antigen, e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE. 
     In certain other embodiments, the TAA or TSA is a carbohydrate or ganglioside, e.g., fuc-GM1, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like. 
     In certain other embodiments, the TAA or TSA is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein Barr virus antigens, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAA0205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein. In another specific embodiment, said tumor-associated antigen or tumor-specific antigen is integrin αvβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Other tumor-associated and tumor-specific antigens are known to those in the art. 
     In certain specific embodiments, the antigen is an antigen not considered to be a TSA or a TAA, but is nevertheless associated with tumor cells, or damage caused by a tumor. In certain specific embodiments, the antigen is a neoantigen. In certain embodiments, for example, the antigen is, e.g., a growth factor, cytokine or interleukin, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines, or interleukins can include, e.g., vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors can also create a hypoxic environment local to the tumor. As such, in other specific embodiments, the antigen is a hypoxia-associated factor, e.g., HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β. Tumors can also cause localized damage to normal tissue, causing the release of molecules known as damage associated molecular pattern molecules (DAMPs; also known as alarmins). In certain other specific embodiments, therefore, the antigen is a DAMP, e.g., a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or can be a deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate. 
     In certain embodiments, the antigen is virus-associated, e.g., HPV associated. In specific embodiments, the antigen is HPV associated. In specific embodiments, the antigen is EBV associated. 
     In certain aspects, the T cell expansion medium comprises 40-60 ng/mL of IL-7, 7-11 ng/mL of IL-15, and 45-65 ng/mL of IL-4. In specific aspects, the T cell expansion medium comprises 50 ng/mL of IL-7, 9 ng/mL of IL-15, and 55 ng/mL of IL-4. 
     In certain aspects, the second T cell expansion medium comprises 40-60 ng/mL of IL-7 and 7-11 ng/mL of IL-15. In specific aspects, the second T cell expansion medium comprises 50 ng/mL of IL-7 and 9 ng/mL of IL-15. 
     In certain aspects, the duration of step (b) is 1-3 days. In specific aspects, the duration of step (b) is 1 day. In certain aspects, the duration of step (d) is 8-16 days. In specific aspects, the duration of step (d) is 12 days. 
     In certain aspects, the blood mononuclear cells, e.g., PBMCs, are isolated from whole blood, buffy coat, or an enriched leukapheresis product. In certain aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded in the APC induction medium at a density of 4-6×10 6 /cm 2 . In specific aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded at a density of 5×10 6 /cm 2 . In specific aspects, the blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are seeded at a density greater than 5×10 6 /cm 2 . 
     In certain aspects, the antigen-specific CD3+ cells are identified by intracellular cytokine staining (ICCS). In certain aspects, the culturing steps are performed in a G-REX® device. In certain aspects, one or more culturing steps are performed in T-flasks. In certain aspects, one or more culturing steps are performed in bags. In specific aspects, the bags are static bags. In certain aspects, the bags are gas-permeable. In certain aspects, one or more culturing steps are performed in a WAVE™ bioreactor. In certain aspects, one or more culturing steps are performed in a spinner flasks. 
     In certain aspects, the APC induction medium further comprises synthetic oligonucleotides with unmethylated CpG dinucleotide motifs. In certain aspects, the APC induction medium further comprises GM-CSF and IFN-α. In certain aspects, the APC induction medium comprises GM-CSF and IFN-α. 
     In a specific embodiment, said T cell population comprises about 70% or more, in some embodiments, 75%, 80%, 85%, 90%, 95%, 98%, or 99% CD3+ cells. In another specific embodiment, said T cell population comprises no less than 80%, 85%, 90%, 95%, 98%, or 99% CD3+ cells. In another specific embodiment, said T cell population comprises between 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-99% CD3+ cells. 
     In certain embodiments, said CD3+ cells in said T cell population comprise CD3+ cells that are additionally CD4+. In certain embodiments, said CD3+ cells in said T cell population comprise CD3+ cells that are additionally CD8+. In certain embodiments, said CD3+ cells in said T cell population comprise CD3+ cells that are additionally CD4+ and CD8+. In certain embodiments, said T cell population comprises CD137+ cells. In certain embodiments, said T cell population comprises CD154+ cells. In certain embodiments, said T cell population comprises cells that express Th1 cytokines. In certain embodiments, said T cell population comprises cells that express IFN-γ. In certain embodiments, said T cell population comprises cells that express TNF-α. In certain embodiments, said T cell population comprises cells that express GM-CSF. In certain embodiments, said T cell population comprises cells that express granzyme B. In certain embodiments, said T cell population comprises CD3+, CD137+, and CD154+ cells. In certain embodiments, said T cell population comprises CD3+, CD4+, CD137+, and CD154+ cells. In certain embodiments, said T cell population comprises CD3+, CD8+, CD137+, and CD154+ cells. In certain embodiments, said T cell population comprises CD3+, CD4+, CD8+, CD137+, and CD154+ cells. In certain embodiments, said T cell population comprises CD3+, CD137+, CD154+ cells, and cells that express one or more Th1 cytokines. In certain embodiments, said T cell population comprises CD3+, CD4+, CD137+, CD154+ cells, and cells that express one or more Th1 cytokines. In certain embodiments, said T cell population comprises CD3+, CD8+, CD137+, CD154+ cells, and cells that express one or more Th1 cytokines. In certain embodiments, said T cell population comprises CD3+, CD4+, CD8+, CD137+, CD154+ cells, and cells that express one or more Th1 cytokines. 
     In certain embodiments, T cells, e.g., the T cells produced by any of the methods disclosed herein, e.g., T cells that are HPV-specific, show increased expression of one or more genes as compared to PBMCs, e.g., matched PBMCs, from which they are produced, wherein said one or more genes are one or more of ACSL5, ACSL6, ACTA2, AHRR, ALDH18A1, ALDH6A1, ANAPC11, AP2B1, BAX, BCL6, BRCA1, C5, CCL13, CCL17, CCL18, CCL20, CCL22, CCL4, CCNA1, CCNA2, CCNB1, CCNB2, CCND3, CCNE2, CD40LG, CD80, CD86, CDC20, CDC25A, CDC25A, CDC25C, CDC45, CDC6, CDK1, CDK1, CDK2, CDK2, CDK4, CDK5, CDK6, CDT1, CHEK2, CKLF, COPG2, CXADR, CXCL16, CXCL2, CXCL8, CXCR5, CXCR5, CYB5A, CYP1A1, CYP1B1, DHFR, E2F1, ESPL1, FADS1, FADS2, FAS, FBX05, FGFR1, FGFR1, FLOT1, FN1, FOS, FYN, FYN, GADD45B, GALK1, GALK2, GMDS, GMPPA, GSTA4, GSTM4, HLA-DMB, HLA-DQA1, HLA-DQB1, HLA-DRA, IFNGR1, IL12RB1, IL12RB2, IL13, IL1B, IL1B, IL21R, IL2RA, IL2RG, IL4, IL5, IL6ST, IRS2, IRS2, ITGA1, ITGA4, ITGA5, ITGA6, ITGAL, ITGAX, ITGB1, ITGB7, JAM3, JUN, KIF11, KIF23, MAP3K2, MCM2, MCM3, MCM4, MCM5, MGST2, MGST3, MMP25, MYL6B, NQ01, NQ02, NRIP1, PCNA, PHGDH, PIK3C2A, PIK3CA, PLK2, PLK3, PLK4, POLA1, PPM1L, PPP2R3B, PRC1, PRIM1, PRKD3, PSAT1, PSPH, PTTG1, RAC2, RAC3, RAD50, RAD51, RARA, RBBP8, RRAS, SDC4, SELPLG, SHMT1, SHMT2, SLC27A2, SMC2, STAT1, TFDP1, TGFB1, TGFBR2, TGM2, TOP2A, TSTA3, UGDH, XCL1, XCL2, ZEB1, and/or ZNF420. In certain embodiments, the increased expression is 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more. In certain other embodiments, T cells, e.g., the T cells produced by any of the methods disclosed herein, e.g., T cells that are HPV-specific, show increased expression of one or more genes as compared to upulsed cells that have otherwise been produced by the methods described herein (that is, T cells not exposed to a PepMix), wherein said one or more genes are one or more of CCL18 (chemokine (C-C motif) Ligand 18); CH13L1 (Chitinase-3-like protein); FN1 (Fibronectin 1); LYZ (Lysozyme); RCHY1 (Ring finger and CHY zinc finger 1); and PALLD (Palladin, cytoskeletal associated protein). Such T cells may be used in, e.g., any of the methods of treatment described herein. 
     5.4 Compositions Comprising T Cells 
     In some embodiments, provided herein is a composition, e.g., a pharmaceutical composition, comprising a T cell population produced using the methods described herein. In a specific embodiment, said T cell population comprises at least 50% of cells in the composition. In another specific embodiment, said T cell population, e.g., CD3+ cells, comprises at least 80%, 85%, 90%. 95%, 98% or 99% of cells in the composition. In certain embodiments, no more than 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% of the cells in said T cell population are CD3+ cells. In certain embodiments, said CD3+ cells are CD4+ and/or CD8+. In certain embodiments, said CD3+ cells are CD137+. In certain embodiments, said CD3+ cells are CD154+. In certain embodiments, said CD3+ cells express Th1 cytokines. In certain embodiments, said CD3+ cells express IFN-γ. In certain embodiments, CD3+ cells express TNF-α. In certain embodiments, CD3+ cells express GM-CSF. In certain embodiments, CD3+ cells express granzyme B. In certain embodiments, said CD3+ cells are CD137+ and CD154+. In certain embodiments, said CD3+ cells are CD4+, CD137+, and CD154+. In certain embodiments, said CD3+ cells are CD8+, CD137+, and CD154+. In certain embodiments, said CD3+ cells are CD4+, CD8+, CD137+, and CD154+. In certain embodiments, said CD3+ cells are CD137+, CD154+, and express one or more Th1 cytokines. In certain embodiments, said CD3+ cells are CD4+, CD137+, CD154+, and express one or more Th1 cytokines. In certain embodiments, said CD3+ cells are CD8+, CD137+, CD154+, and express one or more Th1 cytokines. In certain embodiments, said CD3+ cells are CD4+, CD8+, CD137+, CD154+, and express one or more Th1 cytokines. 
     In a specific embodiment, said T cells in said composition are from the same individual as the individual for whom reintroduction of, e.g., treatment with, the T cells is intended (i.e., the T cells are autologous to the intended recipient). In another specific embodiment, said T cells in said composition are from a different individual than the individual for whom reintroduction of, e.g., treatment with, the T cells is intended (i.e., the T cells are not autologous to the intended recipient). In another specific embodiment, said composition additionally comprises an immunomodulatory compound or thalidomide. In certain embodiments, the immunomodulatory compound is a compound described below. See, e.g., U.S. Pat. No. 7,498,171, the disclosure of which is hereby incorporated by reference in its entirety. In certain embodiments, the immunomodulatory compound is an amino-substituted isoindoline. In one embodiment, the immunomodulatory compound is 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione; 3-(4′aminoisolindoline-1′-one)-1-piperidine-2,6-dione; 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; or 4-Amino-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione. In another embodiment, the immunomodulatory compound is pomalidomide, or lenalidomide. In another embodiment, said immunomodulatory compound is a compound having the structure 
     
       
         
         
             
             
         
       
     
     wherein one of X and Y is C═O the other of X and Y is C═O or CH 2 , and R 2  is hydrogen or lower alkyl, or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. In another embodiment, said immunomodulatory compound is a compound having the structure 
     
       
         
         
             
             
         
       
     
     wherein one of X and Y is C═O and the other is CH 2  or C═O; 
     R 1  is H, (C 1 -C 8 )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 0 -C 4 )alkyl-(C 1 -C 6 )heterocycloalkyl, (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl, C(O)R 3 , C(S)R 3 , C(O)OR 4 , (C 1 -C 8 )alkyl-N(R 6 ) 2 , (C 1 -C 8 )alkyl-OR 5 , (C 1 -C 8 )alkyl-C(O)OR 5 , C(O)NHR 3 , C(S)NHR 3 , C(O)NR 3 R 3′ , C(S)NR 3 R 3′  or (C 1 -C 8 )alkyl-O(CO)R 5 ; 
     R 2  is H, F, benzyl, (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, or (C 2 -C 8 )alkynyl; 
     R 3  and R 3′  are independently (C 1 -C 8 )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 0 -C 4 )alkyl-(C 1 -C 6 )heterocycloalkyl, (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl, (C 0 -C 8 )alkyl-N(R 6 ) 2 , (C 1 -C 8 )alkyl-OR 5 , (C 1 -C 8 )alkyl-C(O)OR 5 , (C 1 -C 8 )alkyl-O(CO)R 5 , or C(O)OR 5 ; 
     R 4  is (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (C 1 -C 4 )alkyl-OR 5 , benzyl, aryl, (C 0 -C 4 )alkyl-(C 1 -C 6 )heterocycloalkyl, or (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl; 
     R 5  is (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, or (C 2 -C 5 )heteroaryl; 
     each occurrence of R 6  is independently H, (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 2 -C 5 )heteroaryl, or (C 0 -C 8 )alkyl-C(O)O—R 5  or the R 6  groups can join to form a heterocycloalkyl group; 
     n is 0 or 1; and 
     * represents a chiral-carbon center; 
     or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. In another embodiment, said immunomodulatory compound is a compound having the structure 
     
       
         
         
             
             
         
       
     
     wherein: 
     one of X and Y is C═O and the other is CH 2  or C═O; 
     R is H or CH 2 OCOR′; 
     (i) each of R 1 , R 2 , R 3 , or R 4 , independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , or R 4  is nitro or —NHR 5  and the remaining of R 1 , R 2 , R 3 , or R 4  are hydrogen; 
     R 5  is hydrogen or alkyl of 1 to 8 carbons 
     R 6  hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro; 
     R′ is R7-CHR 10 —N(R 8 R 9 ); 
     R 7  is m-phenylene or p-phenylene or —(C n H 2n )— in which n has a value of 0 to 4; 
     each of R 8  and R 9  taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R 8  and R 9  taken together are tetramethylene, pentamethylene, hexamethylene, or —CH 2 CH 2 X 1 CH 2 CH 2 — in which X 1  is —O—, —S—, or —NH—; 
     R 10  is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and 
     * represents a chiral-carbon center; 
     or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. 
     In another specific embodiment, the composition additionally comprises one or more anticancer compounds, e.g., one or more of the anticancer compounds described below. In another specific embodiment, the composition additionally comprises an immunomodulatory drug or an epigenetic modifier. In another specific embodiment, the composition comprises an immune checkpoint inhibitor. In more specific embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD1 antibody, and an anti-PD-L1 antibody. 
     In certain embodiments, the antigen-specific T cells are formulated in a pharmaceutically-acceptable solution. In preferred embodiments, the pharmaceutically-acceptable solution is suitable for the delivery of living cells. In specific embodiments, the pharmaceutically-acceptable solution is, for example, saline solution (such as Ringer&#39;s solution), gelatins, carbohydrates (e.g., lactose, amylose, starch, or the like), fatty acid esters, hydroxymethylcellulose, or polyvinyl pyrolidine. In more specific embodiments, the pharmaceutically-acceptable solution is sterilized prior to addition of the cells. In other more specific embodiments, the pharmaceutically-acceptable solution may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring. Pharmaceutical carriers suitable for use in formulating the cells are known in the art and are described, for example, in WO 96/05309. In specific embodiments, the pharmaceutically-acceptable solution comprises one or more of dextran-40, human serum albumin, Plasma Lyte A, sodium chloride, and dimethyl sulfoxide. In certain embodiments, the antigen-specific T cells are formulated in a solution with a cryoprotectant. In specific embodiments, the cryoprotectant is dimethyl sulfoxide (DMSO). 
     In certain embodiments, the antigen-specific T cells produced by the methods described herein are provided in one or more infusion bags. 
     5.5 Uses of Antigen-Specific T Cells 
     In certain embodiments, provided herein are methods of treating a cancer or a viral infection comprising administering to a patient in need thereof a population of antigen-specific T cells produced by the methods described herein, e.g., from isolated blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells. In certain embodiments, the isolated blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are autologous to the patient. In certain embodiments, the isolated blood mononuclear cells, e.g., PBMCs or cord blood mononuclear cells, are not autologous to the patient. 
     The antigen-specific T cells produced using the methods described herein can be used in methods of treating individuals having cancer, e.g., individuals having solid tumor cells and/or blood cancer cells, or persons having a viral infection. The antigen-specific T cells produced using the methods described herein can also be used in methods of treating individuals at risk of developing, or to prevent the development of, cancer. 
     5.5.1 Treatment of Individuals Having Cancer 
     In one embodiment, provided herein is a method of treating an individual having a cancer, for example, a blood cancer or a solid tumor, comprising administering to said individual a therapeutically effective amount of antigen-specific T cells produced using the methods described herein. In one embodiment, provided herein is a method of treating an individual at risk of developing cancer, for example, a blood cancer or a solid tumor, comprising administering to said individual a therapeutically effective amount of antigen-specific T cells produced using the methods described herein. In specific embodiments, the T cells are specific to an antigen expressed by the cancer. In more specific embodiments, the T cells are specific to one or more of the tumor-associated antigens or tumor-specific antigens disclosed in Section 5.3, above. In certain embodiments, the T cells are antigen-specific T cells produced by any of the methods described herein. In certain embodiments, the individual has a deficiency of T cells, e.g., a deficiency of T cells active against the individual&#39;s cancer. In certain embodiments, the individual has too few T cells. In certain embodiments, the individual has T cells with suppressed activity. As used herein, an “effective amount” is an amount that, e.g., results in a detectable improvement of, lessening of the progression of, or elimination of, one or more symptoms of a cancer from which the individual suffers. 
     In specific aspects, the individual has primary ductal carcinoma, leukemia, acute T cell leukemia, chronic myeloid lymphoma, acute myelogenous leukemia, multiple myeloma, chronic myelogenous leukemia, lung carcinoma, colon adenocarcinoma, histiocytic lymphoma, multiple myeloma, retinoblastoma, or colorectal carcinoma. 
     In certain embodiments, provided herein are methods of treating an individual having an HPV positive (HPV+) cancer. In specific embodiments, the HPV+ cancer is head and neck cancer. In more specific embodiments, the HPV+ cancer is squamous cell carcinoma of the head and neck (SCCHN). In specific embodiments, the HPV+ cancer is oropharyngeal cancer. In certain aspects, the HPV+ cancer is penile cancer. In certain aspects, the HPV+ cancer is cervical cancer. In certain aspects, the HPV+ cancer is anal cancer. In certain aspects, the HPV+ cancer is vulval cancer. In certain aspects, the HPV+ cancer is vaginal cancer. In certain aspects, the HPV+ cancer is lung cancer. In specific embodiments, the HPV+ cancer is HPV-16+. In specific embodiments, the HPV+ cancer is HPV-18+. In specific embodiments, the HPV+ cancer is HPV-16+ and HPV-18+. In certain embodiments, the HPV+ cancer is metastatic. In certain embodiments, the HPV+ cancer is recurrent. In certain embodiments, the HPV+ cancer is metastatic and recurrent. In certain embodiments, provided herein are methods of treating an individual who has an HPV+ cancer, as determined by immunohistochemistry. In certain embodiments, provided herein are methods of treating an individual who has an HPV+ cancer, as determined by polymerase chain reaction. In certain embodiments, provided herein are methods of treating an individual who has an HPV+ cancer, as determined by RNA fluorescence in situ hybridization. In certain embodiments, provided herein are methods of treating an individual with an HPV+ cancer who has been previously treated with cetuximab. In certain embodiments, provided herein are methods of treating an individual with an HPV+ cancer who has been previously treated with a platinum-based doublet. In certain embodiments, provided herein are methods of treating an individual with an HPV+ cancer who has been previously treated with cetuximab and a platinum-based doublet. In certain embodiments, the platinum based doublet is cisplatin/5-FU. In certain embodiments, the platinum based doublet is carboplatin/5-FU. In certain embodiments, provided herein are methods of treating an individual with an HPV+ cancer who has been previously treated with a PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is pembrolizumab (KEYTRUDA®; Merck). In certain embodiments, the PD-1 inhibitor is nivolumab (OPDIVO®; Bristol-Myers Squibb). In certain embodiments, provided herein are methods of treating an individual with an HPV+ cancer who has been previously treated with a PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor is durvalumab (MedImmune). 
     In certain aspects, the method further comprises administering to any of the above said individuals an immunomodulatory drug or an epigenetic modifier. In specific aspects, the method further comprises administering to said individual an immune checkpoint inhibitor. In more specific aspects, the immune checkpoint inhibitor is selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD1 antibody, and an anti-PD-L1 antibody. In certain embodiments, the PD-1 inhibitor is pembrolizumab (KEYTRUDA®; Merck). In certain embodiments, the PD-1 inhibitor is nivolumab (OPDIVO®; Bristol-Myers Squibb). In certain other embodiments, the PD-L1 inhibitor is durvalumab (MedImmune). In another specific embodiment, the anti-CTLA4 antibody is ipilimumab (YERVOY®; Bristol-Meyers Squibb). 
     In specific embodiments, administration of an isolated population of T cells or a pharmaceutical composition thereof to a subject is by injection, infusion, intravenous (IV) administration, intrafemoral administration, or intratumor administration. In specific embodiments, administration of an isolated population of T cells or a pharmaceutical composition thereof to a subject is performed with a devise, a matrix, or a scaffold. 
     5.5.2 Treatment of Cancers Using T Cells and Other Anticancer Agents 
     Treatment of an individual having cancer using the antigen-specific T cells produced using the methods described herein can be part of an anticancer therapy regimen that includes one or more other anticancer agents. In addition or alternatively, treatment of an individual having cancer using the T cells produced using the methods described herein can be used to supplement an anticancer therapy that includes one or more other anticancer agents. Such anticancer agents are well-known in the art and include anti-inflammatory agents, immumodulatory agents, cytotoxic agents, cancer vaccines, chemotherapeutics, MAC inhibitors, and siRNAs. Specific anticancer agents that may be administered to an individual having cancer, e.g., an individual having tumor cells, in addition to the antigen-specific T cells produced using the methods described herein include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin; adrucil; aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase (e.g., from  Erwinia  chrysan; Erwinaze); asperlin; avastin (bevacizumab); azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor); Cerubidine; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; Elspar; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; Etopophos; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; Idamycin; idarubicin hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; Proleukin; Purinethol; puromycin; puromycin hydrochloride; pyrazofurin; Rheumatrex; riboprine; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; Tabloid; talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; Toposar; toremifene citrate; trestolone acetate; Trexall; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin hydrochloride. 
     Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-azacytidine; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptosar (also called Campto; irinotecan) camptothecin derivatives; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; CC-122; CC-486; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidenmin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine (e.g., Fludara); fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC®), imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+ estrogen+ progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; anti-EGFR antibody (e.g., Erbitux (cetuximab)); anti-CD19 antibody; anti-CD20 antibody (e.g., rituximab); anti-disialoganglioside (GD2) antibody (e.g., monoclonal antibody 3F8 or ch14&gt;18); anti-ErbB2 antibody (e.g., herceptin); human chorionic gonadotrophin; monophosphoryl lipid A+ myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen (GENASENSE®); O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin (e.g., Floxatin); oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RH retinamide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; Vectibix (panitumumab)velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; Welcovorin (leucovorin); Xeloda (capecitabine); zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. 
     5.5.3 Treatment of Viral Infection 
     In another embodiment, provided herein is a method of treating an individual having a viral infection, e.g., and active viral infection or a dormant viral infection, comprising administering to said individual a therapeutically effective amount of antigen-specific T cells produced using the methods described herein. In certain embodiments, the individual has a deficiency of T cells, e.g., a deficiency of T cells active against the individual&#39;s viral infection. In certain embodiments, the individual has too few T cells. In certain embodiments, the individual has T cells with suppressed activity. In certain other specific embodiments, said administering comprises administering an immunomodulatory compound, e.g., an immunomodulatory compound described above, or thalidomide, to said individual in addition to said antigen-specific T cells produced using the methods described herein, wherein said amount is an amount that, e.g., results in a detectable improvement of, lessening of the progression of, or elimination of, one or more symptoms of said viral infection. In specific embodiments, the viral infection is an infection by a virus of the human papillomavirus (HPV) family. In specific embodiments, the viral infection is HPV. In specific embodiments, the viral infection is a member of the herpes family. In specific embodiments, the viral infection is EBV. In specific embodiments, the viral infection is a member of the hepatitis C virus (HCV) family. In specific embodiments, the viral infection is HCV. 
     In other embodiments, the antigen-specific T cells produced using the methods described herein are administered to an individual having a viral infection as part of an antiviral therapy regimen that includes one or more other antiviral agents. In other embodiments, the antigen-specific T cells produced using the methods described herein are administered to an individual at risk of developing a viral infection as part of an antiviral therapy regimen that includes one or more other antiviral agents. Specific antiviral agents that may be administered to an individual having a viral infection include, but are not limited to: imiquimod, podofilox, podophyllin, interferon alpha (IFNα), reticolos, nonoxynol-9, acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine, rimantadine; ribavirin; zanamavir and oseltaumavir; protease inhibitors such as indinavir, nelfinavir, ritonavir, or saquinavir; nucleoside reverse transcriptase inhibitors such as didanosine, lamivudine, stavudine, zalcitabine, or zidovudine; and non-nucleoside reverse transcriptase inhibitors such as nevirapine, or efavirenz. 
     5.5.4 Administration 
     In certain embodiments, T cells, e.g., antigen-specific T cells, produced using the methods described herein are used, e.g., administered to an individual, in any amount or number that results in a detectable therapeutic benefit to the individual, e.g., an effective amount, wherein the individual has a viral infection, cancer, or tumor cells, for example, an individual having tumor cells, a solid tumor or a blood cancer, e.g., a cancer patient. Such cells can be administered to such an individual by absolute numbers of cells, e.g., said individual can be administered at about, at least about, or at most about, 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , or 1×10 11  T cells produced using the methods described herein. In other embodiments, antigen-specific T cells produced using the methods described herein can be administered to such an individual by relative numbers of cells, e.g., said individual can be administered at about, at least about, or at most about, 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , or 1×10 11  T cells produced using the methods described herein per kilogram of the individual. In other embodiments, antigen-specific T cells produced using the methods described herein can be administered to such an individual by relative numbers of cells, e.g., said individual can be administered at about, at least about, or at most about, 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , or 5×10 8  T cells produced using the methods described herein per kilogram of the individual. 
     In certain embodiments, antigen-specific T cells produced using the methods described herein are administered to a subject according to the subject&#39;s body surface area. In certain embodiments, antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 1×10 7  to 1×10 10  cells/m 2 . In certain embodiments, antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 5×10 7 , 1.5×10 8 , 4.5×10 8 , 1.3×10 9 , or 2×10 9  cells/m 2 . In a specific embodiment, antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 5×10 7  cells/m 2 . In a specific embodiment, antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 1.5×10 8  cells/m 2 . In a specific embodiment, antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 4.5×10 8  cells/m 2 . In a specific embodiment, antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 1.3×10 9  cells/m 2 . In a specific embodiment, antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 2×10 9  cells/m 2 . In other embodiments, the antigen-specific T cells produced using the methods described herein are administered to a subject at a dose of 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , or 1×10 10  cells per kilogram. 
     In certain embodiments, the subject undergoes leukapheresis prior to administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, the subject receives a first therapy prior to administration of antigen-specific T cells produced using the methods described herein. In specific embodiments, the first therapy is chemotherapy. In specific embodiments, the first therapy is 1, 2, 3, 4, 5, or 6 rounds of chemotherapy. In more specific embodiments, the subject receives one round of chemotherapy prior to administration of antigen-specific T cells produced using the methods described herein. In more specific embodiments, the subject receives two rounds of chemotherapy prior to administration of antigen-specific T cells produced using the methods described herein. In more specific embodiments, the subject receives three rounds of chemotherapy prior to administration of antigen-specific T cells produced using the methods described herein. In more specific embodiments, the subject receives four rounds of chemotherapy prior to administration of antigen-specific T cells produced using the methods described herein. In more specific embodiments, the subject receives five rounds of chemotherapy prior to administration of antigen-specific T cells produced using the methods described herein. In more specific embodiments, the subject receives six rounds of chemotherapy prior to administration of antigen-specific T cells produced using the methods described herein. 
     In certain embodiments, after leukapheresis, T cells to be administered to the subject are manufactured, tested, and released for use over the course of 28 days. In certain embodiments, the subject undergoes a lymphodepletion regiment prior to administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, the lymphodepletion regimen is 1, 2, 3, 4, 5, or 6 days. In specific embodiments, the lymphodepletion regimen is 3 days. In certain embodiments, the lymphodepletion regimen is a single agent lymphodepletion regiment. In certain embodiments, the lymphodepletion regimen is a two agent lymphodepletion regiment. In certain embodiments, the single agent is cyclophosphamide. In certain embodiments, the two agents are cyclophosphamide and fludarabine. In certain embodiments, the cyclophosphamide is administered at 300 mg/m 2 /day. In specific embodiments, the cyclophosphamide is administered at 300 mg/m 2 /day for three days. In certain embodiments, the cyclophosphamide is administered at 900 mg/m 2 /day. In specific embodiments, the cyclophosphamide is administered at 900 mg/m 2 /day for three days. In certain embodiments, the cyclophosphamide is administered at 300 mg/m 2 /day and the fludarabine is administered at 30 mg/kg/day. In specific embodiments, the cyclophosphamide is administered at 300 mg/m 2 /day for three days and the fludarabine is administered at 30 mg/kg/day for two days. In certain embodiments, administration of antigen-specific T cells produced using the methods described herein begins 1, 2, 3, 4, or 5 days after the last day of the lymphodepletion regimen. In specific embodiments, administration of antigen-specific T cells produced using the methods described herein begins two days after the last day of the lymphodepletion regimen. 
     In certain embodiments, acetaminophen is administered to the subject prior to administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, diphenhydramine is administered to the subject prior to administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, acetaminophen and diphenhydramine are administered to the subject prior to administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, acetaminophen is administered to the subject after administration of antigen-specific T cells produced using the methods described herein. In specific embodiments, acetaminophen is administered four hours after administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, diphenhydramine is administered to the subject after administration of antigen-specific T cells produced using the methods described herein. In specific embodiments, diphenhydramine is administered four hours after administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, acetaminophen and diphenhydramine are administered to the subject after administration of antigen-specific T cells produced using the methods described herein. In specific embodiments, acetaminophen and diphenhydramine are administered four hours after administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, acetaminophen is administered to the subject prior to and after administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, diphenhydramine is administered to the subject prior to and after administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, acetaminophen and diphenhydramine are administered to the subject prior to and after administration of antigen-specific T cells produced using the methods described herein. In certain embodiments, acetaminophen is administered orally. In certain embodiments, diphenhydramine is administered orally. In certain embodiments, diphenhydramine is administered intravenously. In certain embodiments, diphenhydramine is administered intramuscularly. In certain embodiments, acetaminophen is administered at a dose of 300-1000 mg. In specific embodiments, acetaminophen is administered at a dose of 650 mg. In certain embodiments, diphenhydramine is administered at a dose of 10-40 mg. In specific embodiments, diphenhydramine is administered at a dose of 25 mg. In a specific embodiment, acetaminophen is administered orally at 650 mg and diphenhydramine is administered orally at 25 mg both before and after administration of antigen-specific T cells produced using the methods described herein. 
     The antigen-specific T cells produced using the methods described herein can be administered once to an individual having a viral infection, an individual having cancer, or an individual having tumor cells (whether detectable or undetectable), during a course of anticancer therapy; or can be administered multiple times, e.g., once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 36 or more weeks during therapy. In specific embodiments, T cells produced using the methods described herein are administered once to an individual having a viral infection, an individual having cancer, or an individual having tumor cells, during a course of anticancer therapy. In specific embodiments, T cells produced using the methods described herein are administered once to an individual at risk of developing a viral infection, an individual at risk of developing cancer, or an individual at risk of developing tumor cells, during a course of anticancer therapy. In specific embodiments, T cells produced using the methods described herein are administered once to an individual to prevent recurrence of a viral infection, cancer, or tumor cells, during a course of anticancer therapy. In specific embodiments, antigen-specific T cells produced using the methods described herein can be administered once to an individual having a viral infection every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours. In specific embodiments, antigen-specific T cells produced using the methods described herein can be administered once to an individual having a viral infection every 1, 2, 3, 4, 5, 6 or 7 days. In specific embodiments, antigen-specific T cells produced using the methods described herein can be administered once to an individual having a viral infection every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 36 or more weeks. In specific embodiments, antigen-specific T cells produced using the methods described herein can be administered once to an individual having cancer every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours. In specific embodiments, antigen-specific T cells produced using the methods described herein can be administered once to an individual having cancer every 1, 2, 3, 4, 5, 6 or 7 days. In specific embodiments, antigen-specific T cells produced using the methods described herein can be administered once to an individual having cancer every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 36 or more weeks. In embodiments in which cells and an antiviral compound or anticancer compound are used, the antiviral compound or anticancer compound, and cells, can be administered to the individual together, e.g., in the same formulation; separately, e.g., in separate formulations, at approximately the same time; or can be administered separately, e.g., on different dosing schedules or at different times of the day. The antigen-specific T cells produced using the methods described herein can be administered without regard to whether T cells produced using the methods described herein have been administered to the individual in the past. In one embodiment, the T cells are administered to the individual before a second agent. In another embodiment, a second agent is administered before the T cells. In another embodiment, the T cells and the second agent are administered simultaneously. 
     5.6 Kits 
     Provided herein is a pharmaceutical pack or kit comprising one or more containers comprising one or more of the compositions described herein, e.g., a composition comprising antigen-specific T cells produced by a method described herein. The kit may be usable standalone or may be used with automated cell production machinery in order of produce the T cells described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. 
     The kits encompassed herein can be used in accordance with the methods described herein, e.g., methods of treating cancer and/or methods of treating viral infection. In one embodiment, a kit comprises antigen-specific T cells produced by a method described herein or a composition thereof, in one or more containers. In a specific embodiment, provided herein is a kit comprising a T cell population produced by a method described herein, or a composition thereof. In another embodiment, provided herein is a kit comprising the components, e.g., the media or the components thereof, necessary for producing the antigen-specific T cells by the methods described herein. 
     In one embodiment, a kit comprises (i) T cells produced by a method described herein or a composition thereof, in one or more containers, and (ii) acetaminophen in one or more containers, and/or (iii) diphenhydramine in one or more containers. In one embodiment, a kit comprises (i) T cells produced by a method described herein or a composition thereof, in one or more containers, and (ii) cyclophosphamide in one or more containers, and/or (iii) fludarabine in one or more containers. In one embodiment, a kit comprises (i) T cells produced by a method described herein or a composition thereof, in one or more containers, and (ii) acetaminophen in one or more containers, and/or (iii) diphenhydramine in one or more containers, and (iv) cyclophosphamide in one or more containers, and/or (v) fludarabine in one or more containers. 
     6. EXAMPLES 
     Provided herein are data on varied processes described herein that can be used to establish a wide range of possible conditions described herein. Specific examples provided do not preclude the possibility of other working examples. 
     6.1 Example 1: APC Induction Cocktails 
     Combinations of APC induction cocktails were evaluated in HPV T cell generation (Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Combinations of APC induction cocktails 
               
            
           
           
               
               
               
            
               
                   
                 Condition# 
                 Cytokine Cocktail Description 
               
               
                   
                   
               
               
                   
                 C1 
                 IL-4/CD40L 
               
               
                   
                 C2 
                 IL-4/CD40L/CpG 
               
               
                   
                 C3 
                 GM-CSF/IFN-α 
               
               
                   
                 C4 
                 GM-CSF/IL-4 + TNF-α/IL-1β/IL-6 
               
               
                   
                 C5 
                 GM-CSF/IL-4/CD40L 
               
               
                   
                 C6 
                 GM-CSF/IL-4/CD40L/CpG 
               
               
                   
                 C7 
                 GM-CSF/IFN-α/IL-4/CD40L 
               
               
                   
                 C8 
                 GM-CSF/IFN-α/IL-4/CD40L/CpG 
               
               
                   
                 C9 
                 GM-CSF/IL-4/CD40L + TNF-α/IL-1β/IL-6 
               
               
                   
                 C10 
                 GM-CSF/IL-4/CD40L/CpG + TNF-α/IL-1β/IL-6 
               
               
                   
                   
               
            
           
         
       
     
     PBMCs were cultured in G-REX®-24 from the initial seeding till the final T cell harvest. PGE 2  was eliminated from the process. PBMC were incubated with APC induction cytokine for 3 days prior to HPV PEPMIX™ stimulation. The other process elements in this experiment are listed in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Other parameters for the APC induction cocktail experiment. 
               
            
           
           
               
               
               
            
               
                   
                 Parameters 
                 Condition 
               
               
                   
                   
               
               
                   
                 PBMC 
                 Fresh 
               
               
                   
                 Induction Cytokine 
                 Various 
               
            
           
           
               
               
               
               
            
               
                   
                 IL-4 
                 40 
                 ng/ml 
               
               
                   
                 CD40L 
                 1 
                 μg/ml 
               
               
                   
                 CpG 
                 4 
                 μg/ml 
               
               
                   
                 GM-CSF 
                 800 
                 U/ml 
               
               
                   
                 IFN-α-2b 
                 1000 
                 U/ml 
               
               
                   
                 TNF-α 
                 10 
                 ng/ml 
               
               
                   
                 IL-1β 
                 10 
                 ng/ml 
               
               
                   
                 IL-6 
                 10 
                 ng/ml 
               
            
           
           
               
               
               
            
               
                   
                 Induction Medium 
                 IMDM 
               
               
                   
                 Seeding density 
                 5 × 10e6/well 
               
               
                   
                 Reproducibility 
                 Replicate/condition 
               
            
           
           
               
               
               
               
            
               
                   
                 Induction Duration 
                 3 
                 days 
               
               
                   
                 PEPMIX ™ 
                 1 
                 μg/ml 
               
               
                   
                 Cytokine Feeding Delay 
                 48 
                 hours 
               
               
                   
                 T cell Expansion Cytokine 
               
               
                   
                 IL-4 
                 55 
                 ng/ml 
               
               
                   
                 IL-7 
                 10 
                 ng/ml 
               
               
                   
                 IL-15 
                 1 
                 ng/ml 
               
               
                   
                 Expansion Duration 
                 13 
                 days 
               
               
                   
                   
               
            
           
         
       
     
     The cell number was counted using ViCELL XR after harvest at day 13 post antigen loading. As shown in  FIG. 1 , cells expanded to a different extent under the same APC induction conditions in different donors, ranging from about 2 fold to 6 fold expansion. HPV T cell frequency and the yield are shown in  FIGS. 2 and 3 , respectively. HPV T cell yield takes both total cell yield and HPV T cell frequency into consideration, making it a reasonable factor for culturing condition evaluation. Accordingly, the mean HPV T cell yield is obtained by averaging the T cell yield of the replicates for the same condition. 
     6.2 Example 2: Expansion Cytokines 
     The experimental design of definition of expansion cytokines used the following concentration ranges for each cytokine: IL-4, 15 ng/ml to 55 ng/ml; IL-7, 10 ng/ml to 50 ng/ml; IL-15, 1 ng/ml to 10 ng/ml. No IL-4 (IL-7+1L-15 only) and no IL-15 (IL-4+IL-7 only) were also included. The detailed conditions are listed as Table 4. The process used 1 day APC induction with CpG, sCD40L and IL-4 as shown in Table 5. The dose of PEPMIX™ used was 1 μg/ml. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Expansion cytokine combinations. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Seeding 
                   
                   
                   
                 Expansion 
               
               
                 Runs 
                 APC induction 
                 density 
                 IL-15 
                 IL-4 
                 IL-7 
                 days 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 C1 
                 IL-4 10 
                 ng/ml, 
                 1.75M/CM 2   
                 1 
                 ng/ml 
                 15 
                 ng/ml 
                 10 
                 ng/ml 
                 12 days 
               
               
                 C2 
                 CD40L 1 
                 μg/ml, 
                   
                 1 
                 ng/ml 
                 15 
                 ng/ml 
                 50 
                 ng/ml 
               
               
                 C3 
                 CpG 4 
                 μg/ml 
                   
                 1 
                 ng/ml 
                 35 
                 ng/ml 
                 30 
                 ng/ml 
               
               
                 C4 
                   
                   
                   
                 1 
                 ng/ml 
                 55 
                 ng/ml 
                 10 
                 ng/ml 
               
               
                 C5 
                   
                   
                   
                 1 
                 ng/ml 
                 55 
                 ng/ml 
                 50 
                 ng/ml 
               
               
                 C6 
                   
                   
                   
                 5.5 
                 ng/ml 
                 15 
                 ng/ml 
                 10 
                 ng/ml 
               
               
                 C7 
                   
                   
                   
                 5.5 
                 ng/ml 
                 15 
                 ng/ml 
                 50 
                 ng/ml 
               
               
                 C8 
                   
                   
                   
                 5.5 
                 ng/ml 
                 35 
                 ng/ml 
                 30 
                 ng/ml 
               
               
                 C9 
                   
                   
                   
                 5.5 
                 ng/ml 
                 55 
                 ng/ml 
                 10 
                 ng/ml 
               
               
                 C10 
                   
                   
                   
                 5.5 
                 ng/ml 
                 55 
                 ng/ml 
                 50 
                 ng/ml 
               
               
                 C11 
                   
                   
                   
                 10 
                 ng/ml 
                 15 
                 ng/ml 
                 10 
                 ng/ml 
               
               
                 C12 
                   
                   
                   
                 10 
                 ng/ml 
                 15 
                 ng/ml 
                 50 
                 ng/ml 
               
               
                 C13 
                   
                   
                   
                 10 
                 ng/ml 
                 35 
                 ng/ml 
                 30 
                 ng/ml 
               
               
                 C14 
                   
                   
                   
                 10 
                 ng/ml 
                 55 
                 ng/ml 
                 10 
                 ng/ml 
               
               
                 C15 
                   
                   
                   
                 10 
                 ng/ml 
                 55 
                 ng/ml 
                 50 
                 ng/ml 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 C16 
                   
                   
                   
                 0 
                 55 
                 ng/ml 
                 10 
                 ng/ml 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 C17 
                   
                   
                   
                 5.5 
                 ng/ml 
                 0 
                 30 
                 ng/ml 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 APC induction conditions for the expansion 
               
               
                 cytokine combination experiment. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 2xcytokine 
                   
               
               
                 Reagent 
                 Stock Conc. 
                 Final Conc. 
                 cocktail 
                 Volume/1 ml 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 IL-4 
                 100 
                 μg/ml 
                 20 
                 ng/ml 
                 40 
                 ng/ml 
                 0.4 
                 μl 
               
               
                 CD40L 
                 500 
                 μg/ml 
                 1 
                 μg/ml 
                 2 
                 μg/ml 
                 4 
                 μl 
               
               
                 CpG 
                 4 
                 mg/ml 
                 5 
                 ug/ml 
                 10 
                 μg/ml 
                 2.5 
                 μl 
               
               
                   
               
            
           
         
       
     
     Total cell expansion at harvest illustrated in  FIG. 4 . T cells had significantly lower expansion at lower IL-15 concentration (IL-15=1 ng/ml) than those at higher concentrations (IL-15=5.5 or 10 ng/ml). There was no difference between IL-15 concentration at 5.5 ng/ml and 10 ng/ml. The condition without IL-15 had least expansion, whereas, the one without IL-4 didn&#39;t affect the T cell expansion. 
     T cell frequency was measured by ICCS ( FIG. 5 ). ICCS showed a wide range of variation in T cell frequency cross all the donors in different cytokine expansion conditions. In all donors, the condition without IL-15 successfully generated T cells, but at a lower frequency than in other conditions. The condition with three cytokines in their highest concentrations (IL-4=55 ng/ml, IL-7=50 ng/ml and IL-15=10 ng/ml) had less variation in the duplicates and was on the top list for T cell frequency. 
     Taken both T cell expansion and T cell frequency into consideration, T cell yield was illustrated in  FIG. 6 . The conditions without IL-15 or with lower concentration (1 ng/ml) of IL-15 generated lower HPV T cell in all the donors. The condition with three cytokines in their highest concentrations (IL-4=55 ng/ml, IL-7=50 ng/ml and IL-15=10 ng/ml) had highest T cell yield and less variation in the duplicates. 
     In addition to ICCS assay, secreted cytokines were measured by 8-plex cytometrix beads array (CBA). The panel included Th1 cytokines (IL-2, IFN-γ and TNF-α), Th2 cytokines (IL-5, IL-13), Treg (IL-10) and T cell effector function related cytokines (Granzyme B and GM-CSF). The donor variations were seen in the amount and varieties regarding the secreted cytokines. The condition without IL-4 secreted relatively higher amount of IFN-γ, whereas, the one without IL-15 had the least amount of IFN-γ. The condition with three cytokines in their highest concentrations (IL-4=55 ng/ml, IL-7=50 ng/ml and IL-15=10 ng/ml) was one of the conditions that had higher Th1 cytokines and lower Th2 or Treg cytokines. 
     T cell differentiation markers were also measured in 2 of the 4 donors by antibody staining and flow cytometer analysis. The Tcm, Tem and Teff percentage within CD45R0 positive T cell population were determined as shown in  FIG. 7 . CD45RO+CD62Lhi T cell population was also determined ( FIG. 8 ), since it was reported to have positive correlation with clinical efficacy in adoptive T cell therapy. After 12 day expansion, majority of the T cells were effect T cells in one of the donors. It was difficult to see any difference in their differentiation markers under different cytokine conditions since more than 80% of the T cells were effectors. In the other donor, it clearly showed that higher IL-15 concentration have driven more T cell differentiation. The condition without IL-15 had higher Tcm and less Teff. However, there was no significant difference in the CD45RO+CD62Lhi populations. 
     6.3 Example 3: APC Induction Duration 
     Frozen PBMC are thawed three days in a row and treated with APC induction cocktail as shown in Table 6 for simultaneous HPV PEPMIX™ stimulation post different APC induction durations. PBMC are stimulated with APC induction cocktail for 3 days in the presence or absence of 2 ng/ml IL-7. The APC induction conditions are shown in Table 6, and general experiment conditions are listed in Table 7. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 APC induction conditions. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 APC 
                   
                   
                   
                   
                   
               
               
                   
                 Induction 
               
               
                 CONDITION# 
                 Cocktail 
                 Duration 
                 IL-7 
                 Day −3 
                 Day −2 
                 Day −1 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 IL-4/CD40L 
                 3 
                 days 
                 − 
                 IL-4/CD40L 
                   
                   
               
               
                 2 
                 IL-4/CD40L/CpG 
                 3 
                 days 
                 − 
                 IL-4/CD40L/CpG 
               
               
                 3 
                 GM-CSF/IFN-α 
                 3 
                 days 
                 − 
                 GM-CSF/IFN-α 
               
               
                 4 
                 GM-CSF/IFN-α/ 
                 3 
                 days 
                 − 
                 GM-CSF/IFN-α/ 
               
               
                   
                 IL-4/CD40L/CpG 
                   
                   
                   
                 IL-4/CD40L/CpG 
               
               
                 5 
                 IL-4/CD40L 
                 3 
                 days 
                 + 
                 IL-4/CD40L + 
               
               
                   
                   
                   
                   
                   
                 IL-7 
               
               
                 6 
                 IL-4/CD40L/CpG 
                 3 
                 days 
                 + 
                 IL-4/CD40L/ 
               
               
                   
                   
                   
                   
                   
                 CpG + IL-7 
               
               
                 7 
                 GM-CSF/IFN-α 
                 3 
                 days 
                 + 
                 GM-CSF/IFN-α + 
               
               
                   
                   
                   
                   
                   
                 IL-7 
               
               
                 8 
                 GM-CSF/IFN-α/ 
                 3 
                 days 
                 + 
                 GM-CSF/IFN-α/ 
               
               
                   
                 IL-4/CD40L/CpG 
                   
                   
                   
                 IL-4/CD40L/ 
               
               
                   
                   
                   
                   
                   
                 CpG + IL-7 
               
               
                 9 
                 GM-CSF/IFN-α + 
                 3 
                 days 
                 − 
                 GM-CSF/IFN-α 
                 IL-4/CD40L 
                 CpG 
               
               
                   
                 IL-4/CD40L + 
               
               
                   
                 CpG 
               
               
                 10 
                 GM-CSF/IFN-α + 
                 3 
                 days 
                 + 
                 GM-CSF/IFN-α/ 
                 IL-4/CD40L 
                 CpG 
               
               
                   
                 IL-4/CD40L + 
                   
                   
                   
                 IL-7 
               
               
                   
                 CpG 
               
               
                 11 
                 IL-4/CD40L 
                 2 
                 days 
                 − 
                   
                 IL-4/CD40L 
               
               
                 12 
                 IL-4/CD40L/CpG 
                 2 
                 days 
                 − 
                   
                 IL-4/CD40L/CpG 
               
               
                 13 
                 GM-CSF/IFN- 
                 2 
                 days 
                 − 
                   
                 GM-CSF/IFN-α 
               
               
                   
                 α 
               
               
                 14 
                 GM-CSF/IFN-α/ 
                 2 
                 days 
                 − 
                   
                 GM-CSF/IFN-α/ 
               
               
                   
                 IL-4/CD40L/CpG 
                   
                   
                   
                   
                 IL-4/CD40L/CpG 
               
               
                 15 
                 IL-4/CD40L 
                 1 
                 day 
                 − 
                   
                   
                 IL-4/CD40L 
               
               
                 16 
                 IL-4/CD40L/CpG 
                 1 
                 day 
                 − 
                   
                   
                 IL-4/CD40L/CpG 
               
               
                 17 
                 GM-CSF/IFN-α 
                 1 
                 day 
                 − 
                   
                   
                 GM-CSF/IFN-α 
               
               
                 18 
                 GM-CSF/IFN-α/ 
                 1 
                 day 
                 − 
                   
                   
                 GM-CSF/IFN-α/ 
               
               
                   
                 IL-4/CD40L/CpG 
                   
                   
                   
                   
                   
                 IL-4/CD40L/CpG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Other experimental parameters for the 
               
               
                 APC induction duration experiment. 
               
            
           
           
               
               
               
            
               
                   
                 Parameters 
                 Condition 
               
               
                   
                   
               
               
                   
                 Donor# 
                 4 
               
               
                   
                 PBMC 
                 Frozen 
               
               
                   
                 Induction Cytokine 
                 Various 
               
            
           
           
               
               
               
               
            
               
                   
                 IL-4 
                 20 
                 ng/ml 
               
               
                   
                 CD40L 
                 1 
                 μg/ml 
               
               
                   
                 CpG 
                 4 
                 ug/ml 
               
               
                   
                 GM-CSF 
                 800 
                 U/ml 
               
               
                   
                 IFN-α-2b 
                 1000 
                 U/ml 
               
               
                   
                 IL-7 
                 2 
                 ng/ml 
               
            
           
           
               
               
               
            
               
                   
                 Induction Medium 
                 T cell media 
               
               
                   
                 Seeding density 
                 5 × 10e6/well 
               
               
                   
                 Reproducibility 
                 Replicate/condition 
               
            
           
           
               
               
               
               
            
               
                   
                 Induction Duration 
                 1/2/3 
                 days 
               
               
                   
                 PEPMIX ™ 
                 1 
                 μg/ml 
               
               
                   
                 Cytokine Feeding Delay 
                 48 
                 hours 
               
               
                   
                 Expansion Cytokine 
               
               
                   
                 IL-4 
                 55 
                 ng/ml 
               
               
                   
                 IL-7 
                 50 
                 ng/ml 
               
               
                   
                 IL-15 
                 9 
                 ng/ml 
               
               
                   
                 Expansion Duration 
                 12 
                 days 
               
               
                   
                   
               
            
           
         
       
     
     The cell number was counted using ViCELL XR after harvest at day 12 post antigen loading. As shown in  FIG. 9 , IL-4 and CD40L-treated cells generate highest yield after HPV PEPMIX™ stimulation, whereas GM-CSF and IFN-α-induced conditions show lowest cell expansion, regardless the duration of the APC induction. There was no significant difference in cell expansion capacity between different APC induction durations for a given induction cocktail, except that cell yield of donor #4 after 1-day induction is remarkably lower than that after 2- or 3-day induction ( FIG. 9 ). Sequential addition of components in cocktail IL-4/CD40L/CpG/GMCSF/IFN-α appears to compromise cell expansion compared to concurrent addition. The presence of IL-7 during APC induction minimally improves cell yield, if at all. 
     HPV T cells were identified using ICCS. Opposite to the trend of cell expansion, T cells generated from GM-CSF and IFN-α-induced PBMC showed the highest HPV T cell frequency compared with cells from other APC induction cocktail treatment for a given APC induction duration ( FIG. 10 ). Sequential addition of cocktail component of IL-4/CD40L/CpG/GMCSF/IFN-α improves HPV T cell frequency more or less in the presence or absence of IL-7. 
     HPV T cell yield is shown in  FIG. 11 . The pattern of T cell yield of different conditions is similar to that of T cell frequency as shown in  FIG. 10 , although the differences are generally to a less extent. 
     6.4 Example 4: Growth Curve Definition 
     The experiment was designed to address the following goals by using PBMC from 11 healthy donors: define the growth curve of current T cell process, define the duration of T cell signals, define T cell purity in T cell harvests on different days, explore dynamics of T cell differentiation markers, and generate product materials for microarray analysis and composition IP in multiple donors. 
     To determine the growth curve and cell doubling time, the mean of total cell number from 11 donors±SD was plotted as shown in  FIG. 12 . The Y axis represents the total cell number and X axis represented days post antigen loading. Cell doubling time was calculated by using online software (http://www.doubling-time.com/compute.php?lang=en). 
     Cell viability and cell size were measured by ViCell, and the results as shown in  FIG. 13 . Cell viability started to drop after day 12 and it was much lower on day 16. The cell size also decreased with culture extension. The average diameter of the cells on day 12 were about 9 to 10 μm, it dropped to 8 μm by day 16. 
     The cell number, expansion fold, T cell frequency and T cell yield were shown in  FIG. 14 . Total cell number or fold expansion increased with culture extension from 12 days to 16 days. The average fold change was about 7 on day 12 and it reached more than 10 fold on day 16. There were no significant changes about T cell frequency between day 12 and day 16. The total T cell yield was higher on day 16 than day 12 or day 14 since it had greater cell expansion. 
     T cell purity was defined by antibody staining and flow cytometry analysis, using T cell purity panel of CD19, CD56, CD3, CD11c, CD4, and CD8. The results demonstrated that T cell purity was greater than 85% from all the donors by day 12 and it slightly increased with culture extension. The small percentage of non-T cells are mainly NK and NKT cells. B cells were less than 1% from all the donors. Majority of the T cells generated from this process were CD4 T cells, it accounted about 80%. The CD4/CD8 T cell ratio decreased with culture extension. See  FIG. 15  and  FIG. 16 . 
     The T cell differentiation status was also checked by antibody staining and flow cytometry analysis using T cell differentiation panel of CD3, CD62L, CD4, CD27, CCR7, and CD45RO. From day 12 to day 16, CD45RO+ memory T cell population increased. There was no significant difference in CD45RO+CD62Lhi population between day 12, day 14 and day 16 ( FIG. 17 ). Within the CD45RO+ population, percentage of central memory T cell percentage decreased, while effect T cell population increased ( FIG. 18 ). 
     HPV antigen specific T cells were generated from all 11 donors. The growth curve was determined; the log phase was between 8 to 13 days. The T cell purity increases with culture extension. 10/11 donors purity were more than 85% by day 12. They were more than 90% for all the donors after day 14. The non-T cell populations were mainly NK and NK T cells, less than 1% of B cells. There was significant increase in T cell expansion and T cell yield from day 12 to day 16, however accompanied with the decrease of cell viability and cell size, as well as a more differentiated phenotype. 
     6.5 Example 5: Seeding Density and Cytokine Cocktail Concentrations 
     The purpose of this experiment was to determine the optimal PBMC seeding density and working concentrations of APC induction cytokine cocktail for HPV T cell priming and proliferation. Fresh PBMC are seeded at different density in the wells of G-REX®-24 as shown in Table 8. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 PBMC seeding density conditions. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 DENSITY 
                 IL-4 
                 CD40L 
               
               
                   
                 CONDITION# 
                 (CELL#/WELL) 
                 (ng/ml) 
                 (μg/ml) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 
                 2.50E+06 
                 10 
                 0.25 
               
               
                   
                 2 
                 2.50E+06 
                 10 
                 1 
               
               
                   
                 3 
                 2.50E+06 
                 10 
                 4 
               
               
                   
                 4 
                 2.50E+06 
                 40 
                 0.25 
               
               
                   
                 5 
                 2.50E+06 
                 40 
                 1 
               
               
                   
                 6 
                 2.50E+06 
                 40 
                 4 
               
               
                   
                 7 
                 5.00E+06 
                 10 
                 0.25 
               
               
                   
                 8 
                 5.00E+06 
                 10 
                 1 
               
               
                   
                 9 
                 5.00E+06 
                 10 
                 4 
               
               
                   
                 10 
                 5.00E+06 
                 40 
                 0.25 
               
               
                   
                 11 
                 5.00E+06 
                 40 
                 1 
               
               
                   
                 12 
                 5.00E+06 
                 40 
                 4 
               
               
                   
                 13 
                 1.00E+07 
                 10 
                 0.25 
               
               
                   
                 14 
                 1.00E+07 
                 10 
                 1 
               
               
                   
                 15 
                 1.00E+07 
                 10 
                 4 
               
               
                   
                 16 
                 1.00E+07 
                 40 
                 0.25 
               
               
                   
                 17 
                 1.00E+07 
                 40 
                 1 
               
               
                   
                 18 
                 1.00E+07 
                 40 
                 4 
               
               
                   
                   
               
            
           
         
       
     
     Since the area of the bottom surface of a G-REX®-24 well is 2 cm 2 , the seeding density of 1×10 7 /well, 5×10 6 /well, and 2.5×10 6 /well equals to 5×10 6 /cm 2 , 2.5×10 6 /cm 2 , and 1.25×10 6 /cm 2 , respectively. The cells are treated with APC induction cocktail IL-4 and CD40L at various concentrations for 24 hour prior to HPV PEPMIX™ stimulation. The general experiment conditions are listed in Table 9. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Other parameters for PBMC seeding density experiment. 
               
            
           
           
               
               
               
            
               
                   
                 Parameters 
                 Condition 
               
               
                   
                   
               
               
                   
                 Donor# 
                 3 
               
               
                   
                 PBMC 
                 Fresh 
               
            
           
           
               
               
               
            
               
                   
                 Induction Cytokine 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 IL-4 
                 10 and 40 
                 ng/ml 
               
               
                   
                 CD40L 
                 0.25, 1, and 4 
                 μg/ml 
               
            
           
           
               
               
               
            
               
                   
                 Induction Medium 
                 T cell media 
               
               
                   
                 Seeding density 
                 2.5, 5, 10 × 10e6/well 
               
               
                   
                 Reproducibility 
                 Replicate/condition 
               
            
           
           
               
               
               
               
            
               
                   
                 Induction Duration 
                 1 
                 day 
               
               
                   
                 PEPMIX ™ 
                 1 
                 μg/ml 
               
               
                   
                 Cytokine Feeding Delay 
                 48 
                 hours 
               
            
           
           
               
               
               
            
               
                   
                 T cell Expansion Cytokine 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 IL-4 
                 55 
                 ng/ml 
               
               
                   
                 IL-7 
                 50 
                 ng/ml 
               
               
                   
                 IL-15 
                 9 
                 ng/ml 
               
               
                   
                 T cell Expansion Duration 
                 14 
                 days 
               
               
                   
                   
               
            
           
         
       
     
     All three donors show the same trend that total cell yield is positively proportional to the seeding density, whereas the expansion fold is inversely proportional to the seeding density. No significant impact of different IL-4 and CD40L concentrations on expansion fold change is observed. 
     HPV T cell frequency is also analyzed ( FIG. 19 ). Generally, highest HPV T cell frequency is generated from the condition where the PBMC are seeded at highest density (1×10 7 /well). The HPV T cell frequency is positively proportional to the seeding density. There is no clear pattern regarding the influence of different concentration of IL-4 or CD40L on the HPV T cell frequency across all three donors. 
     HPV T cell yield is shown in  FIG. 20 . The pattern of HPV T cell yield is comparable to HPV T cell frequency. The impact from different seeding density is more pronounced in T cell yield than T cell frequency, attributed to the added effect from both cell yield and T cell frequency. All conditions with seeding density at 1×10 7 /well are superior to other conditions with lower seeding densities in all donors except for one condition from donor #1. Statistical analysis conducted between 6 APC induction conditions with the seeding density at 1×10 7 /well shows no significant difference (P&gt;0.05). 
     The results presented herein show that the seeding density at 5×10 6 /cm 2  (1×10 7 /well) generates highest HPV T cell yield, and the working concentration of IL-4 and sCD40L is 10-40 ng/ml and 0.25-4 μg/ml, respectively. 
     6.6 Example 6: Process Characterization 
     Certain process elements were investigated herein. IMDM and T cell media are used during APC induction for comparison. HPV PEPMIX™ has been used at 1 μg/ml for each of the 4 HPV PEPMIX™ types in previous definition experiments. In this experiment, HPV PEPMIX™ are dosed at 0.5, 1, and 2 μg/ml for optimal dose determination. Timing for initial T cell expansion cytokine supplementation is also defined by delaying the IL-4/IL-7/IL-15 addition until 2 hours, 24 hours, and 48 hours post HPV PEPMIX™ loading. IL-4 presence throughout the T cell expansion, or discontinued after initial feeding is also evaluated when T cell expansion cytokine is added after 48-hour delay. The experiment design is summarized in Table 10. The general experiment conditions are shown in Table 11. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Experimental conditions for overall process. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Cytokine 
                   
               
               
                   
                 APC Induction 
                 PEPMIX ™ 
                 Addition 
               
               
                 CONDITION# 
                 Medium 
                 Dose 
                 Delay 
                 IL-4 Feeding 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 C1 
                 IMDM 
                 1 
                 μg/ml 
                 2 
                 hours 
                 All 
               
               
                 C2 
                 IMDM 
                 1 
                 μg/ml 
                 24 
                 hours 
                 All 
               
               
                 C3 
                 IMDM 
                 1 
                 μg/ml 
                 48 
                 hours 
                 All 
               
               
                 C4 
                 IMDM 
                 1 
                 μg/ml 
                 48 
                 hours 
                 1st feeding 
               
               
                 C5 
                 T cell 
                 1 
                 μg/ml 
                 2 
                 hours 
                 All 
               
               
                 C6 
                 T cell 
                 1 
                 μg/ml 
                 24 
                 hours 
                 All 
               
               
                 C7 
                 T cell 
                 1 
                 μg/ml 
                 48 
                 hours 
                 All 
               
               
                 C8 
                 T cell 
                 1 
                 μg/ml 
                 48 
                 hours 
                 1st feeding 
               
               
                 C9 
                 T cell 
                 0.5 
                 μg/ml 
                 48 
                 hours 
                 All 
               
               
                 C10 
                 T cell 
                 2 
                 μg/ml 
                 48 
                 hours 
                 All 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Other parameters for overall process experiment. 
               
            
           
           
               
               
               
            
               
                   
                 Parameters 
                 Condition 
               
               
                   
                   
               
               
                   
                 Donor# 
                 3 
               
               
                   
                 PBMC 
                 Fresh 
               
            
           
           
               
               
               
            
               
                   
                 Induction Cytokine 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 IL-4 
                 10 
                 ng/ml 
               
               
                   
                 CD40L 
                 1 
                 μg/ml 
               
            
           
           
               
               
               
            
               
                   
                 Induction Medium 
                 IMDM/T cell media 
               
               
                   
                 Seeding density 
                 1 × 10e7/well 
               
               
                   
                 Reproducibility 
                 Replicate/condition 
               
            
           
           
               
               
               
               
            
               
                   
                 APC Induction Duration 
                 1 
                 day 
               
               
                   
                 PEPMIX ™ 
                 0.5, 1, and 2 
                 μg/ml 
               
               
                   
                 1st Cytokine Feeding Delay 
                 2, 24, and 48 
                 hours 
               
               
                   
                 T cell Expansion Cytokine 
               
               
                   
                 IL-4 
                 55 
                 ng/ml 
               
               
                   
                 IL-7 
                 50 
                 ng/ml 
               
               
                   
                 IL-15 
                 9 
                 ng/ml 
               
               
                   
                 T cell Expansion Duration 
                 12 
                 days 
               
               
                   
                   
               
            
           
         
       
     
     For the APC induction cocktail, IL-4 and CD40L are used at 10 ng/ml and 1 μs/ml, respectively. Furthermore, PBMC are seeded at 1×10 7 /well. HPV T cells are expanded for 12 days. 
     In addition to ICCS, cytokine profile is also measured in the T cells upon antigen re-stimulation using cytometric beads array. The measured cytokines are listed in Table 12. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Cytokines measured in the cytokine 
               
               
                 profile upon antigen-stimulation. 
               
            
           
           
               
               
               
            
               
                   
                 Type 
                 Cytokines 
               
               
                   
                   
               
               
                   
                 Th1 
                 IL-2, TNF-α, IFN-γ 
               
               
                   
                 Th2 
                 IL-5, IL-13 
               
               
                   
                 Pro-inflammatory 
                 GM-CSF 
               
               
                   
                 Immune regulatory 
                 IL-10 
               
               
                   
                 Cytotoxic 
                 Granzyme B 
               
               
                   
                   
               
            
           
         
       
     
     Transcription factor for Th1 (T-bet), Th2 (GATA-3), and Treg (FoxP3) are also analyzed in harvested T cell after the completion of process. 
     T cell are harvested at day 12 post HPV PEPMIX™ stimulation. The viable cells are counted using ViCell as described in experiment procedure. The results of cell expansion fold are shown in  FIG. 21 . 
     The results show that higher cell expansion fold is obtained under the condition where T cell media is used as APC induction media across all donors. In addition, cell expansion fold is inversely proportional to the cytokine delay length. Furthermore, IL-4 discontinuation slightly improves cell expansion. In donor #1 and #2, the HPV PEPMIX™ dosed at 0.5 and 1 μg/ml does not show evident effect on cell expansion capacity, whereas 2 μg/ml HPV PEPMIX™ appears to compromise cell yield. The first feeding of T cell expansion cytokine and media to the cells receiving 0.5 and 2 μg/ml PEPMIX™, is skipped in donor #3, therefore the comparison with 1 mg/ml is not conducted in the whole experiment for this donor. 
     The HPV T cell frequency is analyzed and shown in  FIG. 22 . Higher HPV T cell frequency is obtained under many conditions where IMDM media is used as APC induction media across all donors, especially in the case where the HPV T cell frequency is generally low for a given donor, such as Donor #2. In addition, HPV T cell frequency is positively proportional to the cytokine delay length at donor #1. For donor #2 and #3, the differences of HPV T cell frequency between different cytokine delays are not evident except that 24-hr and 48-hr delay generates slightly higher HPV T cell than 2-hr delay in donor #3. IL-4 discontinuation slightly improves HPV T cell frequency where IMDM is used for APC induction, but this improvement is not exhibited under the condition where T cell media is used for APC induction. HPV PEPMIX™ at 2 μg/ml stimulates highest HPV T cell frequency in donor #1 and #2. Stimulation with HPV PEPMIX™ at 0.5 μg/ml generates lowest HPV T cell frequency among all tested conditions in donor #1. 
     HPV T cell yield is shown in  FIG. 23 . The T cell yield shows a similar pattern to HPV T cell frequency. Slightly higher HPV T cell yield is obtained under many conditions where IMDM media is used as APC induction media across all donors, especially in the case where the HPV T cell frequency is generally low for a given donor, such as Donor #2. In addition, HPV T cell yield is positively proportional to the cytokine delay length at donor #1. For donor #2 and #3, the differences of HPV T cell frequency between different cytokine delays are not evident except that 24-hr and 48-hr delay generates slightly higher HPV T cell than 2-hr delay in donor #3. IL-4 discontinuation slightly improves HPV T cell yield where IMDM is used for APC induction, but this improvement is not exhibited under the condition where T cell media is used for APC induction. HPV PEPMIX™ at 2 μg/ml stimulates slightly higher HPV T cell yield than 1 μg/ml counterpart in donor #1 and #2. Stimulation with HPV PEPMIX™ at 0.5 μg/ml generates lowest HPV T cell frequency among all tested conditions in donor #1. 
     Several conditions in this experiment are specifically designed to assess Th1 cytokine IFN-γ production by primed HPV T cell; therefore, in addition to total HPV T cell analysis, we also performed IFN-γ-producing HPV T cell analysis. The results of IFN-γ-producing HPV T cell frequency is shown in  FIG. 24 . Different than the trend in total HPV T cell frequency in  FIG. 22 , most of cells cultured with IMDM for APC induction generate higher IFN-γ-producing T cell frequency than their counterpart culture with T cell media during APC induction. IFN-γ-producing T cell is almost undetectable in cells from donor #2. Within IMDM induction conditions, 24-hr cytokine addition delay shows comparable influence on IFN-γ-producing cell frequency to 48-hr cytokine addition delay, if not any higher. IL-4 withdrawal during T cell expansion in IMDM induction conditions slightly improves IFN-γ-producing HPV T cell frequency in all tested donors. 
     Cytokines secreted by HPV T cell upon antigen re-stimulation are also measured. To discover which T helper cell subset, Th1 or Th2 helper cells, is dominant in HPV T cell product under different culturing conditions, Th1/Th2 ratio is acquired by dividing representative Th1 cytokine IFN-γ level with representative Th2 cytokine IL-5 level ( FIG. 25 ). The higher the ratio of Th1/Th2, the more dominant Th1 response in the microenvironment. Generally, cells cultured with IMDM for APC induction show higher Th1/Th2 than those cultured with T cell media. Cells with expansion cytokine addition delayed till 24 hours later show higher Th1/Th2 ratio than those with 48-hr delay. 
     To further assess the effect of APC induction media and the IL-4 continuous present during T cell expansion on T cell subset differentiation, transcription factors for Th1 (T-bet), Th2 (GATA-3), and Treg (FoxP3) are examined in conditions where T cell expansion cytokine cocktail are not provided until 48 hours post HPV PEPMIX™ stimulation ( FIG. 26 ). Cells cultured with T cell media during APC induction show slightly higher frequency of CD4+T-bet+Th1 cells than those cultured with IMDM. There is no clear pattern as to the impact of APC induction media on frequency of CD4+GATA-3+Th2 cells, therefore the pattern of Th1/Th2 ratio is also compatible with the Th1 frequency. In addition, slightly higher percentage of Treg is obtained in cultured with IMDM as the APC induction media. The effect of IL-4 withdrawal during T cell expansion shows clear improvement of Th1 frequency and reduction of Th2 frequency, resulting in the elevated Th1/Th2 ratio compared with the counterpart condition where IL-4 is present throughout the T cell expansion phase. This suggests that IL-4 removal skews T cell response toward Th1. 
     6.7 Example 7: Example T Cell Production Protocols 
     This Example describes several protocols for producing antigen-specific T cells. 
     Protocol 1: A leukapheresis unit is obtained from an individual. PBMCs are isolated from the unit and frozen in a formulation comprising 40% v/v HSA and 5% DMSO in Plasma-Lyte™. On Day 0, the PBMCs are thawed, and cultured and APC induction is initiated in 6-well plates in IMDM medium comprising IL-4 (10 ng/mL) and CD40L (1 μg/mL). On Day 1, T cells within the PBMCs are activated using T cell medium comprising a PepMix derived from at least one viral or tumor antigen at 1 μg/mL. On Day 2, the T cells are expanded in T cell medium comprising 55 ng/mL IL-4, 50 ng/mL IL-7, and 9 ng/mL IL-15. On Days 5, 8, and 11, the medium is replaced with T cell medium comprising 50 ng/mL IL-7 and 9 ng/mL IL-15. On Day 13, the T cells are collected, concentrated in a centrifuge and washed at least once in Plasma-Lyte™ comprising 10% v/v HSA. The cells are then resuspended in Plasma-Lyte™ comprising 10% v/v HSA and 6% w/v Dextran-40, and diluted to a final cell concentration of 1-20×10 6  cells/mL in a solution comprising RPMI medium (32% v/v), 12.5% w/v HSA, and 5% v/v DMSO. The cells are then frozen for shipping to the point of delivery. 
     Protocol 2: A leukapheresis unit is obtained from an individual. PBMCs are isolated from the unit and frozen in a formulation comprising 40% v/v HSA and 5% w/v DMSO in Plasma-Lyte™. On Day 0, the PBMCs are thawed, and cultured and APC induction is initiated in a G-REX 100 mL culture flask in IMDM medium comprising IL-4 (10 ng/mL) and CD40L (1 μg/mL). On Day 1, T cells within the PBMCs are activated using T cell medium comprising a PepMix derived from at least one viral or tumor antigen at 1 μg/mL. On Day 2, the medium is removed and the T cells are expanded in replacement T cell medium comprising 55 ng/mL IL-4, 50 ng/mL IL-7, and 9 ng/mL IL-15. On Day 5, the medium is replaced with T cell medium comprising 50 ng/mL IL-7 and 9 ng/mL IL-15, and the cells are cultured for an additional seven days. On Day 13, the T cells are collected, concentrated in a centrifuge and washed at least once in Plasma-Lyte™ comprising 10% v/v HSA. The cells are then resuspended in Plasma-Lyte™ comprising 10% v/v HSA and 6% w/v Dextran-40, and diluted to an intermediate cell concentration of 8-35×10 6  cells/mL in the same solution. After passage of the cells through a 60 μm in-line strainer, the cells are brought to a final cell concentration of 1-20×10 6  cells/mL in a solution comprising Plasma-Lyte™ (32% v/v), 0.17% w/v NaCl, 5.5% w/v Dextran-40, 10% w/v HSA, and 5% v/v DMSO. The cells are then frozen for shipping to the point of delivery. 
     Protocol 3: A leukapheresis unit is obtained from an individual. PBMCs are isolated from the unit and washed and frozen in a formulation comprising 40% v/v HSA and 5% w/v DMSO in Plasma-Lyte™. On Day 0, the PBMCs are thawed, and cultured and APC induction is initiated in a WAVE 100 mL bioreactor in IMDM medium comprising IL-4 (10 ng/mL) and CD40L (1 μg/mL). On Day 1, T cells within the PBMCs are activated using T cell medium comprising a PepMix derived from at least one viral or tumor antigen at 1 μg/mL. On Day 2, the medium is removed and the T cells are expanded in replacement T cell medium comprising 55 ng/mL IL-4, 50 ng/mL IL-7, and 9 ng/mL IL-15. On Day 5, the medium is replaced with T cell medium comprising 50 ng/mL IL-7 and 9 ng/mL IL-15, and the cells are cultured for an additional seven days. On Day 13, the T cells are collected, concentrated in a washed at least once in Plasma-Lyte™ comprising 10% v/v HSA using a Lovo Cell Processing System (Fresenius Kabi). The cells are then resuspended in Plasma-Lyte™ comprising 10% v/v HSA and 6% w/v Dextran-40, and diluted to an intermediate cell concentration of 8-35×10 6  cells/mL in the same solution. After passage of the cells through a 60 μm in-line strainer, the cells are brought to a final cell concentration of 1-20×10 6  cells/mL in a solution comprising Plasma-Lyte™ (32% v/v), 0.17% w/v NaCl, 5.5% w/v Dextran-40, 10% w/v HSA, and 5% v/v DMSO. The cells are then frozen for shipping to the point of delivery. 
     Protocol 4: A leukapheresis unit is obtained from an individual. PBMCs are isolated from the unit and washed, using a Cell Saver 5 Centrifuge and frozen in a formulation comprising 10% w/v HSA and 5% v/v DMSO in Plasma-Lyte™ (32% v/v). On Day 0, the PBMCs are thawed, and cultured and APC induction is initiated in a Grex 100 mL culture device in IMDM medium comprising IL-4 (10 ng/mL) and CD40L (1 μg/mL). On Day 1, T cells within the PBMCs are activated in the Grex device using T cell medium comprising a PepMix derived from at least one viral or tumor antigen at 1 μg/mL, e.g., HPV16 and HPV18 E6 and E8 protein PepMixes at 1 μg/mL each. On Day 2, T cells are expanded in replacement T cell medium (added to existing medium) comprising 55 ng/mL IL-4, 50 ng/mL IL-7, and 9 ng/mL IL-15. On Days 5, 8, and 11, the medium is supplemented in the same Grex device with T cell medium comprising 50 ng/mL IL-7 and 9 ng/mL IL-15. On Day 13, the T cells are collected, and washed at least once in Plasma-Lyte™ comprising 10% v/v HSA using a Lovo Cell Processing System (Fresenius Kabi). The cells are then resuspended in Plasma-Lyte™ comprising 10% w/v HSA and 6% w/v Dextran-40, and diluted to an intermediate cell concentration of 8-35×10 6  cells/mL in the same solution. The resulting cell suspension is then filtered through a 60 μm in-line strainer, and diluted to −20×10 6  cells/mL in a solution comprising Plasma-Lyte™ 32% v/v, Dextran-40 5.5% w/v, HAS 10% w/v, NaCl 0.17% w/v, and DMSO 5% v/v. The cell suspension is then transferred to cell a storage bag and frozen for later use. For the foregoing steps, the Grex device may be substituted with a WAVE™ Bioreactor, static bag, or the like. 
     6.8 Example 8: Functional Characterization of T Cells 
     T cells generated as in Example 7 were tested for their functional properties. 
     HPV16/18 in Normal and Cancer Patients. T cells were generated as described in Example 7 from five cervical cancer patients, one healthy donor positive for HPV 16 and HPV 18, and three healthy donors of unknown HPV status. The T cells were than activated with HPV 16 or HPV 18 E6 or E7 antigenic peptides, or E6 and E7 antigenic peptides from both HPV 16 and HPV 18 ( FIG. 27 ). The T cells from the cervical cancer patients responded to both HPV 16 and HPV 18 antigens, but particularly HPV 16 antigens. The T cells from healthy donors responded to both HPV 16 and HPV 18 antigens, but particularly HPV 16 antigens. 
     In Vitro Tumor Killing. T cells generated as described in Example 7 and then tested against autologous targets or HLA-A2 matched targets in vitro at effector to target ratios of 2.5:1, 5:1, 10:1, 20:1, and 40:1 ( FIG. 28 ). When autologous or HLA-A2 matched targets were pulsed with HPV, T cells killed autologous targets ( FIG. 28A ) and HLA-A2 matched targets ( FIG. 28B ). However, when the target cells were not pulsed with HPV, little to no killing was observed. 
     HPV Antigen Specificity and Effector Activity. Cells from seven donors were tested before (PBMCs) and after (T cells) generation of T cells via the protocols described in Example 7. The HPV antigen specificity was tested by measuring surface expression of CD137 ( FIG. 29A ). Effector activity was measured in HPV-stimulated PBMCs and in HPV or non-HPV stimulated T cells by measuring secretion of IFN-γ ( FIG. 29B ), GM-CSF ( FIG. 29C ), and TNF-α ( FIG. 29D ). Only the T cells, and not the PBMCs from which they were derived, secreted pro-inflammatory Th1 cytokines. 
     6.9 Example 9: Gene Expression by T Cells 
     This Example details gene expression differences between PBMCs obtained from patients and the HPV-specific T cells produced by the method of Example 7, above, using a PepMix comprising HPV16 and HPV18 E6 and E7 protein-derived peptides. 
     RNA was isolated from patient PBMCs and from T cells made from PBMCs from the same patient. The data was generated from gene expression array analysis using an Affymetrix U133A Plus 2.0 GeneChip® Array. Briefly, gene expression profiles of 20 lines of T cells (10 from HPV T cell and 10 from their matched PBMC) were generated. The fold changes of gene expression were obtained by normalization with housekeeping genes (beta-2-microglobulin (B2M), hypoxanthine phosphoribosylotransferase (HPRT), and Large Ribosomal Protein (RPLPO)) followed by comparison of expression levels between samples. Pairwise comparison of gene expression between T cells from HPV T cell and that from matched PBMC were performed. Genes that were differentially expressed between the two groups were identified by statistical analysis. Within these identified genes, those that demonstrated more than 5 fold (used as a cut-off to eliminate some genes to meet the software limit for analysis) difference were input into IPA (Ingenuity Pathway Analysis-Biostatistic), which classifies the genes into different pathways based on their knowledge database. 
     Genes overexpressed in the T cells vs. PBMCs are listed in Table 13 below, and are identified by standard gene symbol, Entrez gene name, Affymetrix ID number, and Entrez gene ID. The “Exp Fold Change” column identifies the fold-change in expression of the indicated gene in T cells over PBMCs. Additionally, the genes are grouped according to the particular pathway of which the genes are a part. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Genes overexpressed in the T cells vs. PBMCs 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Entrez 
               
               
                   
                   
                   
                 Exp 
                   
                   
                 Gene 
               
               
                   
                   
                   
                 Fold 
                   
                   
                 ID for 
               
               
                 Symbol 
                 Entrez Gene Name 
                 Affymetrix 
                 Change 
                 Location 
                 Type(s) 
                 Human 
               
               
                   
               
            
           
           
               
            
               
                 CELL CYCLE CONTROL 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 CDC6 
                 cell division cycle 6 
                 203967_at 
                 12.37 
                 Nucleus 
                 other 
                 990 
               
               
                 CDC45 
                 cell division cycle 45 
                 204126_s_at 
                 66.74 
                 Nucleus 
                 other 
                 8318 
               
               
                 CDK2 
                 cyclin dependent kinase 2 
                 211804_s_at 
                 6.86 
                 Nucleus 
                 kinase 
                 1017 
               
               
                 CDK4 
                 cyclin dependent kinase 4 
                 202246_s_at 
                 5.09 
                 Nucleus 
                 kinase 
                 1019 
               
               
                 CDK5 
                 cyclin dependent kinase 5 
                 204247_s_at 
                 46.05 
                 Nucleus 
                 kinase 
                 1020 
               
               
                 CDK6 
                 cyclin dependent kinase 6 
                 235287_at 
                 11.25 
                 Nucleus 
                 kinase 
                 1021 
               
               
                 CDT1 
                 chromatin licensing and 
                 209832_s_at 
                 21.78 
                 Nucleus 
                 other 
                 81620 
               
               
                   
                 DNA replication factor 1 
               
               
                 CHEK2 
                 checkpoint kinase 2 
                 210416_s_at 
                 14.75 
                 Nucleus 
                 kinase 
                 11200 
               
               
                 MCM2 
                 minichromosome 
                 202107_s_at 
                 7.11 
                 Nucleus 
                 enzyme 
                 4171 
               
               
                   
                 maintenance complex 
               
               
                   
                 component 2 
               
               
                 MCM3 
                 minichromosome 
                 201555_at 
                 5.64 
                 Nucleus 
                 enzyme 
                 4172 
               
               
                   
                 maintenance complex 
               
               
                   
                 component 3 
               
               
                 MCM4 
                 minichromosome 
                 222037_at 
                 22.71 
                 Nucleus 
                 enzyme 
                 4173 
               
               
                   
                 maintenance complex 
               
               
                   
                 component 4 
               
               
                 MCM5 
                 minichromosome 
                 201755_at 
                 6.12 
                 Nucleus 
                 enzyme 
                 4174 
               
               
                   
                 maintenance complex 
               
               
                   
                 component 5 
               
               
                 PCNA 
                 proliferating cell nuclear 
                 201202_at 
                 5.56 
                 Nucleus 
                 enzyme 
                 5111 
               
               
                   
                 antigen 
               
               
                 POLA1 
                 DNA polymerase alpha 1, 
                 204835_at 
                 10.84 
                 Nucleus 
                 enzyme 
                 5422 
               
               
                   
                 catalytic subunit 
               
               
                 PRIM1 
                 primase (DNA) subunit 1 
                 205053_at 
                 8.49 
                 Nucleus 
                 enzyme 
                 5557 
               
               
                 TOP2A 
                 topoisomerase (DNA) II 
                 201291_s_at 
                 76.5 
                 Nucleus 
                 enzyme 
                 7153 
               
               
                   
                 alpha 
               
            
           
           
               
            
               
                 T HELPER CELL DIFFERENTIATION 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 BCL6 
                 B-cell CLL/lymphoma 6 
                 203140_at 
                 5.7 
                 Nucleus 
                 transcription regulator 
                 604 
               
               
                 CD80 
                 CD80 molecule 
                 1555689_at 
                 18.46 
                 Plasma 
                 transmembrane receptor 
                 941 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 CD86 
                 CD86 molecule 
                 210895_s_at 
                 8.98 
                 Plasma 
                 transmembrane receptor 
                 942 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 CD40LG 
                 CD40 ligand 
                 207892_at 
                 7.76 
                 Extracellular 
                 cytokine 
                 959 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 CXCR5 
                 C-X-C motif chemokine 
                 206126_at 
                 7.45 
                 Plasma 
                 G-protein coupled receptor 
                 643 
               
               
                   
                 receptor 5 
                   
                   
                 Membrane 
               
               
                 HLA-DMB 
                 major histocompatibility 
                 203932_at 
                 6.2 
                 Plasma 
                 transmembrane receptor 
                 3109 
               
               
                   
                 complex, class II, DM 
                   
                   
                 Membrane 
               
               
                   
                 beta 
               
               
                 HLA-DQA1 
                 major histocompatibility 
                 212671_s_at 
                 8.8 
                 Plasma 
                 transmembrane receptor 
                 3117 
               
               
                   
                 complex, class II, DQ 
                   
                   
                 Membrane 
               
               
                   
                 alpha 1 
               
               
                 HLA-DQB1 
                 major histocompatibility 
                 212998_x_at 
                 6.55 
                 Plasma 
                 other 
                 3119 
               
               
                   
                 complex, class II, DQ beta 
                   
                   
                 Membrane 
               
               
                   
                 1 
               
               
                 HLA-DRA 
                 major histocompatibility 
                 208894_at 
                 6.01 
                 Plasma 
                 transmembrane receptor 
                 3122 
               
               
                   
                 complex, class II, DR 
                   
                   
                 Membrane 
               
               
                   
                 alpha 
               
               
                 IFNGR1 
                 interferon gamma receptor 
                 242903_at 
                 6.43 
                 Plasma 
                 transmembrane receptor 
                 3459 
               
               
                   
                 1 
                   
                   
                 Membrane 
               
               
                 IL4 
                 interleukin 4 
                 207539_s_at 
                 50.34 
                 Extracellular 
                 cytokine 
                 3565 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 IL5 
                 interleukin 5 
                 207952_at 
                 153.37 
                 Extracellular 
                 cytokine 
                 3567 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 IL13 
                 interleukin 13 
                 207844_at 
                 10.3 
                 Extracellular 
                 cytokine 
                 3596 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 IL12RB1 
                 interleukin 12 receptor 
                 206890_at 
                 5.34 
                 Plasma 
                 transmembrane receptor 
                 3594 
               
               
                   
                 subunit beta 1 
                   
                   
                 Membrane 
               
               
                 IL12RB2 
                 interleukin 12 receptor 
                 206999_at 
                 5.88 
                 Plasma 
                 transmembrane receptor 
                 3595 
               
               
                   
                 subunit beta 2 
                   
                   
                 Membrane 
               
               
                 IL21R 
                 interleukin 21 receptor 
                 219971_at 
                 7.08 
                 Plasma 
                 transmembrane receptor 
                 50615 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 IL2RA 
                 interleukin 2 receptor 
                 211269_s_at 
                 24.53 
                 Plasma 
                 transmembrane receptor 
                 3559 
               
               
                   
                 subunit alpha 
                   
                   
                 Membrane 
               
               
                 IL2RG 
                 interleukin 2 receptor 
                 204116_al 
                 9.79 
                 Plasma 
                 transmembrane receptor 
                 3561 
               
               
                   
                 subunit gamma 
                   
                   
                 Membrane 
               
               
                 IL6ST 
                 interleukin 6 signal 
                 212196_at 
                 6.98 
                 Plasma 
                 transmembrane receptor 
                 3572 
               
               
                   
                 transducer 
                   
                   
                 Membrane 
               
               
                 STAT1 
                 signal transducer and 
                 200887_s_at 
                 6.22 
                 Nucleus 
                 transcription regulator 
                 6772 
               
               
                   
                 activator of transcription 1 
               
               
                 TGFB1 
                 transforming growth 
                 203084_at 
                 8.15 
                 Extracellular 
                 growth factor 
                 7040 
               
               
                   
                 factor beta 1 
                   
                   
                 Space 
               
               
                 TGFBR2 
                 transforming growth 
                 207334_s_at 
                 12.04 
                 Plasma 
                 kinase 
                 7048 
               
               
                   
                 factor beta receptor 2 
                   
                   
                 Membrane 
               
            
           
           
               
            
               
                 ARYL HYDROCARBON RECEPTOR SIGNALING 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 AHRR 
                 aryl-hydrocarbon receptor 
                 229354_at 
                 15.64 
                 Nucleus 
                 other 
                 57491 
               
               
                   
                 repressor 
               
               
                 ALDH18A1 
                 aldehyde dehydrogenase 
                 217791_s_at 
                 7.36 
                 Cytoplasm 
                 kinase 
                 5832 
               
               
                   
                 18 family member A1 
               
               
                 ALDH6A1 
                 aldehyde dehydrogenase 6 
                 221590_s_at 
                 5.07 
                 Cytoplasm 
                 enzyme 
                 4329 
               
               
                   
                 family member A1 
               
               
                 BAX 
                 BCL2 associated X, 
                 208478_s_at 
                 18.02 
                 Cytoplasm 
                 transporter 
                 581 
               
               
                   
                 apoptosis regulator 
               
               
                 CCNA1 
                 cyclin A1 
                 205899_at 
                 5.2 
                 Nucleus 
                 other 
                 8900 
               
               
                 CCNA2 
                 cyclin A2 
                 203418_at 
                 134.53 
                 Nucleus 
                 other 
                 890 
               
               
                 CCND3 
                 cyclin D3 
                 1562028_at 
                 10.69 
                 Nucleus 
                 other 
                 896 
               
               
                 CCNE2 
                 cyclin E2 
                 205034_at 
                 7.41 
                 Nucleus 
                 other 
                 9134 
               
               
                 CDK2 
                 cyclin dependent kinase 2 
                 211804_s_at 
                 6.86 
                 Nucleus 
                 kinase 
                 1017 
               
               
                 CDK4 
                 cyclin dependent kinase 4 
                 202246_s_at 
                 5.09 
                 Nucleus 
                 kinase 
                 1019 
               
               
                 CDK6 
                 cyclin dependent kinase 6 
                 235287_at 
                 11.25 
                 Nucleus 
                 kinase 
                 1021 
               
               
                 CHEK2 
                 checkpoint kinase 2 
                 210416_s_at 
                 14.75 
                 Nucleus 
                 kinase 
                 11200 
               
               
                 CYP1A1 
                 cytochrome P450 family 1 
                 205749_at 
                 5.98 
                 Cytoplasm 
                 enzyme 
                 1543 
               
               
                   
                 subfamily A member 1 
               
               
                 CYP1B1 
                 cytochrome P450 family 1 
                 202437_s_at 
                 13.89 
                 Cytoplasm 
                 enzyme 
                 1545 
               
               
                   
                 subfamily B member 1 
               
               
                 DHFR 
                 dihydrofolate reductase 
                 202533_s_at 
                 119.7 
                 Nucleus 
                 enzyme 
                 1719 
               
               
                 E2F1 
                 E2F transcription factor 1 
                 204947_at 
                 6.82 
                 Nucleus 
                 transcription regulator 
                 1869 
               
               
                 FAS 
                 Fas cell surface death 
                 216252_x_at 
                 7.81 
                 Plasma 
                 transmembrane receptor 
                 355 
               
               
                   
                 receptor 
                   
                   
                 Membrane 
               
               
                 FOS 
                 Fos proto-oncogene, AP-1 
                 209189_at 
                 8.18 
                 Nucleus 
                 transcription regulator 
                 2353 
               
               
                   
                 transcription factor 
               
               
                   
                 subunit 
               
               
                 GSTA4 
                 glutathione S-transferase 
                 202967_at 
                 11.95 
                 Cytoplasm 
                 enzyme 
                 2941 
               
               
                   
                 alpha 4 
               
               
                 GSTM4 
                 glutathione S-transferase 
                 204149_s_at 
                 11.02 
                 Cytoplasm 
                 enzyme 
                 2948 
               
               
                   
                 mu 4 
               
               
                 IL1B 
                 interleukin 1 beta 
                 39402_at 
                 17.14 
                 Extracellular 
                 cytokine 
                 3553 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 JUN 
                 Jun proto-oncogene, AP-1 
                 201466_s_at 
                 6.07 
                 Nucleus 
                 transcription regulator 
                 3725 
               
               
                   
                 transcription factor 
               
               
                   
                 subunit 
               
               
                 MGST2 
                 microsomal glutathione S- 
                 204168_at 
                 9.22 
                 Cytoplasm 
                 enzyme 
                 4258 
               
               
                   
                 transferase 2 
               
               
                 MGST3 
                 microsomal glutathione S- 
                 201403_s_at 
                 10.44 
                 Cytoplasm 
                 enzyme 
                 4259 
               
               
                   
                 transferase 3 
               
               
                 NQO1 
                 NAD(P)H quinone 
                 201467_s_at 
                 29.55 
                 Cytoplasm 
                 enzyme 
                 1728 
               
               
                   
                 dehydrogenase 1 
               
               
                 NQO2 
                 NAD(P)H quinone 
                 203814_s_at 
                 5.95 
                 Cytoplasm 
                 enzyme 
                 4835 
               
               
                   
                 dehydrogenase 2 
               
               
                 NRIP1 
                 nuclear receptor 
                 202600_s_at 
                 5.06 
                 Nucleus 
                 transcription regulator 
                 8204 
               
               
                   
                 interacting protein 1 
               
               
                 POLA1 
                 DNA polymerase alpha 1, 
                 204835_at 
                 10.84 
                 Nucleus 
                 enzyme 
                 5422 
               
               
                   
                 catalytic subunit 
               
               
                 RARA 
                 retinoic acid receptor 
                 211605_s_at 
                 6.64 
                 Nucleus 
                 ligand-dependent nuclear 
                 5914 
               
               
                   
                 alpha 
                   
                   
                   
                 receptor 
               
               
                 TFDP1 
                 transcription factor Dp-1 
                 212330_at 
                 10.29 
                 Nucleus 
                 transcription regulator 
                 7027 
               
               
                 TGFB1 
                 transforming growth 
                 203084_at 
                 8.15 
                 Extracellular 
                 growth factor 
                 7040 
               
               
                   
                 factor beta 1 
                   
                   
                 Space 
               
               
                 TGM2 
                 transglutaminase 2 
                 201042_at 
                 14.73 
                 Cytoplasm 
                 enzyme 
                 7052 
               
            
           
           
               
            
               
                 CHK PROTEINS IN CELL CYCLE CHECKPOINT CONTROL 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ANAPC11 
                 anaphase promoting 
                 226414_s_at 
                 5.53 
                 Cytoplasm 
                 enzyme 
                 51529 
               
               
                   
                 complex subunit 11 
               
               
                 CCNB1 
                 cyclin B1 
                 228729_at 
                 109.94 
                 Cytoplasm 
                 kinase 
                 891 
               
               
                 CCNB2 
                 cyclin B2 
                 202705_at 
                 129.25 
                 Cytoplasm 
                 other 
                 9133 
               
               
                 CDC20 
                 cell division cycle 20 
                 202870_s_at 
                 77.39 
                 Nucleus 
                 other 
                 991 
               
               
                 CDC25A 
                 cell division cycle 25A 
                 204695_at 
                 22.51 
                 Nucleus 
                 phosphatase 
                 993 
               
               
                 CDC25C 
                 cell division cycle 25C 
                 217010_s_at 
                 22.79 
                 Nucleus 
                 phosphatase 
                 995 
               
               
                 CDK1 
                 cyclin dependent kinase 1 
                 203213_at 
                 112.57 
                 Nucleus 
                 kinase 
                 983 
               
               
                 CHEK2 
                 checkpoint kinase 2 
                 210416_s_at 
                 14.75 
                 Nucleus 
                 kinase 
                 11200 
               
               
                 ESPL1 
                 extra spindle pole bodies 
                 204817_at 
                 5.39 
                 Nucleus 
                 peptidase 
                 9700 
               
               
                   
                 like 1, separase 
               
               
                 FBXO5 
                 F-box protein 5 
                 218875_s_at 
                 6.72 
                 Nucleus 
                 enzyme 
                 26271 
               
               
                 KIF11 
                 kinesin family member 11 
                 204444_at 
                 34.58 
                 Nucleus 
                 other 
                 3832 
               
               
                 KIF23 
                 kinesin family member 23 
                 204709_s_at 
                 58.62 
                 Cytoplasm 
                 other 
                 9493 
               
               
                 PLK2 
                 polo like kinase 2 
                 201939_at 
                 13.28 
                 Nucleus 
                 kinase 
                 10769 
               
               
                 PLK3 
                 polo like kinase 3 
                 204958_at 
                 10.76 
                 Nucleus 
                 kinase 
                 1263 
               
               
                 PLK4 
                 polo like kinase 4 
                 204887_s_at 
                 13.84 
                 Cytoplasm 
                 kinase 
                 10733 
               
               
                 PPM1L 
                 protein phosphatase, 
                 228108_at 
                 6.25 
                 Cytoplasm 
                 phosphatase 
                 151742 
               
               
                   
                 Mg2+/Mn2+ dependent 
               
               
                   
                 1L 
               
               
                 PPP2R3B 
                 protein phosphatase 2 
                 219264_s_at 
                 13.75 
                 Nucleus 
                 phosphatase 
                 28227 
               
               
                   
                 regulatory subunit B″beta 
               
               
                 PRC1 
                 protein regulator of 
                 218009_s_at 
                 6.28 
                 Nucleus 
                 other 
                 9055 
               
               
                   
                 cytokinesis 1 
               
               
                 PTTG1 
                 pituitary tumor- 
                 203554_x_at 
                 12.57 
                 Nucleus 
                 transcription regulator 
                 9232 
               
               
                   
                 transforming 1 
               
               
                 TGFB1 
                 transforming growth 
                 203084_at 
                 8.15 
                 Extracellular 
                 growth factor 
                 7040 
               
               
                   
                 factor beta 1 
                   
                   
                 Space 
               
            
           
           
               
            
               
                 ATAXIA-TELANGIECTASIA MUTATED (ATM) SIGNALING 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 BRCA1 
                 BRCA1, DNA repair 
                 204531_s_at 
                 79.8 
                 Nucleus 
                 transcription regulator 
                 672 
               
               
                   
                 associated 
               
               
                 CCNB1 
                 cyclin B1 
                 228729_at 
                 109.94 
                 Cytoplasm 
                 kinase 
                 891 
               
               
                 CCNB2 
                 cyclin B2 
                 202705_at 
                 129.25 
                 Cytoplasm 
                 other 
                 9133 
               
               
                 CDC25A 
                 cell division cycle 25A 
                 204695_at 
                 22.51 
                 Nucleus 
                 phosphatase 
                 993 
               
               
                 CDC25C 
                 cell division cycle 25C 
                 217010_s_at 
                 22.79 
                 Nucleus 
                 phosphatase 
                 995 
               
               
                 CDK1 
                 cyclin dependent kinase 1 
                 203213_at 
                 112.57 
                 Nucleus 
                 kinase 
                 983 
               
               
                 CDK2 
                 cyclin dependent kinase 2 
                 211804_s_at 
                 6.86 
                 Nucleus 
                 kinase 
                 1017 
               
               
                 CHEK2 
                 checkpoint kinase 2 
                 210416_s_at 
                 14.75 
                 Nucleus 
                 kinase 
                 11200 
               
               
                 GADD45B 
                 growth arrest and DNA 
                 209304_x_at 
                 5.72 
                 Cytoplasm 
                 other 
                 4616 
               
               
                   
                 damage inducible beta 
               
               
                 JUN 
                 Jun proto-oncogene, AP-1 
                 201466_s_at 
                 6.07 
                 Nucleus 
                 transcription regulator 
                 3725 
               
               
                   
                 transcription factor 
               
               
                   
                 subunit 
               
               
                 RAD50 
                 RAD50 double strand 
                 208393_s_at 
                 8.4 
                 Nucleus 
                 enzyme 
                 10111 
               
               
                   
                 break repair protein 
               
               
                 RAD51 
                 RAD51 recombinase 
                 205024_s_at 
                 28.78 
                 Nucleus 
                 enzyme 
                 5888 
               
               
                 RBBP8 
                 RB binding protein 8, 
                 203344_s_at 
                 9.37 
                 Nucleus 
                 enzyme 
                 5932 
               
               
                   
                 endonuclease 
               
               
                 SMC2 
                 structural maintenance of 
                 204240_s_at 
                 41.75 
                 Nucleus 
                 transporter 
                 10592 
               
               
                   
                 chromosomes 2 
               
               
                 ZEB1 
                 zinc finger E-box binding 
                 212758_s_at 
                 5.16 
                 Nucleus 
                 transcription regulator 
                 6935 
               
               
                   
                 homeobox 1 
               
               
                 ZNF420 
                 zinc finger protein 420 
                 238937_at 
                 5.07 
                 Cytoplasm 
                 other 
                 147923 
               
            
           
           
               
            
               
                 NF-κB ACTIVATION BY VIRUS 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 CXCR5 
                 C-X-C motif chemokine 
                 206126_at 
                 7.45 
                 Plasma 
                 G-protein coupled receptor 
                 643 
               
               
                   
                 receptor 5 
                   
                   
                 Membrane 
               
               
                 FGFR1 
                 fibroblast growth factor 
                 210973_s_at 
                 5.49 
                 Plasma 
                 kinase 
                 2260 
               
               
                   
                 receptor 1 
                   
                   
                 Membrane 
               
               
                 IRS2 
                 insulin receptor substrate 2 
                 209184_s_at 
                 7 
                 Cytoplasm 
                 enzyme 
                 8660 
               
               
                 ITGA1 
                 integrin subunit alpha 1 
                 214660_at 
                 5.14 
                 Plasma 
                 other 
                 3672 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA4 
                 integrin subunit alpha 4 
                 243366_s_at 
                 12.85 
                 Plasma 
                 transmembrane receptor 
                 3676 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA5 
                 integrin subunit alpha 5 
                 201389_at 
                 7.35 
                 Plasma 
                 transmembrane receptor 
                 3678 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA6 
                 integrin subunit alpha 6 
                 201656_at 
                 5.16 
                 Plasma 
                 transmembrane receptor 
                 3655 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGAL 
                 integrin subunit alpha L 
                 1554240_a_at 
                 5.11 
                 Plasma 
                 transmembrane receptor 
                 3683 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGB1 
                 integrin subunit beta 1 
                 1553530_a_at 
                 5.18 
                 Plasma 
                 transmembrane receptor 
                 3688 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 PIK3C2A 
                 phosphatidylinositol-4- 
                 241905_at 
                 6.37 
                 Cytoplasm 
                 kinase 
                 5286 
               
               
                   
                 phosphate 3-kinase 
               
               
                   
                 catalytic subunit type 2 
               
               
                   
                 alpha 
               
               
                 PIK3CA 
                 phosphatidylinositol-4,5- 
                 204369_at 
                 6.08 
                 Cytoplasm 
                 kinase 
                 5290 
               
               
                   
                 bisphosphate 3-kinase 
               
               
                   
                 catalytic subunit alpha 
               
               
                 PRKD3 
                 protein kinase D3 
                 242549_at 
                 5.7 
                 Nucleus 
                 kinase 
                 23683 
               
               
                 RRAS 
                 related RAS viral (r-ras) 
                 212647_at 
                 12.45 
                 Cytoplasm 
                 enzyme 
                 6237 
               
               
                   
                 oncogene homolog 
               
            
           
           
               
            
               
                 SUPERPATHWAY OF SERINE AND GLYCINE BIOSYNTHESIS 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 PHGDH 
                 phosphoglycerate 
                 201397_at 
                 7.01 
                 Cytoplasm 
                 enzyme 
                 26227 
               
               
                   
                 dehydrogenase 
               
               
                 PSAT1 
                 phosphoserine 
                 223062_s_at 
                 81.37 
                 Cytoplasm 
                 enzyme 
                 29968 
               
               
                   
                 aminotransferase 1 
               
               
                 PSPH 
                 phosphoserine 
                 205194_at 
                 24.17 
                 Cytoplasm 
                 phosphatase 
                 5723 
               
               
                   
                 phosphatase 
               
               
                 SHMT1 
                 serine 
                 209980_s_at 
                 23.92 
                 Cytoplasm 
                 enzyme 
                 6470 
               
               
                   
                 hydroxymethyltransferase 
               
               
                   
                 1 
               
               
                 SHMT2 
                 serine 
                 214437_s_at 
                 10.33 
                 Cytoplasm 
                 enzyme 
                 6472 
               
               
                   
                 hydroxymethyltransferase 
               
               
                   
                 2 
               
            
           
           
               
            
               
                 ENDOCYTIC PATHWAYS LINKED WITH VIRAL ENTRY 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ACTA2 
                 actin, alpha 2, smooth 
                 200974_at 
                 5.58 
                 Cytoplasm 
                 other 
                 59 
               
               
                   
                 muscle, aorta 
               
               
                 AP2B1 
                 adaptor related protein 
                 200615_s_at 
                 12.33 
                 Plasma 
                 transporter 
                 163 
               
               
                   
                 complex 2 beta 1 subunit 
                   
                   
                 Membrane 
               
               
                 CXADR 
                 coxsackie virus and 
                 226374_at 
                 5.21 
                 Plasma 
                 transmembrane receptor 
                 1525 
               
               
                   
                 adenovirus receptor 
                   
                   
                 Membrane 
               
               
                 FGFR1 
                 fibroblast growth factor 
                 210973_s_at 
                 5.49 
                 Plasma 
                 kinase 
                 2260 
               
               
                   
                 receptor 1 
                   
                   
                 Membrane 
               
               
                 FYN 
                 FYN proto-oncogene, Src 
                 1559101_at 
                 11.42 
                 Plasma 
                 kinase 
                 2534 
               
               
                   
                 family tyrosine kinase 
                   
                   
                 Membrane 
               
               
                 IRS2 
                 insulin receptor substrate 2 
                 209184_s_at 
                 7 
                 Cytoplasm 
                 enzyme 
                 8660 
               
               
                 ITGA1 
                 integrin subunit alpha 1 
                 214660_at 
                 5.14 
                 Plasma 
                 other 
                 3672 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA4 
                 integrin subunit alpha 4 
                 243366_s_at 
                 12.85 
                 Plasma 
                 transmembrane receptor 
                 3676 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA5 
                 integrin subunit alpha 5 
                 201389_at 
                 7.35 
                 Plasma 
                 transmembrane receptor 
                 3678 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA6 
                 integrin subunit alpha 6 
                 201656_at 
                 5.16 
                 Plasma 
                 transmembrane receptor 
                 3655 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGAL 
                 integrin subunit alpha L 
                 1554240_a_at 
                 5.11 
                 Plasma 
                 transmembrane receptor 
                 3683 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGB1 
                 integrin subunit beta 1 
                 1553530_a_at 
                 5.18 
                 Plasma 
                 transmembrane receptor 
                 3688 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGB7 
                 integrin subunit beta 7 
                 205718_at 
                 6.63 
                 Plasma 
                 transmembrane receptor 
                 3695 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 PIK3C2A 
                 phosphatidylinositol-4- 
                 241905_at 
                 6.37 
                 Cytoplasm 
                 kinase 
                 5286 
               
               
                   
                 phosphate 3-kinase 
               
               
                   
                 catalytic subunit type 2 
               
               
                   
                 alpha 
               
               
                 PIK3CA 
                 phosphatidylinositol-4,5- 
                 204369_at 
                 6.08 
                 Cytoplasm 
                 kinase 
                 5290 
               
               
                   
                 bisphosphate 3-kinase 
               
               
                   
                 catalytic subunit alpha 
               
               
                 PRKD3 
                 protein kinase D3 
                 242549_at 
                 5.7 
                 Nucleus 
                 kinase 
                 23683 
               
               
                 RAC2 
                 ras-related C3 botulinum 
                 207419_s_at 
                 10.13 
                 Cytoplasm 
                 enzyme 
                 5880 
               
               
                   
                 toxin substrate 2 (rho 
               
               
                   
                 family, small GTP binding 
               
               
                   
                 protein Rac2) 
               
               
                 RAC3 
                 ras-related C3 botulinum 
                 206103_at 
                 6.24 
                 Cytoplasm 
                 enzyme 
                 5881 
               
               
                   
                 toxin substrate 3 (rho 
               
               
                   
                 family, small GTP binding 
               
               
                   
                 protein Rac3) 
               
               
                 RRAS 
                 related RAS viral (r-ras) 
                 212647_at 
                 12.45 
                 Cytoplasm 
                 enzyme 
                 6237 
               
               
                   
                 oncogene homolog 
               
            
           
           
               
            
               
                 COLANIC ACID BIOSYNTHESIS 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 GALK1 
                 galactokinase 1 
                 204374_s_at 
                 30.31 
                 Cytoplasm 
                 kinase 
                 2584 
               
               
                 GALK2 
                 galactokinase 2 
                 205219_s_at 
                 5.7 
                 Cytoplasm 
                 kinase 
                 2585 
               
               
                 GMDS 
                 GDP-mannose 4,6- 
                 204875_s_at 
                 7.34 
                 Cytoplasm 
                 enzyme 
                 2762 
               
               
                   
                 dehydratase 
               
               
                 GMPPA 
                 GDP-mannose 
                 218070_s_at 
                 5.74 
                 Cytoplasm 
                 enzyme 
                 29926 
               
               
                   
                 pyrophosphorylase A 
               
               
                 TSTA3 
                 tissue specific 
                 201644_at 
                 5.26 
                 Plasma 
                 enzyme 
                 7264 
               
               
                   
                 transplantation antigen 
                   
                   
                 Membrane 
               
               
                   
                 P35B 
               
               
                 UGDH 
                 UDP-glucose 6- 
                 203343_at 
                 10.71 
                 Nucleus 
                 enzyme 
                 7358 
               
               
                   
                 dehydrogenase 
               
            
           
           
               
            
               
                 LINOLENATE BIOSYNTHESIS 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ACSL5 
                 acyl-CoA synthetase long- 
                 222592_s_at 
                 7.34 
                 Cytoplasm 
                 enzyme 
                 51703 
               
               
                   
                 chain family member 5 
               
               
                 ACSL6 
                 acyl-CoA synthetase long- 
                 229725_at 
                 6.49 
                 Cytoplasm 
                 enzyme 
                 23305 
               
               
                   
                 chain family member 6 
               
               
                 CYB5A 
                 cytochrome b5 type A 
                 215726_s_at 
                 7.26 
                 Cytoplasm 
                 enzyme 
                 1528 
               
               
                 FADS1 
                 fatty acid desaturase 1 
                 208962_s_at 
                 35.99 
                 Plasma 
                 enzyme 
                 3992 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 FADS2 
                 fatty acid desaturase 2 
                 202218_s_at 
                 25.1 
                 Plasma 
                 enzyme 
                 9415 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 SLC27A2 
                 solute carrier family 27 
                 205768_s_at 
                 27.09 
                 Cytoplasm 
                 transporter 
                 11001 
               
               
                   
                 member 2 
               
            
           
           
               
            
               
                 AGRANULOCYTE ADHESION AND DIAPEDESIS 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ACTA2 
                 actin, alpha 2, smooth 
                 200974_at 
                 5.58 
                 Cytoplasm 
                 other 
                 59 
               
               
                   
                 muscle, aorta 
               
               
                 C5 
                 complement component 5 
                 205500_at 
                 6.47 
                 Extracellular 
                 cytokine 
                 727 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 CCL4 
                 C-C motif chemokine 
                 204103_at 
                 6.29 
                 Extracellular 
                 cytokine 
                 6351 
               
               
                   
                 ligand 4 
                   
                   
                 Space 
               
               
                 CCL13 
                 C-C motif chemokine 
                 206407_s_at 
                 5.68 
                 Extracellular 
                 cytokine 
                 6357 
               
               
                   
                 ligand 13 
                   
                   
                 Space 
               
               
                 CCL17 
                 C-C motif chemokine 
                 207900_at 
                 25.39 
                 Extracellular 
                 cytokine 
                 6361 
               
               
                   
                 ligand 17 
                   
                   
                 Space 
               
               
                 CCL18 
                 C-C motif chemokine 
                 209924_at 
                 21.71 
                 Extracellular 
                 cytokine 
                 6362 
               
               
                   
                 ligand 18 
                   
                   
                 Space 
               
               
                 CCL20 
                 C-C motif chemokine 
                 205476_at 
                 18.69 
                 Extracellular 
                 cytokine 
                 6364 
               
               
                   
                 ligand 20 
                   
                   
                 Space 
               
               
                 CCL22 
                 C-C motif chemokine 
                 207861_at 
                 20.55 
                 Extracellular 
                 cytokine 
                 6367 
               
               
                   
                 ligand 22 
                   
                   
                 Space 
               
               
                 CKLF 
                 chemokine-like factor 
                 223451_s_at 
                 30.79 
                 Extracellular 
                 cytokine 
                 51192 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 CXCL2 
                 C-X-C motif chemokine 
                 209774_x_at 
                 94.66 
                 Extracellular 
                 cytokine 
                 2920 
               
               
                   
                 ligand 2 
                   
                   
                 Space 
               
               
                 CXCL8 
                 C-X-C motif chemokine 
                 202859_x_at 
                 27.39 
                 Extracellular 
                 cytokine 
                 3576 
               
               
                   
                 ligand 8 
                   
                   
                 Space 
               
               
                 CXCL16 
                 C-X-C motif chemokine 
                 223454_at 
                 7.09 
                 Extracellular 
                 cytokine 
                 58191 
               
               
                   
                 ligand 16 
                   
                   
                 Space 
               
               
                 FN1 
                 fibronectin 1 
                 211719_x_at 
                 14.42 
                 Extracellular 
                 enzyme 
                 2335 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 IL1B 
                 interleukin 1 beta 
                 39402_at 
                 17.14 
                 Extracellular 
                 cytokine 
                 3553 
               
               
                   
                   
                   
                   
                 Space 
               
               
                 ITGA1 
                 integrin subunit alpha 1 
                 214660_at 
                 5.14 
                 Plasma 
                 other 
                 3672 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA4 
                 integrin subunit alpha 4 
                 243366_s_at 
                 12.85 
                 Plasma 
                 transmembrane receptor 
                 3676 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA5 
                 integrin subunit alpha 5 
                 201389_at 
                 7.35 
                 Plasma 
                 transmembrane receptor 
                 3678 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA6 
                 integrin subunit alpha 6 
                 201656_at 
                 5.16 
                 Plasma 
                 transmembrane receptor 
                 3655 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGB1 
                 integrin subunit beta 1 
                 1553530_a_at 
                 5.18 
                 Plasma 
                 transmembrane receptor 
                 3688 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGB7 
                 integrin subunit beta 7 
                 205718_at 
                 6.63 
                 Plasma 
                 transmembrane receptor 
                 3695 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 JAM3 
                 junctional adhesion 
                 212813_at 
                 12.14 
                 Plasma 
                 other 
                 83700 
               
               
                   
                 molecule 3 
                   
                   
                 Membrane 
               
               
                 MMP25 
                 matrix metallopeptidase 
                 207890_s_at 
                 13.29 
                 Plasma 
                 peptidase 
                 64386 
               
               
                   
                 25 
                   
                   
                 Membrane 
               
               
                 MYL6B 
                 myosin light chain 6B 
                 204173_at 
                 11.23 
                 Cytoplasm 
                 other 
                 140465 
               
               
                 SDC4 
                 syndecan 4 
                 202071_at 
                 5.93 
                 Plasma 
                 other 
                 6385 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 SELPLG 
                 selectin P ligand 
                 209880_s_at 
                 9.3 
                 Plasma 
                 other 
                 6404 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 XCL1 
                 X-C motif chemokine 
                 206366_x_at 
                 18.61 
                 Extracellular 
                 cytokine 
                 6375 
               
               
                   
                 ligand 1 
                   
                   
                 Space 
               
               
                 XCL2 
                 X-C motif chemokine 
                 214567_s_at 
                 24.97 
                 Extracellular 
                 cytokine 
                 6846 
               
               
                   
                 ligand 2 
                   
                   
                 Space 
               
            
           
           
               
            
               
                 CAVEOLAE-MEDIATED ENDOCHE SIGNALING 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ACTA2 
                 actin, alpha 2, smooth 
                 200974_at 
                 5.58 
                 Cytoplasm 
                 other 
                 59 
               
               
                   
                 muscle, aorta 
               
               
                 COPG2 
                 coatomer protein complex 
                 223457_at 
                 5.24 
                 Cytoplasm 
                 transporter 
                 26958 
               
               
                   
                 subunit gamma 2 
               
               
                 FLOT1 
                 flotillin 1 
                 208748_s_at 
                 21.43 
                 Plasma 
                 other 
                 10211 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 FYN 
                 FYN proto-oncogene, Src 
                 1559101_at 
                 11.42 
                 Plasma 
                 kinase 
                 2534 
               
               
                   
                 family tyrosine kinase 
                   
                   
                 Membrane 
               
               
                 ITGA1 
                 integrin subunit alpha 1 
                 214660_at 
                 5.14 
                 Plasma 
                 other 
                 3672 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA4 
                 integrin subunit alpha 4 
                 243366_s_at 
                 12.85 
                 Plasma 
                 transmembrane receptor 
                 3676 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA5 
                 integrin subunit alpha 5 
                 201389_at 
                 7.35 
                 Plasma 
                 transmembrane receptor 
                 3678 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGA6 
                 integrin subunit alpha 6 
                 201656_at 
                 5.16 
                 Plasma 
                 transmembrane receptor 
                 3655 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGAL 
                 integrin subunit alpha L 
                 1554240_a_at 
                 5.11 
                 Plasma 
                 transmembrane receptor 
                 3683 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGAX 
                 integrin subunit alpha X 
                 210184_at 
                 9.34 
                 Plasma 
                 transmembrane receptor 
                 3687 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGB1 
                 integrin subunit beta 1 
                 1553530_a_at 
                 5.18 
                 Plasma 
                 transmembrane receptor 
                 3688 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 ITGB7 
                 integrin subunit beta 7 
                 205718_at 
                 6.63 
                 Plasma 
                 transmembrane receptor 
                 3695 
               
               
                   
                   
                   
                   
                 Membrane 
               
               
                 MAP3K2 
                 mitogen-activated protein 
                 227073_at 
                 5.83 
                 Cytoplasm 
                 kinase 
                 10746 
               
               
                   
                 kinase kinase kinase 2 
               
               
                   
               
            
           
         
       
     
     In a separate experiment, ten lines of HPV T cell and process control cells (T cells not exposed to an HPV PepMix) were generated using PBMC from 10 healthy donors. Pan T cells were purified from HPV T cell, process control cells and their donor matched PBMCs. RNA extraction from the 30 samples was performed. The gene expression profile of the purified T cells was characterized by using Affymetrix U133A Plus 2.0 array. The fold changes of gene expression were obtained by normalization with housekeeping genes (beta-2-microglobulin (B2M), hypoxanthine phosphoribosylotransferase (HPRT), and Large Ribosomal Protein (RPLPO)) followed by comparison of expression levels between samples. Thousands of genes were differentially expressed in HPV T cell compared to their matched PBMCs and/or process control cells. Six of the differentially expressed genes were chosen to be validated by quantitative real-time PCR with TaqMan gene expression assays. As a comparator, unpulsed T cells (that is, T cells generated in the absence of HPV16 and HPV18 E6 and E7 proteins) were used. In this experiment, six differentially-expressed genes were identified: CCL18 (chemokine (C-C motif) Ligand 18); CH13L1 (Chitinase-3-like protein); FN1 (Fibronectin 1); LYZ (Lysozyme); RCHY1 (Ring finger and CHY zinc finger 1); and PALLD (Palladin, cytoskeletal associated protein). CCL18, CHI3L1, FN1 and PALLD were significantly (at least 5-fold) up-regulated in the HPV T cell population, and LYZ and RCHY1 were down-regulated significantly (at least 5-fold), as compared to that in control unpulsed T cells from their matched donors. 
     6.10 Example 10: ELISPOT Assay for T Cells in Patient PBMC Samples 
     This Example describes an assay to quantify HPV-specific T cells in patient PBMC samples. The assay may also be used to reveal frequencies and cytokine signatures of antigen-specific T cells, e.g., cytotoxic T lymphocytes, and to quantify antigen-specific T cells in response to HPV antigen stimulation in PBMC. 
     Materials: Cryopreserved PBMCs from patients; phosphate-buffered saline (PBS), pH7.2 (Invitrogen, Cat #20012027); Tween-20 (Sigma, Cat #P2287); RPMI 1640 Medium (ATCC, Cat #ATCC30-2001); human IFN-γ ELISPOT set (BD Biosciences, Cat #551849)—set components: 51-2555KC Purified NA/LE Anti-Human IFN-γ; 51-1890KC Biotinylated Anti-Human IFN-γ; 51-2447KC BD ELISPOT Plates; and 51-9000209 Streptavidin-HRP; BD™ AEC Substrate Reagent Set (BD Biosciences, Cat #551951); CTL Anti-Aggregate Wash™ Medium (CTL, Cat #CTL-AA-005); CTL-Test Plus Medium (CTL, Cat #CTLTP-005); and human AB serum (Sigma, Cat #:H3667-100ML). 
     Antigens: A TAA antigen solution is made into 100 ug/ml each in distilled water using equal amounts of PepTivator HPV16E6 (Miltenyi, Cat #130-095-998); PepTivator HPV16E7 (Miltenyi, Cat #130-096-000); PepTivator HPV18E6 (Miltenyi, Cat #130-096-006); PepTivator HPV18E7 (Miltenyi, Cat #130-096-007). 
     Equipment: ViCell (Beckman Coulter, Fullerton, Calif.); AID EliSpot Reader (AID GmbH, Strassberg, Germany). 
     Preparation of medium and buffer. Coating Buffer: capture antibody stock is diluted at 1:200 in PBS (Stock: Purified NA/LE Anti-Human IFNγ, 1 mg/ml). Blocking Buffer: 5% v/v human AB serum is prepared in RPMI1640. Wash Buffer I: 1×PBS containing 0.05% Tween-20 is prepared, e.g., by adding 0.5 ml Tween-20 into 1 L PBS. Wash Buffer II: lx PBS. Dilution Buffer: 1×PBS containing 5% human AB serum. Substrate Solution: No more than 15 minutes prior to use, one drop (200) of AEC chromogen is mixed with each 1 ml of AEC substrate, and mixed thoroughly. 
     Experiment procedures: Capture antibodies specific for IFNγ are coated onto a 96-well PVDF plate as follows. Coating buffer is prepared by diluting capture antibody stock at 1:200 in PBS (Stock: Purified NA/LE Anti-Human IFNγ, 1 mg/ml). Plates are coated by adding 100 μl of coating buffer to each well of the 96 well plate. The plate is then stored at 4° C. overnight. The plate is then blocked with blocking buffer. Blocking buffer is prepared as 5% Human AB serum in RPMI1640, e.g., by adding 25 ml of human AB serum into 475 ml of RPMI1640. Excess coating antibody is discarded. 200 μl/well Blocking Solution is added to each well and incubated for 2 hours at room temperature. 
     Cells are then activated with HPV antigen. Plate antigen solution: HPV antigen is diluted into CTL-Test Plus medium at 2 μg/ml, for example, to make 5 ml antigen solution by adding 25 ul antigen stock solution into 5 ml of medium. The blocking buffer is decanted, and 50 μl of antigen solution is added into each well of the 96 well plate, and the plate is incubated at 37° C. for 10-20 minutes before plating cells. PBMCs are plated by thawing the PBMCs in a 50 ml of conical tube, and diluting the cells by slowly adding pre-warmed 37° C. Anti-Aggregate medium. The PBMCs are centrifuged for 5 min at 400 g, the supernatant is decanted, and the resulting cell pellet is resuspended into anti-Aggregate medium. Cell number is determined using the ViCell. PBMC are centrifuged and resuspended into CTL-Test Plus medium at 20 million cells per ml. 1.5-fold serial dilution of PBMC suspension from the highest concentration are made in a new U bottom 96 well plate, as indicated in Table 14 below. Each cell concentration is preferably performed in triplicate. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Serial dilution of PBMC suspension 
               
            
           
           
               
               
               
               
            
               
                 PBMC 
                 Medium 
                 PBMC volume of 
                   
               
               
                 number/50 ul 
                 volume (ul) 
                 serial dilution 
                 Dilution 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.75 
                 0 
                 180 
                 1.5fold 
               
               
                 0.50 
                 60 
                 120 
                 serial 
               
               
                 0.33 
                 60 
                 120 
                 dilution 
               
               
                 0.22 
                 60 
                 120 
               
               
                 0.15 
                 60 
                 120 
               
               
                 0.10 
                 60 
                 120 
               
               
                 0.07 
                 60 
                 120 
               
               
                 0.04 
                 60 
                 120 
               
               
                   
               
            
           
         
       
     
     50 μl of PBMC in a different density is transferred to the assay plate immediately placed into a 37° C. incubator. The plates are then incubated for 48 hours without disturbance. Captured cytokine (IFNAγ) is detected using biotinylated anti-human IFNγ (detection antibody). The cell suspension is aspirated, the wells washed 2× with deionized water, and 3× with 200 μl/well wash buffer I. 1:250 detection antibody is prepared in dilution buffer to make 2 μg/ml detection antibody solution, by adding 400 μl of 0.5 mg/ml detection antibody stock into 100 ml of dilution buffer. 100 μl is added per well and incubated for 2 h at room temperature. 
     Secreted cytokine is visualized by color development using 1:100 enzyme conjugate (Streptavidin-HRP) in Dilution Buffer, adding 100 μl/well diluted enzyme reagent, and incubating for 1 hour at room temperature. The enzyme conjugate solution is then discarded and the wells washed 4× with 200 μl/well wash buffer I, and 2× with 200 μl/well Wash Buffer II. 1000 of Final Substrate Solution is then added to each well, and color development is monitored from 5-60 min. The reaction is stopped by washing wells with DI water, and the plates are air-dried until completely dry. The resulting spots are enumerated with an AID EliSpot Reader with IFNγ spot reader program selected according to manufacturer instructions. 
     6.11 Example 11: Clinical Study Protocol 
     This Example describes a phase 1, multicenter, open-label, dose-escalating safety study of human peripheral blood-derived, culture expanded, autologous, T cell infusion in subjects with HPV+ recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) following first-line chemotherapy with or without radiation therapy. 
     First-line therapy for the purposes of this study is the first treatment administered for the diagnosis of recurrent disease or metastatic disease. The study enrolls up to 84 subjects. The study consists of a Pre Standard of Care Screening Period, a Standard of Care Treatment Period, a T cell Screening Period, a T cell Treatment Period (Day −4 to Day 0), which includes a 3-day conditioning treatment (Day −4 to Day −2), and a T cell infusion on Day 0, and a Follow-up Period beginning on Day 1 to the time of progressive disease (PD). A Long Term Follow-up Period starts after PD or start of new therapy and includes collection of information for survival and additional therapies administered for SCCHN every three months until death, loss to follow-up, or withdrawal of consent, whichever occurs first. The anticipated participation time for subjects is five years. 
     Subjects have recurrent and/or metastatic oropharyngeal cancer that is p16 and/or HPV-16 and/or HPV-18 positive as determined by immunohistochemistry, polymerase chain reaction, or ribonuclic acid fluorescence in situ hybridization. 
     The primary objective of the study is to assess the safety and determine the maximum tolerated dose (MTD) of the T cells administered intravenously (IV) in subjects with HPV+ recurrent and/or metastatic SCCHN as defined by National Comprehensive Cancer Network (NCCN) guidelines. 
     The secondary objectives of the study are to explore potential clinical efficacy by assessing tumor response as measured by the Response Evaluation Criteria in Solid Tumors (RECIST v1.1) guidelines, and by assessing duration and rate of disease control, progression-free survival (PFS) and overall survival (OS), and to evaluate the tolerability of the pre-specified lymphodepletion regimens as measured by safety. 
     The study investigates up to 5 dose-level cohorts utilizing a low cyclophosphamide (LD-Cy) only regimen for lymphodepletion. Once the MTD of the T cells is identified, two alternative lymphodepletion regimens are investigated, high dose cyclophosphamide (HD-Cy) and cyclophosphamide and fludarabine (Cy-Flu). 
     Subjects undergo leukapheresis for the purposes of manufacturing T cells. After the subject undergoes leukapheresis, the subject receives front-line therapy as medically indicated, at the discretion of the treating physician, for example, subjects may receive up to 6 cycles of chemotherapy following standard of care practices. During front-line therapy, the subject undergoes clinical and radiological disease evaluation by the treating physician. Based on the totality of the data treating physician may advise the sponsor to proceed with T cell manufacturing for the subject. 
     Subject-specific T cells are then manufactured, tested and released for use over the course of approximately 28 days. T cell manufacturing is performed according to the methods described herein. Once the T cells are successfully manufactured and released for use, the subject may initiate treatment, the timing of which is at the discretion of the treating physician, but takes place after front-line chemotherapy is completed. In order to accommodate variances in length of first-line therapy and the possibility of early progression, subjects may be enrolled to the study for leukapheresis without a known date of T cell treatment. 
     The study enrolls up to a maximum of 84 T cell-treated subjects. No fewer than 3 dose-limiting toxicity (DLT)-evaluable subjects are investigated at each dose level cohort prior to any recommendation for dose escalation. In the event of the DLT, the cohort is expanded and no fewer than 6 DLT-evaluable subjects are investigated prior to dose level cohort escalation. In the event that 2 DLTs occur, enrollment to that specific cohort is halted at which time MTD recommendation is evaluated. Due to the variability of duration of first-line therapy, and the possibility of subjects not being treated with T cells, no more than 18 subjects may undergo leukapheresis without treatment of T cells or early termination at any given time during the escalation phase of the study. Once the conditioning regimen and MTD of T cells is identified, an 18 subject expansion is enrolled to provide additional safety information and contribute to additional efficacy data. 
     The T cell treatment period starts with a three day lymphodepletion regimen starting on Day −4 to Day −2. Initially a single agent regimen is utilized, specifically LD-Cy which includes cyclophosphamide at 300 mg/m 2 /day administered on Day −4 to −2. T cells are administered on Day 0. Each subject receives a single dose of T cells, which may require multiple infusion bags. 
     Subjects are sequentially assigned to one of up to five dosing cohorts, based on time of entry into the study:
         Dose Level 1: 5.0×10 7  cells/m 2      Dose Level 2: 1.5×10 8  cells/m 2      Dose Level 3: 4.5×10 8  cells/m 2      Dose Level 4: 1.3×10 9  cells/m 2      Dose Level 5: 2.0×10 9  cells/m 2          

     Once the MTD for the T cells is identified, two alternative lymphodepletion regimens are investigated in parallel based on time of treatment period entry. HD-Cy, which includes Cyclophosphamide at a higher dose of 900 mg/m 2 /day for three days starting on Day −4 to −2, or Cy-Flu, which includes cyclophosphamide 300 mg/m 2 /day for three days in combination with fludarabine 30 mg/kg/day for two days starting Day −4 to Day −3. 
     Subjects are pre-medicated with acetaminophen 650 mg orally (P0) and diphenhydramine 25 mg (PO/IV/intramuscularly [IM]) before and approximately 4 hours after the T cell infusion. Subjects are monitored for at least 24 hours post end of T cell infusion. 
     The decision to increase the size of the cohort is based upon the number of DLT events. For the first cohort, not more than 1 subject may begin treatment in any 14 day period. 
     Subjects are closely monitored for adverse reactions throughout the administration of the study treatment regimen. If adverse events (AEs) occur during the T cell infusion, with the potential of becoming severe (defined as National Cancer Institute [NCI]) common terminology criteria for adverse events [CTCAE]) Version 4.03 Grade 3 or above; or urgent intervention indicated to prevent increase in severity with the potential of becoming life threatening), the infusion is stopped and subjects continue to be followed. 
     After completion of the T cell administration, subjects are followed until PD. Thereafter, subjects are followed for survival until death, loss to follow up, or withdrawal of consent, whichever occurs first. 
     EQUIVALENTS 
     The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter provided herein, in addition to those described, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. 
     Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.