bluuebunny/crossref_metadata_2025 huggingface dataset:
Th2 Activities Induced During Virgin T Cell Priming in the Absence of IL-4, IL-13, and B Cells
Authors: Cunningham Adam F. , Fallon Padraic G. , Khan Mahmood , Vacheron Sonia , Acha-Orbea Hans , MacLennan Ian C. M. , McKenzie Andrew N. , Toellner Kai-Michael
Published: April 2014

Virgin T cells being primed to Th2-inducing or Th1-inducing Ags, respectively, start to synthesize IL-4 or IFN-γ as they begin to proliferate. Parallel respective induction of B cells to produce γ1 or γ2a switch transcripts provides additional evidence of early divergent Th activity. This report concerns the roles of IL-4, IL-13, and B cells in these early events in vivo. Th2 responses were induced in lymph nodes against hapten-protein given s.c. with killed Bordetella pertussis adjuvant. In T cell proliferation in wild-type mice, IL-4 message up-regulation and γ1 and ε switch transcript production were underway 48–72 h after immunization. The absence of IL-4, IL-13, or B cells did not alter the early T cell proliferative response. The γ1 and ε switch transcript production was still induced in the absence of IL-4, IL-13, or both, but at a reduced level, while the dominance of switching to IgG1 in the extrafollicular hapten-specific plasma cell response was retained. The up-regulation of IL-4 message was not reduced or delayed in the absence of B cells and was only marginally reduced by the absence of IL-13. It is concluded that signals delivered by dendritic cells, which are not dependent on the presence of IL-4, IL-13, or B cells, can prime virgin T cells and induce the early Th2 activities studied. These early events that direct virgin T cells toward Th2 differentiation contrast with the critical later role of Th2 cytokines in selectively expanding Th2 clones and driving further IL-4 synthesis.
Superantigen Enhancement of Specific Immunity: Antibody Production and Signaling Pathways
Authors: Torres Barbara A. , Perrin George Q. , Mujtaba Mustafa G. , Subramaniam Prem S. , Anderson Amy K. , Johnson Howard M.
Published: April 2014

Superantigens are microbial proteins that induce massive activation, proliferation, and cytokine production by CD4+ T cells via specific Vβ elements on the TCR. In this study we examine superantigen enhancement of Ag-specific CD4+ T cell activity for humoral B cell responses to T-dependent Ags BSA and HIV gp120 envelope, type I T-independent Ag LPS, and type II T-independent Ag pneumococcal polysaccharides. Injection of BSA followed by a combination of superantigens staphylococcal enterotoxin A and staphylococcal enterotoxin B (SEB) 7 days later enhanced the anti-BSA Ab response in mice ∼4-fold as compared with mice given BSA alone. The anti-gp120 response was enhanced ∼3-fold by superantigens. The type II T-independent Ag pneumococcal polysaccharide response was enhanced ∼2.3-fold by superantigens, whereas no effect was observed on the response to the type I T-independent Ag LPS. The superantigen effect was completely blocked by the CD4+ T cell inhibitory cytokine IL-10. SEB-stimulated human CD4+ T cells were examined to determine the role of the mitogen-activated protein (MAP) kinase signal transduction pathway in superantigen activation of T cells. Inhibitors of the mitogen pathway of MAP kinase blocked SEB-induced proliferation and IFN-γ production, while an inhibitor of the p38 stress pathway had no effect. Consistent with this, SEB activated extracellular signal-regulated kinase/MAP kinase as well as MAP kinase-interacting kinase, a kinase that phosphorylates eIF4E, which is an important component of the eukaryotic protein synthesis initiation complex. Both kinases were inhibited by IL-10. Thus, superantigens enhance humoral immunity via Ag-specific CD4+ T cells involving the stress-independent pathway of MAP kinase.
Structural Elements of a Protein Antigen Determine Immunogenicity of the Embedded MHC Class I-Restricted T Cell Epitope
Authors: Paster Wolfgang , Kalat Milena , Zehetner Margit , Schweighoffer Tamás
Published: April 2014

Substantial effort has been invested into optimization of vector structure, DNA formulation, or delivery methods to increase the effectiveness of DNA vaccines. In contrast, it has been only insufficiently explored how the higher order structure of an antigenic protein influences immunogenicity of embedded epitopes in vivo. Potent CD8+ T cell responses specific for a single immunogenic epitope are induced upon electrovaccination with plasmid DNA encoding the full-length heavy chain of the human HLA-Cw3 molecule. Contrary to expectations, a minimal construct, which provoked a substantial release of IFN-γ from specific CTLs in vitro, did not induce a significant response in vivo. Systematically altered variants of the Cw3 molecule were thus tested both in vivo and in vitro to determine which structural parts are responsible for this discrepancy. In complementation experiments the participation of trans-acting helper epitopes was ruled out. Successive C-terminal truncations, human/mouse domain swap variants, and subdomain modifications defined the α3 region of the HLA heavy chain and membrane anchoring as critical elements. Based on these data, refined minimal constructs were engineered that triggered very high in vivo responses. The most advanced variant consisted only of an adenoviral leader, antigenic epitope, α3 domain, and 16 aa of the transmembrane domain. When a tumor Ag epitope was incorporated into one of these high performer minimal constructs, protection against melanoma metastases was attained upon vaccination. Thus, structural elements of the Ag can dominantly influence immunogenicity in vivo. These elements can also markedly improve the immunogenicity of unrelated Ags and may form the basis of a new generation of DNA vaccines.
Recruitment of CTL Activity by Tumor-Specific Antibody-Mediated Targeting of Single-Chain Class I MHC-Peptide Complexes
Authors: Lev Avital , Novak Hila , Segal Dina , Reiter Yoram
Published: April 2014

The MHC class I-restricted CD8 CTL effector arm of the adaptive immune response is uniquely equipped to recognize tumor cells as foreign and consequently initiates the cascade of events resulting in their destruction. However, tumors have developed sophisticated strategies to escape immune effector mechanisms; their most well-known strategy is down-regulation of MHC class I molecules. To overcome this and develop new approaches for immunotherapy, we have constructed a recombinant molecule in which a single-chain MHC is specifically targeted to tumor cells through its fusion to cancer-specific recombinant Ab fragments. As a model we used a single-chain HLA-A2 molecule genetically fused to the variable domains of an anti-IL-2Rα subunit-specific humanized Ab, anti-Tac. The construct, termed B2M-aTac(dsFv), was expressed in Escherichia coli, and functional molecules were produced by in vitro refolding in the presence of HLA-A2-restricted antigenic peptides. Flow cytometry studies revealed the ability to decorate Ag-positive, HLA-A2-negative human tumor cells with HLA-A2-peptide complexes in a manner that was entirely dependent upon the specificity of the targeting Ab fragment. Most importantly, the B2M-aTac(dsFv)-mediated coating of the target tumor cells made them susceptible for efficient and specific HLA-A2-restricted, melanoma gp100 peptide-specific CTL-mediated lysis. These results demonstrate the concept that Ab-guided, Ag-specific targeting of MHC-peptide complexes on tumor cells can render them susceptible and more receptive and thus potentiate CTL killing. This type of approach may open the way for the development of new immunotherapeutic strategies based on Ab targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.
Bcl-2 Controls Dendritic Cell Longevity In Vivo
Authors: Nopora Adam , Brocker Thomas
Published: April 2014

Dendritic cells (DC) were found to down-regulate Bcl-2 protein upon maturation in vivo. Because Bcl-2 has been shown to exert anti-apoptotic functions, down-regulation of Bcl-2 could be a mechanism by which DC longevity is controlled. To dysregulate this potential control system and to study the role of Bcl-2 in DC, we expressed human Bcl-2 under control of the murine CD11c-promoter as a transgene specifically in DC and show that DC frequencies and numbers increase in transgenic mice. In vivo bromodeoxyuridin, as well as adoptive, DC transfer studies show that the relative turnover/survival of mature Bcl-2 transgenic DC is increased. This had a direct impact on CD4+ T cell, as well as humoral immune, responses, which were elevated in transgenic animals. When Bcl-2 transgenic DC were used as DC vaccines, they induced 2- to 3-fold greater expansion of Ag-specific CTL, and stronger in vivo cytotoxicity. Overall, these data indicate that down-regulation of Bcl-2 controls DC longevity, which in turn directly regulates immune responses and the efficacy of DC when used as vaccines.
Active Form of Notch Imposes T Cell Fate in Human Progenitor Cells
Authors: De Smedt Magda , Reynvoet Katia , Kerre Tessa , Taghon Tom , Verhasselt Bruno , Vandekerckhove Bart , Leclercq Georges , Plum Jean
Published: April 2014

The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34+ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19+ B lymphocytes was blocked, the differentiation toward CD14+ monocytes was inhibited, and the differentiation toward CD56+ NK cells was favored. The number of CD7+cyCD3+ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34+ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-γδ T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4+CD8+ TCR-αβ cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.
Regulation of Immunoproteasome Subunit Expression In Vivo Following Pathogenic Fungal Infection
Authors: Barton Lance F. , Cruz Miguel , Rangwala Reshma , Deepe George S. , Monaco John J.
Published: April 2014

The proteasome catalytic β subunits LMP2, LMP7, and MECL-1 and two proteasome activator proteins, PA28 α and β, are induced following exposure to IFN-γ in vitro. Induction of these immunosubunits and the PA28 α/β hetero-oligomer alters proteasome catalytic functions and specificity and enhances production of certain MHC class I epitopes. We sought to determine whether and to what extent proteasome subunit composition is regulated in vivo and to elucidate the mechanisms of such regulation. We analyzed basal expression levels of these inducible genes in normal, IFN-γ-deficient, and Stat-1-deficient mice. Mice of all three genotypes display constitutive expression of the immunosubunits and PA28, demonstrating that basal expression in vivo is independent of endogenous IFN-γ production. However, basal expression levels are reduced in Stat-1−/− mice, demonstrating a role for Stat-1 independent of IFN-γ signaling. To demonstrate that IFN-γ can induce these genes in vivo, mice were infected with Histoplasma capsulatum. Elevated expression of these genes followed the same time course as IFN-γ expression in infected mice. IFN-γ-deficient mice did not display elevated protein expression following infection, suggesting that other inflammatory cytokines produced in infected mice are unable to influence proteasome expression. Cytokines other than IFN-γ also failed to influence proteasome gene expression in vitro in cell lines that had no basal expression of LMP2, LMP7, or MECL-1. Thus, both in vitro and in vivo data demonstrate that IFN-γ is essential for up-regulation, but not constitutive expression, of immunoproteasome subunits in mice.
Gene Conversion-Like Sequence Transfers Between Transgenic Antibody V Genes Are Independent of RAD54
Authors: D’Avirro Nicole , Truong David , Luong Michael , Kanaar Roland , Selsing Erik
Published: April 2014

Homology-based Ig gene conversion is a major mechanism for Ab diversification in chickens and the Rad54 DNA repair protein plays an important role in this process. In mice, although gene conversion appears to be rare among endogenous Ig genes, Ab H chain transgenes undergo isotype switching and gene conversion-like sequence transfer processes that also appear to involve homologous recombination or gene conversion. Furthermore, homology-based DNA repair has been suggested to be important for somatic mutation of endogenous mouse Ig genes. To assess the role of Rad54 in these mouse B cell processes, we have analyzed H chain transgene isotype switching, sequence transfer, and somatic hypermutation in mice that lack RAD54. We find that Rad54 is not required for either transgene switching or transgene hypermutation. Furthermore, even transgene sequence transfers that are known to require homology-based recombinations are Rad54 independent. These results indicate that mouse B cells must use factors for promoting homologous recombination that are distinct from the Rad54 proteins important in homology-based chicken Ab gene recombinations. Our findings also suggest that mouse H chain transgene sequence transfers might be more closely related to an error-prone homology-based somatic hypermutational mechanism than to the hyperconversion mechanism that operates in chicken B cells.
Polymorphism at Position 97 in MHC Class I Molecules Affects Peptide Specificity, Cell Surface Stability, and Affinity for β2-Microglobulin
Authors: Smith Ruth A. , Myers Nancy B. , Robinson Melanie , Hansen Ted H. , Lee David R.
Published: April 2014

The two mouse MHC class I alleles, Ld and Lq, share complete amino acid sequence identity except in the α2 domain, where they differ at six positions. Despite their similarity, Lq has a stronger association with β2-microglobulin (β2m), is expressed at higher levels on the cell surface, demonstrates an increased cell surface half-life, and has fewer open forms on the cell surface than Ld. To determine the basis for their phenotypic differences, Ld molecules containing chimeric Ld-Lq α2 domains were characterized, and these analyses implicated residue 97 (LdTrp and LqArg) as the polymorphic site responsible for the disparity in β2m association between the two alleles. Single substitution analysis at this site (LdW97R and LqR97W) confirmed this. Furthermore, the LdW97R mutant molecule has a longer cell surface half-life than either Lq or Ld, and fewer open forms of LdW97R are observed on the cell surface. In addition, both LdW97R and Lq possess decreased binding affinity for the Ld-restricted tum− P91A14–22 peptide compared with Ld. Collectively, these results and the known location of Trp97 in the peptide binding cleft of Ld strongly suggest that the substitution of Arg for Trp97 in Ld alters the peptide binding cleft, increasing its affinity for endogenous peptides, which results in greater cell surface stability and better retention of β2m. Furthermore, these results imply that Trp97 plays an important role in the ability of Ld to efficiently participate in alternative MHC class I Ag presentation pathways.
Identification of a Novel HLA-B60-Restricted T Cell Epitope of the Minor Histocompatibility Antigen HA-1 Locus
Authors: Mommaas Bregje , Kamp Janine , Drijfhout Jan-Wouter , Beekman Nico , Ossendorp Ferry , van Veelen Peter , den Haan Joke , Goulmy Els , Mutis Tuna
Published: April 2014

The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1H and HA-1R, with a single amino acid difference. Whereas the immunogenicity of the HA-1R allele has not yet been shown, the nonameric HA-1H peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1H or HA-1R associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1H/R peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1H/R peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVLH/RDDL and decameric KECVLH/RDDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1H and HA-1R peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1R peptide did not induce CTLs that recognize the natural HLA-B60/HA-1R ligand. In contrast, dendritic cells pulsed with the nonameric HA-1H peptide induced IFN-γ-secreting T cells specific for the natural HLA-B60/HA-1H ligand in three HLA-B60+ HA-1RR individuals, demonstrating the immunogenicity of the HLA-B60/HA-1H ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.
Peptidic Termini Play a Significant Role in TCR Recognition
Authors: Wang Bo , Sharma Ashawni , Maile Robert , Saad Mohamed , Collins Edward J. , Frelinger Jeffrey A.
Published: April 2014

TCR recognition of class I MHC is dependent on the composition of the antigenic peptide and the MHC. Single amino acid substitutions in either the MHC or the peptide may dramatically alter recognition. While the major interactions between TCR and the peptide/MHC complex appear to be focused on the complementarity-determining region (CDR)3, it is also clear from the cocrystal structure of class I MHC and TCR that the amino and carboxyl ends of the peptide may play a role through interactions with the CDR1. In this work we show that gp33 variants substituted at the peptidic termini at the putative CDR1 contact regions show improved recognition in B6 mice. The rank order of recognition is different using the P14 transgenic T cells, suggesting that one reason for improved recognition is a change in the TCR repertoire that recognizes the peptide. However, the affinity of the TCR by some of the peptide/MHC complex with increased recognition is improved, as shown by increased tetramer binding to P14 T cells. These substitutions at the termini of the peptide-binding cleft cause localized conformational changes as seen by changes in mAb binding and crystallographic structures. The different peptide structures also show different conformations in the center of the peptide, but these are shown to be energetically similar and thus most likely have no significance with respect to TCR recognition. Therefore, small conformational changes, localized to the CDR1 contact regions, may play a significant role in TCR recognition.
MHC Allele-Specific Molecular Features Determine Peptide/HLA-A2 Conformations That Are Recognized by HLA-A2-Restricted T Cell Receptors
Authors: Wang Zichun , Turner Richard , Baker Brian M. , Biddison William E.
Published: April 2014

The structures of αβ TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the α1 and α2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the α1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.
γ/δ T Cell-Deficient Mice Exhibit Reduced Disease Severity and Decreased Inflammatory Response in the Brain in Murine Neurocysticercosis
Authors: Cardona Astrid E. , Teale Judy M.
Published: April 2014

In a recently developed mouse model for neurocysticercosis, the immune response was characterized by a massive influx of γδ T cells and a type 1 pathway of cytokine expression. To understand the role of γδ T cells during this infection, the cellular and cytokine response was analyzed in mice that lack γδ T cells (TCRδ−/−). In TCRδ−/− mice, Mesocestoides corti metacestodes preferentially invaded the extraparenchymal areas of the brain. Furthermore, parasites were able to escape from the brain and establish a systemic infection with liver and peritoneal involvement. Immunopathological studies indicated that TCRδ−/− mice develop little inflammatory response and less neurological symptomatology. Significantly reduced numbers of T cells, macrophages, dendritic cells, and mast cells were present in the brain. The cytokine response in the brain of TCRδ−/− mice appears to be a mixed type1/type 2 response with low levels of IL-2, IL-4, IL-10, IL-12, IL-13, IL-15, and IFN-γ. To further investigate the immunological significance of this cell population, γδ T cells were adoptively transferred into intracranially infected TCRδ−/− mice. γδ T cells were specifically recruited into the CNS in response to this parasitic infection, and they were able to target the infected brain within 12 h after transfer. These results suggest that γδ T cells are key players in the immune response elicited during this CNS infection and direct a type 1 response in wild-type mice upon infection.
A C-Reactive Protein Mutant That Does Not Bind to Phosphocholine and Pneumococcal C-Polysaccharide
Authors: Agrawal Alok , Simpson Melanie J. , Black Steven , Carey Marianne P. , Samols David
Published: April 2014

C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe66 and Glu81 contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe66 and Glu81 to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe66 is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.
Human Papillomavirus Virus-Like Particles Do Not Activate Langerhans Cells: A Possible Immune Escape Mechanism Used by Human Papillomaviruses
Authors: Fausch Steven C. , Da Silva Diane M. , Rudolf Michael P. , Kast W. Martin
Published: April 2014

High-risk human papillomaviruses are linked to several malignancies including cervical cancer. Because human papillomavirus-infected women do not always mount protective antiviral immunity, we explored the interaction of human papillomavirus with Langerhans cells, which would be the first APCs the virus comes into contact with during infection. We determined that dendritic cells, normally targeted by vaccination procedures and Langerhans cells, normally targeted by the natural virus equally internalize human papillomavirus virus-like particles. However, in contrast to dendritic cells, Langerhans cells are not activated by human papillomavirus virus-like particles, illustrated by the lack of: up-regulating activation markers, secreting IL-12, stimulating T cells in an MLR, inducing human papillomavirus-specific immunity, and migrating from epidermal tissue. Langerhans cells, like dendritic cells, can display all of these characteristics when stimulated by proinflammatory agents. These data may define an intriguing immune escape mechanism used by human papillomavirus and form the basis for designing optimal vaccination strategies.
Opsonization of HIV-1 by Semen Complement Enhances Infection of Human Epithelial Cells
Authors: Bouhlal Hicham , Chomont Nicolas , Haeffner-Cavaillon Nicole , Kazatchkine Michel D. , Belec Laurent , Hocini Hakim
Published: April 2014

In the present study we demonstrate that both X4- and R5-tropic HIV-1 strains are able to infect the human epithelial cell line HT-29. Infection was enhanced 2-fold when HIV was added to semen before contact with the cell cultures. The enhancing effect of semen was complement dependent, as evidenced by blockage of generation of C3a/C3adesArg in semen by heat or EDTA treatment of semen and suppression of semen-dependent enhancement with mAbs directed to complement receptor type 3 (CD11b/CD18) and soluble CD16. Infection of HT-29 cells was assessed by the release of p24 Ag in cultures and semiquantitative PCR of the HIV-1 pol gene. Inhibition of infection of HT-29 by stromal cell-derived factor 1 was decreased in the case of semen-opsonized X4- and R5-tropic virus compared with unopsonized virus. In contrast, inhibition of infection by RANTES was increased for opsonized X4-tropic HIV-1 compared with unopsonized virus. Taken together these observations indicate that activation of complement in semen may play an enhancing role in mucosal transmission of HIV-1 by facilitating infection of epithelial cells and/or enhancing infection of complement receptor-expressing target cells in the mucosa.
HIV-1 Transcription and Virus Production Are Both Accentuated by the Proinflammatory Myeloid-Related Proteins in Human CD4+ T Lymphocytes
Authors: Ryckman Carle , Robichaud Gilles A. , Roy Jocelyn , Cantin Réjean , Tremblay Michel J. , Tessier Philippe A.
Published: April 2014

S100A8, S100A9, and S100A12, collectively known as myeloid-related proteins (MRPs), are highly expressed by the myeloid cell lineage and are found in the extracellular milieu during infections and inflammatory conditions. Recent data showed high levels of MRPs in the serum of HIV type 1 (HIV-1)-infected patients which correlated with disease progression and low CD4+ counts. Therefore, we set out to investigate the effect of MRPs on HIV-1 replication. We observed a 4- to 5-fold induction of virus production in J1.1, a human T lymphoid cell line latently infected with HIV-1, following treatment with MRPs. Using luciferase-based reporter gene assays, we demonstrated that MRPs induce a dose- and time-dependent activation of the HIV-1 long terminal repeat promoter region that could be blocked by specific anti-MRP polyclonal Abs and by physical denaturation of these proteins. The MRP-mediated induction was acting through the HIV-1 enhancer sequence and was dependent upon NF-κB activity. These latter results were also confirmed by EMSA experiments conducted in Jurkat cells and freshly isolated PBMCs. In conclusion, we demonstrate that MRPs induce HIV-1 transcriptional activity and viral replication in infected CD4+ T-lymphocytes at concentrations similar to those found in the serum of HIV-1-infected patients.
Adoptive Immunotherapy of Advanced Tumors with CD62 L-Selectinlow Tumor-Sensitized T Lymphocytes Following Ex Vivo Hyperexpansion
Authors: Wang Li-Xin , Chen Bing-Guan , Plautz Gregory E.
Published: April 2014

Tumor-draining lymph nodes (TDLN) contain sensitized T cells with the phenotype CD62 L-selectinlow (CD62Llow) that can be activated ex vivo with anti-CD3 mAb and IL-2 to acquire potent dose-dependent effector function manifested upon adoptive transfer to secondary tumor-bearing hosts. In this study advanced tumor models were used as a stringent comparison of efficacy for the CD62Llow subset, comprising 5–7% of the TDLN cells, vs the total population of TDLN cells following culture in high dose IL-2 (100 U/ml). During the 9-day activation period the total number of CD8+ T cells increased 1500-fold, with equivalent proliferation in the CD62Llow vs the total TDLN cell cultures. Adoptive transfer of activated CD62Llow cells eliminated 14-day pulmonary metastases and cured 10-day s.c. tumors, whereas transfer of maximally tolerated numbers of total TDLN cells was not therapeutic. Despite their propagation in a high concentration of IL-2, the hyperexpanded CD62Llow subset of TDLN cells functioned in vivo without exogenous IL-2, and CD8+ T cells demonstrated relative helper independence. Moreover, the anti-tumor response was specific for the sensitizing tumor, and long term memory was established. The facile enrichment of tumor-reactive TDLN T cells, based on the CD62Llow phenotype, circumvents the need for prior knowledge of the relevant tumor Ags. Coupling the isolation of pre-effector T cells with rapid ex vivo expansion to >3 logs could overcome some of the shortcomings of active immunotherapy or in vivo cytokine treatment, where selective robust expansion of effector cells has been difficult to achieve.
Marked Prolongation of Cardiac Allograft Survival by Dendritic Cells Genetically Engineered with NF-κB Oligodeoxyribonucleotide Decoys and Adenoviral Vectors Encoding CTLA4-Ig
Authors: Bonham C. Andrew , Peng Lansha , Liang Xiaoyan , Chen Zongyou , Wang Lianfu , Ma Linlin , Hackstein Holger , Robbins Paul D. , Thomson Angus W. , Fung John J. , Qian Shiguang , Lu Lina
Published: April 2014

Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-κB-dependent mechanisms, which can be blocked by double-stranded “decoy” oligodeoxyribonucleotides (ODNs) containing binding sites for NF-κB. Herein, we describe the combined use of NF-κB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-κB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.
Restriction of De Novo Pyrimidine Biosynthesis Inhibits Th1 Cell Activation and Promotes Th2 Cell Differentiation
Authors: Dimitrova Petya , Skapenko Alla , Herrmann Matthias L. , Schleyerbach Rudolf , Kalden Joachim R. , Schulze-Koops Hendrik
Published: April 2014

Leflunomide, an inhibitor of de novo pyrimidine biosynthesis, has recently been introduced as a treatment for rheumatoid arthritis in an attempt to ameliorate inflammation by inhibiting lymphocyte activation. Although the immunosuppressive ability of leflunomide has been well described in several experimental animal models, the precise effects of a limited pyrimidine supply on T cell differentiation and effector functions have not been elucidated. We investigated the impact of restricted pyrimidine biosynthesis on the activation and differentiation of CD4 T cells in vivo and in vitro. Decreased activation of memory CD4 T cells in the presence of leflunomide resulted in impaired generation and outgrowth of Th1 effectors without an alteration of Th2 cell activation. Moreover, priming of naive T cells in the presence of leflunomide promoted Th2 differentiation from uncommitted precursors in vitro and enhanced Th2 effector functions in vivo, as indicated by an increase in Ag-specific Th2 cells and in the Th2-dependent Ag-specific Ig responses (IgG1) in immunized mice. The effects of leflunomide on T cell proliferation and differentiation could be antagonized by exogenous UTP, suggesting that they were related to a profound inhibition of de novo pyrimidine biosynthesis. These results indicate that leflunomide might exert its anti-inflammatory activities in the treatment of autoimmune diseases by preventing the generation of proinflammatory Th1 effectors and promoting Th2 cell differentiation. Moreover, the results further suggest that differentiation of CD4 T cells can be regulated at the level of nucleotide biosynthesis.
CD4 T Cell Depletion Is Linked Directly to Immune Activation in the Pathogenesis of HIV-1 and HIV-2 but Only Indirectly to the Viral Load
Authors: Sousa Ana E. , Carneiro Jorge , Meier-Schellersheim Martin , Grossman Zvi , Victorino Rui M. M.
Published: April 2014

The causal relationships among CD4 cell depletion, HIV replication, and immune activation are not well understood. HIV-2 infection, “nature’s experiment” with inherently attenuated HIV disease, provides additional insights into this issue. We report the finding that in HIV-2 and HIV-1 patients with a comparable degree of CD4 depletion the imbalance in the relative sizes of the naive and memory T cell populations and the up-regulation of CD4 and CD8 cell activation markers (HLA-DR, CD38, CD69, Fas molecules) are similar, even though the viral load in the plasma of HIV-2-infected patients is two orders of magnitude lower than in HIV-1 patients and HIV-2 patients are known to have slower rates of CD4 T cell decline and a better clinical prognosis. Moreover, we found a similar increase in the frequency of cycling CD4 T cells (Ki67+), which was in strong correlation with the expression of activation markers. Finally, the level of T cell anergy, as assessed by the proliferative responses to CD3 stimulation and to a panel of microbial Ags, proved to be comparable in HIV-1 and HIV-2 patients with a similar degree of CD4 depletion despite large differences in viral load. Our data are consistent with a direct causal relationship between immune activation and CD4 cell depletion in HIV disease and an only indirect relation of these parameters to the virus replication rate. Invoking the concept of proximal immune activation and virus transmission, which links efficient transmission of virus to local cell activation and proliferation in response to Ags and inflammation, we propose an integrative interpretation of the data and suggest that strongly elevated immune activation induces CD4 cell depletion and not vice versa, with potential implications for the choice of treatment strategies.
Identification of Human Autoantibodies to the DNA Ligase IV/XRCC4 Complex and Mapping of an Autoimmune Epitope to a Potential Regulatory Region
Authors: Lee Kyung-Jong , Dong Xingwen , Wang Jingsong , Takeda Yoshihiko , Dynan William S.
Published: April 2014

The nonhomologous end-joining pathway is the principal mechanism for repair of ionizing radiation-induced, double-strand breaks in mammalian cells. Three polypeptides in this pathway, including the two subunits of Ku protein and the catalytic subunit of the DNA-dependent protein kinase, are known targets of autoantibodies in systemic rheumatic diseases. Here we show that two additional polypeptides in the pathway, DNA ligase IV and XRCC4, are also targets of autoantibodies. These Abs were present in 20% of patients with systemic lupus erythematosus and overlap syndrome. Previous work has shown that XRCC4 is subject to radiation-induced post-translational modification, including phosphorylation by DNA-dependent protein kinase and cleavage by caspase 3. We mapped a major autoimmune epitope in XRCC4 and found that it encompassed a DNA-dependent protein kinase phosphorylation site, which is located at serine 260; that it was adjacent to a site for caspase 3, which cleaves after residue 265; and that it also spanned a site for the inflammatory protease, granzyme B, which cleaves after residue 254. The finding that five different polypeptides in the nonhomologous end-joining pathway are potential targets of autoantibodies together with the observation that one of the autoimmune epitopes in XRCC4 coincides with a sequence that is a nexus for radiation-induced regulatory events suggest that exposure to agents that introduce DNA double-strand breaks may be one of the factors that influences the development of an autoimmune response in susceptible individuals.
Inhibition of Adjuvant Arthritis by a DNA Vaccine Encoding Human Heat Shock Protein 60
Authors: Quintana Francisco J. , Carmi Pnina , Mor Felix , Cohen Irun R.
Published: April 2014

Adjuvant arthritis (AA) is an autoimmune disease inducible in rats involving T cell reactivity to the mycobacterial 65-kDa heat shock protein (HSP65). HSP65-specific T cells cross-reactive with the mammalian 60-kDa heat shock protein (HSP60) are thought to participate in the modulation of AA. In this work we studied the effects on AA of DNA vaccination using constructs coding for HSP65 (pHSP65) or human HSP60 (pHSP60). We found that both constructs could inhibit AA, but that pHSP60 was more effective than pHSP65. The immune effects associated with specific DNA-induced suppression of AA were complex and included enhanced T cell proliferation to a variety of disease-associated Ags. Effective vaccination with HSP60 or HSP65 DNA led paradoxically to up-regulation of IFN-γ secretion to HSP60 and, concomitantly, to down-regulation of IFN-γ secretion to the P180-188 epitope of HSP65. There were also variable changes in the profiles of IL-10 secretion to different Ags. However, vaccination with pHSP60 or pHSP65 enhanced the production of TGFβ1 to both HSP60 and HSP65 epitopes. Our results support a regulatory role for HSP60 autoreactivity in AA and demonstrate that this control mechanism can be activated by DNA vaccination with both HSP60 or HSP65.
T Cell Immunity in Connective Tissue Disease Patients Targets the RNA Binding Domain of the U1-70kDa Small Nuclear Ribonucleoprotein
Authors: Greidinger Eric L. , Foecking Mark F. , Schäfermeyer Kim R. , Bailey Craig W. , Primm Shannon L. , Lee David R. , Hoffman Robert W.
Published: April 2014

Although the T cell dependence of autoimmune responses in connective tissue diseases has been well established, limited information exists regarding the T cell targeting of self Ags in humans. To characterize the T cell response to a connective tissue disease-associated autoantigen, this study generated T cell clones from patients using a set of peptides encompassing the entire linear sequence of the 70-kDa subunit of U1 snRNP (U1-70kDa) small nuclear ribonucleoprotein. Despite the ability of U1-70kDa to undergo multiple forms of Ag modification that have been correlated with distinct clinical disease phenotypes, a remarkably limited and consistent pattern of T cell targeting of U1-70kDa was observed. All tested T cell clones generated against U1-70kDa were specific for epitopes within the RNA binding domain (RBD) of the protein. High avidity binding of the RBD with U1-RNA was preserved with the disease-associated modified forms of U1-70kDa tested. The high avidity interaction between the U1-RBD on the polypeptide and U1-RNA may be critical in immune targeting of this region in autoimmunity. The T cell autoimmune response to U1-70kDa appears to have less diversity than is seen in the humoral response; and therefore, may be a favorable target for therapeutic intervention.
Impaired Effector Function of Hepatitis C Virus-Specific CD8+ T Cells in Chronic Hepatitis C Virus Infection
Authors: Wedemeyer Heiner , He Xiao-Song , Nascimbeni Michelina , Davis Anthony R. , Greenberg Harry B. , Hoofnagle Jay H. , Liang T. Jake , Alter Harvey , Rehermann Barbara
Published: April 2014

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-γ production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.
Cutting Edge: CTLA-4 (CD152) Differentially Regulates Mitogen-Activated Protein Kinases (Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase) in CD4+ T Cells from Receptor/Ligand-Deficient Mice
Authors: Schneider Helga , Mandelbrot Didier A. , Greenwald Rebecca J. , Ng Fai , Lechler Robert , Sharpe Arlene H. , Rudd Christopher E.
Published: April 2014

Although CTLA-4 (CD152) has potent inhibitory effects on T cell function, the signaling events affected by this coreceptor remain to be fully defined. Mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) act as crucial regulators of multiple aspects of cell function. Ab ligation studies have reported an inhibitory effect of CTLA-4 on TCR-induced ERK and JNK activation. In this study, we have re-examined the specificity of CTLA-4 inhibition of MAPKs by using natural ligand with ex vivo-purified CD4+ T cells deficient in CD80 and CD86 (double knockout), or CTLA-4, CD80, and CD86 (triple knockout). Under these conditions, CTLA-4 ligation was found to up-regulate and sustain JNK activation, while inhibiting ERK activity. At the same time, JNK activation could not account for CTLA-4 induction of TGF-β production. Our findings demonstrate that CTLA-4 cosignaling is more complex than previously appreciated, with an ability to differentially regulate members of the MAPK family in T cells.
Fas Resistance of Leukemic Eosinophils Is Due to Activation of NF-κB by Fas Ligation
Authors: Qin Yimin , Camoretti-Mercado Blanca , Blokh Lyubov , Long Catherine G. , Ko Franklin D. , Hamann Kimm J.
Published: April 2014

TNF family receptors can lead to the activation of NF-κB and this can be a prosurvival signal in some cells. Although activation of NF-κB by ligation of Fas (CD95/Apo-1), a member of the TNFR family, has been observed in a few studies, Fas-mediated NF-κB activation has not previously been shown to protect cells from apoptosis. We examined the Fas-induced NF-κB activation and its antiapoptotic effects in a leukemic eosinophil cell line, AML14.3D10, an AML14 subline resistant to Fas-mediated apoptosis. EMSA and supershift assays showed that agonist anti-Fas (CH11) induced nuclear translocation of NF-κB heterodimer p65(RelA)/p50 in these cells in both a time- and dose-dependent fashion. The influence of NF-κB on the induction of apoptosis was studied using pharmacological proteasome inhibitors and an inhibitor of IκBα phosphorylation to block IκBα dissociation and degradation. These inhibitors at least partially inhibited NF-κB activation and augmented CH11-induced cell death. Stable transfection and overexpression of IκBα in 3D10 cells inhibited CH11-induced NF-κB activation and completely abrogated Fas resistance. Increases in caspase-8 and caspase-3 cleavage induced by CH11 and in consequent apoptotic killing were observed in these cells. Furthermore, while Fas-stimulation of resistant control 3D10 cells led to increases in the antiapoptotic proteins cellular inhibitor of apoptosis protein-1 and X-linked inhibitor of apoptosis protein, Fas-induced apoptosis in IκBα-overexpressing cells led to the down-modulation of both of these proteins, as well as that of the Bcl-2 family protein, Bcl-xL. These data suggest that the resistance of these leukemic eosinophils to Fas-mediated killing is due to induced NF-κB activation.