bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA Class I Components Increase Pancreatic Cancer Proliferation and Migration
Authors: Sliker Bailee H , Awaji Mohammad , Nguyen Thanh , Pandey Poomy , Solheim Joyce C
Published: January 2023

Recent studies in transplantation models have implicated HLA class I molecules as important factors in activating downstream pathways that lead to cell proliferation and migration. In a few cancers, β2m alone has been proposed to be high in expression and to increase proliferation and migration. No research to date has reported if β2m and/or the HLA class I heavy chain promote the migration and proliferation of pancreatic cancer cells and, if so, whether β2m and the HLA class I heavy chain work together or as separate entities within the signaling pathways to promote these processes.

Using pancreatic cancer cells, we demonstrate that siRNA knockdown of β2m causes a decrease in cell migration and proliferation without altering viability. This decrease of β2m also leads to a reduction in the expression of the HLA class I heavy chain as well as downstream signaling molecules, pCREB and pFAK. Downregulation of HLA-A leads to decreased pCREB while HLA-B knockdown does not and neither heavy chain isotype has an effect on pFAK expression.

Overall, this study demonstrates the importance of β2m in cancer cell migration and proliferation through signaling pathways involving pCREB and pFAK. We show that HLA class I heavy chains are expressed by pancreatic cancer cells and that HLA-A heavy chains may be more influential in cell migration and proliferation than HLA-B heavy chains. Loss of β2m expression causes a decrease in HLA class I heavy chains, suggesting that β2m may exert some of its effects on growth and migration via the heavy chain. However, the fact that some downstream molecules are altered in cells with β2m reduction, yet no change is observed with the downregulation of either HLA heavy chain isoform, indicates that each may also work in divergent pathways.
Cross-talk between NKT cells and tumor associated macrophages in the tumor microenvironment
Authors: Courtney Amy N , Tian Gengwen , Liu Daofeng , Marinova Ekaterina , Heczey Andras , Xu Xin , Guo Linjie , Gao Xiuhua , Metelitsa Leonid S
Published: January 2023

CD1d-reactive Va24-invariant NKT cells (NKTs) control tumor growth via yet poorly understood interactions with tumor-associated macrophages (TAMs) and other myeloid cells. TAMs comprise M1- and M2-like subsets, but only CD163high M2-like TAMs are associated with poor outcome in neuroblastoma (NB) and other tumors. Here, we demonstrate that NKTs selectively target M2-like TAMs via contact-dependent and independent mechanisms. Upon direct contact with antigen-pulsed M1 or M2 macrophages, NKTs selectively killed the latter. Although the killing was strictly CD1d-dependent, CD1d expression between macrophage subsets was equivalent. In contrast, only M2-polarized macrophages expressed CD204, a scavenger receptor responsible for cellular uptake of CD1d ligands. Anti-CD204 blocking mAb inhibited NKT-cell cytotoxicity against M2 in vitro while lentiviral transduction with CD204 cDNA rendered M1 sensitive to NKT cytotoxicity. We also found that antigen-activated NKTs could reprogram M2- into functional M1-like macrophages via GM-CSF production. Furthermore, adoptive transfer of human NKTs resulted in a M1-like polarization of TAMs in metastatic NB xenografts in humanized NSG mice. However, tumors progressed even after NKT cell treatment, suggesting a tumor escape mechanism. Indeed, we found that exposure to activated NKTs resulted in rapid up-regulation of PD-L1 on both M2 and M1 subsets, which correlated with suppressed NKT proliferation and cytokine production. Thus, our results reveal a novel mechanism of immune regulation, in which NKTs control tumor-supportive inflammation via killing or reprogramming of M2-like TAMs. However NKT function is negatively regulated by TAMs at least in part in a PD-1-dependent manner.
Characterizing the anti-tumor response of T cell receptor gene-modified invariant natural killer T cells
Authors: Fleming-Trujillo Erica Linn , Nagato Kaoru , Scurti Gina , Nishimura Michael I
Published: January 2023

Human invariant NKT cells represent a distinct lymphocyte population that co-express an invariant antigen receptor and NK surface markers. Classic NKT cells express a restricted T cell receptor that recognizes lipids, such as α-Galactosylceramide (α-GalCer), in the context of CD1d. Activated iNKT cells produce a high level of cytokines, such as IFN- γ, thereby bridging innate and acquired immunity through the activation of other effector cells including dendritic cells (DCs), CD8 T cells, and NK cells. One major hurdle to studying the anti-tumor activity of iNKT cells is that they do not recognize antigens presented by classical MHC molecules and they have unknown tumor antigen specificity. Therefore, we expressed the MHC class I-restricted TIL 1383I TCR in human NKT cells to characterize their biology and tumor antigen-specific response. The 1383I TCR is a CD8 independent, high affinity TCR that recognizes the melanoma antigen, tyrosinase, presented by HLA-A2. Here, we report the expression and function of TIL 1383I TCR transduced human iNKT cells. iNKT cells stimulated with α-GalCer- pulsed DCs are transduced with the retroviral vector encoding the TIL1383I TCR. After confirming TIL 1383I TCR expression in iNKT cells by flow cytometry, we assess their anti-tumor activity and polyfunctionality by intracellular cytokine staining. TIL 1383I TCR transduced iNKT cells produced IFN-γ in response to tyrosinase loaded T2 cells and HLA-A2+/tyrosinase+ melanoma. These findings indicate that genetically engineered iNKT cells are useful to study their biology in the context of antigen specificity, as well as to generate new effector populations for the treatment of cancer patients.
Loss of humoral immunity in a mouse model of ovarian cancer
Authors: Riggs James Edward , Torres Gretel , Rodriguez Wilenny , DePierri Kelley , Goldman Naomi
Published: January 2023

Although a low incidence cancer, ovarian carcinoma (OvCa) has a high mortality rate due to late detection. To study the humoral immune response to OvCa we transplant ID8 cells (mouse epithelial carcinoma) into the murine peritoneal cavity (PerC). We found that as the OvCa developed PerC B cells, particularly B-1 B cells, were depleted. To assess the systemic impact of this depletion we assessed humoral immunity. We found a reduction in “natural” IgM and IgG3 production and increased IgA and IgG1. Following immunization, there was loss of the TI-2 response (FITC-dextran, FITC-Ficoll); the TI-1 (FITC-LPS) response was intact until very late stage disease. Since B-1 cells also serve a housekeeping role in apoptotic corpse clearance, we used a FACS assay and a cell-based ELISA to monitor antibodies directed at apoptotic ID8 cells. Mice with OvCa had lower titers of sera that bound apoptotic ID8 cells. Collectively these data validate the systemic loss of B-1 B cells in mice with OvCa. These results could serve to inform strategies designed for early detection of OvCa.
Redirected Adoptive Cell Transfer (ReACT)- A Multi-pronged Strategy to Treat Solid Tumors
Authors: Cui Weiguo , Xin Gang , Schauder David
Published: January 2023

Adoptive cell transfer (ACT) of genetically engineered T cells provides unprecedented opportunities to treat hematological malignancies. However, due to insufficient migration and anti-tumor function of transferred T cells, especially inside the immunosuppressive tumor microenvironment (TME), the efficacy of ACT is much curtailed in treating solid tumors. Pathogen-based cancer vaccines can break immunosuppression in TME, but often fail to mobilize tumor-specific T cell response with ample magnitude to eradicate established tumors. To overcome these challenges, we genetically engineered tumor-specific CD8 T cells in vitro with a second T cell receptor (TCR) that recognizes a bacterial antigen. We then transfer these dual-specific T cells in combination with intratumoral bacteria injection in an approach called redirected-ACT (ReACT) to treat melanoma in mice. The dual-specific CD8 T cells expand robustly in response to bacteria, migrate to the very site of infection/tumor, and confer tumor eradication in the majority of tumor-bearing mice. The mice cured from ReACT also develop immunological memory against subsequent tumor rechallenge. Additionally, we found that this combined approach reverts the immunosuppressive TME and recruits redirected CD8 T cells with enhanced killing ability to the very site of tumor. Overall, we provide the proof-of-principle of a promising strategy to treat various malignancies.
T cell therapy targeting oncogenic antigens in ovarian cancer
Authors: Anderson Kristin G , Bates Breanna M. , Chiu Edison Y. , Greenberg Philip D.
Published: January 2023

Over 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed very little in the last 20 years, highlighting the need for innovative therapies. Reprogramming immune system cells to target and kill ovarian cancer cells represents a potentially promising new treatment strategy. Immune T cells have the potential to control tumor growth without toxicity to healthy tissues if engineered to target proteins uniquely overexpressed in tumors. Recent technological advances have helped identify and validate Wilms’ Tumor Antigen 1 (WT1) and mesothelin (MSLN) as promising ovarian cancer antigen targets; these proteins contribute to malignant and invasive phenotypes and have limited expression in healthy cells. Using patient-derived cell lines and the murine ID8 tumor, we show that T cells engineered to express a high-affinity T cell receptor (TCR) targeting either WT1 or MSLN can kill human and murine ovarian tumor cells in vitro. Moreover, in an in vivo model of established disease with disseminated ID8 tumor, adoptively transferred TCR-engineered T cells infiltrated and preferentially accumulated within ovarian tumors, delayed tumor growth and prolonged survival. However, our data revealed that the tumor microenvironment (TME) limits the survival and killing capacity of these engineered T cells. Cellular and molecular analyses of human tumor specimens have revealed human therapy will face similar obstacles. Therefore, ongoing studies will be discussed that are further exploring strategies to overcome these common elements of the immunosuppressive TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function.
Expanding the scope of WT1- and cyclin A1-specific TCR gene therapy for AML and other cancers
Authors: Perret Rachel , Valliant-Saunders Karine , Cao Jennifer W , Greenberg Philip D
Published: January 2023

TCR gene therapy hastens the reproducible generation of tumor-specific T cells from patients, creating a promising avenue for treating hematological and solid tumors. Several hurdles remain to making this a broadly effective modality, including variable target antigen expression, risk of tumor antigenic escape, and current therapies limited almost exclusively to HLA-A0201+ patients. We previously characterized WT1 and cyclin A1 as favorable T cell immunotherapy targets, based on high expression in malignant versus normal cells and roles in oncogenesis, reducing likelihood of loss variants. We currently have a high-affinity HLA-A0201/WT1-specific TCR in Phase I trials for the treatment of AML and NSCLC, and are now focused on developing WT1- and cyclin A1-specific TCRs for ~7 common HLA types, aiming to cover >90% of patients. T cell lines were generated by stimulating donor PBMC with overlapping peptide libraries to identify new reactive epitopes, and the highest affinity T cell clones for each HLA/antigen combination identified by tetramer binding strength. TCRs were then cloned into lentiviral vectors for expression in CD8 T cells. We are currently in the process of selecting the best HLA-A0201/cyclin A1-specific TCR with which to proceed to clinical development. We are also continuing to optimize our epitope discovery protocol for the improved selection of TCRs for other HLA alleles. Safety and efficacy of selected TCRs are rigorously tested using bioinformatic screens, in vitro assays, and humanized MHC-I mouse models. Our goal is to create a molecular toolbox of off-the-shelf TCR reagents to facilitate rapid treatment of patients, using T cell immunotherapies targeted to their particular tumor antigenic profile and HLA type.
VSV immunotherapy to improve treatment outcomes in a mouse model of metastatic melanoma
Authors: Shim Kevin G. , Thompson Jill , Rajani Karishma , Kottke Tim , Vile Richard
Published: January 2023

Vesicular Stomatitis Virus (VSV) engineered to express tumor-associated antigens (TAA) is a promising systemic therapy against widely disseminated malignancy. Using a mouse model of metastatic melanoma, we have demonstrated that VSV immunotherapy is a rational addition to current treatment strategies. Combining VSV immunotherapy with adoptive T-cell transfer and localized radiation therapy enhances the overall survival and control of disease in mice challenged with disseminated melanoma. Furthermore, our previous data have shown that the use of stereotactic hypofractionated radiotherapy provides control of subcutaneous localized disease while systemically delivered VSV immunotherapies contribute to the control of metastatic disease. From these experiments we have demonstrated that this therapy is mediated by multiple immune subsets including CD4+, CD8+, and NK1.1+ populations. Thus, we hypothesized that the additional combination of the above clinically relevant therapies with adoptive T-cell transfer and immune checkpoint inhibitors would result in distinct and quantifiable improvements in immunological tumor control. Forthcoming data will characterize the expression dynamics of the inhibitory immune receptors PD-1 and TIM3 on effector and naïve populations (as defined by CD44 and CD62L expression) in response to each of the above therapies in order to design rationale treatment strategies against metastatic melanoma.
N-acetylcysteine protects melanoma specific cytotoxic T cells from TCR restimulation induced DNA damage and enhances tumor control in vitro and in vivo.
Authors: Scheffel Matthew J , Scurti Gina , Simms Pat , Nelson Michelle , Garrett-Mayer Elizabeth , Mehrotra Shikhar , Paulos Chrystal M , Nishimura Michael I , Voelkel-Johnson Christina
Published: January 2023

Clinical trials utilizing the adoptive transfer of autologous tumor antigen specific lymphocytes have demonstrated superior clinical efficacy compared to standard-of-care. However, the majority of patients do not achieve a durable complete response requiring further optimization of the therapy. The rapid expansion of therapeutic T cells increases susceptibility to Activation Induced Cell Death (AICD) following TCR restimulation in vitro. Our investigations demonstrate that TCR restimulation enhances phosphorylation of γH2AX and pSMC-1, which are markers of double-strand DNA breaks. Onset of DNA damage was accompanied by the autophosphorylation of ATM, activation of the cell-death regulator p53 via phosphorylation on Ser15, and its accumulation in the nucleus. Directed inhibition of ATM with Caffeine or KU55933 protected cells from AICD. The anti-oxidant N-acetylcysteine (NAC) prevented the onset of DNA damage and subsequent ATM/p53 activation as well as cell death. Expansion of TIL1383I transduced human T cells in NAC reduced levels of γH2AX, PD-1 and CD57 while enhancing killing of MEL624 melanoma cells in vitro. In the Pmel model, expansion of T cells in NAC increased their persistence in blood and spleen and decreased γH2AX expression. NAC treatment also increased the presence of Pmel cells in the tumor, reduced tumor growth and significantly increased survival of B16-F10 challenged mice. Taken together, these data suggest the addition of NAC to current therapeutic T cell expansion protocols has the potential to improve the quality and therapeutic efficacy of the cell product infused into patients.
Comparative genome-wide screens reveal novel regulators of Cytotoxic Lymphocyte-mediated cell death.
Authors: Menasche Bridget L , Davis Eric , Sheppard Jacob , Shen Jingshi
Published: January 2023

Cytotoxic Lymphocytes (CLs) are key players in the effector branch of the immune response, detecting and eliminating transformed and pathogen-infected cells. While the physiological roles of CL-mediated apoptosis are well established, many regulators of these apoptotic pathways in target cells remain poorly understood. We performed comparative genome-wide haploid genetic screens in a human cell culture model for CL killing and resistance in order to identify positive and negative regulators of target cell survival during CL-mediated killing. These regulators include genes encoding glycosylphosphatidylinositol (GPI) anchor synthesis enzymes and immune surveillance receptors. In order to identify further potential components of these pathways, we performed an additional resistance screen in a GPI-anchored protein deficient haploid cell line. We identified both common and disparate regulators between unaltered target cells and GPI-anchored protein deficient target cells. These disparate regulators underline the importance of GPI-anchored proteins in CL target recognition and killing. Some gene knockouts enriched by CL-mediated killing in both screens are also mutated in cancer. Further study of the regulators identified by these screens will advance our understanding of how the many killing pathways used by CLs shape the interaction between the immune system and cancer.
A novel threonine protein kinase regulates prohibitin 2 activity and T cell function
Authors: Robles Elisa , Padilla Diana L. , Kirken Robert A.
Published: January 2023

Hispanic children have the highest rate of leukemia, which causes more deaths than any other cancer among children in the United States. Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer which is characterized by rapid uncontrolled accumulation of immature lymphocytes in the bone marrow and peripheral blood. Our studies have shown that the Prohibitin (Phb) family of proteins, Phb1 and Phb2, were upregulated during normal T-cell activation and likely function to maintain mitochondrial homeostasis and survival. In this work we hypothesized that a novel threonine protein kinase regulates prohibitin 2 (Phb2) activity and T-cell function. To determine the phosphorylation status of prohibitin proteins, phosphoamino acid analysis and mass spectrometry was performed followed by the generation of unique phospho-specific antibodies to characterize the putative regulatory pathways. A novel IL-2 inducible phospho-site in Phb2 was identified and characterized in T-cells. We propose that this novel threonine protein kinase participates in a previously unrecognized IL-2 signaling pathway that regulates T-cell activation and differentiation. The data also suggest that prohibitins are differentially expressed in lymphoid tumor cell lines and may serve as novel cancer biomarkers and possible molecular targets for therapeutic intervention in hematologic malignancies.
Tumor infiltrating T cells have abnormal lipid metabolism that can be modulated by PD-L1 blockade.
Authors: Yin Yiming , Metzger Todd , Bailey-Bucktrout Samantha
Published: January 2023

T cell activation and function are tightly regulated by nutrient availability and cellular metabolism. The solid tumor environment has several attributes that affect the metabolism of tumor infiltrating T cells (TILs). For example, tumor cells compete with T cells for glucose and thereby limit T cell effector function. Moreover, highly hypoxic conditions that are found in the tumor environment impair the oxidative metabolism of T cells. Checkpoint inhibitory and co-stimulatory pathways have been shown to regulate T cell metabolism, indicating that cancer immunotherapies may modulate T cell function via metabolic processes. Here we show that tumor infiltrating T cells have abnormally high fatty acid uptake and lipid content, which is associated with increased expression of CD36, a fatty acid transporter. The abnormal metabolism is associated with the expression of exhaustion markers programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM3) and is due to the cancer microenvironment rather than antigen exposure. Blocking PD-1 signaling in vivo significantly reduced tumor progression and normalized lipid metabolism. In summary, immunometabolism including lipid metabolism can serve as biomarkers of T cell anti-tumor efficacy. Targeting immunometabolism represents a promising venue in cancer immunotherapy.
Coupling metabolic activities to regulate CD8+ T cell differentiation and fate
Authors: Shrikant Protul A
Published: January 2023

Metabolism plays a central role in regulating CD8+ T cell differentiation and clonal expansion. Adoptive cell therapy (ACT) of cancer requires ex vivo generation of large pools of antigen specific effector CD8+ T cells. Since, differentiated effector CD8+ T cells have short life-span, exploiting mechanisms that regulate differentiation and survival can facilitate generation of large pools of effector CD8+ T cells for ACT. Integration of cell extrinsic signals during antigen stimulation (antigen, B7.1 and IL-12) induces mTORC1 activity to regulate transcriptional programs that determine functional outcomes. Our results indicate that antigen stimulation induced mTORC1 induces the mitochondria expressed uncoupling protein 2 (UCP2) to dampen glycolysis and OXPHOS to restrict effector differentiation of CD8+ T cells. Moreover, UCP2 reduces mitochondrial reactive oxygen species (ROS) production to promote clonal expansion by restricting activation induced cell death (AICD). By identifying a regulatory role of UCP2 in metabolic programming of CD8+ T cell differentiation and fate, our studies suggest targeting UCP2 expression and activity for the ex vivo generation of effector CD8+ T cells for ACT of cancer.
Tumor-intrinsic B7-H1 in melanoma and ovarian cancer regulates mTOR, autophagy and tumor growth
Authors: Clark Curtis A , Gupta Harshita B. , Pandeswara Srilakshmi , Sareddy Gangadhara R. , Yuan Bin , Hurez Vincent , Li Rong , Vadlamudi Ratna , Curiel Tyler J
Published: January 2023

Tumor B7-H1 (PD-L1) promotes tumor immune evasion by interaction with T cell PD-1. B16 melanoma and ID8agg ovarian cancer express B7-H1. We genetically made B7-H1lo or B7-H1hi lines to study additional B7-H1 effects. Control B16 and ID8agg grew more rapidly in vitro versus B7-H1lo cells and B7-H1hi B16 cells grew faster than control. In vivo, B16 and ID8agg grew faster than B7-H1lo lines in immunodeficient NSG mice. B16 challenge in NSG mice with B7-H1hi cells produced more lung metastases versus parental, which produced more metastases than B7-H1lo. αB7-H1 significantly reduced tumor growth and metastases. These data support an immune-independent role for tumor B7-H1 in tumor growth and metastatic spread. Control B16 and ID8agg cells had elevated TORC1 and reduced TORC2 versus B7-H1lo cells. The mTOR inhibitor rapamycin reduced cell growth greater in parental versus B7-H1lo cells in vitro. Also in line with reduced TORC1, B7-H1lo cells had increased autophagy (LC3-II/actin ratio and autophagosome formation) compared to parental cells. Notably, autophagy inhibitors chloroquine and 3-methyladenine had a greater inhibitory effect on in vitro proliferation of control B16 versus B7-H1lo cells. Inhibition of autophagy in vivo with either agent significantly reduced tumor growth of B16 but not B7-H1lo cells, suggesting that B7-H1 replete tumors are more dependent on the stress-modulating function of autophagy versus B7-H1lo tumors. Our studies show novel tumor-intrinsic B7-H1 signal effects that regulate mTOR and autophagy signals directly associated with immune-independent tumor growth, supporting a paradigm shifting concept that tumor B7-H1 augments stress response and metabolism through cell-intrinsic mechanisms.
Musocal vaccine for universal influenza A protection
Authors: Phillips Joy A , Tallapaka Shailendra B , Vetro Joseph A , Sanderson Sam D , Thoman Marilyn L
Published: January 2023

The rapid mutation rate of the influenza virus requires that the yearly flu vaccine development cycle start months in advance of the flu season. The search for a single vaccine able to protect against multiple strains of influenza has thus long been the Holy Grail of flu vaccine research. Such a vaccine would require an antigenic target that is highly or completely homologous among all influenza subtypes. There is one such conserved region present on the otherwise highly variable influenza A hemagglutinin protein. This short, linear peptide sequence known as the fusion peptide is highly conserved among influenza A viral subtypes. Up until now, the difficulty generating strong immune responses against short peptide epitopes has prevented exploitation of this conserved region as a vaccine target. We previously developed a novel peptide adjuvant termed EP67 that induces mucosal and systemic adaptive immune responses to conjoined peptide sequences. In this study, we conjugated EP67 to the conserved influenza A fusion peptide sequence. The resulting peptide conjugate was then encapsulated in biodegradable PLGA 50:50 nanoparticles, and the nanoparticle vaccines were delivered to either the lungs or the nasal mucosal tissue. Preliminary testing shows that intranasal administration of our encapsulated peptide vaccine protects young mice from lethal infection with either H1N1 or H3N2 strains of influenza. Aged animals are also protected from lethal infection using this nanoparticle vaccine. These studies support the continued exploration mucosal nanoparticle vaccines containing conserved influenza sequences as a promising strategy for universal influenza protection.
Use of biologic nanolipoprotein particles containing monophosphoryl lipid A as a novel intranasal vaccine platform for Bacillus anthracis
Authors: Weilhammer Dina R , Dunkle Alexis D , Blanchette Craig , Boone Tyler , Gilmore Sean , Corzett Michele , Fischer Nicholas , Lehmann Doerte , Lychak Cheri , Hoeprich Paul , Driks Adam , Rasley Amy
Published: January 2023

There is considerable interest in developing a novel Bacillus anthracis vaccine that improves upon the shortcomings of the licensed vaccine for broader, more rapid protection of the population. Ideally, a vaccine would recognize the spore in addition to having anti-toxin effects as well as induce robust mucosal and lung-associated immunity for early recognition and clearance. Here, we investigate nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs vs. delivery with free MPLA induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. Robust T cell responses to various spore antigens were also detected in the lung. When multiple immunizations were required, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced robust responses to all incorporated antigens, indicating potential for a multivalent vaccine preparation. Experiments to determine the efficacy of multivalent vaccine preparations against inhalational B. anthracis challenge are ongoing. In summary, the NLP platform enhances humoral, cellular and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.
Immune history to influenza infection and vaccination predicts antibody responses to the seasonal influenza vaccine
Authors: Hertz Tomer , Shagal Ayelet , Ohmit Suzanne , Monto Arnold S
Published: January 2023

Vaccination, the most cost-effective public health intervention, stimulates the immune system to generate protective memory responses. A variety of factors impact an individual’s heterogeneity in vaccine induced immune responses, such as age and gender and ‘immunological history’ - the individual’s memory antibody repertoire to previously encountered pathogens and vaccines. Immune history is particularly interesting in influenza since humans are constantly infected and vaccinated annually. To profile the effect of immune history to previously influenza infections, we used samples form an influenza vaccine efficacy trial (FluVacs) in adults aged 18–65 conducted in 2007–2008. Samples were collected at baseline d0, post vaccination d21 and at the end of the season d90. We used antigen microarrays spotted with overlapping 20mer peptides from the HA and NA proteins of the seasonal vaccine to generate antibody profiles at baseline to represent individuals’ immune history. Profiles were also generated post-vaccination. We used unsupervised clustering to cluster profiles of individuals at baseline. We found that individuals clustered into several distinct antibody profiles. Importantly, we found that most individuals within the same baseline clusters produced similar antibody profiles post vaccination. We also compared the pre- to post-vaccination profiles and found that overall the vaccine generated a relatively modest boost of the previous antibody repertoire. Overall our results highlight the importance of profiling immune history to previous influenza infections as a baseline measurement that may be predictive of vaccine induced immune responses and vaccine-induced protection.
An innate helping hand for B cells on the margin during chronic viral infection
Authors: Cranert Stacey A , Moran Michael T , Waggoner Stephen N
Published: January 2023

Marginal zone (MZ) B cells are an anatomically segregated population well-positioned to respond to blood borne pathogens and implicated in defense against encapsulated bacteria. MZ B cells are also an important source of the anti-inflammatory cytokine, IL-10. As such, these cells are poised to prevent secondary bacterial infection or immune pathology during chronic virus infection. Surprisingly, CD169+ MZ macrophages and CD21+CD23neg B cells were lost in mice during persistent clone 13 lymphocytic choriomeningitis virus (LCMV) infection in the absence of natural killer (NK) cells. Confocal staining revealed a near complete absence of the MZ by day 10 of infection in NKdeficient mice, which we previously found contain enhanced populations of antiviral T cells. Intriguingly, depletion of either CD4 or CD8 T cells did not restore the MZ compartment in NKdeficient mice, but combined depletion of both subsets of T cells prevented the loss. This phenomenon was independent of NKT cells. We hypothesize that NK cells play an important role in curtailing harmful inflammation and preventing pathogenic bacterial diseases during chronic viral infection by sustaining the MZ. This discovery has important implications for health, immune function, and viral clearance during persistent infection.
Regulation of effective vs. ineffective B cell responses to chronic viral infections.
Authors: Staupe Ryan P , Wherry E. John
Published: January 2023

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are chronic viral infections that cause significant morbidity and mortality worldwide. Current efforts to vaccinate against these pathogens have been unsuccessful and have failed to induce effective neutralizing antibodies capable of mediating sterilizing immunity. Natural HIV of HCV infection elicits the production of affinity matured class-switched antibodies that target viral proteins. While these antibodies can drive viral evolution, they are ineffective at controlling infection. Moreover, it is now clear that the evolution of these antibody responses, and the B cells that produce them, during chronic infection are radically different than those generated by acute infection or vaccination. Despite these observations, little is known about the underlying mechanisms of B cell differentiation and control of effective antibody production during chronic viral infection. We have developed a novel technique that allows us to track the virus-specific B cell response in the lymphocytic choriomeningitis virus (LCMV) model, a mouse model of chronic viral infection. Using this technique we have found that chronic viral infection does not cause a skewing of the virus-specific B cell response to the plasma cell fate and away from the germinal center fate but induces a robust plasma cell response in an antigen-level and T-cell dependent manner. These results suggest that altered differentiation of virus-specific B cells early during the response to chronic viral infection may contribute to the temporal delay in development of effective neutralizing antibody responses during chronic infection.
Moving in for the kill: natural killer cell migration in regulation of humoral immunity
Authors: Moran Michael T , Rydyznski Carolyn E , Waggoner Stephen N
Published: January 2023

Natural killer (NK) cells are cytotoxic innate lymphocytes that provide immune defense against pathogens and tumors, and act as potent regulators of adaptive immunity. Notably, NK cells inhibit humoral immunity by restricting the magnitude of follicular helper T-cell (TFH) and germinal center (GC) B-cell responses in a perforin-dependent manner. This suppression occurs at an early stage of infection that coincides with an anatomic redistribution of NK cells within secondary lymphoid organs. Specifically, infection with lymphocytic choriomeningitis virus (LCMV) triggers transient NK-cell redistribution from the vascularized red pulp into the white pulp of the spleen. Many of these NK cells localize at the border between T- and B-cell zones where cognate interactions between TFH and B cells facilitates development of GCs. Some NK cells also penetrate deep into the B-cell follicle. We have investigated this phenomenon using a flow cytometry-based intravascular staining method and through confocal microscopy. We have observed a novel subset of NK cells expressing CXCR5 that coincidentally arise with NK-cell redistribution to spleen regions (i.e. B-cell zone) rich in CXCL13. We hypothesize that this subset of NK cells localize in a CXCR5 dependent manner near cells participating in the early steps of GC formation, including TFH, which allows for NK-cell contact and perforin-dependent killing of GC-inducing cells that ultimately dampens the development of immunity to infection. These regulatory NK cells are an innovative target for clinical strategies aimed at subverting or enhancing humoral immune responses.
LAG-3 expressing antigen-specific CD4+ T cells attenuate lung inflammation during acute influenza virus infection
Authors: Dutta Avijit , Huang Ching-Tai , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Yu-Lin
Published: January 2023

The host immune system responds to influenza virus infection for eradication of the virus but the immune response associated inflammation causes tissue injury and damage to the host as well. In our antigen-specific mouse model system for influenza virus infection, influenza hemagglutinin antigen-specific CD4+ T cells responded to acute infection with Th1 effector phenotype. A significant proportion of them were also with concurrent expression of LAG-3, CTLA-4, GITR or CD25. Adoptive transfer of purified LAG-3 expressing hemagglutinin antigen-specific CD4+ T cells controlled lung inflammation with acute influenza virus infection, as represented by the activation of co-transferred influenza hemagglutinin antigen-specific CD8+ T cells. Control of the inflammation by LAG-3 expressing antigen-specific Th1 CD4+ T cells was not necessarily associated with impaired capacity of virus eradication. These results reveal, besides effector function for virus eradication, subsets of CD4+ T cells activated by acute influenza virus infection also acquired regulatory function to attenuate inflammation for reducing the damage to the host. These subsets of CD4+ T cells may be employed as the practical handle for management of severe influenza disease.
Defining limits on efficacy of CD8 T cell-based HIV vaccines
Authors: Ganusov Vitaly V , Yang Yiding
Published: January 2023

It is generally accepted that an effective HIV vaccine should be able to induce neutralizing antibodies and memory CD8 T cells. Yet, basic features of protective CD8 T cell responses remain poorly defined. Using recently published data from a cohort of HIV-infected patients that were followed from the onset of symptoms into the chronic phase, we determined the basic quantitative in vivo features of HIV-specific CD8 T cell responses. First, we found that the number of responses recognizing different viral epitopes (immune response breadth) varied greatly between patients and on average, 8 epitopes were recognized by CD8 T cells. Surprisingly, we found that in most patients the breadth did not change significantly over the course of first year of infection suggesting that most HIV-specific T cell responses were primed in acute infection. There was a strong negative correlation between the predicted precursor frequency and the rate of expansion of epitope-specific T cells suggesting intraclonal competition. Finally, we found that the majority of CD8 T cell populations expanded and contracted in unison; however, about 20% of all CD8 T cell responses had a discordant dynamics suggesting interclonal competition. Taken together, our results suggest that effective control of HIV infection can be achieved with broad CD8 T cell response if vaccine-induced breadth is larger than that induced by a natural infection. However, broader T cell response will likely lead to interclonal competition between responses, and boosting the magnitude of epitope-specific T cell responses may reduce the expansion rate of these responses due to intraclonal competition. Our results thus suggest potential limits on the efficacy of CD8 T cell-based HIV vaccines.
Myelin-specific CD8+ T cells exacerbate CNS autoimmunity
Authors: Wagner Catriona , Goverman Joan
Published: January 2023

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). Although the pathogenic pathways of MS are not fully understood, myelin-specific T cells are hypothesized to initiate disease. Myelin-specific CD4+ T cells have been the main focus of MS research and animal models of MS have relied exclusively on the activity of CD4+ T cells. However, a substantial body of data from MS patients indicate that CD8+ T cells are also involved in MS pathogenesis. We hypothesized that recruitment of myelin-specific CD8+ T cells to the site of inflammation initiated by CD4+ T cells could influence disease. To test our hypothesis, we use a mouse model of MS, experimental autoimmune encephalomyelitis (EAE) in which adoptive transfer of CD4+ T cells specific for myelin oligodendrocyte glycoprotein (MOG) induces CNS autoimmunity. To determine the influence of CD8+ T cells, we introduce CD8+ T cells from a TCR-transgenic mouse model expressing a TCR specific for a MHC class I-restricted epitope of myelin basic protein (MBP) into the periphery of mice just after disease induction. We found that the recruitment of the MBP-specific but not control CD8+ T cells exacerbated CD4+ T cell-initiated EAE. Furthermore, recruitment of MBP-specific CD8+ T cells increases the number of MOG-specific CD4+ T cells within the CNS at peak disease. Interestingly, mice that received MBP-specific CD8+ T cells exhibited an increase in symptoms associated with brain inflammation. These data suggest that the interplay between CD4+ and CD8+ T cells specific for two different myelin proteins is critical for determining the manifestation of CNS autoimmune disease.
Th17 cell differentiation increases aquaporin-3 expression, which is further increased by treatment with NaCl.
Authors: Zhou Xiaoming , Nurmukhambetova Saule , Kim Yong-Chan , Adair Patrick , Wu Chuan , Scott David W , Kuchroo Vijay K , Lees Jason R
Published: January 2023

Aquaporin3 (AQP3) is one of 13 members of the aquaglyceroporin family that transports water and other small molecules across the cell membrane. In the immune system, AQP3 is important in macrophage phagocytosis, T cell migration, and dendritic cell activation. Th17 cells are thought to play a pivotal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), which is sensitive to salt intake. Through microarray, we previously demonstrated that in vitro differentiation of mouse Th17 cells increases AQP3 mRNA abundance over that seen in Th0 T cells. Utilizing both Western and flow cytometric analyses, we now show that this mRNA increase is correlated with an increase in AQP3 protein abundance, whereas Th1 differentiation has no significant effect on AQP3 protein abundance as compared with Th0 cells. Moreover, increased concentration of NaCl (40 mM over standard) to the medium further augments AQP3 protein abundance, but only within Th17 differentiated cells. We have also extended these studies to human T cells and found that human CCR6 positive cells, previously characterized as IL-17 producing cells constitutively express more AQP3 protein than human CCR6 negative cells. To test whether Th17 cells express more AQP3 protein than Th1 cells in vivo, we induced EAE by immunization of the mice with MOG35–55 with the mice injected with PBS as a control. We analyzed AQP3 protein abundance in splenic T cells directly ex vivo with flow cytometry and found that Th17 cells express more AQP3 protein than Th1 cells under both conditions. We conclude that Th17 cells express more AQP3 than Th1 cells both in vitro and in vivo, and that treatment with NaCl in vitro further increases AQP3 protein abundance only in Th17 cells. Supported by NMMS PP3448.
Myeloid deletion of SIRT1 suppresses collagen-induced arthritis in mice by modulating dendritic cell maturation
Authors: Ji Woo Seong , Jang Hyun-Young , Lee Sang-Il , Lee Sang-Myeong , Park Byung-Hyun
Published: January 2023

Objective

The type III histone deacetylase SIRT1 is an enzyme critical in the modulation of immune and inflammatory responses. However, data on its role in rheumatoid arthritis (RA) are limited and controversial. To better understand how SIRT1 regulates adaptive immune responses in RA, we evaluated collagen-induced arthritis (CIA) in myeloid cell-specific SIRT1 knockout (mSIRT1 KO) mice.

Methods

CIA was induced in mSIRT1 KO and wild-type (WT) mice. Arthritis severity was gauged by clinical, radiographic, and pathologic scores. Levels of cytokines and transcription factors were determined by real-time RT-PCR and ELISA. Flow cytometry was used to quantify T cells and dendritic cells (DCs). T cell response was also investigated when co-cultured with antigen-pulsed DCs.

Results

Compared with WT counterparts, arthritis in mSIRT1 KO mice was less severe and proved less destructive to joints. Expression levels of inflammatory cytokines, matrix metalloproteinases, and ROR-γT in mSIRT1 KO mice were also reduced by comparison, paralleled by reductions in Th1 and Th17 cells and CD80- or CD86-positive DCs. In addition, impaired maturation of DCs and resultant declines in Th1/Th17 immune response were observed in mSIRT1 KO mice. In co-culture, DCs of mSIRT1 KO mice were less adept at driving T cell proliferation and Th1/Th17 immune response.

Conclusion

Myeloid cell-specific deletion of SIRT1 appears to suppress CIA through modulation of DC maturation. Thus, careful investigation of DC-specific SIRT1 downregulation is needed to weigh the therapeutic utility of agents targeting SIRT1 in RA.
CD70 methylation of a regulatory region increases with age in lupus patients.
Authors: Ray Donna , Harlow Sioban , Strickland Faith , Orlowski Rachel , Marder Wendy , McCune W. Joseph , Somers Emily C
Published: January 2023

T cell DNA hypomethylation contributes to systemic lupus erythematosus (SLE), although the mechanisms are poorly understood. Previous work has shown that CD70, a B cell costimulatory ligand, is hypomethylated and overexpressed in lupus T cells, and that the overexpression leads to increased B cell costimulation and concomitant immunoglobulin overproduction. Recent evidence suggests that the methylation status of certain genes may characterize ‘epigenetic aging’, and that epigenetic age may be a stronger predictor than chronological age of health outcomes. CD70 has not been examined in this context, therefore we sought to characterize the methylation status of CD70 in lupus patients across age. CD4+ T cells were obtained from peripheral blood of 89 consecutive lupus patients enrolled in a population-based cohort. CD70 methylation of a regulatory region was assessed by bisulfite conversion and pyrosequencing. Mean methylation across 10 sites known to be hypomethylated in lupus patients was measured and correlated with age. CD70 methylation increased linearly with increasing age of lupus patients. These results suggest that CD70 may be involved in “epigenetic aging”, and that the increased hypomethylation of CD70 in younger patients may contribute to the earlier onset and increased severity of disease. Further studies of aging effects on methylation of immune genes involved in lupus may improve our understanding of the variability in susceptibility and onset of the disease.
Dendritic cells as potential mediators of Mycobacterium tuberculosis infection of the central nervous system in a MHC class II-deficient mouse model.
Authors: Mergaert Aisha M , Hernandez Gianna , Heninger Erika , Gerhart Christian , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Mycobacterium tuberculosis (Mtb) infection of the central nervous system (CNS) in a rare, but deadly form of TB affecting immune-compromised individuals. Due to the HIV-AIDS epidemic this population has recently increased, however the pathway by which Mtb gains access to the CNS is poorly understood. Our work seeks to understand the potential ability of dendritic cells to traffic Mtb to the CNS. Preliminarily, we established the importance of mycobacterium infected dendritic cells to the formation of nervous system infection in MHC class II-deficient mice. In this model we used bone marrow derived dendritic cells (BMDC) from a dendritic cell reporter mouse strain (CD11c-eYFP). These cells increased the dissemination of Mtb 6020 to the brain by one log compared to Mtb 6020 alone, though the systemic infection remained the same. When GM-CSF BMDCs were infected with tdTomato H37Rv Mtb and injected IV into MHC class II-deficient mice CFU was detected in the brain, cervical lymph nodes and lung with no Mtb observed in the liver and superficial lymph nodes. After infection with tdTomato Mtb, BMDCs matured using GM-CSF or FLT3 had increased expression of CD11c, eYFP, CD40 and CD86 as detected by flow cytometry. These immunocompromised mice, whose phenotype reflects aspects of immunodeficiency seen in human populations, have a predisposition to CNS infection when BMDCs are directly infected with mycobacterium, suggesting the ability of dendritic cells to facilitate the movement of Mtb across the BBB or choroid plexus to initiate infection. These data support the use of MHC class II-deficient mice as a model for future work.