bluuebunny/crossref_metadata_2025 huggingface dataset:
Human tumor-infiltrating Th17 cells have the capacity to differentiate into IFN-gamma+ and FOXP3+ T cells with potent suppressive function (66.27)
Authors: Peng Guangyong , Ye Jian , Su Xinming , Hsueh Eddy , Zhang Yanping , Koenig Joyce , Hoft Daniel
Published: January 2023

Accumulating evidence suggests that T-helper 17 (Th17) cells and regulatory T (Treg) cells may exhibit development plasticity, and that CD4+ Tregs can differentiate into IL-17-producing T cells; however, whether Th17 cells can reciprocally convert into Tregs has not been described. In this study, we generated Th17 clones from tumor-infiltrating T lymphocytes (TILs). We showed that Th17 clones generated from TILs can differentiate into IFN-gamma-producing and FOXP3+ cells after in vitro stimulation with OKT3 and allogeneic peripheral blood mononuclear cells (PBMCs). We further demonstrated that T-cell receptor (TCR) engagement was responsible for this conversion, and that this differentiation was due to the epigenetic modification and reprogramming of gene expression profiles, including lineage-specific transcriptional factor and cytokine genes. In addition to expressing IFN-gamma and FOXP3, we showed that these differentiated Th17 clones mediated potent suppressive function after repetitive stimulation with OKT3, suggesting that these Th17 clones had differentiated into functional Tregs. We further demonstrated that the Th17-derived Tregs, unlike naturally occurring CD4+CD25+ Tregs, did not reconvert back into Th17 cells even under Th17-biasing cytokine conditions. These results provide the critical evidence that human tumor-infiltrating Th17 cells can differentiate into Tregs and indicate a substantial developmental plasticity of Th17 cells.
Tumor cell Programmed Death Ligand-1-mediated T cell suppression is overcome by co-expression of CD80 (66.29)
Authors: Haile Samuel , Bosch Jacobus , Zeender Annette , Srivastava Minu , Rodel Sabine , Wolf Julie , Ksander Bruce , Ostrand-Rosenberg Suzanne
Published: January 2023

Programmed death ligand 1 (PDL1 or B7-H1) is expressed constitutively or is induced by IFNγ on the cell surface of most human cancer cells and acts as a “molecular shield” by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma) we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1- mediated immune suppression by tumor cells and restores the ability of tumor cells to activate tumor-specific T lymphocytes. Mechanistically, CD80 mediates its effects through its extracellular domain which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating anti-tumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.
Natriuretic peptide receptor A knock out restricts tumorigenesis through ablation of myeloid derived suppressor cells expansion (66.3)
Authors: Nelson Allison , Mohapatra Subhra , Mohapatra Shyam , Nagaraj Srinivas
Published: January 2023

Recent data from our group and a number of others have demonstrated that expansion and activation of Myeloid Derived Suppressor Cells(MDSC) play a pivotal role in tumorigenesis. The receptor for atrial natriuretic peptide, natriuretic peptide receptor A (NPRA) has been implicated in cancers. We have shown that NPRA expression and signaling is important for tumor growth. We hypothesize a novel link between NPRA expression, altered differentiation of myeloid cells and tumor formation. MDSC population consists of two major subsets of Ly6G+ granulocytic and Ly6C+ monocytic cells. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. MDSC isolated from tumor bearing mice expressed high levels of NPRA. Next we hypothesized that NPRA knock out would result in restricted tumor growth. Using NPRA deficient mice, we report that these mice have altered differentiation of myeloid cells. As a consequence tumor cells injected subcutaneously to NPRA KO mice failed to grow. We investigated the nature of cells responsible for tumorigenesis and proved that ablation of a subset of MDSC cells restricts tumor formation. This work may indicate key roles for NPRA in myeloid cell differentiation and cancer formation.
Macrophage polarization in the tumor following radiation therapy is mediated by NFκB p50 (66.30)
Authors: Gough Michael , Nguyen Cynthia , Cottam Benjamin , Savage Talicia , Crittenden Marka
Published: January 2023

By causing the death of cancer cells, radiation therapy has been proposed to provide both tumor antigen and endogenous immune adjuvants to initiate de novo tumor-specific immune responses. However, our experiments have shown that following radiation therapy of tumors in mice there is a rapid influx of tumor macrophages that polarize towards immune suppression. In this study we investigate the mechanisms by which newly recruited macrophages are polarized following exposure to irradiated cancer cells. Macrophage exposure to irradiated cancer cells causes a switch from TNFα to IL-10 secretion following LPS stimulation. Using luciferase reporter assays we identify that this is a transcriptional switch consistent with the action of NFκB p50 homodimers. Using macrophages from knockout mice we confirm that the immune suppressive polarization of macrophages following exposure to irradiated cancer cells requires NFκB p50. To identify the source of the upstream signal causing macrophage polarization, we determined that combined blockade of the TGFβ receptor and the Mer receptor for Gas6 bound to exposed phosphatidylserine was able to restore TNFα secretion on LPS stimulation. We propose that wound repair following radiation therapy limits control of residual disease by increasing immune suppression in the tumor. Directing the polarization of newly recruited tumor macrophages following radiation therapy represents a strategy to enhance adaptive immune control of residual cancer cells.
A molecular mechanism of tumor mediated suppression of dendritic cell differentiation (66.41)
Authors: Farren Matthew , Carlson Louise , Lee Kelvin
Published: January 2023

Cancer-induced immunosuppression contributes to tumor outgrowth and occurs, in part, via tumor-mediated dysregulation of DC-differentiation, resulting in immunosuppressive immature myeloid cells and fewer dendritic cells (DCs). Tumors impair DC-differentiation via factors that hyperactivate STAT3 in DC progenitors, though the mechanisms by which this inhibits DC-differentiation are poorly defined. PKCβII is essential in DC-differentiation. Here, we hypothesize that tumor driven STAT3 activity inhibits DC-differentiation by downregulating PKCβII expression. In most breast cancer patients tested, circulating myeloid cells had significantly decreased PKCβII expression (p<0.03). Myeloid cells from tumor bearing mice also had decreased PKCβII expression. In vitro, culture in tumor conditioned media (TCM) decreased PKCβII protein levels (~50%), significantly decreased PKCβII mRNA levels (p<0.01) and decreased phorbol ester driven DC-differentiation (p<0.01) in a human model (KG1). TCM also significantly reduced PKCβ promoter activity (p<0.01). TCM drove rapid STAT3 activation and STAT3 binding to the PKCβ promoter, suggesting that STAT3 downregulates PKCβII promoter activity. Clones stably expressing a constitutive active STAT3 construct had significantly decreased PKCβII expression (p<0.05), an effect blocked by STAT3 inhibitors. This argues that tumor-driven STAT3 hyperactivation downregulates PKCβII expression in DC progenitor cells, inhibiting DC-differentiation.
Dual TCR/common gamma chain cytokine signaling regulates OX40 expression and synergizes with OX40 ligation to augment anti-tumor immunity (66.42)
Authors: Redmond William , Triplett Todd , Floyd Kevin , Weinberg Andrew
Published: January 2023

The provision of T cell co-stimulation via members of the TNFR super-family, including OX40 (CD134) and 4-1BB (CD137), provides critical signals that promote T cell survival and differentiation. Recent studies have demonstrated that ligation of the OX40 receptor can augment T cell-mediated anti-tumor immunity in pre-clinical models and more importantly, several agonist OX40-specific agents are under clinical development for cancer immunotherapy. OX40 is of particular interest as a therapeutic target as it is not expressed on naïve T cells but rather, is transiently up-regulated following TCR stimulation. Although TCR engagement is necessary for inducing OX40 expression, whether additional downstream signals are needed to regulate OX40 remains unclear. In this study, we demonstrate that OX40 expression is regulated through a TCR and common gamma chain cytokine-dependent signaling cascade that requires JAK3-mediated activation of the downstream transcription factors STAT3 and STAT5. Furthermore, combined treatment with an agonist anti-OX40 mAb and IL-2R-directed cytokine therapy synergized to augment anti-tumor immunity by reviving the anti-tumor efficacy of anergic tumor-reactive CD8 T cells in vivo. Together, these data reveal the ability of TCR/common gamma chain cytokine signaling to regulate OX40 receptor expression and demonstrate a novel means of augmenting cancer immunotherapy by restoring the function of tumor-specific T cells.
CpG ODN-induced alteration of MDSC during immunotherapy of advanced renal cell carcinoma (66.44)
Authors: James Britnie , Kucaba Tamara , Norian Lyse , Griffith Thomas
Published: January 2023

Myeloid derived suppressor cells (MDSC) are immature, activated myeloid cells that can suppress T cell function via multiple mechanisms and dampen antitumor immunity. Using an orthotopic model of advanced renal cell carcinoma (RCC), in which lung metastases arise spontaneously from the primary renal tumors, we have been evaluating the efficacy of an antitumor therapy consisting of a recombinant adenovirus encoding TRAIL (Ad-TRAIL) combined with CpG ODN. In the present study, we examined the extent to which MDSC suppress antitumor immunity after Ad-TRAIL/CpG ODN therapy. We found a high frequency of CD11b+ CD11- Gr-1+ cells at the primary tumor site; these MDSC suppress T cell proliferation in vitro. Ad-TRAIL/CpG ODN therapy given at the primary tumor site decreased MDSC numbers at the primary tumor site, metastatic lung, and spleen, and also induced systemic CD8 T cell-mediated antitumor immunity and clearance of primary RCC tumors and lung metastases. Subsequent analysis revealed that CpG ODN alone is able to modulate MDSC frequency, phenotype, and function at the primary tumor site, metastatic lung, and spleen. Current studies are investigating the mechanism by which CpG ODN, either alone or in combination with Ad-TRAIL, modulates the frequency and function of MDSC. We are also investigating the ability of Ad-TRAIL/CpG ODN, administered in combination with MDSC-depleting anti-Gr-1 mAb, to further reduce tumor progression and MDSC numbers.
RNA aptamer against the IL-10 receptor block IL-10 function in vitro and in vivo (66.46)
Authors: Berezhnoy Alexey , Stewart Charles , Giagrande Paloma , McNamara James , Trinchieri Giorgio , Gilboa Eli
Published: January 2023

IL-10 is a key mediator of immune suppression in malignancies and during chronic infections. In mice, the inability of the immune system to clear viral infections and tumor growth can be reversed by antibody-mediated blockade of IL-10 action. Given the limited availability of clinical grade anti-IL-10 or anti-10 receptor antibodies, we have developed an RNA-based, nuclease-resistant, aptamer that binds to the murine IL-10 receptor (IL-10R). The IL-10R aptamer was isolated using a modified SELEX protocol starting from a random 1014 library of 80nt long oligonucleotides containing pyrimidines with 2’fluoro-modified bases. After 5 rounds of selection followed by high-throughput Solexa sequencing we generated several IL10 receptor binding aptamers with Kd ranging from 12 to 35 nM. The best candidate was further truncated to 48 nt - size suitable for chemical synthesis - which also led to a further increase in its affinity, Kd~3 nM. The IL-10R aptamer binds to IL-10 receptor on the cell surface and blocks IL-10 function in vitro, as shown by its ability to inhibit the IL-10 dependent proliferation of MC9 cells and rescue LPS mediated IL-12 secretion by DC in the presence of IL-10. Preliminary experiments suggest that intratumoral as well as systemic administration of the IL-10R aptamer was capable of inhibiting tumor growth in mice. On a molar basis the IL-10R aptamer was at least as potent as a corresponding antibody, both in vitro and in vivo.
Interleukin 21-activated B cells can kill tumor cells in a granzyme B-dependent manner (66.5)
Authors: Brüggemann Sabine , Kaltenmeier Christof , Hagn Magdalena , Sontheimer Kai , Dahlke Karen , Beyer Thamara , Lunov Oleg , Fabricius Dorit , Tron Kyrylo , Nienhaus Uli , Simmet Thomas , Jahrsdörfer Bernd
Published: January 2023

Interleukin 21 (IL-21) is a promising cytokine for the treatment of tumors and infections. Recently, IL-21 was identified as an inducer of plasma cells when combined with CD40 ligand (CD40L). Using various immunological methods including FACS analysis, ELISA, ELISpot and fluorescence microscopy, we demonstrate here that in the absence of CD40L B cells (BC) rather differentiate into granzyme B (GzmB)-secreting cytotoxic cells instead of plasma cells. GzmB induction in BC requires IL-21 and BC receptor cross-linking, and is associated with phosphorylation of SYK, JAK1/3 and STAT1/3, signaling molecules which are also involved in the activation of CTL and NK cells. CD40L effectively suppresses GzmB in BC, suggesting that GzmB-secreting BC play a role early during inflammatory responses, before CD40L-expressing T cells are present. BC-derived GzmB is enzymatically active and induces apoptosis in tumor cell lines including HeLa cells and PC3 cells. Importantly, induction of apoptosis by GzmB-secreting B cells can be suppressed by neutralization of GzmB using the substrate inhibitor Ac-IEPD-CHO as well as a novel small molecule inhibitor of GzmB identified in our laboratory. Our data reveal a novel role of IL-21-activated BC, which involves GzmB secretion and cytotoxicity. Our findings may have implications for the understanding of tumor immunosurveillance and early anti-viral immune responses, and may open novel approaches for the immunotherapy of neoplastic and viral diseases.
Sindbis virus suppresses Akt/mTOR signaling late during infection and induces mTOR independent antiviral response in HEK293T cells (67.12)
Authors: Mohankumar Vidyarani , Dhanushkodi Nisha , Raju Ramaswamy
Published: January 2023

Sindbis (SIN), an alphavirus has potent oncolytic properties and mTOR specific inhibitors are tested in combination with oncolytic viruses. Our aim was to study the effect of SIN replication on PI3K/Akt/mTOR signaling and its possible implications on innate antiviral response. In the presence and absence of PI3K/ mTOR inhibitors, HEK 293T cells were infected with SIN and UV-SIN. Replication of SIN was unaltered by the presence of inhibitors Ly294002, rapamycin and torin1 and rather suppressed phosphorylation of Akt, mTOR, S6 and 4E-BP1 late during infection. Currently, we are focusing on the effect of SIN infection on Akt phosphorylation in response to a subsequent growth stimulus in serum starved cells. PI3K/Akt pathway is required for IRF3 phosphorylation and expression of antiviral genes, which led us to study the regulation of these factors during SIN infection in the presence of inhibitors. Increased IRF3 phosphorylation and higher mRNA levels for ISG56 and IP-10 were seen with high intracellular viral RNA late during infection. Rapamycin and torin1 upregulated these antiviral responses, whereas prolonged Ly treatment partially blocked them. These results suggest that activation of IRF3 by SIN may be independent of mTOR, but partially dependent on PI3K/Akt. The potent inhibition of Akt/mTOR signaling by SIN may find applications in counteracting cellular drug resistance to kinase inhibitors and foster studies on engineered SIN genotypes in combination therapies.
Role of IL-12b in the regulation of viral pathogenesis in the respiratory tract (67.15)
Authors: Chakraborty Krishnendu , Lancelin Wallissa , Zhou Zehua , Wakamatsu Nobuko , Guerrero Plata Antonieta
Published: January 2023

Human metapneumovirus (hMPV), a newly-recognized member of the Paramyxoviridae family, mediate serious lower respiratory tract illness in very young children, the elderly and immunocompromised patients. The mechanisms that regulate the host immune response to hMPV are largely unknown. IL-12b is an important mediator in limiting lung inflammation and Th2 responses. However, the relative contributions of IL-12b to viral pathogenesis have not been extensively studied. We have observed that hMPV infection induces a robust and sustained production of IL-12b in the lung of infected mice beyond the resolution phase, suggesting that this cytokine plays an important role in hMPV infection. Using an IL-12b deficient (IL-12 KO) mouse model of infection we determined the role of IL-12b in hMPV pathogenesis. Our data indicate that the absence of IL-12b increased the disease severity in the course of hMPV infection as indicated by a significant increase in body weight loss (>20%) in deficient mice when compared to WT animals. In addition, a three-fold increase in airway resistance, measured in artificially ventilated conditions by the Flexivent system was observed in the IL-12b KO mice. Furthermore, relative to WT mice, an altered CD4+ and CD8+ T cell responses in lung and draining lymph nodes was observed in hMPV-infected IL-12b KO mice. In conclusion, these findings indicate that IL-12b plays a regulatory role in disease pathogenesis and immune responses in the course of hMPV infection.
PD-1 blockage reverses immune dysfunction and viral persistence to Hepatitis B virus infection in mouse animal model (67.16)
Authors: Hsu Ping-Ning , Tzeng Horng-Tay , Liao Hsiu-Jung
Published: January 2023

Recent studies in animal models of virus infection, indicate that the interaction between the inhibitory receptor programmed death (PD)-1 on lymphocytes and its ligands plays a critical role in T-cell exhaustion by inducing T-cell inactivation, and high PD-1 expression levels on peripheral T-lymphocytes as well as the possibility to improve the T-cell function by blocking PD-1-mediated signaling to confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1/PD-L1 blockade on intrahepatic infiltrating T cells in our recently developed mouse model of hepatitis B virus (HBV) persistence with hydrodynamic injection of HBV DNA. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1 expressing CD8+ T cells and increased CD4+ FoxP3+ T cells in the mice with HBV persistence. Blockade of PD-1/PD-L1 interaction increased HBcAg-specific IFN-gamma production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with its ligand PD-L1 by anti-PD-1 mAb reversed the viral persistence to clearance of HBV viral constructs in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence to HBV infection in mouse animal model, suggesting anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.
Differential effects of blocking PD-1/PDL1 interaction in enhancing virus specific CD8 T cell response in young and aged mice (67.4)
Authors: Channappanavar Rudragouda , Twardy Brandon , Suvas Susmit
Published: January 2023

In this report, we compared the impact of blocking PD-1/PDL1 interaction to the virus specific CD8 T cell function in young and old C57BL/6 mice. Anti-PDL1 mAb treatment at the time of foot-pad HSV-1 infection to young C57BL/6 mice resulted in significantly increased proportion of SSIEFARL specific effector CD8 T cells in popliteal lymph node (PLN), spleen and PBMC. The frequency of IFN-γ, TNF-α and IL2, secreting SSIEFARL specific CD8 T cells was also increased in anti-PDL1 treated mice in comparison to isotype treated groups. An increased proportion of granzyme B expressing CD8 T cells were determined in PLN and spleen of anti-PDL1 treated mice. Interestingly, blocking of PD-1/PDL1 interaction at the time of CD8 T cell priming in young mice also improved the quality of HSV-1 specific memory CD8 T cell pool as noted by an enhanced recall response to SSIEFARL encoding vaccinia virus. On the contrary, blocking PD-1/PDL-1 interaction in aged mice increased SSIEFARL specific CD8 T cells only in PLN but not in spleen and PBMC. Moreover, frequency of effector molecules (IFN-γ, TNF-α, IL-2 and Granzyme B) expressing CD8 T cells did not change significantly in any of the collected tissues. Additionally, unlike in young mice, α-PDL1 treatment during priming failed to improve the quality of memory CD8 T cell response in aged mice. Taken together, we suggest that blocking of PD-1/PDL-1 interaction during priming enhance HSV-1 specific CD8 T cell response in young but not in aged mice.
Protective effect of IL-2 gene transfected cells against Salmonella infection in mice. (99.17)
Authors: Saito-Taki Tatsuo , Shimura Sayoko , Kanesaka Tomoyo , Onogawa Tsuyoshi , Kitasato Hidero
Published: January 2023

It is very hard to recover from lethal Salmonella infection in immunocompromized hosts by chemotherapy. Since the anti-bacterial activity of immunocompromized hosts is suppressed, immunomodulative therapy by immunostimulants such as adjuvant, cytokine, et al., is required. In this study, we used interleukin 2 (IL-2) as immunomodulator because IL-2 enhance cellular immune response via interferon γ production. As intracellular pathogen, Salmonella Typhimurium LT-2 infection induced Typhoid fever in mice. murine IL-2 gene transfered cell (C57/mIL-2) was prepared. Immunocompromized mice were inoculated with C57/mIL-2 cells before, simultaneous or after S. Typhimurium LT2 infection, and observed for 2 weeks about their viability. Protective effect of C57/mIL-2 was only observed cells were inoculated before LT2 infection. Now, more detail experiments to make clear the protective mechanisms by C57/mIL-2.
UVB-induced suppression of resistance to Mycobacterium tuberculosisin guinea pigs after BCG vaccination: cellular and molecular changes in the skin (99.19)
Authors: Jeevan Amminikutty , Formichella Cathryn , Russell Karen , Dirisala Vijaya
Published: January 2023

Previously, we reported that ultraviolet-B radiation (UVR) suppressed BCG vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs as it induced immuno-suppression and increased pathogenesis. In this study, we investigated the cellular and molecular changes within the guinea pig epidermis.The dorsal skin of guinea pigs was exposed to 5.6 kJ/m2 UVR and the epidermal cells were harvested 24 h later. Although UVR did not induce any gross morphological changes in the epidermis, a significant increase in the angular nucleated keratinocytes and a reduction in the number of cells with high nuclear to cytoplasmic ratio and binucleated cells were observed. Flow cytometry studies indicated that the proportions of CD3+ and CD4+ T cells increased in the epidermis while spleen showed a decrease in the CD3+, CD4+, CD8+, CD25+, MHC class II+ and CD14+ cells. Upregulation of IL-10, IL-4, IL-12p40, TGF-β, IL-1β and INOS mRNA expression was observed in the UV-irradiated epidermal cells. In addition, IL-4, IL-12p40, IFN-γ, and IL-1β mRNAs were increased in the spleen cells after UVR. Furthermore, culture supernatants from in vitro UV-irradiated keratinocytes reduced the MHC class II expression in the peritoneal cells and reduced the spleen cell proliferation to ConA. Future studies will address the identity of the suppressive factors induced by UVR and the systemic effect of culture supernatants from UV-irradiated epidermal cells on M. bovis BCG or M. tuberculosis infection.
Epitope-specific CD4+ T cell response to Group A Streptococcal infection (99.27)
Authors: Dileepan Thamotharampillai , Linehan Jonathan , Moon James , Pepper Marion , Jenkins Marc , Cleary Patrick
Published: January 2023

Group A Streptococcus (GAS) is the cause of a variety of clinical conditions, ranging from pharyngitis to autoimmune disease. Peptide-major histocompatibility complex class II (pMHCII) tetramers have recently emerged as a highly sensitive means to characterize pMHCII-specific CD4+ helper T cells. In lieu of identifying an immunodominant peptide expressed by GAS, a surrogate peptide (2W) was fused to the highly expressed M1 protein on the surface of GAS to allow in-depth analysis of the CD4+ helper T cell response in C57BL/6 mice. Following intranasal inoculation with GAS-2W, naïve 2W:I-Ab specific CD4+ T cells proliferated in the nasal-associated lymphoid tissues (NALT) and rapidly differentiated into cells that produced IL-17A. The acquisition of IL-17A production by 2W:I-Ab-specific T cells and the capacity of mice to survive infection depended on the innate cytokine IL-6. IL-6-deficient mice that survived infection became long-term carriers despite the presence of abundant IFN-γ-producing 2W:I-Ab specific CD4+ T cells. Experiments also revealed a unique consequence of multiple or recurrent infections; 2W:I-Ab T cells shifted from the IL-17A+ phenotype to a IL-17A+ IFN-γ+ (double positive) phenotype, cells associated with autoimmune disease in humans and rodent models; therefore, these findings could be key to understanding the chronic carrier stages and autoimmune complications to GAS infections in humans.
B cells promote CD4 T cell expansion and T cell mediated host defense against the fungal pathogen Pneumocystis (99.3)
Authors: Opata Michael , Garvy Beth
Published: January 2023

Host defense against the opportunistic fungal pathogen Pneumocystis (Pc) is dependent upon T cells, B cells and TNFα. Preliminary studies from our laboratory have demonstrated that CD4 T cells undergo increased apoptosis in the absence of B cells suggesting that B cells are critical for increased expansion and survival of Pc-specific effecter CD4 T cells. In this study, we observed increased TNFα secretion by the lymph node B cells isolated from Pc infected mice and stimulated with Pc ex vivo. B cell-derived TNFα enhanced T cells expansion in vivo and host defense against the pathogen. We further confirmed if this survival advantage elicited by B cells was through a direct or indirect contact with the CD4 T cells. We observed increased proliferation of CD4 T cells ex vivo if B cells, T cells and dentritic cells (Dcs) were co-cultured together as compared to cultures with T cells and Dcs alone. There was no difference in CD4 T cell proliferation if B cells were in direct contact with the T cells or were separated by a transwell. Taken together, our data suggests that B cells are capable of producing a soluble factor that can directly act to enhance CD4 T cell function or can indirectly increase CD4 T cell activity by enhancing the dentritic cell functions. We are currently determining whether this factor is B cell-derived TNFα, or some other soluble factor.
A synopsis of the existing knowledge of immunoreactivity against hepatitis C virus (HCV); a meta-analysis of the IEDB data (105.17)
Authors: Vaughan Kerrie , Kim Yohan , Greenbaum Jason , Peters Bjoern , Grey Howard , Sette Alessandro
Published: January 2023

HCV represents a major global health challenge, with ~3% of the world population infected. A large amount of immunological data related to T cell and antibody epitopes currently exists in the HCV literature, dispersed over almost 400 different scientific reports. It is therefore challenging for the scientific community to access this information in a comprehensive yet compact format. Analysis of all HCV-related epitope hosted in the IEDB, a repository of freely accessible epitope data, quantified this challenge: more than 3,300 different structures recognized by the immune system have been reported for this organism. Considering that the HCV genome itself encodes only ~3,000 amino acids in a single open reading frame, this large number likely reflects overlapping or closely related structures from the large number of HCV genotypes and quasi-species. To meet this challenge we developed a novel strategy that combines large sets of epitope data, assessing immunoreactivity by mapping structures onto reference genomes and displaying the frequency of positive responses. This allows for the simultaneous compilation and visualization of the HCV immune reactivity from hundreds of different studies, painting an unprecedented complete and complex picture of HCV immune epitope data to date. The results provide a wealth of information inventoried and cataloged for easy access by the HCV community, highlight interesting findings and underscoring areas for future study.
The role of soluble IL-7 receptor α in murine CD8+ T-cell responses (105.22)
Authors: Cote Sandra , Crawley Angela , Sad Subash , Angel Jonathan
Published: January 2023

The expression of membrane-bound IL-7 receptor α (CD127) is reduced in many viral infections. In HIV infection, this downregulation may be explained in part by increased plasma levels of a soluble form of CD127 (sCD127). We and others have shown that the activity of human IL-7 and cytotoxic CD8+T-cell (CTL) function is decreased by sCD127 in vitro. Observed impairment of IL-7 activity in HIV infection may contribute to the decline of CTL function. The role of sCD127 in such immunopathogenesis is not known. Determining the role of sCD127-mediated immune modulation necessitates the use of an in vivo model. The effect of sCD127 on the IL-7 response of murine splenic CD8+T-cells was evaluated. CFSE-labeled naïve CD8+T-cells were incubated with medium, IL-7 or pre-complexed IL-7 + sCD127 and cell proliferation was assessed. Unlike human T-cells, the survival of murine T-cells in vitro is dependent on the addition of γ-chain receptor cytokines to the culture medium. Cells cultured with sCD127 + IL-7 were less viable and proliferated less (as measured by cell division) compared to IL-7 alone. Our results indicate that sCD127 inhibits the activity of IL-7 on murine naïve CD8+T-cells in vitro, confirming previous results in human T-cell culture. These data suggest that the biological activity of sCD127 on CD8+T-cells is similar between mice and humans, thus providing rationale for the further study of sCD127 in established murine models of CD8+T-cell-mediated responses to infection.
Cornea-derived DC are critical for T cell expansion following HSV-1 corneal infection (105.32)
Authors: Buela Kristine-Ann , Hendricks Robert
Published: January 2023

The cornea possesses a resident population of DCs, but their contribution to T cell expansion in draining lymph nodes (DLN) following corneal infection is unknown. Using CD11c-DTR-EGFP mice we were able to locally deplete cornea-resident DC (in cornea before infection) and cornea-derived DC (coming from the cornea after infection (subconjunctival DT) or systemically deplete both corneal and draining lymph node (DLN) resident DC intraperitoneal DT). Depletion of cornea-resident DC did not affect T cell expansion in the DLN. Local depletion of cornea-derived DC through 3 days post infection (dpi) abrogated early CD8+ T cell expansion at 3 dpi, but only reduced CD4+ T cell expansion by 50%. Systemic depletion through 3 dpi completely abrogated early (3 dpi) expansion of both CD4 and CD8 T cells. Depletion of cornea-derived DC through 7 dpi resulted in complete abrogation of both CD4 and CD8 T cell expansion at 7 dpi. We conclude that CD8+ T cells can only be activated by DC that derive from the cornea. CD4+ T cells can be expanded by both cornea-derived and DLN resident DC up to 3 dpi. Both CD4 and CD8+ T cell expansion is dependent on DC transport of viral proteins from the cornea after 3 dpi when free virus is presumably no longer reaching the DLN. Neither local nor systemic DC depletion beyond 7 dpi influenced the development of CD4+ T cell-mediated inflammation in the cornea, suggesting that DC are dispensable for re-stimulation of CD4+ T cells in the cornea
Hepatitis C Virus Genotype 1b Vaccine can Benefit Egyptians, where Genotype 4a prevails (105.39)
Authors: Abdelwahab Sayed , Fathy Iman , Zakaria Zainab , Waked Imam , Mahmoud Mohamed , Capone Stefania , Nicosia Alfredo , Folgori Antonella , Refaat Ahmed , Osman Ahmed , Strickland G. Thomas , Cortese Riccardo
Published: January 2023

Hepatitis C Virus (HCV) infection is a global health burden specially in Egypt, where HCV genotype 4a predominates. Control of HCV infection will remain a challenge until the development of an effective vaccine that protects against different genotypes. There are several HCV genotype 1 vaccines in the pipelines. Differences in antigenic structure among genotypes could make cross protection against other genotypes unlikely. To assess whether Egyptians can benefit from genotype 1-based vaccines, HCV-specific T cell responses were examined in 37 HCV genotype 4a-infected patients using an interferon gamma (IFNγ) ELISpot assay in response to 7 HCV overlapping 15mer peptide pools derived from both genotype 1b and 4a that cover most of the HCV genome. Twenty two (59%) and 16 (43%) subjects responded to at least one peptide pool derived from genotype 1b and 4a, respectively, with 39% exact match in responses. Importantly, responses in different genome segments showed ≥55% exact match in response to the pools tested except the NS5a region (25%). The average IFNγ total spot forming cells (SFC)/106 PBMC (+SE) in the responding subjects for genotype 1b and 4a was 484±89 and 544±77, respectively (p=0.6). There was no significant differences in the responses of those who resolved HCV infection or not (p=0.6). In conclusion, no significant differences were found in the T cell responses of Egyptians to genotype 1b and 4a antigens suggesting that they can benefit from a genotype 1b vaccine.
Lack of SLP-76 reduces homeostatic turnover without effecting persistence of antigen specific CD4 memory T cells (105.42)
Authors: Rodgers Evann , Wiehagen Karla , Staub Elizabeth , Maltzman Jonathan
Published: January 2023

CD4 memory T cells are generated in response to infection or vaccination, provide protection to the host against re-infection, and persist through a combination of enhanced survival and slow homeostatic turnover. We developed a system to study the role of tonic TCR signaling in turnover and persistence of memory cells by timed deletion of the TCR-signaling adaptor SLP-76 following acute infection. In previously published studies, SLP-76 deficient memory CD4 T cells generated by in-vitro stimulation have decreased homeostatic turnover and reduced persistence. To extend these studies and directly assess antigen-specific (AgSp) memory cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) Armstrong or Listeria monocytogenes expressing GP61-80 (LM-GP61). SLP-76 was deleted 30 days post-infection, and CD4 memory T cells are identified by staining with I-Ab:GP61 tetramer. SLP-76 deficient I-Ab:GP61+ cells generated in response to either pathogen are unable to participate in recall responses and have reduced steady-state BRDU incorporation. Interestingly, despite decreased turnover, loss of SLP-76 in GP61+AgSp memory cells has no effect on persistence at 30 weeks post deletion following LCMV, and the rate of memory cell decline is fixed. These studies confirm that CD4 memory T cells rely on SLP-76 dependent signals for normal homeostatic turnover and suggest that persistence of CD4 memory subpopulations differentially require tonic TCR signaling.
ICAM-1 interactions enhance effector cell contraction and memory T cell function following acute infection. (105.5)
Authors: Cox Maureen , Bullard Daniel , Zajac Allan
Published: January 2023

CD8 T cell responses are critical for protection from intracellular pathogens, and one hallmark of effective CD8 T cell responses is the development of a memory T cell pool which can rapidly respond to reinfection. Interactions mediated by intercellular adhesion molecule 1 (ICAM-1) have been suggested to be critical in the formation of memory CD8 T cells, as these interactions enhance both antigenic and cytokine signals. We have investigated the effect of ICAM-1 on cellular immune responses to acute infections. Surprisingly, elevated numbers of virus-specific CD8 T cells were maintained in ICAM-1null mice following LCMV infection, and this increase in the virus-specific T cell pool represents enhanced retention of “effector-like” CD8 T cells (CD127lo KLRG-1hi IL-2-) into the memory phase of the response. The development of memory phenotype CD8 T cells (CD127hi KLRG-1lo IL-2 producing) however, was not impaired by ICAM-1 deficiency. Moreover, ICAM-1null LCMV immune mice were able to efficiently control secondary infections despite proliferative defects observed in their memory T cells. The enhanced maintenance of effector phenotype cells appears to be the result of ICAM-1 deficiency on non-T cell subsets, suggesting a role for ICAM-1 mediated interactions with the APC in programming efficient contraction of effector and proliferative potential of memory CD8 T cells.
Differential ability of H-2 class Ia genes to effectively direct anti viral immunity (105.53)
Authors: Pease Larry , Pavelko Kevin , Mendez-Fernandez Yanice , Johnson Aaron , Rodriguez Moses
Published: January 2023

MHC class Ia molecules are regulators of antiviral immunity and are thought to function similarly in the presentation of self and antigenic peptides to T cells. However, differences in natural polymorphisms found among class Ia loci in humans and mice indicate these genes may not function equivalently. We used transgenic mice to evaluate the equivalence of MHC Ia gene function in directing cellular immunity against the picornavirus, Theiler’s encephalomyelitis virus, and found that effective antiviral immunity was promoted by H-2D transgenes but not by H-2K in this system. Because transgenes evaluated in our study encoded the peptide binding domain of the Db antigen presenting molecule expressed in the context of the Db or Kb genetic framework, our findings indicate that structural features distinguishing these genes mapping outside of these coding sequences determine ability to direct effective viral immunity. Irrespective of which transgene was expressed, the mice developed robust anti viral T cell responses against the immunodominant TMEV epitope and the T cells were recruited to infected tissue. However, only when the Db antigen binding domain was expressed in the context of the H-2D genetic framework was virus cleared. Our findings suggest that differences in tissue specific class I gene expression at the site of infection is a key factor in this differential function of class Ia genes. Such difference could underlie natural variations in class Ia polymorphism.
Immunomodulatory potential of CD3+ CD4-CD8- double negative (DN) T cells in sooty mangabeys (105.7)
Authors: Rout Namita , Else Jim , Kaur Amitinder
Published: January 2023

Simian immunodeficiency virus (SIV) infection in natural hosts like sooty mangabeys (SM) is typically non-pathogenic and characterized by the absence of chronic immune activation. In contrast, SIV-infected Asian macaques, such as rhesus macaques (RM), progress to AIDS. CD3+ CD4-CD8- double negative (DN) T cells comprise <5% of circulating T cells in AIDS-susceptible species such as RM, but are present at significantly higher proportions (mean 20%) in SM. To investigate the significance of this difference, we compared the phenotype and function of DN T cells in SM and RM. γδ T cells, Tregs and NKT cells accounted for <15% of DN T cells in both SM and RM. SM DN T cells contained a significantly higher frequency of CXCR5-positive and CCR6-positive cells indicating ability to home to lymphoid tissue and mucosal sites of inflammation. Mitogen-stimulated DN T cells in both SM and RM were functionally similar to CD4+ T cells with regards to upregulation of CD40L and expression of Th1 (IFN-γ, IL-2), Th2 (IL-13) and Th17 (IL-17) cytokines. However, SM DN T cells produced significantly higher levels of IL-13 and IL-17, and contained significantly higher frequencies of Tregs compared to RM suggesting more potent immunomodulatory function. These data raise the possibility that the Treg and Th17 functionality of SM DN T cells are instrumental in regulating immune activation and gut mucosal integrity, features that help to prevent immunodeficiency in SIV-infected SM.
Autophagy is implicated in MHC class I antigen cross-presentation mediated by potexvirus VLP (106.10)
Authors: Hanafi Laila-Aicha , Leclerc Denis , Lapointe Rejean
Published: January 2023

The development of vaccination platforms capable of triggering an efficient cytotoxic-T-lymphocyte (CTL) response is a priority for eliciting a protective cellular immune response. The proposed technology is based on the use of a new adjuvant composed of a virus-like particle (VLP) with the chimeric coat protein of papaya mosaic virus (PapMV). This VLP can trigger a CTL response through MHC class I antigenic cross-presentation of inserted epitopes. We have wanted to establish the cellular pathway leading to cross-presentation by those VLP. First, we chemically inhibit different molecular path that can be important for cross-presentation. We observed that cathepsin S and autophagy are implicated in cross-presentation by PapMV VLP. Also, we localized the VLP by fluorescence microscopy in antigen presenting cells (APC). To confirm the role of autophagy, we observed its induction by LC3 western blot and microscopy. When the APC are incubated with VLP we observed an activation of autophagy by LC3 lipidation. Finally for more specific inhibition, we used shRNA against ATG5 and cathepsin S in APC. Both ATG5 and cathepsin S reduced the cross-presentation by VLP In conclusion, the cross-presentation mediated by PapMV VLP is dependant on autophagy and degradation by cathepsin S. A better understanding of this mechanism would lead to the development of an efficient vaccine platform against cancer and infectious diseases
Cryptic fate of gliadin epitopes: implication for celiac disease (P730) (106.26)
Authors: Hou Tieying , Wang Nan , Qiao Shuo-Wang , Jin Xi , Sidney John , Sollid Ludvig , Mellins Elizabeth
Published: January 2023

Epitopes of an intact antigen that elicit potent T cell activation are classified as immunodominant epitopes. By contrast, epitopes that fail to trigger a T cell response, but can bind to class II, are called cryptic epitopes. HLA-DM (DM), an important regulator of antigen presentation, determines the immunodominant and cryptic fate of epitopes. Notably, however, we previously reported that DM interacts weakly with HLA-DQ2 (DQ2), a genetic risk factor for celiac disease (CD). Here, using the T x B hybrid cell line, T2, transfected with DQ2 with or without higher than physiological levels of DM, we have investigated the contribution of DM in the presentation of different gliadin epitopes (VII). The presentation of digested gluten requires internal processing and was dramatically inhibited when APCs were fixed. Cells lacking DM efficiently presented all four epitopes to gliadin-specific T cells. Interestingly, cells expressing DM were significantly compromised in their ability to display the above epitopes despite the higher level of surface DQ2 in DM+ cells. We conclude that all fourgliadin peptides were DM-suppressed epitopes. We hypothesize that in vivo the inefficient interaction between DQ2 and DM influences the fate of these epitopes, changing them from cryptic to immunodominant, and inducing a gliadin-specific immune response in CD patients expressing the DQ2 allele.
Suppression of antigen presentation by the adenovirus E3-19K protein; a crystallographic analysis of E3-19K/MHC I interaction (106.37)
Authors: Li Lenong , Muzahim Yasameen , Bouvier Marlene
Published: January 2023

The cell-surface presentation of viral antigens by MHC class I molecules is critical for eliminating infected cells. In turn, viruses have evolved strategies to interfere with the process of antigen presentation. The adenovirus (Ad) E3-19K protein binds to and retains MHC class I molecules in the endoplasmic reticulum (ER), blocking their egress to the cell surface. We characterized for the first time interaction between Ad type 2 (Ad2) E3-19K and HLA-A2 using x-ray crystallography. Our structure to 2.1Å resolution reveals that Ad2 E3-19K interacts with the N-terminus of the α1-helix and C-terminus of the α2-helix of HLA-A2, i.e., the C-terminal end of the groove. E3-19K binds away from the opening of the groove. This mode of binding is entirely consistent with our biochemical studies of E3-19K/MHC I interaction, including the ability of E3-19K to associate with mature and immature MHC class I molecules. Importantly, the Ad2 E3-19K/HLA-A2 structure reveals how E3-19K uses it variable and conserved regions to bind to MHC I, and how it accommodates polymorphism in MHC I. Our structure also allows to understand the ability of E3-19K of different Ad serotypes and subgroups to associate with MHC I in an allele- and locus-specific manner. Overall, the Ad2 E3-19K/HLA-A2 structure reveals determinants in E3-19K that are critical for targeting MHC class I molecules. It also permits to better understand how E3-19K has evolved to subvert antigen presentation.
A CD1 lipidomic analysis determines the structures of human CD1b scaffold lipids and its function to enhance mycobacterial antigen presentation (106.43)
Authors: Huang Shouxiong , Cheng Tan-Yun , Young David , Layre Emilie , Madigan Cressida , Shires John , Cerundolo Vincenzo , Altman John , Moody Branch
Published: January 2023

Unlike the dominant role of one class II invariant chain peptide (CLIP) in blocking MHC class II, comparative lipidomics analysis shows that human CD1a, CD1b, CD1c and CD1d proteins bind lipids corresponding to hundreds of diverse accurate mass retention time values. Although most ions were observed in association with several CD1 proteins, ligands binding selectively to one CD1 isoform allowed the study of how differing antigen binding grooves influence lipid capture. The CD1b groove is distinguished by its unusually large volume, however, the average mass of compounds eluted from CD1b was similar to that of lipids from other CD1 proteins. Elution of small ligands from the large CD1b groove might be explained, if two small lipids bind simultaneously in the groove. In the context of the known CD1b crystal structures with scaffold lipids seated below the antigen, we also found that ligands selectively associated with CD1b lacked the hydrophilic head group, which is generally needed for antigen recognition, but interferes with scaffold function. Furthermore, we identified the scaffolds as deoxyceramides and diacylglycerols and directly demonstrated a function in augmenting presentation of a mycobacterial antigen, the short chain glucose monomycolate, to T cells. Thus, unlike MHC class II, CD1 proteins capture highly diverse ligands in the secretory pathway. CD1b has a mechanism for binding two small lipids to enhance bacterial antigen presentation and T cell activation.