bluuebunny/crossref_metadata_2025 huggingface dataset:
WC1 functions as a pathogen-associated molecular pattern receptor and co-receptor for γδ T cells (P6083)
Authors: Telfer Janice , Hsu Haoting , Chen Chuang , Baldwin Cynthia
Published: January 2023

WC1 proteins, expressed on bovine γδ T cells, are members of the scavenger receptor cysteine-rich (SRCR) superfamily and are coded by 13 genes (WC1-1 to WC1-13). Phosphorylation of a tyrosine in the WC1 cytoplasmic domain upon co-ligation of WC1 and TCR is required for WC1 co-receptor activity. Serologically-defined WC1.1+ γδ T cells specifically respond to the spirochete Leptospira; in contrast, WC1.2+ γδ T cells respond to the rickettsiales Anaplasma. Silencing via shRNA suggests that only cells expressing the WC1.1 type gene products WC1-1, WC1-2 or WC1-3 contribute to the γδ T cell response to the spirochete Leptospira, suggesting that expression of individual WC1 receptors encodes antigen specificity through ligation of bacteria. Comparing a representative of WC1 proteins expressed by WC1.1+ Leptospira-responsive cells (i.e., WC1-3) to a representative of WC1.2+ Leptospira-nonresponsive cells (i.e., WC1-4), we found that five out of eleven WC1-3 SRCR domains and none of the eleven WC1-4 SRCR domains bound to two Leptospira species, L. borgpetersenii and L. interrogans. Mutational analysis of WC1-3 SRCR domains suggests that the active site for bacterial binding is different from that previously reported for other SRCR proteins that bind bacteria and also that it is affected by single amino acid changes. Taken together, our data suggest that co-ligation of specific WC1 proteins and the γδ TCR by pathogen-associated molecular patterns (PAMPs) induces γδ T activation.
High level of CD8+ cytotoxic T lymphocyte is essential for eliminating mesothelioma (P2125)
Authors: Tan Zhiwu , Zhou Jingying , Cheung Allen , Liu Li , Chen Zhiwei
Published: January 2023

Mesothelioma presents an aggressive human cancer in the lungs of people exposed to asbestos. Although immunotherapy is a way of treatment, the type of immune response that eliminates mesothelioma remains poorly defined. Here, we developed a quantitative mouse model using mesothelioma AB1 cell line that stably expresses HIV-1 Gag fused with firefly luciferase. This AB1-Gag-Luc cell line shows identical growth kinetics in mice compared to parental AB1 cell. We then investigate a novel DNA vaccine to particularly stimulate antigen specific CD8+ T cell responses, which involves the fusion of soluble PD-1 protein with a HIV Gag to target dendritic cell effectively. As compared with traditional DNA vaccination, preventive use of the new vaccine elicited immunity that significantly reduced the tumor growth post AB1-Gag-Luc challenge due to Gag-specific cytotoxocity activity detected in both CD4+ and CD8+ T cell subsets. Moreover, therapeutic use of the new vaccine significantly reduced tumor growth and improved tumor-free survival in tumor bearing BALB/c mice. Importantly, adoptive transfer and T cell depletion experiments indicated that Gag-specific CD8+ T cells was more effective than CD4+ T cells in suppression of AB1-Gag-Luc cells in SCID and BALB/c mice, respectively. Our findings demonstrated that high level of antigen specific CD8+ cytotoxic T lymphocyte is essential for eliminating mesothelioma.
Involvement of IgG derived from cancer in tumor immune escape (P2147)
Authors: Qiu Xiaoyan , Shao Wenwei , Xiao Lin , Li Guohui , Huang Jing , Fu Ning
Published: January 2023

As we known that IgG have served as prototypical effector molecules of immune defense. However, monomeric IgG, such as the IVIG purified from the serum of thousands of healthy donors, has been demonstrated as an immune inhibitor at high doses as a commonly administered for the treatment of a number of autoimmune diseases. Igs has long been considered to be only produced by B cells, however, by a series of experimental evidences from ours and others, it has been found that other types of non-hematopoietic cells, especially cancer cells could also produce IgG, and the IgG is involved in the cell survival and carcinogenesis. In this study, we unexpectedly found that like the IVIG, epithelial cancer-derived IgG, but not the normal IgG, also showed strong immune inhibitory effect to CD4+ or CD8+ T lymphocytes at low dose. More importantly, the inhibitory effect of cancer-derived IgG was stronger than IVIG. Subsequently, we have analyzed the mechanism of the immune inhibition. Our results suggested that cancer-derived IgG can not directly inhibit the activation and proliferation of CD4+ or CD8+ T cells, their immune inhibitory effect should be dependent on myeloid cells, at least partly, dependent on the monocytes. In addition, we found that the immune inhibitory effect of cancer-derived IgG was not dependent on the Treg. Although the mechanism is remain unclear, we believe that the IgG is so far unknown arms of tumor cells to against the immune system for their immune escape.
Exosome from EBV-positive cell reduces vitamin D receptor expression on multiple myeloma (P2151)
Authors: Kim Hae jin , Kim Ja-young , Choi Yunock , Park Ga Bin , Kim Seonghan , Kim Bomi , Hur Dae Young , Kim Yeong Seok
Published: January 2023

Epstein-Barr Virus (EBV) is causative agent of cancer, and most people in the world are infected with EBV. EBV is deeply associated B cell lymphoma, nasopharyngeal carcinoma and gastric carcinoma. Some reported that EBV is linked to multiple myeloma. The therapy of multiple myeloma has been reported that it has effect on several anticancer drug and vitamin D. In this study, we studied whether EBV-infected cells or EBV itself modulate vitamin D receptor on multiple myeloma and resulting drug resistance. We add B95-8 cell supernatant including EBV particles to a multiple myeloma cell line NCI-H929 and we found that VDR mRNA and protein were decreased. However, there was no mRNA related with EBV genes. Experiments for cell proliferation and apoptosis showed that NCI-H929 have a resistance to vitamin D toxicity after adding with B95-8 cell supernatant. We also found that vitamin D receptor expression was decreased in NCI-H929 when we treated the exosome from EBV-infected B cell lymphoma or B95-8. As a result, exosomal components in EBV-positive cells may modulate susceptibility of vitamin D treatment on multiple myeloma. It can present the theoretical basis for vitamin D resistance appeared in multiple myeloma caused by the EBV-infected cells.
Myd88 signaling blockade induces antitumor effects by skewing the immunosuppressive function of MDSCs (P2155)
Authors: Ko Hyun-jeong , Lee Bora , Hong Eun-Hye
Published: January 2023

Myd88 is an important adaptor molecule for the activation of NADPH oxidase and arginase-1, which are responsible for the suppressive function of myeloid-derived suppressor cells (MDSCs). When wild-type and Myd88-/- mice were s.c. injected with CT26 colon cancer cells expressing human Her-2/neu, tumor growth was retarded in Myd88-/- mice than in wild-type mice. Although the generation of CD11b+Gr-1+ MDSCs was less in Myd88-/- mice than in wild-type mice, Myd88-/- mice having tumor masses still had significant quantities of MDSCs, suggesting that MDSC generation might be independent of Myd88 signaling. However, MDSCs obtained from tumor-bearing Myd88-/- mice failed to suppress antigen-specific proliferation of CD8+ T cells and CD4+ T cells, whereas MDSCs from wild-type mice significantly suppressed both types of T cells. Consistent with this, we found that the levels of costimulatory molecules and MHC class II were significantly increased in MDSCs obtained from Myd88-/- mice compared to wild-type mice after tumor challenge. Finally, the blockade of Myd88 signaling by treatment with Myd88 inhibitory peptide, during later tumor stages, significantly inhibited the growth of immunogenic tumors. Overall, these data suggest that signaling through the Myd88 adaptor molecule is critical for the direct suppressive function of MDSCs, and approaches to block Myd88-mediated signaling in MDSCs might be effective to inhibit the immunosuppressive function of MDSCs.
Eicosapentaenoic acid induces apoptosis in human pancreatic cancer cells: Role of ROS accumulation, caspase 8 activation, and autophagy induction (P2161)
Authors: Fukui Masayuki , Umemura Masayuki , Matsuzaki Goro
Published: January 2023

In a recent study, we showed that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two common omega-3 fatty acids, can cause ROS accumulation and subsequently induce caspase-8-dependent apoptosis in human breast cancer cells in vitro and in vivo. In this study, we showed that the pancreas has a unique ability to accumulate EPA at a level markedly higher than several other tissues analyzed. Based on this finding, we sought to further investigate the anticancer actions of EPA and its analog DHA in human pancreatic cancer cells using both in vitro and in vivo models. EPA and DHA were found to induce ROS accumulation and caspase-8-dependent cell death in human pancreatic cancer cells (MIA-PaCa-2 and Capan-2) in vitro. Feeding animals with a diet supplemented with 5% fish oil, which contains high levels of EPA and DHA, also strongly suppresses the growth of MIA-PaCa-2 human pancreatic cancer xenografts in athymic nude mice, by inducing oxidative stress and cell death. In addition, we showed that EPA can concomitantly induce autophagy in these cancer cells, and the induction of autophagy diminishes its ability to induce apoptotic cell death. It is therefore suggested that combination of EPA with an autophagy inhibitor may be a useful strategy in increasing the therapeutic effectiveness in pancreatic cancer.
Integrins and chemokine receptors control maturation and duration of the cytotoxic immune synapse between CTL and tumor cells (P2171)
Authors: Mami-Chouaib Fathia , Boutet Marie , Le Floc'h Audrey , Franciszkiewicz Katarzyna
Published: January 2023

Cytotoxic T lymphocytes (CTL) exert their lytic activity through the polarized release of cytotoxic granules at the immune synapse (IS) formed with specific target cells. Integrins and their cognate ligands on target cells play a major role in T-cell receptor (TCR)-mediated functions. We show here that cytotoxicity towards epithelial cancer cells requires, along with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1 thereby enhancing the strength of the T-cell/target cell adhesion. Integrin-dependent mature cytotoxic IS (mcIS) displays a cohesive ultrastructure characterized by polarized degranulation and chemokine release associated with target cell killing and chemokine receptor induction on effector cells. Our results provide new insights into molecular mechanisms regulating antitumor CTL responses and the role of integrins and chemokine receptors on the formation and duration of the cIS.
BTLA: new biomarker for a highly proliferative CD8+ TIL subset associated with melanoma regression during adoptive cell therapy (P2189)
Authors: Haymaker Cara , Wu Richard , Bernatchez Chantale , Hwu Patrick , Radvanyi Laszlo
Published: January 2023

Adoptive T cell therapy using tumor-infiltrating lymphocytes (TIL) expanded ex vivo with high-dose IL-2 is a promising approach for the treatment of metastatic melanoma. Recently, our lab demonstrated the importance of CD8+ T cells expressing B and T lymphocyte attentuator (BTLA) with a positive clinical response. This suggests that functional differences may exist between CD8+BTLA+ and CD8+BTLA- cells resulting in the differential therapeutic potency of TIL in treated patients. Here, we isolated BTLA+ and BTLA- CD8+ TIL from metastatic melanoma patients accrued in a Phase II clinical trial at MD Anderson and performed functional assays measuring proliferation, apoptosis, cytokine production, and CTL activity as well as differences in global gene expression profiles between the subsets using microarray analysis. Overall, BTLA appears to be a new and powerful biomarker distinguishing a less differentiated, more highly active and polyfunctional CD8+ T cell subset with high responsiveness to IL-2. As BTLA ligation by its binding partner HVEM (which is expressed on melanoma cells) results in a decreased proliferative responsiveness to anti-CD3 stimulation, paradoxically, BTLA signaling however may still serve as a negative co-inhibitory molecule. Our results also underscore that increased expression of T cell co-inhibitory molecules may not be necessarily markers of “exhaustion”, but markers of more highly-activated, responsive T cells susceptible to negative regulation.
Murine prostate tumors recruit and expand thymic-derived regulatory T cell populations (P2191)
Authors: Malchow Sven , Leventhal Daniel , Nishi Saki , Fischer Benjamin , Socci Nicholas , Savage Peter
Published: January 2023

For some human cancers, elevated densities of Foxp3+ regulatory T cells (Tregs) in the tumor tissue correlate with poor prognosis, suggesting that Tregs may play a functional role in cancer progression. Despite considerable interest in the modulation of tumor-associated Tregs for therapeutic benefit, little is known about the developmental origins of these cells and the nature of the antigens that they recognize. By a TCR sequencing approach of T cells isolated from tumors of mice with oncogene-driven prostate cancer (TRAMP), we identified an endogenous population of antigen-specific Tregs found recurrently enriched in the tumors. These Tregs were not reactive to a tumor-specific antigen, but instead recognized a prostate-associated antigen that is present in tumor-free mice. Transgenic T cells expressing this Treg-derived TCR underwent Treg development in the thymus, but inefficient development in the periphery, indicating a thymic origin of these cells. Taken together, our data support a model in which organ-specific Tregs reactive to peripheral tissue antigens develop as Tregs in the thymus and are co-opted by tumors arising within the associated organ.
T cells dominate the immune infiltrate in human pancreatic cancer. (P2195)
Authors: Pillarisetty Venu , Shibuya Kendall
Published: January 2023

The absence of an effective immune response to pancreatic cancer is thought to contribute to the poor prognosis of this devastating disease. Recent studies using murine models of pancreatic cancer suggest that regulatory cells within the tumor microenvironment prevent effector CD8 T cells from entering and eliminating tumors. In order to gain a detailed understanding of the immune response to the human disease, we performed flow cytometry to study fresh tumor and blood from patients undergoing surgical resection for pancreatic cancer. Within the dense immune tumor infiltrate, a majority of the cells expressed CD3 (68.2 +/- 13.5%). The distribution of CD4 and CD8 T cells was, on average, comparable to the blood. However, we found significantly elevated levels of inflammatory IL-17 producing CD4 Th17 cells regulatory and CD25+FoxP3+ T cells within the tumor. Although there was a higher percentage of Tregs among the CD4 T cells in the tumor than in the blood, we found that the CD4 and CD8 T cells within the tumor were as capable of producing the inflammatory cytokines IFN-gamma (CD8: tumor 73.2% +/- 17.2%, blood 60 +/- 26%; CD4: tumor 33.3% +/- 12.3%, blood 24.2% +/- 13.9%) and IL-17 (CD4: tumor 4.3% +/- 4.2%, blood 1.35% +/- 1.3%) as their counterparts in the blood. In order to determine the relationship between the T cells and cancer cells, we performed CD3 immunohistochemistry and determined that infiltrating CD3+ cells are typically in close proximity to cancer cells.
Levels of murine, but not human, CXCL13 are greatly elevated in a NOD-SCID xenograft model of AIDS-associated Burkitt lymphoma (P2203)
Authors: Martinez-Maza Otoniel , Magpantay Larry , Widney Daniel
Published: January 2023

In these studies, a novel mouse/human xenograft model of AIDS-associated Burkitt lymphoma (AIDS-BL) was created by injecting cells of the AIDS-BL cell line, 2F7, intraperitoneally into NOD-SCID mice. Mice developed tumors in the peritoneal cavity, with metastases to the spleen, thymus, and mesenteric lymph nodes. Expression of the chemokine receptor, CXCR5, was greatly elevated in vivo on BL tumor cells in this model, as shown by flow cytometry. The chemokine ligand for CXCR5 is CXCL13, and serum and ascites levels of murine, but not human, CXCL13 showed a striking elevation in tumor-bearing mice, with levels as high as 200,000 pg/ml in tumor ascites, as measured by ELISA. As shown by immunohistochemistry, tumors were extensively infiltrated with F4/80+ histiocyte-like cells, many of which also stained positively for murine CXCL13. Blocking CXCR5 on 2F7 cells with neutralizing antibodies prior to injection into the mice substantially delayed tumor formation. The marked elevations in tumor cell CXCR5 expression and in murine CXCL13 levels seen in the model may identify an important link between tumor-interacting histiocytes and tumor cells in AIDS-BL, and also confirm previous results suggesting that CXCL13 is a potential biomarker for AIDS-NHL. This mouse model may be useful for future studies on the interactions of the innate immune system and AIDS-BL tumor cells, as well as for the identification of other potential tumor biomarkers for AIDS-NHL.
Homogeneity of the adaptive immune repertoire of primary and metastatic ovarian tumors (P2205)
Authors: Sherwood Anna , Emerson Ryan , Desmarais Cindy , Tewari Muneesh , Guenthoer Jamie , Carlson Chris , Drescher Charles , Robins Harlan
Published: January 2023

The presence, abundance, population, and diversity of Tumor Infiltrating Lymphocytes (TILs) have been identified as prognostic indicators in several cancers. Emerging cancer therapeutics including immunomodulators and adoptive T-cell therapy highlight the need to better understand and track this population of T cells. We have developed a multiplex PCR amplification assay for the rearranged T-cell receptor Beta (TCRB), followed by deep sequencing to characterize the adaptive immune response spatially within ovarian tumor. Specifically, we used our assay to characterize the heterogeneity of ovarian tumor TIL populations. We sequenced 10 primary and metastatic ovarian tumors collected from 5 patients. Each tumor was divided into a grid pattern with 8-22 sections (tumor size dependent). For each tumor section we collected data on the number, diversity, and the unique CDR3 sequences carried by the TILs, to characterize the intra-tumor heterogeneity of TIL count, diversity, and T cell clone overlap. In addition, we sequenced the immune repertoire from a paired peripheral blood sample to identify if the immune response was tumor specific. We found that the immune response within peripheral blood was significant and quantifiably different than the tumor repertoire. TIL repertoire within sections of a tumor showed high similarity to each other. These data indicate that the TCR repertoire of the ovarian tumor environment is tumor specific and homogeneous.
NLRP3 inflammasome independent release of IL-1β by tumor cell lysates stimulated dendritic cells (P2215)
Authors: Wang Minan , Gollnick Sandra
Published: January 2023

Photodynamic therapy (PDT) has long been known for capable of killing tumor cells and induces anti-tumor immunity. The mechanism of how PDT can enhance anti-tumor response is not clear. We tested the hypothesis that bone marrow derived dendritic cells (BMDCs) activated by PDT-generated tumor cell lysates (PDTTCL) can induce anti-tumor response in Lewis lung carcinoma (LLC), a poorly immunogenic tumor model. We found that LLC tumor growth was retarded by immunization with BMDCs pulsed by PDTTCL. Furthermore, therapeutic effects are observed in preexisting LLC tumor when PDT is combined with PDTTCL treated BMDCs and R848 (TLR7/8 ligand) therapy. This study aims to understand how PDT enhances tumor immunogenicity and provides insight to improve both in situ PDT and PDT vaccine efficacy in combating secondary disease. We found that stimulation of BMDCs with PDTTCL leads to dramatic increase in pro-inflammatory cytokine production, including IL-1β, which plays a critical role in PDT efficacy. Previous studies have shown that production and activation of IL-1β require both TLR signaling and activation of NLRP3 inflammasome. IL-1β production induced by PDTTCL in BMDCs is MyD88 dependent, but the activation of IL-1β appears to be dependent upon elastase and proteinase 3 rather than NLRP3 inflammasome. Together, our findings reveal a novel mechanism of IL-1β activation in BMDCs that does not require NLRP3 inflammasome in response to danger signals from tumor cell lysates.
Withaferin A, a potent and abundant component of Withania somnifera root extract, reduces myeloid-derived suppressor cell function (P2103)
Authors: Sinha Pratima , Ostrand-Rosenberg Suzanne
Published: January 2023

Myeloid cells play a crucial role in growth and metastasis of malignant tumors. Tumor infiltrating myeloid cells include myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages. These cells infiltrate into tumor and suppress tumor immunity by their inherent immune suppressive activity which is enhanced by interactions with each other (cross-talk). The root extract of the plant Withania somnifera (WRE) has been reported to reduce tumor cell proliferation and angiogenesis. We hypothesize that WRE or its constituents impact tumor infiltrating myeloid cells and thereby boost anti-tumor immunity. HPLC and MS analysis revealed that Withaferin A (WA) is the most abundant constituent of WRE. A prominent effect of MDSC is their production of IL-10 which increases upon cross-talk with macrophages, promoting type-2 immunity. WA reduces constitutive and cross-talk induced IL-10 secretion from MDSC. Macrophage secretion of IL-6 and TNFa cytokines that are characteristic of M1-type macrophages and that also increase MDSC accumulation and function, are also reduced by WA. Much of the T cell suppressive activity of MDSC is due to MDSC production of reactive oxygen species (ROS), and WA significantly reduces MDSC production of ROS. Thus adjunctive treatment with WA has the potential to concomitantly reduce myeloid cell mediated immune suppression, to polarize immunity towards a tumor-rejecting type I phenotype, and to facilitate the development of anti-tumor immunity.
IFN-γ activates p300 to over-ride silencing effects of DNA methylation to activate GPR109A and SLC5A8 transcription (P2105)
Authors: Bardhan Kankana , Martin Pamela , Browning Darren , Ganapathy Vadivel , Robertson Keith , Liles Georgia , Lee Jeffery , Liu Kebin
Published: January 2023

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress inflammation-mediated diseases such as inflammatory bowel disease and colorectal cancer. GPR109A and SLC5A8, receptor and transporter, respectively, for short chain fatty acids, are often silenced in human colon carcinoma cells. We demonstrated that the GPR109A and SLC5A8 promoter DNA is hypermethylated in human colon carcinoma cells in vitro and in vivo. Strikingly, IFN-γ, a cytokine secreted by activated cytotoxic T lymphocytes, effectively activated GPR109A and SLC5A8 transcription without altering their promoter DNA methylation levels. Analysis of the IFN-γ signaling pathways indicated that IFN-γ-activated pSTAT1 binds to the promoter of immediate early target gene p300 to rapidly activate p300 transcription. p300 then binds to the GPR109A and SLC5A8 promoters and induces chromatin hyperacetylation, resulting in chromatin remodeling in the hypermethylated GPR109A and SLC5A8 promoter regions. pSTAT1 then directly binds to the remodeled but still hypermethylated GPR109A and SLC5A8 promoters to activate transcription of secondary response genes GPR109A and SLC5A8. Taken together, our data suggest that the host immune system might use IFN-γ to counteract DNA methylation-mediated silencing of tumor suppressor genes as a mechanism to suppress tumor development.
Cytolytic capacity of KIR2DS1+ decidual natural killer cells in the fetal-maternal interface (P3203)
Authors: Crespo Angela , Tilburgs Tamara , Strominger Jack
Published: January 2023

Decidual natural killer cells (dNK) are the most abundant lymphocytes at the fetal-maternal interface. dNK have been shown to be less cytotoxic but to secrete more cytokines and growth factors that may help trophoblast invasion than peripheral blood NK cells (pNK). Previous studies have shown that mothers expressing the activating Killer Immunoglobulin-like Receptor-S1 (KIR2DS1) are protected from pregnancy complications when carrying a fetus expressing more HLA-C2 group alleles than them (Hiby et al, 2010). However, no experimental studies have addressed the mechanisms involved in this beneficial effect. In this study, we demonstrate that a significantly higher percentage of dNK express KIR2DS1 and KIR2DL1 than pNK cells. dNK and pNK express similar levels of perforin and granzyme B but dNK contain increased granulysin levels. Although pNK are more cytotoxic than dNK against HLA negative targets, the total pNK cytotoxicity is efficiently inhibited by HLA-C1+ and HLA-C2+ targets. In contrast, HLA-C2+ targets only inhibit the cytotoxicity of total dNK from KIR2DS1- but not KIR2DS1+ mothers. Assessment of single KIR2DL1+ and KIR2DS1+ pNK and dNK shows that HLA-C2 efficiently inhibits KIR2DL1+ but not KIR2DS1+ cell cytotoxicity. These results suggest a dominant role for KIR2DS1+ cells in the total dNK population. Future studies will focus on the influence of the fetal/maternal HLA-C genotype on KIR2DS1+ cell function and the identification of cytokines secreted by KIR2DS1+ dNK.
Antigen persistence augments maintenance of effector-like non-lymphoid T cells but may impair protective efficacy (P3207)
Authors: Beura Lalit , Anderson Kristin , Schenkel Jason , Locquiao Jeremiah , Fraser Katy , Vezys Vaiva , Masopust David
Published: January 2023

The innovative concept of developing persistent vaccines is predicated on the hypothesis that tonic antigen stimulation may maintain abundant effector-like memory CD8 T cells in tissues. We wished to test the impact of persistent antigen stimulation on tissue localized CD8 T cell distribution, differentiation, and function in a reductionist model. To this end, we compared T cell responses in mice infected with strains of LCMV that cause acute versus persistent infections. In contrast to previous reports in this model, persistent infection resulted in the maintenance of ~30 fold more virus-specific lung CD8 T cells relative to acute infection. This interpretation was dependent on the use of refined in vivo labeling techniques that allowed discrimination between T cells present within tissue parenchyma and abundant populations that fail to be removed from vascular compartments despite perfusion. Persistent infection also maintained higher granzyme B expression in lung and greater accumulation of virus-specific T cells in the large intestine, liver, kidney, and female reproductive tract. Surprisingly, quantity and granzyme B expression did not correlate with protective capacity against a local respiratory infectious challenge. In fact, memory CD8 T cells established via acute infection afforded better protection. These data illustrate that protective efficacy cannot be predicted based solely on effector-like CD8 T cell quantity in tissues and phenotype.
Defective induction of regulatory B cells by decidua stromal cells underlies the pathogenesis of preterm birth (P3213)
Authors: Wang Zhao-Yuan , Mathiarasu Aditya , Hong Peng , Xu Yi , Li Aiqun , Bluth Martin , Lum Lawrence , Huang Bihui , Romero Roberto , Cerutti Andrea , Chen Kang
Published: January 2023

Preterm birth is the leading cause of mortality and morbidity of newborns. Many maternal autoimmune conditions that predispose to preterm birth involve the dysregulation of autoreactive B cells that contribute to disease pathogenesis. Despite the fundamental roles of B cells as effectors and regulators of immunity, their functions in pregnancy are poorly understood. We found that human B cells undergo activation, class switching, memory and plasmacytoid differentiation in the decidua in situ. Preterm decidua harbors increased numbers of total B cells and B-1 cells but diminished numbers of IL-10-producing regulatory B cells in vivo. Furthermore, stromal cells in preterm decidua, as compared to those in term decidua, stimulate increased B cell activation and the differentiation of potentially autoreactive B cells, but are defective in inducing IL-10 expression by B cells. These results demonstrate that preterm birth involves abnormal B cell functions at the maternal-fetal interface, whereby decidua stromal dysfunction engenders unique populations of B cells with increased autoreactivity but diminished regulatory functions, and suggest that regulatory B cells are important for the maintenance of normal pregnancy. These studies provide insights into the functions of B cells at the maternal-fetal interface during pregnancy and inform the design of B cell-targeted therapies to treat preterm birth and other pregnancy complications involving B cell dysfunction.
Persistent enteric murine norovirus infection is associated with functionally suboptimal virus-specific CD8 T cell responses (P3187)
Authors: Tomov Vesselin , Osborne Lisa , Dolfi Douglas , Sonnenberg Gregory , Monticelli Laurel , Mansfield Kathleen , Virgin Herbert , Artis David , Wherry E.
Published: January 2023

Norovirus (NV) gastroenteritis is a major contributor to global morbidity and mortality, yet little is known about immune mechanisms leading to NV control. Previous studies using the murine norovirus (MNV) model have established a key role for T cells in MNV clearance. Despite these advances, important questions remain regarding the magnitude, location, and dynamics of the MNV-specific T cell response. To address these questions, we identified MNV-specific MHC class I immunodominant epitopes by conducting an overlapping peptide screen. Using MHC class I-peptide tetramers, we tracked MNV-specific CD8 T cells in lymphoid and mucosal sites during infection with two strains with distinct biological behavior: acutely-cleared CW3 and persistent CR6. Here, we show that enteric MNV infection elicited robust T cell responses primarily in the intestinal mucosa, and that MNV-specific T cells dynamically regulated the expression of surface molecules associated with activation, differentiation, and homing. Furthermore, compared to MNV-CW3, chronic infection with MNV-CR6 resulted in fewer and less functional CD8 T cells. Finally, MNV-specific CD8 T cells were capable of reducing viral load in persistently infected Rag1-/- mice, consistent with a crucial role for these cells in vivo. Collectively, these data provide fundamental new insights into the adaptive immune response to NV and bring us closer to understanding the correlates of protective antiviral immunity in the intestine.
GM-CSF control of macrophage differentiation is a critical effector of Th17 responses in vivo (P4180)
Authors: Barin Jobert , Baldeviano G. , Talor Monica , Stickel Natalie , Bedja Djahida , Gabrielson Kathleen , Rose Noel , Cihakova Daniela
Published: January 2023

Like many models of tissue-localized autoimmune disease, experimental autoimmune myocarditis (EAM) is mediated by Th17-directed pathology. The composition of cardiac infiltrates in EAM is predominantly monocytic, indicating that these cells are likely to be major inflammatory effectors in this disease process. In common with other Th17 autoimmune diseases, IFNγ-deficient mice develop very severe disease. We observed enhanced GM-CSF production from autoantigen-specific IFNγ-/- CD4+ cells, derived from the heart or spleen, suggesting that the induction of GM-CSF by Th17 cells controls the involvement of monocytes and macrophages in disease. After generating IFNγ-/-IL17A-/- double-knockout mice, we determined that the production of GM-CSF in CD4+ is driven by IL17 signaling. Towards this end, we generated bone marrow-derived macrophages (BMMϕ) in vitro, in the presence of GM-CSF, rather than M-CSF. Following adoptive transfer, recipients of GM-CSF BMMϕ developed more severe disease than controls. This severe disease was accompanied by enhanced fibrosis and compromised cardiac function. This novel report links GM-CSF control of macrophage differentiation to the functional consequences of Th17-mediated autoimmunity.
Identification of posttranslational modified autoantigens in Type 1 diabetes. (P4152)
Authors: Yang Mei-Ling , Connolly Sean , Wen Li , Herold Kevan , Mamula Mark
Published: January 2023

Autoimmune diseases such as lupus, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes (T1D) frequently target self proteins that undergo posttranslational protein modifications. Oxidative stress, which is implicated in the pathogenesis of T1D, leads to various protein modifications including isoaspartyl (isoAsp) and carbonyl formation. We have previously identified that isoAsp protein modification can trigger T cell autoimmunity in both human and murine models of lupus. However, the role of oxidative stress-induced posttranslational modifications in T1D is still poorly understood. Herein, we demonstrate that isoAsp modification and oxidative carbonylation accumulate in pancreas and/or pancreatic islet proteins from spontaneous diabetes-prone NOD mice. Specifically, isoAsp content were elevated in both target organ (pancreas) and peripheral erythrocytes of diabetic NOD mice compared to pre-diabetic NOD mice. Moreover, we identified the beta subunit of prolyl-4-hydroxylase (P4hb) as a protein that is abundantly carbonylated in pancreatic islet. Sera from NOD mice and early onset diabetic patients contain antibodies that recognized isoAsp- and/or carbonyl-modified pancreas proteins including P4hb protein. These studies provide a novel insight into the mechanism of breaking immune tolerance and the diagnostic and/or therapeutic targets associated with isoAsp and carbonylated posttranslational protein modifications in T1D.
The critical role of Th1 cytokine polarization in murine primary biliary cirrhosis. (P4154)
Authors: Tsuda Masanobu , Zhang Weici , Yang Guo-Xiang , Ando Yugo , Kawata Kazuhito , Tanaka Hajime , Leung Patrick , Lian Zhe-Xiong , Gershwin M. Eric
Published: January 2023

Primary biliary cirrhosis (PBC) is a female predominant organ specific autoimmune disease with specific destruction of intrahepatic small biliary epithelial cells (BECs). Patients develop a multilineage response to the E2 component of pyruvate dehydrogenase (PDC-E2) involving autoantibody. We previously reported that IL-12p40 deficiency eliminated biliary disease in dnTGFβRII mice, suggesting that IL-12 and IL-23 are important in the development of biliary disease. IL-12 induced Th1 and IL-23 induces Th17, which express both IFN-\#933; and IL-17, but the role of IL-12p40 in PBC remains unidentified. To further investigate the role of the IL-12p40 in this murine model of autoimmune biliary disease, we immunized IL-12p40-/-, IFN-\#933;-/- and IL-17-/- mice with a xenobiotic mimic of PDC-E2 , 2-octynoic acid (2-OA), coupled to bovine serum albumin. The PBC-related autoepitope of PDC-E2 contains lipoic acid, and previous work has demonstrated that mimics of lipoic acid following immunization of mice lead to a PBC-like disease. Importantly IL-12p40-/- and IFN-\#933;-/- mice immunized with 2-OA did not manifest autoimmune biliary disease. In contrast to IL-12p40-/- and IFN-\#933;-/- mice, IL-17-/- mice developed autoimmune biliary disease. These data suggest that signaling by way of Th1 polarizaion via IL-12p40 is an essential requirement for the development of autoimmune cholangitis. These data have implications for the biologic therapy of patients with primary biliary cirrhosis.
The development of tertiary lymphoid organs in the central nervous system facilitates determinant spreading of the MP4-specific T cell response (P4164)
Authors: Kuerten Stefanie , Schickel Achim , Kerkloh Christian , Recks Mascha , Addicks Klaus , Ruddle Nancy , Lehmann Paul
Published: January 2023

Accumulating evidence indicates that B cells aggregate into tertiary lymphoid organs (TLOs) in the central nervous system (CNS) in the human autoimmune disease multiple sclerosis (MS). In patients with secondary-progressive MS the presence of ectopic B cell aggregates has been linked to more severe clinical disease and cortical pathology. Here we demonstrate the formation of TLOs in myelin basic protein (MBP)-proteolipid protein (PLP) fusion protein MP4-induced experimental autoimmune encephalomyelitis (EAE) of C57BL/6 mice. TLOs were a characteristic feature of the chronic disease stage and most prevalent in the cerebellum, while also being present in the spinal cord and cerebrum, but absent in the brain stem. TLOs were characterized by B and T cell compartmentalization, the presence of high endothelial venules and germinal center formation. An association with TH17, but not TH1 cells was evident. In addition, results obtained from ELISPOT analysis of the antigen-specific T cell response suggest that TLOs facilitate determinant spreading of the MP4-specific T cell response to myelin oligodendrocyte glycoprotein (MOG). Our data add novel insights into the contribution of TLOs to disease progression in the context of CNS autoimmunity. We suggest that the blockade of TLO development is a therapeutic strategy that needs to be carefully considered in future studies.
Inflammation regulates the differentiation of CD4 T cells with cytolytic potential (P6088)
Authors: Brown Deborah , Workman Aspen , Vogel Alexander , Condon Shirley
Published: January 2023

Cytolytic CD4 T cells have been described during viral infections; however, the factors necessary for driving the cytolytic phenotype have not been identified. Our data indicate IL-2 regulates granzyme B (GrB) expression in CD4 T cells but IL-12 and IL-6 enhance GrB expression and promote perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of inflammation in CD4 CTL generation in vivo, we used an adoptive transfer system and infection with highly pathogenic influenza virus PR8, compared to x31 with lower pathogenicity. At relatively low doses (1000 EID50), Ova specific CD4 cells expressing GrB differentiated in PR8/Ova infected mice but not x31/Ova infected mice. This pattern was observed at 10-fold higher dose of x31/Ova compared to PR8/Ova. In addition, CD4 T cell influx to the lung and IFN-γ expression was increased in mice infected with PR8/Ova compared to x31/Ova. Importantly, these differences were seen in the endogenous, polyclonal population of CD4 T cells. Inflammatory mediators including IL-6, CXCL-9, CXCL-10 and MIP1β were 3-5 fold higher in PR8/Ova infected lungs at day 4 post infection compared to x31/Ova infected lungs. Whether these inflammatory cytokines are responsible for inducing CD4 CTL is being investigated. These data suggest that CD4 CTL are induced in vivo by strong inflammatory responses and may provide an extra layer of protection against highly pathogenic IAV infection.
Dynamic regulation correlates with variable inhibitory function of CD244 on antiviral CD8 T cells (P6117)
Authors: Pacheco Yovana , McLean Anna , Rohrbach Janine , Porichis Filippos , Kaufmann Daniel , Kavanagh Daniel
Published: January 2023

Antigen-experienced T cells express a wide variety of costimulatory molecules, some of which can contribute to activating, inhibitory, or exhaustion pathways depending on context. In particular 2B4 (CD244/ SLAMf4) has been shown to be capable of either inhibitory or enhancing effects upon engagement of its ligand CD48 (SLAMf2). We examined the phenotypes of CD8 T cell lines and clones derived from HIV-infected or -uninfected subjects, and specific for HIV or for respiratory syncytial virus. Antiviral CTL expressed varying levels of PD-1, 2B4, and TIM-3. Cognate peptide induced rapid downregulation of both 2B4 and TIM-3, but not PD-1 on cultured CD8 T cells. 2B4 downregulation also occurred directly ex vivo and required simultaneous signaling via both TCR and 2B4, as downregulation was not observed in the absence of antigen or in the presence of CD48-blocking antibody. 2B4 downregulation was not induced by PMA or prevented by PI3-kinase inhibition, implicating a TCR-proximal signaling mechanism. Downregulation occurred rapidly upon stimulation with a low dose of peptide, concomitant with internalization of CD3 and CD8. The degree of 2B4 downregulation varied among CD8 T cells of different specificities and HLA restrictions, and the degree of downregulation correlated with the respective enhancement or suppression of antiviral IFNγ production in response to CD48 blockade. Our results indicate a role for 2B4 downregulation in modulation of antiviral CD8 T cell responses.
Herpesvirus latency differentially impacts primary versus secondary memory T cell pools (P6119)
Authors: Grayson Jason , Jennings Ryan , Rajkarnikar Sujana , Barton Erik
Published: January 2023

Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of herpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were infected with murine Gammaherpesvirus 68 (MHV68), a model of Epstein-Barr infection, and then after latency was established, infected with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). This resulted in increased LCMV-specific effector CD4+ T cells compared to mice infected only with LCMV. Although the number of LCMV-specific effector CD8+ T cells was not altered, they were skewed to a memory phenotype. When the memory phase was reached, mice that were infected with both viruses had a memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8+ T cells had altered CD27 and KLRG1 expression. Upon secondary challenge the LCMV-specific effector CD8+ and CD4+ T cell pools expanded and cleared the infection. When the secondary memory pool was established, mice that were also infected with MHV68 had decreased numbers of LCMV-specific CD8+ T cells and these cells were skewed to an effector-memory phenotype. Taken together these results demonstrate that acute viral infection in the context of ongoing latency affects the number and phenotype of primary versus secondary memory CD8+ T cells.
Function and regulation of human NK cells in an acute viral infection model (P6127)
Authors: Marquardt Nicole , Ivarsson Martin , Blom Kim , Braun Monika , Gonzalez Veronica , Falconer Karolin , Sandberg Johan , Michaelsson Jakob
Published: January 2023

Natural killer (NK) cells play an important role in shaping the immune response by releasing proinflammatory cytokines and direct lysis of malignant or virus-infected cells. However, very little is known about the earliest phases of the human NK cell response after acute infection in vivo. Here we made use of the yellow fever virus (YFV) vaccine strain 17D as an in vivo model of an acute viral infection in humans. Vaccination with the live attenuated 17D yellow fever strain induces a measurable viral load and strong T cell activation. YFV vaccination activated NK cells in vivo with distinct kinetics for different functions. NK cell activation as measured by upregulation of Ki67 peaked at day 10, whereas increased degranulation and cytokine production by NK cells peaked at day 6, and coincided with a peak in type I interferons and viral load. Interestingly, within the CD56dim NK cell population, activation was strongest in the least differentiated subset, but was independent of NK cell education via killer cell Ig-like receptors (KIRs). In conclusion, vaccination with 17D allows a detailed longitudinal analysis of NK cell activation after an acute viral infection in humans, and reveals a hierarchy of activation among different NK cell subsets.
Severely exhausted CD8 T cells are refractory to rescue by PD-1 blockade, but re-invigorate following concomitant CD4 T cell depletion in vivo (P6097)
Authors: Penaloza-MacMaster Pablo , Barouch Dan
Published: January 2023

Blockade of the inhibitory PD-1 receptor during chronic infections and cancers has been promising at restoring CD8 T cell function, but it is currently unknown if this functional rescue is optimal during a specific stage of the process of T cell exhaustion. In a model of lifelong LCMV infection in mice, we showed that there is an early phase when PD-1 blockade is able to induce robust rescue of exhausted CD8 T cell responses. As the infection progresses, however, most of the severely exhausted cells become refractory to rescue by PD-1 blockade. This indicated a continuous accumulation of more terminally differentiated virus-specific T cells that underwent a T cell exhaustion program that was regulated by additional inhibitory pathways. This progressive decrease in CD8 T cell rescue potential was associated with a gradual increase in CD4+ Tregs. Interestingly, PD-1 blockade together with transient CD4 depletion during the late stage of chronic infection was able to re-invigorate severely exhausted CD8 T cells and enhance antiviral control. Importantly, whole CD4 depletion did not result in fulminant autoimmune-mediated death that is typically observed following specific Treg ablation. These data suggest that a crucial interplay exists between the PD-1 pathway and CD4+ Tregs at modulating CD8 T cell exhaustion. Our findings may also help to improve immune prophylaxis regimens in patients who remain refractory to PD-1 blockade therapy during chronic infections and cancer.
DNAM-1 is required for expansion of effector NK cells and generation of memory NK cells during mouse cytomegalovirus infection (P6100)
Authors: Nabekura Tsukasa , Lanier Lewis
Published: January 2023

Recent studies have demonstrated that natural killer (NK) cells have adaptive immune features including antigen-specific expansion and subsequent generation of a memory subset. However, the precise signaling pathways that promote the expansion and the generation of memory NK cells remain to be elucidated. Using mixed bone marrow chimeric mice, we investigated the role of DNAM-1, an activating NK cell receptor, in expansion and memory generation of NK cells in a mouse model of cytomegalovirus infection (MCMV). We demonstrated that DNAM-1-deficient MCMV-specific Ly49H+ NK cells exhibit not only a poor expansion following MCMV infection, but also defects in both memory NK cell generation and secondary expansion after re-challenge with MCMV, as compared with wild type Ly49H+ NK cells. The frequency of DNAM-1- NK cells bearing Ly49H gradually increase and the long-lived Ly49H+ NK cells do not express DNAM-1 in MCMV-infected wild type mice. Interestingly, DNAM-1-Ly49H+ effector NK cells are more proliferative and less sensitive to cell death than DNAM-1+Ly49H+ NK cells during MCMV infection. These observations suggest that cooperative signaling through DNAM-1 and Ly49H induces downmodulation of DNAM-1 on Ly49H+ NK cells, resulting in predominant expansion and persistence of DNAM-1-Ly49H+ NK cells during MCMV infection. Therefore, DNAM-1 is a key molecule for optimal effector response and memory generation of virus-specific NK cells.