bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse strain differences in macrophage regulation of peritoneal T cells (100.22)
Authors: Riggs James , Composto Gabriella , Bartlett Thomas , Chaviano Gabriella , Gonzalez Diana
Published: January 2023

Murine peritoneal cavity (PerC) T cells from C57BL/6J mice respond poorly to CD3 ligation due to the high concentration of macrophages (mf) found in this anatomic location. Mfs suppressed T cell activation by their catabolism of ARG by iNOS. To further investigate PerC mf-T cell interaction, we characterized the immunobiology of these cells from C57BL/6J, BALB/c, BALB.xid, and DBA/2J mice. C57BL/6J mice had the greatest number of PerC T cells and the lowest CD4:CD8 ratio. Comprehensive FACS analysis revealed an increased frequency of activated (CD44hi, CD62lo, CD45Rblo, GITRhi) T cells in the PerC relative to conventional lymphoid (lymph node, spleen) tissue. Although there were significant numbers of CD25+, GITR+ T cells in the PerC, Foxp3+ Treg cells were not enriched relative to LN or SP cells. The degree of suppression of T cell activation varied among the strains as well as the mechanism involved. Immunosuppressive prostaglandin production was a modest factor in all strains. Suppression best correlated with the number of IFNg-secreting cells: PerC cells from the “Th1 strain” C57BL/6J and the “Th2 strain” BALB/c were the most and least suppressed, respectively. These results reveal strain-specific differences in PerC mf suppression of T cells. Supported by NIH AREA R15-CA136901-01.
Cancer cells utilize a novel regulatory subset of B cells to promote escape and metastasis (100.26)
Authors: Olkhanud Purevdorj , Damdinsuren Bazarragchaa , Sen Ranjan , Gress Ronald , Biragyn Arya
Published: January 2023

B cells play an important role in antigen presentation and antibody production. However, they appear to exhibit suppressive function inhibiting autoimmune responses in mice. Yet, it remains unknown whether regulatory B cells exist or participate in cancer progression. Here, we demonstrate that a unique B cel subset is induced from resting B cells by cancer cells. These B cells, designated as tumor Bregs (tBregs), are poorly proliferative and express high level of IL-2Rα. The major function of tBregs is to suppress T cell responses, as they efficiently and in dose dependent manner inhibits a proliferation of T cells stimulated with anti-CD3/28 beads. The suppressive activity of tBregs is a cell contact-dependent, but Fas/FasL- and PD1/B7H1-independent, and unlike any other reported regulatory cells, does not require soluble factors such as IL-2, IL-10, TGFβ, IL-27 and IL-35. Importantly, we show for the first time, tBregs induce a conversion of Tregs from non-regulatory CD4+ T cells. Taken together, tBregs are an important and distinct subset of regulatory cells which promote cancer escape and metastasis through direct and indirect (via Tregs) suppression of cellular responses. This work was supported by the Intramural Research Program of the National Institute on Aging, NIH.
Advanced liver disease due to hepatitis C infection is associated with reduced CD19+CD27+ memory B-cells (100.29)
Authors: Kaplan David , Iyer Tara , Carpenter Erica , Vonderheide Robert
Published: January 2023

Advanced neoplasia is associated with defects in B-cell memory. Hepatocellular carcinoma (HCC) frequently arises in the setting of hepatitis C-induced cirrhosis. We hypothesized that memory B-cells from patients with HCC would be reduced in frequency relative to non-cancer bearing cirrhotics and non-cirrhotic HCV. We studied PBMC samples from HCC+ HCV+ cirrhotic patients (n=12), HCC- HCV+ cirrhotic patients (n=12), HCC- HCV+ non-cirrhotic patients (n=10), and HCV- normal donors (n=10) for surface expression of CD19, CD21, CD27, and FcRL4. The frequency of CD27+ memory cells among CD19+ B-cells was significantly lower in patients with cirrhosis (± HCC) compared to HCV+ non-cirrhotic patients (mean ± SE, 16.0 ± 2.7 vs. 29.7 ± 3.8%, p = 0.007) and normal donors (p=0.034). The frequency of atypical memory B-cells (CD27-CD21-/CD19+) was significantly greater among HCC+ patients (31.1 ± 3.7%) compared to HCC- cirrhotics (19.0 ± 3.5%, p=0.023), non-cirrhotic HCV+ patients (16.4 ± 3.9%, p=0.009) and normal donors (17.3 ± 3.9%, p=0.014). Among HCC patients, there was a positive correlation between CD27-CD21- B-cell frequency and AFP levels (R2 = 0.55, p=0.04). Conclusions: HCV-induced cirrhosis is associated with loss of CD19+CD27+CD21+ memory B-cells, while HCC is associated with an increase in CD19+CD27-CD21- atypical memory B cells. These findings identify a phenotype that may be associated with immunodeficiency in cirrhosis as well as with tumor-induced immune suppression in HCC.
Tumor-derived mesenchymal stem cells enhance tumor development via nitric oxide (100.33)
Authors: Ren Guangwen , Zhang Liying , Wen Ting , Zhao Xin , Yuan Zeng-rong , Zhang Jimin , Ling Weifang , L'huillie Andrew , Shao Changshun , Shi Yufang
Published: January 2023

Mesenchymal stem cells (MSCs) have been widely applied in treating immune disorders due to their immunosuppressive capacity. However, a major concern for the clinical application of MSCs is their risk to promote tumorigenesis. In the meantime, there are increasing reports about the identification of MSCs from various tumor tissues. In this study, we compared the tumor-derived MSCs with bone marrow-derived MSCs (BM-MSCs) in vitro and in vivo. We found that only tumor-derived MSCs, but not BM-MSCs enhance tumor development. In vitro, tumor-derived MSCs had a similar capacity to suppress T cell receptor-induced T cell proliferation via nitric oxide (NO), but exhibited a more vigorous inhibition on the differentiation and maturation of dendritic cells, compared to BM-MSCs. Moreover, co-culture with tumor-derived MSCs led bone marrow cells to differentiate into more CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs), another NO-producing immunosuppressive cell population. In vivo, more MDSCs were also detected after administration of tumor-derived MSCs, but not BM-MSCs in tumor-bearing mice. Furthermore, tumor-derived MSCs, but not BM-MSCs significantly enhanced tumor growth, which was partially dependent on NO. Therefore, NO-dependent tumor promoting effect is likely not mediated by MSCs themselves, but through the indirect action of MDSCs. Our findings indicate a novel role of MSCs in tumor progression and provide new information for the clinical application of MSCs.
Response Gene to Complement 32 regulates chromatin assembly in colon cancer (100.35)
Authors: Tegla Cosmin , Vlaicu Sonia , Cudrici Cornelia , Fosbrink Matthew , Nguyen Vingh , Azimzadeh Philippe , Rus Violeta , Shamsuddin Abulkalam , Rus Horea
Published: January 2023

Response Gene to Complement 32 (RGC-32) is induced by complement activation and plays an important role in cell cycle activation. In this study we investigated the role of RGC-32 in colon cancer cell cycle. Our data indicate that RGC-32 expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages than in precancerous states or the initial stages of colon cancer. In addition C5b-9 neoantigens deposits were found to be present on the colon tumor cells expressing RGC-32. In order to identify the genes that are regulated by RGC-32, we used gene array to investigate the effect of RGC-32 knockdown on gene expression in the SW480 cell line. Of the 230 genes that were differentially modulated by RGC-32, a group of genes involved in chromatin assembly were the most significantly regulated. RGC-32 knockdown induced an increase in acetylation of histones H2B lysine 5 (H2BK5), H2BK15, H3K9, H3K18, and H4K8, a decreased expression of SIRT1 and trimethylation of histone H3K27. In addition, RGC-32 knockdown caused a significantly higher percentage of SW480 cells to enter S phase, and subsequently G2/M. These results strongly suggest that silencing of the RGC-32 is associated with chromatin remodeling and activation of cell cycle. Since RGC-32 is induced by C5b-9 is possible that activation of complement has a benefic effect in colon cancer by inducing the expression of this tumor suppressor gene.
Tumor-secreted PGE2 inhibits CCL5 production from activated macrophages through cAMP signaling pathway (100.6)
Authors: Liu Jianguo , Qian Xuesong
Published: January 2023

It is well known that tumor cells could evade immuosurveillance by dampening host immune responses and shaping host stromal cells to favor its growth. CCL5 has been shown to play important roles in T cell proliferation, IFN-g and IL-2 production, which promotes differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. Here we report that the CCL5 mRNA expression and protein production were significantly reduced in tumor-bearing mice compared with tumor-free mice during endotoxic shock. Our data further demonstrate that soluble factors secreted from tumor cells inhibited CCL5 mRNA expression and protein production in LPS-stimulated macrophages. The inhibition of CCL5 expression was regulated at the level of post-transcription. NS398, a selective inhibitor of enzymatic activity of cyclooxygenase 2 in PGE2 synthesis, could reverse the inhibitory effect of tumor cells on LPS-induced CCL5 expression. Moreover, PGE2 and cAMP analog Forskolin could mimic the inhibitory effect of tumor cells on CCL5 expression and blockade of cAMP signaling by H89 completely reversed the inhibitory effect. Taken together, our data indicate that PGE2 secreted from tumor cells suppresses CCL5 expression in LPS-activated macrophages through a cAMP signaling pathway, which may result in suppression of host immune responses against subsequently bacterial infection.
In vitro enhancing oxidative phosphorylation by rapamycin in proliferative CD8+ T cells during antigenic priming augments the generation of long-lived memory T cells in vivo (101.11)
Authors: He Shan , Kato Koji , Jiang Jiu , Wahl Daniel , Mineishi Shin , Cui Shuaiying , Glick Gary , Murasko Donna , Zhang Yi
Published: January 2023

Oxidative phosphorylation is critical to the transition of effectors to memory T cells. However, whether increasing oxidative phosphorylation in proliferative CD8+ T cells during antigenic priming can augment memory T cell generation remains unknown. Using T cell receptor transgenic CD8+ T cells, we found that rapamycin significantly increased the oxidative phosphorylation in antigen-primed naïve CD8+ T cells in vitro without inhibiting their activation and proliferation. Augmented oxidative phosphorylation endowed the rapamycin-treated T cells great ability to survive after IL-2 withdrawal, whereas all the untreated control cells rapidly diminished. Rapamycin-treated CD8+ T cells expressed high levels of CD62L and CD127 but low levels of KLRG-1, PD-1 and IFN-γ. When adoptively transferred in vivo, CD8+ T cells derived from rapamycin-treated cultures generated 5-fold more memory T cells over a period of 6 months than control cells. These long-lived memory T cells had potent ability to produce IFN-γ and proliferate when reexposed to the specific antigen both in vivo and in vitro. These data indicate that augmentation of oxidative phosphorylation in T cells by rapamycin in vitro during antigenic priming dramatically increases both the number and quality of memory precursors, thereby enhancing the generation of long-lived memory T cells. Our findings may have significant implications in optimizing T cell properties to improve the efficacy of adoptive immunotherapy.
Many chronic lymphocytic leukemia antibodies bind apoptotic cells with exposed non-muscle myosin heavy chain IIA in a similar manner as natural antibodies (101.13)
Authors: Chu Charles , Catera Rosa , Zhang Lu , Didier Sebastien , Agagnina Briana , Kaufman Matthew , Kolitz Jonathan , Allen Steven , Rai Kanti , Chiorazzi Nicholas
Published: January 2023

Chronic lymphocytic leukemia (CLL) mAbs containing an IGHV1-69, IGHD3-16 and IGHJ3 rearrangement with a nearly identical or stereotyped sequence (subset 6) recognize non-muscle myosin heavy chain IIA (MYHIIA). Immunohistochemistry showed that MYHIIA becomes exposed on the surface of apoptotic cells. Flow cytometry with 7-amino-actinomycin D and Annexin V-Phycoerytherin showed that MYHIIA exposure (and concomitant subset 6 CLL mAb binding) occurs in a subgroup of both early and late apoptotic cells. Because other CLL mAbs bind apoptotic cells, the binding of MYHIIA exposed apoptotic cells (MEACs) to a panel of CLL mAbs, only two from subset 6, was measured by flow cytometry. Most CLL mAbs (16/26) bound MEACs well. The MEAC binding level inversely correlated with the degree of IGHV mutation. CLL patients with unmutated IGHV (≤ 2%) tend to have shorter survival times. Similarly, CLL mAbs with high binding to MEACs correlated with shorter patient survival. Natural antibodies are characterized by unmutated IGHV and binding to apoptotic cells. Thus, binding of normal serum antibodies to MEACs was tested. Serum samples had reactivities comparable to CLL mAbs with high binding to MEACs, whereas an antigen-selected human antibody specific for CD20 had little MEAC reactivity. Thus, many CLL leukemic clones may derive from natural antibody-expressing B cells, such as B-1 cells, and stimulation by MEACs could lead to development and/or expansion of CLL leading to poor patient outcome.
Interleukin-18 regulates transferrin-induced cell proliferation in MCF-7, breast cancer cell line. (101.14)
Authors: Park Sunyoung , Cho Dae-Ho
Published: January 2023

Interleukin-18 (IL-18) has been reported to enhance cell growth and proliferation in cancer. Transferrin is an iron carrier protein that is responsible for cell growth in both normal and highly proliferative cells like cancers. Although increased serum levels of IL-18 and transferrin in cancer patients correlate with malignancy, little is known about the role of IL-18 and transferrin in the cellular response. Here, we found that exogenous transferrin induces cell growth in human breast cancer cell line, MCF-7. The proliferation rate of MCF-7 cells was reduced when endogenous transferrin expression was inhibited by trasferrin-siRNA transfection. Additionally, we discovered that the expression of endogenous transferrin lies under the control of IL-18 via the MAPK pathway. In conclusion, our data indicate that IL-18 has a pro-cancer effect of accelerating cell proliferation rate by increasing production of endogenous transferrin through the MAPK pathway. Thus, we suggest IL-18 as an inducer of endogenous transferrin expression in breast cancer.
Detection of RET/PTC-associated class I and II-restricted phosphopeptides as tumor specific antigens. (101.25)
Authors: Wixted Josephine , Eisenlohr Laurence
Published: January 2023

Cancer immunotherapy continues to be a viable potential therapy for many tumor types, however, discovering the existence of and breaking tolerance to tumor specific antigens remains a challenge. In our strategy we will take advantage of a common theme of all cancer cell types - the constitutive activation of signal transduction members that promote cell survival and proliferation. Post-translational modification of tyrosine, serine, and threonine via the addition of a phosphate group can both activate signal transduction pathways and generate the production of “neophosphoantigens”. Here we use both sodium vanadate and a model fusion oncogene, RET/PTC, to transform fibroblasts and examine the immunoreactivity of phosphoantigens derived from class I and class II.
T-bet and Eomesodermin are required for adaptive immunity against cancer (101.27)
Authors: Zhu Yibei , Ju Songguang , Chen Elizabeth , Dai Shao , Li Changyou , Liu Lin , Zhang Xueguang , Lu Binfeng
Published: January 2023

Cell-mediated adaptive immunity is very important in tumor immune surveillance and tumor vaccination. However, the genetic program underlying an effective adaptive anti-tumor immunity is elusive. T-bet and Eomes have been suggested to be master regulators of Th1 cells and CD8+ T cells. Whether they are important for anti-tumor immunity is controversial. Here we show that lack of T-bet and Eomes has resulted in profound defects in adaptive anti-tumor immune responses in both prophylactic and therapeutic settings. T-bet and Eomes are also important for CD8+ T cell activation without affecting T cell clonal expansion, the gaining of effector phenotype and cytolytic function. Unexpectedly, T-bet and Eomes are dispensable for IFNg production by CD8+ T cells under optimal Th1 differentiation conditions. However, they are necessary for suppressing alternative fates of CD8+ T cells. They drive Tc1 differentiation by preventing alternative CD8+ T cell differentiation to Tc17 or Tc2 cells. Surprisingly, T-bet and Eomes are not critical for the generation of systemic CTL activities against tumor cells. Instead, T-bet and Eomes are crucial for T cell migration and retention in the tumor site and elimination of solid tumors by CD8+ T cells through regulating chemokine receptors such as CXCR3 and IFNg expression in tumor microenvironment. Therefore, our study establishes T-bet and Eomes as key regulators of adaptive cell-mediated immunity against cancer.
Cytotoxic mechanisms of IL-4 polarized CTLs (101.28)
Authors: Hudig Dorothy , McKinney Christopher , Alves Bryce , Tamang David , Redelman Doug , Singer Cherie , Elliott Viki
Published: January 2023

IL-4 down-regulates perforin and granzymes and depresses rapid (4 hr) cytotoxic T lymphocyte (CTL) killing. Curiously, IL-4 can increase anti-tumor immunity in animal models. We wanted to know how IL-4 polarized CTLs kill and found that they have potent delayed cytotoxicity. We cultured BALB/c mouse splenocytes with conA and 500 units/ml mouse IL-4 or IL-2 for 3 days and then re-cultured the cells for 3 days with cytokines without conA. The IL-4 CTLs were polarized as indicated by expression of the IL-4 inducible lipase PLRP2. By flow cytometry, IL-4 CTLs expressed Grz B at lower levels than IL-2 CTLs. We assayed cytotoxicity using anti-CD3-redirected death of P815 tumor cells. Death was measured at 48 hrs by loss of EGFP+ P815 cells using flow cytometry with cell count beads to account for dead cell disintegration. Cytotoxicity by the IL-4 CTLs was 80% at effector lymphocyte to P815 (E:T) ratios of 1:10. IL-4 CTLs had perforin-independent mechanisms, with similar activity for WT and Prf1-/- CTLs. We monitored T cells for death receptor ligands. Quantitative RT-PCR indicated that IL-4 cells maintained comparable levels of mRNA for LTβ, LIGHT, TRAIL and FasL, but had lower levels of TNFα and TNFβ, than IL-2 CTLs. We conclude that IL-4 polarized CTLs have potent cytotoxicity which could be mediated by death receptor ligands and/or by novel IL-4 inducible potential cytotoxins. Supported in part by NIH grants R01CA38942, T32CA09563, P20 RR-016464 & P20-RR015581.
Role of CD91 in HSP-mediated innate immune responses (101.29)
Authors: Pawaria Sudesh , Binder Robert
Published: January 2023

Immunogenic Heat shock proteins (HSPs) are known to chaperone antigenic peptides and elicit strong immunity against tumors and infectious agents. The peptide bound form of the HSP, gp96, interacts with the antigen presenting cells (APCs) through the receptor, CD91 (LRP1), resulting in endocytosis of the complex and the presentation of the peptides on MHC I and MHC II to T-cells. Independent of the peptides, the binding of HSPs to the APCs results in the translocation of NF-kB into the nucleus and the elaboration of a number of pro-inflammatory cytokines. Binding of gp96, HSP70 or calreticulin to CD91 results in the phosphorylation of its beta-chain, association with the Shc adaptor molecule and activation of NF-kB. By selective mutation of the two phosphorylation sites of CD91, we observed that HSPs appear to utilize different sites resulting in distinct downstream signaling pathways. The cytokine profiles resulting from these signals are unique. These data shed light on the responses of CD91-expressing antigen presenting cells to immunogenic HSPs released from dying cells or introduced by vaccination.
Diphenyl difluoroketone (EF-24) modulates dendritic cell responses. (101.30)
Authors: Vilekar Prachi , Awasthi Vibhudutta , Lagisetty Pallavi , Natarajan Aravindan , Awasthi Shanjana
Published: January 2023

Dendritic cells (DCs) are the most potent antigen-presenting cells that link the antigen-specific innate host responses with adaptive immunity. They recognize pathogens, pathogen-ligands and inflammatory signals via pattern recognition receptors. Inflammatory signals induce DC maturation and release of cytokines, chemokines and chemical mediators. We synthesized EF24, a known more potent analog of curcumin, and studied its immunomodulatory properties using immortalized murine DCs (JAWS II). We hypothesized that pharmacological downregulation of proinflammatory pathway by EF24 may suppress lipopolysaccharide (LPS)-induced phenotypic and functional changes in DCs. Murine bone marrow-derived JAWSII DCs were incubated with 1-10 µM EF24 with or without LPS (100 ng/ml). We investigated cell viability, morphology, expression of DC-specific markers and TLR4, NF-κB activity and cytokines. The short- and long-term treatment with EF24 suppressed the LPS-induced expression of DC-markers (CD80, CD86, MHC class II). Immunohistochemical expression of TLR4 in the cells was significantly reduced. Electrophoretic-mobility shift assay showed inhibition of LPS-induced NF-κB activation. EF24 also reduced the expression of IL-6, TNF-α and MCP-1 in DCs. There was no effect of EF24 on viability of DCs in the concentrations used. Overall, our results suggest that the immunomodulatory properties of EF24 may be of therapeutic value in inflammatory conditions, such as septic shock.
Amplifying TLR-MyD88 signals within tumor-specific T-cells enhances antitumor activity to low concentrations of subdominant tumor-antigens (101.35)
Authors: Davila Eduardo , Geng Degui , Srivastava Ratika , Riker Adam , Markovic Svetomir
Published: January 2023

There is a dire need to understand the cellular signals that can potentiate T-cell responses to weakly immunogenic tumor antigens (TAg) and to tumors expressing suboptimal levels of these antigens. We examined the anti-tumor activity and survival of TLR2-MyD88-stimulated tumor-specific CD8 T-cells derived from melanoma patients and TAg-specific T-cell receptor transgenic pmel mice. TLR2 engagement on pmel CD8 T-cells, but not on TLR2-/-pmel or MyD88-/-pmel T-cells, reduced the activation threshold to a subdominant TAg, resulting in increased production of effector molecules and cytotoxicity. Wild-type or MyD88-/- mice treated with pmel T-cells and TLR2 ligand, but not TLR2-/-pmel or MyD88-/-pmel T-cells, showed significant tumor regression of an established melanoma tumor. Over-expressing TLR2 in pmel T-cells eradicated established tumors and four-times fewer cells were needed to generate anti-tumor responses. The enhanced anti-tumor activity was associated with improved survival and increased effector function of TLR2-MyD88-stimulated T-cells. Activating TLR-MyD88 signals in patient-derived T-cells reduced the activation threshold to several weakly immunogenic TAgs, resulting in increased cytokine production, expansion and cytotoxicity. These data highlight the physiological importance of activating TLR-MyD88 signals within tumor-reactive T-lymphocytes for enhancing their longevity and augmenting effector function against suboptimal levels of weakly-immunogenic antigens.
Vascular Disrupting Agent, DMXAA, Enhances Therapeutic HPV Vaccine-Induced CTL responses and Antitumor Effects (101.37)
Authors: Zeng Qi , Peng Shiwen , Monie Archana , Hung Chien-Fu , Wu T.-c
Published: January 2023

Antigen-specific immunotherapy has emerged as an attractive approach for the treatment of cancers. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 peptide-based vaccination for their ability to enhance the E7-specific CD8+ T cell immune responses and antitumor effects in treated mice. We found that the combination of DMXAA treatment with E7 long peptide (aa43-62) vaccination mixed with polyI:C generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes and tumor-infiltrating lymphocytes (TILs) of tumor bearing mice. Furthermore, we found that the DMXAA-mediated enhancement of E7-specific CD8+ T cell immune responses was dependent on the timing of administration of DMXAA and was also applicable to other antigen-specific vaccines. We further tested the combination of DMXAA with E7 peptide vaccination in a vaginal TC-1 tumor growth model and observed a significant enhancement in the antitumor effects in treated mice. Taken together, our data demonstrated that administration of the vascular disrupting agent, DMXAA, enhances therapeutic HPV vaccine-induced CTL responses and antitumor effects against E7-expressing tumors in two different locations. Our study has significant implications for future clinical translation.
Programmed Death Ligand-1 expression on the cell surface is regulated by costimulatory molecule CD80 expressed by cancer vaccines (101.4)
Authors: Haile Samuel , Bosch Jacobus , Agu Nnenna , Ostrand-Rosenberg Suzanne
Published: January 2023

Programmed Death Ligand 1 (PDL1) is a major obstacle in the development of cancer immunotherapies. PDL1 is constitutively expressed or induced by IFNγ on many tumor cells, resulting in the prevention of T cell activation or the apoptotic cell death of activated T cells. Cell-based vaccines previously developed in our lab were made by modifying tumor cells to express Major Histocompatibility Complex Class II (MHCII) and costimulatory B7-1 (CD80) molecules. These cell-based vaccines do not express PDL1, even after IFNγ stimulation. As determined by flow cytometry using three anti-PDL1 monoclonal antibodies that detect distinct PDL1 epitopes, and human lung, uveal and cutaneous melanoma, and breast cancer cells, the over-expression of CD80 blocks constitutive and IFNγ-induced cell surface expression of PDL1. Quantitative analysis of CD80 and PDL1 expression indicates an inverse relationship between PDL1 and CD80. CD80+ tumor cells contain intracellular PDL1, as assessed by flow cytometry, suggesting that CD80 regulates PDL1 at the protein level. Tumor cells transfected with CD80 constructs lacking a cytoplasmic domain or with an irrelevant non-signaling cytoplasmic domain, retain the ability to block PDL1 expression at the cell surface indicating that CD80 does not modulate PDL1 transcription. These results suggest that in addition to serving as a costimulatory molecule, CD80 facilitates T cell activation by blocking coinhibitory signals and preventing T cell apoptosis.
Ovarian tumor infiltrating lymphocytes B have pro angiogenic properties (101.7)
Authors: Nunez-Cruz Selene , Guerra Matthew , Connolly Denise , Coukos George , Gimotty Phyllis , Scholler Nathalie
Published: January 2023

Despite extensive studies of the role of tumor-infiltrating lymphocytes-T and macrophages, the role of tumor-infiltrating lymphocytes-B (TIL-B) during cancer progression remains poorly understood. To address this issue, we characterized the phenotype and function of TIL-B in a transgenic mouse model of epithelial ovarian cancer that resembles human serous ovarian cancer. We first observed that CD45+ CD19+ TIL-B cells are one of first populations to infiltrate ovarian tumors and their percentages decreased over time, from 32±5% at 13 weeks to 16±3% at 26 weeks. Our microarrays results revealed that TIL-B transcripts segregated with normal splenocytes, and significantly up-regulated Angiopoietin2, platelet derived growth factor (PDGF), Midkine, Insulin-like growth factor binding proteins and Transforming growth factors beta. In addition, multiplex cytokine assays showed that TIL-B spontaneously secreted PDGF-bb, CXCL9, Leukemia inhibitory factor and Macrophage-colony stimulating factor. Finally, we found that TIL-B secreted the suppressive cytokine IL-10 and attracted endothelial cells in vitro. Our results demonstrated for the first time that TIL-B beared different functional capabilities than normal B cells, in particular TIL-B synthesized angiogenic factors and attracted endothelial cells. These results suggest that TIL-B could promote angiogenesis and thus represent an attractive therapeutic target for solid tumor therapy.
Exosome-mediated MHCII cross-presentation substantially enhanced by the receptor binding activity of influenza hemeagglutinin (130.10)
Authors: Testa James , Apcher Geraud , Eisenlohr Laurence
Published: January 2023

T cell responses to viral infection often depend upon cross-presentation, a process in which antigen is transferred from an infected cell to a professional antigen presenting cell. This process has been extensively studied with respect to MHC class I-restricted activation of CD8+ T cells, with several mechanisms having been reported including transfer of peptide-loaded heat shock proteins, apoptotic bodies and exosomes. In contrast, little work has been done on MHCII-restricted cross-presentation. Using infectious influenza virus and its glycoproteins expressed in antigen presenting cells, we readily observed MHCII cross-presentation, but only for one of the three epitopes that we routinely study, the “S1” epitope of the hemagglutinin glycoprotein (HA). This selectivity was explained by subsequent experiments that identified the process to be exosome-mediated. While exosomes are naturally endocytosed at a basal level, blocking antibodies and neuraminidase treatment of acceptor APCs revealed that cross-presentation was substantially enhanced by the sialic acid binding activity of the HA that had been incorporated into exosomes produced by the donor cells. This mechanism could provide an explanation for the skewed CD4+ responses to influenza that have been reported and may be an important component of the immune response to other enveloped viruses.
The effects of HLA-DO on peptide binding by MHC Class II HLA-DR1 (130.14)
Authors: Poluektov Yuri , Kim AeRyon , Khoruzhenko Stanislav , Sadegh-Nasseri Scheherazade
Published: January 2023

Ever since the discovery of the HLA-DOA and HLA-DOB genes in the HLA Class II region, controversy has surrounded their true function. Studies resulting in transcriptional modification of the HLA-DO gene analogs in mice (H-2O) support the general theory that DO decreases the presentation of antigens through the MHC class II system by inhibiting the effects of the MHC class II accessory molecule HLA-DM. The mechanism of such inhibition, however, still remains a mystery. To shed some light on this issue, we have constructed a recombinant form of soluble HLA-DO, without its cytoplasmic anchors, and conducted peptide binding experiments with soluble forms of recombinant HLA-DM and HLA-DR1. Our experiments indicate that DO can have both a negative and a positive effect on peptide presentation by the MHC Class II molecule HLA-DR1, depending on the quality of the anchoring residues of the peptide. In addition, HLA-DO only affects peptide association to the MHC Class II molecules and has virtually no effect on peptide dissociation.
Mapping TCR recognition of a beryllium antigen with implications for chronic beryllium disease (130.17)
Authors: Bowerman Natalie , Falta Michael , Kappler John , Fontenot Andrew
Published: January 2023

Exposure to the metal beryllium in the workplace can result in chronic beryllium disease (CBD), which is associated by the accumulation of large expansions of beryllium-specific CD4+ Th1 cells in the lung. Several T cell clones have been isolated from the lung of a CBD patient that recognize beryllium in the context of HLA-DPB1*0201/peptide. Sequence comparison of the TCR complementarity determining regions (CDRs) has revealed nearly identical Vβ and CDR3 Vα domains, yet different CDR1-2 Vα regions (due to expressing different TCRAV gene segments). Hence, we propose an atypical docking footprint for recognition of the beryllium antigen by these specific TCRs, with CDR1α and CDR2α not significantly contributing to T cell recognition. To test this hypothesis, we have focused on a particular Vα22+/Vβ5+ beryllium-specific TCR derived from a T cell clone designated JK28. Single-site alanine substitutions have been introduced across the CDRs of this TCR in order to delineate amino acids that are critical for recognition of the HLA-DP2/Be2+/peptide complex. TCR variants containing alanine substitutions were expressed on a murine α-β- T cell hybridoma also transduced with human CD4. Assays comparing IL-2 secretion when T cell hybridomas containing wildtype and mutated TCRs are stimulated with beryllium support the notion that the Vβ region contributes most to antigen recognition by this TCR. Supported by grants from the NIH, HL062410 and HL092997.
Quantitative analysis of immune synapse formation using imaging flow cytometry. (130.18)
Authors: George Thaddeus , Spurkland Anne , Sundvold-Gjerstadt Vibeke , Burbach Brandon , Shimizu Yoji , Hall Brian , Friend Sherree
Published: January 2023

Antigen-specific immune responses are initiated via direct cellular contact between an antigen presenting cell (APC) and an effector cell. Early events in the interaction between these two cells involve reorganization of the actin cytoskeleton and recruitment of adhesive and signaling molecules to the immunological synapse (IS), which ultimately results in effector cell activation. Because manual image acquisition and quantitative image analysis are time consuming processes, microscopic analyses of protein recruitment to the IS have remained either qualitative or statistically limited, despite the rich information content inherent in high resolution digital images. Here we describe an objective, statistically robust microscopy-based method for quantifying molecular recruitment to the IS using multispectral imaging flow cytometry, which enables quantitative image analysis of large populations of automatically collected images. The analysis technique uses morphology-based features to identify conjugates, followed by measurement of fluorescence specifically at the conjugate junction. Two models are described, including: 1) actin recruitment to the IS following T cell activation by artificial APC coated with anti-CD3; 2) antigen-specific recruitment of ADAP to the IS within TCR transgenic T cells. These data outline an objective and statistically robust method to rapidly quantify molecular recruitment to the immune synapse using automated high speed microscopy.
Endogenous MR1 is transiently expressed on the cell surface and can be stabilized with a unique mAb for activation of mucosal-associated invariant T cells (130.19)
Authors: Chua Weijen , Yu Lawrence , Myers Nancy , Kim Sojung , Huang Shouxioung , Hansen Ted
Published: January 2023

MHC-related protein 1 (MR1) is highly conserved among mammals and restricts the development of mucosal-associated invariant T (MAIT) cells. MAIT cells bear an invariant TCRα chain preferentially paired with limited β chains. MAIT cells reside predominantly in the mucosal tissues but are absent in mice lacking commensal bacteria or B cells. Upon activation, MAIT cells rapidly secret cytokines including IFN-γ, IL-4, and IL-10. Surprisingly, endogenous MR1 has not been detected at the cell surface; but, surface MR1 is detected on cells overexpressing MR1 by transfection or transduction. To better characterize endogenous MR1 expression, we generated mAbs to mouse MR1. Using these mAbs, we show that select B cell lines express low levels of endogenous MR1 at the cell surface. Interestingly, a unique mAb to MR1 was found to enhance MAIT cell activation, whereas other mAbs to MR1 blocked MAIT cell activation. The enhancing and the blocking mAbs recognize different MR1 epitopes mapped by a panel of MR1 mutants and by mAb competition assays. Mechanistically, the enhancing mAb stabilizes MR1 at the cell surface suggesting it either promotes assembly of the MR1 complex or allosterically induces a more favorable conformation. These findings demonstrate that endogenous MR1 transiently visits the cell surface of APCs, and that induced levels of MR1/ligand complexes are required for MAIT cell activation.
Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphinogolipids presented by CD1d (130.22)
Authors: Muindi Karen , Cernadas Manuela , Watts Gerald , Royle Louise , Neville David , Dwek Raymond , Besra Gurdyal , Rudd Pauline , Butters Terry , Brenner Michael
Published: January 2023

CD1d molecules present lipid antigens that activate CD1d-restricted natural killer T (NKT) cells. NKT cell activation leads to potent downstream activation of other immune cells through cell-cell interactions and rapid production of large quantities of cytokines. Endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of TLR induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The repertoire of GSL bound to CD1d expressed in myeloid antigen presenting cells at steady state and in the setting of activation has not been delineated. We have identified the range of GSL bound to soluble CD1d molecules that sample the ER/secretory routes and cell surface cleaved CD1d that also samples the endocytic system. CD1d bound specific GSL preferentially as the CD1d GSL profile did not solely reflect cellular GSL abundance. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, while GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation the quantities of soluble CD1d bound GM3 and GM1a markedly increased. A unique a-galactose-terminating GSL was also found to be preferentially bound to CD1d at steady state and increased with APC activation. Our studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation. In press PNAS
Ovalbumin-derived precursor peptides are transferred sequentially from gp96 and calreticulin to MHC I in the endoplasmic reticulum (130.26)
Authors: Kropp Laura , Garg Manish , Binder Robert
Published: January 2023

Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC I are associated with several chaperones. Hsp70, hsp90, and the TCP1-ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC I and hence presentation of the MHC 1-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC I heavy chains and beta2-microglobulin as a trimolecular complex.
Surface peptide-MHC generation from Listeria-derived recombinant proteins is independent of protein half-life (130.30)
Authors: Wolf Benjamin , Kumagai Chiharu , Guterman Jacqueline , Princiotta Michael
Published: January 2023

The generation of surface peptide-MHC (pMHC) complexes from virally infected cells is dependant on the degradation of newly synthesized proteins, with the amount of pMHC complexes generated directly correlating with the amount of protein degraded. pMHC complex generation from cells infected with the bacteria Listeria monocytogenes, on the other hand, is not as well understood. While it is known that the generation of pMHC complexes from Listeria-infected cells is dependant on newly synthesized bacterial proteins, it is not known whether the amount of pMHC on the cell surface correlates with the amount of protein degraded. To determine whether there is such a correlation, we generated Listeria strains that produce model recombinant proteins containing the minimal Kb binding peptide SIINFEKL. These recombinant proteins are nearly identical (1 amino acid difference) yet differ in half-life once in the eukaryotic cell cytosol. By changing the half-life of the recombinant protein and then comparing Kb-SIINFEKL generation, we have found that full-length proteins secreted into the host cell generate similar amounts of Kb-SIINFEKL regardless of their protein half-life. These data suggest that full-length proteins secreted into the host cell by Listeria follow distinct processing pathways for the production of pMHC complexes.
Direct presentation regulates the magnitude of the CD8+ T cell response to cell-associated antigen through prolonged T cell proliferation (130.32)
Authors: Tatum Angela , Schell Todd
Published: January 2023

The magnitude and complexity of antigen-specific CD8+ T cell responses is determined by both intrinsic properties of the immune system and extrinsic factors such as vaccination. We evaluated mechanisms that regulate the CD8+ T cell response to two distinct determinants derived from the same protein antigen, SV40 T antigen (T Ag), in C57BL/6 mice following immunization with T Ag transformed cells. We found that the immunodominance hierarchy was reversed in a dose-dependent manner by addition of excess naïve T cells targeting the subdominant epitope. However, competition by T cells targeting distinct determinants played only a minor role in limiting T cell accumulation under physiological conditions. In contrast, the magnitude of the T cell response was regulated by the ability of T Ag transformed cells to directly present the T Ag determinants. The hierarchy of the CD8+ T cell response was maintained when antigen presentation in vivo was restricted to cross-presentation, but immunization with T Ag transformed cells capable of direct presentation dramatically enhanced T cell accumulation at the peak of the response. Enhanced accumulation was due to a prolonged period of T cell proliferation, resulting in a delay in T cell contraction. Our findings reveal that direct presentation by non-professional antigen presenting cells can dramatically enhance accumulation of CD8+ T cells during the primary response, revealing a potential strategy to enhance vaccination approaches.
Molecular mechanisms of Th1-mediated antigen cross-presentation: roles of Iigp1 and ingenol. (130.34)
Authors: Kariyone Ai , Ikutani Masashi , Nagai Yoshinori , Takatsu Kiyoshi
Published: January 2023

Antitumor immunity requires activation of CD8+ cytotoxic T cells (CTL) by antigen-presenting cells (APCs) through interaction with tumor antigen peptides on MHC class I molecules. Intracellular mechanisms on MHC class I-restricted soluble antigen presentation (cross-presentation) remain elusive. Peptide-25 of Ag85B induces Th1 cell response in I-Ab mice that facilitate CTL generation against unrelated Ovalbumin (OVA) peptide when co-immunized with OVA, indicating that Peptide-25 augments cross-presentation by APCs. In this presentation we will discuss about roles of Iigp1 and effects of various natural products on Th1-mediated enhancement of the cross-presentation. We evaluated the cross-presentation by 2-step culture system. At first, APCs were cultured with P25 TCR-Tg CD4+ T cells, Peptide-25, OVA or natural products for overnight. After the culture the OVA-pulsed APCs were harvested and cultured with OVA specific OT-I Tg CD8+ T cells for 3 days. The cross-presentation activity was evaluated by OT-1 proliferation and IFN-γ production. Results revealed an indispensable roles of IFN-γ and IFN-γ-inducible genes such as Iigp1 in the Th1-mediated cross-presentation. We also found that some of natural products such as ingenol derivatives augment the cross-presentation in an Th1-independent manner.
Quantitative assessment of soluble B7RP-1 in bound and unbound state in normal and Systemic Lupus Erythematosus patients’ plasma (130.4)
Authors: Bautista Ami , Zhou Lei , Zhang Ming , Sullivan Barbara , Belouski Shelley , Starcevic Marta , Kaliyaperumal Arunan , Chirmule Naren , Jawa Vibha
Published: January 2023

Membrane associated receptors can undergo shedding following cross-linking with ligand. B7RP-1 receptor is present on B-cells and macrophages and binds to the inducible costimulatory (ICOS) molecule on activated and memory T cells. ICOS binding can induce B7RP-1 receptor shedding. An electrochemiluminescence assay (ECLA) on the Meso Scale Discovery Sector Imager 6000 was validated to quantitate the soluble B7RP1 (sB7RP-1) levels in plasma from normal and Systemic Lupus Erythematosus (SLE) subjects. Comparing results of untreated samples or those pretreated with acetic acid enabled the detection of free sB7RP-1 and complexed sB7RP-1 (bound with ICOS or anti-B7RP-1 antibody). Using a bivalent Fc fused B7RP-1 receptor as the standard, the assay range was validated from 0.55 to 40 ng/mL with less than 30% total bias (%relative error plus %CV) at each level. Repeat measures of 51 subjects (normal and SLE) over day 1, 8, and 15 were analyzed. Utilizing the ratio of the sB7RP-1 levels quantified at day 8 or 15 compared to day 1 (post to pre), the natural variability between subjects and over time was statistically determined. No significant differences were noted between SLE and normal subjects, hence, the post to pre ratios were pooled and the range was set between 0.63 to 1.47, representing the 2.5% and 97.5% distributions, respectively. Hence, this assay can help monitor the extent of B7RP-1 shedding following ligand binding and it's potential to form an immunocomplex.
Neutrophils markedly influence the immune response to protein antigens in adjuvant (130.45)
Authors: Yang Chiao-Wen , Unanue Emil
Published: January 2023

Neutrophils (PMN) have a crucial role in innate immunity against microbial infections. In vitro experiments indicated that PMN dampened T cell proliferation. However, it is unclear if PMN affect adaptive immunity in vivo. Here we used protein immunization in various adjuvants as a model system to study how PMN modulate T cell priming. After immunization with Freund’s adjuvants or alum, PMN rapidly entered the draining lymph nodes (dLN), localizing to the cortical sinus where antigen accumulated. In mice lacking PMN, due to antibody-mediated depletion or genetic ablation, CD4 T cell priming was enhanced by 2-3 fold. We observed enhanced CD4 T cell responses to three different protein antigens (hen-egg white lysozyme, ovalbumin and listeriolysin-O); this effect was independent of the adjuvant and antigen dose. PMN depletion in mice lacking NADPH oxidase or inducible NO synthase still showed enhanced CD4 T cell responses. Dendritic cells (DCs) and macrophages in the dLN contained more antigen and presented more peptide-MHC complexes in the absence of PMN, suggesting antigen competition between PMN and antigen presenting cells (APCs). Finally, PMN-depletion enhanced CD4 T cell responses in mice immunized with antigen-loaded DCs, indicating that PMN migrating to the dLN had a negative effect on antigen bearing APCs. Together, these experiments showed that PMN influenced adaptive immune responses via modulation of antigen presentation.