bluuebunny/crossref_metadata_2025 huggingface dataset:
The upregulation of the inhibitory receptors BTLA and PD-1 defines a novel subset of dysfunctional tumor antigen-specific CD8+ T cells (127.19)
Authors: Fourcade Julien , Sun Zhaojun , Pagliano Ornella , Guillaume Philippe , Luescher Immanuel , Sander Cindy , Kirkwood John , Olive Daniel , Kuchroo Vijay , Zarour Hassane
Published: January 2023

Cytotoxic T cells present at the periphery or at tumor sites recognize tumor epitopes presented by melanoma cells but fail to induce tumor rejection in melanoma patients. Inhibitory receptors including PD-1 and Tim-3 expressed by spontaneous CD8+ T cells directed against the cancer-germline antigen NY-ESO-1 play a role in promoting tumor-induced T cell dysfunction. In the present study, we show that BTLA is upregulated on spontaneous NY-ESO-1-specific CD8+ T cells and that BTLA-expressing PD-1+Tim-3- CD8+ T cells represent the largest subset of NY-ESO-1-specific CD8+ T cells in melanoma patients. These cells were partially dysfunctional, producing less IFN-γ than BTLA- T cells, but more IFN-γ, TNF and IL-2 than the highly dysfunctional subset expressing all three receptors. Unlike PD-1, BTLA expression by NY-ESO-1-specific CD8+ T cells does not increase with higher T cell dysfunction or upon cognate antigen stimulation, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Finally, BTLA blockade adds to PD-1 and Tim-3 blockades, enhancing NY-ESO-1-specific CD8+ T cell expansion, proliferation and cytokine production. Collectively, our findings support the targeting of BTLA, PD-1 and Tim-3 pathways to reverse tumor-induced T cell dysfunction in patients with advanced melanoma.
Effects of a multi-targeted receptor tyrosine kinase inhibitor on radiosentization of head and neck squamous cell carcinoma. (127.21)
Authors: Mirshahidi Saied , Hsu Heng- Wei , Chen Chien-Shing
Published: January 2023

Targeted biological therapies that selectively interfere with cancer cell growth signals may improve patient survival by enhancing the effects of radiation, with the added benefit of reduced systemic toxicity. Head and Neck Squamous Cell Carcinoma (HNSCC) is the most common epithelial malignancy arising in the upper aerodigestive tract. It is the sixth most common cancer worldwide. Signal transducers and activators of transcription 3 (STAT3), is present in a number of different cancer cells including head and neck cancer and is related to chemoresistance and radioresistance, therefore is a potential novel molecular target for therapy to improve survival of the patients. In the present study, we report that linifanib (ABT-869), is a novel multi-targeted receptor tyrosine kinase (RTK) inhibitor of members of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor families, has anti-proliferative effects in HNSCC cell lines in a dose dependent manner. It inhibits STAT3 signaling and related proteins and arrests cell cycle at G2/M phase, induces apoptosis, inhibits colony formation efficiency of the cells. In addition, when this multi-targeted receptor tyrosine kinase inhibitor is combined with radiation, significant antitumor effects are achieved in the cell lines. Thus, it may provide a therapeutic strategy and improves efficacy of radiation against HNSCC.
Natural CXCR3+ FOXP3+ Treg selectively accumulate in human ovarian carcinomas to control type I immunity (127.27)
Authors: Raffin Caroline , Redjimi Nassima , Odunsi Kunle , Valmori Danila , Ayyoub Maha
Published: January 2023

Anti-tumor type I T cell responses are critical to control cancer but their efficacy is limited by immunoregulatory mechanisms, including the action of immunosuppressive FOXP3+ Treg. Recent studies have revealed a significant heterogeneity in FOXP3+ Treg, that, similar to conventional CD4+ T cells, include subsets co-expressing lineage-specific transcription factors, that specifically control over the corresponding conventional effector subset. FOXP3+ Treg expressing CXCR3 have been recently described as a subset specialized in the control of type I T cell responses in vivo. We found that CXCR3+ Treg are highly enriched in ovarian carcinomas (OC), particularly in solid tumor masses, where they represent the majority of Treg. CXCR3+ Treg in OC express T-bet but do not secrete IFN-γ ex vivo and suppress proliferation and IFN-γ secretion of T effectors. OC CXCR3+ Treg are Helios+, suggesting that they originate from natural rather than from adaptive Treg, In support of this, CXCR3+ T-bet+ Helios+ IFN-γ- Treg are efficiently generated upon stimulation of natural naïve Treg under TH1 polarizing conditions. Finally, we show that the presence of CXCR3+ Treg is directly correlated with that of CXCR3+ T effectors and is consistent with expression of CXCR3 ligands at OC sites. Together, our findings support the concept that natural CXCR3+ Treg selectively accumulate in OC tumors to control type I T cell responses, which results in the limitation of efficient anti-tumor immunity.
IL-12 and Type I IFN differentially program CD8 T cells for PD-1 expression levels and tumor control (127.32)
Authors: Heltemes Harris Lynn , Gerner Michael , Mescher Matthew
Published: January 2023

Naïve CD8 T cells proliferate in response to signals through TCR and CD28, but require IL-12 or Type I IFN to undergo differentiation and develop optimal effector functions. CTLs generated in vitro in response to IL-12 or IFN-alpha displayed comparable cytolytic activity and IFN-gamma production at day 3. Despite similar day 3 activity, IL-12-stimulated cells were much more effective in controlling tumor growth in an adoptive immunotherapy model. IL-12-stimulated cells maintained high numbers and function in the tumors, while IFN-alpha-stimulated cells declined in number and became exhausted. Consistent with this, IFN-alpha-stimulated cells expressed significantly higher levels of the PD-1 inhibitory receptor than did IL-12-stimulated cells. The efficacy of IFN-alpha-stimulated CTL in adoptive immunotherapy was strongly increased, and became comparable to that of IL-12-stimulated cells when a blocking antibody specific for PD-L1, the ligand of PD-1, was administered. Thus, IL-12 and IFN-alpha differentially program CD8 T cells to re-express distinct levels of PD-1 when the cells re-encounter antigen, resulting in IL-12-stimulated cells being less susceptible to exhaustion in the face of sustained tumor antigen.
Characterization of human B cells and macrophages infiltrating ovarian cancers. (127.33)
Authors: Nunez-Cruz Selene , Walter Marc , Danet-Desnoyers Gwenn , Scholler Nathalie
Published: January 2023

Suitable tumor xenograft models represent a significant improvement to the understanding of ovarian cancer. However phenotype and function of tumor-associated lymphocytes and macrophages in different tumor sites has not been reported. We characterized the ovarian tumor microenvironment in NSG mice reconstituted with human immune system using cord blood-derived CD34+ cells. Mice were injected with OVcar5 human ovarian tumor cells orthotopically (io), subcutaneously (sc) or both (io/sc) 12-16 weeks post CD34-transplant. Our findings indicated that 4 weeks after implantation, all human tumors were infiltrated with B cells, T cells and macrophages, regardless of the tumor site. However, sc tumors contained proportionally more B cells and macrophages than io tumors. Macrophages infiltrating sc and io tumors exhibited an IL10high immunosuppresive phenotype. Io macrophages expressed higher levels of the Fc receptor of high affinity (FcgRI) and of low affinity (FcgRII) and mannose receptor compared to macrophages infiltrating sc or io/sc tumors. Finally, io tumors were infiltrated by more B cells than all other tumors. B cells from tumor-bearing mice presented an altered phenotype compared to non tumor-bearing animals, characterized by the upregulation of plasma and activation markers and IgM and IgG production. Our results suggested that the location of the tumor implantation impacts the phenotype and function of human tumor infiltrate and may influence the tumor development.
Tumor-induced myeloid-derived suppressor cell function is independent of IFNγ and IL-4Rα (127.37)
Authors: Sinha Pratima , Parker Katherine , Ostrand-Rosenberg Suzanne
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and experimental animals where they exert a profound immune suppression and are a significant obstacle to immunotherapy. IFNγ and IL-4Rα have been implicated as essential molecules for MDSC development and immunosuppressive function. If IFNγ and IL-4Rα are critical regulators of MDSC, then they are potential targets for preventing MDSC accumulation or inhibiting MDSC function. Because data supporting a role for IFNγ and IL-4Rα are not definitive, we have examined tumor-induced MDSC from IFNγ-deficient, IFNγR-deficient, and IL-4Rα-deficient mice. Although MDSC express functional IFNγR and IL-4Rα and signal via the STAT1 and STAT6 pathways, respectively, neither IFNγ nor IL-4Rα impacts the phenotype, accumulation, or T cell suppressive potency of MDSC, although IFNγ and IL-4Rα modestly alter macrophage -induced MDSC production of IL-10. Therefore, neither IFNγ nor IL-4Rα is a key regulator of MDSC and targeting these molecules is unlikely to significantly alter MDSC function.
Resveratrol abrogates tumor-evoked Bregs conversion by targeting PPAR-alpha pathway (127.41)
Authors: Lee-Chang Catalina , Wejksza Katarzyna , Bodogai Monica , Malchinkhuu Enkhol , Ghosh Paritosh , Martin-Montalvo Sanchez Alejandro , de Cabo Rafael , Biragyn Arya
Published: January 2023

We recently reported that breast cancer cells induce the generation of a regulatory B-cell subset (tumor-evoked Bregs, tBregs) from naïve B cells to promote cancer escape and lung metastasis. One of the mechanisms involved is the ability of tBregs to promote Treg expansion. Thus, aiming to understand this process we screened several compounds that could block the generation of tBregs. We found that resveratrol (RSV), a natural polyphenol with chemopreventice properties, specifically blocked the ability of cancer cells to convert B cells into tBregs. This blockage results in the inability of tBregs to suppress T-cell proliferation and promote tumor growth and lung metastasis. The molecular mechanism of this process is that RSV targeted and inactivated PPAR-alpha, a nuclear receptor implicated in several processes such as lipid metabolism, proliferation or differentiation. Thus, our data show the importance of PPAR-alpha in tBreg generation, and one mechanism of the anti-tumor properties of RSV is the inactivation of this pathway in tBregs. This work was entirely supported by the Intramural Research Program of the National Institutes of Health.
Regulation of B cell development and immune tolerance by the MiTF family of transcription factors (159.12)
Authors: Lopez Ocasio Maria , Huan Chongmin , Gottesman Susan , Roman Christopher
Published: January 2023

B cell activation is tightly regulated by transcription factors (TFs) that control gene expression programs determining plasma and memory cell development. Dysregulation of these programs can lead to autoantibody production. MiTF, a member of the MiT family of transcription factors that includes TFEC and the CD40L regulators TFE3 and TFEB, is important in restraining plasma cell development by repressing IRF4. This was demonstrated in MiTFmi/mi bone marrow chimeras being necessary because the mi allele is a dominant negative mutation that causes osteopetrosis due to defective MiTF-dependent osteoclast development. However, because the MiTFmi protein could inhibit other MiTF family members and affect other hematopoietic cells, we are developing B cell specific MiT family inactivation models that obviate bone marrow transplantation. One, a single transgene model, exploits B cell-specific expression of an artificial trans-dominant negative protein (TDN) that forms inactive heterodimers with all MiT members. In preliminary data, we observed several defects in these mice, including splenomegaly, elevated numbers of spontaneous Germinal Centers and plasma cells, and perturbations in the distribution of B cell subsets not observed in the transplantation model. These data show a B cell subset specific dependency on the MiT family and the possible contribution of other MiT family members.
Significant association of specific DRB1 & DQB1 sequence feature variant types with systemic sclerosis autoantibodies types and racial groups (159.20)
Authors: Scheuermann Richard , Marthandan Nishanth , Guidry Paula , Thomson Glenys , Arnett Frank , Karp David
Published: January 2023

The sequence feature variant type (SFVT) approach to genetic association analysis has previously identified parts of HLA proteins that are associated with development of a variety of autoimmune diseases. Using the SFVT approach, we analyzed DRB1 and DQB1 typing data in a cohort of patients with Systemic Sclerosis (SSc). While no traditional class II alleles were significant across three racial groups (African American, Caucasian and Hispanic), 23 related DRB1 SFVTs were found in common. We also found that while more than 80% of the same sequence features (SFs) were significantly associated with the presence of either anti-centromere or anti-topoisomerase antibodies for each of the loci, only 2 (3%) and 5 (8%) of the risk-associated variant types (VTs) for DRB1 and DQB1, respectively, were shared, indicating that the same SFs but different VTs are associated with the presence of the different autoantibodies. Intriguingly, several protective anti-topoisomerase VTs corresponded to the risky anti-centromere VTs. We found that SFVTs that differ by only a few amino acids can give rise to different autoantibodies in SSc patients (e.g. association of the antigen binding motif 67L_70Q_71R with anti-centromere, versus association of 67F_70D_71R with anti-topoisomerase). Tools in ImmPort (www.immport.org) for the conversion of four-digit HLA typing data into an SFVT data matrix and the calculation of chi-squared association statistics were used for the analysis.
Localized and peripheral autoantibodies: evidence of humoral autoimmunity in patients with inclusion body myositis (159.21)
Authors: Ray Arundhati , Willis Simon , Bradshaw Elizabeth , Goldstein Jonathan , Lundberg Ingrid , Nowak Richard , Ploegh Hidde , O'Connor Kevin
Published: January 2023

Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The disease is viewed to be T cell mediated. However, emerging data suggests that B cells may play a role in IBM. To further the understanding of IBM immunopathology and explore the role of the humoral autoimmune response in IBM, we searched for autoantibodies both in the periphery and at the site of tissue damage. Peripheral autoantibodies that recognized muscle-derived cells were demonstrated in IBM serum using both immunohistochemistry and flowcytometry. To focus on the site of tissue injury, we examined the immunoglobulin-secreting plasma cells that infiltrate IBM muscle. A series of single, tissue-associated plasma cells were isolated from IBM muscle. Recombinant immunoglobulins (rIgG) produced from these cells were used as molecular tools to search for autoantigens. Flow cytometry and immunoblotting revealed that these tissue-derived rIgGs recognized human muscle tissue-associated antigens. To identify the target of the rIgGs, a multi-step protein separation scheme and mass spectrometry were employed. Desmin, a major intermediate filament protein expressed in muscle, was identified as a candidate autoantigen for one IBM rIgG. Specificity to desmin was confirmed with a solid-phase assay. Collectively, these data indicate that IBM autoimmunity includes a humoral component.
Investigating the role of MZB and B1 Cells in Sjögren’s Disease (159.22)
Authors: Shen Long , Xuan Jingxiu , Suresh Lakshmanan , Ambrus, Jr. Julian
Published: January 2023

We have previously demonstrated that IL14a transgenic mice (IL14aTG) reproduce features of Sjogren’s disease (SD) seen in patients in the same relative time frame, including autoantibodies, early destruction of the salivary and lachrymal glands, infiltration of the salivary and lachrymal glands with lymphocytes, lymphocytic pneumonia, mild renal disease and eventually lymphoma (J.Immunol. 177: 5676, 2006 and Clin. Immunol. 130:304, 2009). We have more recently demonstrated that lymphotoxin is an important early mediator of salivary gland injury in IL14aTG mice and patients with SD (J.Immunol. 185: 6355, 2010). In our current studies we investigate the role of MZB and B1 cells in SD in IL14aTG mice. We demonstrate that IL14aTG mice lacking MZB cells (IL14aTG.CD19Cre.RBPj-/-) do not develop any features of SD up to the ages of 18 months. However, IL14aTG mice lacking Bruton’s tyrosine kinase (btk; IL14aTG.btk-/-), and hence B1 cells, develop more severe and accelerated SD with B cell lymphomas by 10 months of age, about 3 months earlier than IL-14a TG mice. Our hypothesis is that MZB and MZB precursors (MZBP) trafficking in the salivary and lachrymal glands are critical for inducing SD. Mice lacking B1 cells via the deletion of Bruton’s tyrosine kinase (btk) have accelerated SD because of increased numbers of MZB and MZBP along with decreased numbers of regulatory B cells. Current studies are evaluating these hypotheses.
A unifying principle for autoantigenicity: Transforming self-molecules into autoantigens by association with dermatan sulfate polysaccharide (159.25)
Authors: Wang Julia , Roehrl Michael
Published: January 2023

Autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus are chronic and disabling diseases that affect hundreds of millions of people worldwide. Despite great advances in biomedical research, etiology and pathogenesis of systemic autoimmune diseases have remained mysterious. In particular, the question of why and how a small, seemingly disparate subset of self-molecules become autoantigenic holds a key to understanding the basic principles of autoimmunity. Based on our recent data, we propose a unifying principle for autoantigenicity. We discovered that dermatan sulfate (DS) is an important functional link between cell turnover, autoantigens (autoAgs), autoAbs, autoreactive B-1a cells, and autoimmunity. By selectively interacting with autoAgs released from apoptotic/dead cells, DS can tether many autoAg molecules along its polymeric chain and convert singular self-molecules into multivalent polymer-like antigens, thus changing the “self”-nature of this molecule. Polymeric DS-autoAg complexes are potent stimulators of autoreactive B-1a cells that secrete autoAbs and present autoAgs to autoreactive T cells. This principle provides a mechanistic explanation for how only certain self-molecules derived from dead cells become autoantigenic. This principle also explains how microbial pathogens may convert self-molecules to autoantigens and provides clues to how environmental factors may contribute to autoimmunity and trigger autoimmune diseases.
The accumulated VH replacement products contribute to the generation of auto/polyreactive antibodies in systemic lupus erythematosus (P1721) (159.3)
Authors: Li Song , Yu Yangsheng , Fan Run , Liao Hongyan , Wang Zhixin , Yue Yinshi , Huang Lin , Guo Sara , Hearth-Holmes Michelene , Cannella Amy , Chatham W. Winn , Kimberly Robert , Klassen Lynell , O’Dell James , Carter Robert , Su Kaihong , Zhang Zhixin
Published: January 2023

Systemic lupus erythematosus (SLE) is characterized by the over-production of high affinity autoantibodies. However, it is not clear how these autoantibodies are generated and selected. VH replacement is originally considered as a receptor editing process to change unwanted non-functional immunoglobulin heavy chain (IgH) genes or IgH genes encoding self reactive BCRs. Our sequence analysis shows that VH replacement products are highly enriched in IgH genes derived from different autoimmune diseases, including SLE. In this study, we performed single cell RT-PCR to study the functional antibody repertoires in active SLE patients. Analysis of 180 recombinant antibodies derived from SLE patients showed that 19.0% of the antibodies derived from naïve B cells and 56.9% of the antibodies derived from plasmablasts are autoreactive. In contrast, only 7.7% and 17.4% of the antibodies derived from naïve B cells and plasmablast B cells are autoreactive in healthy donors. The VH replacement products are significantly increased in IgH genes obtained from SLE patients (20%) compared to that in healthy controls (10%). Importantly, the accumulated VH replacement products in SLE patients have long CDR3 regions enriched with positively charged amino acids; 74% of them directly encode anti-nuclear antigen or polyreactive antibodies. Based on these results, we conclude that the accumulated VH replacement products in SLE patients contribute to the generation of auto/polyreactive antibodies in SLE.
Mechanisms for non-antigen specific rheumatoid factor B cell activation in the NZM2410 mouse model of lupus (159.9)
Authors: Sang Allison , Morel Laurence
Published: January 2023

Systemic lupus erythematosus is characterized by the presence of autoantibodies, including rheumatoid factor (RF), due to a breakdown in B cell tolerance. In the AM14 model, the AM14 transgenic heavy chain combined to Vκ8 produces a RF specific for IgG2aa. Contrary to the MRL/lpr lupus model, RF B cell activation required the presence of chromatin-IgG immune complexes (ICs), but could occur in the absence of the IgG2aa autoantigen in the B6.NZM2410.Sle1.Sle2.Sle3 (TC) strain, which contains the three major NZM2410-derived lupus susceptibility loci (Sle1, Sle2, Sle3). Treatment of young TC.AM14 and control B6.AM14 mice with a TLR7/9 antagonist did not decrease RF B cell activation after immunization with antigen non-specific anti-chromatin ICs. Splenic DCs from mice injected with anti-chromatin hybridomas showed elevated expression of IL-6 and IFN-γ mRNA in TC mice in comparison to B6 controls. B cell co-cultures with TC-derived BMDCs induced greater B cell proliferation than B6-derived BMDCs. Finally, B cell activation of aged AM14 mice expressing Sle1 or Sle2 showed that while Sle1 promoted antigen specific activation, Sle2 induced B-cell activation is polyclonal in nature. Therefore, our results show that while cognate-antigen independent RF B cell activation is not affected by inhibition of TLR7/9 signaling, it is enhanced by DC-derived cytokines and the expression of the Sle loci.
A role for immune deficiency in the mass mortality of the Caribbean sea urchin Diadema antillarum-a?(160.10)
Authors: Beck Gregory , Miller Robert , Ebersole John
Published: January 2023

Immune responses and diseases of marine invertebrates in natura are not well understood, and studies linking this knowledge to ecological events are rare. An epidemic nearly eliminated the long spined sea urchin Diadema antillarum-a from the Caribbean in 1983, with limited recovery since. Loss of this keystone grazer is a major factor in the ensuing shift from living coral to algae-covered rock on Caribbean reefs. Our studies of immune responses of four Caribbean sea urchin species suggest that a deficiency in the Diadema immune system may have played a role in the mass mortality. Using coelomocytes, we tested humoral responses of the urchins with classic stimulators in several assays. All urchin coelomocytes responded to stimulators with one statistically significant exception: Diadema did not respond as vigorously to lipopolysaccharide. This suggests a defect in immune response that is specific to Diadema and independent of stressors associated with particular environments. This inability of Diadema to respond to lipopolysaccharide provides the first evidence of a deficient immune response in a natural population of a marine invertebrate, and may help explain why Caribbean Diadema antillarum-a was vulnerable to an epidemic. Recovery of Diadema allows us to addresses questions on how recovery may be influenced by immunological processes. Further investigation of phylogenetic and geographic patterns in immunity of marine organisms may help us predict future epidemics.
Immunogenicity evaluation of in silico identified Mycobacterium avium subsp paratuberculosis recombinant proteins that were upregulated under stress conditions (160.3)
Authors: Gurung Ratna , Purdie Auriol , Begg Douglas , Whittington Richard
Published: January 2023

Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne’s disease (JD) in ruminants. It is known to enter a dormant phase outside the host typically on soil and survive inside the host macrophage. In vitro experiments have reported differential regulation of certain MAP proteins when exposed to stressors similar to dormancy. It is believed that in vivo regulation of dormancy genes and associated proteins by MAP may play an important role in evading the host defence mechanisms and the host may also mount an immune response against these dormancy related proteins. Evaluation of such proteins may provide insight to host-pathogen interaction during the course of MAP infection and may be of diagnostic interest. A group of dormancy genes upregulated under stress conditions were examined using in silico analysis to identify B and T-cell epitopes. Five potential candidate genes based on epitope prediction results were selected and cloned. Recombinant proteins were produced, purified and evaluated for their immunogenicity using a panel of sera from sheep with a spectrum of JD and sheep free of MAP infection by detection of serum antibodies. Individually, the five proteins were found to have the ability to partially discriminate between sera from sheep unexposed and exposed to MAP infections. This study showed that the MAP proteins upregulated under in vitro stress conditions were recognised by host immune system.
The pig heavy and light chain immunoglobulin repertoires provide insights into the adaptive immune response against porcine reproductive and respiratory syndrome virus (PRRSV). (160.4)
Authors: Schwartz John , Murtaugh Michael , Abrahante Juan
Published: January 2023

Antibody responses are critical to effective immunity to viral infections. Thus, extensive efforts have been directed to characterize antibody responses, including neutralizing antibodies, to PRRSV infection, in the expectation of elucidating key insights into protective and cross-protective immunity. Despite these efforts, the role of antibody responses in PRRSV immune protection remains poorly understood due, in part, to the lack of specific information about porcine immunoglobulin structures. To address this limitation, we characterized the expressed heavy and light chain immunoglobulin repertoires in healthy and PRRSV-infected pigs using amplicon-based 454 high-throughput sequencing. Bioinformatic analysis of approximately 450,000 heavy chain reads revealed differential heavy chain variable gene (IGHV) usage specific to PRRSV infection, including several exceptionally common immunoglobulin sequences representing clonally expanded B cell populations. The total richness of the heavy chain repertoire was approximately 3.5x105, equivalent to that reported in humans, suggesting that the swine antigen-binding repertoire is similarly complex, despite lack of diversity in the porcine IGHV framework regions. Initial analysis of over 13,000 light chain reads confirms that pigs use only two lambda J-C cassettes and are heavily reliant on a single kappa J gene. Further analysis of the light chain repertoire and its response to PRRSV infection is pending.
Cellular responses of the long-spined sea urchin (Diadema antillarum-a)(160.9)
Authors: DeFilippo John , Ebersole John , Beck Gregory
Published: January 2023

The long-spined sea urchin (Diadema antillarum-a) began dying on coral reefs near the Panama Canal in 1983, and over the next year they virtually disappeared from the Caribbean Sea. Present evidence suggests that a water-borne pathogen was responsible. Since their die-off Diadema densities remain low, and as they facilitate coral recruitment and persistence by controlling macroalgae, its loss has contributed to the transformation of living coral reefs in the Caribbean to barren algae-covered rock. Our preliminary on-site studies of the humoral responses of the urchins showed that Diadema coelomocytes did not respond to lipopolysaccharide as vigorously as did other common Caribbean sea urchins (Tripneustes ventricosa, Echinometra lucunter, and E. viridis). We then studied the cellular immune responses of coelomocytes and coelomic fluid of Diadema and those of the other urchins unaffected by die-off. Light microscopy revealed differences in coelomocyte populations of the predominant urchins of the Caribbean. We used flow cytometry to investigate differences in cellular composition and responses of coelomocytes in phagocytosis assays. It is hoped that this research will increase insight into one of the most devastating and ecologically important die-offs ever recorded, as well as provide the information required to understand whether weakened immunity was involved in the mass mortality of Diadema antillarum-a, and whether strengthening of immunity has occurred since then.
Loss of IFN-gamma 3’untranslated region AU-rich element affects B220+ B cell and plasmacytoid dendritic cell populations in novel murine lupus model (161.3)
Authors: Hodge Deborah , Berthet Cyril , Coppola Vincenzo , Shirota Hidekazu , Reynolds Della , Klinman Dennis , Young Howard
Published: January 2023

Interferon-gamma (IFN-g) is a key player in immunoregulation, inflammation and autoimmunity. We created a mouse with a162 nt deletion of an AU-rich element (ARE) region in the 3’UTR of the IFN-g gene. The ARE-deleted (ARE-Del) mice have chronic circulating serum IFN-g levels and develop a lupus-like disease characterized by DNA and nuclear antigen-specific autoantibodies and glomerulonephritis. B220+ B cells and plasmacytoid dendritic cells (pDCs) play a prominent role in systemic lupus erythematosus (SLE). We find alterations in both cell types. Greater B cell percentages are found in lymph nodes and thymus with a concomitant reduction in spleen. The B cells have an activated phenotype with increased CD69 and/or CD86 expression. The ARE-Del thymic B cell population is partially composed of B1a and B1b B cells and is correlated with increased thymic mRNA expression of the B cell chemoattractant, CXCL13. Splenic B cells down-regulate CD23 and CD21 with no change in surface markers CD19, B220, IgM, and IgD. The percentage of CD11c+, pDCA1+, B220+ Siglec H+ pDCs is increased in bone marrow and spleens from AU-deleted mice. Current studies are underway to further characterize the pDC population and to investigate expression of bone marrow-specific factors important for pDC development. Overall, these data demonstrate that low chronic IFN-g levels are sufficient to alter the phenotype and potentially the development of cells important for SLE disease onset and progression.
Chronic alcohol consumption increases and activates iNKT cells in B16BL6 melanoma-bearing mice (162.21)
Authors: Zhang Hui , Zhu Zhaohui , Meadows Gary
Published: January 2023

Drinking alcohol is a risk factor for cancer. High alcohol consumption is immunosuppressive, and relatively little is known about how it regulates antitumor immunity. Herein, we examined the effects of chronic high alcohol consumption on natural killer (NK) T cells since they play important roles in shaping antitumor immunity. We utilized a well-established mouse chronic alcohol consumption and B16BL6 melanoma transplantation model to study NKT cell regulation. We found that chronic alcohol consumption dramatically increases NK1.1+ invariant NKT (iNKT) cells in the blood of melanoma-bearing mice that were fed 20% (w/v) alcohol for 3-6 months before subcutaneous tumor inoculation. The percentage of NK1.1- iNKT cells decreased 2-4 fold in the alcohol-consuming mice compared to their water-drinking counterparts. Alcohol consumption increased the IL-4/IFN-γ ratio in iNKT cells activated in vitro with αGalCer compared to control. IFN-γ concentration in the blood 14 hours after in vivo activation of iNKT cells with αGalCer was 2-fold higher than in the blood of controls, and the expression of CD80 and CD86 on B cells and dendritic cells, and CD69 on CD4+ and CD8+ T cells was up regulated in alcohol-consuming mice compared to control. . These results indicate that chronic alcohol consumption facilitates the activation of iNKT cells in B16BL6 melanoma-bearing mice.
Aging-associated B7-DC+ B cells enhance anti-tumor immunity (162.24)
Authors: Shin Tahiro , Ludwig Sara , Zhang Bin , Curiel Tyler , Tomihara Kei
Published: January 2023

Because most cancer patients are aged and because immunological functions are altered during aging, it is important to account for aging-associated immunological alterations in the design of new cancer immunotherapies. We thus compared immune populations in young and aged mice and found that B7-DC+ (PD-L2/CD273) B cells, a minor population in young mice, were significantly increased in aged mice. Induction of both Th1 and Th17 cells was significantly augmented by B7-DC+ B cells from aged mice and this effect was blocked with anti-B7-DC antibodies in vitro and in vivo. Moreover, retardation of tumor growth in aged mice was largely B7-DC-dependent. Tumor growth in young mice was significantly inhibited by immunization with B7-DC+ B cells from aged mice due to increased induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs). These data indicate that B7-DC+ B cells could play an important role in aging-associated cancer immunopathology as well as other aging-associated-diseases, and further suggest that B7-DC+ B cells have potential for future cancer immunotherapy.
Multipotent Vδ2-negative γδT-cells after CMV-reactivation in allogeneic stem cell transplantation (162.36)
Authors: Kuball Jürgen , Scheper Wouter , van Dorp Suzanne , Kersting Sabina , Pietersma Floor , Hol Samantha , Marcu-Malina Victoria , Becke Sabine , Plachter Bodo , van Baarle Debbie
Published: January 2023

Human cytomegalovirus (CMV) infections and relapse of disease remain major problems after allogeneic stem cell transplantation (allo-SCT). Great promise as an alternative strategy, with a potential use for very broad patient population, arises from the non-MHC-restricted target-recognition of cancer cells and virally-infected cells by γδT-cells. Therefore, we investigated the role of γδT-cells after allo-SCT and CMV infection. An increase in selectively Vδ1-positive γδT-cells was observed in CMV-reactivating patients from CMV-positive conventional donors. Even more important, γδT-cell expansions were also observed after CMV-negative cordblood transplantation. However, in contrast to expansions from CMV-positive donors, γδT-cells from cordblood donors did not only include Vδ1-positive but also Vδ3-positive cells. Expanded Vδ2-negative γδT-cells were not only able to lyse CMV-infected fibroblasts, but also to partially mature dendritic cells (DCs) and to kill primary leukemic blasts. CMV- and leukemia-reactivity were restricted to the same clonal population and separated from DC-maturation capacities of Vδ2-negative T-cells. Vδ2-negative γδTCRs selectively mediated anti-leukemia-reactivity. This makes CMV-reactive γδT-cells e.g. from cordblood donors as well as individual Vδ2-negative γδTCRs interesting candidates for adoptive immunotherapy strategies
The role of regulatory B cells during the progression of human hepatocellular carcinoma (162.39)
Authors: Shao Yan , Li Chang Xian , Ma Yuen Yuen , Liu Xiao bing , Chu Chi Yuen , Ling Chang Chun , Yeung Wai Ho , Lee Kin Wah , Fan Sheung Tat , Lo Chung Mau , Man Kwan
Published: January 2023

Y SHAO1, CX LI1, YY MA1, XB LIU1, CY CHU2, CC LING1, WH Yeung1, KW Lee3, ST FAN1,2, CM LO1,2, K MAN1,2 1.Department of Surgery 2.Centre for Cancer Research 3. Department of Pathology, The University of Hong Kong, Hong Kong Human regulatory B cells (hBregs) have been shown to play an important role in autoimmune diseases, but their function during human cancer progression remains elusive. In this study, we aim to unveil the underlying mechanism of hBregs regulating human hepatocellular carcinoma (HCC) growth. B cells in HCC liver tumor were examined by immunohistochemistry. We found a tendency that the amount of B cells increased with HCC progression (p=0.001), and it’s in the marginal region of HCC increased (P<0.001) more dramatically. In vitro, human HCC cell line MHCC-97L cells were cocultured with hBregs. hBregs promoted HCC cells proliferation (P=0.011) and migration (p=0.006) and decreased HCC cells apoptosis (p=0.078). HCC cells also promoted hBregs proliferation and migration (p=0.003). hBregs could interact with HCC cells directly through CD40-CD154 signaling. Tail vein injection of hBreg cells into SCID mice increased the size of HCC xenograft tumor (4 weeks, p=0.0221). In vivo imagining showed that hBreg cells could migrate into tumor. Our findings indicate that hBregs could promote HCC progression.
Role of resveratrol and nitric oxide in the modulation of murine prostate cancer cells (162.9)
Authors: Mishra Manoj , Eroglu Erdal , Singh Udai , Nyairo Elijah , Singh Shree
Published: January 2023

Resveratrol (RES), a polyphenolic compound found in the skin of red fruits, exhibits anti-inflammatory, anti-oxidative, anti-cancer, and anti-proliferative characteristics. RES is known to affect nitric oxide (NO) production. NO is produced by nitric oxide synthase, a regulator of innate and adaptive immunity and displays cytostatic and cytotoxic activity against pathogens and cancer cells. Prior reports suggest that RES and NO mediate the killing of cancer cells in vivo as well as in vitro. In this study, we hypothesized that the RES and NO, either alone or synergistically, modulates the molecular mechanisms in murine prostate cancer (PCa) cells. This hypothesis has been tested on mouse PCa cell lines, derived from transgenic adenocarcinoma of mouse prostate (TRAMP), in C57/B6 mice. Interestingly, TRAMP-C1 and TRAMP-C2 are tumorigenic while TRAMP-C3 is non-tumorigenic. TRAMP cells were treated with different concentrations of RES/NO at different time points and analyzed for cell viability/proliferation, cytokines and growth factors production. The results show that the RES/NO-induced molecular mechanisms in TRAMP-C1 and C2 are significantly different while compared to TRAMP-C3 suggesting that TRAMP-C3 cells may use distinct pathways that regulate “eat me” signal in comparison to TRAMP-C1 and C2. This data also demonstrates that RES/NO mode of action is very distinct and exclusive for each TRAMP cell lines in the modulation of molecular pathways.
Foxp4 is dispensable for T cell development, but required for Interferon gamma recall responses during chronic Toxoplasmosis (163.12)
Authors: Wiehagen Karla , Corbo-Rodgers Evann , Li Shanru , Staub Elizabeth , Hunter Christopher , Morrisey Edward , Maltzman Jonathan
Published: January 2023

Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are fundamental for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, another member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4+ and CD8+ T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore Foxp3+ T regulatory cells develop normally in Foxp4 deficient animals, and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii infection, IFNγ production during an antigen specific recall response is reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for T cell cytokine recall responses to antigen in chronically infected mice.
PPARγ activation drives Th17 cells into a Treg phenotype (163.7)
Authors: Carbo Adria , Hontecillas Raquel , Hoops Stefan , Kronsteiner-Dobramysl Barbara , Lu Pinyi , Wendelsdorf Katherine , Mei Yongguo , Eubank Stephen , Marathe Madhav , Bassaganya-Riera Josep
Published: January 2023

Th17 cells mediate inflammatory and effector responses during infectious and immune-mediated diseases. However, the mechanisms of action modulating the plasticity between Th17 and Treg are incompletely understood. To gain a better understanding of the differentiation process we have constructed a computational and mathematical model in COPASI that mimics CD4+ T cell differentiation and contemplates the plasticity between effector Th17 and Treg. Our computer simulations predicted that activation of peroxisome proliferator activated receptor gamma (PPARγ) induces a switch from already differentiated Th17 into Treg. Conducted experimental validation approaches support in silico experimentation. Naive CD4+ T cells obtained from spleens of T cell-specific PPARγ null, RORcGFP+/+ or wild-type donor mice, all in a C57BL6 background, were transferred to SCID and RAG2-/- recipients to induce CD4+ T cell-driven colitis. Our data demonstrate a significant decrease in the expression of FOXP3 and increase of IL-17A and RORγt in recipients of PPARγ null naïve CD4+ T cells when compared to recipients of naïve CD4+ T cells from wild-type mice. Furthermore, administration of PPARγ agonists to recipient mice with colitis decreased the percentages of GFP+ Th17 cells at the gut mucosa. In conclusion, PPARγ promotes differentiation of naïve CD4+ T cells into Treg, downregulates Th17 differentiation and favors phenotype switch from Th17 into Treg CD4+ T cells.
Signals through GITR program CD4+ T cells to induce interleukin-9 (163.9)
Authors: Kim Il-Kyu , Seok Jae-Won , Kim Byung-Seok , Lee Ka-Eun , Choi Gye-Young , Kang Chang-Yuil
Published: January 2023

Glucocorticoid-induced TNF receptor family-related protein (GITR) is expressed on various immune cells and well known as one of the co-stimulatory molecules to promote T helper (Th) type 2 responses. In this study, we show that an agonistic anti-GITR mAb (clone DTA-1) induced IL-9 production on the committing Th2 and regulatory T (Treg) cells, as well as enhanced IL-9 production from naive CD4+ T cells under Th9 skewing condition. Whereas, DTA-1 did not induce IFN-γ or IL-17A, lineage-specific cytokines of Th1 or Th17, respectively and was resistant to produce IL-9 from naive CD4 T cells under Th1 or Th17 polarizing conditions. Furthermore, GITR stimulation promoted reprogramming of regulatory T cells to produce IL-9+Foxp3+ Treg and effector Th9 cells. We found transcription factor Interferon-regulatory factor4 (IRF4) was up-regulated by GITR ligation at the early stage of Th9 differentiation and contributed to enhance IL-9 production. Also, when GITR signaling was activated, p65, activated by NFκB signaling pathways, directly interacted with Il9 locus including promoter region at the later stage of Th9 differentiation. Consistent with in vitro study, in vivo administration of DTA-1 enhanced IL-9 at the transcript level and the roles of IL-9 induced by GITR ligation in vivo are remained to be investigated for further studies.
The negative impact of helminth infection on CD8+ T cell immunity (164.11)
Authors: Marple Andrew , Wu Wenhui , Grotenbreg Gijsbert , Gause William , Yap George
Published: January 2023

Upon infection the protozoan parasite Toxoplasma gondii induces a strongly polarized Th1 cytokine response, necessary for effector differentiation of CD8 lymphocytes. Conversely, pre-infection with an intestinal nematode, such as Heligmosomoides polygyrus has been shown to attenuate immune responses to intracellular pathogens and inhibit the development of Th1 responses. Previous studies have shown that mice co-infected with H. polygyrus and T. gondii exhibit suppressed CD8+ T cell responses against T. gondii, however the precise mechanism by which co-infection impinges upon the priming and differentiation of antigen-specific T cells remains to be determined. Using a novel Kb-peptide tetramer-binding assay for Tgd057-reactive CD8+ T cells, we show that co-infected mice develop a diminished antigen-specific response to T. gondii. Co-infected mice develop fewer antigen-specific CD8+ T cells, a decreased fraction of terminally differentiated effector cells, and reduced effector IFN-γ production. Furthermore, terminally differentiated antigen-specific effector T cells (CD62Llo, KLRG1+) from co-infected mice retain an intrinsic defect in cytokine production. Helminth infection blocked IL-12 responses of CD8α+ dendritic cells to T. gondii, indicating that rather than inhibiting CD8+ T cell priming itself, the principal effect of helminth infection is to attenuate the differentiation of effector lymphocytes by inhibiting critical innate cytokine responses.
Vitamin D regulation of Th2 Immune responses to Aspergillus fumigatus in mice and humans: role of OX40L (164.19)
Authors: Nguyen Nikki , Chen Kong , Alcorn John , Kolls Jay
Published: January 2023

4-15% of cystic fibrosis (CF) patients develop Allergic Bronchopulmonary Aspergillosis (ABPA) characterized by heightened Th2 responses to Aspergillus fumigatus (Af). Colonization rates with Af in CF can approach 40-50% suggesting immune tolerance may prevent ABPA. We have previously shown that CD11c dendritic cells (DCs), OX40 Ligand, Vitamin D play a role in sensitization versus tolerance to Af in CF patients. We have set up an animal model to further explore the roles of CD11c DCs, OX40L, and Vitamin D in response to Af. Vitamin D deficient mice (by nutritional diet) and Vitamin D normal mice where sensitized and challenged with Af. In addition, mice were given anti-OX40L or PBS control 4 hours prior to Af challenge. Lung, lymph nodes (LN), and bronchoalveolar lavage (BAL) fluid where analyzed for Th2 cytokine production. Vitamin D deficient mice have significantly increased Th2 cytokine response to Af in lung, BAL, and LN. In addition, OX40L mRNA levels are increased in Vitamin D deficient mice compared to Vitamin D normal mice. When given anti-OX40L prior to Af challenge, Th2 response in both Vitamin D deficient and Vitamin D normal mice are attenuated confirming the critical role of OX40L driven Th2 response in Af sensitization. This suggests a critical role of OX40L in Vitamin D mediated Th2 suppression. These results support therapeutic trial of Vitamin D and/or anti-OX40L to treat ABPA in CF and suggest Vitamin D plays a role in immune tolerance to Af.