Abstract Introduction We recently generated T cell receptor (TCR) transgenic (Tg) mice specific to cardiac myosin heavy chain-α (Myhc-α 334—352) on both myocarditis-resistant (C57BL/6) and susceptible (A/J) genetic backgrounds. While functional antigen-specific TCRs were expressed in both CD4+ and CD8+ T cells, the T cells from naïve Tg C57BL/6 mice did not respond to Myhc-α 334-352. In contrast, both cell types from A/J mice spontaneously responded to the antigen, suggesting their underlying molecular mechanisms might differ. Methods Using flow cytometrically sorted cells from naïve C57BL/6 and A/J Tg mice, we performed bulk RNA sequencing on CD4+ and CD8+ T cell subsets. Differentially expressed genes (DEGs), gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways, Gene set enrichment analysis (GSEA) of GO and KEGG, and transcription factor (TF) network analyses were performed to identify pathways and regulators of immune responses. Results Principal component analysis distinguished CD4+ from CD8+ T cells and revealed distinct transcriptomic profiles between C57BL/6 and A/J mice. KEGG enrichment analysis indicated downregulation of pathways related to viral myocarditis and autoimmune conditions in C57BL/6 compared to A/J mice. GSEA of GO revealed that negative regulation of heart contraction and positive regulation of cardiac muscle hypertrophy were negatively enriched in CD4+ T cells from C57BL/6 mice. TF network analysis revealed unique TFs that have a role in autoimmunity, T cell activation, tolerance, and regulatory T cells. Conclusion Our data provide new insights into the transcriptomic profiles that may contribute to the genetic resistance mechanisms for developing cardiac autoimmunity. Funding Source 5R21AI142281-02 Topic Categories Basic Autoimmunity (BA)

Background: We recently generated T cell receptor (TCR) transgenic (Tg) mice specific to cardiac myosin heavy chain-α (Myhc-α 334–352) on both myocarditis-resistant (C57BL/6) and susceptible (A/J) genetic backgrounds. We noted that the antigen-specific TCRs were expressed in CD4+ and CD8+ T cells in both strains, but their responses differed. While the T cells from naïve Tg C57BL/6 mice do not respond to Myhc-α 334–352, whereas those from A/J mice spontaneously respond to the antigen, suggesting their underlying molecular mechanisms might differ. Methods: To investigate the mechanisms of differences in the antigen-responsiveness between the Tg C57BL/6 and A/J mice, we performed bulk RNA sequencing on CD4⁺ and CD8⁺ T cells sorted by flow cytometry. Differentially expressed genes, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, gene set enrichment analysis (GSEA) of GO and KEGG, and transcription factor (TF) network analyses were performed to identify pathways and regulators of immune responses. Results: First, the principal component analysis of the transcriptomic profiles distinguished CD4+ from CD8+ T cells, which also differed between the two strains. Second, the differentially expressed cytokine and cytotoxicity genes revealed similar patterns between CD4+ and CD8+ T cells. Importantly, KEGG enrichment analysis revealed downregulated pathways in both CD4+ and CD8+ T cells that are associated with viral myocarditis, and various autoimmune conditions in C57BL/6 as compared to A/J mice. Similarly, the GSEA of GO revealed negative regulation of heart contraction and positive regulation of cardiac muscle hypertrophy processes were negatively enriched in CD4+ T cells of C57BL/6 mice. Finally, by generating the transcription factor (TF) networks, 22 TFs were found common to both CD4+ and CD8+ T cells, whereas eight TFs were unique to CD4+ or CD8+ T cells that have a role in T cell activation, tolerance, and T regulatory cells. Conclusions: Our data provide new insights into the transcriptomic profiles that may contribute to the genetic resistance mechanisms for developing cardiac autoimmunity.

Haploidentical hematopoietic cell transplantation (haplo-HCT) is associated with an increased risk of allograft rejection. Here, we employed a major histocompatibility complex (MHC)-mismatched allogeneic HCT (allo-HCT) murine model to better understand the role of Gal-1 in immune tolerance. Transplanted mice were classified into either rejected or engrafted based on donor chimerism levels. We noted significantly higher frequencies of CD4+ T cells, CD8+ T cells, natural killer cells, IFN-γ and TNF-α producing CD4+ T cells, and IFN-γ producing dendritic cells and macrophages in rejected mice. Conversely, we found significantly increased frequencies of regulatory T cells (Tregs), predominantly Helios+, IL-10-producing CD4+ T cells, type 1 regulatory (Tr1) cells, and the proportion of Tr1+Gal-1+ cells in engrafted mice. Further, Gal-1 specific blockade in Tregs reduced suppression of effector T cells in engrafted mice. Lastly, effector T cells from engrafted mice were more prone to undergo apoptosis. Collectively, we have shown that Gal-1 may favor HSC engraftment in an MHC-mismatched murine model. Our results demonstrate that Gal-1-expressing Tregs, especially at earlier time points post-transplant, are associated with inducing immune tolerance and stable mixed chimerism after HCT.

Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative option for patients with certain non-malignant hematological diseases. High-dose post-transplant cyclophosphamide (PT-Cy) (200 mg/kg) and sirolimus (3 mg/kg), (HiC) synergistically induce stable mixed chimerism. Further, sirolimus and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin (CTLA4-Ig), also known as Abatacept (Aba), promote immune tolerance and allograft survival. Here, in a major histocompatibility complex (MHC)-mismatched allo-HCT murine model, we combined Aba and/or T-cell depleting anti-Thy1.2 (Thy) with a lower dose of PT-Cy (50 mg/kg) and Sirolimus (3 mg/kg), (LoC). While mice in the LoC group showed graft rejection, the addition of Thy to LoC induced similar donor chimerism levels when compared to the HiC group. However, the addition of Aba to LoC led to graft acceptance only in younger mice. When Thy was added to the LoC+Aba setting, graft acceptance was restored in both age groups. Engrafted groups displayed significantly reduced frequencies of recipient-specific interferon-γ-producing T cells as well as an increased frequency in regulatory T cells (Tregs) except in the LoC+Aba group. Splenocytes from engrafted mice showed no proliferation upon restimulation with Balb/c stimulators. Collectively, in combination with Aba or Thy, LoC may be considered to reduce graft rejection in patients who undergo allo-HCT.

Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans.

Group B Coxsackieviruses (CVB) contains six serotypes, namely CVB1 through CVB6, affecting various organs. But, no vaccines are currently available to prevent these infections. We recently derived a live attenuated vaccine virus termed mutant (Mt) 10 encoding a single amino acid substitution H790A within the viral protein 1 (VP1) of the CVB3 viral canyon region. This virus could prevent myocarditis and pancreatitis caused by both homologous (CVB3) and heterologous (CVB4) serotypes in A/J mice. Mechanistically, we noted that the vaccine virus induces cross-reactive neutralizing antibodies that skewed towards mainly IgG isotypes. Similarly, by using major histocompatibility complex class II dextramers and tetramers for various VP1 epitopes, we demonstrated that the vaccine recipients develop antigen-specific T cell responses producing preferentially interferon-γ responses. Furthermore, our preliminary studies revealed that the vaccine virus could prevent the progression of diabetes-induced by CVB4 in the non-obese diabetic mice. Together, our data suggest that the Mt10 vaccine can prevent infections caused by multiple CVB serotypes, paving the way for developing monovalent CVB vaccines to prevent heart and pancreatic diseases of enteroviral origin. This work was supported by The Transformational Grant from the American Heart Association (18TPA34170206)

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.

BACKGROUND:Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition.METHODS:Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment.RESULTS:We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit.CONCLUSION:Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

Abstract T cells play a central role in cancer immunosurveillance and current cancer immunotherapies, including adoptive cell transfer (ACT), T cell receptor or chimeric antigen receptor (CAR) T cell therapies and immune checkpoint blockade. Understanding the factors regulating T cell function is hence critical for improving the success of these immunotherapies. It has been recognized that metabolism can greatly affect different aspects of T cell function, including differentiation, cytokine production, longevity and exhaustion. Here we integrate genome-scale metabolic modeling (GEM) with biological experiments to discover novel metabolic determinants of T cell function. Previously we developed the metabolic transformation algorithm (MTA), a GEM method that was successfully applied to identify driving factors and targets for different diseases. Here applying MTA to data on CAR-T cell gene expression and patient response to anti-CD19 CAR-T therapy, we predicted mitochondrial metabolite transport and specifically proton transport in mitochondrial uncoupling, as key determinants of CAR-T therapy response. Mitochondrial uncoupling is also important for the in vivo persistence of adoptively transferred tumor-infiltrating lymphocytes, as further confirmed by analyzing their gene expressions from a KRAS-targeting ACT dataset. Focusing on the mitochondrial uncoupling protein 2 (Ucp2), which is abundantly expressed in T cells, we experimentally validated that it is required for T cell longevity and anti-tumor function. Specifically, the loss of Ucp2 either via knock-out (KO) or treatment by genipin (a Ucp2 inhibitor) in mice T cells results in accelerated differentiation into “terminal effector cells”, as shown by increased levels of T cell cytotoxicity and exhaustion markers, and decreased levels of central memory and stemness markers. Adoptive transfer of Ucp2-KO Pmel-1 T cells to mice bearing B16 melanomas displayed poorer anti-tumor efficacy and worse survival than the transfer of Ucp2-wildtype T cells. We find that Ucp2 modulates oxidative stress and DNA damage by regulating the levels of mitochondrial superoxide. Reducing mitochondrial reactive oxygen species was sufficient to rescue the loss of Ucp2-mediated effector T cell differentiation, senescence, cytokine production and anti-tumor activity Ucp2-KO Pmel-1 T cells. Tumor-specific CD8+ T cells could be metabolically reprogrammed by Ucp2 overexpression, which improved T cell longevity and anti-tumor function in the Pmel-1/B16 ACT mice model. In sum, our study establishes a novel role of Ucp2 in regulating T cell longevity and anti-tumor activity by repressing increased ROS levels accompanying mitochondrial dysfunction in differentiated and exhausted cells, suggesting that manipulating Ucp2 levels in T cells can be exploited to enhance T cell-based cancer immunotherapies. Citation Format: Madhusudhanan Sukumar, Kuoyuan Cheng, Arunakumar Gangaplara, Yogin Patel, Suman K. Vodnala, Rafiqul Islam, Arash Eidizadeh, Carolyn Subramaniam, Ping Lee, Rigel Kishton, Amanda N. Henning, Michael J. Kruhlak, Zhiya Yu, Ethan M. Shevach, Toren Finkel, Eytan Ruppin, Nicholas P. Restifo. Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1527.

Abstract Haploidentical hematopoietic stem cell transplantation is one of the most widely available curative options for patients with sickle cell disease (SCD). Our previous non-myeloablative haploidentical peripheral blood stem cell transplantation (PBSCT) trial showed improved engraftment with post-transplant cyclophosphamide, but with a high incidence of allograft rejection. Here, our aim was to evaluate mechanisms of engraftment in SCD patients who underwent PBSCT. We analyzed and compared the following cells (Treg, Tr1, Th1, Th17, NK, Breg, pmnMDSCs, mMDSCs, eMDSCs, mDCs, and pDCs) in peripheral blood samples obtained from 20 transplanted patients at baseline (BSL), post-transplant day (PT-D) 30, 60, 100, and 180 using flow cytometry. Because we previously showed that 20% donor myeloid chimerism (DMC) is sufficient to reverse SCD, we classified patients at each time point as engrafted (DMC ≥20%) and rejected (DMC< 20%). Early MDSC frequencies were significantly elevated in engrafted patients (PT-D30). Patients were also evaluated based on engrafted/rejected at the end of study and mMDSC and Th17 cell frequencies were significantly elevated at PT-D30 and PT-D60 respectively in engrafted patients. All other cell populations were not statistically different. Tregs showed an elevated trend in engrafted patients and exhibited direct proportionality in patients with varying DMC levels. Our results reveal that eMDSC and mMDSC frequencies were elevated in engrafted patients and decreasing Tregs were associated with decreasing DMC. Taken together, eMDSCs, mMDSCs and Tregs may have a synergistic association in favoring the development of a tolerogenic immune milieu that promotes stable mixed chimerism after PBSCT in SCD patients.

Group B coxsackieviruses (CVBs) belonging to the genus, Enterovirus and contain six serotypes that induce various diseases, whose occurrence may involve the mediation of more than one serotype. We recently identified immunogenic epitopes within coxsackieviruses B3 (CVB3) viral protein 1 that induce anti-viral T cell responses in mouse models of CVB infections. In our investigations to determine the protective responses of the viral epitopes, we unexpectedly noted that animals immunized with complete Freund's adjuvant (CFA) alone and later challenged with CVB3 were completely protected against myocarditis. Similarly, the pancreatitis-inducing ability of CVB3 was remarkably reduced to only 10% in the CFA group as opposed to 73.3% in the control group that received no CFA. Additionally, no mortalities were noted in the CFA group, whereas 40% of control animals died during the course of 21 days post-infection with CVB3. Taken together, our data suggest that the adjuvant effects of CFA may be sufficient for protection against CVB infections. These observations may provide new insights into our understanding of the occurrence of viral infections.

Coxsackievirus group B (CVB) contains six serotypes that can affect various organs. Some of these organ-specific diseases such as myocarditis and pancreatitis can be caused by more than one serotype. Thus, development of immunological tools common to multiple serotypes is desired. This is especially critical for analyzing antigen-specific T cell responses at a single cell level. To this end, we made efforts to identify the immunogenic epitopes of CVB3 leading us to localize three T cell epitopes within the viral protein 1 (VP1) namely, VP1 681–700, VP1 721–740 and VP1 771–790. First, we confirmed their immunogenicity in the immunization settings. Second, we sought to verify the ability of VP1 epitopes to bind major histocompatibility complex (MHC) class II (IAk) molecules. Third, we created MHC class II (IAk) dextramers and tetramers and ascertained the T cell responses to be antigen-specific. Fourth, we analyzed the T cell responses in animals infected with CVB3 and noted the magnitude of antigen-specific T cell responses occurring in the order of VP1 721–740 and VP1 681–700 followed by VP1 771–790 as verified by proliferation assay and IAk tetramer staining. All epitopes induced interferon (IFN)-γ as a major cytokine. Finally, we investigated whether the VP1 tools generated for CVB3 can also be used to verify T cell responses in infections caused by other serotypes. To this end, we established the CVB4 infection model in A/J mice and found that the CVB4 infection led to the induction of IFN-γ-producing T cell responses primarily for VP1 721–740 and VP1 681–700. Thus, the VP1-specific tools, particularly IAk tetramers can be used to monitor anti-viral T cell responses in multiple CVB serotypes.

Abstract INTRODUCTION Despite multi-model therapies that include maximal surgical resection, radiation, chemotherapy, and tumor treating fields, the median survival of Glioblastoma (GBM) patients is around 15 months. WASP-family verprolin homologous protein 1 (WAVE1) is a downstream effector that receives signals from small GTPases to regulate the actin cytoskeleton. WAVE1’s interaction with arp2/3 modulates critical roles, such as cell motility and morphologic changes. Expression of WAVE1 has been implicated in leukemia, ovarian, and prostate cancer. In this study, we tested the role of WAVE1 in GBM tumor biology. METHODS Expression of WAVE1 in normal brain and GBM tumor specimens was assessed by immunohistochemistry (IHC). The relevance of targeting WAVE1 for GBM therapy was evaluated in vitro by western blot, proliferation assay, cell cycle analysis, apoptosis assay, migration assay, and neurosphere formation assay using scrambled and target specific WAVE1-siRNA in patient-derived primary GBM neurospheres (GBMNS). RESULTS IHC data shows that the expression of WAVE1 is higher in GBM tumor samples than in low-grade gliomas and normal brain tissues. WAVE1 expression is upregulated in GBMNS compared to normal human astrocytes. WAVE1 knockdown significantly decreased the proliferation, migration, and self-renewal of GBMNS without affecting its cell cycle progression. Furthermore, WAVE1 depletion did not show any alterations in the apoptotic cell population, indicating that WAVE1 knockdown has a cytostatic effect on GBMNS. CONCLUSION Expression of WAVE1 positively correlates with GBM, and its knockdown imparts antitumor efficacy through cytostasis.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is associated with increased risk of allograft rejection. Understanding the mechanisms of graft rejection and tolerance induction are critical for improving HSCT outcome. Recently, we performed a proteomic analysis using plasma samples from sickle cell disease (SCD) patients who underwent haplo-HSCT and found that galectin-1 (Gal-1) levels were significantly higher in engrafted patients. Here, we studied major histocompatibility complex (MHC)-mismatched allo-HSCT murine model to better understand the role of Gal-1 in immune tolerance. Transplanted mice were classified into either rejected (cyclophosphamide/sirolimus alone) or engrafted (cyclophosphamide and sirolimus) based on our previous findings. In rejected mice we noted significantly higher frequencies of CD4+T, CD8+T, NK cells, IFN-γ TNF-α producing CD4+T cells, and also IFN-γ producing DCs and macrophages. Importantly, in engrafted mice we found significantly increased frequencies of Tregs and IL-10 producing CD4+T and Tr1 cells, and the proportion of these cells expressing Gal-1 was significantly higher. Further, we detected a significant increase in Gal-1 levels in plasma of engrafted mice, and this corroborated with our data from engrafted patients. Here, we have shown for the first time that not only Gal-1 favors engraftment in SCD patients who underwent haplo-HSCT, but Gal-1 also contributes to HSC engraftment in our MHC-mismatched murine model. Taken together, our results demonstrate that Gal-1 expressing Tregs and Tr1 cells may play an essential role in inducing immune tolerance and stable mixed chimerism after HSCT.

Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971-990, and cardiac myosin heavy chain-alpha (Myhc) 334-352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21-40 dextramer(+) T cells were inconsistently detected, the beta 1-adrenergic receptor 181-200/211-230 or branched chain alpha-ketoacid dehydrogenase kinase 111-130 dextramer(+) cells were absent. Interestingly, SERCA2a 971-990, Myhc 334-352 and ANT 21-40 dextramer(+) cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971-990-reactive T cells generated in CVB3 infection could transfer disease to naive mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.

NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I-specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti-MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.

Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in T-regulatory (Treg) cells. However, the role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated mice with a conditional knock out of Eos in Treg cells (lkzf4fl/fl X Foxp3YFP−cre, Eos cKO). Deletion of Eos in Treg resulted in activation of CD4+Foxp3- and CD8+ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyletis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-γ/TNF-α production by CD8+ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7–8 months, respectively). Taken together, Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an as yet unknown role in the function of CD4+Foxp3- and CD8+ T cells and potentially in non-T cells.

Sickle cell disease (SCD) is an inherited blood disorder in which red blood cells are sickle-shaped as a result of an amino acid alteration from glutamate to valine at the sixth position of the β-globin chain. Hematopoietic stem cell transplant (HSCT) is a curative option for SCD, with both HLA-matched and haploidentical transplant being viable approaches. Stable mixed chimerism and tolerance induction are sufficient to reverse the sickle phenotype. Graft rejection and graft versus host disease may occur following nonmyeloablative haploidentical peripheral blood stem cell transplantation (PBSCT). Various regulatory cytokines have been identified as biomarkers of engraftment in graft transplantation. Previously, we observed that TGF-β, interleukin (IL)-7, and IL-10 cytokines were elevated in engrafted SCD patients as compared to those who rejected their grafts following haploidentical PBSCT. Now, we aim to identify the immune cell populations responsible for producing the above cytokines in both engrafted and rejected patients. The plasma samples of 21 SCD patients who underwent nonmyeloablative haploidentical PBSCT at the National Institutes of Health between March 2010 and September 2015 were evaluated previously by multiplexed magnetic bead enzyme linked immune sorbent assay. Here, we analyzed various immune cells which produce the above regulatory cytokines through multi-color flow cytometry in 8 out of the 21 patients (four engrafted and four rejected). We evaluated the cytokine producing capabilities via intracellular cytokine detection among T regulatory (Treg, CD3+CD4+CD25+Foxp3+) cells, Type 1 regulatory (Tr1, CD3+CD4+Foxp3-CD45RA-CD49+LAG3+) T cells, T helper 1 (Th1, CD3+CD4+Foxp3-CD45RO+CXCR3+) cells, T helper 17 (Th17, CD3+CD4+Foxp3-CD45RO+CCR6+) cells, natural killer (NK, CD3-CD56+NKG2D+) cells, B regulatory (Breg, CD3-CD56-CD19+CD24hiCD38hi)cells, polymorphonuclear-myeloid derived suppressor cells (PMN-MDSCs, CD45+CD14-CD15+CD11b+), monocytic-MDSCs (CD45+CD14+CD15-HLADRlow), early-MDSCs (CD45+Lin-HLADR-CD11b+CD33+), myeloid dendritic cells (mDCs, CD45+CD3-CD56-HLADR+CD11c+CD123-), and plasmacytoid dendritic cells (pDCs, CD45+CD3-CD56-HLADR+CD11c-CD123+). Cytokine producing cell frequencies were measured at five timepoints: baseline (day 0) and days 30, 60, 100, and 180 post-transplant (PT). Percentages of intracellular cytokine and cell population frequencies were assessed through flow cytometry analysis and statistical significance was determined by Student's t-test. We noted IL-10-producing Tregs were significantly elevated in engrafted patients at PT day 100 (p< 0.01, see Figure 1). Further, IL-10-producing Th1 cells were significantly higher in engrafted patients on PT day 30 (p< 0.01) and day 100 (p< 0.01). However, rejected patients had significantly higher amounts of IL-10-producing PMN-MDSCs (p< 0.01), and myeloid DCs (p< 0.01, see Figure 2), but their levels were lower than IL10-producing Tregs and Th1 cells of engrafted patients. There were no significantly different alterations of IL-10-producing cell frequencies among other cell populations (Tr1, Th17, NK, Breg, early-MDSCs, monocytic-MDSCs, and pDCs). In addition, there were no significant variations in TGF-β- and IL-7-producing cell frequencies in any of the immune cell populations tested. Overall, the engrafted patients showed higher IL-10-producing Tregs and Th1 cells at PT day 30 and/or day 100, which suggest that the immunosuppressive cytokine IL-10 may play an important role in engraftment. Further analyses with a larger sample size are indicated to evaluate whether IL-10 producing immune regulatory cells may serve as a biomarker to predict transplant outcome. Taken together, continuous regulatory cytokine analysis at serial time points pre- and post-transplant may aid in establishing mechanisms of successful engraftment and tolerance induction. Disclosures No relevant conflicts of interest to declare.