Immune checkpoint protein V-domain immunoglobulin suppressor of T-cell activation (VISTA) controls antitumor immunity and is a valuable target for cancer immunotherapy. This study identified a role of VISTA in regulating Toll-like receptor (TLR) signaling in myeloid cells and controlling myeloid cell-mediated inflammation and immunosuppression. VISTA modulated the polyubiquitination and protein expression of TRAF6. Consequently, VISTA dampened TLRmediated activation of MAPK/AP-1 and IKK/NF-κB signaling cascades. At cellular levels, VISTA regulated the effector functions of myeloid-derived suppressor cells (MDSCs) and tolerogenic DC subsets. Blocking VISTA augmented their ability to produce proinflammatory mediators and diminished their T cell-suppressive functions. These myeloid cell-dependent effects resulted in a

Many autoimmune diseases are characterized by the production of autoantibodies. The current view is that CD4+ T follicular helper (Tfh) cells are the main subset regulating autoreactive B cells. Here we report a CXCR5+PD1+ Tfh subset of CD8+ T cells whose development and function are negatively modulated by Stat5. These CD8+ Tfh cells regulate the germinal center B cell response and control autoantibody production, as deficiency of Stat5 in CD8 T cells leads to an increase of CD8+ Tfh cells, resulting in the breakdown of B cell tolerance and concomitant autoantibody production. CD8+ Tfh cells share similar gene signatures with CD4+ Tfh, and require CD40L/CD40 and TCR/MHCI interactions to deliver help to B cells. Our study thus highlights the diversity of follicular T cell subsets that contribute to the breakdown of B-cell tolerance.

Key Points B-cell tolerance plays a critical role in controlling production of PF4/heparin-specific antibodies.

Antibodies specific for platelet factor 4 (PF4)/heparin complexes are central to the pathogenesis of heparin-induced thrombocytopenia. Marginal zone B cells appear to be the source of such antibodies, but whether T-cell help is required is unclear. Here, we showed that induction of PF4/heparin-specific antibodies by PF4/heparin complexes was markedly impaired in mice depleted of CD4 T cells by anti-CD4 antibodies. Furthermore, Rag1-deficient recipient mice produced PF4/heparin-specific antibodies upon PF4/heparin challenge when reconstituted with a mixture of wild-type splenic B cells and splenocytes from B-cell-deficient (mMT) mice but not splenocytes from T-and B-cell-deficient (Rag1 knockout) mice. Lastly, mice with B cells lacking CD40, a B-cell costimulatory molecule that helps T-cell-dependent B-cell responses, displayed a marked reduction of PF4/ heparin-specific antibody production following PF4/heparin challenge. Together, these findings show that helper T cells play a critical role in production of PF4/heparin-specific antibodies. (Blood. 2015;125(11):1826-1829 Introduction Heparin-induced thrombocytopenia (HIT) is the most common druginduced, antibody-mediated thrombocytopenia and occurs 3 to 6 days following heparin treatment.1,2 HIT patients develop antibodies quickly, however, which are typically undetectable in a few months. Platelet factor 4 (PF4)/heparin-specific antibodies, central to the pathogenesis of HIT, are predominantly of the immunoglobulin G1 (IgG1) isotype with some IgG2 in humans.2-4 IgG/PF4/heparin immune complexes bind FcgRIIA on the platelet surface and induce platelet activation, leading to thrombocytopenia and a high risk of arterial and/or venous thrombosis/thromboembolism. 5,6 Long-lived mature B cells comprise 3 subsets: marginal zone (MZ), B1, and follicular B cells. 7,8 The MZ subset has been shown to be critical for production of PF4/heparin-specific antibodies.9 Typically, MZ B cells produce IgM or IgG antibodies independent of T-cell help. [10][11][12] Indeed, HIT patients have features of a T-cell-independent humoral immune response, characterized by rapid onset and decline of antibodies and apparent absence of immunologic memory.1 However, patients with severe HIT possess T cells that have a T-cell receptor with highly restricted complementarity determining region 3 regions and are responsive to PF4/heparin, suggesting a role of T cells in HIT pathogenesis. 13,14 Nonetheless, direct evidence for a role of T cells in HIT pathogenesis has not been reported. Here, we describe studies to define the role of T-cell help in regulating production of PF4/heparin-specific antibodies. Study design MiceEight-to 10-week-old Rag1-deficient, CD40-deficient, mMT, and wild-type C57BL/6 mice from The Jackson Laboratory were maintained in the Biological Resource Center at the Medical College of Wisconsin (MCW). Animal protocols were approved by the MCW Institutional Animal Care and Use Committee. In vivo depletion of CD4 T cellsWild-type C57BL/6 mice were injected intraperitoneally with ...

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin, and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here, we show that anti-PF4/heparin antibodies are readily generated in wild-type mice on challenge with PF4/heparin complexes, and that antibody production is severely impaired in B-cell-specific Notch2-deficient mice that lack marginal zone (MZ) B cells. As expected, Notch2-deficient mice responded normally to challenge with T-cell-dependent antigen nitrophenyl-chicken g globulin but not to the T-cell-independent antigen trinitrophenylFicoll. In addition, wild-type, but not Notch2-deficient, B cells plus B-cell-depleted wild-type splenocytes adoptively transferred into B-cell-deficient mMT mice responded to PF4/heparin complex challenge. PF4/heparin-specific antibodies produced by wild-type mice were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular, B cells adoptively transferred into B-cell-deficient mMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells are critical for PF4/heparin-specific antibody production. (Blood. 2013;121(17):3484-3492)

The precise molecular mechanism underlying the regulation of early B cell lymphopoiesis is unclear. The PLCγ signaling pathway is critical for antigen receptor-mediated lymphocyte activation, but its function in cytokine signaling is unknown. Here we show that PLCγ1/PLCγ2 double deficiency in mice blocks early B cell development at the pre-pro-B cell stage and renders B cell progenitors unresponsive to IL-7. PLCγ pathway inhibition blocks IL-7-induced activation of mTOR, but not Stat5. The PLCγ pathway activates mTOR through the DAG/PKC signaling branch, independent of the conventional Akt/TSC/Rheb signaling axis. Inhibition of PLCγ/PKC-induced mTOR activation impairs IL-7-mediated B cell development. PLCγ1/PLCγ2 double-deficient B cell progenitors have reduced expression of genes related to B cell lineage, IL-7 signaling, and cell cycle. Thus, IL-7 receptor controls early B lymphopoiesis through activation of mTOR via PLCγ/DAG/PKC signaling, not via Akt/Rheb signaling.

1175 Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here we show that upon challenge with PF4/heparin complexes, anti-PF4/heparin antibody production is severely impaired in B cell-specific Notch2-deficient mice (CD19CreNotch2fl/fl) that specifically lack marginal zone (MZ) B cells, and that antibody production is readily generated in wild-type mice (CD19CreNotch2+/+). As expected, Notch2-deficient mice responded normally to challenge with T cell-dependent antigen NP-CGG but not T cell-independent antigen TNP-Ficoll, in agreement with the lack of MZ B cells in the mutant mice. PF4/heparin-specific antibodies produced by wild-type mice on a C57BL/6 background were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells from wild-type C57BL/6 mice were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular (FO), B cells adoptively transferred into B cell-deficient muMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells play a critical role in production of PF4/heparin-specific antibodies. Disclosures: Arepally: Teva Pharmaceuticals: Research Funding.

Immune thrombocytopenia (ITP) is a common platelet disorder in pediatric patients. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF-antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF-antigen in these patients. The O-glycan sialyltransferase St3gal1 add sialic acid specifically on the TF-antigen. To understand if TF-antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF-antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon (IFN-I) secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following and inhibition of interferon and Siglec H receptors. Single cell RNAseq determined that TF-antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release IFN-I. Single cell RNAseq further revealed a new population of immune cells with a plasmacytoid dendritic cell (pDC)-like signature and concomitant upregulation of immunoglobulin re-arrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF-antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.-