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Heparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a pro-thrombotic state. While a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet-activating. A close correlation between platelet-activating antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies but how platelet-activating (presumptively pathogenic) and non-activating (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned seven platelet-activating (PA) and 47 non-activating (NA) PF4/H-binding antibodies from six HIT patients and characterized their structural and functional properties. Findings made showed that PA clones differed significantly from NA clones in possessing one of two heavy chain complementarity-determining region 3 (HCDR3) motifs - RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5) - in an unusually long CDR3 region (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their platelet-activating activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become platelet-activating. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in HIT than in non-HIT patients given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.

Immune thrombocytopenia (ITP) is a platelet disorder. 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 adds 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 secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK−/− BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell–like signature and concomitant upregulation of the immunoglobulin rearrangement 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.

The CARD11-Bcl10-Malt1 (CBM) signalosome controls TAK1 activation to regulate B-cell receptor (BCR)-induced NF-κB activation and B cell biology. The biological function of caspase recruitment domain family member 19 (CARD19), originally identified as a BCL10-interacting CARD protein (BinCARD), is not known. Here we found CARD19 strongly interacted with TAK1 but not BCL10 or other CBM components and prevented TAK1's association with TAB2, thereby inhibiting TAB2-mediated TAK1 ubiquitination and activation and subsequent NF-κB activation. CARD19 was ubiquitously expressed in hematopoietic lineages but its deficiency in mice had no effect on hematopoiesis, including B cell development and humoral immune response. CARD19 deficiency enhanced clonal deletion, receptor editing and anergy of self-reactive B cells, thus reducing autoantibody production in vivo. Mechanistically, CARD19 deficiency led to an increase of BCR/TAK1-mediated NF-κB activation. Activation of NF-κB, such as c-Rel, was responsible for the up-regulation of BCR-induced expression of the transcription factor early growth response genes 2 and 3 (Egr2, Egr3) and the E3 ubiquitin ligases, c-Cbl and Cbl-b, the important inducers of B-cell tolerance in B cells. Further, high-throughput RNA sequencing analysis revealed that CARD19 deficiency did not affect the overall antigen-induced gene expression in naïve B cells but suppressed BCR signaling to increase hyporesponsiveness of self-reactive B cells. Consequently, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus (SLE) and autoimmunity in a B cell-intrinsic manner. Taken together, CARD19 negatively regulates BCR-induced NF-κB activation via blocking TAK1/TAB2 interaction and its deficiency leads to NF-κB-induced expression of Egr2/3 and c-Cbl/Cbl-b in self-reactive B cells, which enhances B-cell tolerance and thus prevents autoimmunity. Disclosures No relevant conflicts of interest to declare.

Heparin-induced thrombocytopenia (HIT) is a serious reaction to heparin treatment characterized by antibodies that recognize a complex formed between heparin and platelet factor 4 (PF4/H) and are capable of activating platelets and inducing a pro-thrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset of these antibodies are platelet-activating (pathogenic) and capable of causing HIT. We previously reported that we cloned B cells from six patients experiencing HIT and identified two types of PF4/H-binding antibodies: seven platelet-activating (PA) and 48 non-activating (NA). Comparison of the structural features in the PA, NA, and non PF4/H-binding (NB) clones showed that the length and the number of basic amino acid and tyrosine residue in the heavy chain complementarity determining region 3 (HCDR3) were significantly different, and was in the order of PA>NA>NB. Most significantly, the seven platelet-activating antibodies each have one of the two pathogenic motifs: RX1-2 R/KX1-2 R/H and YYYYY in an unusually long HCDR3 (≥ 20 residues). In the current study, we attempt to understand the origin of the B cells that produce the PA and NA antibodies and the nature of the immune response in HIT through analyzing somatic hypermutation and biological property of such antibodies. Longer HCDR3 and more basic Aas and Tyr residues in the HCDR3 are features of autoreactive and polyreactive antibodies. With this in mind, we tested PA and NA clones in a standard antinuclear antibody (ANA) assay and found that these clones were significantly more reactive than NB antibodies, and the plasma of HIT patients were significantly more reactive than normal plasma (Figure1). We then compared reactions of PA, NA and NB clones against a group of self and foreign antigens commonly used in polyreactivity assays: dsDNA, ssDNA, LPS, insulin, and keyhole limpet hemocyanin (KLH). About 90% of PA and NA clones were reactive to at least two antigens, this was true of only 20% of the NB clones, and the latter is consistent with the frequency of polyreactive clones in the IgG+ B cells (Figure2). Taken together, these data indicate that PA and NA antibodies are largely polyreactive. We then investigated the development of the PA and NA B cells through analyzing somatic hypermutation in the antibodies. Through analyzing the HCDR3 nucleotide insertion, trimming and VDJ segment usage, we found that longer HCDR3 typical of PF4/H-binding clones and the RKH and Y5 motifs identified in PA clones were the result of original recombination not somatic hypermutation. Consistently, the average number of nucleotide mutations in the VH genes of the binding clones was lower (PA and NA, 9.4 ± 9.5) compared to that of peripheral blood IgG+ memory B cells in healthy subjects (~18) (Figure3). Total mutation frequency in the VH and Vk CDRs of the PF4/H-binding PA and NA clones was comparable to that of the framework regions. This finding contrasts with findings made in peripheral blood IgG+ memory B cells of healthy subjects showing that the mutation frequencies are much higher in the CDRs than in the FRs of VH. Taken together, these findings suggest that affinity maturation plays a limited role in the evolution PF4/H-binding antibodies during the immune response that leads to HIT. In this study, we showed thay PF4/H-binding PA and NA IgGs are largely polyreactive antibodies and contain lower levels of mutations compared to IgG+ memory B cells. B1 and MZ B cells are innate B cells that are main producers of polyreactive natural antibodies and can respond to toll-like receptor signaling, quickly differentiate into antibody-secreting cells, and undergo IgG class switch extrafollicularly. Polyreactivity identified in the PF4/H-binding PA and NA IgGs supports the possibility that human B cells producing PF4/H-binding antibodies are innate B cells akin to MZ B cells shown to be a source of PF4/H antibodies in mice. A mutation rate lower than that of IgG+ memory cells in the PF4/H-binding IgGs is also consistent with an extrafollicular response typical of innate B cells. These observations would help to improve our understanding of the immunological responses and B cell origin in HIT patients. Disclosures Padmanabhan: Retham Technologies: Current equity holder in private company; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Blood Research Institute: Patents & Royalties.

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 (μMT) 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.

Heparin-induced thrombocytopenia (HIT) is a common adverse drug reaction associated with frequent life-threatening thrombotic complications. The hallmark of HIT is polyclonal antibodies (Abs) that recognize platelet alpha granule chemokine PF4 when it binds to heparin (PF4/H). These Abs can be detected in solid phase assays that use PF4/H as a target (PF4 ELISA), but only a minority of patients testing positive actually have HIT, i.e., most heparin-induced Abs are non-pathogenic. In patients who have clinical HIT, Abs that activate platelets can be detected using a platelet-activation assay such as the serotonin release assay, or the PF4-dependent p-selectin expression assay (PEA) (Chest 2016; 150:506). Thus, there are at least two distinct types of heparin-induced Abs - those that react only in PF4 ELISA and are seemingly "non-platelet-activating" and "non-pathogenic" and those that are "platelet-activating" and "pathogenic". To date, the molecular basis for the differing clinical and serologic behaviors of pathogenic and non-pathogenic Abs is uncertain. To address this issue, we performed single cell cloning to clone B cell receptors from IgG1+ B cells from HIT patients. We deposited single B cells (CD19+IgG1+) from 6 patients with "classical" and 2 patients with "spontaneous" HIT into 96 well plates containing feeder cells (from G Kelsoe, Duke U) that support B cell proliferation and Ab secretion (Immunity 2018;48:174). Clones secreting IgG were first screened in PF4 ELISA and positive results were obtained with 55 clones from 6 patients. Further screening showed that 7 of these clones (from 4 patients) were also PEA-positive (platelet-activating). Clones positive only in PF4 ELISA, positive in both PF4 ELISA and PEA, or negative in PF4 ELISA were designated NP (non-pathogenic), PA (platelet-activating) and NB (non-binding), respectively. H and L chain variable regions were defined in 7 PA, 42 NP and 34 NB clones. The following findings were made when sequences in the 3 clonal groups were compared: PA clones preferentially used JH6 (p=0.002) and the VH3/JH6 combination (p=0.0003)The PA and NP Abs all employed κ chains, whereas κ chain usage for NB clones was 61% (p Utilization of nearly identical H and L chains within 3 groups of clones and of shared H chains within 3 groups of clones (both PA and NP) was observed in multiple patients. Moreover, utilization of a shared H chain was observed within 3 NP clones from two unrelated patients. These findings indicate clonal amplification and convergence of the B cell (both PA and NP) response, likely in response to a common antigen. High throughput sequencing of IgG H chains were performed on peripheral blood mononuclear cells (PBMC) from 7 HIT patients and 3 healthy donors. Eleven of 1585 H chain sequences (0.69%) from HIT patients contained the RKH and 18 (1.1%) contained the Y5 motif. In 3 healthy donors, 4 of 1418 H chain sequences (0.28%) contained RKH and none (0%) contained Y5. The findings reflect amplification of B cells with receptors containing RKH and Y5 motifs in HIT patients (p=0.1 for excess RKH and p These observations provide the first characterization of Ig structural motifs that are favored for selection in the humoral immune response leading to HIT and suggest that the RKH and Y5 CDR3 motifs in particular may contribute importantly to Ab pathogenicity. Findings made are expected to facilitate further work to define features specific to "pathogenic" HIT Abs and, possibly, to identify genetic variants that predispose individuals to experience HIT. Disclosures Padmanabhan: Terumo BCT: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Versiti Wisconsin: Patents & Royalties: Related to HIT patents; Retham Technologies: Equity Ownership; Janssen R&D: Consultancy.

ATP-dependent chromatin remodeling complexes, including BAF (BRG1 associated factor) and PBAF (Polybromo-associated BRG1 associated factor), play major roles in development and disease. These complexes contain multiple subunits and utilize ATP to remodel nucleosomes and, as a result, affect downstream processes. Subunits of the BAF and PBAF complexes are frequently mutated in various cancers, including in acute myeloid leukemia. However, the role of distinct members of these complexes in leukemogenesis is poorly understood. BAF and PBAF complexes are distinguished from each other by unique AT-rich interacting domain (ARID) subunits, ARID1A/B and ARID2, respectively. Recently, Arid2 was suggested to have tumor suppressive functions in MLL-AF9 leukemogenesis, however the role of Arid1b is unknown. In this study, we further evaluated the tumor suppressor roles of Arid2 and Arid1b in MLL-AF9 leukemogenesis. To study the effect of the loss of Arid2 and Arid1b on MLL-AF9 leukemia in vitro, hematopoietic stem and progenitor cells from Arid1bfl/fl, Arid2fl/fl and wild type (control) mice were transformed by retroviral expression of MLL-AF9. Arid1b or Arid2 was then deleted by retroviral delivery of Cre-recombinase and cellular phenotypes were characterized by flow cytometry. In vitro characterization revealed Arid1b-/-and Arid2-/-MLL-AF9 cells have similar growth rates to control cells and no changes in cell cycle status and apoptosis. To study leukemogenesis in vivo, two hematopoietic specific conditional knockout mouse models, Vav1Cre, a constitutively active Cre-recombinase, and Mx1Cre, an inducible Cre-recombinase, were used. Primary and secondary transplant recipient mice were assessed for survival, disease burden, and leukemia stem cell frequency. In primary transplantation, leukemic initiation was not altered by the loss of the Arid2. Overall survival of primary recipients and disease burden in moribund mice (percentage of GFP positive MLL-AF9 cells in bone marrow/spleen and spleen weight) were similar to control counterparts. However, in limiting dilution secondary transplantation of Arid2fl/flVav1Cre (Arid2-/-) and wild type Vav1Cre (control) MLL-AF9, median survival of the Arid2-/- cohort decreased 1 to 2 weeks in comparison to the control cohort and there was a two-fold increase in leukemia initiating cell frequency upon deletion of Arid2. Together these results support the previous indication that Arid2 is a tumor suppressor in MLL-AF9 leukemic initiation. To gain insight into the function of Arid1b in MLL-AF9 leukemogenesis, we performed primary transplantation of Arid1bfl/flcells transduced with MLL-AF9. Our data showed that loss of Arid1b enhanced leukemic initiation in the MLL-AF9 leukemia model. One-month post induction with pIpC, Arid1bfl/fl Mx1Cre MLL-AF9 cells accumulated faster in the peripheral blood and primary recipients exhibited decreased median survival compared with wild type Mx1Cre controls. Percentage of GFP+ leukemia cells was higher in the spleen of moribund mice in the Arid1bfl/fl Mx1Cre cohort, compared with the control cohort. Taken together, our data suggests Arid1b, in addition to Arid2, acts as a potential novel tumor suppressor in MLL-AF9 leukemogenesis. To study the mechanism of Arid1b and Arid2's function in MLL-AF9 leukemogenesis, RNA-sequencing of leukemic bone marrow cells was conducted. Consistent with the previous study, Arid2 null leukemia cells had increased Gata2 expression and decreased p57 expression. Gene Set Enrichment Analysis of Arid1b null cells showed enrichment of oncogenic signatures including ATM, Cyclin D and macrophage development as potentially contributing to the underlying tumor suppressive mechanism of Arid1b. In summary, our study uncovered Arid1b, in addition to Arid2, as a potential novel tumor suppressor in MLL-AF9 leukemia. Our study, together with published studies on the requirement of other subunits (SMARCA4, SMARCD2 and DPF2) of the BAF and PBAF complexes in MLL-AF9 leukemia, reveal opposing roles of the components of the BAF and PBAF complexes in leukemogenesis, the mechanism of which require further investigation. Disclosures No relevant conflicts of interest to declare.

MLL-rearranged leukemias are found in 5-10% of adult leukemias and over 70% of infant leukemias and are associated with intermediate to poor prognosis. We have recently shown that JMJD1C, a Jumonji domain containing lysine demethylase (KDM), is important for leukemia stem cell (LSC) function in MLL-AF9 and HOXA9 leukemias but dispensable for normal hematopoietic stem cell function, therefore a potential therapeutic target for acute myeloid leukemia (AML). To determine which domains within JMJD1C are essential for its function in AML, we performed a CRISPR/Cas9 negative selection screen against its coding sequence using guide RNAs (gRNA) tiled at ~ 30bp intervals. Specifically, gRNAs were cloned into a lenti-viral vector with a TdTomato marker gene, transduced into a clonal Cas9 expressing mouse MLL-AF9 leukemia and percentage of gRNA positive cells was analyzed by flow cytometry in a time course. JMJD1C Jumonji and Zinc finger domain (ZFD) and surrounding regions exhibited the most depletion at day 14 compare to day 2, comparable or higher than that of the positive control. To investigate the effect of mutating Jumonji and ZFD on MLL-AF9 leukemogenesis, we subject gRNA transduced MLL-AF9 Cas9 cells to colony forming cell assay. Mutating Jumonji or ZFD impaired colonegenic potential of MLL-AF9 Cas9 cells as evident by a significant reduction in the number of colonies. The resulting colonies appeared smaller and diffused, indicative of cell differentiation. Consistently, we observed impaired proliferation, increased apoptosis and increased expression of the myeloid differentiation marker Mac1 upon mutating these domains. Moreover, mutating Jumonji and ZFD prolonged survival of recipient mice compare to control in transplantation experiment. Finally, we performed chromatin-immunoprecipitation followed by sequencing (ChIP-seq) on H3K27, K36 and K9 methylation in JMJD1C knockout versus floxed MLL-AF9 leukemia cells to identify histone marks that correlate with JMJD1C activity. We observed increased H3K36 trimethylation (me3) level on JMJD1C regulated genes loci in the knockout cells compare to controls. Similar increase was observed upon mutating Jumonji and ZFD using CRISPR/Cas9 in ChIP assay. Using Gene Set Enrichment Analysis (GSEA), we found enrichment of JMJD1C regulated genes in the knockout cells in H3K36me3 ChIP-seq data but not H3K27me3 or H3K9 methylation data. Together, these data suggest that Jumonji and ZFD of JMJD1C are most critical for its function in AML and that methylated H3K36 can be used as a marker for JMJD1C activity at gene loci. Next, we performed single cell RNA-seq to examine the transcriptome change upon mutating the catalytic Jumonji domain. Our analysis showed a hierarchy within control leukemia cells with one cluster bearing more LSC property than the other. Upon mutating Jumonji domain, leukemia cells are driven into differentiated states that bear either more granulocyte or monocyte gene signatures. Using MAST, a single cell transcriptome GSEA tool, we observed enrichment of KRAS signatures in Jumonji mutated cells. This observation in addition to the increased expression of the IL-3 receptor subunit genes Csf2ra/b led us to examine whether the IL-3 signaling pathway is affected in these cells. Western blotting of IL-3 down signaling molecules ERK1/2 and STAT5 showed elevated phosphorylation in Jumonji mutated cells compare to controls both at basal level and in response to IL-3 stimulation. Consistently with this finding, Jumonji mutated cells showed increased sensitivity to inhibitors against RAS (Tipifarnib), MEK (Selumetinib) and JAK (Tofacitinib and Ruxolitinib). Finally, by mining published shRNA and gRNA screen, we observed that mouse as well as human MLLr leukemia cell lines with activating RAS mutations showed decreased or diminished response to knockdown or mutation of JMJD1C suggesting that the presence of RAS mutations render them resistant to JMJD1C modulation. Overall, we show that enzymatic domain of JMJD1C, the Jumonji domain, mediates its function in AML leukemogenesis, therefore can be targeted for potential therapeutic intervention. In addition, we uncovered a novel interplay between JMJD1C and IL-3 signaling pathway. Disclosures No relevant conflicts of interest to declare.

Small GTPases of the Ras subfamily regulate multiple signaling pathways and control numerous biological functions. Although the three major Ras members, Kras, Hras and Nras, are highly homologous, individual Ras gene can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological role of Kras. Here, we generated and examined mice with hematopoietic- and B cell-specific deletion of Kras. In VavCreKrasfl/fl mice with hematopoietic deletion of Kras, the populations of bone marrow (BM) pre-, immature and mature B cells were reduced. The population of peripheral follicular (FO), marginal zone (MZ) and B1 mature B cells were also reduced in VavCreKrasfl/fl relative to control mice. In addition, BM chimeric mice with B cell-specific deficiency of Kras generated by transplantation of T- and B-null Rag1-deficient mice with a mixture of BM cells from VavCreKrasfl/fl mice and B cell-deficient μMT mice displayed a marked reduction of pre-, immature and mature B cells in the BM and mature B cells in the spleen. Thus, Kras deficiency intrinsically impairs early B cell development and late B cell maturation. Further, the effect of Kras deficiency on B-cell proliferation and survival was examined. The 3H-thymidine incorporation rate of Kras-deficient, relative to control, mature B cells in response to anti-IgM or anti-IgM plus IL-4 was markedly reduced. In addition, Kras-deficient B cells displayed a marked decrease of cell cycle entry and increase of cell apoptosis upon anti-IgM or anti-IgM plus IL-4 stimulation. Thus, Kras deficiency impairs BCR-induced B cell proliferation and survival. Lastly, the role of Kras in BCR signaling was studied. The level of total BCR-activated Ras (Ras-GTP) was largely reduced in Kras-deficient B cells. BCR-induced Ca2+ flux was comparable between Kras-deficient and control B cells. BCR-induced phosphorylation of Akt and IkBa was normal in mutant relative to control B cells. However, BCR-induced activation of ERK1/2 but not JNK or p38 was impaired in Kras-deficient relative to control B cells. Consistently, BCR-induced activation of Raf-1 and MEK1/2, the upstream activators of ERK1/2, was markedly reduced in mutant B cells. In addition, pre-BCR-induced ERK1/2 activation was impaired in Kras-deficient pre-B cells. Pre-BCR-induced activation of AP-1, the transcription factors downstream of ERK1/2, was decreased in mutant pre-B cells. Thus, Kras plays an important role in BCR- and pre-BCR-mediated activation of the ERK pathway in B cells. Taken together, these findings demonstrate that Kras is the Ras family member that critically regulates early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway. Disclosures No relevant conflicts of interest to declare.

Small GTPases regulate multiple signaling pathways and individual Ras member can have distinct biological function. To overcome embryonic lethality of Kras-deficient mice, we generated and examined mice with hematopoietic- and T cell-specific deletion of Kras. In VavCreKrasfl/fl mice with hematopoietic deletion of Kras, thymic T-cell development was normal based on the presence of normal populations of total, CD4- CD8-, CD4+ CD8+, CD4+ and CD8+ thymocytes. The populations of splenic CD4+ and CD8+ T cells were also comparable between VavCreKrasfl/fl relative to control mice. In addition, no consistent defects in the 3 H-thymidine incorporation rate of Kras-deficient splenic CD4+ or CD8+ T cells in response to anti-CD3 or anti-CD3 plus IL-2 was detected. Nonetheless, we studied the effect of Kras deficiency on CD8 T-cell immune response to acute infection of the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). Sub-lethally irradiated Rag1-deficient mice transplanted with bone marrow (BM) cells from VavCreKrasfl/fl or control mice were subjected to LCMV infection. Infection-induced expansion of CD8 T cells and generation of LCMV epitope gp33-specific CD8 T cells were markedly reduced in the recipients that received the BM from VavCreKrasfl/fl relative to control mice. Following in vitro stimulation with the LCMV epitope gp33, the induction of IFNg-expressing CD8 T cells from LCMV-infected recipients that received the BM from VavCreKrasfl/fl mice was dramatically reduced. Further, BM chimeric mice with CD8 T cell-specific deficiency generated by transplantation of lethally irradiated CD8 T cell-depleted CD45.1 congenic mice with a mixture of BM cells from VavCreKrasfl/fl mice and BM cells from CD8 T cell-deficient mice exhibited an impaired CD8 T-cell immune response to LCMV infection. Lastly, we examined the role of Kras in TCR signaling. The level of total TCR-activated Ras (Ras-GTP) was markedly reduced in Kras-deficient relative to control CD8 T cells. Importantly, TCR-induced ERK1/2 activation was impaired in Kras-deficient relative to control CD8 T cells. Consistently, TCR-induced activation of Raf-1 and MEK1/2 was markedly reduced in mutant CD8 T cells. However, TCR-induced JNK and p38 activation as well as Ca2+ flux were normal in Kras-deficient CD8 T cells. Of note, TCR-induced activation of Ca2+ flux, JNK and p38 as well as ERK1/2, MEK1/2 and Raf1 was normal in Kras-deficient relative to control CD4 cells. Taken together, these data demonstrate that Kras is dispensable for T cell development or TCR-induced proliferation of CD4 or CD8 T cells in vitro, but regulates TCR-induced activation of the Raf-1/MEK/ERK pathway in CD8 but not CD4 T cells and intrinsically controls CD8 T-cell immune response to viral infection. Disclosures No relevant conflicts of interest to declare.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that can cause arterial or venous thrombosis/thromboembolism, and platelet factor 4 (PF4)/ heparin-reactive antibodies are essential to the pathogenesis of HIT. Our recent studies have demonstrated that marginal zone (MZ) B cells play a major role in production of PF4/heparin-specific antibodies. However, the role of T cells in production of these pathogenic antibodies is not clear. Here we showed that PF4/heparin complex-induced production of PF4/heparin-specific antibodies was markedly impaired in mice, in which CD4 T cells were depleted by administration of GK1.5 anti-CD4 monoclonal antibody. As expected, the CD4 T cell-depleted mice responded normally to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, in agreement with the lack of CD4 T cells in these GK1.5-treated mice. Further, following adoptive transfer of a mixture of wild-type splenic B cells and splenocytes from B cell-deficient μMT mice, T and B cell-deficient Rag1 knockout mice responded to PF4/heparin complex challenge to produce PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received a mixture of wild-type splenic B cells and splenocytes from Rag1-deficient mice barely produced PF4/heparin-specific antibodies upon PF4/heparin complex challenge. These data suggest that T cells are required for production of PF4/heparin-specific antibodies. Consistent with this concept, mice with B cells lacking CD40 molecule, 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 complex challenge. Also as expected, mice with CD40-deficient B cells were able to respond to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, consistent with the lack of T-cell help in these mice. Taken together, these findings demonstrate that T cells play an essential role in production of PF4/heparin-specific antibodies by MZ B cells. Disclosures No relevant conflicts of interest to declare.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis. Disclosures: White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.

Abstract 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.