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Background Flow cytometry immunophenotyping (FCIP) can improve diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), although its application is challenging due to difficulties in standardization, complexity of antibody panels and subjective interpretation of data. Since blasts are invariably affected in these disorders, we developed a FCIP approach for detailed and objective analysis of the blast population. Methods FCIP using a one-tube 10-color (13-marker) antibody panel was performed on bone marrow samples from 23 MDS and 8 MDS/MPN patients, 21 cytopenic patients non-diagnostic for MDS (Non-MDS), and 16 Control samples. Results MDS and MDS/MPN cases demonstrated one to several immunophenotypic abnormalities including: increased myeloblasts, decreased stage-1 hematogones, aberrant stem cells, abnormal myeloblast heterogeneity/divergence from normal, increased or decreased CD45 intensity, increased CD117 or CD123 intensity, decreased CD38 intensity, and aberrant expression of lineage markers (CD5, CD19, CD56). A Blast score was developed that showed sensitivity of 80.6% and specificity of 90.5% for immunophenotypic diagnosis of MDS and MDS/MPN. Expression levels of CD45RA and CD371 were used to evaluate abnormal myeloblast heterogeneity and stem cell aberrancy. Both these features were, for the first time, incorporated into a scoring system and resulted in 19% increase in the sensitivity of the assay for lower-risk MDS. Conclusion Deep immunophenotypic analysis of the blast population is valuable for diagnosis of MDS and MDS/MPN and can potentially provide sensitivity and specificity figures comparable to those previously described using more comprehensive panels that assess maturing myelomonocytic and erythroid elements in addition to progenitor cells.

High-grade B cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (“double-hit” lymphoma (DHL)/“triple-hit” lymphoma (THL)) are mature B cell neoplasms with highly aggressive clinical behavior and poor response to therapy. Lymphoblastic transformation of mature B cell neoplasms is an uncommon event that is best recognized for follicular lymphoma (FL). To our knowledge, only one case of “B lymphoblastic” transformation of de novo DHL has been reported in the literature. Here, we describe another case of such transformation. The patient was a 57-year-old man who presented with hypercalcemia, high lactate dehydrogenase (LDH), and multiple lytic bone lesions and was diagnosed with high-grade B cell lymphoma with MYC and BCL2 rearrangements. The neoplastic cells were positive for CD45, CD19, CD20, CD79a, PAX5, CD10, BCL6, and BCL2 and negative for CD34, CD99, and terminal deoxynucleotidyl transferase (TdT). The patient received chemotherapy followed by autologous stem cell transplant and achieved complete remission. Nine months after the initial presentation, he developed general weakness and found to have cytopenias and circulating blasts. Bone marrow examination revealed extensive involvement by a high-grade B cell neoplasm co-expressing PAX5, CD10, and BCL2, in addition to precursor markers TdT and CD99. There was loss of CD20, CD79a, and BCL6 expression, and CD34 remained negative. A diagnosis of B lymphoblastic leukemia/lymphoma (B-LBL) was established. Similar MYC and BCL2 rearrangements were identified. IGH gene rearrangement studies confirmed clonal relatedness. The patient passed away 3 days after bone marrow examination. This case represents an extremely rare case of DHL transformation to B-LBL.

Background Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)–B cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)–MALT1 paracaspase (MALT1) (CBM) complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B-cell development, signaling, and function is incompletely understood. Objectives This study sought to determine the cellular, immunological, and biochemical basis of disease for 2 unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. Methods Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signaling assays by immunoblot, and transcriptome profiling by RNA-sequencing. Results Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837∗) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of nuclear factor-κB, c-Jun N-terminal kinase, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naive and type 1 transitional B-cell stage and impaired circulating T follicular helper cell (cTFH) development, which was associated with impaired antibody responses and absent germinal center structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signaling is essential for immune signaling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations, which led to functional normalization. Conclusions Complete human CARD11 deficiency causes profound CID by impairing naive/type 1 B-cell and cTFH cell development and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. Hematopoietic stem cell transplantation functionally restores impaired signaling pathways.

Alpha-synuclein is highly expressed in the central nervous system and plays an important role in pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia. Previous studies have demonstrated the expression of α-synuclein in hematopoietic elements and peripheral blood mononuclear cells, although its roles in hematopoiesis and adaptive immunity are not studied. Using an α-synuclein knock out (KO) mouse model, we have recently shown that α-synuclein deficiency is associated with a mild defect in late stages of hematopoiesis. More importantly, we demonstrated a marked defect in B lymphocyte development and IgG, but not IgM production in these mice. Here we show a marked defect in development of T lymphocytes in α-synuclein KO mice demonstrated by a significant increase in the number of CD4 and CD8 double negative thymocytes and significant decreases in the number of CD4 single positive and CD8 single positive T cells. This resulted in markedly reduced peripheral T lymphocytes. Interestingly, splenic CD4(+) and CD8(+) T cells that developed in α-synuclein KO mice had a hyperactivated state with higher expression of early activation markers and increased IL-2 production. Moreover, splenic CD4(+) T cells from α-synuclein KO mice produced lower levels of IL-4 upon antigenic stimulation suggesting a defective Th2 differentiation. Our data demonstrate an important role for α-synuclein in development of T lymphocytes and regulation of their phenotype and function.

We investigated whether a prevalent epitope of the β-cell–specific autoantigen islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP206–214) reaches regional Ag-presentation pathways via unprocessed polypeptide chains, as free IGRP206–214 peptide or via preformed IGRP206–214/Kd complexes. This was accomplished by expressing bacterial artificial chromosome transgenes encoding wild-type (stable) or ubiquitinated (unstable) forms of IGRP in IGRP-deficient NOD mice carrying MHC class I–deficient β-cells, dendritic cells, or B cells. We investigated the ability of the pancreatic lymph nodes of these mice to prime naive IGRP206–214-reactive CD8+ T cells in vivo, either in response to spontaneous Ag shedding, or to synchronized forms of β-cell necrosis or apoptosis. When IGRP was made unstable by targeting it for proteasomal degradation within β-cells, the cross-priming, autoimmune-initiating potential of this autoantigen (designated autoantigenicity) was impaired. Yet at the same time, the direct presentation, CTL-targeting potential of IGRP (designated pathogenicity) was enhanced. The appearance of IGRP206–214 in regional Ag-presentation pathways was dissociated from transfer of IGRP206–214 or IGRP206–214/Kd from β cells to dendritic cells. These results indicate that autoantigenicity and pathogenicity are separable and inversely related properties and suggest that pathogenic autoantigens, capable of efficiently priming CTLs while marking target cells for CTL-induced killing, may have a critical balance of these two properties.

IL-2 plays a critical role in both effector T-cell development and FoxP3+CD4+ Treg-cell homeostasis. A reduction in Il2 transcription results in impaired FoxP3+CD4+ Treg-cell recruitment and function, and accounts for the association between murine Il2 and type 1 diabetes (T1D). The progression of T1D elicits a disease-countering negative feedback regulatory loop that involves the differentiation of low-avidity autoreactive CD8+ T cells into memory-like autoregulatory T cells in a CD4+ Th-dependent manner. Since these auto-regulatory T cells express IL-2Rβ (CD122), we hypothesized that their development might also be regulated by IL-2. Here, we investigate the effects of differences in IL-2 expression on this autoregulatory subset. We show that decreased IL-2 production impairs the regulatory capacity of memory-like autoregulatory CD8+CD122+ T cells. Surprisingly, we also find that a reduction in IL-2 production capacity increases memory autoregulatory CD8+ T-cell formation indirectly, by decreasing the development and function of FoxP3+ Treg cells in nonobese diabetic mice. These results illustrate a complex homeostatic interplay between IL-2, CD4+ Th cells, FoxP3+CD4+ Treg cells and autoregulatory CD8+ T-cell memory whereby IL-2 controls the function of both Treg-cell subsets, but IL-2-potentiation of FoxP3+CD4+ Treg-cell function results in the suppression of CD4+ Th-cell activation and autoregulatory memory CD8+ T-cell formation.

A current paradigm states that non-antigen-specific inflammatory cues attract noncognate, bystander T cell specificities to sites of infection and autoimmune inflammation. Here we show that cues emanating from a tissue undergoing spontaneous autoimmune inflammation cannot recruit naive or activated bystander T cell specificities in the absence of local expression of cognate antigen. We monitored the recruitment of CD8+T cells specific for the prevalent diabetogenic epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214in gene-targeted nonobese diabetic (NOD) mice expressing a T cell “invisible” IGRP206–214sequence. These mice developed islet inflammation and diabetes with normal incidence and kinetics, but their inflammatory lesions could recruit neither naive (endogenous or exogenous) nor ex vivo-activated IGRP206–214-reactive CD8+T cells. Conversely, IGRP206–214-reactive, but not nonautoreactive CD8+T cells rapidly homed to and accumulated in the inflamed islets of wild-type NOD mice. Our results indicate that CD8+T cell recruitment to a site of autoimmune inflammation results from an active process that is strictly dependent on local display of cognate pMHC and suggest that CD8+T cells contained in extralymphoid autoimmune lesions are largely autoreactive.

Summary Blunting autoreactivity without compromising immunity remains an elusive goal in the treatment of autoimmunity. We show that progression to autoimmune diabetes results in the conversion of naive low-avidity autoreactive CD8 + T cells into memory-like autoregulatory cells that can be expanded in vivo with nanoparticles coated with disease-relevant peptide-major histocompatibility complexes (pMHC-NP). Treatment of NOD mice with monospecific pMHC-NPs expanded cognate autoregulatory T cells, suppressed the recruitment of noncognate specificities, prevented disease in prediabetic mice, and restored normoglycemia in diabetic animals. pMHC-NP therapy was inconsequential in mice engineered to bear an immune system unresponsive to the corresponding epitope, owing to absence of epitope-experienced autoregulatory T cells. pMHC-NP-expanded autoregulatory T cells suppressed local presentation of autoantigens in an interferon-γ-, indoleamine 2,3-dioxygenase-, and perforin-dependent manner. Nanoparticles coated with human diabetes-relevant pHLA complexes restored normoglycemia in a humanized model of diabetes. These observations expose a paradigm in the pathogenesis of autoimmunity amenable for therapeutic intervention.

Intact tolerogenic mechanisms preclude effective immunity against tumors, as most tumor Ags differ little from normal host Ags. In contrast, when tolerance fails, the immune system becomes inappropriately activated against an autoantigen. We postulated that CD8+ T cells activated during autoimmunity are capable of protecting against tumors that express the targeted autoantigen. To test this hypothesis, double-transgenic 8.3-NOD-RIPTAg mice were developed (where NOD is nonobese diabetic, RIP is rat insulin promoter, and TAg is large T Ag). In this model, individuals with the RIPTAg transgene develop insulinoma; those expressing a transgenic TCR (8.3-TCR) recognizing the islet-specific glucose 6 phosphatase catalytic subunit-related protein (IGRP) harbor a peripheral immune system dominated by diabetogenic CD8+ T cells. Although tumor emergence was significantly slower in 8.3-NOD-RIPTAg mice compared with NOD-RIPTAg mice, all 8.3-NOD-RIPTAg mice eventually developed insulinoma. Tumor emergence was not secondary to clonal deletion or anergy. Ag loss and MHC down-regulation were not apparent. Endogenous 8.3-TCR CD8+ T cells were recruited to the tumor site and proliferated upon arrival to the tumor, although they were notably absent from the central parts of more advanced tumors. These results demonstrate that a breakdown of tolerance capable of causing autoimmune disease is insufficient for effective tumor immunity. Alterations in the tumor microenvironment may inhibit efficient and comprehensive delivery of CD8+ T cells to all regions of the tumor. These data suggest that any immunotherapeutic strategy for cancer must involve enhancement of a proinflammatory tumor microenvironment in addition to inhibition of tolerogenic mechanisms.

Autoreactive CD8 + T cells are emerging as important players in several animal models of autoimmunity; their roles in the human autoimmune diseases are only beginning to be understood. Autoreactive CD8 + T cells can act as pathogenic effector cells and mediate tissue damage, while other autoreactive CD8 + T cells may have regulatory properties and serve to protect self against autoimmune attacks. Recent advances made in the field include a better understanding of their activation, mechanism of action, negative regulation, and antigenic specificities. Importantly, advances have been made in the development of strategies that detect these antigenic specificities in human patients and at risk individuals. Together, these findings foster hope for the development of novel antigen-specific therapeutic strategies that target the CD8 + T cell compartment of autoimmune processes.

α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein(-/-) (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220(+)IgM(+) B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×10(5) vs. KO: 27±5×10(5)). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response.

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8+ cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8+ T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8+ T cell memory is CD4+ T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti–4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4+ T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4+ T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2−/− mice, but inhibited it in low-affinity TCR-TG NOD.Rag2−/− mice. Thus, in chronic autoimmune responses, CD4+ Th cells contribute to both promoting and suppressing pathogenic autoimmunity.

Adenosine has been established as an important regulator of immune activation. It signals through P1 adenosine receptors to suppress activation of T cells and professional APCs. Adenosine deaminase (ADA) counters this effect by catabolizing adenosine. This regulatory mechanism has not been tested in a disease model in vivo. Questions also remain as to which cell types are most sensitive to this regulation and whether its dysregulation contributes to any autoimmune conditions. We approached this issue using the NOD model. We report that ADA is upregulated in NOD dendritic cells, which results in their exuberant and spontaneous activation. This, in turn, triggers autoimmune T cell activation. NOD DCs deficient in ADA expression have a greatly reduced capacity to trigger type I diabetes. We also provide evidence that although many cell types, particularly T cells, have been implicated as the suppression targets by adenosine in an in vitro setting, DCs also seem to be affected by this regulatory mechanism. Therefore, this report illustrates a role of ADA in autoimmunity and suggests a potential target for therapeutic intervention.

Diabetogenic CD8+ T cells are primed in the pancreatic lymph nodes (PLNs) by dendritic cells (DCs) carrying islet cell Ags. TLR signaling modifies DC function. The goal of this study was to determine the effect of TLR9 signaling on diabetogenic CD8+ T cell activation and the course of type 1 diabetes. We explored the effects of CpG oligonucleotide, TLR9 antagonists, and genetic TLR9 deficiency on the activation of diabetogenic CD8+ T cells. NOD bone marrow-derived DCs pulsed with freeze-thawed insulinoma cells in the presence of TLR9 agonist CpG and CD40 agonist induced diabetogenic CD8+ T cell activation. The addition of TLR9 antagonist oligodeoxynucleotide or chloroquine inhibited bone marrow-derived DCs activation and CD8+ T cell priming in response to CpG. CpG alone or with CD40 agonist induced CTL activity that triggered diabetes development in 8.3-TCR transgenic NOD mice. Oligodeoxynucleotide treatment of 8.3-TCR transgenic NOD mice delayed spontaneous diabetes development. Chloroquine treatment delayed the spontaneous onset of diabetes in NOD mice, coincident with the decreased activation of PLN DCs. TLR9−/− NOD mice had delayed onset of diabetes compared with TLR9−/+ NOD littermates. TLR9−/− NOD mice had lower levels of IFN-α in PLNs and decreased frequencies of plasmacytoid DCs and diabetogenic CD8+ T cells compared with NOD mice. We propose that TLR9 activation contributes to the spontaneous onset of diabetes in NOD mice by increasing IFN-α and promoting diabetogenic CD8 T cell activation.

Type 1 diabetes (T1D), an autoimmune disease once thought to be mediated exclusively by beta cell-specific CD4+ T cells, is now recognized as one in which autoreactive CD8+ T cells play a fundamental role. In the nonobese diabetic (NOD) mouse model, CD8+ T effector cells take centre stage in the destruction of pancreatic beta cells and contribute to sustaining islet inflammation. Recent investigations have elucidated the mechanisms underlying the activation, homing, and beta cell destructive properties of this type of cells. Another important area is the development and testing of novel preemptive or therapeutic "vaccines" that, by targeting effector and/or regulatory autoreactive CD8+ T cell specificities may be able to induce immunological tolerance to beta cells. In humans, our understanding of the role of CD8+ T cells in T1D is also growing, through genetic linkage analyses, as well as epitope identification and characterization of disease-relevant CD8+ T cell responses in patient blood samples. The following review discusses these important advances and how they can converge towards the goal of developing an antigen-specific immunotherapy for T1D.

Adenosine is a suppressive agent that protects the host from excessive tissue injury associated with strong inflammation. In tissue stress, higher levels of adenosine signal through adenosine receptors to exert strong anti-inflammatory effects, and thus protect host cells. Existing evidence also suggests that elevated adenosine potently down-regulates the activation of lymphocytes during inflammation. This notion, however, is in contrast with another basic observation that the immune system is highly activated precisely under the same circumstances against pathogens. In this study, we show that inflammatory responses of dendritic cells (DCs) are highly sensitive to adenosine suppression. However, they intrinsically carry high adenosine deaminase activity, which in turn degrades and removes adenosine from the surroundings, cutting off DCs from the suppression. This regulatory mechanism is important in DC responses to pathogen-associated molecular patterns and their activation of T cells. Our findings suggest a mechanism that DCs maintain their hyperreactive state in inflammation despite the general state of suppression, and reveal a regulatory role of adenosine deaminase in DC innate immune responses.

Synucleins (including α-, β- and γ-synucleins) are a group of proteins that are highly expressed in the central nervous system. Alpha-synuclein, in particular, has been implicated in the pathogenesis of neurodegenerative disorders known as synucleinopathies. The function of these proteins in other organ systems is largely unknown. Some studies have demonstrated expression of α-synuclein on peripheral blood mononuclear cells (PBMC), including B and T lymphocytes, NK cells and monocytes; and its expression has been shown to be higher in PBMCs of individuals with Parkinson’s compared to healthy controls. We have recently shown that α-synuclein-deficiency is associated with marked defect in development of mature B and T lymphocytes. In particular, we showed enhanced negative selection of developing thymic T cells in the absence α-synuclein. Furthermore, we demonstrated that α-synuclein-deficiency is associated with an impaired IgG response to T cell-dependent antigens. Here we used age and sex-matched α-synuclein knock-out (KO) and wild type (WT) mice to further investigate the lineage differentiation and activation of T cells. We found that few splenic T cells that develop in KO mice contain a higher percentage of CD8+ T cell expressing early activation markers CD69 (7.6 ± 0.09 for KO vs. 5.3 ± 0.23 for WT, p=0.005, figure 1A) and CD49d (12.97 ± 0.3 for KO vs. 7.32 ± 0.6 for WT, p=0.006, figure 1A). A similar trend was noted for CD4+ CD49d+ T cells, although the difference did not reach statistical significance (23.90 ± 3.48 for KO vs.13.23 ± 0.73 for WT, p=0.086, figure 1A). No difference was noted in the expression of late activation marker CD44, and lymph node homing marker CD62L. This was associated with significantly increased IL-2 production from KO CD4+ T cells (OD 2.70 ± 0.12 for KO vs.1.05 ± 0.39 for WT, p=0.002, figure 1B) and a trend for increased IFN-γ production from KO CD4+ T cells (OD 2.89 ± 0.33 for KO vs.2.12 ± 0.59 for WT, p=0.12) after in vitro activation with anti-CD3/anti-CD28 beads. Interestingly, In vitro activation of splenic CD4+ T cells resulted in significantly reduced IL-4 production from KO T cells (OD 0.20 ± 0.14 for KO, vs. 0.74 ± 0.31 for WT, p=0.05, Figure 2) suggesting a defective Th2 differentiation in KO CD4+ T cells. Further flow cytometric analysis of T cells showed that while thymic Foxp3+ CD4+ regulatory T cells are significantly reduced in KO mice (3.07 ±0.35 for KO vs. 5.00 ± 0.87 for WT, p=0.02, Figure 3), the percentage of splenic Foxp3+ CD4+ T cells is higher in the KO mice compared to WT mice (18.33 ± 4.90 for KO vs.10.33 ± 1.39 for WT, p=0.05, Figure 3). No difference was noted among NK cells from KO and WT mice. In summary, we demonstrate a role for α-synuclein in lineage differentiation and function of T cells. While α-synuclein-deficiency leads to a significant defect in development of mature T cells, the small population of cells that do mature, express higher levels of early activation markers, and produce higher levels of IL-2 upon antigenic stimulation. Of interest, these cells are defective in IL-4 production. Additionally, we also show that α-synuclein-deficiency is associated with a higher percentage of peripheral CD4+ Foxp3+ T cells, a finding that might be explained by higher levels of IL-2 production by α-synuclein-deficient CD4+ T cells, although a direct effect of α-synuclein on the survival of regulatory T cells cannot be excluded. The underlying mechanism for the function α-synuclein in development and function of T cells is subject of future studies. Disclosures No relevant conflicts of interest to declare.

Synucleins (including α-, β- and γ-synucleins) are a group of proteins that are expressed at high levels in the central nervous system. The physiologic function of these proteins is unknown. Alpha-synuclein has been implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia, as it is highly expressed in the Lewy bodies from both disorders. The expression of α-synuclein in hematopoietic system has been shown in erythroid precursors and megakaryocytes in bone marrow, as well as erythrocytes and platelets in peripheral blood. Moreover, some studies demonstrated the expression of α-synuclein on peripheral blood mononuclear cells (PBMC), including B and T lymphocytes, NK cells and monocytes; and its expression is shown to be higher in PBMCs of individuals with Parkinson's disease compared to healthy controls. In order to study the role of α-synuclein in development of different hematopoietic elements, we compared bone marrow, peripheral blood and lymphoid organs of age and sex-matched α-synuclein knock-out (KO) mice and wild type (WT) animals of the same genetic background (n=10). Flow cytometric analysis of bone marrow elements did not show differences in the percentages and absolute numbers of erythroid, megakryocytic and myeloid lineages (data not shown). However, differential complete blood cell count (CBC) showed statistically significant decrease in red blood cell (RBC) count, hemoglobin (Hb) and hematocrit (Hct) in KO mice compared to WT mice. No difference was noted in other RBC indices (Table 1). However, platelets were smaller in KO mice as measured by the mean platelet volume (MPV). There was no difference in the number of platelets and white blood cell (WBC) counts. There was a significant reduction in the percentage of circulating lymphocytes, and associated increase in the percentage of neutrophils and monocytes in KO mice compared to WT mice, although the difference in the number of lymphocytes did not reach statistical significance (Table 1). Flow cytometric analysis of T lymphocytes in thymus and peripheral lymphoid organs demonstrated marked defect in development of mature T cells. There was a significant increase in the number of double negative thymocytes in KO mice associated with significant decrease in the number of single positive T cells. Furthermore, splenic CD4+ and CD8+ T cells were markedly decreased in KO mice, indicating that α-synuclein is required for T cell development (Table 2). In summary, our findings indicate an absolute requirement for α-synuclein in development of mature T lymphocytes. The underlying mechanism for this function is subject of future studies. Moreover, while α-synuclein-deficiency does not affect the development of myeloid lineage and platelets, lack of this protein is associated with lower number of erythrocytes, suggesting its role in development and/or survival red blood cells. Disclosures: No relevant conflicts of interest to declare.

Introduction Synucleins comprise a family of small proteins that were first identified in normal and neoplastic brain tissues. As a key component of the Lewy body, a-synuclein plays crucial roles in Parkinson disease and other dementias, and mediates neurotransmitter trafficking. The role of a-synuclein in hematopoiesis is largely unknown; however, in the hematopoietic system, a-synuclein is present in megakaryocytes, platelets, erythroid precursors and erythrocytes. In addition, it has also been detected by RT-PCR in monocytes, T-, B-, and NK-cells. Of interest, there is reduced expression of a-synuclein in the megakaryocytes of myeloproliferative neoplasm (MPN), but not normal reactive marrow or myelodysplastic syndrome, suggesting that a-synuclein could play an important role in the pathogenesis of MPN; while there is increased expression in blasts of megakaryoblastic leukemia. In this study, we utilized a-synuclein-/- mice as a model to investigate the role of a-synuclein in hematopoiesis. We identified an unexpected role of a-synuclein in B cell development and maturation. Methods Age- and sex-matched a-synuclein-/- mice and wild type mice (6-week-old; N=10 each group) were purchased from The Jackson Laboratory (Bar Harbor, ME). Bone marrow cells, splenocytes, and lymph nodes were harvested and flow cytometry analysis performed looking at B cell markers. Histological examination of bone marrow, lymph nodes, and spleen were also performed. Results B220lo immature B cells were comparable between WT and KO mice (WT: 2.62±0.30% vs. KO: 3.55±0.67%, Figure1 Aand Table 1); similarly, pre-B cells identified as B220loCD43+ population were comparable between the two groups (Figure 1B). However, when IgD was applied to separate B220hi population into IgD+ and IgD- subsets, circulating B cells (B220hiIgD+ subset), were significantly reduced by 5-fold in KO mice compared to WT (KO: 0.12±0.05% vs. WT: 0.59±0.37%, p=0.02), whereas B220hiIgD- subset representing transitional B cells was similar between WT and KO mice (WT: 1.54±0.22% vs. KO: 2.09±0.70%, Figure1 A and Table 1). Therefore, although early B cell development is not affected, the number of mature B cells in bone marrow is reduced in a-synuclein deficient mice. In spleen, there was a marked reduction in the number of B cells compared to WT: 5.5+1.6% vs. 14.0+1.9%, respectively (Figure 2A and Table 1). The absolute number of B cells was more drastically reduced in KO mice as the total number of splenocytes was only half of that in WT (Figure 2B and data not shown). Histologically, white pulp areas in KO mice were disorganized compared to WT mice (Figure 2B). These results collectively show that the number and distribution of B cells in spleen is regulated by a-synuclein. On the other hand, though the percentage of B cells in lymph nodes was comparable between WT and KO mice, the absolute number of B cells was lower in KO mice and morphologically the lymph nodes from KO mice were smaller than those from WT mice (Table 1, Figure 3A and data not shown). Normal lymph node cortical/ follicular architecture was missing in KO mice compared to WT controls (Figure 3B). The number of follicles in KO mice was 5-fold lower than that WT controls (WT: 5±0.2/HPF vs. KO: 1±0.3/HPF, p=0.001). Conclusion Our data shows that the number and localization of mature B cells in spleen and lymph nodes is in part regulated by a-synuclein. This is the first report to implicate an important role of a-synuclein in B cell development. The mechanism of a-synuclein regulation in B cells is under investigation. Disclosures: No relevant conflicts of interest to declare.

Engagement of peptide-MHC by the TCR induces a conformational change in CD3ε that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3ε ITAM, has been implicated in synapse formation and T cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3ε PRS independent. In this study, we show that the CD3ε PRS is necessary for peptide-MHC-induced phosphorylation of CD3ε and for recruitment of protein kinase Cθ to the immune synapse in differentiated CD8+ T lymphocytes. However, whereas these two events are dispensable for functional T cell responsiveness to high-avidity ligands, they are required for responsiveness to low-avidity ones. Thus, in at least certain T cell clonotypes, the CD3ε PRS amplifies weak TCR signals by promoting synapse formation and CD3ε phosphorylation.