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The caspase recruitment domain family member (CARD)11-Bcl10-Malt1 signalosome controls TGF-β–activated kinase 1 (TAK1) activation and regulates BCR-induced NF-κB activation. In this study, we discovered that CARD19 interacted with TAK1 and inhibited TAB2-mediated TAK1 ubiquitination and activation. Although CARD19 deficiency in mice did not affect B cell development, it enhanced clonal deletion, receptor editing, and anergy of self-reactive B cells, and it reduced autoantibody production. Mechanistically, CARD19 deficiency increased BCR/TAK1-mediated NF-κB activation, leading to increased expression of transcription factors Egr2/3, as well as the E3 ubiquitin ligases c-Cbl/Cbl-b, which are known inducers of B cell tolerance in self-reactive B cells. RNA sequencing analysis revealed that although CARD19 deficiency did not affect the overall Ag-induced gene expression in naive B cells, it suppressed BCR signaling and increased hyporesponsiveness of self-reactive B cells. As a result, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus. In summary, CARD19 negatively regulates BCR/TAK1-induced NF-κB activation and its deficiency increases Egr2/3 and c-Cbl/Cbl-b expression in self-reactive B cells, thereby enhancing B cell tolerance.

C-type lectin like molecule-1 (CLL1) is preferentially expressed on acute myeloid leukemia (AML) stem cells and AML blasts, and can be considered as AML-associated antigen. Anti-CLL1-based CAR-T cells exhibited effective tumor killing capacity in vitro and in AML-bearing mouse model. In this report, eight children with relapsed or refractory AML (R/R-AML) were recruited for a phase 1/2 clinical trial of autologous anti-CLL1 CAR-T cell immunotherapy. The objectives of this clinical trial were to evaluate the safety and the anti-AML responses after CLL1-CAR-T cell treatment, with long-term prognosis within those patients who did not receive allogeneic hematopoietic stem cells transplantation (allo-HSCT) as an additional aim. These R/R-AML patients received one dose of autologous CLL1-CAR-T cells after lymphodepletion conditioning. Grade 3–4 hematologic adverse events were observed post CAR-T cell infusion. Meanwhile, grade 1–2 cytokine release syndrome (CRS) was observed but without any lethal events. 4 out of 8 AML patients achieved incomplete remission (CRi) and minimal residual disease (MRD) negativity, 2 patients with CRi but MRD positivity, and 2 patients with decreased AML burden and CLL1 positive AML blast clearance. These results suggested that anti-CLL1-based CAR-T cell immunotherapy can be considered as a well-tolerated and effective option for treating children with R/R-AML.

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 TLR-mediated activation of MAPK/AP-1 and IKK/NF-κB signaling cascades. At cellular levels, VISTA regulated the effector functions of myeloid-derived suppressor cells and tolerogenic dendritic cell (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 stimulatory tumor microenvironment that promoted T-cell infiltration and activation. We conclude that VISTA is a critical myeloid cell–intrinsic immune-checkpoint protein and that the reprogramming of tolerogenic myeloid cells following VISTA blockade promotes the development of T cell–mediated antitumor immunity.

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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.

The switch/sugar nonfermenting (SWI/SNF) family of chromatin remodeling complexes have been implicated in normal hematopoiesis. The ARID2 protein is a component of the polybromo-associated BAF (PBAF), one of the two main SWI/SNF complexes. In the current study, we used a conditional Arid2 knockout mouse model to determine its role in normal hematopoiesis. We found that the loss of Arid2 has no discernable effects on steady-state hematopoiesis, with the exception of a modest effect on erythropoiesis. On bone marrow transplantation, however, the loss of Arid2 affects HSC differentiation in a cell-autonomous manner, resulting in significant decreases in the ability to reconstitute the lymphoid lineage. Gene expression analysis of Arid2 knockout cells revealed enrichment of myeloid-biased multipotent progenitor (MPP) cell signatures, while the lymphoid-biased MPPs are enriched in the wild type, consistent with the observed phenotype. Moreover, Arid2 knockout cells revealed enrichment of inflammatory pathways with upregulation of TLR receptors, as well as downstream signaling cascade genes. Furthermore, under lymphocyte-biased growth conditions in vitro, Arid2 null bone marrow cells have significantly impaired proliferation, which decreased further on lipopolysaccharide stimulation. Overall, these data suggest that the loss of Arid2 impairs HSC differentiation ability, and this effect may be mediated through upregulation of inflammatory pathways.

Recently, two types of common sensors, LiDAR and Camera, show significant performance on all tasks in 3D vision. LiDAR provides accurate 3D geometry structure, while camera captures more scene context and semantic information. The fusion of two different sensor becomes a fundamental and common idea to achieve better performance. To give a thorough cognition of the complementary and boosting about two kind of sensors. This paper briefly reviews the fusion and enhancement systems between both two sensors in the field of depth completion, 3D object detection, 2D\3D semantic segmentation and 3D object tracking. Meanwhile, the state of art fusion algorithms is quantitatively demonstrated, in this paper, based on the in KITTI widely-used public dataset. Furthermore, the technical challenge and the future potential of LiDAR and camera fusion are also discussed.

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6–mediated Th17 cell differentiation and impaired IL-6–induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD.

Developing solid polymer electrolytes (SPEs) is a promising approach to realize practical dendrite-free lithium metal batteries (LMBs). Tuning the nanoscale polymer network chemsitry is of critical importance for SPE design. In this work, we took lessons from the rubber chemistry and developed a series of comb-chain crosslinker-based SPEs (ConSPEs) using a preformed polymer as the multifunctional crosslinker. The high-functionality crosslinker increased the connectivity of nanosized cross-linked domains, which led to a robust network with dramatically improved toughness and superior lithium dendrite resistance even at a current density of 2 mA cm

Compared with linear homopolymer and block copolymer electrolytes, network-based solid polymer electrolytes (SPEs) demonstrated much improved lithium dendrite resistance and cell cyclability in lithium metal batteries (LMBs), suggesting that polymer chain architecture plays a pivotal role in SPE design. To further improve the state-of-the-art performance of SPEs, in this work, we report a novel interpenetrating network (IPN) SPE that is comprised of poly (ethylene oxide) and poly (propylene carbonate) (PPC) chains. The synthesis of the SPE is based on the crosslinking of octakis(3-glycidyloxypropyldimethylsiloxy)octasilsesquioxane and amine-terminated polyethylene glycol while incorporating linear PPC with a controlled concentration. The IPN SPEs showed improved ionic conductivity and a nearly threefold increase of lithium ion transference number. The optimized SPE could be stably cycled for over 300 ​h ​at a harsh current density of 1.5 ​mA ​cm−2 in the galvanostatic symmetrical cell plating/stripping experiment. Scanning electron microscopy (SEM) revealed that incorporation of PPC could effectively reduce the uneven lithium deposition while X-ray photoelectron spectroscopy (XPS) was used to confirm the active components that are associated with the stable solid electrolyte interface. Full LMBs fabricated using the IPN SPE, a lithium metal anode and a LiFePO4 cathode delivered high specific capacity of over 160 ​mAh g−1 at 0.1 C and over 95 ​mAh g−1 at 2 ​C with an excellent Coulombic efficiency and lifetime. This work demonstrated a new pathway of SPE molecular design towards high performance SPEs for LMB applications.

Replacing liquid electrolytes with solid-state electrolytes is a promising approach to achieving practical applications of lithium metal batteries (LMBs). In this work, ionic liquid N-methyl-N-propyl-pyrrolidinium bis(fluorosulfonyl)imide (Pyr13FSI) was introduced into a hybrid network to obtain a series of gel polymer electrolytes (GPEs). Mechanical and electrochemical properties of the GPEs were tuned through controlling the network structure and ionic liquid contents, and ionic conductivity higher than 1 ​mS ​cm-1 at room temperature was achieved. The newly developed GPEs are flame-retardant and show excellent thermal and electrochemical stability as well as ultra-stability with lithium metal anode. Symmetrical lithium cells with the GPEs exhibit a stable cycling over 6800 ​h ​at a current density of 0.1 ​mA ​cm-2 and stable lithium stripping-plating at 1 ​mA ​cm-2, the highest current density reported for ionic liquid-based GPEs. Detailed correlation between mechanical properties, ionic conductivity and cell short-circuit time is discussed. Moreover, Li/LiFePO4 batteries with the obtained GPEs exhibit desirable cycling stability and rate performance over a wide temperature range from 0 ​°C to 90 ​°C, further suggesting that this new hybrid-network/ionic liquid GPE system has great potential for practical applications in next generation LMBs.

Acute myeloid leukemia (AML) is driven by mutations that occur in numerous combinations. A better understanding of how mutations interact with one another to cause disease is critical to developing targeted therapies. Approximately 50% of patients that harbor a common mutation in NPM1 (NPM1cA) also have a mutation in the cohesin complex. As cohesin and Npm1 are known to regulate gene expression, we sought to determine how cohesin mutation alters the transcriptome in the context of NPM1cA. We utilized inducible Npm1(cAflox/+) and core cohesin subunit Smc3(flox/+) mice to examine AML development. While Npm1(cA/+);Smc3(Δ/+) mice developed AML with a similar latency and penetrance as Npm1(cA/+) mice, RNA-seq suggests that the Npm1(cA/+); Smc3(Δ/+) mutational combination uniquely alters the transcriptome. We found that the Rac1/2 nucleotide exchange factor Dock1 was specifically upregulated in Npm1(cA/+);Smc3(Δ/+) HSPCs. Knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis only in Npm1(cA/+);Smc3(Δ/+) AML. Higher Rac activity was also observed in Npm1(cA/+);Smc3(Δ/+) vs. Npm1(cA/+) AMLs. Importantly, the Dock1/Rac pathway is targetable in Npm1(cA/+);Smc3(Δ/+) AMLs. Our results suggest that Dock1/Rac represents a potential target for the treatment of patients harboring NPM1cA and cohesin mutations and supports the use of combinatorial genetics to identify novel precision oncology targets.

Natural killer (NK) cells generate proinflammatory cytokines that are required to contain infections and tumor growth. However, the posttranscriptional mechanisms that regulate NK cell functions are not fully understood. Here, we define the role of the microRNA cluster known as Mirc11 (which includes miRNA-23a, miRNA-24a, and miRNA-27a) in NK cell–mediated proinflammatory responses. Absence of Mirc11 did not alter the development or the antitumor cytotoxicity of NK cells. However, loss of Mirc11 reduced generation of proinflammatory factors in vitro and interferon-γ–dependent clearance of Listeria monocytogenes or B16F10 melanoma in vivo by NK cells. These functional changes resulted from Mirc11 silencing ubiquitin modifiers A20, Cbl-b, and Itch, allowing TRAF6-dependent activation of NF-κB and AP-1. Lack of Mirc11 caused increased translation of A20, Cbl-b, and Itch proteins, resulting in deubiquitylation of scaffolding K63 and addition of degradative K48 moieties on TRAF6. Collectively, our results describe a function of Mirc11 that regulates generation of proinflammatory cytokines from effector lymphocytes.

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. Regulatory T (Treg) cells are a subpopulation of CD4 T cells that modulate immune system by suppressing or down-regulating immune response and play an important role in immune homeostasis. However, the role of Treg cells in controlling PF4/heparin-specific antibody production is not known. Here we found that FoxP3-deficient mice, which have no functional Treg cells, spontaneously generated PF4/heparin-specific antibodies as early as three weeks after birth. Similarly, following transplantation with bone marrow cells from FoxP3-deficient mice, Rag1-deficient mice that have no endogenous B cells and T cells spontaneously produced PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received wild-type bone marrow cells failed to produce PF4/heparin-specific antibodies. In addition, adoptively transferred Treg cells prevented spontaneous production of PF4/heparin-specific antibodies in FoxP3-deficient mice. Adoptively transferred Treg cells, not conventional CD4 T cells, also suppressed PF4/heparin complex-induced production of PF4/heparin-specific IgGs in wild-type mice. Treg cells suppress immune response mainly through releasing anti-inflammatory cytokines, such as interleukin-10 (IL-10). Deficiency of IL-10 led to spontaneous production of PF4/heparin-specific antibodies in mice. Moreover, BM chimeric mice with CD4 T cell-specific deletion of IL-10 increased PF4/heparin-specific IgG production following PF4/heparin complex challenge. Taken together, these findings demonstrate that Treg cells play an important role in suppressing PF4/heparin-specific antibody production in mice. Disclosures Haribhai: AbbVie Inc.: Employment. Aster:BloodCenter of Wisconsin: Patents & Royalties.

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 TLR-mediated activation of MAPK/AP-1 and IKK/NF-κB signaling cascades. At cellular levels, VISTA regulated the effector functions of myeloid-derived suppressor cells and tolerogenic dendritic cell (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 stimulatory tumor microenvironment that promoted T-cell infiltration and activation. We conclude that VISTA is a critical myeloid cell–intrinsic immune-checkpoint protein and that the reprogramming of tolerogenic myeloid cells following VISTA blockade promotes the development of T cell–mediated antitumor immunity.

JMJD1C, a member of the lysine demethylase 3 family, is aberrantly expressed in mixed lineage leukemia (MLL) gene-rearranged (MLLr) leukemias. We have shown previously that JMJD1C is required for self-renewal of acute myeloid leukemia (AML) leukemia stem cells (LSCs) but not normal hematopoietic stem cells. However, the domains within JMJD1C that promote LSC self-renewal are unknown. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) negative-selection screening and identified a requirement for the catalytic Jumonji (JmjC) domain and zinc finger domain for leukemia cell survival in vitro and in vivo. In addition, we found that histone H3 lysine 36 methylation (H3K36me) is a marker for JMJD1C activity at gene loci. Moreover, we performed single cell transcriptome analysis of mouse leukemia cells harboring a single guide RNA (sgRNA) against the JmjC domain and identified increased activation of RAS/MAPK and the JAK-STAT pathway in cells harboring the JmjC sgRNA. We discovered that upregulation of interleukin 3 (IL-3) receptor genes mediates increased activation of IL-3 signaling upon JMJD1C loss or mutation. Along these lines, we observed resistance to JMJD1C loss in MLLr AML bearing activating RAS mutations, suggesting that RAS pathway activation confers resistance to JMJD1C loss. Overall, we discovered the functional importance of the JMJD1C JmjC domain in AML leukemogenesis and a novel interplay between JMJD1C and the IL-3 signaling pathway as a potential resistance mechanism to targeting JMJD1C catalytic activity.

Severe COVID-19 is associated with unprecedented thromboembolic complications. We found that hospitalized COVID-19 patients develop immunoglobulin Gs (IgGs) that recognize a complex consisting of platelet factor 4 and heparin similar to those developed in heparin-induced thrombocytopenia and thrombosis (HIT), however, independent of heparin exposure. These antibodies activate platelets in the presence of TLR9 stimuli, stimuli that are prominent in COVID-19. Strikingly, 4 out of 42 antibodies cloned from IgG1+ RBD-binding B cells could activate platelets. These antibodies possessed, in the heavy-chain complementarity-determining region 3, an RKH or Y5 motif that we recently described among platelet-activating antibodies cloned from HIT patients. RKH and Y5 motifs were prevalent among published RBD-specific antibodies, and 3 out of 6 such antibodies tested could activate platelets. Features of platelet activation by these antibodies resemble those by pathogenic HIT antibodies. B cells with an RKH or Y5 motif were robustly expanded in COVID-19 patients. Our study demonstrates that SARS-CoV-2 infection drives the development of a subset of RBD-specific antibodies that can activate platelets and have activation properties and structural features similar to those of the pathogenic HIT antibodies.

The resistance of synovial sublining macrophages to apoptosis has a crucial role in joint inflammation and destruction in rheumatoid arthritis (RA). However, the underlying mechanism is incompletely understood. Here we report that inactivation of the pro-apoptotic BCL-2 family protein BAD is essential for survival of synovial sublining macrophage in RA. Genetic disruption of Bad leads to more severe joint inflammation and cartilage and bone damage with reduced apoptosis of synovial sublining macrophages in collagen-induced arthritis (CIA) and TNFα transgenic (TNF-Tg) mouse models. Conversely, Bad3SA/3SA mice, in which BAD can no longer be inactivated by phosphorylation, are protected from collagen-induced arthritis. Mechanistically, phosphorylation-mediated inactivation of BAD specifically protects synovial sublining macrophages from apoptosis in highly inflammatory environment of arthritic joints in CIA and TNF-Tg mice, and in patients with RA, thereby contributing to RA pathology. Our findings put forward a model in which inactivation of BAD confers the apoptosis resistance on synovial sublining macrophages, thereby contributing to the development of arthritis, suggesting that BAD may be a potential therapeutic target for RA.

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 transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through AktS473-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-AktS473-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells.

Solid polymer electrolytes (SPEs) are one of the most promising solutions to the safety issues of lithium batteries. Understanding the morphology and dynamic effects on the ion transport properties of SPEs would be essential for future SPE design. In this article, using poly(ethylene oxide) (PEO) as an example, we focus on morphology control in semicrystalline SPEs. We show that the effect of semicrystallinity can be quantitatively separated into volume, structure and dynamic effects. We further demonstrate that morphological control plays an important role in ion transport control in semicrystalline SPE systems.

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.

Aim The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered. Main methods We detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups. Key findings Here, we determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, we found that miR-315 regulates synaptic structure and transmission by targeting dfmr1. Significance These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression.

The Thomsen-Friedenreich antigen (TF-antigen) occurs during exposure of the underlying Core-1 disaccharide (Gal-beta(1,3)GalNAc) through the loss of its capping sialic acid (Sia). Exposure of the cryptic TF-antigen occurs during inflammation, during acute infections with influenza viruses or bacteria, in malignancies, and is associated with thrombocytopenia. Exposure of the TF-antigen on circulating blood cells, including platelets and red blood cells (RBC), can lead to severe thrombocytopenia or hemolysis in hemolytic uremic syndrome and other immune diseases. Recent data suggest that altered Sia may cause platelet destruction because treatment with the sialidase inhibitor Tamiflu increases platelet count in healthy and thrombocytopenic patients. In humans, genetic mutations involving Sia synthesis and transport, and atypical cell surface sialylation, unrelated to any genetic mutation, are associated with reduced platelet count, supporting the role of Sia in regulating platelet count. Immune cells, including classical dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs), and subsets of T cells (CD8+, CD4+, and Treg cells) can also affect immune thrombocytopenia pathogenesis. Like many other immune cells, cDCs, and pDCs express Siglecs (sialic-acid-binding immunoglobulin-like lectins), which often contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that act as immunosuppressors. Whether BM immune cells monitor MKs via glycan-lectin receptors, including Siglecs and Sia interactions, to control platelet production is unclear. To investigate the role of the TF-antigen in thrombopoiesis, we generated St3gal1MK-KO mice (Pf4-Cre) that display increased TF-antigen specifically in megakaryocytes (MK) and platelets. St3gal1MK-KO mice developed significant thrombocytopenia, but had normal platelet half-life, suggesting that the TF-antigen affected BM thrombopoiesis. In vitro MK maturation and proplatelet production from primary ST3Gal1MK-KO mouse BM cells were also normal, pointing to extrinsic factors in the BM environment affecting thrombopoiesis. Platelet counts of St3gal1MK-KO mice were restored to wild-type levels by 1) crossing St3gal1MK-KO mice with Jak3KO mice that have impaired of lymphoid cell development, 2) by treatment with anti-inflammatory dexamethasone, and 3) treatment with a depleting anti-CD4 antibody. Immunofluorescence staining of the St3gal1MK-KO BM revealed proplatelet structures positive for GPIba+ and the TF-antigen, being infiltrated by mononuclear cells resembling lymphocytes. We speculated that immune cells surveil megakaryocytes to control thrombopoiesis. Bulk RNAseq of CD4+ cells in St3gal1MK-KO BM confirmed a population bias for Type I interferon (IFN-I)-releasing pDCs, a cell type regulated by unique sialic acid binding lectins (Siglecs). Inhibition of IFN-I activity, by a blocking receptor antibody improved platelet counts in St3gal1MK-KO mice. Co-cultures of pDCs with MKs show inhibited pro-platelet formation when TF-antigen is present on MKs with elevated IFN-I levels. Gene set enrichment analysis of BM pDCs single cell RNASeq (scRNAseq) data further confirmed that TF-antigen exposure by MKs up-regulates IFN-I transcripts. scRNAseq also reveals a new population of immune cells with pDC transcript signature and concomitant upregulation of immunoglobulin re-arrangement gene transcripts Igkc and Ighm. In conclusion, the data shows that recognition of aberrant MK sialylation by pDCs regulates thrombopoiesis through IFN-I secretion. Disclosures No relevant conflicts of interest to declare.

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.

In this paper, cost based droop schemes are proposed, to minimize the total active power generation cost in an islanded microgrid (MG), while the simplicity and decentralized nature of the droop control are retained. In cost based droop schemes, the incremental costs of distributed generators (DGs) are embedded into the droop schemes, wherethe incremental cost is a derivative of the DG cost function with respect to output power. In the steady state, DGs share a single common frequency, and cost based droop schemes equate incremental costs of DGs, thus minimizing the total active power generation cost, in terms of the equal incremental cost principle. Finally, simulation results in an islanded MG with high penetration of intermittent renewable energy sources are presented, to demonstrate the eectiveness, as well as plug and play capability of the cost based droop schemes.

Pre-T cell receptor (TCR) signaling is required for pre-T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre-TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, that are important to mediate PKC activation and intracellular Ca2+ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR-mediated signaling, development and T-cell activation, but the role of PLCγ1 in pre-TCR signal transduction and pre-T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre-T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre-TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre-T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre-TCR mediated Ca2+ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre-TCR mediated signal transduction and pre-T cell development.

The three major Ras members, Kras, Hras, and Nras, are highly homologous and individual Ras genes can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological functions of this Ras family member. In this study, we generated and examined mice with hematopoietic-specific deletion of Kras and bone marrow (BM) chimeric mice with B cell–specific targeted deletion of Kras. Hematopoietic-specific deletion of Kras impaired early B cell development at the pre–B cell stage and late B cell maturation, resulting in the reduction of BM pre–, immature, and mature B cells and peripheral follicular, marginal zone, and B1 mature B cells. In contrast, Kras deficiency did not affect T cell development. Studies of BM chimeric mice with B cell–specific deletion of Kras demonstrated that Kras deficiency intrinsically impaired B cell development. Kras deficiency reduced BCR-induced B cell proliferation and survival. Furthermore, Kras deficiency specifically impaired pre–BCR- and BCR-induced activation of the Raf-1/MEK/ERK pathway in pre–B and mature B cells, respectively. Thus, Kras is the unique Ras family member that plays a critical role in early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway.

Solid polymer electrolytes (SPEs) are promising for the next-generation all-solid-state lithium metal battery (LMB) applications. The effect of semicrystalline structure on ion transport is two-fold: the crystalline domains provide a tortuous diffusion pathway (structural effect) while the tethered chains can cause sluggish ion diffusion (dynamic effect). These two effects are intertwining in a bulk semicrystalline SPE system. In this work, we fabricate a series of polymer single crystal (PSC)-based SPEs by infiltrating lithium bis(trifluoromethylsulfonyl) imide (LiTFSI) into poly(ethylene oxide) (PEO) PSC films. The obtained SPE films show anisotropic conducting behavior with the in-plane conductivity being one to three orders of magnitudes greater than the through-plane conductivity, which can be attributed to the PSC-guided ion transport. The room temperature in-plane conductivity of the PSC SPE can be comparable to its amorphous counterpart, indicating that the tortuosity effect plays a major role in the reduced ionic conductivity of semicrystalline SPEs. PSC-LiTFSI superstructure is observed in the PSC SPEs due to selective perforation of the crystalline PEO lamellae by LiTFSI salts. This work demonstrates the importance of the crystalline morphology on the ion transport in semicrystalline SPEs.

Introduction: V-domain Ig suppressor of T-cell activation (VISTA, gene Vsir) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T-cell activation. Vsir-/- mice developed chronic inflammatory phenotypes, and Vsir-/- CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Our recent study (Li et al., Sci Rep 2017) has identified a novel role of VISTA as a critical regulator of IL-23/IL-17 inflammatory axis induced by Toll-like receptor (TLR) stimulation. The molecular mechanisms by which VISTA inhibits TLR signaling remain to be elucidated. Methods: Peritoneal macrophages from WT or Vsir-/- mice were isolated and stimulated with TLR agonists. Alternatively, human monocyte THP-1 cells overexpressing VISTA were stimulated by TLR2 agonist Pam3CSK4. The activation of TLR signaling pathways and the production of inflammatory cytokines were examined by Western blotting, gel shift assay, or ELISA. Tumor-bearing mice were treated with VISTA-specific monoclonal antibody (mAb) and a peptide vaccine containing TLR agonists. The production of inflammatory cytokines and chemokines was examined via RT-PCR and ELISA. Results: VISTA downregulates Toll-like receptor (TLR)/TRAF6/TAK1-mediated signaling pathway via promoting K48-linked polyubiquitination and proteasomal degradation of TRAF6 and inhibiting K63-linked polyubiquitination and activation of TRAF6. VISTA blockade by an antibody or genetic deletion augments the activation of MAPKs/AP-1 and IKK/NF-kB signaling cascades in myeloid cells and induces the accumulation of inflammatory cytokines and chemokines within tumor tissues. Inflamed tumor tissues promote the infiltration and effector function of tumor-reactive CD8+ T cells. TLR/TRAF6-mediated inflammatory responses promote the antitumor efficacy of VISTA-blocking antibodies and contribute to a synergistic outcome when VISTA blockade is combined with a TLR agonistic vaccine. Conclusions: Our study establishes that VISTA critically regulates the inflammatory responses of myeloid cells mediated by TLR signaling. Unlike targeting other immune checkpoint proteins, the therapeutic efficacy of VISTA inhibition benefits from the activation of myeloid cells and early induction of inflammatory cytokines may predict positive clinical responses. Citation Format: Wenwen Xu, Yongwei Zheng, Juan Zhou, Ying Yuan, Hieu Minh Ta, Jun Dong, Halli E. Miller, Michael Olson, Kamalakannan Rajasekaran, Marc S. Ernstoff, Demin Wang, Subramaniam Malarkannan, Li Wang. Immune checkpoint protein VISTA controls antitumor immunity via regulating Toll-like receptor signaling and myeloid cells-mediated inflammation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A82.

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., B cell response and antibody production are generally facilitated by CD4+ follicular helper (Tfh) cells. Here the authors identify a subset of CXCR5+PD1+CD8+ Tfh cells that is normally suppressed by STAT5 signaling, so that STAT5 deficiency in mice increases the number of these CD8+ Tfh cells and induces concomitant production of autoantibodies.

Nanocomposite polymer electrolytes (CPEs) are promising materials for all-solid-state lithium metal batteries (LMBs) due to their enhanced ionic conductivities and stability to the lithium anode. MXenes are a new two-dimensional, 2D, family of early transition metal carbides and nitrides, which have a high aspect ratio and a hydrophilic surface. Herein, using a green, facile aqueous solution blending method, we uniformly dispersed small amounts of Ti3C2Tx into a poly(ethylene oxide)/LiTFSI complex (PEO20-LiTFSI) to fabricate MXene-based CPEs (MCPEs). The addition of the 2D flakes to PEO simultaneously retards PEO crystallization and enhances its segmental motion. Compared to the 0D and 1D nanofillers, MXenes show higher efficiency in ionic conductivity enhancement and improvement in the performance of LMBs. The CPE with 3.6 wt% MXene shows the highest ionic conductivity at room temperature (2.2 × 10−5 S m−1 at 28 °C). An LMB using MCPE with only 1.5 wt% MXene shows rate capability and stability comparable with that of the state-of-the-art CPELMBs. We attribute the excellent performance to the 2D geometry of the filler, the good dispersion of the flakes in the polymer matrix, and the functional group-rich surface.

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.

We prepared light-responsive shape memory polymer (SMP) micropillars (10 μm in diameter, 40 μm in height, and 20 μm in pillar-to-pillar distance) in a hexagonal array mixed with 0.1–0.2 mol% gold nanorods (AuNRs) by replica molding from a poly(dimethylsiloxane) (PDMS) mold. The addition of AuNRs did not change the glass transition temperature (Tg) of SMPs much, for example, from 47.8 °C (pristine SMP) to 42.1 °C (SMP with 0.2 mol% AuNRs). The pillars were bent to different angles by controlling the force applied on top of the sample above its Tg, followed by cooling down to room temperature to lock the deformed structures. The bent pillars were nearly completely recovered to the straight ones when exposed to a visible laser. Recovery time as small as 5 s from 0.3 W green laser was demonstrated, depending on the loading of AuNRs, the bending angle, the Tg of composite material, and exposure dosage. Between bent and straight pillars, we demonstrated tunable transmittance in the visible wavelength and water contact angle from 160° (straight pillars) to 100° (completely collapsed pillars).

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., IL-7R activation drives early B cell development, but the signalling is unclear. Here the authors show PLCγ is involved in IL-7R-induced mTOR activation via a DAG/PKC-dependent pathway, and that double deficiency of the two PLCγ isoforms arrests B cell development at the pre-pro-B stage in mice.