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Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid,massively parallel profiling of transcriptomes. However,assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool,demuxlet,that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data,we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples,demuxlet correctly recovers the sample identity of<99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples. View Publication

Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively,an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with β5i and β6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells,and to a lesser extent in activated human T cells. Reversible,β5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells. View Publication

Mobile sensing based on the integration of microfluidic device and smartphone,so-called MS2 technology,has enabled many applications over recent years,and continues to stimulate growing interest in both research communities and industries. In particular,it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction,in this paper,we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit,which includes microfluidic chips,a smartphone-based imaging platform,the phone apps for image capturing and data analysis,and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore,neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications,we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus,this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. View Publication

Mucosal Langerhans cells (LCs) originate from pre-dendritic cells and monocytes. However,the mechanisms involved in their in situ development remain unclear. Here,we demonstrate that the differentiation of murine mucosal LCs is a two-step process. In the lamina propria,signaling via BMP7-ALK3 promotes translocation of LC precursors to the epithelium. Within the epithelium,TGF-β1 finalizes LC differentiation,and ALK5 is crucial to this process. Moreover,the local microbiota has a major impact on the development of mucosal LCs,whereas LCs in turn maintain mucosal homeostasis and prevent tissue destruction. These results reveal the differential and sequential role of TGF-β1 and BMP7 in LC differentiation and highlight the intimate interplay of LCs with the microbiota. View Publication

MicroRNAs play an important role in T cell responses. However,how microRNAs regulate CD8 T cell memory remains poorly defined. Here,we found that miR-150 negatively regulates CD8 T cell memory in vivo. Genetic deletion of miR-150 disrupted the balance between memory precursor and terminal effector CD8 T cells following acute viral infection. Moreover,miR-150-deficient memory CD8 T cells were more protective upon rechallenge. A key circuit whereby miR-150 repressed memory CD8 T cell development through the transcription factor c-Myb was identified. Without miR-150,c-Myb was upregulated and anti-apoptotic targets of c-Myb,such as Bcl-2 and Bcl-xL,were also increased,suggesting a miR-150-c-Myb survival circuit during memory CD8 T cell development. Indeed,overexpression of non-repressible c-Myb rescued the memory CD8 T cell defects caused by overexpression of miR-150. Overall,these results identify a key role for miR-150 in memory CD8 T cells through a c-Myb-controlled enhanced survival circuit. View Publication

We report that SHIP(-/-) mice,compared to SHIP(+/+) mice,are Th2 skewed with elevated serum IgE and twice as many splenic CD4(+) Th2 cells that,when stimulated with anti-CD3,produce more IL-4 and less IFN-$\gamma$. Exploring the reason for this Th2 skewing,we found that freshly isolated SHIP(-/-) splenic and bone marrow basophils are present in elevated numbers and secrete far more IL-4 in response to IL-3 or to Fc$\epsilon$RI stimulation than do WT basophils. These SHIP(-/-) basophils markedly skew wild-type macrophage colony stimulating factor-derived macrophages toward an M2 phenotype,stimulate OT-II CD4(+) Th cells to differentiate into Th2 cells,and trigger SHIP(+/+) B cells to become IgE-producing cells. All these effects are completely abrogated with neutralizing anti-IL-4 Ab. Exploring the cell signaling pathways responsible for hyperproduction of IL-4 by SHIP(-/-) basophils,we found that IL-3-induced activation of the PI3K pathway is significantly enhanced and that PI3K inhibitors,especially a p110$\alpha$ inhibitor,dramatically suppresses IL-4 production from these cells. In vivo studies,in which basophils were depleted from mast cell-deficient SHIP(+/+) and SHIP(-/-) mice,confirmed the central role that basophils play in the Th2 skewing of naive SHIP-deficient mice. Taken together,these studies demonstrate that SHIP is a potent negative regulator of IL-4 production from basophils and thus may be a novel therapeutic target for Th1- and Th2-related diseases. View Publication

Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis,induced by injection of the TLR-2 ligand,zymosan,we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein,we show that only in low-dose injected mice,the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose,the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells. View Publication

Background: B cells play an important role in the development and maintenance of rheumatoid arthritis (RA). Although IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) able to suppress autoimmune and inflammatory responses,recent reports showed that B cell-mediated immune suppression may also occur independent of IL-10. For instance,B cells can modulate T cell immune responses through the expression of regulatory molecules such as PD-L1. So far,PD-L1-expressing B cells have not been analyzed in RA patients. Objective: To analyze the frequency of PD-L1-expressing B cells in the peripheral blood of RA patients compared to healthy controls (HC) matched for sex and age,their function on T cell response and their changes in response to therapy. Methods: Fresh peripheral blood B cells from RA patients and HC were characterized by flow cytometry and their functionality assessed in a co-culture system with autologous T cells. Results: The frequencies of CD19+PD-L1+ B cells,CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells were significantly lower in untreated RA patients than in HC. In a follow-up study,the frequencies of PD-L1+ B cells (CD19+PD-L1+ B cells,CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells) increased significantly after treatment in good responder patients,although the frequency of total CD24hiCD38hi B cells decreased. CD19+ B cells from untreated RA patients and HC upregulated PD-L1 expression similarly upon stimulation with CpG plus IL-2 and were able to suppress,in vitro,CD8+ T cell proliferation and cytokine production in a PD-L1-dependent manner. Conclusions: Our results show that PD-L1+ B cells exhibiting T cell suppressive capacity are significantly decreased in untreated RA patients but increase in response to successful treatment. PD-L1 expression on B cells from RA patients can be modulated in vitro and PD-L1+ B cells could thus provide new perspectives for future treatment strategies. View Publication

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports,we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing,IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably,Bcl6hi B cells can form in the combined absence of IL-21R- and STAT6-derived signals; however,these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6,aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus,IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals. View Publication

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226,with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells,with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore,inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors. View Publication

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment,not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver,including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a,M2b,and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions,respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics,such as increased CD86 expression and IL-10 secretion,compared to uninfected controls. In contrast,M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-gamma. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls,M1 macrophages alone significantly increased CD8+ T cell IFN-gamma expression in a cytokine-independent and cell-contact-dependent manner. However,M1 macrophages from HCV-infected individuals significantly decreased IFN-gamma expression in CD8+ T-cells. Therefore,altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals,and aid in the mitigation of associated negative clinical outcomes. View Publication

Pathobiology of several chronic inflammatory disorders,including ulcerative colitis and Crohn's disease is related to intermittent,spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). In this report,we show that TNFalpha promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor,Src and Rac1 signaling to promote wound closure. Consistent with these findings,delayed colonic mucosal repair was observed after administration of a neutralizing TNFalpha antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa,the pro-inflammatory milieu containing TNFalpha and PAF sets the stage for reparative events mediated by PAFR signaling. View Publication