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BACKGROUND Microglia,the principle immune cells of the brain,play important roles in neuronal development,homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease,we,and others,have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology,labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore,we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS iPSCs are first differentiated toward a mesodermal,hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF,IL-34,and TGF$\beta$-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation,complex media formulation,FACS sorting,or co-culture,thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia,we performed RNA-sequencing,functional testing,and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGF$\beta$ signaling (IDE1) can be used to replace recombinant TGF$\beta$1,further reducing costs,we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach,although transcriptional differences do occur that should be considered. CONCLUSION We anticipate that this new and greatly simplified protocol will enable many interested labs,including those with little prior stem cell or flow cytometry experience,to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation,the field will continue to improve our understanding of microglial biology and their important roles in human development,homeostasis,and disease. View Publication

Protective antibody responses to vaccination or infection depend on affinity maturation,a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind,gather,and present antigen to T follicular helper (Tfh) cells. Here,we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with na{\{i}}ve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of na{\"{i}}ve B cells. Thus intrinsic properties of human GC B cells set thresholds for affinity selection.""" 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

Vascular-deposited IgG immune complexes promote neutrophil recruitment,but how this process is regulated is still unclear. Here we show that the CD18 integrin Mac-1,in its bent state,interacts with the IgG receptor Fc$\gamma$RIIA in cis to reduce the affinity of Fc$\gamma$RIIA for IgG and inhibit Fc$\gamma$RIIA-mediated neutrophil recruitment under flow. The Mac-1 rs1143679 lupus-risk variant reverses Mac-1 inhibition of Fc$\gamma$RIIA,as does a Mac-1 ligand and a mutation in Mac-1's ligand binding $\alpha$I-domain. Sialylated complex glycans on Fc$\gamma$RIIA interact with the $\alpha$I-domain via divalent cations,and this interaction is required for Fc$\gamma$RIIA inhibition by Mac-1. Human neutrophils deficient in CD18 integrins exhibit augmented Fc$\gamma$RIIA-dependent recruitment to IgG-coated endothelium. In mice,CD18 integrins on neutrophils dampen IgG-mediated neutrophil accumulation in the kidney. In summary,cis interaction between sialylated Fc$\gamma$RIIA and the $\alpha$I-domain of Mac-1 alters the threshold for IgG-mediated neutrophil recruitment. A disruption of this interaction may increase neutrophil influx in autoimmune diseases. View Publication

T-cell responses in the lung are critical for protection against respiratory pathogens. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However,whether these signals take place mainly during priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under conditions of competition,GITR provides a T-cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue,as well as for the formation of CD4 and CD8 tissue-resident memory T cells during respiratory influenza infection in mice. In contrast,under non-competitive conditions,GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T-cell response partially compensated for GITR deficiency by expansion of higher affinity T cells; whereas,the polymerase-specific response was less flexible and more GITR dependent. Following influenza infection,GITR is expressed on lung T cells and GITRL is preferentially expressed on lung monocyte-derived inflammatory antigen presenting cells. Accordingly,we show that GITR+/+ T cells in the lung parenchyma express more phosphorylated-ribosomal protein S6 than their GITR-/- counterparts. Thus,GITR signaling within the lung tissue critically regulates effector and tissue-resident memory T-cell accumulation. View Publication

During chronic viral infection,the inflammatory function of CD4 T-cells becomes gradually attenuated. Concurrently,Th1 cells progressively acquire the capacity to secrete the cytokine IL-10,a potent suppressor of antiviral T cell responses. To determine the transcriptional changes that underlie this adaption process,we applied a single-cell RNA-sequencing approach and assessed the heterogeneity of IL-10-expressing CD4 T-cells during chronic infection. Here we show an IL-10-producing population with a robust Tfh-signature. Using IL-10 and IL-21 double-reporter mice,we further demonstrate that IL-10+IL-21+co-producing Tfh cells arise predominantly during chronic but not acute LCMV infection. Importantly,depletion of IL-10+IL-21+co-producing CD4 T-cells or deletion of Il10 specifically in Tfh cells results in impaired humoral immunity and viral control. Mechanistically,B cell-intrinsic IL-10 signaling is required for sustaining germinal center reactions. Thus,our findings elucidate a critical role for Tfh-derived IL-10 in promoting humoral immunity during persistent viral infection. View Publication

Common variable immunodeficiency disorders (CVID) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and impaired specific Ab response,resulting in recurrent infections due to dysfunctional immune response. The specific mechanisms mediating immune deficiency in CVID remain to be determined. Previous studies indicated that immune dysregulation in CVID patients is associated with chronic microbial translocation,systemic immune activation,and altered homeostasis of lymphocytic and myeloid lineages. A detailed phenotypic,functional characterization of plasma markers and immune cell populations was performed in 46 CVID patients and 44 healthy donors. CVID patients displayed significantly elevated plasma levels of a marker of neutrophil activation neutrophil gelatinase-associated lipocalin. Neutrophils from CVID patients exhibited elevated surface levels of CD11b and PD-L1 and decreased levels of CD62L,CD16,and CD80,consistent with a phenotype of activated neutrophils with suppressive properties. Neutrophils from CVID patients actively suppressed T cell activation and release of IFN-$\gamma$ via the production of reactive oxygen species. Furthermore,CVID was associated with an increased frequency of low-density neutrophils (LDNs)/granulocytic myeloid-derived suppressor cells. LDN/granulocytic myeloid-derived suppressor cell frequency in CVID patients correlated with reduced T cell responsiveness. Exogenous stimulation of whole blood with bacterial LPS emulated some but not all of the phenotypic changes observed on neutrophils from CVID patients and induced neutrophil population with LDN phenotype. The presented data demonstrate that neutrophils in the blood of CVID patients acquire an activated phenotype and exert potent T cell suppressive activity. Specific targeting of myeloid cell-derived suppressor activity represents a novel potential therapeutic strategy for CVID. View Publication

Plasminogen activator inhibitor-1 (PAI-1) has a pro-tumorigenic function via its pro-angiogenic and anti-apoptotic activities. Here,we demonstrate that PAI-1 promotes the recruitment and M2 polarization of monocytes/macrophages through different structural domains. Its LRP1 interacting domain regulated macrophage migration,while its C-terminal uPA interacting domain promoted M2 macrophage polarization through activation of p38MAPK and nuclear factor $\kappa$B (NF-$\kappa$B) and induction of an autocrine interleukin (IL)-6/STAT3 activation pathway. We then show in several experiments in mice that expression of PAI-1 is associated with increased tumorigenicity,increased presence of M2 macrophages,higher levels of IL-6,and increased STAT3 phosphorylation in macrophages. Strong positive correlations between PAI-1,IL-6,and CD163 (M2 marker) expression were also found by meta-analysis of transcriptome data in many human cancers. Altogether,these data provide evidence for a mechanism explaining the paradoxical pro-tumorigenic function of PAI-1 in cancer. View Publication

Vaccines are among the most effective public health tools for combating certain infectious diseases such as influenza. The role of the humoral immune system in vaccine-induced protection is widely appreciated; however,our understanding of how antibody specificities relate to B cell function remains limited due to the complexity of polyclonal antibody responses. To address this,we developed the Spec-seq framework,which allows for simultaneous monoclonal antibody (mAb) characterization and transcriptional profiling from the same single cell. Here,we present the first application of the Spec-seq framework,which we applied to human plasmablasts after influenza vaccination in order to characterize transcriptional differences governed by B cell receptor (BCR) isotype and vaccine reactivity. Our analysis did not find evidence of long-term transcriptional specialization between plasmablasts of different isotypes. However,we did find enhanced transcriptional similarity between clonally related B cells,as well as distinct transcriptional signatures ascribed by BCR vaccine recognition. These data suggest IgG and IgA vaccine-positive plasmablasts are largely similar,whereas IgA vaccine-negative cells appear to be transcriptionally distinct from conventional,terminally differentiated,antigen-induced peripheral blood plasmablasts. View Publication

While it is now acknowledged that CD4+ T cells expressing CD25 and Foxp3 (Treg cells) regulate immune responses and,consequently,influence the pathogenesis of infectious diseases,the regulatory response mediated by Treg cells upon infection by Trypanosoma cruzi was still poorly characterized. In order to understand the role of Treg cells during infection by this protozoan parasite,we determined in time and space the magnitude of the regulatory response and the phenotypic,functional and transcriptional features of the Treg cell population in infected mice. Contrary to the accumulation of Treg cells reported in most chronic infections in mice and humans,experimental T. cruzi infection was characterized by sustained numbers but decreased relative frequency of Treg cells. The reduction in Treg cell frequency resulted from a massive accumulation of effector immune cells,and inversely correlated with the magnitude of the effector immune response as well as with emergence of acute immunopathology. In order to understand the causes underlying the marked reduction in Treg cell frequency,we evaluated the dynamics of the Treg cell population and found a low proliferation rate and limited accrual of peripheral Treg cells during infection. We also observed that Treg cells became activated and acquired a phenotypic and transcriptional profile consistent with suppression of type 1 inflammatory responses. To assess the biological relevance of the relative reduction in Treg cells frequency observed during T. cruzi infection,we transferred in vitro differentiated Treg cells at early moments,when the deregulation of the ratio between regulatory and conventional T cells becomes significant. Intravenous injection of Treg cells dampened parasite-specific CD8+ T cell immunity and affected parasite control in blood and tissues. Altogether,our results show that limited Treg cell response during the acute phase of T. cruzi infection enables the emergence of protective anti-parasite CD8+ T cell immunity and critically influences host resistance. View Publication

Pathogen immune responses are profoundly attenuated in fetuses and premature infants,yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic,metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-$\gamma$-regulated metabolic pathways,limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast,they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants,and suggest that the fetal immune system is metabolically programmed to avoid energetically costly,dispensable and potentially harmful immune responses during ontogeny. 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