The Src-family kinase Lyn plays a critical role in establishing and maintaining B cell anergy by suppressing PI3K-dependent signaling.

Although the Src family kinase (SFK) Lyn is known to be involved in induction and maintenance of peripheral B cell tolerance, the molecular basis of its action in this context remains unclear. This question has been approached using conventional as well as B cell-targeted knockouts of Lyn, with varied conclusions likely confused by collateral loss of Lyn functions in B cell and myeloid cell development and activation. Here we utilized a system in which Lyn gene deletion is tamoxifen inducible and B cell restricted. This system allows acute elimination of Lyn in B cells without off-target effects. This genetic tool was employed in conjunction with immunoglobulin transgenic mice in which peripheral B cells are autoreactive. DNA reactive Ars/A1 B cells require continuous inhibitory signaling, mediated by the inositol phosphatase SHIP-1 and the tyrosine phosphatase SHP-1, to maintain an unresponsive (anergic) state. Here we show that Ars/A1 B cells require Lyn to establish and maintain B cell unresponsiveness. Lyn primarily functions by restricting PI3K-dependent signaling pathways. This Lyn-dependent mechanism complements the impact of reduced mIgM BCR expression to restrict BCR signaling in Ars/A1 B cells. Our findings suggest that a subset of autoreactive B cells requires Lyn to become anergic and that the autoimmunity associated with dysregulated Lyn function may, in part, be due to an inability of these autoreactive B cells to become tolerized.

Failed Downregulation of PI3K Signaling Makes Autoreactive B Cells Receptive to Bystander T Cell Help.

The role of T cell help in autoantibody responses is not well understood. Because tolerance mechanisms govern both T and B cell responses, one might predict that both T cell tolerance and B cell tolerance must be defeated in autoantibody responses requiring T cell help. To define whether autoreactive B cells depend on T cells to generate autoantibody responses, we studied the role of T cells in murine autoantibody responses resulting from acute B cell-specific deletion of regulatory phosphatases. Ars/A1 B cells are DNA reactive and require continuous inhibitory signaling by the tyrosine phosphatase SHP-1 and the inositol phosphatases SHIP-1 and PTEN to maintain unresponsiveness. Acute B cell-restricted deletion of any of these phosphatases results in an autoantibody response. In this study, we show that CD40-CD40L interactions are required to support autoantibody responses of B cells whose anergy has been compromised. If the B cell-intrinsic driver of loss of tolerance is failed negative regulation of PI3K signaling, bystander T cells provide sufficient CD40-mediated signal 2 to support an autoantibody response. However, although autoantibody responses driven by acute B cell-targeted deletion of SHP-1 also require T cells, bystander T cell help does not suffice. These results demonstrate that upregulation of PI3K signaling in autoreactive B cells, recapitulating the effect of multiple autoimmunity risk alleles, promotes autoantibody responses both by increasing B cells' cooperation with noncognate T cell help and by altering BCR signaling. Receptiveness to bystander T cell help enables autoreactive B cells to circumvent the fail-safe of T cell tolerance.

Polymorphic KIR3DL3 expression modulates tissue-resident and innate-like T cells.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

Failed down-regulation of PI3K signaling makes autoreactive B cells receptive to bystander T cell help.

The role of T cell help in autoantibody responses is not well understood. Since tolerance mechanisms govern both T and B cell responses, one might predict that both T cell tolerance and B cell tolerance must be defeated in autoantibody responses requiring T cell help. To define whether autoreactive B cells depend on T cells to generate autoantibody responses, we studied the role of T cells in autoantibody responses resulting from acute cell-specific deletion of regulatory phosphatases. Ars/A1 B cells are DNA-reactive and require continuous inhibitory signaling by the tyrosine phosphatase SHP-1 and the inositol phosphatases SHIP-1 and PTEN to maintain unresponsiveness. Acute B cell-restricted deletion of any of these phosphatases results in an autoantibody response. Here we show that CD40-CD40L interactions are required to support autoantibody responses of B cells whose anergy has been compromised. If the B cell-intrinsic driver of loss of tolerance is failed negative regulation of PI3K signaling, bystander T cells provide sufficient CD40-mediated signal 2 to support an autoantibody response. However, while autoantibody responses driven by acute B cell-targeted deletion of SHP-1 also require T cells, bystander T cell help does not suffice. These results demonstrate that upregulation of PI3K signaling in autoreactive B cells, recapitulating the effect of multiple autoimmunity risk alleles, promotes autoantibody responses both by increasing B cells’ cooperation with non-cognate T cell help, as well as by altering BCR signaling. Receptiveness to bystander T cell help enables autoreactive B cells to circumvent the fail-safe of T cell tolerance. Significance: Phosphatase suppression of PI3K signaling is an important mechanism by which peripheral autoreactive B cells are kept in an unresponsive/anergic state. Loss of this suppression, due to genetic alleles that confer risk of autoimmunity, often occurs in autoreactive B cells of individuals who develop autoimmune disease. Here we demonstrate that de-repression of PI3K signaling promotes autoantibody responses of a DNA-reactive B cell clone by relaxing dependence of autoantibody responses on T cell-derived helper signals. These results suggest that impaired regulation of PI3K signaling can promote autoantibody responses in two ways: by restoring antigen receptor signaling and by enabling autoreactive B cells to circumvent restrictions imposed by T cell tolerance mechanisms.

Comparative analysis of the repertoire of insulin-reactive B cells in type 1 diabetes-prone and resistant mice.

Seropositivity for autoantibodies against multiple islet antigens is associated with development of autoimmune type 1 diabetes (T1D), suggesting a role for B cells in disease. The importance of B cells in T1D is indicated by the effectiveness of B cell-therapies in mouse models and patients. B cells contribute to T1D by presenting islet antigens, including insulin, to diabetogenic T cells that kill pancreatic beta cells. The role of B cell receptor (BCR) affinity in T1D development is unclear. Here, we employed single cell RNA sequencing to define the relationship between BCR affinity for insulin and B cell phenotype during disease development. We utilized immunoglobulin (Ig) heavy chain (VH125) mouse models in which high-affinity insulin-reactive B cells (IBCs) were previously shown to be anergic in diabetes-resistant VH125.C57BL/6-H2g7 and activated in VH125. NOD mice developing disease. Here, high-affinity IBCs were found in the spleen of prediabetic VH125. NOD mice and exhibited marginal zone or follicular phenotypes. Ig light chains expressed by these B cells are unmutated and biased toward Vκ4-74 and Vκ4-57 usage. Receptors expressed by anergic high-affinity IBCs of diabetes-resistant VH125.C57BL/6-H2g7 are also unmutated; however, in this genetic background light chains are polymorphic relative to those of NOD. Light chains derived from NOD and C57BL/6-H2g7 genetic backgrounds conferred divergent kinetics of binding to insulin when paired with the VH125 heavy chain. These findings suggest that relaxation of tolerance mechanisms in the NOD mouse leads to accumulation and partial activation of B cells expressing germline encoded high-affinity BCRs that support development of autoimmunity.

Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

Role of inhibitory signaling in peripheral B cell tolerance.

At least 20% of B cells in the periphery expresses an antigen receptor with a degree of self-reactivity. If activated, these autoreactive B cells pose a risk as they can contribute to the development of autoimmune diseases. To prevent their activation, both B cell-intrinsic and extrinsic tolerance mechanisms are in place in healthy individuals. In this review article, I will focus on B cell-intrinsic mechanisms that prevent the activation of autoreactive B cells in the periphery. I will discuss how inhibitory signaling circuits are established in autoreactive B cells, focusing on the Lyn-SHIP-1-SHP-1 axis, how they contribute to peripheral immune tolerance, and how disruptions of these circuits can contribute to the development of autoimmunity.

B cells promote CD8 T cell primary and memory responses to subunit vaccines.

The relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called "helper" cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Inhibitory Receptor Trap: A Platform for Discovery of Inhibitory Receptors That Utilize Inositol Lipid and Phosphotyrosine Phosphatase Effectors.

Among the areas of most impactful recent progress in immunology is the discovery of inhibitory receptors and the subsequent translation of this knowledge to the clinic. Although the original and canonical member of this family is FcγRIIB, more recent studies defined PD1 as an inhibitory receptor that constrains T cell immunity to tumors. These studies led to development of "checkpoint blockade" immunotherapies (CBT) for cancers in which PD1 interactions with its ligand are blocked. Unfortunately, although very effective in some patients, only a small proportion respond to this therapy. This suggests that additional as yet undescribed inhibitory receptors exist, which could be exploited. Here, we describe a new platform, termed inhibitory receptor trap (IRT), for discovery of members of this family. The approach takes advantage of the fact that many of the known inhibitory receptors mediate signaling by phospho-immunoreceptor tyrosine-based inhibition motif (ITIM) mediated recruitment of Src Homology 2 (SH2) domain-containing phosphatases including the SH2 domain-containing inositol phosphatase SHIP1 encoded by the INPP5D gene and the SH2 domain-containing phosphotyrosine phosphatases SHP1 and SHP2 encoded by the PTPN6 and PTPN11 genes respectively. Here, we describe the IRT discovery platform in which the SH2 domains of inhibitory phosphatases are used for affinity-based isolation and subsequent identification of candidate effectors via immunoblotting and high sensitivity liquid chromatography-mass spectrometry. These receptors may represent alternative targets that can be exploited for improved CBT. Salient observations from these studies include the following: SH2 domains derived from the respective phosphatases bind distinct sets of candidates from different cell types. Thus, cells of different identity and different activation states express partially distinct repertoires of up and downstream phosphatase effectors. Phosphorylated PD1 binds not only SHP2 but also SHIP1, thus the latter may be important in its inhibitory function. B cell antigen receptor signaling leads predominantly to CD79 mono-phosphorylation as indicated by much greater binding to LynSH2 than Syk(SH2)2. This balance of ITAM mono- versus bi-phosphorylation likely tunes signaling by varying activation of inhibitory (Lyn) and stimulatory (Syk) pathways.

Selective Loss of Responsiveness to Exogenous but Not Endogenous Cyclic-Dinucleotides in Mice Expressing STING-R231H.

Stimulator of interferon genes (STING) plays a central role in innate immune responses to viral and intracellular bacterial infections, and cellular damage. STING is a cytosolic sensor of cyclic dinucleotides (CDNs) including those produced by pathogenic bacteria and those arising endogenously as products of the DNA sensor cGAS (e.g., 2'3' cGAMP). The two most common alternative allelic variants of STING in humans are STING-R71H-G230A-R293Q (STING-HAQ) and STING-R232H that are found in 20.4% and 13.7-17.6% of the population, respectively. To determine the biologic consequences of these genotypic variations, we generated knock-in mice containing the murine equivalents of each variant and studied their responsiveness to CDNs. Homozygous STING-HAQ (R71H-I229A-R292Q) and STING-R231H mice were found to be unresponsive to all exogenous CDNs tested (ci-di-GMP, ci-di-AMP, 3'3' cGAMP and Rp,Rp-CDA). Responses of homozygous STING-HAQ mice to endogenous 2'3' cGAMP was also greatly impaired. However, homozygous STING-R231H mice are fully responsive to 2'3' cGAMP. Analysis of heterozygous mice revealed reduced responsiveness to exogenous and endogenous CDNs in mice carrying a single copy of STING-HAQ, while STING-R231H heterozygous mice exhibit reduced responsiveness to exogenous but not endogenous CDNs. These findings confirm and extend previous reports by demonstrating differing impact of allelic variation of STING on the ability to sense and respond to exogenous vs. endogenous CDNs. Finally, the STING-R231H variant mouse represents a useful tool with which to examine the relative contributions of STING sensing of exogenous and endogenous CDNs in the context of bacterial infections and CDN-based cancer immunotherapeutics.

γδ T cells shape memory-phenotype αß T cell populations in non-immunized mice.

Size and composition of γδ T cell populations change dramatically with tissue location, during development, and in disease. Given the functional differentiation of γδ T cell subsets, such shifts might alter the impact of γδ T cells on the immune system. To test this concept, and to determine if γδ T cells can affect other immune cells prior to an immune response, we examined non-immunized mice derived from strains with different genetically induced deficiencies in γδ T cells, for secondary changes in their immune system. We previously saw extensive changes in pre-immune antibodies and B cell populations. Here, we report effects on αß T cells. Similarly to the B cells, αß T cells evidently experience the influence of γδ T cells at late stages of their pre-immune differentiation, as single-positive heat stable antigen-low thymocytes. Changes in these and in mature αß T cells were most prominent with memory-phenotype cells, including both CD8+ and CD4+ populations. As previously observed with B cells, most of the effects on αß T cells were dependent on IL-4. Unexpectedly, IL-4 seemed to be produced mainly by αß T cells in the non-immunized mice, albeit strongly regulated by γδ T cells. Similarly to our findings with B cells, changes of αß T cells were less pronounced in mice lacking all γδ T cells than in mice lacking only some, suggesting that the composition of the γδ T cell population determines the nature of the γδ-influence on the other pre-immune lymphocytes.

Protective role of B cells in sterile particulate-induced lung injury.

Susceptibility to chronic beryllium (Be) disease is linked to HLA-DP molecules possessing a glutamic acid at the 69th position of the ß-chain (ßGlu69), with the most prevalent ßGlu69-containing molecule being HLA-DP2. We have previously shown that HLA-DP2 transgenic (Tg) mice exposed to Be oxide (BeO) develop mononuclear infiltrates in a peribronchovascular distribution and a beryllium-specific, HLA-DP2-restricted CD4+ T cell response. In addition to T cells, B cells constituted a major portion of infiltrated leukocytes in the lung of BeO-exposed HLA-DP2 Tg mice and sequester BeO particles within ectopic lymphoid aggregates and granulomas. B cell depletion was associated with a loss of lymphoid aggregates and granulomas as well as a significant increase in lung injury in BeO-exposed mice. The protective role of B cells was innate in origin, and BeO-induced B cell recruitment to the lung was dependent on MyD88 signaling. Similar to BeO-exposed HLA-DP2 mice, B cells also accumulate in the lungs of CBD subjects, located at the periphery and surrounding the granuloma. Overall, our data suggest a novel modulatory role for B cells in the protection of the lung against sterile particulate exposure, with B cell recruitment to the inflamed lung occurring in an antigen-independent and MyD88-dependent manner.

A Precision B Cell-Targeted Therapeutic Approach to Autoimmunity Caused by Phosphatidylinositol 3-Kinase Pathway Dysregulation.

The inositol lipid phosphatases PTEN and SHIP-1 play a crucial role in maintaining B cell anergy and are reduced in expression in B cells from systemic lupus erythematosus and type 1 diabetes patients, consequent to aberrant regulation by miRNA-7 and 155. With an eye toward eventual use in precision medicine therapeutic approaches in autoimmunity, we explored the ability of p110δ inhibition to compensate for PI3K pathway dysregulation in mouse models of autoimmunity. Low dosages of the p110δ inhibitor idelalisib, which spare the ability to mount an immune response to exogenous immunogens, are able to block the development of autoimmunity driven by compromised PI3K pathway regulation resultant from acutely induced B cell-targeted haploinsufficiency of PTEN and SHIP-1. These conditions do not block autoimmunity driven by B cell loss of the regulatory tyrosine phosphatase SHP-1. Finally, we show that B cells in NOD mice express reduced PTEN, and low-dosage p110δ inhibitor therapy blocks disease progression in this model of type 1 diabetes. These studies may aid in the development of precision treatments that act by enforcing PI3K pathway regulation in patients carrying specific risk alleles.

Non-Antibody-Secreting Functions of B Cells and Their Contribution to Autoimmune Disease.

B cells play multiple important roles in the pathophysiology of autoimmune disease. Beyond producing pathogenic autoantibodies, B cells can act as antigen-presenting cells and producers of cytokines, including both proinflammatory and anti-inflammatory cytokines. Here we review our current understanding of the non-antibody-secreting roles that B cells may play during development of autoimmunity, as learned primarily from reductionist preclinical models. Attention is also given to concepts emerging from clinical studies using B cell depletion therapy, which shed light on the roles of these mechanisms in human autoimmune disease.

Elevated PTEN expression maintains anergy in human B cells and reveals unexpectedly high repertoire autoreactivity.

It has been reported that 2.5%-30% of human peripheral CD27- B cells are autoreactive and anergic based on unresponsiveness to antigen receptor (BCR) stimulation and autoreactivity of cloned and expressed BCR. The molecular mechanisms that maintain this unresponsiveness are unknown. Here, we showed that in humans anergy is maintained by elevated expression of PTEN, a phosphatidylinositol 3,4,5P-3-phosphatase. Upregulation of PTEN was associated with reduced expression of microRNAs that control its expression. Pharmacologic inhibition of PTEN lead to significant restoration of responsiveness. Consistent with a role in conferring risk of autoimmunity, B cells from type 1 diabetics and autoimmune thyroid disease patients expressed reduced PTEN. Unexpectedly, in healthy individuals PTEN expression was elevated in on average 40% of CD27- B cells, with levels gradually decreasing as IgM levels increase. Our findings suggest that a much higher proportion of the peripheral repertoire is autoreactive than previously thought and that B cells upregulate PTEN in a manner that is proportional to the recognition of autoantigens of increasing avidity, thus tuning BCR signaling to prevent development of autoimmunity while providing a reservoir of cells that can be readily activated to respond when needed.

The c-Myc/miR17-92/PTEN Axis Tunes PI3K Activity to Control Expression of Recombination Activating Genes in Early B Cell Development.

Appropriate PI3K signals generated by the antigen receptor are essential to promote B cell development. Regulation of recombination activating gene (RAG)-1 and RAG-2 expression is one key process that is mediated by PI3K to ensure developmental progression and selection. When PI3K signals are too high or too low, expression of RAGs does not turn off and B cell development is impaired or blocked. Yet, the mechanism which tunes PI3K activity to control RAG expression during B cell development in the bone marrow is unknown. Recently we showed that a c-Myc/miR17-92/PTEN axis regulates PI3K activity for positive and negative selection of immature B cells. Here, we show that the c-Myc/miR17-92/PTEN axis tunes PI3K activity to control the expression of RAGs in proB cells. Using different genetically engineered mouse models we show that impaired function of the c-Myc/miR17-92/PTEN axis alters the PI3K/Akt/Foxo1 pathway to result in dis-regulated expression of RAG and a block in B cell development. Studies using 38c-13 B lymphoma cells, where RAGs are constitutively expressed, suggest that this regulatory effect is mediated post-translationally through Foxo1.

B Cell-Intrinsic STING Signaling Triggers Cell Activation, Synergizes with B Cell Receptor Signals, and Promotes Antibody Responses.

Generation of protective immune responses requires coordinated stimulation of innate and adaptive immune responses. An important mediator of innate immunity is stimulator of IFN genes (STING, MPYS, MITA), a ubiquitously but differentially expressed adaptor molecule that functions in the relay of signals initiated by sensing of cytosolic DNA and bacterial cyclic dinucleotides (CDNs). Whereas systemic expression of STING is required for CDN-aided mucosal Ab responses, its function in B cells in particular is unclear. In this study, we show that B cells can be directly activated by CDNs in a STING-dependent manner in vitro and in vivo. Direct activation of B cells by CDNs results in upregulation of costimulatory molecules and cytokine production and this can be accompanied by caspase-dependent cell death. CDN-induced cytokine production by B cells and other cell types also contributes to activation and immune responses. Type I IFN is primarily responsible for this indirect stimulation although other cytokines may contribute. BCR and STING signaling pathways act synergistically to promote Ab responses independent of type I IFN. B cell expression of STING is required for optimal in vivo IgG and mucosal IgA Ab responses induced by T cell-dependent Ags and cyclic-di-GMP but plays no discernable role in Ab responses in which alum is used as an adjuvant. Thus, STING functions autonomously in B cells responding to CDNs, and its activation synergizes with Ag receptor signals to promote B cell activation.

Silencing of high-affinity insulin-reactive B lymphocytes by anergy and impact of the NOD genetic background in mice.

AIMS/HYPOTHESIS: Previous studies have demonstrated that high-affinity insulin-binding B cells (IBCs) silenced by anergy in healthy humans lose their anergy in islet autoantibody-positive individuals with recent-onset type 1 diabetes, and in autoantibody-negative first-degree relatives carrying certain risk alleles. Here we explore the hypothesis that IBCs are found in the immune periphery of disease-resistant C57BL/6-H2g7 mice, where, as in healthy humans, they are anergic, but that in disease-prone genetic backgrounds (NOD) they become activated and migrate to the pancreas and pancreatic lymph nodes, where they participate in the development of type 1 diabetes. METHODS: We compared the status of high-affinity IBCs in disease-resistant VH125.C57BL/6-H2g7 and disease-prone VH125.NOD mice. RESULTS: Consistent with findings in healthy humans, high-affinity IBCs reach the periphery in disease-resistant mice and are anergic, as indicated by a reduced expression of membrane IgM, unresponsiveness to antigen and failure to become activated or accumulate in the pancreatic lymph nodes or pancreas. In NOD mice, high-affinity IBCs reach the periphery early in life and increase in number prior to the onset of hyperglycaemia. These cells are not anergic; they become activated, produce autoantibodies and accumulate in the pancreas and pancreatic lymph nodes prior to disease development. CONCLUSIONS/INTERPRETATION: These findings are consistent with genetic determination of the escape of high-affinity IBCs from anergy and their early contribution to the development of type 1 diabetes.

High-efficiency RNA-based reprogramming of human primary fibroblasts.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient's fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming.

Impaired B cell function during viral infections due to PTEN-mediated inhibition of the PI3K pathway.

Transient suppression of B cell function often accompanies acute viral infection. However, the molecular signaling circuitry that enforces this hyporesponsiveness is undefined. In this study, experiments identify up-regulation of the inositol phosphatase PTEN (phosphatase and tensin homolog) as primarily responsible for defects in B lymphocyte migration and antibody responses that accompany acute viral infection. B cells from mice acutely infected with gammaherpesvirus 68 are defective in BCR- and CXCR4-mediated activation of the PI3K pathway, and this, we show, is associated with increased PTEN expression. This viral infection-induced PTEN overexpression appears responsible for the suppression of antibody responses observed in infected mice because PTEN deficiency or expression of a constitutively active PI3K rescued function of B cells in infected mice. Conversely, induced overexpression of PTEN in B cells in uninfected mice led to suppression of antibody responses. Finally, we demonstrate that PTEN up-regulation is a common mechanism by which infection induces suppression of antibody responses. Collectively, these findings identify a novel role for PTEN during infection and identify regulation of the PI3K pathway, a mechanism previously shown to silence autoreactive B cells, as a key physiological target to control antibody responses.