Summary Anergic B cells are characterized by impaired signaling and activation following aggregation of their antigen receptors (BCR). The molecular basis of this impairment is not understood. In studies reported here Src homology-2 (SH2)-containing inositol 5-phosphatase SHIP-1 and its adaptor Dok-1 were found to be constitutively phosphorylated in anergic B cells, and activation of this inhibitory circuit was dependent on Src-family kinase activity and consequent to biased BCR immunoreceptor tyrosine-based activation motif (ITAM) monophosphorylation. B cell-targeted deletion of SHIP-1 caused severe lupus-like disease. Moreover, absence of SHIP-1 in B cells led to loss of anergy as indicated by restoration of BCR signaling, loss of anergic surface phenotype and production of autoantibodies. Thus chronic BCR signals maintain anergy in part via ITAM monophosphorylation-directed activation of an inhibitory signaling circuit involving SHIP-1 and Dok-1.

SummaryA byproduct of the largely stochastic generation of a diverse B-cell specificity repertoire is production of cells that recognize autoantigens. Indeed, recent studies indicate that more than half of the primary repertoire consists of autoreactive B cells that must be silenced to prevent autoimmunity. While this silencing can occur by multiple mechanisms, it appears that most autoreactive B cells are silenced by anergy, wherein they populate peripheral lymphoid organs and continue to express unoccupied antigen receptors yet are unresponsive to antigen stimulation. Here we review molecular mechanisms that appear operative in maintaining the antigen unresponsiveness of anergic B cells. In addition, we present new data indicating that the failure of anergic B cells to mobilize calcium in response to antigen stimulation is not mediated by inactivation of stromal interacting molecule 1, a critical intermediary in intracellular store depletion-induced calcium influx. Keywords B cell; tolerance; anergy; PI3K Modes of B-cell toleranceThe ability of the adaptive immune system to provide protection against pathogens depends upon a diverse repertoire of antigen receptors that enable recognition of a seemingly infinite range of foreign protein and carbohydrate antigens. Diversity is generated early in lymphocyte development by random rearrangement of immunoglobulin (Ig) V, D, and J region gene segments. The random nature of this process leads inevitably to generation of receptors that recognize self-antigen. It is estimated that as much as 70% of early immature human B cells are self-reactive (1). It appears that in about a third of these self-reactive immature B cells autoreactivity is eliminated by receptor editing, wherein new Ig gene rearrangement generates an alternate light chain that pairs with the existing Ig heavy chain, altering specificity (2,3). If editing fails to eliminate reactivity, the B cell is deleted by apoptosis (4). Experimental evidence shows that only a few percent of B cells meets this fate (5).Although these modes of 'central' tolerance silence B cells in the bone marrow (BM), many self-reactive B cells escape to the periphery where they must be silenced by alternate mechanisms. Studies in the 1970s demonstrated that B cells from peripheral lymphoid organs can be rendered tolerant by exposure to antigen in vitro. In these early studies, the mechanism by which B cells were rendered tolerant was often referred to as clonal inactivation/abortion based on the fact that the frequency of antigen-specific, antibody-secreting cells fell drastically following tolerizing treatments. However, these experiments did not distinguish between the Correspondence to: John C.

Cambier et al. show that the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 are required to maintain B cell anergy.

Summary Receptors for immunoglobulin Fc regions play multiple critical roles in the immune system, mediating functions as diverse as phagocytosis, triggering degranulation of basophils and mast cells, promoting immunoglobulin class switching and preventing excessive activation. Transmembrane signaling associated with these functions is mediated primarily by two amino acid sequence motifs, ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) and ITIMs (Immunoreceptor Tyrosine-based Inhibition Motifs) that act as the receptors’ interface with activating and inhibitory signaling pathways, respectively. While ITAMs mobilize activating tyrosine kinases and their consorts, ITIMs mobilize opposing tyrosine and inositol-lipid phosphatases. In this review we will discuss our current understanding of signaling by these receptors/motifs and their sometimes blurred lines of function.

IgE Abs, passively administered together with their specific Ag, can enhance the production of Abs recognizing this Ag by >100-fold. IgE-mediated feedback enhancement requires the low affinity receptor for IgE, CD23. One possible mechanism is that B cells take up IgE-Ag via CD23 and efficiently present Ag to Th cells, resulting in better Ab responses. To test whether IgE Abs have an effect on Th cells in vivo, mice were adoptively transferred with CD4+ T cells expressing a transgenic OVA-specific TCR, before immunization with IgE anti-TNP (2,4,6-trinitrophenyl) plus OVA-TNP or with OVA-TNP alone. IgE induced a 6- to 21-fold increase in the number of OVA-specific T cells. These cells acquired an activated phenotype and were visible in splenic T cell zones. The T cell response peaked 3 days after immunization and preceded the OVA-specific Ab response by a few days. Transfer of CD23+ B cells to CD23-deficient mice rescued their ability to respond to IgE-Ag. Interestingly, in this situation also CD23-negative B cells produce enhanced levels of OVA-specific Abs. The data are compatible with the Ag presentation model and suggest that B cells can take up Ag via “unspecific” receptors and activate naive T cells in vivo.

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.

Antibodies administered in vivo together with the antigen they are specific for can regulate the immune response to that antigen. This phenomenon is called antibody‐mediated feedback regulation and has been known for over 100 years. Both passively administered and actively produced antibodies exert immunoregulatory functions. Feedback regulation can be either positive or negative, resulting in >1000‐fold enhancement or >99% suppression of the specific antibody response. Usually, the response to the entire antigen is up‐ or downregulated, regardless of which epitope the regulating antibody recognizes. IgG of all isotypes can suppress responses to large particulate antigens like erythrocytes, a phenomenon used clinically in Rhesus prophylaxis. IgG suppression works in mice lacking the known Fc‐γ receptors (FcγR) and a likely mechanism of action is epitope masking. IgG1, IgG2a and IgG2b administered together with soluble protein antigens will enhance antibody and CD4+ T‐cell responses via activating FcγR, probably via increased antigen presentation by dendritic cells. IgG3 as well as IgM also enhance antibody responses but their effects are dependent on their ability to activate complement. A possible mechanism is increased B‐cell activation caused by immune complexes co‐crosslinking the B‐cell receptor with the complement‐receptor 2/CD19 receptor complex, known to lower the threshold for B‐cell activation. IgE‐antibodies enhance antibody and CD4+ T‐cell responses to small soluble proteins. This effect is entirely dependent on the low‐affinity receptor for IgE, CD23, the mechanism probably being increased antigen presentation by CD23+ B cells.

A number of studies in experimental animal models point to an important role of FcγRs in autoimmunity and allergy. In this study, we investigate how the production of IgG, an early step in the chain of events leading to inflammation, is regulated by activating and inhibitory FcγRs. IgG Abs are known to feedback-enhance Ab responses to soluble Ags, and this effect requires activating FcγRs. To test proliferation of Th cells, mice were adoptively transferred with CD4+ T cells expressing a transgenic OVA-specific TCR before immunization with IgG2a anti-2,4,6-trinitrophenyl (TNP) plus OVA-TNP or with OVA-TNP alone. IgG2a induced a significant increase in OVA-specific T cell numbers, which preceded the OVA-specific Ab response and was dependent on the FcRγ chain. The role of the inhibitory FcγRIIB in Ab responses was studied in mice lacking this receptor. Although IgG2a enhanced primary Ab responses, development of germinal centers, and immunological memory in wild-type mice, enhancement was markedly stronger in FcγRIIB−/− mice. The presented data are compatible with the hypothesis that the mechanism behind IgG2a-mediated up-regulation of Ab responses involves increased Ag presentation to CD4+ T cells by FcγR+ APCs. Our observations also illustrate the intricate immunoregulatory role of IgG Abs. On the one hand, they enhance Ab responses via activating FcγRs, and on the other hand, they set an upper limit for the same Ab response via FcγRIIB.