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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.

Background New strategies to inhibit acute rejection are needed for further applications of composite tissue allotransplantation. The nuclear receptor subfamily 4 group A member 1 (NR4A1) is considered a key controller of maintaining tolerance homeostasis. However, the effect of NR4A1 in suppressing rejection responses after allotransplantation remains unknown. Methods Brown Norway rat groin flaps were transplanted into Lewis rat recipients. The recipients were administrated cytosporone B, an NR4A1 activator. NR4A1 expression and graft survival time were assessed. T helper type 1 and regulatory T cell populations in the second lymphoid organ were detected by flow cytometry. Furthermore, a retrovirus containing NR4A1 was constructed and transfected to T cells in vitro. After stimulation, interleukin 2 and interferon gamma secretions were detected in the T cells. Results Administration of cytosporone B activated NR4A1 expression in allotransplant recipients and was associated with prolonged survival time of the vascularized free flap allograft. T helper type 1 cells in the recipient secondary lymphoid organs were decreased, whereas the population of regulatory T cells did not change. Interleukin 2 and interferon gamma were suppressed in vitro in the T cells overexpressing NR4A1. Conclusions We demonstrated that the overexpressed NR4A1 is associated with suppressed graft rejection response. The suppression effect may attribute to induction of T-cell anergy and blockade of key immunologic cytokines.

The origin of the impaired CD4 T-cell response and immunodeficiency of HIV-infected patients is still only partially understood. We recently demonstrated that PLA2G1B phospholipase synergizes with the HIV gp41 envelope protein in HIV viremic plasma to induce large abnormal membrane microdomains (aMMDs) that trap and inactivate physiological receptors, such as those for IL-7. However, the mechanism of regulation of PLA2G1B activity by the cofactor gp41 is not known. Here, we developed an assay to directly follow PLA2G1B enzymatic activity on CD4 T-cell membranes. We demonstrated that gp41 directly binds to PLA2G1B and increases PLA2G1B enzymatic activity on CD4 membrane. Furthermore, we show that the conserved 3S sequence of gp41, known to bind to the innate sensor gC1qR, increases PLA2G1B activity in a gC1qR-dependent manner using gC1qR KO cells. The critical role of the 3S motif and gC1qR in the inhibition of CD4 T-cell function by the PLA2G1B/cofactor system in HIV-infected patients led us to screen additional microbial proteins for 3S-like motifs and to study other proteins known to bind to the gC1qR to further investigate the role of the PLA2G1B/cofactor system in other infectious diseases and carcinogenesis. We have thus extended the PLA2G1B/cofactor system to HCV and Staphylococcus aureus infections and additional pathologies where microbial proteins with 3S-like motifs also increase PLA2G1B enzymatic activity. Notably, the bacteria Porphyromonas gingivalis, which is associated with pancreatic ductal adenocarcinoma (PDAC), encodes such a cofactor protein and increased PLA2G1B activity in PDAC patient plasma inhibits the CD4 response to IL-7. Our findings identify PLA2G1B/cofactor system as a CD4 T-cell inhibitor. It involves the gC1qR and disease-specific cofactors which are gC1qR-binding proteins that can contain 3S-like motifs. This mechanism involved in HIV-1 immunodeficiency could play a role in pancreatic cancer and several other diseases. These observations suggest that the PLA2G1B/cofactor system is a general CD4 T-cell inhibitor and pave the way for further studies to better understand the role of CD4 T-cell anergy in infectious diseases and tumor escape.

Background Diffuse large B cell lymphoma (DLBCL) is the commonest lymphoma that is highly aggressive where one-third of the patients relapse despite effective treatment. Interaction between the lymphoma cells and the non-clonal immune cells within the bone marrow microenvironment is thought to play a critical role in the pathogenesis of DLBCL. Methods We used flow cytometry to characterize the proportion of B cell subpopulations in the bone marrow (N = 47) and peripheral blood (N = 54) of 75 DLBCL patients at diagnosis and study their impact on survival. Results Anergic B cells in the bone marrow (BM), characterized as having CD21(−/low)/CD38- expression, influenced survival with high numbers (defined as > 13.9%) being associated with significantly shorter overall survival (59.7 months vs 113.6 months, p = 0.0038). Interestingly, low numbers of anergic B cells in the BM (defined as ≤13.9%) was associated with germinal center B cell type of DLBCL (p = 0.0354) that is known to have superior rates of survival when compared to activated B cell type. Finally, Cox regression analysis in our cohort of patients established that the inferior prognosis of having high numbers of anergic B cells in the bone marrow was independent of the established Revised International Prognostic Index (R-IPI) score. Conclusions High proportion of anergic B cells in the BM characterized by CD21(−/low)/CD38- expression predicts poor survival outcomes in DLBCL.

Impaired NK cytotoxicity has been linked to poor cancer prognosis, but its mechanisms are not clearly established. Increasing data demonstrate that NK cells lose cytotoxicity after interaction with NK cell-sensitive tumor cells. In this paper, we provide evidence that the human adenocarcinoma cell line MiaPaCa2 and TNFα and TGFβ-treated MiaPaCa2 cultures (MiaPaCa2-TT) induced functional anergy of NK cells

CD8+ T cells responding to chronic infections or tumors acquire an 'exhausted' state associated with elevated expression of inhibitory receptors, including PD-1, and impaired cytokine production. Exhausted T cells are continuously replenished by T cells with precursor characteristics that self-renew and depend on the transcription factor TCF1; however, their developmental requirements are poorly understood. In the present study, we demonstrate that high antigen load promoted the differentiation of precursor T cells, which acquired hallmarks of exhaustion within days of infection, whereas early effector cells retained polyfunctional features. Early precursor T cells showed epigenetic imprinting characteristic of T cell receptor-dependent transcription factor binding and were restricted to the generation of cells displaying exhaustion characteristics. Transcription factors BACH2 and BATF were key regulators with opposing functions in the generation of early precursor T cells. Overall, we demonstrate that exhaustion manifests first in TCF1+ precursor T cells and is propagated subsequently to the pool of antigen-specific T cells.

The BCR recognizes foreign Ags to initiate humoral immunity that needs isotype-switched Abs generated via class switch recombination (CSR); however, stimulating the BCR in the absence of costimulation (e.g., CD40) does not induce CSR; thus, it remains elusive whether and how the BCR induces CSR mechanistically. Autoreactive B cells can maintain anergy via unresponsiveness of their BCRs to self-antigens. However, it remains unknown what molecule(s) restrict BCR signaling strength for licensing BCR-induced CSR and whether deficiency of such molecule(s) disrupts autoreactive B cell anergy and causes B cell–mediated diseases by modulating BCR signaling. In this study, we employ mouse models to show that the BCR’s capacity to induce CSR is restrained by B cell–intrinsic checkpoints TRAF3 and TRAF2, whose deletion in B cells enables the BCR to induce CSR in the absence of costimulation. TRAF3 deficiency permits BCR-induced CSR by elevating BCR-proximal signaling intensity. Furthermore, NF-κB2 is required for BCR-induced CSR in TRAF3-deficient B cells but not for CD40-induced or LPS-induced CSR, suggesting that TRAF3 restricts NF-κB2 activation to specifically limit the BCR’s ability to induce CSR. TRAF3 deficiency also disrupts autoreactive B cell anergy by elevating calcium influx in response to BCR stimulation, leading to lymphoid organ disorders and autoimmune manifestations. We showed that TRAF3 deficiency-associated autoimmune phenotypes can be rectified by limiting BCR repertoires or attenuating BCR signaling strength. Thus, our studies highlight the importance of TRAF3-mediated restraint on BCR signaling strength for controlling CSR, B cell homeostasis, and B cell–mediated disorders.

Purpose of review The immune determinants of granuloma formation and disease progression in sarcoidosis have not been completely disclosed, and the role of both innate and the adaptive immunity is still under investigation. Recent findings M2 macrophage polarization, previously thought to be a specific feature of a progressing and fibrosing disease, has been related to the initial steps of granuloma formation both in animal and in-vitro models. The dysregulation of specific metabolic pathways and autophagy has been associated with disease activity and progression. T cells have been reported to be strongly influenced by a macrophage-driven microenvironment and more dangerous when acquiring hybrid phenotypes (e.g. Th17.1) or even becoming anergic, leading to disease chronicization. Locally released serum amyloid A was suggested to induce a more pro-inflammatory Th17 transcription program. The possible role of in-situ humoral immunity and bone marrow-derived mesenchymal stromal cells has also been highlighted. Summary Evidence points at microenvironment and cell functional features rather than cell polarization or differentiation as determinants of pathogenesis. In terms of therapeutic implications, future advances will rely on molecular disease profiling, aiming at personalized and combined therapeutic approaches.

TNF‐blockade has shown clear therapeutic value in rheumatoid arthritis and other immune‐mediated inflammatory diseases, however its mechanism of action is not fully elucidated. We investigated the effects of TNF‐blockade on CD4+ T cell activation, maturation, and proliferation, and assessed whether TNF‐inhibitors confer regulatory potential to CD4+ T cells. CyTOF and flow cytometry analysis revealed that in vitro treatment of human CD4+ T cells with the anti‐TNF monoclonal antibody adalimumab promoted IL‐10 expression in CD4+ T cells, whilst decreasing cellular activation. In line with this, analysis of gene expression profiling datasets of anti‐TNF‐treated IL‐17 or IFN‐γ‐producing CD4+ T cells revealed changes in multiple pathways associated with cell cycle and proliferation. Kinetics experiments showed that anti‐TNF treatment led to delayed, rather than impaired T‐cell activation and maturation. Whilst anti‐TNF‐treated CD4+ T cells displayed some hyporesponsiveness upon restimulation, they did not acquire enhanced capacity to suppress T‐cell responses or modulate monocyte phenotype. These cells however displayed a reduced ability to induce IL‐6 and IL‐8 production by synovial fibroblasts. Together, these data indicate that anti‐TNF treatment delays human CD4+ T‐cell activation, maturation, and proliferation, and this reduced activation state may impair their ability to activate stromal cells., Through deep phenotyping using CyTOF and functional assays we have shown that TNF inhibition using adalimumab altered the phenotype and function of multiple populations of human CD4+ T‐cells, leading to an overall decreased pro‐inflammatory potential. These results provide insight into the anti‐TNF mechanism of action in human CD4+ T‐cells.

A considerable proportion of peripheral B cells is autoreactive, and it is unclear how the activation of such potentially harmful cells is regulated. In this study, we show that the different activation thresholds or IgM and IgD BCRs adjust B cell activation to the diverse requirements during development. We rely on the autoreactive 3-83 model BCR to generate and analyze mice expressing exclusively autoreactive IgD BCRs on two different backgrounds that determine two stages of autoreactivity, depending on the presence or absence of the cognate Ag. By comparing these models with IgM-expressing control mice, we found that, compared with IgM, IgD has a higher activation threshold in vivo, as it requires autoantigen to enable normal B cell development, including allelic exclusion. Our data indicate that IgM provides the high sensitivity required during early developmental stages to trigger editing of any autoreactive specificities, including those enabling weak interaction with autoantigen. In contrast, IgD has the unique ability to neglect weakly interacting autoantigens while retaining reactivity to higher-affinity Ag. This IgD function enables mature B cells to ignore autoantigens while remaining able to efficiently respond to foreign threats.

Mammalian group 2 innate lymphoid cells (ILC2s) are responsible for the early production of type 2 cytokines at mucosal barriers upon exposure to allergen. Inflammatory tissue environmental cues can influence ILC2 activity, and this cellular population can be further categorized into subtypes with additional or alternative functions. Subtypes can include trained (or 'memory-like') ILC2s, which recall previous allergic inflammation, inflammatory ILC2s, which acquire the ability to produce IL-17, and ex-ILC2s, which produce ILC1 cytokines. However, the functional states of ILC2s at sites of chronic or severe inflammation are not well characterized. Here, we discuss the emergence of ILC2s with 'exhausted'-like signatures, and argue that their hyporesponsiveness to stimulation and expression of inhibitory receptors is relevant in mammalian chronic allergic inflammation.

The random nature of immunoglobulin gene segment rearrangement inevitably leads to the generation of self-reactive B cells. Avoidance of destructive autoimmune reactions is necessary in order to maintain physiological homeostasis. However, current central and peripheral tolerance concepts fail to explain the massive number of autoantibody-borne autoimmune diseases. Moreover, recent studies have shown that in physiological mouse models autoreactive B cells were neither clonally deleted nor kept in an anergic state, but were instead able to mount autoantibody responses. We propose that activation of autoreactive B cells is induced by polyvalent autoantigen complexes that can occur under physiological conditions. Repeated encounter of autoantigen complexes leads to the production of affinity-matured autoreactive IgM that protects its respective self-targets from degradation. We refer to this novel mechanism as adaptive tolerance. This article discusses the discovery of adaptive tolerance and the unexpected role of high affinity IgM autoantibodies.

Despite the existence of central tolerance mechanisms, including clonal deletion and receptor editing to eliminate self-reactive B cells, moderately self-reactive cells still survive in the periphery (about 20% of peripheral B cells). These cells normally exist in a functionally silenced state called anergy; thus, anergy has been thought to contribute to tolerance by active-silencing of potentially dangerous B cells. However, a positive rationale for the existence of these anergic B cells has recently been suggested by discoveries that broadly neutralizing antibodies for HIV and influenza virus possess poly- and/or auto-reactivity. Given the conundrum of generating inherent holes in the immune repertoire, retaining weakly self-reactive BCRs on anergic B cells could allow these antibodies to serve as an effective defense against pathogens, particularly in the case of pathogens that mimic forbidden self-epitopes to evade the host immune system. Thus, anergic B cells should be brought into a silenced or activated state, depending on their contexts. Here, we review recent progress in our understanding of how the anergic B cell state is controlled in B cell-intrinsic and B cell-extrinsic ways.

Superantigens are unconventional antigens which recognise immune receptors outside the usual binding sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulin receptors on B-cells affecting opsonisation, IgG-mediated phagocytosis, and drive B-cells into apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

The bone marrow transplantation (BMT) between haplo-identical combinations (haploBMT) could cause unacceptable bone marrow graft rejection and graft-versus-host disease (GVHD). To cross such barriers, Johns Hopkins platform consisting of haploBMT followed by post-transplantation (PT) cyclophosphamide (Cy) has been used. Although the central mechanism of the Johns Hopkins regimen is Cy-induced tolerance with bone marrow cells (BMC) followed by Cy on days 3 and 4, the mechanisms of Cy-induced tolerance may not be well understood. Here, I review our studies in pursuing skin-tolerance from minor histocompatibility (H) antigen disparity to xenogeneic antigen disparity through fully allogeneic antigen disparity. To overcome fully allogeneic antigen barriers or xenogeneic barriers for skin grafting, pretreatment of the recipients with monoclonal antibodies (mAb) against T cells before cell injection was required. In the cells-followed-by-Cy system providing successful skin tolerance, five mechanisms were identified using the correlation between super-antigens and T-cell receptor (TCR) Vβ segments mainly in the H-2-identical murine combinations. Those consist of: 1) clonal destruction of antigen-stimulated-thus-proliferating mature T cells with Cy; 2) peripheral clonal deletion associated with immediate peripheral chimerism; 3) intrathymic clonal deletion associated with intrathymic chimerism; 4) delayed generation of suppressor T (Ts) cells; and 5) delayed generation of clonal anergy. These five mechanisms are insufficient to induce tolerance when the donor-recipient combinations are disparate in MHC antigens plus minor H antigens as is seen in haploBMT. Clonal destruction is incomplete when the antigenic disparity is too strong to establish intrathymic mixed chimerism. Although this incomplete clonal destruction leaves the less-proliferative, antigen-stimulated T cells behind, these cells may confer graft-versus-leukemia (GVL) effects after haploBMT/PTCy.

B cell expansion with NF-κB and T cell anergy (BENTA) is a rare primary immunodeficiency disorder caused by gain-of-function (GOF) mutations in the CARD11 gene. Affected patients present with persistent B cell lymphocytosis in early childhood paired with lymphadenopathy and splenomegaly. Until now only six activating mutations from 14 patients have been reported in CARD11. Here we report a patient from China with polyclonal B cell lymphocytosis and frequent infections in early life. A heterozygous mutation (c.377G>A, G126D) in exon 5 of CARD11 gene (NM_032415) was identified by whole exome sequencing. In vitro functional studies showed that the G126D mutation is associated with increased expression of CARD11 and NF-κB activation in Hela cells. Flow cytometry analysis indicated NK cell activity and CD107a degranulation of the patient were decreased. RNA sequencing analysis showed that a number of genes in NF-κB pathway increased while those involved in NK cell activity and degranulation were down-regulated. In summary, our work identified a de novo germline GOF mutation in CARD11 with functional evidence of BENTA.