Search

The absence of alloantibodies is a feature of transplantation tolerance. Although the lack of T cell help has been evoked to explain this absence, herein we provide evidence for B cell-intrinsic tolerance mechanisms. Using a murine model of heart tolerance, we showed that alloreactive B cells were not deleted but rapidly lost their ability to differentiate into germinal center B cells and secrete donor-specific antibodies. We inferred that tolerant alloreactive B cells retained their ability to sense alloantigen because they continued to drive T cell maturation into CXCR5+PD-1+ T follicular helper cells. Unexpectedly, dysfunctional alloreactive B cells acquired the ability to inhibit antibody production by new naive B cells in an antigen-specific manner. Thus, tolerant alloreactive B cells contribute to transplantation tolerance by foregoing germinal center responses while retaining their ability to function as antigen-presenting cells and by actively suppressing de novo alloreactive B cell responses.

Summary Molecular-level understanding of plasma cell (PC) differentiation has been modeled using lipopolysaccharide (LPS) stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)—a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins. For further details on the use and execution of this protocol, please refer to Sciammas et al. (2011) and Ochiai et al. (2018, 2020)., Graphical abstract, Highlights • In vitro plasma cell differentiation system via B-cell receptor signaling • Antibody conjugation of protein A or G beads for immunoprecipitation • Purification of endogenous protein complexes and chromatin-associated proteins • Gel extraction and digestion of proteins in preparation for LC-MS/MS analysis, Molecular-level understanding of plasma cell (PC) differentiation has been modeled using LPS stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)—a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins.

The germinal center (GC) reaction is a coordinated and dynamic ensemble of cells and processes that mediate the maturation and selection of high-affinity GC B cells (GCBs) from lower-affinity precursors and ultimately results in plasma cell and memory cell fates that exit the GC. It is of great interest to identify intrinsic and extrinsic factors that control the selection process. The transcription factor IRF4, induced upon BCR and CD40 signaling, is essential for the acquisition of plasma cell and GCB cell fates. We hypothesized that beyond this early requirement, IRF4 continuously operates at later phases of the B cell response. We show that IRF4 is expressed in GCBs at levels greater than seen in resting cells and plays a role in efficient selection of high-affinity GCBs. Halving Irf4 gene copy number in an Ag-specific murine B cell model, we found that Ag presentation, isotype switching, GC formation and zonation, somatic hypermutation rates, and proliferation were comparable with cells with a full Irf4 allelic complement. In contrast, Irf4 haploinsufficient GCBs exhibited impaired generation of high-affinity cells. Mechanistically, we demonstrate suboptimal Blimp-1 regulation among high-affinity Irf4 haploinsufficient GCBs. Furthermore, in cotransfer settings, we observed a marked disadvantage of Irf4 haploinsufficient cells for GC entry, evidential of ineffective recruitment of T cell help. We propose that, analogous to its role in early GC entry, IRF4 continues to function in the late phase of the Ab response to promote productive T follicular helper cell interactions and to activate optimal Blimp-1 expression during GC selection and affinity maturation.

Summary The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.

Interruption of the programmed death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway is an established and effective therapeutic strategy in human oncology and holds promise for veterinary oncology. We report the generation and characterization of monoclonal antibodies specific for canine PD-1 and PD-L1. Antibodies were initially assessed for their capacity to block the binding of recombinant canine PD-1 to recombinant canine PD-L1 and then ranked based on efficiency of binding as judged by flow cytometry. Selected antibodies were capable of detecting PD-1 and PD-L1 on canine tissues by flow cytometry and Western blot. Anti-PD-L1 worked for immunocytochemistry and anti-PD-1 worked for immunohistochemistry on formalin-fixed paraffin embedded canine tissues, suggesting the usage of this antibody with archived tissues. Additionally, anti-PD-L1 (JC071) revealed significantly increased PD-L1 expression on canine monocytes after stimulation with peptidoglycan or lipopolysaccharide. Together, these antibodies display specificity for the natural canine ligand using a variety of potential diagnostic applications. Importantly, multiple PD-L1-specific antibodies amplified IFN-γ production in a canine peripheral blood mononuclear cells (PBMC) concanavlin A (Con A) stimulation assay, demonstrating functional activity.

Cyclic AMP (cAMP) is involved in multiple biological processes. However, little is known about its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent) regulates conventional type-2 Dendritic Cells (cDC2s), but not cDC1s and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors (TFs) for Th2 induction. Genetic loss of IRF4 phenocopies the effects of cAMP signaling on Th17-induction, indicating that the cAMP effect is secondary to repression of IRF4. Moreover, signaling in cDC2s by a PRR-dependent microbial product, curdlan, represses IRF4 and KLF4, resulting in a pro-Th17 phenotype. These results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the novel cDC2 and cDC17 classification. In addition, the data reveal that cAMP signaling can alter DCs function and fate by repressing IRF4 and KLF4, a pathway that can be harnessed for immuno-regulation.

Veselits et al. show that Igβ ubiquitination activates PI3K and the accumulation of PIP3 on BCR-associated endosomal membranes, which is necessary and sufficient for sorting into classical antigen-processing compartments. Surprisingly, proper BCR sorting is critical for endosomal TLR activation yet dispensable for T-dependent humoral immunity., A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor–containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell–dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.

FcμR is present on the cell surface of B cells as well as other antigen presenting cells where they bind the secreted form of IgM (sIgM). Our previous studies have shown that follicular B cells internalize sIgM continuously in vivo and that lack of sIgM or the FcμR on B cells results in diminished IgG responses following influenza virus infection. We aim to determine the mechanisms underlying the need for sIgM in the development of IgG responses. For this we generated HELB-FcmR−/− and HELB-FcmR+/+ mice. In vitro stimulation of purified B cells from these mice with HEL-OVA conjugate in the presence of OVA-specific OTII CD4 T cells showed an antigen-dose-dependent stimulation of both types of B cells. However, FcmR−/− B cells proliferated less at intermediate and low doses of antigen compared to controls, while they proliferated similar as wild type B cells following high-dose antigen stimulation. Intravenous injection of CD45.1-expressing C57BL/6 mice with B cells from either FcmR-deficient or -sufficient CD45.2+ HELB mice together with sheep red blood cells conjugated to HEL showed no significant differences in HEL-specific serum IgM or IgG, or frequencies of HEL-specific B cells between mice receiving either type of B cell, as assessed by ELISA and FACS, respectively. Ongoing studies are aimed at determining whether antigen-dose affects the need for FcmR expression in vivo. For that we have created mixed bone marrow (BM) irradiation chimeras with 2% BM from CD45.2 HELB or HELB-FcmR−/− mice, and 98% CD45.1 wild type BM. To-date our studies suggest that FcmR-IgM interaction enhances B cell proliferation when antigen is limiting. Future studies are aimed at testing this conclusion and explore how sIgM-FcmR interaction enhanced B cell responses.

Plasma cells (PCs) are terminally differentiated B cells which secrete high levels of immunoglobulin (Ig). While Ig is essential for neutralizing and clearing pathogens, an excess of Ig is detrimental in autoimmune conditions. The transcription factor Blimp-1 is necessary and sufficient for this transition to PCs and is known to regulate Ig secretion. PC differentiation is reliant on multiple levels of regulation - epigenetic, post-transcriptional, and translational, which we hypothesize are dependent on methyltransferase activity for dynamic methylation. PCs also upregulate high levels of CD98, which functions as a membrane transporter for amino acids such as methionine, and thus initialization of the methionine cycle. In this cycle, methionine adenosyltransferase (MAT) coordinates the production of the methyl donor S-adenosylmethionine (SAM) from methionine. We are interested in determining whether this strong expression of CD98 is dependent on Blimp-1 or regulated by PCs, and to understand the role of SAM is this differentiation event. Utilizing a Blimp-1-YFP reporter mouse line, we are able to detect the transition to PCs in-vitro and study the requirement for SAM during this transition. Depletion of the precursor to SAM, methionine, resulted in a loss of PC number and secretory capacity. To specifically target the role of SAM for PCs, we treated in-vitro cultured PCs with inhibitors of MAT2A. Preliminary data provides evidence that inhibition of SAM reduces PC phenotype, function, and viability. We aim to determine the role of SAM in Ig secretion through transcriptional analysis and metabolomics, and hypothesize that Ig post-transcriptional regulation in PCs is controlled by methyltransferase activity.

Summary The transcription factor IRF4 regulates immunoglobulin class switch recombination and plasma cell differentiation. Its differing concentrations appear to regulate mutually antagonistic programs of B and plasma cell gene expression. We show IRF4 to be also required for generation of germinal center (GC) B cells. Its transient expression in vivo induced the expression of key GC genes including Bcl6 and Aicda . In contrast, sustained and higher concentrations of IRF4 promoted the generation of plasma cells while antagonizing the GC fate. IRF4 cobound with the transcription factors PU.1 or BATF to Ets or AP-1 composite motifs, associated with genes involved in B cell activation and the GC response. At higher concentrations, IRF4 binding shifted to interferon sequence response motifs; these enriched for genes involved in plasma cell differentiation. Our results support a model of "kinetic control" in which signaling-induced dynamics of IRF4 in activated B cells control their cell-fate outcomes.

Much of the research on the humoral response to allografts has focused on circulating serum antibodies and the long-lived plasma cells that produce these antibodies. In contrast, the interrogation of the quiescent memory B cell compartment is technically more challenging and thus has not been incorporated into the clinical diagnostic or prognostic toolkit. In this review, we discuss new technologies that have allowed this heretofore enigmatic subset of B cells to be identified at quiescence and during a recall response. These technologies in experimental models are providing new insights into memory B cell heterogeneity with respect to their phenotype, cellular function, and the antibodies they produce. Similar technologies are also allowing for the identification of comparable memory alloreactive B cells in transplant recipients. Although much of the focus in transplant immunology has been on controlling the alloreactive B cell population, long-term transplant patient survival is also critically dependent on protection by pathogen-specific memory B cells. Techniques are available that allow the interrogation of memory B cell response to pathogen re-encounter. Thus, we are poised in our ability to investigate how immunosuppression affects allospecific and pathogen-specific memory B cells, and reason that these investigations can yield new insights that will be beneficial for graft and patient survival.

Despite recent evidence of improved graft outcomes and safety, the high incidence of early acute cellular rejection with belatacept, a high-affinity CTLA4-Ig, has limited its use in clinical transplantation. Here we define how the incomplete control of endogenous donor-reactive memory T cells results in belatacept-resistant rejection in an experimental model of BALB/c.2W-OVA donor heart transplantation into C57BL/6 recipients presensitized to donor splenocytes. These sensitized mice harbored modestly elevated numbers of endogenous donor-specific memory T cells and alloantibodies compared with naive recipients. Continuous CTLA4-Ig treatment was unexpectedly efficacious at inhibiting endogenous graft-reactive T cell expansion but was unable to inhibit late CD4+ and CD8+ T cell infiltration into the allografts, and rejection was observed in 50% of recipients by day 35 after transplantation. When CTLA4-Ig was combined with the sphingosine 1-phosphate receptor-1 (S1PR1) functional antagonist FTY720, alloantibody production was inhibited and donor-specific IFN-γ-producing T cells were reduced to levels approaching nonsensitized tolerant recipients. Late T cell recruitment into the graft was also restrained, and graft survival improved with this combination therapy. These observations suggest that a rational strategy consisting of inhibiting memory T cell expansion and trafficking into the allograft with CTLA4-Ig and FTY720 can promote allograft survival in allosensitized recipients.

Clinical observations that kidney transplant recipients receiving belatacept who experienced T cell-mediated acute rejection can be successfully treated and subsequently maintained on belatacept-based immunosuppression suggest that belatacept is able to control memory T cells. We recently reported that treatment with CTLA4-Ig from day 6 post-transplantation successfully rescues allografts from acute rejection in a BALB/c to C57BL/6 heart transplant model, in part by abolishing B cell germinal centers and reducing alloantibody titers. Here, we show that CTLA4-Ig is additionally able to inhibit established T cell responses independently of B cells. CTLA4-Ig inhibited the in vivo cytolytic activity of donor-specific CD8+ T cells, and the production of IFNγ by graft-infiltrating T cells. Delayed CTLA4-Ig treatment did not reduce the numbers of graft-infiltrating T cells nor prevented the accumulation of antigen-experienced donor-specific memory T cells in the spleen. Nevertheless, delayed CTLA4-Ig treatment successfully maintained long-term graft acceptance in the majority of recipients that had experienced a rejection crisis, and enabled the acceptance of secondary BALB/c heart grafts transplanted 30 days after the first transplantation. In summary, we conclude that delayed CTLA4-Ig treatment is able to partially halt ongoing T cell-mediated acute rejection. These findings extend the functional efficacy of CTLA4-Ig therapy to effector T cells, and provide an explanation for why CTLA4-Ig-based immunosuppression in the clinic successfully maintains long-term graft survival after T cell-mediated rejection.

BackgroundThe dual role of B cells as drivers and suppressors of the immune responses have underscored the need to trace the fate of B cells recognizing donor major histocompatibility complex class I and class II after allograft transplantation.MethodsIn this study, we used donor class II tetramers to trace the fate of I-E-specific B cells after immunization with BALB/c spleen cells or cardiac transplantation, in naive or sensitized C57BL/6 recipients. We combined this approach with genetic lineage tracing of memory B cells in activation-induced cytidine deaminase regulated Cre transgenic mice crossed to the ROSA26-enhanced yellow fluorescent protein reporter mice to track endogenous I-E-specific memory B cell generation.ResultsImmunization with BALB/c splenocytes or heart transplantation induced an expansion and differentiation of I-E-specific B cells into germinal center B cells, whereas BALB/c heart transplantation into sensitized recipients induced the preferential differentiation into antibody-secreting cells. A 10.8-fold increase in the frequency of I-E-specific memory B cells was observed by day 42 postimmunization. Treatment with CTLA4-Ig starting on day 0 or day 7 postimmunization abrogated I-E-specific memory B cell generation and sensitized humoral responses, but not if treatment commenced on day 14.ConclusionsThe majority of donor-specific memory B cells are generated between days 7 and 14 postimmunization, thus revealing a flexible timeframe whereby delayed CTLA4-Ig administration can inhibit sensitization and the generation of memory graft-reactive B cells.

The contribution of T cells and graft-reactive antibodies to acute allograft rejection is widely accepted but the role of graft-infiltrating B and plasma cells is controversial. We examined 56 consecutive human renal transplant biopsies classified by Banff schema into T cell-mediated (N=21), antibody-mediated (N=18) and mixed (N=17) acute rejection, using standard immunohistochemistry for CD3, CD20, CD138, and CD45. In a predominantly African-American population (75%), neither Banff classification nor C4d deposition predicted the return to dialysis. Immunohistochemical analysis revealed CD3+ T cells as the dominant cell type, followed by CD20+ B cells and CD138+ plasma cells in all acute rejection types. Using univariate Cox Proportional Hazard analysis, plasma cell density significantly predicted graft failure while B cell density trended towards significance. Surprisingly T cell density did not predict graft failure. The estimated glomerular filtration rate (eGFR) at diagnosis of acute rejection also predicted graft failure, while baseline eGFR ≥6 months prior to biopsy did not. Using multivariate analysis, a model including eGFR at biopsy and plasma cell density was most predictive of graft loss. These observations suggest that plasma cells may be a critical mediator and/or an independently sensitive marker of steroid-resistant acute rejection.

While most blood lineages are assumed to mature through a single cellular and developmental route downstream of HSCs, dendritic cells (DCs) can be derived from both myeloid and lymphoid progenitors in vivo. To determine how distinct progenitors can generate similar downstream lineages, we examined the transcriptional changes that accompany loss of in vivo myeloid potential as common myeloid progenitors differentiate into common DC progenitors (CDPs), and as lymphoid-primed multipotent progenitors (LMPPs) differentiate into all lymphoid progenitors (ALPs). Microarray studies revealed that IFN regulatory factor 8 (IRF-8) expression increased during each of these transitions. Competitive reconstitutions using Irf8−/− BM demonstrated cell-intrinsic defects in the formation of CDPs and all splenic DC subsets. Irf8−/− common myeloid progenitors and, unexpectedly, Irf8−/− ALPs produced more neutrophils in vivo than their wild-type counterparts at the expense of DCs. Retroviral expression of IRF-8 in multiple progenitors led to reduced neutrophil production and increased numbers of DCs, even in the granulocyte-macrophage progenitor (GMP), which does not normally possess conventional DC potential. These data suggest that IRF-8 represses a neutrophil module of development and promotes convergent DC development from multiple lymphoid and myeloid progenitors autonomously of cellular context.

Confirmation of clinical tolerance requires the cessation of immunosuppressive drugs, which evokes immune reactivation and allograft rejection in all but the rare individuals that successfully transition into a state of operational transplantation tolerance. Therefore the safe conduct of trials in transplantation tolerance requires two conditions: a sensitive and reliable means to identify individuals still being maintained on immunosuppression that are most likely to exhibit tolerance after immunosuppression is withdrawn, and a non-invasive means that assesses the quality or robustness of the tolerant state. Two recent studies attempting to identify a gene signature in peripheral blood of spontaneously tolerant kidney transplant recipients made the unexpected observation that tolerant, but not immune suppressed transplant recipients, exhibited enriched B cells and B cell transcripts in their blood. In concert with the emerging appreciation of a specialized subset of regulatory B cells that possess immunomodulatory function, these observations raise the possibility that regulatory B cells play a critical role in the maintenance of tolerance to renal allografts in transplant patients. This review summarizes these recent findings and speculates on the relationship of regulatory B cells to the maintenance of transplantation tolerance.

PD-1 is an inhibitory receptor that binds to PD-L1 on the surface of tumor and antigen presenting cells in the tumor microenvironment, causing suppression of T-cell activity and tumor progression. Human clinical trials with anti PD-1 monoclonal antibodies, nivolumab and pembrolizumab, showed an objective response rate (ORR) of 30% to 40% with melanoma and 87% with relapsed or refractory Hodgkin’s lymphoma. Here, we have explored the development of canine PD-1/PD-L1 reagents for potential veterinary applications. An S2 insect cell expression system was utilized to produce recombinant canine PD-1Ig and PD-L1Ig and antigen-specific B cells were enriched using PD-1 and PD-L1 tetramers. Enriched B cells were fused and resulting hybridomas were screened for specific reactivity to PD-1 and PD-L1. Then, antibodies capable of blocking PD-1Ig and PD-L1Ig were selected. A total of 5 monoclonal antibodies were generated from the two rounds of immunizations and are being studied for in vitro activity in T cell assays. These canine reagents have multiple diagnostic and therapeutic applications in the context of canine oncology.

Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses towards antibody production via Tfh cells or inflammation by Teff cells. Tfh–Teff fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6–Blimp-1 counter-antagonism. We found that the TCR-signal induced transcription factor IRF4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised IRF4 amounts and promoted Teff fates at the expense of Tfh ones. Importantly, orthogonal induction of IRF4 expression redirected Tfh fate trajectories towards those of Teff and this occurred independently of IL-2 signals. Mechanistically, we linked greater IRF4 abundance with its recruitment towards low affinity binding sites within Teff cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the “reader” of TCR signal strength, in turn, concentration-dependent activity of IRF4 “writes” T helper fate choice.

Sensitized recipients with pretransplant donor-specific Abs are at higher risk for Ab-mediated rejection than nonsensitized recipients, yet little is known about the properties of memory B cells that are central to the recall alloantibody responses. Using cell enrichment and MHC class I tetramers, C57BL/6 mice sensitized with BALB/c splenocytes were shown to harbor H-2Kd–specific IgG+ memory B cells with a post–germinal center phenotype (CD73+CD273+CD38hiCD138−GL7−). These memory B cells adoptively transferred into naive mice without memory T cells recapitulated class-switched recall alloantibody responses. During recall, memory H-2Kd–specific B cells preferentially differentiated into Ab-secreting cells, whereas in the primary response, H-2Kd–specific B cells differentiated into germinal center cells. Finally, our studies revealed that, despite fundamental differences in alloreactive B cell fates in sensitized versus naive recipients, CTLA-4Ig was unexpectedly effective at constraining B cell responses and heart allograft rejection in sensitized recipients.

CD4+ Tcells orchestrate immune responses and destruction of allogeneic organ transplants, but how this process is regulated on a transcriptional level remains unclear. Here, we demonstrated that interferon regulatory factor 4 (IRF4) was a key transcriptional determinant controlling Tcell responses during transplantation. IRF4 deletion in mice resulted in progressive establishment of CD4+ Tcell dysfunction and long-term allograft survival. Mechanistically, IRF4 repressed PD-1, Helios, and other molecules associated with Tcell dysfunction. In the absence of IRF4, chromatin accessibility and binding of Helios at PD-1 cis-regulatory elements were increased, resulting in enhanced PD-1 expression and CD4+ Tcell dysfunction. The dysfunctional state of Irf4-deficient Tcells was initially reversible by PD-1 ligand blockade, but it progressively developed into an irreversible state. Hence, IRF4 controls a core regulatory circuit of CD4+ Tcell dysfunction, and targeting IRF4 represents a potential therapeutic strategy for achieving transplant acceptance.

Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses toward antibody production via Tfh cells or inflammation by Teff cells. Tfh-Teff cell fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6-Blimp-1 counter-antagonism. We found that the TCR-signal-induced transcription factor Irf4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised Irf4 amounts and promoted Teff cell fates at the expense of Tfh ones. Importantly, orthogonal induction of Irf4 expression redirected Tfh cell fate trajectories toward those of Teff. Mechanistically, we linked greater Irf4 abundance with its recruitment toward low-affinity binding sites within Teff cell cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the "reader" of TCR signal strength, and in turn, concentration-dependent activity of Irf4 "writes" T helper fate choice.

T cell dysfunction has emerged as a key event leading to failure in the control of persistent infections and tumors. However, the transcriptional determinants for T cell dysfunction remain unknown. Here we show that IRF4 is a key transcriptional determinant of T cell dysfunction by restraining the dysfunctional differentiation process. We use a heart transplantation model to decipher the molecular basis of T cell dysfunction. Heart allografts survived indefinitely in Irf4-deficient mice. This stable engraftment is due to the progressive establishment of effector T cell dysfunction. Mechanistically, IRF4 represses PD-1, Helios, and other molecules associated with T cell dysfunction. In particular, IRF4 has a profound impact on epigenetic accessibility of PD-1 cis-regulatory elements. In the absence of IRF4, chromatin accessibility as well as binding of Helios at PD-1 cis-regulatory elements are markedly increased in effector T cells, resulting in enhanced expression of PD-1 and T cell dysfunction. Intriguingly, although the dysfunctional state of Irf4-deficient T cells is initially reversible by blockade of PD-1/PD-L1 pathway, it progressively evolves into a “terminal” irreversible state within 30 days post-transplant. Lastly, we revealed that the MEK1/2 inhibitor, trametinib, dramatically decreases IRF4 expression in T cells, abrogates EAE development, and prolongs heart allograft survival. Our results reveal a previously unappreciated aspect of T cell dysfunction, namely the repression of the dysfunctional differentiation of T cells by IRF4. Central control of T cell dysfunction by IRF4 has a significant potential impact on the course of future work in modulating T cell dysfunction

BackgroundThe recommendation by the American Society of Transplantation for annual trivalent inactivated influenza vaccination greater than 3 to 6 months post-kidney transplantation provides a unique opportunity to test the in vivo impact of immunosuppression on recall T- and B-cell responses to influenza vaccination.MethodsThis study took advantage of recent breakthroughs in the single-cell quantification of human peripheral blood B-cell responses to prospectively evaluate both B- and T-cell responses to the seasonal (2010 and 2011) influenza vaccine in 23 stable renal transplant recipients and 22 healthy controls.Results and conclusionThe results demonstrate that the early B-cell response to influenza vaccination, quantified by the frequency of influenza-specific antibody-secreting cells (ASC) in peripheral blood, was significantly reduced in stable transplant recipients compared to healthy controls. The magnitude of the seroresponse and the rate of seroconversion were also blunted. The influenza-specific interferon-gamma (IFNγ) T-cell response was significantly reduced in transplant recipients; however, there was no correlation between the magnitude of the influenza-specific IgG ASC and IFNγ responses. The induction of memory T- and B-cell responses to influenza vaccination supports the recommendation to vaccinate while the blunted responses demonstrate the efficacy of immunosuppression in controlling memory responses individual transplant recipients.

T-cell receptor gammadelta cells (TCRgammadelta) are often found in increased numbers during the course of several viral infections in humans. Although these findings suggest an important role for this unique subset, their precise function has not been ascertained. Recent studies in murine models of both RNA and DNA virus infections have begun to shed new light on the potential function for TCRgammadelta cells in antiviral immunity. It is clear that TCRgammadelta cells participate in the immune response to human immunodeficiency virus (HIV), influenza, Sendai, coxsackie, vaccinia, vesicular stomatitis virus (VSV), and herpes simplex virus-1 (HSV-1) viral infections since they become activated and home to the sites of viral replication. In this review we will summarize current efforts to dissect the role of TCRgammadelta cells in these disease settings, emphasizing the effector functions utilized, the TCR repertoire, and the antigens recognized. Particular focus will be placed on HSV-1 infections where we have begun to address these issues and have shown that TCRgammadelta cells are sufficient for protection from lethal infection and are able to recognize the herpes virus antigen glycoprotein I.

Despite the description of numerous antigenic ligands recognized by TCRγδ cells, detailed information concerning the structural nature of these antigenic epitopes is lacking. In addition, the recent descriptions of human TCRγδ cells recognizing mycobacterium-derived low m.w. lipid molecules confirms that the spectrum and nature of biologic structures that are capable of being recognized by TCRγδ cells are unclear. We have previously described a murine TCRγδ cell clone, TgI4.4, that is reactive to herpes simplex virus (HSV)-1 glycoprotein I (gI). Unlike TCRαβ-mediated, MHC-restricted Ag recognition but similar to Ig Ag recognition, TgI4.4 recognizes purified gI directly, in the absence of Ag processing or presentation. Since gI is a complex glycoprotein, the nature of the antigenic epitope was investigated. First, gI recognition by TgI4.4 is conformationally dependent, as revealed by denaturation and proteolytic experiments. Secondly, the epitope recognized by TgI4.4 was mapped to the amino terminus by using insertion mutants of gI. Lastly, TgI4.4 recognizes the gI protein directly since completely deglycosylated forms of gI are efficiently recognized. Therefore, TCRγδ cells are capable of recognizing a variety of molecular structures, including proteins. The ability of TgI4.4 to recognize a nonglycosylated form of gI suggests that HSV-1 recognition by TCRγδ cells in vivo is not limited by cell-specific glycosylation patterns or glycosylation-dependent conformational influences.

Increased numbers of T cell receptor (TCR)-gamma/delta cells have been observed in animal models of influenza and sendai virus infections, as well as in patients infected with human immunodeficiency virus and herpes simplex virus type 1 (HSV-1). However, a direct role for TCR-gamma/delta cells in protective immunity for pathogenic viral infection has not been demonstrated. To define the role of TCR-gamma/delta cells in anti-HSV-1 immunity, TCR-alpha-/- mice treated with anti- TCR-gamma/delta monoclonal antibodies or TCR-gamma/delta x TCR-alpha/beta double-deficient mice were infected with HSV-1 by footpad or ocular routes of infection. In both models of HSV-1 infection, TCR-gamma/delta cells limited severe HSV-1-induced epithelial lesions and greatly reduced mortality by preventing the development of lethal viral encephalitis. The observed protection resulted from TCR-gamma/delta cell-mediated arrest of both viral replication and neurovirulence. The demonstration that TCR-gamma/delta cells play an important protective role in murine HSV-1 infections supports their potential contribution to the immune responses in human HSV-1 infection. Thus, this study demonstrates that TCR-gamma/delta cells may play an important regulatory role in human HSV-1 infections.

Balancing immunogenicity with inflammation is a central tenet of vaccine design, especially for subunit vaccines that utilize traditional pro-inflammatory adjuvants. Here we report that by using a nanoparticulate peptide-based vaccine, immunogenicity and local inflammation could be decoupled. Self-assembled β-sheet-rich peptide nanofibers, previously shown to elicit potent antibody responses in mice, were found to be non-cytotoxic in vitro and, remarkably, elicited no measurable inflammation in vivo —with none of the swelling at the injection site, accumulation of inflammatory cells or cytokines, or production of allergic IgE that were elicited by an alum-adjuvanted vaccine. Nanofibers were internalized by dendritic cells and macrophages at the injection site, and only dendritic cells that acquired the material increased their expression of the activation markers CD80 and CD86. Immunization with epitope-bearing nanofibers elicited antigen-specific differentiation of T cells into T follicular helper cells and B cells into germinal center cells, as well as high-titer, high-affinity IgG that cross-reacted with the native protein antigen and was neutralizing in an in vitro influenza hemagglutination inhibition assay. These responses were superior to those induced by alum and comparable to those induced by complete Freund's adjuvant. Thus, nanoparticulate assemblies may provide a new route to non-inflammatory immunotherapies and vaccines.

Atopic asthma is an inflammatory pulmonary disease associated with Th2 adaptive immune responses triggered by innocuous antigens. While dendritic cells (DCs) are known to shape the adaptive immune response, the mechanisms by which DCs promote Th2 differentiation remain elusive. Herein we demonstrate that Th2-promoting stimuli induce DC expression of IRF4. Mice with conditional deletion of Irf4 in DCs show a dramatic defect in Th2-type lung inflammation, yet retain the ability to elicit pulmonary Th1 antiviral responses. Using loss- and gain-of-function analysis, we demonstrate that Th2 differentiation is dependent on IRF4 expression in DCs. Finally, IRF4 directly targets and activates the Il-10 and Il-33 genes in DCs. Reconstitution with exogenous IL-10 and IL-33 recovers the ability of Irf4-deficient DCs to promote Th2 differentiation. These findings reveal a regulatory module in DCs by which IRF4 modulates IL-10 and IL-33 cytokine production to specifically promote Th2 differentiation and inflammation.

We have previously shown that mice deficient in protein kinase C theta (PKCθ) have the ability to reject cardiac allografts, but are susceptible to tolerance induction. Here we tested role of B cells in assisting alloimmune responses in the absence of PKCθ. Mouse cardiac allograft transplantations were performed from Balb/c (H-2d) to PKCθ knockout (PKCθ(-/-)), PKCθ and B cell double-knockout (PBDK, H-2b) mice and wild-type (WT) C57BL/6 (H-2b) mice. PBDK mice spontaneously accepted the allografts with the inhibition of NF-κB activation in the donor cardiac allograft. Anti-B cell antibody (rituximab) significantly delayed allograft rejection in PKCθ(-/-), but not in WT mice. Co-transfer of PKCθ(-/-) T plus PKCθ(-/-) B cells or primed sera triggered allograft rejection in Rag1(-/-) mice, and only major histocompatibility complex class II-enriched B cells, but not class I-enriched B cells, were able to promote rejection. This, together with the inability of PKCθ(-/-) and CD28(-/-) double-deficient (PCDK) mice to acutely reject allografts, suggested that an effective cognate interaction between PKCθ(-/-) T and B cells for acute rejection is CD28 molecule dependent. We conclude that T-B cell interactions synergize with PKCθ(-/-) T cells to mediate acute allograft rejection.

Alloantibodies mediate acute antibody-mediated rejection as well as chronic allograft rejection in clinical transplantation. To better understand the cellular dynamics driving antibody production, we focused on the activation and differentiation of alloreactive B cells in the draining lymph nodes and spleen following sensitization to allogeneic cells or hearts. We used a modified staining approach with a single MHC Class I tetramer (K(d)) bound to two different fluorochromes to discriminate between the Class I-binding and fluorochrome-streptavidin-binding B cells with a high degree of specificity and binding efficiency. By Day 7-8 postsensitization, there was a 1.5- to 3.2-fold increase in the total numbers of K(d) -binding B cells. Within this K(d) -binding B cell population, approximately half were IgD(low) , MHC Class II(high) and CD86(+), 30-45% expressed a germinal center (Fas(+) GL7(+)) phenotype and 3-12% were IRF4(hi) plasma cells. Remarkably, blockade with anti-CD40 or CTLA-4Ig, starting on Day 7 postimmunization for 1 or 4 weeks, completely dissolved established GCs and halted further development of the alloantibody response. Thus MHC Class I tetramers can specifically track the in vivo fate of endogenous, Class I-specific B cells and was used to demonstrate the ability of delayed treatment with anti-CD154 or CTLA-4Ig to halt established allo-B cell responses.

The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.

γδ cells participate in pathogenic infections and autoimmune conditions, yet, almost a decade after their discovery, little is known regarding their TCR repertoire or effector functions. Unlike MHC-restricted antigen recognition employed by TCRαβ cells, TCRγδ cells can recognize whole unprocessed antigens in an MHC-independent manner. The nature of positive and negative selection used to shape the repertoire of TCRγδ cells is unclear, especially in the nonlymphoid tissues where these cells predominate. While TCRγδ cells express an activated phenotype and are present in pathological conditions, their roles in immunological protection is unknown. This review will focus on our efforts to study these issues of TCRγδ biology.

Motivation: Identifying the target genes regulated by transcription factors (TFs) is the most basic step in understanding gene regulation. Recent advances in high-throughput sequencing technology, together with chromatin immunoprecipitation (ChIP), enable mapping TF binding sites genome wide, but it is not possible to infer function from binding alone. This is especially true in mammalian systems, where regulation often occurs through long-range enhancers in gene-rich neighborhoods, rather than proximal promoters, preventing straightforward assignment of a binding site to a target gene. Results: We present EMBER (Expectation Maximization of Binding and Expression pRofiles), a method that integrates high-throughput binding data (e.g. ChIP-chip or ChIP-seq) with gene expression data (e.g. DNA microarray) via an unsupervised machine learning algorithm for inferring the gene targets of sets of TF binding sites. Genes selected are those that match overrepresented expression patterns, which can be used to provide information about multiple TF regulatory modes. We apply the method to genome-wide human breast cancer data and demonstrate that EMBER confirms a role for the TFs estrogen receptor alpha, retinoic acid receptors alpha and gamma in breast cancer development, whereas the conventional approach of assigning regulatory targets based on proximity does not. Additionally, we compare several predicted target genes from EMBER to interactions inferred previously, examine combinatorial effects of TFs on gene regulation and illustrate the ability of EMBER to discover multiple modes of regulation. Availability: All code used for this work is available at http://dinner-group.uchicago.edu/downloads.html Contact: dinner@uchicago.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Immunologic tolerance is the ultimate goal of organ transplantation yet, is rarely attainable and an infrequent event in humans. Even with the use of conventional immunosuppression, which has successfully improved short-term allograft survival, long-term allograft survival has remained static and is complicated by serious side effects secondary to the long-term use of immunosuppressive agents. Accordingly, over the past several decades, there has been a push to fully understand both the cellular and molecular mechanisms that play a role in the induction and maintenance of tolerance, with recent data implicating B cells and donor specific alloantibody as a barrier to and potential mediator of allograft tolerance. The study of B cells and alloantibody in transplant tolerance has evolved over recent years from using rodent models to large animals to non-human primate models. This review will discuss the role of B cells and alloantibody as antagonists and facilitators of transplantation tolerance, and highlight the experimental models developed for elucidating the mechanisms of B cell tolerance to alloantigen.