ABSTRACT
B cells play a critical role in host defense, producing antibodies in response to microbial infection. An inability to produce an effective antibody response leaves affected individuals prone to serious infection; therefore, proper B-cell development is essential to human health. B-cell development begins in the bone marrow and progresses through various stages until maturation occurs in the spleen. This process involves several sequential, complex events, starting with pre- and pro-B cells, which rearrange the heavy and light chain genes responsible for producing clonally diverse immunoglobulin (Ig) molecules. These cells then differentiate into immature B cells, followed by mature B cells. The bone marrow is a complex ecological niche of supporting stromal cells, extracellular matrix components, macrophages, and hematopoietic precursor cells influencing B-cell development, maturation, and differentiation. Once fully mature, B cells circulate in peripheral lymphoid organs and can respond to antigenic stimuli. As specific cell surface markers are expressed during each stage of B-cell development, researchers use flow cytometry as a powerful tool to evaluate developmental progression. In this protocol, we provide a step-by-step method for bone marrow isolation, cell staining, and data analysis. This tool will help researchers gain a deeper understanding of the progression of B-cell development and provide a pertinent flow gating strategy.
ABSTRACT
CD4 tissue-resident memory T cells (TRMs) allow robust protection of barrier surfaces against pathogens. We investigated the role of T-bet in the formation of liver CD4 TRMs using mouse models. T-bet-deficient CD4 T cells did not efficiently form liver TRMs when compared with wild-type (WT). In addition, ectopic expression of T-bet enhanced the formation of liver CD4 TRMs, but only when in competition with WT CD4 T cells. Liver TRMs also expressed higher levels of CD18, which was T-bet dependent. The WT competitive advantage was blocked by Ab neutralization of CD18. Taken together, our data show that activated CD4 T cells compete for entry to liver niches via T-bet-induced expression of CD18, allowing TRM precursors to access subsequent hepatic maturation signals. These findings uncover an essential role for T-bet in liver TRM CD4 formation and suggest targeted enhancement of this pathway could increase the efficacy of vaccines that require hepatic TRMs.
Subject(s)
CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Animals , Mice , Immunologic Memory , Liver , Memory T Cells , CD18 AntigensABSTRACT
Class switch recombination generates distinct antibody isotypes critical to a robust adaptive immune system, and defects are associated with autoimmune disorders and lymphomagenesis. Transcription is required during class switch recombination to recruit the cytidine deaminase AID-an essential step for the formation of DNA double-strand breaks-and strongly induces the formation of R loops within the immunoglobulin heavy-chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here, we report that cells lacking two enzymes involved in R loop removal-senataxin and RNase H2-exhibit increased R loop formation and genome instability at the immunoglobulin heavy-chain locus without impacting its transcriptional activity, AID recruitment, or class switch recombination efficiency. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking senataxin or RNase H2B alone. We propose that senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.
The immune system is a complex network of cells and molecules, which helps to protect the body from invaders. The adaptive immune system can recognise millions of assailants, kill them, and 'learn' from this experience to mount an even quicker defence the next time the body is infected. To achieve this level of protection, specific immune cells, called B cells, divide when they come into contact with a molecule from a foreign particle, the antigen. The cloned B cells then produce millions of protective proteins, the antibodies, which patrol the blood stream and tag harmful particles for destruction. An antibody resembles a Y-shaped structure that contains a 'variable' region, which gives it the specificity to interact with an antigen, and a 'constant' region, which interacts with components of the immune system and determines the mechanisms used to destroy a pathogen. Based on the constant region, antibodies can be divided into five main classes. B cells are able to switch their production from one antibody class to another in an event known as class switch recombination, by making changes to the constant region. They do this by cutting out a portion of the genes for the constant region from their DNA and fusing the remaining DNA. The resulting antibodies still recognise the same target, but interact with different components of the immune system, ensuring that all the body's forces are mobilised. R-loops are temporary structures that form when a cell 'reads' the instructions in its DNA to make proteins. R-loops provide physical support by anchoring the transcription template to the DNA. They help control the activity of genes, but if they stay on the DNA for too long they could interfere with any form of. DNA repair including the cutting and fusing mechanisms during class switch recombination. To find out more about this process, Zhao et al. used B-cells from mice lacking two specific proteins that usually help to remove R-loops. Without these proteins, the B cells generated more R-loops than normal. Nevertheless, the B-cells were able to undergo class switch recombination, even though their chromosomes showed large areas of DNA damage, and DNA sections that had been repaired contained several mistakes. Errors that occur during class switch recombination have been linked to immune disorders and B cell cancers. The study of Zhao et al. shows that even if R-loops do not affect some processes in B cells, they could still impact the overall health of their DNA. A next step would be to test if an inability to remove R-loops could indeed play a role in immune disorders and B-cell cancers.
Subject(s)
Recombination, Genetic , Ribonucleases , Humans , Ribonucleases/genetics , Immunoglobulin Class Switching/genetics , Endoribonucleases/genetics , Immunoglobulin Isotypes/genetics , Genomic Instability , Cytidine Deaminase/geneticsABSTRACT
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.
Subject(s)
B-Lymphocytes , Haploinsufficiency , Animals , B-Lymphocytes/metabolism , Cell Differentiation/genetics , Germinal Center/metabolism , Mice , Plasma Cells/metabolismABSTRACT
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).
Subject(s)
Cell Differentiation , Chromatin/metabolism , Plasma Cells/cytology , Proteins/isolation & purification , Receptors, Antigen, B-Cell/metabolism , Animals , Chromatography, Liquid/methods , Culture Media , Mice , Mice, Transgenic , Proteins/metabolism , Receptors, Antigen, B-Cell/genetics , Tandem Mass Spectrometry/methodsABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , Ikaros Transcription Factor/metabolism , Interferon Regulatory Factors/metabolism , Transcription Factors/metabolism , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Cell Differentiation/physiology , Cell Line, Tumor , Humans , Mice , Mice, TransgenicABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , B7-H1 Antigen/immunology , Programmed Cell Death 1 Receptor/immunology , T-Lymphocytes/immunology , Animals , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Dogs , Interferon-gamma/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Peptidoglycan/pharmacology , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes/metabolismABSTRACT
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.
Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Isoantibodies/immunology , Isoantigens/immunology , T-Lymphocytes, Helper-Inducer/immunology , Transplantation Tolerance , Animals , B-Lymphocytes/pathology , Female , Germinal Center/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , T-Lymphocytes, Helper-Inducer/pathologyABSTRACT
Control of diverse pathogens requires an adaptive antibody response, dependent on cellular division of labor to allocate antigen-dependent B- and CD4+ T-cell fates that collaborate to control the quantity and quality of antibody. This is orchestrated by the dynamic action of key transcriptional regulators mediating gene expression programs in response to pathogen-specific environmental inputs. We describe a conserved, likely ancient, gene regulatory network that intriguingly operates contemporaneously in B and CD4+ T cells to control their cell fate dynamics and thus, the character of the antibody response. The remarkable output of this network derives from graded expression, designated by antigen receptor signal strength, of a pivotal transcription factor that regulates alternate cell fate choices.
Subject(s)
Antibody Formation , B-Lymphocytes , Gene Regulatory Networks , Interferon Regulatory Factors , T-Lymphocytes, Helper-Inducer , Animals , Antibody Formation/genetics , B-Lymphocytes/immunology , Cell Differentiation , Gene Expression Regulation , Humans , Interferon Regulatory Factors/immunology , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.
Subject(s)
Interferon Regulatory Factors , Receptors, Pattern Recognition , Signal Transduction/immunology , Th17 Cells , Th2 Cells , Animals , Cell Line, Tumor , Cyclic AMP/immunology , Cyclic AMP/metabolism , Cytokines , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Interferon Regulatory Factors/antagonists & inhibitors , Interferon Regulatory Factors/immunology , Interferon Regulatory Factors/metabolism , Kruppel-Like Factor 4 , Mice , Receptors, Pattern Recognition/immunology , Receptors, Pattern Recognition/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
CD4+ T cells 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 T cell responses during transplantation. IRF4 deletion in mice resulted in progressive establishment of CD4+ T cell dysfunction and long-term allograft survival. Mechanistically, IRF4 repressed PD-1, Helios, and other molecules associated with T cell 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+ T cell dysfunction. The dysfunctional state of Irf4-deficient T cells 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+ T cell dysfunction, and targeting IRF4 represents a potential therapeutic strategy for achieving transplant acceptance.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Graft Rejection/immunology , Graft Survival , Heart Transplantation , Interferon Regulatory Factors/immunology , Animals , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Differentiation , Cell Movement , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Gene Expression Profiling , Gene Expression Regulation , Graft Rejection/genetics , Graft Rejection/mortality , Graft Rejection/pathology , Granzymes/genetics , Granzymes/immunology , Interferon Regulatory Factors/deficiency , Interferon Regulatory Factors/genetics , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-17/genetics , Interleukin-17/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/immunology , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/immunology , Signal Transduction , Survival Analysis , Transcription Factors/genetics , Transcription Factors/immunology , Transplantation, HomologousABSTRACT
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.
Subject(s)
CD79 Antigens/metabolism , Endosomes/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Signal Transduction , Ubiquitination , Animals , B-Lymphocytes/metabolism , Endocytosis , Histocompatibility Antigens Class II/metabolism , Immunity, Humoral , Male , Mice, Inbred C57BL , Phosphatidylinositol Phosphates/metabolism , Receptors, Antigen, B-Cell/metabolism , Toll-Like Receptors/metabolism , Ubiquitin/metabolismABSTRACT
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 posttransplantation 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.
ABSTRACT
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.
Subject(s)
Gene Expression Regulation , Gene Regulatory Networks , Interferon Regulatory Factors/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Animals , Antigens/immunology , Binding Sites , Cell Differentiation/immunology , Cell Line , Female , Gene Expression Profiling , Humans , Immunization , Interferon Regulatory Factors/genetics , Interleukin-2/metabolism , Male , Mice , Mice, Knockout , Nucleotide Motifs , Protein Binding , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes, Helper-Inducer/cytologyABSTRACT
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.
ABSTRACT
BACKGROUND: The 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. METHODS: In 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. RESULTS: Immunization 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. CONCLUSIONS: The 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.
Subject(s)
Abatacept/administration & dosage , B-Lymphocytes/drug effects , Cell Lineage , Cell Proliferation/drug effects , Graft Rejection/prevention & control , Heart Transplantation/adverse effects , Histocompatibility Antigens Class II/immunology , Immunologic Memory , Immunosuppressive Agents/administration & dosage , Lymphocyte Activation/drug effects , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Cell Tracking/methods , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Disease Models, Animal , Drug Administration Schedule , Genotype , Graft Rejection/blood , Graft Rejection/genetics , Graft Rejection/immunology , Graft Survival/drug effects , Histocompatibility Antigens Class II/blood , Integrases/genetics , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , RNA, Untranslated/genetics , Time FactorsABSTRACT
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-2K(d)-specific IgG(+) memory B cells with a post-germinal center phenotype (CD73(+)CD273(+)CD38(hi)CD138(-)GL7(-)). These memory B cells adoptively transferred into naive mice without memory T cells recapitulated class-switched recall alloantibody responses. During recall, memory H-2K(d)-specific B cells preferentially differentiated into Ab-secreting cells, whereas in the primary response, H-2K(d)-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.
Subject(s)
Abatacept/pharmacology , B-Lymphocytes/drug effects , Graft Survival , Heart Transplantation , Immunologic Memory , 5'-Nucleotidase/analysis , Allografts , Animals , B-Lymphocytes/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Programmed Cell Death 1 Ligand 2 Protein/analysisABSTRACT
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.
Subject(s)
B-Lymphocytes/immunology , Immunity, Humoral , Immunologic Memory , Organ Transplantation , Transplantation Tolerance , Allografts , Animals , B-Lymphocytes/drug effects , Graft Rejection/blood , Graft Rejection/immunology , Graft Rejection/prevention & control , Graft Survival , HLA Antigens/immunology , Humans , Immunity, Humoral/drug effects , Immunologic Memory/drug effects , Immunosuppressive Agents/therapeutic use , Isoantibodies/blood , Organ Transplantation/adverse effects , Phenotype , Transplantation Tolerance/drug effects , Treatment OutcomeABSTRACT
Casitas B-lineage lymphoma-b (Cbl-b) is a ubiquitin ligase (E3) that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR) and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA) domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9) into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Antigens/immunology , Endosomes/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Receptors, Antigen, B-Cell/metabolism , Toll-Like Receptor 9/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Animals , Endocytosis , Humans , Mice, Inbred C57BL , Mutant Proteins/metabolism , Protein Structure, Tertiary , Protein Transport , Proto-Oncogene Proteins c-cbl/chemistry , Spleen/enzymology , Ubiquitin-Protein Ligases/metabolismABSTRACT
BACKGROUND: The 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. METHODS: This 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 CONCLUSION: The 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.