Follicular dendritic cells restrict IL4 availability in germinal centers and foster memory B cell generation
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ABSTRACT: Germinal centers (GCs) are sites of antibody affinity maturation and memory B cell generation. Follicular dendritic cells (FDCs) form a dense stromal network within GCs, but the full extent of their supportive activity is not understood. Here we showed that FDCs expressed IL4Ra and they reduced IL4 availability in GCs. We used scRNA-seq to examine IL4 actions within the GC and identified a subset of light zone cells marked by high IL4Ra and CD23 and lack of a Myc gene-expression signature. Trajectory inference analysis suggested this population could differentiate into pre-memory and pre-plasma cells. Genetic experiments showed that IL4Ra and STAT6 acted intrinsically to restrain pre-memory B cell generation. By reducing IL4 availability, FDCs enhanced GC B cell selection and fostered memory cell generation. This work suggests that GC FDCs provide a niche that limits bystander IL4 activity, focusing IL4 action on B cells undergoing selection and enhancing memory cell differentiation
Project description:Germinal centers (GCs) are clusters of activated B cells built on stromal cells known as follicular dendritic cells (FDCs). In the Peyerâs patches (PPs), GCs are chronically induced by bacteria and are the major sites for generation of gut IgA immune responses. Whether FDCs directly contribute to the IgA production in PP GCs is unknown. To investigate the role FDCs in gut immune system, we examined comprehensive gene profiles of FDCs purified from PPs or perypheral lymph nodes (pLNs) with or without immunization. We also tried to reconstitute the PP FDC signature in vitro by pulsed or continuous stimulation of pLN FDCs through TLRs, RARs or simultaneously through TLRs and RARs. The number of samples is as follows; ex vivo naïve pLN FDC=3, ex vivo immunized pLN FDC=2, ex vivo PP FDCs=3, in vitro without stimulation=2, in vitro with LPS stimulation for 5 hours=1, in vitro with LPS stimulation for 72h=1, in vitro with Pam2CSK4 (Pam) stimulation for 96h=2, in vitro with retinoic acid(RA) stimulation for 96h=2,in vitro with RA+Pam stimulation for 96h=2. In addition, ex vivo FDCs with or without immunizations were compared (3 samples), or in vitro cultured FDCs with or without various stimulations were compared (6 samples)
Project description:Germinal centers (GCs) are clusters of activated B cells built on stromal cells known as follicular dendritic cells (FDCs). In the Peyer’s patches (PPs), GCs are chronically induced by bacteria and are the major sites for generation of gut IgA immune responses. Whether FDCs directly contribute to the IgA production in PP GCs is unknown. To investigate the role FDCs in gut immune system, we examined comprehensive gene profiles of FDCs purified from PPs or perypheral lymph nodes (pLNs) with or without immunization. We also tried to reconstitute the PP FDC signature in vitro by pulsed or continuous stimulation of pLN FDCs through TLRs, RARs or simultaneously through TLRs and RARs.
Project description:Comparison of follicular dendritic cell-enriched versus -depleted splenocytes. Affinity maturation and Ab class switches occur in lymphoid germinal centers (GCs), in which differentiation and maintenance depend on lymphotoxin (LT) signaling and include differentiation of follicular dendritic cells (FDCs). The events leading to FDC and GC maturation are poorly defined. Using several approaches of functional genomics, we enumerated transcripts affected in mice by suppressing LT receptor (LTR) signaling and/or overrepresented in FDC-enriched GC isolates. Protein expression analysis of 3 of 12 genes both enriched in FDCs and down-regulated by LTR signaling suppression validated them as FDC markers. Functional analysis of one of these three, clusterin, suggests a role as an FDC-derived trophic factor for GC B cells. Hence, the set of genes presented in this study includes markers emanating from LTR signaling and transcripts relevant to GC and FDC function.
Project description:Follicular helper T cells (T FH ) mediate B cell selection and clonal expansion in germinal centers (GCs), and follicular regulatory T cells (T FR ) prevent the emergence of self-reactive B cells and help to extinguish the reaction. Here we show that GC reactions continually recruit T cells from both the naïve conventional and naive thymic regulatory T cell (Treg) repertoires. In the early GC, newly recruited T cells develop into T FH , whereas cells entering during the contraction phase develop into T FR cells that contribute to GC dissolution. The T FR fate decision is associated with decreased antigen availability and is modulated by slow antigen delivery or mRNA vaccination. Thus, invasion of ongoing GCs by newly developing T FH and T FR from naïve conventional and Treg repertoires, helps remodel the GC based on antigen availability.
Project description:Common variable immunodeficiency (CVID), characterized by recurrent infections, low serum class-switched immunoglobulin isotypes and poor antigen specific antibody responses, comprises a heterogeneous patient population in terms of clinical presentation and underlying etiology. The diagnosis is regularly associated with a severe decrease of germinal center (GC) derived B-cell populations in peripheral blood. However, data from B-cell differentiation within GC is limited. We present a multiplex approach combining histology, flow cytometry and B-cell receptor repertoire analysis of sorted GC B-cell populations allowing the modelling of distinct disturbances in GCs of three CVID patients. Our results reflect pathophysiological heterogeneity underlying the reduced circulating pool of post-GC memory B cells and plasmablasts in the three patients. In patient 1, quantitative and qualitative B-cell development in GCs is relatively normal. In patient 2, irregular shaped GCs are associated with reduced somatic hypermutation (SHM), antigen selection and class-switching, while in patient 3, high SHM, impaired antigen selection and class-switching with large single clones imply increased re-cycling of cells within the irregular shaped GCs. In lymph nodes of patients 2 and 3, only limited numbers of memory B cells and plasma cells are formed. While reduced numbers of circulating post GC B cells is a general phenomenon in CVID, the integrated approach exemplified distinct defects during GC maturation ranging from near normal morphology and function to severe disturbances with different facets of impaired maturation of memory B cells and/or plasma cells. Integrated dissection of disturbed GC B-cell maturation by histology, flowcytometry and BCR repertoire analysis reveals essential steps during memory formation.
Project description:Stromal-derived follicular dendritic cells (FDCs) are essential for germinal centers (GCs), the site where B cells maturate their antibodies. FDCs present native antigen to B cells and maintain a CXCL13 gradient to form the B cell follicle. Yet despite their essential role, the transcriptome of human FDCs remains undefined. Using single-cell RNA sequencing and microarray, we provided the transcriptome of these enigmatic cells as a comprehensive resource. Key genes were validated by flow cytometry and microscopy. Surprisingly, marginal reticular cells (MRCs) rather than FDCs expressed B cell activating factor (BAFF). Furthermore, we found that human FDCs expressed TLR4 and can alter antigen availability in response to pathogen-associated molecular patterns (PAMPs). High expression of PD-L1 and PD-L2 on FDCs activated PD1 on T cells. In addition, we found expression of genes related to T cell regulation, such as HLA-DR, CD40, and others. These data suggest intimate contact between human FDCs and T cells.
Project description:Stromal-derived follicular dendritic cells (FDCs) are essential for germinal centers (GCs), the site where B cells maturate their antibodies. FDCs present native antigen to B cells and maintain a CXCL13 gradient to form the B cell follicle. Yet despite their essential role, the transcriptome of human FDCs remains undefined. Using single-cell RNA sequencing and microarray, we provided the transcriptome of these enigmatic cells as a comprehensive resource. Key genes were validated by flow cytometry and microscopy. Surprisingly, marginal reticular cells (MRCs) rather than FDCs expressed B cell activating factor (BAFF). Furthermore, we found that human FDCs expressed TLR4 and can alter antigen availability in response to pathogen-associated molecular patterns (PAMPs). High expression of PD-L1 and PD-L2 on FDCs activated PD1 on T cells. In addition, we found expression of genes related to T cell regulation, such as HLA-DR, CD40, and others. These data suggest intimate contact between human FDCs and T cells.
Project description:Ig diversification occurs in peripheral lymphoid organs after establishment of central tolerance during B cell development. In germinal centers (GCs), somatic hypermutation of Ig genes occurs in dark zones, followed by selection of mutated clones in light zones (LZs). This generates high-affinity Ig receptors to pathogens but can also produce autoreactive Ig receptors, which are removed by selection mechanisms that are incompletely understood. The ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop potentially fatal autoimmune diseases; yet, how Itch regulates GC B cells is not well understood. By studying Itch-deficient mice, we have recently shown that Itch directly limits the magnitude of GC responses. Proteomic profiling of GC B cells uncovered that Itch-deficient cells exhibit high mTORC1 and Myc activity, hallmarks of positive selection. Bone marrow chimera and adoptive transfer experiments revealed that B cell Itch restricts noncycling LZ cells. These results support, t our knowledge, a novel role for Itch in skewing selection of GC B cells to restrict LZ accumulation and shape GC-derived humoral immunity. Determining how B cells integrate cues within GCs to navigate through LZs and dark zones will aid in understanding how autoreactive clones emerge from GCs in people with autoimmune disease.
Project description:Germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B-cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh differentiation, although some malaria-infected B-cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through non-canonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC leading to BCL6 suppression, therefore failed to form GC. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon re-infection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.
Project description:The expression of the Nerve growth factor receptor (NGFR) was described in follicular dendritic cells (FDCs), the major lymphoid stromal cell (LSC) compartment regulating B-cell activation within germinal centers (GCs). However, the role of NGFR in humoral response is not well defined. In this work, we have studied the effect of Ngfr KO in LNs organization and function. Ngfr KO led to spontaneous GC formation and expansion of GC B-cell compartment that were related on Ngfr depletion in non-hematopoietic radioresistant compartment. In agreement, Ngfr KO mice showed alterations in LSC with an increased frequency of FDCs harboring an activated phenotype characterized by the overexpression of CD21/35, MAdCAM-1, and VCAM-1. Moreover, Ngfr KO mice showed GC ectopic location, loss of polarization, impaired high-affinity antibody production, and increased circulating autoantibodies. In addition, Ngfr KO/Bcl2 Tg mice displayed increased levels of autoantibodies, higher incidence of autoimmunity, and decreased overall survival. Our work shows that NGFR maintains GC structure and functionality being involved in the regulation of antibody production and immune tolerance.