Project description:Purpose: The goal of this study was to assess if TLR9 deficiency (TLR9-/-) or TLR9 incapacity to signal through MyD88 would differentially influence the gene programs of B cell subsets, in a B cell intrinsic manner. Methods : Mixed bone marrow chimeras of one-third each TLR9+/+ CD45.1/2, TLR9-/- CD45.1/1 and TLR9P915H/P915H CD45.2/2 were generated on the MRL/lpr background. From each recipient, three B cell subsets, FO, MZ and ABC were sorted with respect to the CD45 congenic markers at [x weeks] post chimerism. RNA was isolated using the RNeasy Plus Micro Kit (QIAGEN). Samples were sequenced using NovaSeq 6000 flowcell (Illumina, Inc, California, USA) with 100 bp paired-end reads (20 million reads per sample) and aligned to the mm10 genome using the STAR aligner. Results: ABCs showed the most transcriptional differences among TLR9 genotypes. TLR9P915H and TLR9WT ABC transcriptomes were much more similar to each other than to those of TLR9-/- mice, sharing 246 of 381 DEGs, even though TLR9P915H B cells could not signal via MyD88. These included a number of genes encoding for inhibitory or anti-inflammatory pathways, suggesting that TLR9 could induce a regulatory signal independently of MyD88.
Project description:We compared in vitro mTECs-high stimulated with TLR9 ligand CpG ODN (1826) or non stimulated from MyD88 -/- and MyD88 +/+ mice: Thymi were enzymaticaly digested, cells were MACS enriched for CD45- fraction, and FACS sorted using BD Influx sorter. mTECs-high were gated as EpCAM+CD11c-Ly51-MHCII+CD80+. Cell from MyD88 -/- and MyD88 +/+ mice were incubated in RPMI with or without CpG ODN (1826) for 24 hours. Total RNA was isolated using RNeasy Plus Micro Kit (Qiagen). 4 samples per condition were used.
Project description:CD4 T cell help is critical for both the generation and maintenance of germinal centers, and T follicular helper (TFH) cells are the CD4 T cell subset required for this process. SAP (SH2D1A) expression in CD4 T cells is essential for germinal center development. However, SAP-deficient mice have only a moderate defect in TFH differentiation as defined by common TFH surface markers. CXCR5+ TFH cells are found within the germinal center as well as along the boundary regions of T/B cell zones. Here we show that germinal center associated T cells (GC TFH) can be identified by their co-expression of CXCR5 and the GL7 epitope, allowing for phenotypic and functional analysis of TFH and GC TFH populations. Here we show GC TFH are a functionally discrete subset of further polarized TFH cells, with enhanced B cell help capacity and a specialized ability to produce IL-4 in a TH2-independent manner. Strikingly, SAP-deficient mice have an absence of the GC TFH subset and SAP- TFH are defective in IL-4 and IL-21 production. We further demonstrate that SLAM (Slamf1, CD150), a surface receptor that utilizes SAP signaling, is specifically required for IL-4 production by GC TFH. GC TFH cells require IL-4 and IL-21 production for optimal help to B cells. These data illustrate complexities of SAP-dependent SLAM family receptor signaling, revealing a prominent role for SLAM receptor ligation in IL-4 production by germinal center CD4 T cells but not in TFH and GC TFH differentiation. Analysis of in vivo polyclonal GC Tfh vs Tfh vs Non-Tfh eight days after LCMV viral infection. Analysis of in vivo follicular helper CD4 T cells (CXCR5high GL7low), versus germinal center follicular helper CD4 T cells (CXCR5hi GL7hi), versus non-follicular helper CD4 T cells (CXCR5low) eight days after viral infection.
Project description:We have recently demonstrated that the function of T follicular helper (Tfh) cells obtained from lymph nodes (LN) of HIV-infected individuals is impaired. We found that these cells were unable to provide proper help to germinal center (GC)-B cells, as observed by altered and inefficient anti-HIV antibody response and premature death of memory B cells. The underlying molecular mechanisms of this dysfunction remain poorly defined. Herein, we have used a unique transcriptional approach to identify these molecular defects. We consequently determined the transcriptional profiles of LN GC-Tfh cells following their interactions with LN GC-B cells from HIV-infected and HIV-uninfected individuals, rather than analyzing resting ex-vivo GC-Tfh cells. We observed that proliferating HIV-infected GC-Tfh cells were transcriptionally different than those from HIV-uninfected individuals, displaying a significant downregulation of immune- and GC-Tfh-associated pathways and genes compared to cells from uninfected individuals. Our results strongly demonstrated that MAF (coding for the transcription factor c-Maf) and its upstream signaling pathway mediators (IL6R and STAT3) were significantly downregulated in HIV-infected subjects, which could contribute to the impaired GC-Tfh and GC-B cell functions reported during infection. We further showed that c-Maf function was associated with the adenosine pathway and that the signaling upstream c-Maf could be partially restored by adenosine deaminase -1 (ADA-1) supplementation. Overall, we identified a novel mechanism that contributes to GC-Tfh impairment during HIV infection. Understanding how GC-Tfh function is altered in HIV is crucial and could provide critical information about the mechanisms leading to the development and maintenance of effective anti-HIV antibodies.
