Using Gene Set Enrichment Analysis (GSEA) and looking for defined genelists, three genesets were found enriched in FL R5-PD1dim: PID Notch pathway, KEGG cell adhesion molecules and Biocarta Integrin Pathway (Figures 2DCF); the latter two comprising all described adhesion molecules and integrin downstream molecules, respectively

Using Gene Set Enrichment Analysis (GSEA) and looking for defined genelists, three genesets were found enriched in FL R5-PD1dim: PID Notch pathway, KEGG cell adhesion molecules and Biocarta Integrin Pathway (Figures 2DCF); the latter two comprising all described adhesion molecules and integrin downstream molecules, respectively. and for stromal cells. Proliferation and Survival Assays For proliferation assays, sorted tonsil R5-PD-1dim and GC-Tfh subsets were stained with CFSE and cultured in 10% FCS-RPMI 1640 alone or with pre-seeded TSCs or FRCLs (5:1 ratio) for 4 days with anti-CD3 (0.2 ug/ml) and anti-CD28 (0.2 ug/ml) stimulating antibodies (Sanquin). Cells were then trypsinized and stained with CD2 and CD105 to analyze CFSE+CD2+CD105- T cells. For survival assays, sorted tonsil R5-PD-1dim and GC-Tfh subsets were cultured in 10% FCS-RPMI 1640 alone or with preseeded TSCs or FRCLs (5:1 ratio) for 5 days, followed by CD2, CD105 and active caspase-3 staining according to the manufacturers instructions. Percentage of active caspase-3 unfavorable cells was evaluated on CD2+CD105- T cells. Cytokine Secretion Assay Sorted tonsil or FL R5-PD-1dim and GC-Tfh were cultured for 3 days in 10% FCS-RPMI 1640 with pre-seeded TSCs or FRCLs (5:1 ratio) in presence of anti-CD3 (0.2 ug/ml) and anti-CD28 (0.2 ug/ml) stimulating antibodies. After 3 days, a restimulation step was done with 100 ng/ml phorbol myristate acetate and 750 ng/ml ionomycin for 6?h, supplemented with GolgiPlug (Becton Dickinson) for the last 4?h. For inhibition experiments, Notch chemical inhibitor L685,458 (Sigma Aldrich) or blocking antibodies (bAbs) (Supplemental Table 1) were used. The percentage of singlet viable T cells producing IL-4, IL-21, and IFN- was determined by staining with live/lifeless fixable yellow lifeless cell stain (Thermo Fisher Scientific) and CD2, followed by fixation in paraformaldehyde 4% for 15min, permeabilization with saponin 0.5%, and staining for intracellular cytokines. Statistical Analysis Statistical analyses were performed Bucetin with Graphpad Prism 6 software suite (GraphPad Software) using non-parametric Wilcoxon test for matched pairs, or Mann Whitney U test. Results FRCs Stimulate the Growth of Follicular CXCR5+ CD4+ T-Cell Compartments Having identified two subsets of human CXCR5+CD4+ follicular T cells based on their differential expression of CXCR5 and PD-1 (Supplemental Physique 1), we decided to explore the impact of FRCs on both GC-Tfh and R5-PD1dim cells. Indeed, FRCs express high levels of adhesion molecules, extracellular matrix components, and LN chemokines, and promote B and T cell recruitment, adhesion, and survival (7, 21, 22) in both Bucetin T-cell zone, inter-follicular area, and at follicle border, the place of T-cell priming for Tfh differentiation. In addition, FRCLs obtained by differentiation of uncommitted TSCs have been proposed as a good model to perform functional FRC evaluation (16, 23). Tonsil R5-PD1dim and GC-Tfh were Bucetin prone to die when removed from their microenvironment and were efficiently rescued from death by coculture with Bucetin both TSCs and FRCLs (Figure 1A). In addition, TSCs and FRCLs similarly enhanced the proliferation of R5-PD1dim and GC-Tfh (Figure Rabbit Polyclonal to POU4F3 1B). FRCLs and TSCs displayed thus similar capacities to sustain the growth of R5-PD1dim and GC-Tfh. In order to decipher the specific impact of FRCLs on follicular CD4+ T cells, we then compared their gene expression profile (GEP) with those of TSCs. Unsupervised Pearson correlation performed on the top 20% most variable transcripts adequately segregated TSCs and FRCLs (Figure 1C). We then focused on genes overexpressed in FRCLs (Supplemental Table 3). Unexpectedly, pathway enrichment analysis using REACTOME database revealed a strong enrichment of FCRL signature for Notch-1 and Noctch-2 signaling. Moreover, several genes known to be involved in adhesion and antigen presentation to T cells were found in this FRCL signature and could impact CD4+ T-cell behavior. Among 733 genes, the adhesion molecule ICAM1 was the most upregulated gene. ICAM1 and CD58, which was also overexpressed in FRCL, are two molecules involved in adhesion process through binding of LFA-1 and CD2, respectively. Several inflammatory chemokines, such as CCL2, CCL5, CCL11, and CXCL10 were also found overexpressed, and could be involved in the recruitment of CD4+ activated T cells expressing CCR1, CCR2, CCR3, CCR4, CCR5, or CXCR3 (Table 1). In agreement to the previously demonstrated antigen-presenting cell properties of mouse LN stromal cells (8), we also observed an overexpression of CD74, which is Bucetin involved in the formation and transport of MHC class II protein (24), as well as CD83 which is known to deliver costimulatory signals for naive and memory T-cell activation (25). We also revealed a high expression of immunosuppressive molecules such as HLA-G and CD274, in agreement with the recently proposed role of FRCs in immune tolerance (26C28). Finally, we found an overexpression of cytokines involved.