Differentially expressed genes were filtered by keeping transcripts with at least 1 read from each population significant at FDR-adjusted value, with the exact value presented within each figure legend. Supplementary Material 1Click here to view.(6.2M, docx) 2Click here to view.(59K, pdf) ACKNOWLEDGEMENTS The authors acknowledge members of our departments for critical review of the manuscript. signals provided by CD4+ follicular helper T (TFH) cells1, including interleukin 21 (IL-21) and costimulatory molecules such as CD40L (CD40 ligand) 2-5. The signals provided by TFH cells include cytokines shared by other Rabbit polyclonal to NGFRp75 TH cell subsets, such as IL-4 and interferon- (IFN-), which promote B cell isotype switching BMS-5 appropriate to pathogen challenge 3,6-8. TFH cell-derived IL-21 is a key regulator of the GC as, in its absence, B cells display defects in affinity maturation and generation of long-lived plasma cells 4,5. IL-4 also promotes the GC response as mice deficient in this cytokine or its high affinity receptor IL-4R have compromised immunoglobulin IgG1 and IgE responses 7,9,10, and its deletion results in defective GC B cell expansion 7. IL-4 secretion, together with CD40-CD40L signaling, enables TFH cells to induce the enzyme activation-induced cytidine deaminase (AID) in B cells, necessary for class switch recombination (CSR) and Ig affinity maturation 6,11. The interplay of IL-21 and IL-4 signals shapes the humoral response, with IL-21-deficiency in mice resulting in increased IL-4-driven IgE switching, with their combined deficiency leading to an impairment in GC formation and antibody responses that exceeds that of either alone 12,13. Interactive engagement between TFH cells and GC B cells entails repeated short-lived cellular contacts 14. Chronological accumulation of T cell-derived signals results in the development of B cells expressing high affinity Ig receptors 15, and their differentiation into antibody secreting cells (ASCs) 16. Conversely, repetitive cognate T-GC B cell interactions result in TCR-dependent changes in Ca+ and in cytokine expression in T cells 17, with B cell-derived ICOS signals promoting proper positioning of TFH cells within the B cell follicle and GC 18 and upregulation of CD40L on TFH cells 19, necessary for GC B cell selection 20. Here we show that as a consequence of T-B cell interactions, TFH cell function evolved during the GC response, with these changes critical for B cell maturation. TFH cells differentiated from an IL-21+ TFH population observed proximally to the GC dark zone, the site of Ig gene hypermutation, early after immune challenge to an IL-4+ TFH cell population robustly expressing CD40L that developed later and resided more distal to the dark zone. Modulation of the TFH cell phenotype within the GC was dependent upon cell division and occurred in concert with alterations in gene expression. These distinct TFH cell populations were responsible for unique effects on B cell maturation, with the IL-21+ BMS-5 TFH cells enabling selection of high-affinity clones and IL-4+ TFH cells facilitating differentiation of antibody-secreting plasma cells. Thus, after entering the GC, TFH cells undergo progressive maturation to regulate GC B cell differentiation. RESULTS IL-4 and IL-21 expression define three populations of TFH cells Disruption of signaling by either IL-21 or IL-4 results in defective humoral responses 4,5,7,12,21. The non-redundant functions of IL-21 or IL-4 22 suggest that TFH cells producing these cytokines are discrete, differing in their ability to regulate GC B cells. To explore this possibility, we generated C57BL/6 (B6) bicistronic (Kat) reporter mice (infection of (Kat?GFP+), (Kat+GFP+), and (Kat+GFP?) CD4+ cells, respectively. (e) Flow cytometry of CellTrace Violet labeled donor CD4+Thy1.2+ < 0.05; **< 0.01; ***< 0.001 (Student's begins in lymph nodes (LNs) of the mediastinum, followed by those in the mesentery, and then the spleen 28. In the mediastinal LNs of and following transfer of CellTrace Violet? dye labeled ovalbumin (OVA)-specific Thy1.2+CD4+OT-II TCR transgenic T cells from combined with 4-hydroxy-3-nitrophenylacetyl-OVA (NP-OVA), followed by a single intravenous (i.v.) injection of NP-OVA two days post-infection, to ensure Ag persistence and enable tracking of Ag-specific T and B cells. plus NP-OVA injection we found infection. Although we detected three TFH cell populations expressing and mRNA between days 5 and 8 during our initial time-course experiment, intracellular cytokine staining after stimulation with phorbol 12-myristate 13-acetate and ionomycin at these time points indicated that TFH cells primarily BMS-5 produced either IL-4 or IL-21 (Supplementary Fig. 4a). Similar observations were made after i.p. immunization of wild type mice.