Supplementary Materials2. possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibited negative autoregulation and controlled pleiotropic attributes of TFH differentiation and function, including migration, costimulation, inhibitory receptors, and cytokines, via multiple repressor-of-repressor gene circuits. INTRODUCTION The formation of germinal centers (GCs) is essential for the development of high-affinity memory B cells and antibody secreting long-lived plasma cells in response to pathogen infections or vaccinations1. Follicular helper T cells (TFH) provide key signals to antigen-specific B cells for the development of germinal center B (BGC) cells1,2. CD4+ T cells receiving TFH inductive signals upregulate Bcl-6, the lineage defining transcription factor (TF) of TFH cells3C5. Upregulation of Bcl-6 is associated with expression of the chemokine receptor CXCR5 and reduction of CCR7 and PSGL1, among other molecules, allowing for migration to the T-B border and GCs1, the sites where TFH and then GC-TFH cells interact with antigen-specific B cells. TFH and GC-TFH cells express many surface and secreted molecules that serve as positive markers and contribute THIQ to the differentiation (ICOS, IL-6R, PD-1), migration (CXCR5, CD69), and function (IL-21, IL-4, CXCL13, SAP, ICOS, PD-1, CD200, CD40L) of TFH and GC-TFH cells. GC-TFH cells provide IL-21, IL-4, and CD40L that are required for BGC cell survival, proliferation, and somatic hypermutation1,2,6. Bcl-6 function is critical in TFH differentiation3C5. Multiple TFs in addition to Bcl-6 have been identified that regulate TFH differentiation2,7C18. Inhibition of Blimp-1 (encoded by TFH cells in response to acute LCMV infection. Expression of TFH signature surface proteins was dysregulated (Fig.1c), indicating that Bcl-6 has important functions in gene regulation beyond repression of Blimp-1 that are necessary for TFH differentiation in both immunization and viral infection contexts. Open in a separate window Figure 1. TFH differentiation is not the default pathway.a, Schematic of the SMARTA cell transfer Rabbit Polyclonal to Synapsin (phospho-Ser9) system used for KLH-gp61 immunization. wild-type, for instructing functional GC-TFH and GC development. Bcl-6 is an autoregulatory repressor in CD4+ T cells In B cells, Bcl-6 is generally considered an obligate repressor of transcription, but Bcl-6 mechanisms of action have been controversial in CD4+ T cells. While Bcl-6 expression positively correlates with expression of many genes in TFH cells, including genes with Bcl-6 binding sites24,25, a mechanistic connection between Bcl-6 binding and gene regulation has been lacking. One example target gene of interest is itself. Bcl-6 binds to its own promoter in human and mouse GC-TFH cells24,25. This Bcl-6 binding site (Promoter Site 1; BPS1) sequence is conserved among mammals (Extended Data Fig.2a). Given that Bcl-6 expression positively correlates with TFH differentiation, Bcl-6 has been considered a plausible candidate for positive regulation by Bcl-6. In contrast, there is evidence THIQ in B cell tumor lines that BCL-6 exhibits negative autoregulation30. To test whether Bcl-6 acts as a repressor or an activator of its own expression in CD4+ T cells, we first utilized a self-inactivating (SIN) retroviral vector (RV) to measure promoter activity THIQ (Fig.2a and Extended Data Fig.2b). SMARTA cells were transduced with the wild-type Thy1.1-RV (an RV construct containing the proximal promoter upstream of a Thy1.1 reporter) or BPS1 Thy1.1-RV (a mutated promoter construct with an 8-nt deletion mutation), transferred to recipient mice, and promoter activity was analyzed in TFH and TH1 cells after acute LCMV infection (Extended Data Fig.2cCd). Wild-type promoter activity (Thy1.1 expression) was reduced in TFH cells compared to TH1 cells. BPS1 promoter activity was increased in TFH cells in comparison to the wild-type promoter (Fig.2b). Thus, Bcl-6 appears to repress promoter activity in TFH cells by binding of Bcl-6 to the BPS1 locus. Open in a separate window Figure 2. Bcl-6 exhibits direct negative autoregulatory feedback.a, Schematic diagram of promoter RV plasmid. Wild-type or BPS1 promoter Thy1.1-RV were generated based on pQdT.