Extended Data Fig. 9: Identification of Klf2 as a repressor, acting downstream of Bcl-6 regulating major of T_FH genes.

Related to Fig. 8 a-c, WT or Bcl6^f/fPrdm1^f/f Cre^Cd4 SMARTA CD4 T cells transfected with crCd8 or crKlf2 were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMV_Arm, and analyzed 6-7 days later (n=4-5 mice per group). (a) Frequency of CD45.1⁺ SMARTA cells among total CD4 T cells from spleen of LCMV_Arm infected mice. (b) Representative flow cytometry of gp₆₆-restimulated IL-21⁺ SMARTA cells. Quantification of results from left panels. (c) Quantification of expression of Tcf-1, T-bet, GATA-3, and Maf, gated on SMARTA cells. Three independent experiments were performed; each dot represents one mouse (a, n = 4; b-c, n = 5). Data are mean ± s.d., unpaired two-tailed Student’s t-test. Related to Fig. 8a-c. d, Schematic of the CRISPR/Cas9-mediated gene knockdown of SMARTA cell system used for testing Klf2 in KLH-gp₆₁ immunization. WT or Bcl6^f/fPrdm1^f/f Cre^CD4 SMARTA CD4 T cells transfected with crCd8 or crKlf2 were transferred to C57BL/6 host mice, followed by immunization of the host mice with KLH-gp₆₁ and alum + cGAMP, and analyzed 8 days later. Related to Fig. 8d-f. e,g, Genome-browser tracks depict ATAC–seq chromatin accessibility and TF occupancy. Peak calls indicated below each track. * and ** indicate DEseq2 raw p-val ≤ 0.05 and ≤ 0.01, respectively, in comparison between Bcl6^f/fPrdm1^f/f Cre^CD4 T_FH-like and Prdm1^f/f Cre^CD4 T_FH. Gene expression from RNA-seq data of LCMV_Arm infected mice and KLH-gp₆₁ immunized mice are graphed. f, Genome-browser tracks depict Tcf1 ChIP–Seq peaks at Pdcd1 and IL6ra loci from murine T_FH cells. h, Gene expression of Tox and Tox2 from RNA-seq data of LCMV_Arm infected mice or KLH-gp₆₁ immunized mice. i, Representative flow cytometry of T_FH cells, gated on SMARTA cells from spleens of LCMV_Arm infected mice (n=4 mice per group) in Fig. 8a. Quantification of results from left panels.