Fig 2: Expansion of TFH cells in DKO mice is cell-intrinsic. Analysis of mixed bone marrow chimeric mice (n = 8) 8–10 weeks after the reconstitution in Rag-1-deficient hosts. Chimeric mice were generated with 1:1 ratio of wild-type CD45.1 and wild-type CD45.2 bone marrow cells (control) or wild-type CD45.1+ and DKO CD45.2+ bone marrow cells (experimental) or only DKO CD45.2+ bone marrow cells (reference). a–c FACS plots for CXCR5 and PD1 a or CXCR5 and Bcl6 b or ICOS and PD1 c expression gated on CD4+ T cells. d, e Frequencies of TFH cells in the chimeric mice of the indicated genotypes. f Frequencies of GC B cells in the chimeric mice of the indicated genotypes. PLN peripheral lymph nodes. Each dot represents an individual mouse. Error bars indicate mean ± s.d., P-value by two-tailed t-test. Combined data from two independent experiments

Fig 3: Spontaneous expansion of TFH cells in DKO mice. Flow cytometric analysis of TFH cells in the spleens from young (6–11-week old) a, b and aging (>24-week old) c, d wt or DKO (Def6 tr/tr Swap70 -/-) mice. a, c Representative FACS plots for CXCR5 and PD1 (upper panel) or CXCR5 and Bcl6 (lower panel) expression gated on CD4+ T cells. b, d Quantification of the frequencies and numbers of TFH cells (CD4+CXCR5hiPD1hi) (n = 10–13). e, f Flow cytometric analysis of TFH cells in wt, Def6 tr/tr, Swap70 -/-, and DKO mice. e Representative FACS plots for CXCR5 and PD1 (upper panel) or CXCR5 and Bcl6 (lower panel) expression gated on CD4+ T cells in the spleens from mice of the indicated genotype (>18 weeks). f Quantification of TFH cells (CD4+CXCR5hiPD1hi) (n = 5–7). Combined data from three independent experiments. g Representative immunofluorescence images of splenic sections from wt unimmunized mice, wt mice 8 days post-immunization with NP-CGG and DKO unimmunized mice (two independent experiments, n = 2–3 each). PNA, green; PD1, red; CD3, magenta; Scale bar, 100 µm. h, i Quantification of CD4+ T cells producing IL-17, IL-21, and IFN-? in the spleens of aging wt and DKO mice (n = 4–8) analyzed by intracellular flow cytometry. Each dot represents an individual mouse. Error bars indicate mean ± s.d., P-value by two-tailed t-test. Data were combined from at least two independent experiments

Fig 4: De novo protein synthesis of Bcl6 is increased in DKO T cells. a FACS-sorted naive T cells (CD4+CD25-CD44-CD62L+) from wt and DKO mice were stimulated with anti-CD3 and anti-CD28 for 3 days. Nuclear extracts were evaluated by western blotting with antibodies to Bcl6 and IRF4. Lamin B was used as a loading control. Data are representative of three independent experiments. b Quantitative RT–PCR analysis of the expression of Bcl6 mRNA in sorted naive T cells cultured for 2 or 3 days as indicated. The data were normalized relative to ß-Actin mRNA expression. Data are representative of two independent experiments. c Assessment of Bcl6 protein stability was conducted by exposing wt or DKO T cells cultured as in a to either vehicle control or cycloheximide (100 µg/ml) for the indicated times during the final hours of the 3-day culture. Nuclear extracts were assessed by western blotting. IRF4 and Lamin B were used as control. Data are representative of two independent experiments. d Quantification of Bcl6 signal intensity by densitometry over the time course of cycloheximide treatment. The levels of Bcl6 in the vehicle-treated controls were set as 100%. e Autoradiography of SDS-PAGE gel to detect [35S]-labeled Bcl6 protein immunoprecipitated from nuclear extracts of wt or DKO T cells cultured as in a followed by metabolic labeling with [35S]methionine/cysteine for 1 h. Data are representative of two independent experiments. NE nuclear extract, CE cytoplasmic extract. Error bars indicate mean ± s.d., P-value by two-tailed t-test

Fig 5: Expression of Bcl6 protein in DKO T cells is mTOR dependent. a Naive T cells from wt and DKO mice were cultured as described in Fig. 3 and treated with vehicle control or rapamycin (20 nM) for the final 24 h of the 3-day culture. Bcl6 expression was assessed by western blotting using nuclear extracts. Levels of IRF4 and Lamin B were evaluated as control. b Western blotting analysis of the levels of p-4E-BP in extracts from wt or DKO T cells. c Representative FACS plots for CXCR5 and PD1 on splenic CD4+ T cells from young (4–14-week old) wt, DKO, and BXSB-Yaa mice. F female, M male. Data are representative of two independent experiments (n = 3–4). d Representative FACS plots for BTLA and PD1 gated on CD4+CD44+ T cells from DKO or BXSB-Yaa mice. e Representative FACS histograms showing p-4E-BP levels in TFH effector (CD4+BTLAhiPD1hiCD44+) and non-TFH effector (CD4+BTLA-PD1-CD44+) cells of the indicated genotypes. Rapa rapamycin. f Western blotting analysis of the levels of Bcl6 in nuclear extracts from naive wt or DKO T cells cultured as in Fig. 3 and treated with vehicle control or the indicated doses of 4EGI-1 for the final 24 h of the 3-day culture. Levels of IRF4 and HDAC1 were evaluated as control. Data are representative of two independent experiments