Nature Immunology: doi: /ni Supplementary Figure 1. Gene expression profile of CD4 + T cells and CTL responses in Bcl6-deficient mice.

Transcription

1 Supplementary Figure 1 Gene expression profile of CD4 + T cells and CTL responses in Bcl6-deficient mice. (a) Gene expression profile in the resting CD4 + T cells were analyzed by an Affymetrix microarray chip (MouseGenome array). Differential expressions were found in 10 genes that indicated. (b) Gene expression profile of OT-II derived CD4 + T cells in the response to OVA. The heat map represents expression of signature genes of T H 1, T H 2, T FH, and T H 17 subsets. (c) Flow cytometry analysis of NP tetramer binding CD8 + T cells. Spleen cells were obtained from PR8-infected WT and Bcl6 T mice at day10 post infection and were cultured with NP peptide pulsed EL-4 cells in presence of IL-2. After 5-day culture, cells were stained with NP tetramer and anti-cd8 mab. The graph shows the percentage of NP tetramer binding CD8 cells in WT and Bcl6 T mice (n=4). (d) For the killing assay, NP peptide pulsed EL-4 cells were labeled with CFSE and then co-cultured with spleen cells. After 6hr-culture, killing rate was determined with the EL-4 cell number. The percent lysis was calculated based on the background lysis of EL-4 cells without NP peptide. Mean of the specific lysis were indicated at different effector/target ratios (n=4). Error bars represent S.D.

2 Supplementary Figure 2 Persistent protective response and GC-independent IgG2 memory B formation in Bcl6-deficient mice. (a) Naive mice were intravenously treated with sera from WT, or Bcl6 T mice collected at 2 weeks or 1 year after vaccination and then infected with a lethal dose of Narita (2.5 LD 50 /mouse). Body weight was measured in the mice receiving sera from the unvaccinated C57BL/6 mice (UV) (closed circle), the WT mice at 2-week (2wk) (open circles) (n=5) and 1-year (1y) (filled blue circle) (n=4) post vaccination, and Bcl6 T mice at 1-year post vaccination (filled red circles) (n=4). Statistical analysis was performed using Mann-Whitney U test. **P<0.01. *P<0.05. (b) Circles represent IgG2b + B cell number of HA binding CD38 + memory cells and GL-7 + GC B cells. (c and d) The memory B cells were transferred into Rag1-deficient mice with Narita primed T cells. Circles represent the frequency of virus-specific IgG2b (c) and IgG2c (d) in the spleen at day 7 after IAV challenge (n = 4).

3 Supplementary Figure 3 Generation of HA-specific IgG-producing plasmablasts in Bcl6-deficient mice. (a) Flow cytometry analysis of HA-binding CD138 + B cells. Spleen cells from Narita immunized WT, Bcl6 ΔT and Bcl6 ΔB mice were stained with B220, and CD138 antibodies and APC labeled HA at the indicated days after immunization. HA + CD138 + B cells were sorted for the IgG sequencing analysis. (b) The bar graphs show the absolute number of HA + (top) and CD138 + cells (bottom) (n = 3).

4 Supplementary Figure 4 Gene signature of CXCR3 + CXCR5 + T FH cells and the expression of CXCR3 and CXCR5 in IFN- + cells (a) Construction map of the Venus-targeted Ifng locus (Top). The IRES-Venus cassette was inserted immediately after the stop codon of Ifng gene. Splenocytes from Ifng Venus reporter mice were stimulated with anti-tcr antibody in the presence of IL- 12 and anti-il-4 antibody (for T H 1), or IL-4 and anti-ifn- antibody (for T H 2). Five days after the stimulation, ex-vivo induced T H 1 and T H 2 cells were re-stimulated with anti-tcr antibody, and intracellular staining of IFN- and IL-4 (bottom) were carried out. (b) Heatmap of T H 1, T H 2, T H 17, and T FH signature genes, and heatmap of common signature genes of T H 1 and T FH in CXCR5 + and CXCR3 + CXCR5 + cells. CXCR5 single positive and CXCR3 CXCR5 double positive CD4 + T cells were sorted from vaccinated C57BL/6 mice at day14 post vaccination. The RNA sequencing analysis of sorted populations was carried out by Hiseq. (c) Comparison analysis of CXCR3 + CXCR5 + and CXCR5 + T FH cells. Fold-change of gene expression in CXCR3 + CXCR5 + versus CXCR5 + cells are plotted as histogram. Major T FH signature genes are shown in red. (d) Flow cytometry analysis of Venus expression was examined in splenic CD4 + T cells from vaccinated ifng Venus reporter mice at 14 days after immunization. The CD4+ T cells were separated into 3 fractions based on the magnitude of Venus expression (Negative (Neg), intermediated (Med) and High). Each population was analyzed for PD-1, CXCR5, and CXCR3 expression. The bar graph shows cell number of T FH and CD4 + T cells in Neg, Med, and High fractions (n=3).

5 Supplementary Figure 5 Gene signature of CXCR3 + CXCR5 + T cell population (a) Flow cytometry analysis of PD-1 and CXCR5 expression (top) and CXCR3 and CXCR5 expression (bottom) by CD4 + T cells at the indicated time points. Splenic CD4 + T cells from vaccinated WT or Bcl6 T mice at the indicated days after vaccination were stained for PD-1, CXCR3, and CXCR5. (b) Heatmap of T H 1, T H 2, T H 17, and, T FH signature genes in CXCR5 +, CXCR3 + CXCR5 -, and CXCR3 + CXCR5 dull cells. The indicated populations were sorted from the vaccinated WT or Bcl6 T mice at day14 post vaccination. The RNA sequencing analysis of sorted populations were carried out by Illmina Hiseq.

6 Supplementary Figure 6 Establishment of and characterization of the Il21 ΔT mice (a) Construction of the LoxP-flanked Il21 allele (Il21 f/f ) mice. Exons 1 and 2 of the Il21 gene locus were flanked by LoxP sites (open triangles) by homologous recombination. The flanked region was removed from the mouse germline by crossing with Cd4-cre mice. Arrows show binding sites of PCR primers. (b) Electrophoretic analysis of PCR fragments of DNA in the Il21 locus of the sorted CD4 + T cells from WT and Il21 T mice. Arrows indicate PCR products derived from the Il21 locus. (c) Percentage of CD8 + and CD4 + T cells in thymus of Il21 T mice. (d) The expression of Il21 mrna in splenic CD4 + T cells and Peyer s patches. Il21 mrna expression was measured in CD4 + T cells from Il21 T mice by qpcr. (e) Expression of GL-7 and Fas were analyzed in B220 + B cells from WT or Il21 T mice immunized with Narita. The bar graph shows the percentage of GC B cells in the B220 + population (n=5).

7

8 Supplementary Table 1 The effects of Bcl6 deficiency in T or B cells are limited and not directory related to TH1 function. CXCR3 + CXCR5 - cells were sorted from splenic CD4 + cells of vaccinated WT, Bcl6 T and Bcl6 B mice. After the RNA sequencing analysis, the genes indicating two fold l ower FPKM value in Bcl6 T or Bcl6 B than WT (See Figure 4e) were analyzed with Ingenuity Pathway Analysis. Pathways with a p-value of <0.05 are listed.

9 Supplementary Table 2 List of primers used in this study.