Fig 2: Tfh cell responses in P. chabaudi infection are abrogated in Bcl6fl/flCD4-cre+/- but not in SAP-deficient mice. Bcl6fl/flCD4-cre+/- and Sh2d1a-/- mice were infected with P. chabaudi via intra peritoneal injection of 105 iRBC, and the occurrence of Tfh cells in the spleen was determined by flow cytometry at days 0, 8 and 14 post-infection. (a) Representative plots of naïve (top row) and infected (8 days post-infection, bottom row) Bcl6fl/flCD4-cre+/- and wt control mice. Gates show frequency of CD3+ CD4+ CD44hi cells expressing CXCR5 and PD-1. (b) Total numbers of Tfh cells, defined as CD3+ CD4+ CD44hiCXCR5hiPD-1hi, in Bcl6fl/flCD4-cre+/- and wt control mice. (c) Representative plots of naïve (top row) and infected (8 days post-infection, bottom row) Bcl6fl/flCD4-cre+/- and wt control mice. Gates show frequency of CD3+ CD4+ cells expressing CXCR5 and Bcl6. (d) Total numbers of Tfh cells, defined as CD3+ CD4+ CXCR5+ Bcl6+, in Bcl6fl/flCD4-cre+/- and wt control mice. (e) Representative plots of naïve and infected Sh2d1a-/- and wt C57BL/6 control mice. (f) Total numbers of Tfh cells, defined as CD3+ CD4+ CD44hiCXCR5hiPD-1hi, in Sh2d1a-/- and wt C57BL/6 control mice. (g) Representative plots of naïve and infected Sh2d1a-/- and wt C57BL/6 control mice. (h) Total numbers of Tfh cells, defined as CD3+ CD4+ CXCR5+ Bcl6+, in Sh2d1a-/- and wt C57BL/6 control mice. Bars represent median values. Data are representative or pooled from at least two independent experiments and were obtained in groups of 3–8 mice per time point. Statistical significance was obtained using Two-Way ANOVA with Tukey's multiple comparisons test. ND: Not done.

Fig 3: Bcl6fl/flCD4-cre+/- mice have increased numbers of ?dT cells in the spleen during chronic P. chabaudi infection. Bcl6fl/flCD4-cre+/- and Sh2d1a-/- mice were infected with P. chabaudi via intra peritoneal injection of 105 iRBC, and the number of ?dT cells in the spleen was determined by flow cytometry at days 0, 8, 14 and 120 post-infection. (a) Representative plots of naïve (top row) and infected (14 days post-infection, bottom row) Bcl6fl/flCD4-cre+/- and wt control mice. Upper right quadrants show frequency of CD3+ ?d+ T cells. (b) Total numbers of ?dT cells in Bcl6fl/flCD4-cre+/- and wt control mice. (c) Representative plots of naïve and infected Sh2d1a-/- and wt C57BL/6 control mice. (d) Total numbers of ?dT cells in Sh2d1a-/- and wt C57BL/6 control mice. Bars represent median values. Data are representative or pooled from at least two independent experiments and were obtained in groups of 3–5 mice per time point. Statistical significance was obtained using Two-Way ANOVA with Tukey's multiple comparisons test.

Fig 4: The maturation of DCs in tumors and lymph nodes. (A–C) Cell suspensions from treated Pan02_HVEM tumors were analyzed by flow cytometry after stained with indicated fluorescence-conjugated antibodies. The tumor infiltrating immune cells were presented as the percentages of CD45+ cells in total viable cells (A). DCs (B) and T cells (C) were expressed as the percentages of CD11c+ and CD3+ in CD45+ cells, respectively (n=5). (D–G) Pan02_HVEM cells were mock-infected (black bars), or infected with oHSV (blue bars) or oHSV-CD40L (red bars) at MOI=1. At 12 hpi, bone marrow-derived DCs (BM-DCs) were added and co-cultured for 12 hours. The expression of CD40 (D), CD80 (E), CD86 (F), and MHC-II (G) was analyzed by flow cytometry and presented as the percentages of CD11c+ cells, respectively. (H) The concentration of IL-12 in above co-culture media was quantitated by ELISA and plotted in columns. (I–L) The percentages of CD40+ (I), CD80+ (J), CD86+ (K), and MHC-II+ (L) in CD11c+ cells from treated tumors (on day 20) were plotted in scatter graphs (n=5). (M–P) Cell suspensions from tumor-draining lymph node (tdLN) of treated mice were analyzed by flow cytometry after stained with indicated fluorescence-conjugated antibodies on day 20. The DCs among tdLNs were defined as CD11c+ in CD45+ cells (M), and the percentages of CD40+ (N), CD80+ (O), and CD86+ (P) in CD11c+ cells were plotted in scatter graphs (n=5). (Q) Mice were sacrificed on day 3 or day 6 after completion of treatment. The concentration of IL-12 in the tumors was determined by ELISA (n=3). (R) Mice-bearing Pan02_HVEM tumors received PBS or oHSV-CD40L treatment, and IL-12 was blocked using anti-IL-12 antibody before each virus treatment. The survival of treated mice was plotted using Kaplan-Meier analysis and log-rank test (n=5). Other values were presented as mean±SD and statistics were performed using two-tailed Student’s t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. DC, dendritic cell; IL, interleukin; MHC, major histocompatibility complex; oHSV, oncolytic herpes simplex virus-1; PBS, phosphate buffered saline.

Fig 5: Phenotypic Differences between CD122HI V?7+ IELs and Other IEL Subsets, Related to Figure 1(A) Cell surface phenotype of V?7+, V?7- (CD3+TCRß-V?7-) and aß (TCRß+) IEL from 3-5 week old (W3-5) C57Bl/6 (WT) mice (n = 7). (B) Cell surface phenotype of WT V?7+CD122HI versus V?7+CD122LO IEL (n = 7). (C) Heat map of genes differentially expressed (log-2-FoldChange) between V?7+CD122hi and V?7+CD122lo IEL sorted from D14-D17 WT mice. Data generated by RNA sequencing (‘cell cycle’ & ‘cell surface’ GO terms annotated). Values scaled to their median value across the samples. (D) 3hr EdU incorporation in vivo in V?7+ versus V?7- IEL from D28 WT mice assessed by flow cytometry in indicated IEL subsets (V?7- are CD3+TCRß-V?7-). Data are representative of 1 (C) or = 3 (A,B) independent experiments. Panel (D) presents data pooled from 3 independent experiments. All error bars represent mean ± SD. Related to Figure 1.