Fig 2: T Cell-Specific Tbx21 Deficiency Promotes TFH Differentiation following Influenza InfectionCD4cre and Tbx21?T mice were infected with influenza, and dLN CD4+CD44+ polyclonal and antigen-specific cells were analyzed at d8 p.i.(A–D) Representative flow cytometry plots of polyclonal CD4+CD44+ (A and C) with frequency and total number (B and D) of natural repertoire of TH1 (CD44+PSGL-1+Ly6c+) (A and B) and TFH (CD44+PSGL-1-Ly6c-PD1+CXCR5+) (C and D).(E and F) Plots (E) and frequency and total number (F) of NP311 CD4+ T cells.(G–J) Plots (G and I) and frequency and total number (H and J) of TH1 (NP311+CD44+PSGL-1+Ly6c+) (G and H) and TFH (NP311+ PSGL-1-Ly6c-PD-1+CXCR5+) (I and J).Data are representative of three independent experiments, n = 3–5 mice/group. Data are mean ± SEM.

Fig 3: Combined Blockade of NKG2A and PD-1/PD-L1 Promotes Anti-Tumor Immunity in RMA Rae-1ß Tumor-Bearing C57BL/6J Mice(A) RMA Rae-1ß tumor cells were injected subcutaneously into C57BL/6J mice. Flow cytometry characterization of NK and CD8+ TILs 12 days post-injection, with the spleen used as the standard. Upper panels: representative FACS profiles of PD-1 and NKG2A expression at the surface of NK and CD8+ T cells in the spleen and the tumor bed. Lower panels: pie chart analysis (mean ± SD). The data presented are the pooled results of two independent experiments (n = 8 mice).(B) RMA Rae-1ß tumor-bearing C57BL/6J mice were treated with IC antibodies, anti-NKG2A, anti-PD-L1, or a combination of these last two mAbs. Graphs show tumor growth in each individual mouse and combined survival curves. The data presented are the pooled results of four independent experiments. Complete regressions are indicated. log rank test, ****p < 0.0001.(C) Experiment similar to that in (B), except that the mice were treated with anti-NK1.1 mAb or anti-aCD8a mAb 1 day before the initiation of immunotherapy with the combination of anti-NKG2A and anti-PD-L1 mAbs. Graphs show tumor growth in each individual mouse and combined survival curves. Complete regressions are indicated. log rank test, **p = 0.0024.(D) Upper left panels: FACS profiles of CD44 and CD62L expression on CD8+ T cells in the spleen of naive (no tumor) mice, mice receiving their first injection of RMA Rae-1ß tumor cells (RMA Rae-1ß), and mice previously injected with RMA Rae-1ß tumors, cured by immunotherapy and rechallenged (RMA Rae-1ß + mAbs rechallenged). Percentages of naive (CD44-CD62L+), central memory (TCM, CD44+CD62L+), effector memory (TEM, CD44+CD62L-) and effector CD8+ T cells (eff, CD44-CD62L-) are indicated. Upper right panel: absolute numbers of effector memory CD8+ T cells in the spleen are shown. Lines represent medians. Lower panels: RMA Rae-1ß tumor-bearing C57BL/6J mice were treated with IC antibody or with a combination of anti-NKG2A and anti-PD-L1 mAbs. Mice cured by immunotherapy (n = 13) were rechallenged subcutaneously with RMA-Rae-1ß tumor cells after 70 days. Untreated C57BL/6J mice (n = 15) also received injections of RMA-Rae-1ß cells as a control. The graphs show tumor growth in each individual mouse. The data presented are the pooled results of two independent experiments.See also Figure S3.

Fig 4: The Combined Blockade of NKG2A and PD-1/PD-L1 Promotes Anti-tumor Immunity in A20 Tumor-Bearing BALB/c Mice, Related to Figure 2(A) A20 tumor cells were engrafted in BALB/c mice. Tumor-bearing mice were then treated at three- to four-day intervals with isotype control (IC) antibody, anti-NKG2A antibody, anti-PD-1 antibody or a combination of these last two antibodies. Graphs show tumor growth in each individual and combined survival curves. The data presented are the pooled results of two independent experiments. Log-rank test, **p = 0.0087, ***p = 0.0001, ****p < 0.0001.(B) Experiment similar to that described in (A) but with treatment of the mice with an anti-asialo-GM1 pAbs or an anti-CD8a mAb one day before the initiation of immunotherapy. Graphs show tumor growth in each individual and combined survival curves. Log Rank test, *p < 0.0016, **p < 0.01, ***p = 0.0001.

Fig 5: T and B GC Cells Express T-bet Following Influenza InfectionZsGreen_T-bet reporter mice were infected with influenza and dLNs analyzed.(A–C) Representative confocal micrograph at indicated days p.i. T-bet+ cells (yellow) in GC LZ (IgD-GL7+CD35+) and DZ (IgD-GL7+CD35+). Arrow indicates T-bet+GL7+ cell at d4 p.i. Dashed lines mark GC regions, bottom panels show T-bet+ cells without GC markers (A). Day 8 p.i. T-bet+ GC co-stained with CD4 (cyan) (B) and co-stained with B220 (cyan) (C).(D) Representative flow cytometry plots showing frequency of T-bet+ TFH (red, CD44+PSGL-1- Ly6c- PD-1+ CXCR5+) and T-bet+ TH1 (blue, CD44+ PSGL-1+Ly6c+) at indicated days p.i.(E and F) Frequency (E) and total numbers (F) of T-bet+ TFH and TH1 cells.(G) Flow cytometry plots showing frequency of T-bet+ GC B cells (red, B220+IgD-CD95+CD38-) and plasma cells (blue, B220lo CD138+).(H and I) Frequency (H) and total numbers (I) of T-bet+ GC B and plasma cells.In (E), (F), (H), and (I), data are pooled from three independent experiments. Data are mean ± SEM.