Figure 2.

PD-1-mediated cell intrinsic signaling regulates Tc17 differentiation. (A) Naïve PD-1^+/+ and PD-1^-/- OT-1 CD8⁺ T cells were labelled with CFSE (5μM) before priming them with OVA_257–264 loaded APCs and cultured under Tc17 conditions. Proliferation of PD-1^+/+ and PD-1^-/- Tc17 cells was evaluated by CFSE dilution at indicated time points. (B) Naïve PD-1^+/+ and PD-1^-/-OT-1 CD8⁺ T cells were labelled with CFSE (5μM) or CTV (2.5μM) or vice versa. CFSE and CTV stained cells were combined 1:1 prior to adding OVA_257–264 loaded APCs. At day 3 after primary activation under Tc17 conditions cells were analyzed by flow cytometry for expression of IL-17. Dot plot representing IL-17 expression (dashed gates) in PD-1^+/+ and PD-1^-/- Tc17 cells within each generation (solid gates) of proliferating cells. (C) CD8⁺ T-cells from C57BL/6 mice were stimulated with microspheres immobilized with anti-CD3, anti-CD28 and rPD-L1 (+rPD-L1) or IgG (αCD3/αCD28) under Tc17 polarizing conditions. Three days after primary stimulation, IL-17 and IFN-γ expression in these cells was measured by flow cytometry and are presented in the bar graph. (D) CD8⁺ T-cells from PD-1^+/+ and PD-1^-/- mice were stimulated with microspheres immobilized with anti-CD3, anti-CD28 and rPD-L1 under Tc17 conditions and at day three IL-17 and IFN-γ expression in these cells was measured by flow cytometry. The data is representative of two to five independent experiments. Data points represent individual experiments with mean+SD. ***P<0.001, calculated by Welch’s t-test.