Fig. 5: Constitutive TGFβR signaling rescues the anti-PD-1-dependent inhibition of T_RM cell formation and engraftment in a cell-autonomous manner.

a, Schematic showing Thy1.1⁺ or tdTomato⁺ OT-I cell transfer into Thy1.2⁺ C57BL/6J mice at day −1, VACV-OVAss immunization at day 0, treatment with anti-PD-1 or isotype control at days 0, 3, 6 and 9 after infection and isolation of ear skin at days 10–12 after infection (top) and quantification (bottom left) and mean number of cells per experiment (bottom right) of skin CD69⁺CD103^+/–CD8⁺ OT-I cells from anti-PD-1- or isotype (Iso)-treated mice at days 10–12 after infection. Data were pooled from six independent experiments (n = 29 total per group). b, Schematic showing transfer of Thy1.1⁺/Thy1.2⁺ E8i^CreERT2Tgfbrca^fl/+ OT-I cells into Thy1.2⁺ C57BL/6J mice as in a that were also intraperitoneally injected with tamoxifen at days 0–4 after infection, ear skin was collected at days 10–12 after infection (top) and quantification of donor E8i^CreERT2Tgrbrca^fl/+ CD8⁺CD69⁺CD103^+/− OT-I cells in the skin of isotype- and anti-PD-1-treated mice at days 10–12 after infection; isotype, n = 24; anti-PD-1, n = 22. Data were pooled from four independent experiments. c, Schematic showing the activation of Thy1.1⁺ or tdTomato⁺ OT-I splenocytes in vitro by culture with SIINFEKL peptide and IL-2 for 3.5 days and in vivo adoptive transfer of activated OT-I cells into Thy1.2⁺ C57BL/6J mice by intravenous tail vein injection, topical application of 0.5% DNFB on depilated flank skin at day 0, intraperitoneal injection with either anti-PD-1 or isotype control at days 0, 3, 6 and 9 after DNFB application, administration (or no administration) of 0.5 µg of TGFβ1 daily at days 1–9 and isolation of flank skin tissue at day 10 (top) and quantification of donor skin CD8⁺CD69⁺CD103^+/− OT-I cells in anti-PD-1- or isotype-treated mice that received TGFβ1 at day 10 after DNFB or not (bottom); isotype, n = 21; anti-PD-1, n = 23. Data were pooled from six independent experiments in mice without TGFβ1; isotype, n = 17; anti-PD-1, n = 17. Data were pooled from three independent experiments in mice with TGFβ1. The differences between anti-PD-1- and isotype-treated mice or between mice treated with or without TGFβ1 were modeled using MEM on original-scale (a and d) or log-transformed data (c; see Methods, statistics). d, Schematic showing the transfer of Thy1.1⁺/Thy1.2⁺ E8i^CreERT2Tgfbrca^fl/+ OT-I cells as in c, but without TGFβ treatment and with tamoxifen treatment on days 0–5 after DNFB (top) and quantification of donor skin E8i^CreERT2Tgfbrca^fl/+ CD69⁺CD103^+/–CD8⁺ OT-I cells in anti-PD-1- or isotype-treated mice on day 10 (bottom); isotype, n = 13; anti-PD-1, n = 13. Data were pooled from three independent experiments. Bars and error bars show mean ± s.e.m. Statistics were estimated using a linear mixed-effects model with group by treatment interaction, anatomic site when relevant, as fixed effects and a random intercept for each mouse or replicate; *P ≤ 0.05; ****P ≤ 0.0001.