Figure 2. Mouse 4PD1^hi limit T-cell effector functions.

(A) Schema of in vitro suppression assay of CD45.1⁺ T cells (target) with 4PD1^hi, 4PD1⁻ or PD-1⁻Tregs FACS-sorted from spleens of naive Foxp3-GFP mice (CD45.1⁻, effectors). (B) CTV dilution and frequency of CD44⁺CD25⁺ in total CD45.1⁺CD4⁺ target T cells in the indicated co-cultures at the indicated effector:target ratios after 48-hr incubation (mean ± SD; n=2; 2-way ANOVA, 4PD1^hi and Tregs vs 4PD1⁻). (C) Quantification of IFN-γ, TNF-α and IL-2 by FACS-based bead immunoassay in culture supernatants of CD4⁺ cells alone or co-cultured with the indicated cells for 48 hr (ratio 1:1; mean ± SD; n=2; unpaired t test). (D) Foxp3, CD25 and PD-1 MFI in effector CD45.1⁻CD4⁺ T-cell subsets co-cultured with CD45.1⁺CD4⁺ target T cells (ratio 1:1) for 48 hr (mean ± SD; n=2; unpaired t test). (E) In vivo suppression assay with 4PD1^hi or Tregs FACS-sorted from B16-bearing Foxp3-GFP mice and co-transferred with CFSE-labeled Pmel/gp100-TCR-specific CD8⁺ T cells (Pmels) (1:1 ratio) into irradiated CD45.1⁺ recipients and stimulated in vivo with irradiated B16 cells (schema). Proliferation (CFSE dilution) and activation (CD44 and CD25 expression) of CD45.1⁻Thy1.1⁺CD8⁺ Pmels in recipient spleens (mean ± SEM; n=4–5; unpaired t test). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S2.