PD-1 inhibition promoted CD4 Tcon expansion and suppressed recovery of Tregs. (A) Sublethally irradiated BDF1 recipient mice received transplants of 5 × 10⁶ PD-1^+/+ B6 or PD-1^−/− B6 SPs and 5 × 10⁶ Ly 5.1 B6 TCD-BM cells (n = 5 per group). Recipient mice were euthanized on days 3, 7, and 14 after HSCT, and spleens were harvested. (B) Representative lymphocyte gates of the spleen for identification of CD4 and CD8 T-cell subsets and chimerism. (C) Numbers of donor spleen–derived CD4 Tcons and donor spleen–derived Tregs after HSCT (CD4 Tcons, PD-1^+/+ vs PD-1^−/−, P < .01 on day 7, P < .0001 on day 14; Tregs, PD-1^+/+ vs PD-1^−/−, P < .01 on day 7, P < .0001 on day 14). (D) Annexin V expression of spleen-derived T cells on day 14 after HSCT. Data are shown as the mean ± standard error of the mean. *P < .05, **P < .01, ****P < .0001. FACS, fluorescence-activated cell sorting.