Transplantation was performed as in Figure 1. PTCy was 25 mg/kg/d on days +3 and +4 where not otherwise specified. (A) High-level proliferation (Ki-67⁺) of liver-infiltrating alloreactive (Vβ6⁺) CD4⁺CD25^–Foxp3^– donor (H2k^k+) conventional T cells at days +21 and +200. (B–F) At day +21, liver-infiltrating cells were isolated and restimulated (stim) in vitro with irradiated (30 Gy) donor-parental (C3H) or host-parental (DBA/2) splenocytes at 2 × 10⁵ each of donors and stimulators per well. (B) Donor (H2k^k+H2k^b+) T cells from PTCy-treated mice had low proliferation to C3H at 5 days. (C) Proliferation was robust to alloantigen (DBA/2), although CD8⁺ T cell proliferation was reduced. (D) Cytokine production at 24 hours to alloantigen by PTCy-treated cells was markedly lower. (E and F) Procedures similar to those shown in B–D were followed, except responder T cells were flow cytometrically purified including removing CD4⁺CD25⁺ T cells (Tregs) prior to coculture. Similarly impaired (E) proliferation and (F) cytokine production by PTCy-treated T cells were seen. (G) The procedures in B–D were repeated at day +150. PTCy-treated T cells continued to proliferate and produce IFN-γ and IL-2 in response to alloantigen but not self-antigen; these differences were not due to disparities in the CD4⁺CD25⁺Foxp3⁺ T cell content of the restimulated cells (P = 0.21; not shown). Combined results from 2 independent experiments are shown for all parts. n = 8 mice/group for all parts except the 100 mg/kg PTCy group shown in the left panel in A (n = 5), the right panel in A (which shows all mice from Figure 1A surviving to day 200), E–F (n = 5-6/group), and the 25 mg/kg PTCy groups in G (n = 7). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, unpaired t test with Welch’s correction. ND, not detectable. Statistical testing of cytokines was adjusted for multiple comparisons by the Holm-Šidák method.