Signaling through the high-affinity IL-2R, whose expression is inhibited by PD-L1:PD-1 engagement, is required for FH cell survival, but not proliferation. (A-B) FH cells were transferred into tyrosinase⁺ mice treated with (A) blocking anti-PD-L1 (■), agonistic anti-4-1BB and –OX40 (▴), or left untreated (•) and (B) tyrosinase⁺ mice (•), tyrosinase⁺ mice treated with agonistic anti-4-1BB and –OX40 (■), anti-4-1BB only (▴), or anti-OX40 only (▾). CD25 expression on proliferating FH cells was examined 3 days later. Data represent the percentage of FH cells that have undergone the indicated number of divisions that express CD25 3 days after transfer. (A) Data represents 4 to 9 mice per condition from 4 to 9 independent experiments (*anti-PD-L1, #anti-4-1BB and –OX40) (B) Data represent 3 mice per condition (*anti-4-1BB + –OX40, #anti-4-1BB). #, *, P < .05; # #, **, P < .01; # # #, ***, P < .005 (2-way ANOVA, Bonferonni posttest). (C) Tyrosinase⁺ mice that had been adoptively transferred with CTV-labeled FH cells were left untreated (•) or treated with blocking anti-CD25 (■) beginning on day 0 and every 24 hours thereafter. Proliferation was evaluated 3 days posttransfer by flow cytometry. Data represent 3 to 6 mice per condition from 3 independent experiments. **P = .0052. (D) PD-L1^−/− recipients and recipients treated with agonistic anti-4-1BB and –OX40 were treated with anti-CD25 beginning on day 0 and every 24 hours thereafter. LNs were harvested 7 days later and the percentage of FH cells of total T_CD8 cells was determined. Data represent 3 untreated and 3 treated PD-L1^−/− mice from one experiment and 6 mice treated with anti-4-1BB⁺–OX40 and 2 mice treated additionally with anti-CD25 from 2 independent experiments (C-D: 2-tailed, unpaired t test). Error bars (A-D), SEM.