PD-L1 engages PD-1, which is expressed more rapidly and at a higher level following presentation of tyrosinase by LECs because of deficient costimulation, leading to FH cell deletion. (A) FH cells were transferred into tyrosinase⁺ recipients treated with antibodies against PD-L1 that block interactions with PD-1 or CD80 and LNs harvested 7 days after transfer. Data represent 5 to 8 mice per condition from 5 to 6 independent experiments, respectively. **P = .0011, #P = .0262 (2-tailed, unpaired t test). (B) PD-1^+/+, PD-1^+/neg, or PD-1^−/− FH cells were transferred into tyrosinase⁺ or albino recipients and LN harvested 3 or 7 days after transfer. Data represent 5 to 7 mice per condition from 3 independent experiments for day 3. Day 7 data represents 8 to 9 mice per condition from 3 to 5 independent experiments (*compared with tyrosinase⁺, #compared with albino). Day 3: *P = .0222, **P = .0057, #P = .0381, ##P = .0061. Day 7: ***P = .0006, ##P = .0028 (nonparametric Mann-Whitney test). (C) PD-1 expression was determined on naive (shaded) and CD69⁺ FH cells 24 hours after transfer into tyrosinase⁺ recipients (solid black line) or into albino recipients infected with VACV-tyr (dashed line). Data are representative of mice from 2 independent experiments. (D) Data represent the percentage of FH cells that have undergone the indicated number of divisions that express PD-1 and their level of PD-1 expression by MFI 3 days after transfer. Data are from 5 to 8 independent experiments using tyrosinase⁺ mice (•), tyrosinase⁺ mice treated with agonist anti-4-1BB and –OX40 (▴), and albino mice infected with VACV-Tyr (▾). *P < .05, **P < .01, ***P < .005 (2-way ANOVA, Bonferonni posttest). (E) FH cells were transferred into tyrosinase⁺ mice, albino mice infected with VACV-tyr, or antigen-free albino mice. LNs were harvested 7 days postadoptive transfer and the percentage of FH cells determined. Data represent 4 to 6 mice per condition from 5 independent experiments (*compared with tyrosinase⁺, #compared with albino); **P = .0056, #P = .0259 (2-tailed, unpaired t test). (F) Tyrosinase⁺ recipients were left untreated or treated with agonist anti-4-1BB and –OX40, anti-4-1BB only, or anti-OX40 only. Cumulative data represents 3 to 7 mice per condition from 3 independent experiments. *P = .0167 (nonparametric Mann-Whitney test). (G) Data represent the percentage of FH cells that have undergone the indicated number of divisions that express PD-1 3 days posttransfer. Data are from one experiment using 3 untreated tyrosinase⁺ mice (•), tyrosinase⁺ mice treated with agonist anti-4-1BB and –OX40 (■), agonist anti-4-1BB only (▴), or agonist anti-OX40 only (▾). #,*P < .05; ##,**P < .01; ###,***P < .005 (2-way ANOVA, Bonferonni posttest). Error bars (A,B,D-G), SEM.