Extended Data Fig. 3: LN early BrdU, Ki67, and live/dead comparisons; early anti-PD1 effects on circulating memory in spleen & LN.

a. Schematic showing mixed adoptive transfer of 1 × 105 each of CD45.1 + /Thy1.1+ or Thy1.1 + WT and Thy1.1+ or CD45.1 + /Thy1.1 + KO OT-I cells into Thy1.2 + C57BL/6J recipients at day −1, followed by VACV-OVAss immunization on ear and tail at day 0 (top). BrdU was given to mice starting on day 0 and provided on consecutive days between days 0–2, 0–4 or 0–6 until harvest on day 3, 5, and 7 respectively. Data were pooled from 2 independent experiments each for day 3 (n = 12), 5 (n = 12), and 7 (n = 12). Mixed pairs were distinguished using CD45.1, CD45.2, Thy 1.2 and/or Thy 1.1 congenic markers. Percent (left) and total counts (right) of PD-1 +/+ OT-I and PD-1−/− donor OT-I cells are given with a line to connect the paired samples by mouse. b. Percent (left) and total counts (right) of BrdU+ cells from the PD-1 +/+ OT-I and PD-1−/− donor OT-I. c. Percent (left) and total counts (right) of Ki67+ cells from PD-1 +/+ OT-I and PD-1−/− donor OT-I. d. Percent (left) and total counts (right) of Live/Dead Blue+ cells from PD-1 +/+ OT-I and PD-1−/− donor OT-I. e. Schematic showing tdTomato+ or Thy1.1 + OT-I cells were transferred into Thy1.2 + C57BL/6J recipients at day −1, followed by VACV-OVAss immunization at day 0, and treatment with either PD-1 Ab or isotype administered on days 0, 3, 6, and 9 post-infection (top). FTY720 was given every 2 days starting at day 35. At day 42, contralateral R and L depilated flank sites of the same mouse were challenged with topical PBS or SIINFEKL. dLN and spleen were harvested at day 49. Quantification of total KLRG1-CD62L + OT-I TCM numbers (log) from the dLN of the PBS-challenged skin site versus those from the dLN of the SIINFEKL-challenged skin site (bottom, left). Quantification of spleen KLRG1-CD62L + TCM (bottom, middle) and KLRG1-CD62L- TEM (bottom, right). A paired t-test was performed for a-d. Elsewhere, statistics were estimated using a linear mixed-effects model with group and by treatment challenge, as fixed effects and a random intercept for each mouse. Comparisons of KLRG1-CD62L + TCM numbers in PBS versus SIINFEKL-dLN of the same mouse, are shown in red or blue. Data were pooled from 4 independent experiments (Isotype, n = 13; anti-PD-1, n = 18). Bars and error bars show mean ± SEM. +p ≤ 0.1 * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.