FIGURE 3: Inflammation-induced PD-L1-specific T cells in vivo. (A) Experimental timeline: C56BL/6 mice were injected twice with 1 µg IFN-y in 200 µl PBS IP (or no injections as a control) on day 0 and day 2. On day 6 the mice were sacrificed, and spleens were analyzed for PD-L1 reactivity. (B) Examples of IFN-y-ELISPOT experiments with splenocytes from a control mouse (top) or an IFN-y-injected mouse (bottom) with or without the mPD-L1long peptide (MRIFAGIIFTACCHLLRA). (C) In total, splenocytes from the ten IFN-y-injected mice and the five control mice were examined for reactivity towards the mPD-L1long peptide (MRIFAGIIFTACCHLLRA) by IFN-y- ELISPOT. The average number of mPD-L1long spots (after subtraction of the spots without peptide) was calculated per 9×10⁵ splenocytes for each mouse. Experiments were performed in triplicates. (D) Experimental timeline: DNFB or as control OOA was painted on the dorsal side of both ears of C57Bl/6 mice on three consecutive days. On day 5, the mice were sacrificed and spleens and dLNs were removed for further analysis in IFN-y-ELISPOT. (E) Examples of IFN-y-ELISPOT experiments with cells from a dLN (top) or Spleen (bottom) from an OOA-treated control mouse or a DNFB-treated mouse with or without the mPD-L1long peptide (MRIFAGIIFTACCHLLRA). (F) In total, cells from dLNs and splenocytes from twelve DNFB-treated mice and twelve OOA-treated control mice were examined for reactivity towards the mPD-L1long peptide (MRIFAGIIFTACCHLLRA) by IFN-y-ELISPOT. The average number of mPD-L1long spots (after subtraction of the spots without peptide) was calculated per 9×10⁵ cells for each mouse. (G) Experimental timeline: C56BL/6 mice were injected twice with 1 µg IFN-y in 200 µl PBS IP (or no injections as a control) on day 0,2 and 5. On day 7 the mice were sacrificed, and spleens were analyzed for PD-L1 reactivity. (H) Examples of IFN-y-ELISPOT experiments with splenocytes from a control mouse (top) or an IFN-y-injected mouse (bottom) with or without the short murine peptide, mPD-L1-01 (GIIFTACCHL). (I) In total, splenocytes from the six IFN-y-injected mice and the six control mice were examined for reactivity towards the short murine peptide, mPD-L1-01 (GIIFTACCHL) by IFN-y-ELISPOT. The average number of spots with and without mPD-L1-01 (GIIFTACCHL) was counted for each mouse. Experiments were performed in triplicates.