To investigate whether PD-1+Tim-3+ CD8+ TILs acquire pMHCs from APCs in a Tim-3–mediated fashion to become the target of tumor-reactive PD-1+Tim-3– CD8+ TILs, we sorted PD-1+Tim-3+ and PD-1–Tim-3– CD8+ TILs from either human or YUMMER 1.7 melanoma, labeled cells with PKH26, coincubated cells with PKH67-labeled PD-1+Tim-3– CD8+ TILs, and performed flow cytometry. Here, CD8A is linked to melanoma.