<jats:title>Abstract</jats:title> <jats:p>Stem-like CD8+ T cells constitute the major proliferative cytotoxic CD8+ T cell subset during checkpoint therapy. While it is increasingly appreciated that tumor draining LNs (tdLN) serve as reservoirs from which intratumoral stem-like CD8+ T cells can be replenished, how activated CD8+ T cells interact with the tdLN environment and differentiate into stem-like T cells remain unresolved.</jats:p> <jats:p>Using multiplexed 3D tissue microscopy, we identified that during late differentiation (Days 4-8+), TCF-1+ PD-1+ stem-like CD8+ T cells formed distinct clustering niches with XCR1+ dendritic cells (cDC1), which serve as sites of ongoing antigen presentation in the tdLN that drive continuous stem-like T cell expansion. Intriguingly, PD-1 expression among polyclonal TCF-1+ stem-like cells is positively correlated with tetramer binding, suggesting that the PD-1/cDC1 axis promotes selective enrichment and expansion of high affinity stem-like cells during effector differentiation. In vivo blockade of PD-1/PD-L1 signaling resulted in the loss of high affinity stem-like clones in the tdLN, along with a phenotypic shift towards terminally differentiated effector cells.</jats:p> <jats:p>Our findings raise questions about whether prolonged use of PD-1 checkpoint therapy in cancer patients interferes with maintenance of a key precursor population necessary for the robust effector generation needed to achieve full tumor regression.</jats:p> <jats:p>This work was supported by the Intramural Research program of NIAID, NIH.</jats:p>