550 PD-1-mediated inhibition is critical to the development and maintenance of high affinity stem-like CD8+ T cells in the tumor draining lymph node

<h3>Background</h3> Stem-like CD8+ T cells constitute a subset of activated cytotoxic T cells with high proliferative potential critical for successful checkpoint immunotherapy.¹ Emerging evidence suggests that tumor-draining lymph nodes (tdLN) can serve as reservoirs that support the generation of TCF-1+ stem-like cells that continuously replenish the tumor sites to sustain ongoing anti-tumor activity and responsiveness to checkpoint therapy.^{2 3} However, how activated CD8+ T cells retain their stemness in a highly antigenic and inflammatory tdLN microenvironment, especially those of the highest affinity for the presented tumor antigen, has not been determined. <h3>Methods</h3> OVA-expressing mouse Kras/p53 adenocarcinoma tumor cells were intradermally implanted into mice. Optically cleared tdLN harvested late in the immune response (Days 4–8+ post-tumor initiation) were analyzed with an advanced multiplexed 3D tissue microscopy technique that enables single-cell level quantification of immune cell functional states <i>in situ</i>. Antigen-specific polyclonal cells were also tracked using tetramer staining via flow cytometry from animals treated with anti-PD-L1/2 or anti-PD-1 checkpoint therapies. <h3>Results</h3> 3D tissue microscopy of tdLN identified a PD-1+ SLAMF6-hi stem-like CD8+ subset that is uniquely retained in the tdLN, forms distinct niches with cross-presenting XCR1+ dendritic cells (cDC1) and engages in prolonged antigen stimulation beyond the initial priming phase. <i>In vivo</i> ablation of cDC1 during late stage significantly reduced TCF-1+ stem-like cell expansion, suggesting that late antigen presentation in the tdLN sustains a mode of lymphocyte proliferation that also retains stemness. These PD-1+ SLAMF6-hi stem-like clones comprise the highest affinity antigen-specific CD8+ T cell population, as measured using tetramer binding normalized to TCR expression, indicating that cDC1 likely serve as antigen presentation niches that promote the expansion and affinity evolution of stem-like T cells over time. Co-localization of PD-1 molecules with their ligands at the T-DC interface suggests that inhibitory PD-1 pathway restrains effector differentiation <i>in vivo</i> through attenuating TCR-dependent signaling on high affinity clones during their competition for antigen presented by cDC1. <i>In vivo</i> PD-1 checkpoint blockade led to the loss of high affinity stem-like clones in the tdLN, thus highlighting the central role of PD-1 in the maintenance of the high affinity stem-like population. <h3>Conclusions</h3> Our findings reveal an unexpected relationship between TCR ligand affinity recognition, negative feedback regulatory loops, and T cell stemness across space and time. Importantly, these findings raise the clinical question of whether prolonged use of PD-1 checkpoint therapy provides short-term anti-tumor effects at the expense of long-term maintenance of high affinity stem-like precursors, which subsequently diminishes treatment efficacy. <h3>Acknowledgements</h3> This work was supported by the Division of Intramural Research of NIAID, NIH and the Intramural Research Program of the Center for Cancer Research of NCI, NIH. We would also like to thank Tsuneyasu Kaisho (RIKEN Research Center for Allergy and Immunology) for providing the transgenic mouse strains for this study. <h3>References</h3> Zehn D, Thimme R, Lugli E, de Almeida GP, Oxenius A. ‘Stem-like’ precursors are the fount to sustain persistent CD8+ T cell responses. <i>Nat Immunol</i> 2022 Jun;<b>23</b>(6):836–847. Connolly KA, Kuchroo M, Venkat A, Khatun A, Wang J, William I, Hornick NI, Fitzgerald BL, Damo M, Kasmani MY, Cui C, Fagerberg E, Monroy I, Hutchins A, Cheung JF, Foster GG, Mariuzza DL, Nader M, Zhao H, Cui W, Krishnaswamy S, Joshi NS. A reservoir of stem-like CD8+ T cells in the tumor-draining lymph node preserves the ongoing antitumor immune response. <i>Sci Immunol</i> 2021 Oct;<b>6</b>(64):eabg7836. Schenkel JM, Herbst RH, Canner D, Li A, Hillman M, Shanahan SL, Gibbons G, Smith OC, Kim JY, Westcott P, Hwang WL, Freed-Pastor WA, Eng G, Cuoco MS, Rogers P, Park JK, Burger ML, Rozenblatt-Rosen O, Cong L, Pauken KE, Regev A, Jacks T. Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes. <i>Immunity</i> 2021 Oct 12;<b>54</b>(10):2338–2353.e6.