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Background CD8 + resident memory (Trm) cells, identified by CD69 and CD103 expression, persist long-term within tissues where they mediate sustained anti-cancer immunity.1 2 Our laboratory identified melanoma antigen specific Trm cells in tumor draining lymph node (tdLN) of melanoma tumor-excised mice with vitiligo, where they provided durable protection against lymph node metastasis.3 However, the signals supporting tdLN Trm maintenance in the setting of cancer and autoimmunity remain unclear, as does the function, plasticity, and therapeutic potential of this T cell subset.Methods TdLN Trm cells with specificity for melanoma differentiation antigens gp100 and TRP-2, were generated using an established B16 melanoma-induced vitiligo model. 4 5 To determine Trm requirement for persistent antigen exposure, we employed T cell receptor-alpha (Trac) and beta-2 microglobulin (B2m) inducible knockout mice. To assess the role of homeostatic cytokines, mice were treated with neutralizing antibodies against IL-7R alpha, IL-15 and IL-15R alpha. Ex vivo expansion potential and function of tdLN Trm cells was assessed by fluorescence assisted cell sorting of CD8+CD103+CD69+ T cells from draining lymph nodes, followed by in vitro restimulation with anti-CD3/CD28 beads and culture with IL-7 and IL-15 for 7 days. To assess anti-tumor function and plasticity, ex vivo expanded Trm cells were adoptively transferred in lymphodepleted mice harboring large established B16F10 tumors based on an established adoptive cell therapy regimen.6 7 Finally, ex vivo expansion was validated using human CD8+CD103+CD69+ Trm isolated from regional lymph nodes (LNs) of patients with non-small cell lung cancer.Results Knockdown of either TCR or B2m in mice with established Trm populations revealed significant reduction of the response, revealing dependence of tdLN Trm on ongoing antigen exposure. Additionally, blockade of both IL-7 and IL-15 signaling led to significant reduction in tdLN Trm cell populations, indicating co-dependence on homeostatic cytokines. Purified CD8 +Trm cells isolated from tdLNs of mice and patients were capable of recall expansion ex vivo. Importantly, adoptive transfer of only 20,000 LN Trm cells per mouse was sufficient to significantly restrict the growth of large established B16 tumors. Analysis of T cell engraftment in treated mice revealed that transferred Trm cells persisted long term in tumors and tdLNs, where they differentiated into central memory, effector, and Trm populations.Conclusions These studies reveal mechanistic insights into requirements for long-term maintenance of LN Trm cells and demonstrate that ex vivo expanded Trm cells from tdLNs can be leveraged as a potent new form of adoptive cell therapy.References Beumer-Chuwonpad A, Taggenbrock RLRE, Ngo TA, Van Gisbergen KPJM. The potential of tissue-resident memory T cells for adoptive immunotherapy against Cancer. Cells. 2021;10(9):2234.Ramirez DE, Mohamed A, Huang YH, Turk MJ. In the right place at the right time: tissue-resident memory T cells in immunity to cancer. Current Opinion in Immunology 2023;83:102338.Molodtsov AK, Khatwani N, Vella JL, Lewis KA, Zhao Y, Han J, et al. Resident memory CD8+ T cells in regional lymph nodes mediate immunity to metastatic melanoma. Immunity 2021;54(9):2117-2132.e7.Byrne KT, Côté AL, Zhang P, Steinberg SM, Guo Y, Allie R, et al. Autoimmune melanocyte destruction is required for robust CD8+ memory T cell responses to mouse melanoma. J Clin Invest. 2011;121(5):1797–809.Malik BT, Byrne KT, Vella JL, Zhang P, Shabaneh TB, Steinberg SM, et al. Resident memory T cells in the skin mediate durable immunity to melanoma. Sci Immunol. 2017;2(10):eaam6346.Nelson MH, Kundimi S, Bowers JS, Rogers CE, Huff LW, Schwartz KM, et al. The inducible costimulator augments Tc17 cell responses to self and tumor tissue. The Journal of Immunology 2015;194(4):1737–47.Muranski P, Boni A, Antony PA, Cassard L, Irvine KR, Kaiser A, et al. Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood 2008;112(2):362–73.