Abstract In this study, we evaluated the feasibility of expanding tumor infiltrating lymphocytes (TIL) from surgically resected renal cell carcinoma (RCC) tumors. Tumors were collected from 43 patients undergoing surgery to remove primary kidney tumors. Tumor types included clear cell RCC (86.0%), papillary RCC (11.6%) as well as chromophobe RCC (2.3%). Tumors were minced into fragments, placed in individual wells of a 24-well plate containing media with high dose IL-2 (6000 IU/mL), and cultured for four weeks. Successful expansion was considered when at least one fragment expanded to a minimum of 2 wells. Successfully expanded TIL were then evaluated for T-cell phenotypes as well as reactivity to the autologous tumor. Of all RCC tumors collected, TIL were successfully grown for 79% of the samples (34/43). The T-cell phenotype of these TIL was highly variable across samples, although there was a higher percentage of CD4+ T-cells relative to CD8+ T-cells. The reactivity of expanded TIL was then assessed by IFNγ release. We found that TIL secreted IFNγ in response to autologous tumor in 71% of the samples. Together, these results demonstrated the feasibility of expanding functional tumor reactive TIL from RCC. Given that hypoxia plays an important role in the development of RCC tumors and is associated with poor prognosis, we then evaluated the ability to expand TIL under hypoxic conditions. To this end, primary TIL were cultured at various O2 levels (20% O2, 5% O2 or 1% O2) and the cell yield as well as the T-cell memory phenotypes were evaluated. We found that the yield of TIL grown in hypoxic conditions was lower when compared to normoxic conditions (20% O2), suggesting a reduction of TIL proliferation in hypoxia. TIL grown in hypoxic conditions displayed increased percentage of central memory T-cells compared to TIL cultured at 20% O2, as evaluated by CD45RA and CCR7 expression (CD45RA-CCR7+). Primary TIL were then expanded to larger numbers using a rapid expansion protocol (REP) by stimulation with anti-CD3 antibody in the presence of allogenic feeder cells. REP of TIL was carried out at either atmospheric O2 (20% O2) or at a hypoxic O2 level (5% O2), and the T-cell phenotype as well as reactivity to the autologous tumor were assessed. Unlike pre-REP TIL, TIL that underwent REP were able to expand in hypoxic conditions (5% O2) and displayed increased percentage of tissue resident memory T-cells (CD69+CD103+; 19%) when compared to the starting TIL population (0.11%) or TIL expanded at atmospheric O2 (0.17%). When co-cultured with the autologous tumor, hypoxic TILs displayed increased IFNγ, TNFα as well as increased Granzyme B release (p<0.001) when compared to TIL expanded in normoxia (20% O2). Collectively, these results support the advantage of adapting TIL to hypoxic conditions in the production of tumor-reactive TIL. Citation Format: Mohammed Alkhouli, Veronica Martinez-Brockhus, Matthew S. Beatty, Robert J. Gillies, Shari Pilon-Thomas, Jad Chahoud. Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 575.