Background/Aim: To determine if tumor-infiltrating T lymphocytes (TIL T) harvested from EBV-positive Nasopharyngeal cancer (NPC) is an effective source of cellular based immunotherapy.

Methods: HLA-A02 Peripheral blood (PB; n=6; paired) and NPC tissue biopsies (n=10; undifferentiated, non-keratinizing and EBV positive) were collected from patients with written consent. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat after density gradient centrifugation of PB. Tissue biopsies were minced, dissociated with human tumour dissociation kit (Miltenyi Biotec) and passed through nylon mesh to single cell suspension. Both PBMC and dissociated tumour were used for T cell expansion by incubation with anti-CD3/CD28 microbeads (Thermo Fisher Scientific) in RPMI-1640 (Hyclone) supplemented with 10% Fetal Bovine Serum (Hyclone) and 20IU/mL recombinant IL-2 (Miltenyi Biotec). TIL T cells were isolated with pan T cell isolation kit (Miltenyi Biotec) after expansion. Functionality of both PB T and TIL T cells were assessed using flow cytometry, trans-well assay and cytotoxicity assay against C666-A2 (NPC cell line expressing HLA-A02) thereafter.

Results: Successful isolation and expansion of NPC TIL T cells was achieved using 1-2 fresh NPC biopsy samples (2-3 mm); with fold increase of 210 to 867 after 28 days (1.7-4.5 × 107 cells). Comparison of primary TIL and expanded TIL T demonstrated that the former has greater percentage of CD4+ (p<0.006) while the latter has more CD8+ cells (p<0.003) cells, suggesting preferential expansion of cytotoxic T cells. Expanded TIL T cells also shown greater cytotoxicity against NPC cell line (C666-A2; E:T =1:20) than paired, expanded PB T cells (19.5% vs 4.9%, p=0.02). Furthermore, the cytotoxicity against C666-A2 was even greater (p<0.015) after incubating the TIL T cells with EBV peptides (LMP-1, LMP-2, EBNA-1). Primed TIL T cells also demonstrated greater lytic cell degranulation (CD107a), higher T cell activation (CD137) and IFN-γ secretion than non-primed, expanded TIL T cells. Importantly, expanded TIL T cells also demonstrated increased migration towards C666-A2 than expanded PB T cells (p=0.002). Greater anti-tumour effector function was observed with expanded TIL T despite positive expression of T inhibitory molecules (PD-1, CTLA-4 and TIM-3).

Conclusion: TIL T cells can be expanded reliably from NPC biopsies after 28-day culture. Expansion protocol demonstrated preferential expression of cytotoxic T cells over CD4 T cells. The greater anti-tumour effect of expanded TIL T over paired, expanded PB T suggested greater inherent anti-tumour T cells present in NPC TIL despite positive expression of T cell exhaustion markers. This warrant further investigations to explore therapeutic potential of TIL T in NPC.

Citation Format: Yitian Png, Audrey Zhi-Yi Yang, Louise Soo-Yee Tan, See-Voon Seow, Swarnalatha Lucky Asidharan, Jamie Mong, Min-Han Tan, Han ChongohOH, Chwee Ming Lim. Re-invigorating tumor infiltrating T lymphocytes against EBV positive nasopharyngeal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO075.