IDX-treated cancer cells induce CD8+ T cell activation concomitantly with enhanced cytotoxicity and degranulation
Given that EBV infection is a critical event in NPC tumorigenesis, we then focused the subsequent functional assays on authentic NPC cell lines positive with EBV (C17 and NPC43+ve). To investigate whether IDX exerts an immunomodulatory effect that drives adaptive immune responses in NPC, we performed in vitro coculture of tumor cells with peripheral blood mononuclear cells (PBMCs) from healthy donors, with particular focus on T-cell functionality (Figure 2A). IDX modestly promoted CD4+ and CD8+ T cell proliferation in PBMCs alone, as evaluated by Ki67 labeling. This up-regulatory effect was significantly enhanced on CD8+, but not CD4+ T-cells, when PBMCs were cocultured with IDX-pretreated cancer cells (Figure 2B). Surprisingly, the highest concentration of IDX (4 µM) greatly impaired CD8+ T-cell proliferation, suggesting that IDX doses ≥4 µM were toxic to T cells. This was consistent with Georgaki’s finding where a high concentration of phenoxodiol was demonstrated to be toxic to PBMCs (Georgaki et al., 2009). To assess IDX’s ability to induce cytolytic PBMC responses we examined granzyme B production in the T cells. Granzyme B was significantly increased in IDX-pretreated cancer cells at low-doses (1 and 2 µM), while a high dose of IDX markedly downregulated that expression, normalizing to baseline levels (Figure 2C). To confirm that we were measuring newly produced granzyme B, rather than residual unreleased granzyme B, within responding cells, we co-stained the cells with anti-CD107a to assess degranulation. The up-regulation of CD107a expression mirrored that of granzyme B (Figure 2D).