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).