Background
T cell checkpoint receptors are expressed when T cells are activated, and modulation of checkpoint receptor expression can alter the function of T cells and their anti-tumor efficacy. We have previously found that murine CD8+ T cells activated with cognate antigen increase the expression of PD-1, and this can be attenuated in the presence of Toll-like receptor (TLR) agonists. In the current report, we sought to investigate whether single or multiple TLR agonists can affect the expression of T cell checkpoint receptors on human CD8+ T cells and improve their functionality.
Methods
Cryopreserved human PBMCs containing CD8+ T cells reactive to CMV or EBV were thawed, then stimulated with HLA-A2 specific peptides (CMV: NLVPMVATV , EBV: FLYALALLL) in the presence of TLR agonists. Antigen-specific CD8+ T cells were collected and evaluated for the expression of T cell checkpoint receptors (PD-1, CLTA-4, CD160, CD244, LAG-3, TIGIT, and VISTA) by flow cytometry. PBMCs from HLA-A2+ patients previously treated with a tetanus vaccine and an antitumor DNA vaccine (pTVG-HP, encoding prostatic acid phosphatase, PAP) were stimulated with tetanus toxoid or PAP-derived HLA-A2 peptide epitopes (TLMSAMTNL, ALDVYNGLL) for 72 hours in the presence of TLR agonists, and culture supernatant was evaluated for the presence of IFN by ELISA. Immature dendritic cells (iDCs) were stimulated with TLR agonists for 24 hours, and culture supernatant was evaluated for the presence of IL-12 p70 by ELISA.
Results
Activation of human CD8+ T cells in the presence of TLR agonists did not show further expansion of tetramer-positive CD8+ T cells and showed no significant change in PD-1 expression. However, CTLA-4 expression was downregulated on human CD8+ T cells in the presence of TLR3, TLR7, TLR8, and TLR9 agonists. Stimulation of PBMCs with tetanus toxoid or PAP peptides showed increase in IFN secretion in the presence of TLR8 agonists. Dendritic cells were found to secrete IL-12 in response to stimulation with several TLRs.
Conclusions
TLR agonists can support vaccine-mediated activation of human CD8+ T cells and modulate the expression of T cell checkpoint receptors, including CTLA-4. These data provide the rationale for choosing the optimal TLR agonists to improve the efficacy of human anti-cancer vaccines.