In vitro conditioning of antigen-reactive CD8+ T cells with toll-like receptor agonists enhances their expansion in vivo in an adoptive transfer mouse model

Background: Anti-cancer adoptive T cell therapy has shown significant anti-tumor responses in cancer patients. This therapy is based on in vitro activation of autologous T cells harvested from a cancer patient and infusing them back through blood. The efficacy of adoptively transferred T cells depends on the in vitro conditioning regimen in particular the type of used cytokines. Aim: This study aimed to use toll like receptors (TLRs) ligands (TLRLs) to condition T cells in vitro as a novel approach instead of cytokines. Methods: Spleen cells were harvested from TCR transgenic C57BL/6 pmel-1 mice, in which CD8+ T cells are engineered to recognize melanoma MHC class-I peptides.Unfractionated splenocytes were cultured for 24 hours in vitro with media or melaoma peptide plus or minus IL-12 (10ng/ml), poly(I:C) (TLR3L; 25ug/ml) or CpG (TLR9L; 10ug/ml). Then, activation, proliferation and cytokine production of the cultured cells were assessed. In another set of experiments, the cultured cells were harvested and infused into syngeneic B6 mice followed by vaccination to evaluate their antigen-specific expansion and contraction. Results: Conditioning of donor splenocytes in vitro with the TLR3L or TLR9L during antigen stimulation increased the antigen specific activation, proliferation, and cytokine production. Interestingly, in vitro treatment of the unfractionated splenocytes with TLR9L enhanced the activation and proliferation of B cells regardless antigen stimulation. Adoptive transfer of TLRL-conditioned peptide stimulated CD8+ T cells into naïve mice showed better survival and higher expansion in response to concomitant vaccination with peptide. Interestingly, in vitro conditioning of CD8+ T cells with TLR9L and in vivo conditioning with TLR3L resulted in the best antigen specific expansion of these cells upon their adoptive transfer into recipient mice. Conclusion: These results show that provision of CD8+ T cells in vitro and in vivo with certain TLR agonists can markedly enhance their antigen specific responses upon their adoptive transfer, opening a potential application of this approach in anti-cancer adoptive immunotherapy.