Jing Liu

Serum cytokines from Luminex analysis not included in the main figures and cytokine levels in tumor interstitial fluid

Flow gating strategy and TIL phenotypes not included in the main figures

Appending CD3ζ:MyD88 to the TCR α-chain and the α- and β chain concurrently prevents the TCR from being expressed and appending CD3ζ:MyD88 to the TCR β chain increases IFNg production following TCR stimulation

Flow gating strategy and tumor-infiltrating myeloid cell phenotypes not included in the main figures

Introduced TCRs are not expressed on all primary human T cells but the β:CD3ζ:Myd88 TCR-expressing T cells are selectively expanded after repeat stimulations

The recent FDA approval of the first T-cell receptor–engineered (TCR) T-cell cancer therapy has solidified the treatment as a viable therapeutic strategy. However, only a fraction of patients who received this treatment achieved substantial and long-lasting responses, highlighting the need to understand the molecular underpinnings of these responses and develop more effective strategies to improve this therapy. Previous research has demonstrated that the activation of myeloid differentiation primary response 88 (MyD88) signaling in tumor-specific T cells functions as a robust costimulatory signaling pathway that promotes effective antitumor T-cell responses. In this study, we developed a strategy for improving TCR T-cell therapies by creating a synthetic TCR in which the intracellular domain of CD3ζ and a modified MyD88 are appended to the β-chain of a tumor-reactive TCR. The β:CD3ζ:MyD88 TCR activated MyD88 signaling in a tumor antigen/TCR-specific manner, and β:CD3ζ:MyD88 TCR T cells exhibited enhanced functionality and a reduced propensity to become exhausted. Furthermore, MyD88 signaling promoted T-cell persistence and expansion in vitro. In vivo, tumor-infiltrating β:CD3ζ:MyD88 TCR T cells skewed the immunosuppressive tumor microenvironment toward a less immunosuppressive state. Finally, β:CD3ζ:MyD88 TCR T cells substantially delayed tumor growth kinetics in a melanoma model. These findings introduce an approach for improving TCR-dependent T-cell responses by directly appending signaling domains to the full-length TCR and exemplify the utility of activating MyD88 signaling in tumor-specific T cells.Significance:A chimeric β:CD3ζ:MyD88 TCR reduces exhaustion and improves antitumor T-cell responses, overcoming the limited efficacy of current TCR-engineered T-cell cancer therapies.

Fewer β:CD3ζ:MyD88 TCR-J76 cells activate NFAT and the enhancing effect of the β:CD3ζ:MyD88 TCR is generalizable to multiple tumor-reactive TCRs in J76 cells

The enhancing effect of β:CD3ζ:Myd88 TCR is generalizable to multiple tumor-reactive TCRs in primary human T cells

The β:CD3ζ:MyD88 pMel-1 TCR augments pMel-1 T cell cytokine production but not cytotoxicity ability

Flow gating strategy and tumor-infiltrating NK phenotypes not included in the main figures