Background

CD8+ T cells are a highly diverse population of cells with distinct phenotypic functions that can influence immunotherapy outcomes. Further insights on the roles of CD8+ specificities and TCR avidity of naturally arising tumor-specific T cells, where both high and low avidity T cells recognizing the same peptide-major histocompatibility complex (pMHC) coexist in the same tumor, are crucial for understanding T cell exhaustion and resistance to PD-1 immunotherapy.

Methods

CT26 models were treated with anti-PD-1 on days 3, 6 and 9 following subcutaneous tumor implantation generating variable responses during early tumor development. Tetramer staining was performed to determine the frequency and avidity of CD8+ T cells targeting the tumor-specific epitope GSW11 and confirmed with tetramer competition assays. Functional characterization of high and low avidity GSW11-specific CD8+ T cells was conducted using flow cytometry and bulk RNA-seq. In vitro cytotoxicity assays and in vivo adoptive transfer experiments were performed to determine the cytotoxicity of high and low avidity populations.

Results

Treatment success with anti-PD-1 was associated with the preferential expansion of low avidity (Tet lo) GSW11-specific CD8+ T cells with Vβ TCR expressing clonotypes. High avidity T cells (Tet hi), if present, were only found in progressing PD-1 refractory tumors. Tet lo demonstrated precursor exhausted or progenitor T cell phenotypes marked by higher expression of Tcf-1 and T-bet, and lower expression of the exhaustion markers CD39, PD-1 and Eomes compared with Tet hi, whereas Tet hi cells were terminally exhausted. Transcriptomics analyses showed pathways related to TCR signaling, cytotoxicity and oxidative phosphorylation were significantly enriched in Tet lo found in both regressing and progressing tumors compared with Tet hi, whereas genes related to DNA damage, apoptosis and autophagy were downregulated. In vitro studies showed that Tet lo exhibits higher cytotoxicity than Tet hi. Adoptive transfer of Tet lo showed more effective tumor control than Tet hi, and curative responses were achieved when Tet lo was combined with two doses of anti-PD-1.

Conclusions

Targeting subdominant T cell responses with lower avidity against pMHC affinity neoepitopes showed potential for improving PD-1 immunotherapy. Future interventions may consider expanding low avidity populations via vaccination or adoptive transfer.