4-1BB+ Tregs and inhibitory progenitor exhausted T cells confer resistance to anti-PD-L1 and anti-CTLA-4 combination therapy

Predictors of immune checkpoint inhibitor (ICI) response among cancer patients are unclear despite active research into their tumor microenvironment. Moreover, mechanisms of combinatory ICI treatment are not understood, leading to ineffective clinical trials. There is currently an urgent need to stratify and select cancer patients for implementing successful immunotherapy. In this study we leverage the advantages of neoadjuvant immunotherapy phase II window of opportunity study, collecting paired samples of Durvalumab or Durvalumab and Tremelimumab treated head and neck cancer patients. Single-cell multi-omics profiling of TCR or RNA was performed for 57 biopsies including paired baseline prior to ICI treatment and were annotated for early ICI pathologic response. Samples were analyzed using state-of-the-art bioinformatic approaches using non-negative matrix factorization (NMF), cell-type specific gene networks, and a transformer-based foundation model. We found signatures associated with ICI sensitivity and immune activation that predict ICI response at baseline, validated using foundation model pretrained on millions of single cells. Moreover, we found that Tremelimumab therapy effect is inhibited via upregulation of IL2 signaling by 4-1BB+ (TNFRSF9+) regulatory T cells, and ICI response is distinguished by 4-1BB activity of effector CD8+ T cells. Importantly, we report a novel subset of tumor-specific precursor exhausted CD8+ T (Tpex) state distinguished with high expression of KLRB1 and IL17R that is associated with ICI resistance. The balance of the heterogeneous precursor subsets predicts immunotherapy response for multiple cancer types. Our findings provide important cellular and genetic insights into the immunological bases for predicting response via malignant-CD8+ T cell axis. Evidence of transcriptional regulation governing Tpex transition toward immune suppression necessitates future development of clinical strategies for maintaining ICI response and suppressing relapse. 57 HNSCC tumor biopsies, extracted from various tumor primary sites, were sequenced for TCR or GEX at single cell resolution. Samples are either treated with immune checkpoint inhibitor (Durvalumab or Durvalumab and Tremelimumab) or at extracted at baseline (pre-treatment).