Background: Bladder cancer is characterized by a poor prognosis, with muscle-invasive cases harboring a 34-76% 10-year recurrence-free survival rate [1]. Neoadjuvant PD-1/PD-L1 blockade strategies have recently been approved by the US Food and Drug Administration for bladder cancer treatment, yet only achieving a complete response rate of 31-37%, thereby suggesting additional mechanisms of resistance [2]. HLA-E is a known inhibitor of NKG2A+ CD8 T cells and NK cell responses. A monoclonal antibody binding to the NKG2A receptor has been developed and proven to restore CD8 T cell and NK cell responses in head and neck cancer, with ongoing clinical trials across multiple tumor indications [3,4]. We evaluated the potential role of the HLA-E/NKG2A inhibitory pathway in modulating T cell immunity in bladder cancer. Methods: CyTOF was performed on CD8+ T cells from fresh bladder tumors (n=6), as well as on expanded CD8+ T cells from bladder-draining lymph nodes (n=11) and tumors (n=8). Flow cytometry (n=25) and single-cell RNA-sequencing (scRNAseq) (n=13) were performed on cells from fresh bladder tumors. Results: Mechanisms of tumor escape from CD8+ T cell recognition include impairment of antigen presentation. Accordingly, we found a significant reduction of HLA class I expression on tumors. However, expression of DNAM-1-activating ligands (e.g. CD112,CD155) on bladder tumors was retained, indicating a possible role for TCR-independent activation pathways traditionally ascribed to natural killer (NK) cells. Using mass cytometry and scRNAseq, we observed that acquisition of NKG2A on tumor-derived PD-1+ CD8+ T cells promotes tissue-resident memory features alongside diminished CD28 expression and significantly weaker sensitivity to CD3/CD28-signaling. However, NKG2A+ CD8 T cells possess a proliferative advantage with enhanced expression of DNAM-1 and cytolytic machinery. Strikingly, we found that NKG2A+PD-1+ CD8 T cells are strongly activated in response to HLA class I-deficient tumors compared to their NKG2A PD-1+ CD8 T cell counterparts. TCR-independent NK-like function by NKG2A+ CD8 T cell is partly mediated by the DNAM-1 pathway and inhibited by HLA-E. NKG2A+ CD8 T cell functions are restored upon NKG2A blockade, where efficiency positively correlates with HLA-E expression on bladder tumors. Discussion: Collectively, our data indicate that NKG2A+ CD8 T cells display a strong capacity for TCR-independent activation that enables them to circumvent bladder tumor evasion mechanisms. NKG2A+ CD8 T cells lack expression of CD28 suggesting a lower susceptibility to PD-1-mediated inhibition. Our data suggest a need for thorough reappraisal of current protocols that assess CD8 T cell exhaustion and for strategies to restore their antitumor functions. References: 1. Sanli, O. et al., Nat Rev Dis Primers, 2017 2. Rouanne, M. et al., Eur Urol Oncol, 2020 3. André, P. et al., Cell, 2018 4. Van Hall, T. et al., J Immunother Cancer, 2019

Citation Format: Bérengère Salomé, John P. Sfakianos, Jorge Daza, Andrew Charap, Adam M. Farkas, Daniel Geanon, Geoffrey Kelly, Ronaldo M. De Real, Brian Lee, Kristin G. Beaumont, Sanjana Shroff, Ying-Chih Wang, Yuan-Shuo A. Wang, Li Wang, Robert P. Sebra, Alice O. Kamphorst, Karl J. Malmberg, Emanuela Marcenaro, Pedro Romero, Rachel Brody, Yuko Yuki, Maureen Martin, Mary Carrington, Reza Mehrazin, Peter Wiklund, Jun Zhu, Matthew D. Galsky, Nina Bhardwaj, Amir Horowitz. NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P046.