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314 NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer
  1. Berengere Salome1,
  2. John Sfakianos1,
  3. Andrew Charap1,
  4. Adam Farkas1,
  5. Daniel Geanon1,
  6. Geoffrey Kelly1,
  7. Ronaldo de Real1,
  8. Brian Lee1,
  9. Kristin Beaumont1,
  10. Sanjana Shroff1,
  11. Ying-Chih Wang1,
  12. Yuan-Shuo Wang1,
  13. Li Wang1,
  14. Robert Sebra1,
  15. Alice Kamphorst1,
  16. Karl-Johan Malmberg2,
  17. Emanuela Marcenaro3,
  18. Pedro Romero4,
  19. Rachel Brody1,
  20. Yuko Yuki5,
  21. Maureen Martin5,
  22. Mary Carrington5,
  23. Reza Mehrazin1,
  24. Peter Wiklund1,
  25. Jun Zhu1,
  26. Matthew Galsky1,
  27. Nina Bhardwaj1 and
  28. Amir Horowitz1
  1. 1Icahn School of Medicine at Mount Sinai, New York, NY, USA
  2. 2University of Oslo, Oslo, Norway
  3. 3Department of Experimental Medicine, Geno, Italy
  4. 4Department of Oncology UNIL CHUV, Epalinges, Switzerland
  5. 5Frederick National Laboratory for Cancer Research, Frederick, MD, USA


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.

Conclusions 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 inhibiton. 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.


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  2. Rouanne M, Bajorin DF, Hannan R, Galsky MD, Williams SB, Necchi A, Sharma P, Powles T. Rationale and outcomes for neoadjuvant immunotherapy in urothelial carcinoma of the bladder. Eur Urol Oncol 2020 December;3(6):728–738. doi: 10.1016/j.euo.2020.06.009. Epub 2020 Nov 8. PMID: 33177001.

  3. André P, Denis C, Soulas C, Bourbon-Caillet C, Lopez J, Arnoux T, Bléry M, Bonnafous C, Gauthier L, Morel A, Rossi B, Remark R, Breso V, Bonnet E, Habif G, Guia S, Lalanne AI, Hoffmann C, Lantz O, Fayette J, Boyer-Chammard A, Zerbib R, Dodion P, Ghadially H, Jure-Kunkel M, Morel Y, Herbst R, Narni-Mancinelli E, Cohen RB, Vivier E. Anti-NKG2A mAb is a checkpoint inhibitor that promotes anti-tumor immunity by unleashing both T and NK Cells. Cell 2018 December 13;175(7):1731–1743.e13. doi: 10.1016/j.cell.2018.10.014. Epub 2018 Nov 29. PMID: 30503213; PMCID: PMC6292840.

  4. van Hall T, André P, Horowitz A, Ruan DF, Borst L, Zerbib R, Narni-Mancinelli E, van der Burg SH, Vivier E. Monalizumab: inhibiting the novel immune checkpoint NKG2A. J Immunother Cancer 2019 October 17;7(1):263. doi: 10.1186/s40425-019-0761-3. PMID: 31623687; PMCID: PMC6798508.

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