Article Text

Download PDFPDF

621 NKG2A and HLA-E define a novel mechanism of resistance to immunotherapy with M. bovis BCG in non-muscle-invasive bladder cancer patients
  1. Amir Horowitz1,
  2. Jorge Daza1,
  3. Y Alice Wang1,
  4. Daniel Ranti1,
  5. Berengere Salome1,
  6. Elliot Merritt1,
  7. Julie-Ann Cavallo-Fleming1,
  8. Everado Hegewisch-Solloa2,
  9. Emily Mace2,
  10. Adam Farkas1,
  11. Sanjana Shroff1,
  12. Michelle Tran1,
  13. Jingjing Qi1,
  14. Manishkumar Patel1,
  15. Daniel Geanon1,
  16. Geoffrey Kelly1,
  17. Ronaldo de Real1,
  18. Brian Lee1,
  19. Seunghee Kim-Schulze1,
  20. Tin Htwe Thin1,
  21. Monica Garcia-Barros1,
  22. Kristin Beaumont1,
  23. Ying-Chih Wang1,
  24. Li Wang1,
  25. Dominic LaRoche3,
  26. Yong Lee3,
  27. Robert Sebra1,
  28. Rachel Brody1,
  29. Reza Mehrazin1,
  30. Jun Zhu1,
  31. Anna Tocheva1,
  32. Benjamin Hopkins1,
  33. Peter Wiklund1,
  34. Matthew Galsky1,
  35. Nina Bhardwaj1 and
  36. John Sfakianos1
  1. 1Icahn School of Medicine at Mount Sinai, New York City, NY, USA
  2. 2Columbia University, New York, NY, USA
  3. 3HTG Molecular Diagnostics, Tucson, AZ, USA


Background 75% of diagnosed bladder tumors are non-muscle-invasive (NMIBC)[1, 2]. Most require intravesical instillation of M.bovis Bacillus Calmette-Guérin (BCG). Recurrence after immunotherapy occurs in ~50% patients. Development of treatments for BCG-resistant disease has lagged partly because few studies have attempted to understand the relationship between timing of tumor recurrence, reasoning for the recurrence, and the state of immune system at the time of recurrence.Immune exhaustion is observed following microbial infections, cancers and chronic inflammation [3–5]. Natural Killer (NK) cells are among the earliest responders[6–8] and undergo a similar program of exhaustion as T cells[9]. HLA-E strongly inhibits NKG2A-expressing NK and CD8+T cells and is commonly upregulated on tumors[10]. We evaluated the potential restorative capacity of NKG2A and PD-L1-blockade for reinvigorating NK and CD8+T cell antitumor functions in in BCG-resistant bladder cancer.

Methods mRNA analysis of 2,892 genes was performed on tumor tissue of NMIBC patients before and after BCG therapy (n=35). Immunostaining (serial-IHC,immunofluorescence,imaging-mass cytometry) was performed on consecutive tissue sections. Single-cell-RNA-sequencing (scRNAseq) was performed on fresh bladder tumors (NMIBC,n=4; MIBC,n=9). OLink Proteomics (”Inflammation” panel) was performed longitudinally on plasma/urine from a prospective cohort of NMIBC patients. Patient tumors (n=3) were expanded as organoids and co-cultured with autologous tumor-derived NK and CD8+T cells in presence/absence of anti-PD-L1/NKG2A antibodies.

Results We demonstrate a robust local TME and systemic response to BCG that correlates with chronic inflammation and adaptive resistance rather than with preventing tumor recurrence. This resistance is mediated through IFN-γ-production by tumor-infiltrating NKG2A+NK and NKG2A+PD-1+CD8+T cells and results in increased HLA-E and PD-L1 on recurring tumors. Co-culture of treatment-naïve NMIBC tumors with recombinant IFN-gamma directly enhanced expression of PD-L1 and HLA-E. Longitudinal analysis of plasma before and during BCG immunotherapy revealed an inflammatory signature, including but not limited to IFN-gamma, that is maintained throughout treatment.Immunostaining and scRNAseq of NMIBC specimens revealed highly enriched infiltration by NKG2A+NK and NKG2A+CD8+T cells in HLA-EBrightPD-L1+ tumors and were spatially organized relative to tumors in a manner suggesting direct inhibition. Tumor-derived NK and CD8+T cells from BCG-resistant patients were co-cultured with autologous tumor organoids. Preliminary analyses demonstrated an improved anti-tumor response in presence of NKG2A/PD-L1-blockade.

Conclusions Our data support a model of BCG-resistance that points to a novel checkpoint axis that contributes to BCG-resistance: HLA-E/NKG2A. New insights into this axis in NMIBC and how it is altered with repeated BCG exposure will enable us to explore combination therapies (PD-L1/NKG2A-blockade) that may reduce BCG-resistance and provide durable response.


  1. Eidinger D, Morales A: Discussion paper: treatment of superficial bladder cancer in man. Ann N Y Acad Sci 1976, 277:239–240.

  2. Morales A, Eidinger D, Bruce AW: Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. J Urol 1976, 116:180–183.

  3. Blank CU, Haining WN, Held W, Hogan PG, Kallies A, Lugli E, Lynn RC, Philip M, Rao A, Restifo NP et al: Defining ‘T cell exhaustion’. Nat Rev Immunol 2019, 19:665–674.

  4. Hashimoto M, Kamphorst AO, Im SJ, Kissick HT, Pillai RN, Ramalingam SS, Araki K, Ahmed R: CD8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions. Annu Rev Med 2018, 69:301–318.

  5. McLane LM, Abdel-Hakeem MS, Wherry EJ: CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol 2019, 37:457–495.

  6. Lanier LL: NK cell receptors. Annu Rev Immunol 1998, 16:359–393.

  7. Biron CA, Gazzinelli RT: Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome. Curr Opin Immunol 1995, 7:485–496.

  8. Welsh RM, Jr.: Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. I. Characterization of natural killer cell induction. J Exp Med 1978, 148:163–181.

  9. da Silva IP, Gallois A, Jimenez-Baranda S, Khan S, Anderson AC, Kuchroo VK, Osman I, Bhardwaj N: Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade. Cancer Immunol Res 2014, 2:410–422.

  10. van Hall T, Andre 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, 7:263.

Ethics Approval Primary urothelial bladder cancer tumor tissue was obtained after obtaining informed consent in the context of an Institutional Review Board (IRB)-approved genitourinary cancer clinical database and specimen collection protocol (IRB #10-1180) at the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai (New York, NY).

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.