A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy
Balamayoora Theivanthiran, … , Alisha Holtzhausen, Brent A. Hanks
Balamayoora Theivanthiran, … , Alisha Holtzhausen, Brent A. Hanks
Published February 4, 2020
Citation Information: J Clin Invest. 2020;130(5):2570-2586. https://doi.org/10.1172/JCI133055.
View: Text | PDF
Research Article Immunology Oncology

A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti–PD-1 immunotherapy

Abstract

An in-depth understanding of immune escape mechanisms in cancer is likely to lead to innovative advances in immunotherapeutic strategies. However, much remains unknown regarding these mechanisms and how they impact immunotherapy resistance. Using several preclinical tumor models as well as clinical specimens, we identified a mechanism whereby CD8+ T cell activation in response to programmed cell death 1 (PD-1) blockade induced a programmed death ligand 1/NOD-, LRR-, and pyrin domain–containing protein 3 (PD-L1/NLRP3) inflammasome signaling cascade that ultimately led to the recruitment of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) into tumor tissues, thereby dampening the resulting antitumor immune response. The genetic and pharmacologic inhibition of NLRP3 suppressed PMN-MDSC tumor infiltration and significantly augmented the efficacy of anti–PD-1 antibody immunotherapy. This pathway therefore represents a tumor-intrinsic mechanism of adaptive resistance to anti–PD-1 checkpoint inhibitor immunotherapy and is a promising target for future translational research.

Authors

Balamayoora Theivanthiran, Kathy S. Evans, Nicholas C. DeVito, Michael Plebanek, Michael Sturdivant, Luke P. Wachsmuth, April K.S. Salama, Yubin Kang, David Hsu, Justin M. Balko, Douglas B. Johnson, Mark Starr, Andrew B. Nixon, Alisha Holtzhausen, Brent A. Hanks

×

Figure 5

CD8+ T cells trigger a PD-L1/NLRP3 signaling pathway to drive PMN-MDSC recruitment to the tumor.

Options: View larger image (or click on image) Download as PowerPoint
CD8+ T cells trigger a PD-L1/NLRP3 signaling pathway to drive PMN-MDSC r...
(A) Western blots for HSP70 supernatant, caspase-1 p20, and Wnt5a in BRAFV600E PTEN–/– melanoma cells treated with anti–PD-L1 Ab with or without IFN-γ. (B) Immunoprecipitation (IP) of NLRP3 after treatment of BRAFV600E PTEN–/– melanoma cells with IFN-γ, anti–PD-L1, or both followed by Western blotting for ASC and NLRP3. IgG-IP, IP control; ATP, positive control. (C) Left: ASC polymerization assay following treatment of BRAFV600E PTEN–/– melanoma cells with IFN-γ, anti–PD-L1, or both. Right: ASC polymerization assay following treatment of Pdl1-silenced and NTC BRAFV600E PTEN–/– melanoma cells with IFN-γ. (D) Coculture of OT-I CD8+ T cells with OVA-expressing BRAFV600E PTEN–/– melanoma cells, with or without anti–PD-1 Ab alone or anti–PD-1 Ab plus anti–IFN-γ–blocking Ab, was followed by Western blotting for HSP70 and caspase-1 p20. (E) Coculture of OT-I CD8+ T cells with BRAFV600E PTEN–/– OVA melanoma cells, with or without anti–PD-1 Ab alone or anti–PD-1 Ab plus NLRP3 inhibitor, was followed by Western blots for caspase-1 p20, HSP70, and Wnt5a. (F) Western blots for caspase-1 p20, HSP70, and Wnt5a Western blots in BRAFV600E PTEN–/– OVA melanoma cells following coculture with OT-I CD8+ T cells after genetic silencing of either Nlrp3 (NRLP3KD) or Pdl1 (PD-L1KD). (G) IP of NLRP3 after treatment of BRAFV600E PTEN–/– melanoma cells with IFN-γ, anti–PD-L1, or both, followed by Western blotting for PKR and NLRP3. (H) Western blots for p-PKR and total PKR in control and Pdl1-silenced BRAFV600E PTEN–/– melanoma cells. GAPDH was used as a cytoplasmic loading control and laminin B as a nuclear loading control. (I) Western blotting for STAT3, p-PKR, and total PKR in control and Pdl1-silenced BRAFV600E PTEN–/– melanoma cells. (J) Western blots for caspase-1 p20 and Wnt5a in WT and STAT3CA-expressing BRAFV600E PTEN–/– melanoma cells following treatment with IFN-γ, anti–PD-L1, or both. (K) Schematic diagram depicting the PD-L1/STAT3/PKR/NLRP3 signaling axis in tumor cells. cyt, cytoplasm. All Western blots are representative of 2–3 independent experiments. See also Supplemental Figure 6.