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.
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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

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Figure 3

HSP70-TLR4 induces Wnt5a expression in response to anti–PD-1 Ab immunotherapy.

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HSP70-TLR4 induces Wnt5a expression in response to anti–PD-1 Ab immunoth...
(A) RNA-Seq GSEA showing top 12 pathways enriched in autochthonous BRAFV600E PTEN–/– melanomas following escape from anti–PD-1 Ab therapy. Arrows indicate pathways associated with cellular stress (n = 3/group). (B) SILAC-AHA LC-MS/MS secretome analysis of resected autochthonous BRAFV600E PTEN–/– melanoma tissues following anti–PD-1 Ab therapy versus IgG isotype control. Secreted protein levels were normalized to the number of cells (n = 3/group). (C) Plasma HSP70 ELISA analysis following anti–PD-1 versus IgG isotype control treatment of autochthonous BRAFV600E PTEN–/– melanoma-bearing mice (n = 6). (D) qRT-PCR analysis of TLR expression in BRAFV600E PTEN–/– melanoma cells. Data were normalized to Tlr9 expression levels (n = 3). (E) Treatment of BRAFV600E PTEN–/– melanoma cells with titrated concentrations of recombinant HSP70 (rHSP70) followed by Wnt5a Western blot analysis of total cell lysates and supernatant (SNT). Blots are representative of 2 independent experiments. (F) Treatment of BRAFV600E PTEN–/– melanoma cells with titrated concentrations of the HSP70 inhibitor VER155008 (HSP70i). Blots are representative of 2 independent experiments. (G) Treatment of BRAFV600E PTEN–/– NTC cells with rHSP70 with or without the TLR4 inhibitor CLI-095 (TLR4i) and treatment of Tlr4-silenced BRAFV600E PTEN–/– melanoma cells (TLR4KD) with HSP70 followed by Western blotting for Wnt5a. Blots are representative of 3 independent experiments. (H) BRAFV600E PTEN–/– melanoma growth curve following treatment with TLR4 siRNA versus control siRNA (n = 5). (I) Whole-tissue Western blot analysis of Wnt5a, CXCL5, and β-actin in TLR4 siRNA–treated and control siRNA–treated BRAFV600E PTEN–/– melanomas. Data are representative of 2 independent experiments. (J) Top: PMN-MDSC flow cytometric analysis of TLR4 siRNA– and control siRNA–treated BRAFV600E PTEN–/– melanomas (n = 4). Bottom: CD8+ T cell flow cytometric analysis of TLR4 siRNA– and control siRNA–treated BRAFV600E PTEN–/– melanomas (n = 4). *P < 0.05, by Student’s t test for comparison of treatment groups. See also Supplemental Figure 4.