Sustained Type I interferon signaling as a mechanism of resistance to PD-1 blockade

Abstract

PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy. Delineating mechanisms of secondary resistance to this strategy is increasingly important. Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb). Sustained IFNβ transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC). Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation. Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature. Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy.

Conflict of interest statement

G.K. reports grants and personal fees from Bayer Healthcare, grants from Genentech, grants from Glaxo Smyth Kline, grants and personal fees from Lytix Pharma, grants from PharmaMar, grants from Sotio, grants from Vasculox, other from Bristol Myers Squibb Foundation France, other from everImmune, other from Samsara Therapeutics, outside the submitted work. L.Z. has the following financial relationships to disclose, none of which deals with the submitted work: Scientific advisory boards at Lytix Pharma, EpiVax, NeoVax and Tusk Pharma; Administrative board: Transgene; Grant/Research support from Glaxo Smith Kline, Merus, Tusk Roche and Incyte; Founder and shareholder: everImmune. M.C.A. reports honorarium, travel support and advisory board participation from Merck, unrelated to the current work. J.W. and V.G. are inventors on a US patent application (PCT/US17/53.717) submitted by the University of Texas MD Anderson Cancer Center that covers methods to enhance immune checkpoint blockade responses by modulating the microbiome, unrelated to this work submitted. J.W. reports compensation for speaker’s bureau and honoraria from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, MedImmune and Bristol-Myers Squibb; consultant/advisory board membership for AstraZeneca, Merck, Biothera Pharmaceuticals and Microbiome DX; consultant/advisory board membership and research support from GlaxoSmithKline, Roche/Genentech, Bristol-Myers Squibb, and Novartis. V.G. reports consultancy fees from MicrobiomeDX and ExpertConnect, and honoraria from ExcelCME and Kansas Society of Clinical Oncology. M.J.S. has scientific research agreements with Bristol Myers Squibb and Tizona Therapeutics and is an advisory board member for Compass Therapeutics and Tizona Therapeutics. A.M.E. declares receipt over the last 2 years of Honoraria for Scientific Advisory Boards and Data Monitoring Safety Board from the following companies: BMS, GSK, Incyte, IO Biotech, ISA-Pharmaeuticals, MedImmune, Merck-Serono, MSD, Novartis, Pfizer, Sanofi, Sellas, SkylineDx. P.A.A. has/had a consulting or advisory role for BMS, Roche-Genentech, MSD, Array, Novartis, Merck Serono, Pierre Fabre, Incyte, Genmab, Newlink Genetics, Medimmune, AstraZeneca, Syndax, Sun Pharma, Sanofi, Idera, Ultimovacs. Sandoz, Immunocore. He also received research funds from BMS, Roche-Genentech, Array, and travel support from MSD. V.B. reports advisory board participation from ITeos Therapeutics Sa, Tusk Therapeutics Ltd, Io Biotech ApS, Xios Therapeutics, Codiak BioSciences. Over the last 5 years, J.-C.S. has received consultancy fees from AstraZeneca, Astex, Clovis, GSK, GamaMabs, Lilly, MSD, Mission Therapeutics, Merus, Pfizer, PharmaMar, Pierre Fabre, Roche/Genentech, Sanofi, Servier, Symphogen, and Takeda. He has been a full-time employee of MedImmune since September 2017. He is a shareholder of AstraZeneca and Gritstone. L.V. reports personal fees from Adaptherapy, personal fees from Pierre-Fabre, grants from Bristol-Myers Squibb, outside the submitted work. The remaining authors declare that they have no conflict of interest.

Figures

Fig. 1

“Sensitive”, “eventually resistant” and “innately resistant” tumor models to PD-1 blockade. a and e Therapeutic antitumor protocols based on anti-PD-1 or its isotype control. When tumors reached 20–25 mm2 (indicated by an arrow), anti-PD-1 mAb (or its isotype control) alone a or together with anti-CTLA-4 mAb e were administered i.p. every 3 days for 4 a to 6 e injections (as described in Material and Methods). b–d, f MCA205WT sarcoma b, f, MC38 colon c or AT3 breast d cancer cells were injected subcutaneously. Tumor growth kinetics (left panels), survival curves (middle panels) and tumor sizes after sequential injections of isotype or anti-PD-1 mAb (right panels) are depicted. Each line or dot corresponds to one animal. Each graph represents 1 experiment f, a pool of 2 d to 3 b, c experiments with 5–6 animals per group and per experiment. For tumor growth and Kaplan–Meier curves, statistical analyses were performed using the specific software detailed in the Material and Methods. Unpaired t-tests were used in b, c, d and f, right panels. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant. Means ± SEM are represented

Fig. 2

Host and tumor IFNAR1 involved in secondary resistance to PD-1 blockade. a MCA205WT growth kinetics (top panels) and Kaplan–Meier survival curves (bottom panel) of WT and Ifnar1−/− C57BL/6 mice treated with anti-PD-1 mAb (or its isotype control) as described in Fig. 1a. b and c Representative gating strategy b and flow cytometry analyses c of tumor-infiltrating leukocytes after 4 injections and the proportions of CD45+ (left panel), ICOS+ cells in the CD8+T cell gate and FOXP3+ cells in the CD4+T cell gate (middle panels) and the ratio CD8+/CD4+FOXP3+ (right panel) are depicted. d Tumor growth kinetics and Kaplan–Meier survival curves of WT mice inoculated with two different clones of Ifnar1−/− MCA205. Each line or dot represents one animal. The graphs represent 1 experiment (c) or depict pooled data from 2 a and d independent experiments encompassing 4–7 mice/group. For tumor growth and Kaplan–Meier curves, statistical analyses were performed using the specific software detailed in the Material and Methods. ANOVA statistical tests and pairwise comparisons with Bonferroni adjustment were used in c. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant. Means ± SEM are represented

Fig. 3

Sources and kinetics of Type I IFN in the TME during PD-1 inhibition. a and b In vitro assays. Relative expression of Ifnβ1 quantified by qRT-PCR following stimulations of various tumor cell lines or BMDCs and BMMCs with IFNα, IFNγ or LPS. Each dot represents one sample and graphs represent 1 experiment or are the pool of 2 to 3 independent experiments including biological replicates for each experiment. Unpaired t-tests were used to compare 2 groups. ANOVA statistical tests and pairwise comparisons with Bonferroni adjustment were adopted for more than 2 groups. c–h In vivo studies. Flow cytometry sorting of CD45+ live fractions from the TME of MCA205WT c–e or MC38 f–h tumors 48 h after 1, 2, 3 or 4 i.p. administrations of anti-PD-1 (or isotype control) mAb. Relative expression of Ifnβ1c and f and IFN-sensitive gene products d, e, g, h quantified by qRT-PCR. Unpaired t-tests were used to compared transcription levels between the anti-PD-1 and isotype control treated groups for each time point. Each dot represents 1 mouse with 5 mice per time point per experiment. Graphs represent 1 representative experiment out of 2–3 independent experiments (MC38, time points 1 and 2, d, e), 1 experiment (MC38, time points 3 and 4) or are the pool of 2–3 independent experiments c. *p < 0.05, **p < 0.01, n.s.: not significant. Mean ± SEM are represented

Fig. 4

Secondary resistance to PD-1 blockade is PD-L1 independent. a Effects of neutralizing anti-PD-L1 mAb co-administered early or late following initiation of anti-PD-1 mAb in MCA205WT tumor bearers. b Effects of anti-PD-1 mAb treatment on tumor growth against MCA205WT versus two different clones of Pd-l1−/− MCA205. c Equivalent schema as in a using anti-CD80 mAb. Each line or dot represents 1 animal. Mean ± SEM are represented. Tumor growth kinetics and Kaplan–Meier survival curves are shown. The graphs depict tumor growth kinetics of 1 (a, anti-PD-L1 early; c, anti-CD80 early and late) or 2 independent experiments encompassing 5–8 mice/group and per experiment. Statistical analyses were performed using the specific software detailed in the Material and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant

Fig. 5

Type I IFN-induced Nos2 expression post- PD-1 blockade in the TME. a Microarray analysis and qRT-PCR analyses of TILs in MC38 during PD-1 blockade. CD45+ cells from MC38 tumors were cell-sorted 48 h post-1, 2 injections of anti-PD-1 or isotype control mAbs to perform trancriptomic analyses. Heat-map depicting the shared significant downregulated and upregulated genes in the anti-PD-1 treated groups compared with the isotype control treated groups across time points 1 and 2. b Relative expression of Nos2 in CD45+ cells from MC38 tumors 48 h after 1, 2, 3 and 4 injections of anti-PD-1 or Isotype mAbs using qRT-PCR analyses. c and g Same as b with MCA205WT tumor bearers after treatment with 1, 2, 3 and 4 injections of mAbs evaluated in both CD45+c and CD45−g fractions. d and h Representative gating strategy and flow cytometric analyses of NOS2 protein expression in CD45+ cells d and in the CD45- fraction h, 48 h after the fourth injection of anti-PD-1 or its isotype control mAbs. e, f Relative expression of Nos2 quantified by qRT-PCR following stimulations of BMDCs and BMMCs e or various tumor cell lines f with either IFNα, IFNγ or LPS. Each dot corresponds to one stimulated sample or 1 mouse with 2 or more biological replicates per experiment and 5 mice per group per time point per experiment. Graphs depict 1 experiment (b, time points 3 and 4, d and h), are representative of 1 experiment out of 2–3 performed (b, time points 1 and 2), or are the pool of 2–3 independent experiments c, e–g including biological replicates for each experiment. Unpaired t-tests were used to compare two groups (b–d, f for AT3 tumor model and g, h). ANOVA statistical tests and pairwise comparisons with Bonferroni adjustment were adopted for more than 2 groups e, f. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant. Means ± SEM are represented

Fig. 6

Pharmacological inhibition of NOS2 ameliorated the efficacy of PD-1 blockade in various tumor models. Concomitant blockade and inhibition of PD-1 and NOS2 with L-NAME in established MCA205WT a and MC38 b tumors. Tumor growth kinetics (left and middle panels) and survival curves (right panels) are depicted for each group. L-NAME treatment (1 g/L) was started one day prior to anti-PD-1 infusion and was maintained until the end of the experiment. The graphs depict tumor growth kinetics of a pool of 2–3 independent experiments encompassing 5–9 mice per group and per experiment. c Flow cytometry analyses of TILs after 4 injections focusing on the proportion of FOXP3+ cells among CD4+ T cells and the ratio of CD8+ T cells/CD4+ FOXP3+ Treg cells are depicted. Each dot represents 1 animal and graphs depict the pool of 3 independent experiments. Means ± SEM are represented. Statistical analyses were performed using ANOVA statistical tests and pairwise comparisons with Bonferroni adjustment. d Effect of L-NAME in anti-PD-1-treated MCA205WT tumors inoculated into WT or Nos2−/− mice. The graphs depict a pool of 2 independent experiments including 5–7 mice/group and per experiment. Statistical analyses were performed using the specific software detailed in the Material and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant

Fig. 7

Type I IFN and NOS2 are associated with the secondary resistance of anti-PD-1 + anti-CTLA-4 therapy in patients. a Volcano plot of NanoString gene expression analysis in tumor biopsies harvested prior to initiation of combination anti-CTLA-4 and anti-PD-1 immune checkpoint blockade comparing responders (R) versus non-responders (NR). b Heatmap of differentially-expressed genes (FDR < 0.10) in pre-treatment samples comparing R versus NR patients, indicating RECIST-based best overall response (CR = complete response, PR = partial response, PD = progressive disease), and receipt of prior melanoma-directed systemic immunotherapy (cytokine, checkpoint blockade agent). c Boxplots of NOS2 gene expression stratified by response to combination immune checkpoint blockade (R = responder, NR = non-responder) and prior immunotherapy status (Yes/No) demonstrating numerically higher NOS2 levels in NR patients with prior immunotherapy exposure