In contrast, we observed significant twofold and 1.3-fold increases in Th17 and T17 cell activity, respectively, in mice treated with i.t., but not i.p., antibody (Fig.1b). tumor biopsy samples from non-small cell lung cancer (NSCLC) patients revealed that pre-therapy intratumoral CD8+/RORc+cell ratio correlated with response to immune checkpoint blockade (ICB). These findings provide the initial evidence for a new mechanism of ICB resistance in lung cancer. == Electronic supplementary material == The online version of this article (10.1007/s00262-020-02795-2) contains supplementary material, which is available to authorized users. Keywords:Anti-PD-1, NSCLC, IL-17, Checkpoint blockade resistance, CD4 T-cell, Th17 cell == Introduction == Immune checkpoint inhibitors, in particular anti-PD-1 antibodies, represent a new paradigm in the management of NSCLC [1]. At the same time, while effective in a subset of patients as first or second line therapy, anti-PD-1 still fails in 5075% of NSCLC patients [1]. High tumor PD-L1 expression and neoantigen burden correlate with anti-PD-1 responsiveness, and in part may explain the suboptimal response rates [1,2]. However, 3040% of PD-L1-positive BEC HCl and high neoantigen burden tumors still do not respond while up to 14% of low neoantigen tumors do respond [3] suggesting that other, yet unidentified, factors contribute to resistance. To this end, the broader tumor immune signature, the functional ontogeny of tumor-infiltrating CD8+T-cells as well as the commensal microbiota have been identified as other contributors to responsiveness, but are yet to provide specific measurable prognostic markers in the clinical setting [2]. To gain further insight into this conundrum we investigated the therapeutic potential of anti-PD-1 antibody in the checkpoint blockade-resistant, low-neoantigen burden LSL-K-rasG12Dmurine spontaneous lung cancer model [46]. Anti-PD-1 antibody had no detectable therapeutic benefit in LSL-K-rasG12Dmice, confirming previous findings [6]. Phenotypic and functional analyses of lung T-cell populations revealed that anti-PD-1-mediated activation of lung-intrinsic Type 17 T-cells (T17 cells) directly interfered with the ability of the antibody to activate antitumor CD8+T-cells, and that post-therapy lung Th17 cell prevalence was predictive of therapeutic outcome in individual mice. Consistent with these findings, preliminary analysis of NSCLC patient tumor biopsies revealed a correlation between intratumoral CD8+/RORc+cell ratio and tumor responsiveness to ICB. This is the first report of a potential role for anti-PD1-mediated T17 cell activation in resistance to ICB. == Materials and methods == == Mice and tumor model == LSL-K-rasG12D(B6.129S4-Krastm4Tyj/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Tumors were induced as described previously [7]. All experiments were approved by the University IACUC. == Patient samples == De-identified NSCLC patient tumor biopsy specimens were obtained from the Brown Cancer Center Biorepository, University of Louisville. Tissue sections BEC HCl prepared from formalin-fixed, paraffin-embedded tumor samples were processed for RNAscope. The study was approved by the University Institutional Review Board. == Microsphere preparation and treatment == Anti-PD-1 antibody-encapsulated biodegradable polylactic acid microspheres were prepared as previously described [7]. Two formulations were produced: (1) control (no antibody) and (2) anti-mouse CD279 (PD-1) antibody (clone J43, BioXCell, West Lebanon, NH) with a loading of 8 g antibody/mg of particles. Control or anti-PD-1 microspheres (0.125 mg particles in 35 l sterile water) were administered via intubation-mediated intratracheal instillation (IMIT) 2x/week for 4 weeks starting 6 weeks after adenoviral infection [7]. Soluble antibody (200 g in 0.2 ml saline) was administered i.p. 3x/week. == In vivo antibody-mediated leukocyte subset depletion and cytokine neutralization == Depletion was performed by i.p. injection of 250 BEC HCl g of anti-mouse CD4 (clone GK1.5), CD8 (clone 53-6.72, BioXCell) or via i.v. injection of TCR antibody (clone UC7-13D5, BioXCell or Leinco Technologies) 3x/week for 4 weeks. For in vivo neutralization of IL-17, 100 g anti-mouse IL-17A (clone 17F3; BioXCell) was administered i.p. 3x/week for 4 weeks. == Tumor quantification == Lung tumor burden was quantified by digital imaging analysis of H&E-stained serial lung sections as described previously [7]. QuPath open source software was used to quantify lesion Rabbit Polyclonal to OR10J3 vs total lung area per section. == Isolation of lung mononuclear cells == Lung mononuclear cells were isolated as previously described [7]. == Antibodies and flow cytometry == Fluorescence-conjugated anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-TCR (GL3), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-Ly-6G (1A8), anti-IL-17A (TC11-18H10.1), anti-IFN- (XMG1.2) and anti-RORt (Q31-378), were purchased from BioLegend (San Diego, CA), eBioscience (Waltham,.