Patient enrollment and response evaluation
A total of 45 patients diagnosed with NSCLC and treated with anti-PD-1 inhibitors from January 2018 to December 2019 at the Cancer Hospital and Institute, Chinese Academy of Medical Sciences (CICAMS, Beijing, China) were included in the present study. Enrolled patients received immunotherapy once every 2 or 3 weeks (nivolumab was administered once every 2 weeks, other anti-PD-1 drugs were given once every 3 weeks). Radiologic assessments, including computed tomography, were conducted every 6 weeks. If necessary, head magnetic resonance imaging was simultaneously performed. The tumor response was assessed according to the Response Evaluation Criteria in Solid Tumors (version 1.1) and was categorized as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Progression-free survival (PFS) and OS were defined as the time from the initiation of ICIs administration to the time of PD and from the beginning of ICIs to death, respectively. The last follow-up assessment was performed on January 2, 2021. The Ethics Committee of CICAMS approved this study (approval number 19/147-1925). Clinical characteristics of these NSCLC patients are described in Additional file 1: Table S1.
Sample collection and cytokine assessment
Blood samples were collected in an EDTA tube before the administration of anti-PD-1 immunotherapy. All samples were processed within 2 h of collection, and centrifuged at 3000 rpm for 10 min at 4 °C. The upper plasma fraction was stored at − 80 °C until assayed. Plasma samples were obtained from NSCLC patients to perform the cytokine assessment. The levels of secreted cytokines, including IL-17A, TNF-α, IL-2, IL-10, IL-4, IFH-γ, IL-6, and granzyme B, were quantified using a custom LEGENDplex™ Human Multi-Analyte Flow Assay kit (Cat# 740267; BioLegend). Data were gained via the Flow Cytometer (BD Biosciences) with the manufacturer’s instructions. Furthermore, the human IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Cat# ab46042; Abcam) was applied to measure the levels of IL-6.
Specimen collection and immunohistochemistry (IHC)
We collected tissue specimens and archived the samples via formalin fixation paraffin embedding (FFPE) before the initiation of anti-PD-1 immunotherapy. FFPE samples from 25 patients with NSCLC were examined for protein levels of IL-6. IHC was used to assess the expression of IL-6 with an anti-IL-6 monoclonal antibody (mAb; Cat# 12153; CST, USA). The staining score of IL-6 in every tissue sample was calculated using the following formula: IHC score of IL-6 = staining intensity × percentage of positive tumor cells × 100. The staining intensity score was divided into four grades: no color was zero (negative), light yellow was 1 (weakly positive), yellow was 2 (moderately positive), and brownish yellow was 3 (strongly positive). Ten fields were randomly selected under a high-power microscope (× 400). The average value was taken to calculate the percentage of tumor cells that stain positively compared to all tumor cells in view. All slides were assessed by two pathologists independently based on the evaluation criteria of the previously published method . These pathologists were blinded to the patients’ clinical characteristics.
Clinical cohort and TIME analysis
Tumor samples were resected from 196 patients with NSCLC, including 137 patients diagnosed with lung adenocarcinoma (LUAD) and 59 with lung squamous carcinoma (LUSC), between January 2012 and December 2016. These specimens were analyzed for IL-6 expression, PD-L1 expression, and numbers of CD8+ T cells, myeloid-derived suppressor cells (MDSCs), M2 macrophages, and regulatory T cells (Treg cells). Using IHC, tumor sections were stained for PD-L1 (pre-diluted anti-human PD-L1 mAb, clone SP263, Cat# 740-4907; Ventana, USA) using an automated Ventana Benchmark XT instrument, CD163 (pre-diluted anti-human CD163 mAb, Cat# ZM-0428; Zsbio Tech, China), Foxp3 (anti-human Foxp3 mAb, Cat# ab20034; Abcam, UK), and CD8 (pre-diluted anti-human CD8 mAb, Cat# ZA-0508; Zsbio Tech, China) [23, 24]. MDSCs were analyzed for CD11b expression (anti-human CD11b mAb, Cat# ab52478; Abcam, UK) and CD33 expression (anti-human CD33 mAb, Cat# ab30371; Abcam, UK) by double immunofluorescence . The PD-L1 tumor proportion score and the proportion of immune cells were assessed according to the evaluation criteria of the previously published approach .
Cell lines and reagents
Immortalized human bronchial epithelial cells (Beas-2B cells) were cultured in BEGM media (Cat# CC-3170; Lonza, Switzerland) containing 10% fetal bovine serum (FBS, Cat# 35-081-CV, Corning, USA). Human LUAD cell lines (H358, A549, PC-9, and H1975) were cultured in RPMI-1640 media (Cat# 10-040-CV, Corning, USA) containing 10% FBS and an antibiotic mixture (100 U/mL penicillin and 100 μg/mL streptomycin, Cat# 15140-122, Gibco, USA). Human LUSC cell lines (H226 and H1703) were cultured in RPMI-1640 media containing 10% FBS and an antibiotic mixture. Mouse LA795 cells (clones from the lung neoplasm of a 615 mouse with LUAD) were cultured in RPMI-1640 media containing 10% FBS and an antibiotic mixture. Mouse KLN205 cells (clones from the lung neoplasm of a DBA-2 J mouse with LUSC) were cultured in DMEM media (Cat# 10-013-CVR, Corning, USA) containing 10% FBS and an antibiotic mixture. Beas-2B, H358, A549, H1975, H226, and KLN205 were purchased from American Type Culture Collection. PC-9, H1703, and LA795 were purchased from Cell Resource Center in the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
The reagents used in this study included human recombinant IL-6 (Cat# 200-06-20, PeproTech, USA), human recombinant IL-2 (Cat# 200-02-50, PeproTech, USA), and JAK1/2 inhibitor (ruxolitinib, Cat# S1378, Selleck, China). The antibodies for western blot analysis used in this study are listed as follows: IL-6 (Cat# 12153; CST, USA), PD-L1 (Cat# 13684 T; CST, USA), JAK1 (Cat# 3344; CST, USA), phospho-JAK1 (Cat# 3331; CST, USA), Stat3 (Cat# 9139; CST, USA), phospho-Stat3 (Cat# 4113; CST, USA), and GAPDH (Cat# ab8245, Abcam, UK).
Stable lentiviral IL-6 overexpression
Full-length IL-6 cDNA was ligated into the pHS-BVC-LW334 vector. According to the manufacturer’s guidelines, HEK-293 T cells were co-transfected with the IL-6 vector and packaging plasmid (pLP1, pLP2, and pLP/VSVG) using Lipofectamine 3000 (Cat# L3000015, Invitrogen, USA) to produce lentivirus carrying the IL-6 gene. A549 and H1703 cells were then infected with harvested lentiviruses and exposed to 5 μg/mL polybrene (Cat# P4505, Sigma-Aldrich, USA) and selected for mCherry expression using flow cytometry. The expression levels of IL-6 in the infected cells were confirmed by western blot analysis after selection.
In vitro tumoricidal activity assays
Human peripheral blood mononuclear cells (PBMCs) were extracted by Ficoll centrifugation (Cat# 17-1440-02, GE Healthcare, USA) from peripheral blood provided by healthy donors. We then isolated CD8+ T cells from PBMCs using the human CD8+ T Cell Isolation kit (Cat# 130-096-495, Miltenyi Biotec, Germany). Isolated CD8+ T cells were activated by the addition of human anti-CD3/CD28 Dynabeads (Cat# 40203D, Thermo Fisher Scientific, USA) for 3 days and cultured in RPMI-1640 media containing 10% FBS, an antibiotic mixture, and 200 U/mL IL-2. After adherent overnight, control or IL-6-overexpressing A549 and H1703 cells were incubated with activated CD8+ T cells for 48 h, and exposed to either the isotype control or durvalumab (1500 μg/mL). In addition, after adherent overnight, A549 and H1703 cells were incubated with activated CD8+ T cells for 48 h with or without exposure to the IL-6 (20 ng/mL) isotype control or durvalumab (1,500 μg/mL). The ratio of CD8+ T cells to cancer cells was 5:1. After the removal of T cells and cellular debris, living cancer cells were then quantified using the Cell Counting Kit-8 (Cat# CK04, DOJINDO, Japan). The tumoricidal activity was calculated using the following formula: killing activity (%) = [1 − (optical density value of experimental group − optical density value of T cells group)/optical density value of tumor cells group] × 100%.
In vivo mouse experiments
Female 615 mice (SPF, 5–6 weeks old) were purchased from the Institute of Hematology, Chinese Academy of Medical School (Tianjin, China). Male DBA-2 J mice (SPF, 5–6 weeks old) were purchased from Huafukang Bioscience Co., Ltd. (Beijing, China). The mice were housed under standard conditions in the animal care facility at the Center of Experimental Animals of CICAMS. The temperature and humidity were maintained at 26 °C–28 °C and 60 ± 5%, respectively. All procedures were approved by the Animal Care and Use Committee of CICAMS. As shown in Figs. 4A and 5A, to establish the LUAD and LUSC mouse models to study the respective immune systems, 615 mice and DBA-2 J mice were subcutaneously implanted with murine LA795-derived xenografts and KLN205-derived xenografts, respectively. We measured tumor size using a caliper every three days and calculated the tumor volume based on the formula V = L × W
2/2 (V: tumor volume; L: tumor length; W: tumor width). The mice were randomly placed into five groups with five mice/group and given different treatments when the maximum tumor diameter reached 7 mm. The treatment regimens involved anti-PD-L1 mAb (10 mg/kg three times a week, BioXCell, USA), anti-IL-6 mAb (10 mg/kg three times a week, BioXCell, USA), a combination of anti-PD-L1 and anti-IL-6 drugs, or a combination of an anti-PD-L1 drug and paclitaxel (20 mg/kg twice a week, CSPC Inc., China) as described in Figs. 4A and 5A. The endpoints were defined when the maximum tumor diameter reached 20 mm, the weight loss was greater than 2 g, or death. All mice were sacrificed by carbon dioxide asphyxiation to harvest tumors.
Harvested tumors were fixed in formalin, embedded in paraffin, and sectioned (4 μm). Using IHC, tumor sections were stained for CD8 (anti-mouse CD8 antibody, Cat# 98941; CST, USA), CD163 (anti-mouse CD163 antibody, Cat# ab182422; Abcam, UK), Foxp3 (anti-mouse Foxp3 antibody, Cat# ab215206; Abcam, UK), and PD-L1 (anti-mouse PD-L1 antibody, Cat# ab238697; Abcam, UK). MDSCs were stained for CD11b (anti-mouse CD11b antibody, Cat# ab133357; Abcam, UK) and Ly6G (anti-mouse Ly6G antibody, Cat# GB11229; Servicebio, China) using double immunofluorescence analysis. The staining score of PD-L1 in mouse samples was calculated using the following formula: IHC score of PD-L1 = staining intensity× percentage of positive tumor cells × 100. The proportion of immune cells was assessed according to the evaluation criteria of the previously published approach . Ten fields were randomly selected under a high-power microscope (× 400) in subcutaneous tumor nodule per mouse. The average value was taken to calculate the percentage of immune cells that stain positively compared to all immune cells in view. All slides were assessed by two experienced pathologists blinded to the clinical parameters.
Data analysis was conducted using R software (version 3.6.0), SPSS (version 22.0; IBM, New York, USA), and GraphPad Prism software (version 8.0, Graph Pad, San Diego, CA, USA). Differences between independent variables were evaluated using the Kruskal-Wallis H test or Mann-Whitney U test. Fisher’s exact test was used to analyze categorical variables. Correlation coefficients were obtained using Pearson correlation analysis. Survival was assessed with the log-rank test and Kaplan-Meier analysis. Furthermore, Cox regression was conducted for univariate and multivariate analyses of prognosis. Notably, all factors with a P value less than 0.15 in the log-rank test were analyzed in the multivariate Cox regression analysis. All statistical analyses were double-sided, and statistical significance was considered as P values less than 0.05.