Journal: Cell Reports Medicine

Article Title: CD8 + T cell-intrinsic IL-6 signaling promotes resistance to anti-PD-L1 immunotherapy

doi: 10.1016/j.xcrm.2022.100878

Figure Lengend Snippet: IL6 expression associates with poor clinical outcome in atezolizumab-treated patients with metastatic RCC (A–C) RNA-seq analysis of pre-treatment tumor samples from the atezolizumab monotherapy arm of IMmotion150. (A) Differential gene expression analysis (Limma-voom), comparing PD (progressive disease) versus SD (stable disease), PR (partial response), or CR (complete response). Nominal p values are shown. (B) Differentially expressed cytokine and chemokine genes. (C) Pearson correlation of IL6 with SOCS3 and STAT3 . ∗∗∗∗p < 0.0001. (D) ISH analysis of IL6 mRNA in RCC tumors from IMmotion150 (n = 59). Black arrows, epithelial cell expression; arrowheads, stromal cell expression. Scale bar, 50 μm. Pie chart: proportions of tumors with IL6 expression (staining in ≥1% of cells) in epithelial cells only (yellow), stromal cells only (blue), or both epithelial and stromal cells (red). (E) Association of tumor IL6 mRNA with overall survival (OS) in IMmotion150. (F) Association of tumor IL6 mRNA with OS in patients with high intratumoral T cell signature expression (>median). In (E) and (F), HR (+/− 95% CI) and p values were adjusted after multivariate analysis including the following co-variates: MSKCC (Memorial Sloan Kettering Cancer Center) prognostic risk score, previous nephrectomy, and liver metastasis.

Article Snippet: Renal cell cancer tissue blocks from IMmotion150 , Genentech , N/A.

Techniques: Expressing, RNA Sequencing, Gene Expression, Staining

Journal: Cell Reports Medicine

Article Title: CD8 + T cell-intrinsic IL-6 signaling promotes resistance to anti-PD-L1 immunotherapy

doi: 10.1016/j.xcrm.2022.100878

Figure Lengend Snippet: IL-6 inhibits anti-PD-L1 efficacy and anti-tumor CTL response (A–H) Treatment of EMT6 tumor-bearing mice with antibodies targeting PD-L1 and/or IL6R. (A) Individual tumor growth curves (n = 10 per group) from one of three independent studies. PD, progressive disease; PR, partial response; CR, complete response. (B) Progression-free survival (time to 5x increase in tumor volume) of mice pooled from three independent studies, analyzed using log rank test. (C) Cellular composition of CD45 + tumor-infiltrating leukocytes, from one of three experiments. (D) Balance of CD4 + and CD8 + T cells (CTLs) among total TCRβ + tumor-infiltrating T cells. Data are concatenated from n = 5 mice per group, from one of three studies. (E) Relative abundance (normalized to tumor weight) of CTLs, conventional CD4 + T cells, and regulatory (CD4 + Foxp3 + ) T cells in EMT6 tumors, pooled from three experiments. Groups compared using one-way ANOVA with Holm-Sidak’s multiple comparisons test. (F) Effector phenotype of tumor-infiltrating CTLs after re-stimulation with PMA/ionomycin. (G) Frequencies of polyfunctional cells among tumor-infiltrating CTLs (left panel), and their absolute abundance relative to isotype control (right panel). Data pooled from three experiments. Groups compared using one-way ANOVA with Holm-Sidak’s multiple comparisons test. (H) Relative abundance (mean ± SEM) of polyfunctional CTLs and IFN-γ + TNF + CD4 + T cells (multifunctional cells) vs. dysfunctional cells (IFN-γ − TNF − GzmB − CD8 + T cells, or IFN-γ − TNF − CD4 + T cells), from one of three experiments with n = 3–5 per group. Groups compared using one-way ANOVA with Holm-Sidak’s multiple comparisons test. ∗p = 0.0143, ∗∗∗p = 0.0001, ∗∗∗∗p < 0.0001. (I–K) Single-cell RNA-seq analysis of pre-treatment peripheral blood CD8 + T cells from patients with RCC (from IMmotion150) or UC (from IMvigor210). Differential expression analysis was performed on CD8 + T cells from patients with high (>10 pg/mL) versus low plasma IL-6 (separate analyses for each cancer type). Genes identified as concordant in both cohorts (“consensus genes”) were evaluated for Reactome pathway enrichment (I). Volcano plots of differential gene expression are shown for UC and RCC samples in (J) and (K), respectively.

Article Snippet: Renal cell cancer tissue blocks from IMmotion150 , Genentech , N/A.

Techniques: Control, RNA Sequencing, Quantitative Proteomics, Clinical Proteomics, Gene Expression

Journal: Cell Reports Medicine

Article Title: CD8 + T cell-intrinsic IL-6 signaling promotes resistance to anti-PD-L1 immunotherapy

doi: 10.1016/j.xcrm.2022.100878

Figure Lengend Snippet: IL-6 blocks CTL effector differentiation (A) Splenocytes from OT-I mice were stimulated with SIINFEKL peptide +/− IL-6 and analyzed by flow cytometry on day 7. Boolean analysis of IFN-γ, TNF, and GzmB expression in CTLs is shown. Groups (mean +/− SEM of n = 4 technical replicates) were compared by t test and represent one of three independent experiments. (B) Cytokine secretion (measured by Luminex multiplex assay) by FACS-purified CTLs activated with anti-CD3/CD28 +/− IL-6 for 3 days ∗p < 0.05 (t test; n = 3 technical replicates per condition). (C) OT-I CTLs were activated as described in (A) and co-cultured with SIINFEKL-pulsed MC38.GFP cells at a 5:1 effector/target ratio. MC38 destruction was quantified using Incucyte live-cell imaging. Groups compared using two-way ANOVA (n = 4 technical replicates per condition); data indicate mean ± SEM, and are representative of three independent experiments. (D–I) OT-I splenocytes activated with SIINFEKL peptide in the presence of IL-6, control IgG, or anti-IL6R antibody. CTLs were FACS-purified for RNA-seq analysis after 2 and 7 days (n = 3 technical replicates per condition/time point). Principal components analysis (PCA) is shown in (D). (E) Differentially expressed genes (FDR <0.05 and absolute fold change >1.5). (F) Heatmap of representative differentially expressed genes (day 7). (G) Boolean flow cytometry analysis of OT-I CTLs at day 7. Groups (mean +/− SEM of n = 4 technical replicates) compared using t tests, from one of three independent experiments. (H) Reactome pathway analysis of differentially expressed genes between IL-6- and anti-IL6R-treated cells at day 7. (I) Distribution of differentially expressed genes (day 7) among differentiation modules from Best et al. ∗p = 0.0239, ∗∗∗∗p < 0.0001 (Fisher’s exact test). Fold differences refer to IL-6 versus anti-IL6R treatment. (J) Tumor RNA-seq analysis from IMmotion150. Gene modules (average Z scores) associated with effector or naive-like CTLs were used to calculate an effector/naive-like (Eff/N) ratio. IL6 expression across Eff/N quartiles (mean +/− SEM, n = 65–66 per group) was compared using one-way ANOVA.

Article Snippet: Renal cell cancer tissue blocks from IMmotion150 , Genentech , N/A.

Techniques: Flow Cytometry, Expressing, Luminex, Multiplex Assay, Purification, Cell Culture, Live Cell Imaging, Control, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: CD8 + T cell-intrinsic IL-6 signaling promotes resistance to anti-PD-L1 immunotherapy

doi: 10.1016/j.xcrm.2022.100878

Figure Lengend Snippet:

Article Snippet: Renal cell cancer tissue blocks from IMmotion150 , Genentech , N/A.

Techniques: Control, Recombinant, Cell Stimulation, Staining, Cell Isolation, cDNA Synthesis, Expressing, CRISPR, Negative Control, In Situ Hybridization, Software

Journal: Molecular Oncology

Article Title: Obesity alters the fitness of peritumoral adipose tissue, exacerbating tumor invasiveness in renal cancer through the induction of ADAM12 and CYP1B1

doi: 10.1002/1878-0261.13782

Figure Lengend Snippet: Adipose peritumoral tissue affects the expression of inflammatory genes by renal cell carcinoma (RCC) cells. (A) Relative mRNA expression of the different tumor suppressor genes in the healthy and tumor kidney tissues of RCC patients ( n = 10), including VHL , PBRM1 , SETD2 , and BAP1 , was measured by real‐time PCR. Cyclophilin‐A was used as an internal control. Data are shown as mean ± SD from at least 3 separate experiments. P ‐values were determined by Student's t ‐test. (B) Schematic figure illustrating the protocol for differentiation of adipocytes from peritumoral adipose tissue (AT) of lean, overweight, and obese RCC patients. (C) Relative mRNA expression of the leptin and adiponectin of the differentiated adipocytes among the lean, overweight, and obese groups ( n = 6). Data are shown as mean ± SD from at least 3 separate experiments. P ‐values were determined by one‐way ANOVA. (D) Schematic figure of the conditioned medium (CM) experiment, showing RCC cells treated with conditioned media (CMs) of lean, overweight, and obese differentiated adipocytes. (E) Relative mRNA expression of the IL6 , CXCR4 , SDF1 , and BAFFR of the A498 cells treated with CMs collected from differentiated adipocytes derived from the lean, overweight, and obese groups ( n = 6). Data are shown as mean ± SD from at least 3 separate experiments. P ‐values were determined by one‐way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The human renal cancer cell line (A498) was grown in DMEM (Microgem); supplemented with 10% FBS, 1% Pen/Strep, and 1% L‐Glutamine, at 37 °C in 5% CO 2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Derivative Assay