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: For polyclonal T cell activation, bulk splenocytes or CD8 + T cells isolated using the EasySep CD8 + T cell Isolation Kit (STEMCELL Technologies, Cambridge, MA) were plated in T cell media containing 10 ng/mL human IL-2 at 0.2 million cells per well in Falcon flat bottom 96 well plates (Corning Life Sciences, Corning, NY) coated overnight with 5 μg/mL anti-CD3 antibody (BD Biosciences, San Jose, CA, clone 145-2C11) and 2.5 μg/mL anti-CD28 antibody (BD Biosciences, San Jose, CA, clone 37.51).

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

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 regulates CTLs via STAT3-dependent BATF induction (A–C) RNA-seq analysis of WT naive CTLs stimulated with IL-6 +/− anti-CD3/CD28 antibodies for 4 h. (A) Differentially expressed genes (FDR <0.05 and absolute fold change >2). (B) IL-6-regulated genes with potential roles in CTL differentiation, and their functional categorization (C). (D) IFN-γ expression in WT or STAT3.ko CTLs (from CD4-Cre x Il6r loxP /loxP mice) activated with anti-CD3/CD28 +/− IL-6 or hyper-IL-6 for 3 days. Groups (mean ± SEM of n = 4 technical replicates) compared by t test; data representative of three independent experiments. (E) Batf mRNA expression (qRT-PCR) in WT or STAT3.ko CTLs activated +/− IL-6. ∗∗p < 0.01, ∗∗∗p < 0.001 (WT vs. STAT3.ko; t tests). Data points indicate mean +/− SEM of n = 4 technical replicates, from one of two independent experiments. (F) Western blot of BATF and p-STAT3 (Y705) in activated WT or STAT3.ko CTLs. Data represent one of two independent experiments. (G) BATF CRISPR-ko or control CTLs were activated +/− IL-6 and analyzed on day 3 (groups compared by t test; mean ± SEM of n = 4 technical replicates). Data representative of two independent experiments.

Article Snippet: For polyclonal T cell activation, bulk splenocytes or CD8 + T cells isolated using the EasySep CD8 + T cell Isolation Kit (STEMCELL Technologies, Cambridge, MA) were plated in T cell media containing 10 ng/mL human IL-2 at 0.2 million cells per well in Falcon flat bottom 96 well plates (Corning Life Sciences, Corning, NY) coated overnight with 5 μg/mL anti-CD3 antibody (BD Biosciences, San Jose, CA, clone 145-2C11) and 2.5 μg/mL anti-CD28 antibody (BD Biosciences, San Jose, CA, clone 37.51).

Techniques: RNA Sequencing Assay, Functional Assay, Expressing, Quantitative RT-PCR, Western Blot, CRISPR

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: For polyclonal T cell activation, bulk splenocytes or CD8 + T cells isolated using the EasySep CD8 + T cell Isolation Kit (STEMCELL Technologies, Cambridge, MA) were plated in T cell media containing 10 ng/mL human IL-2 at 0.2 million cells per well in Falcon flat bottom 96 well plates (Corning Life Sciences, Corning, NY) coated overnight with 5 μg/mL anti-CD3 antibody (BD Biosciences, San Jose, CA, clone 145-2C11) and 2.5 μg/mL anti-CD28 antibody (BD Biosciences, San Jose, CA, clone 37.51).

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