Fig 1: Distinct chemotactic SMCs express high levels of MΦ chemoattractants, ameliorating atheroprogression(A–C) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780. (A) Violin plots (calculated on all cells expressing detectable baseline levels of the respective gene) of highly expressed cytokines and chemokines in chemotactic SMCs. Dots represent single cells, only cells exhibiting detectable expression of the particular gene are included (B) interactome depicting cell-cell interactions between MΦ and SMC subsets, prominent SMC → MΦ interactions are depicted in red. Intensity of red color depicts the respective portion of the CCL2-CCR2 axis for the concrete interaction (the darker the red color, the more the CCL2-CCR2 axis accounts for the respective inter-cluster interplay among all detected chemokine-receptor interactions). (C) Heatmap further unraveling SMC → MΦ chemokine:chemokine-receptor interactions. Blue box depicts interactions of chemotactic SMC subset, red box depicts CCL2-mediated interactions between SMC and MΦ subsets.(D) Ccl2 and Mif expression in Ng2+ SMCs FACS-sorted from western-diet fed atherosclerotic MCRFP-rep mice compared to chow-diet fed non-atherosclerotic control mice. n = 3–4 mice per group.(E) Representative images of BCA sections from Ccl2SMC +/+ and Ccl2SMCΔ/Δ littermates after 14 weeks of western diet stained for ACTA2 (green), LGALS3 (red), and Hoechst (blue). Scale bars, 100 μm.(F and G) Morphometric analysis of plaque size (F) and vascular remodeling (G) from BCA sections at three consecutive locations from Ccl2SMC +/+ (n = 11) and Ccl2SMCΔ/Δ (n = 10) littermates.(H and I) Quantification of ACTA2+ smooth muscle cell content as ACTA2+ area in percentage of total plaque area and percentage of 30 μm plaque surface area in valves (H) and in the BCA at three consecutive locations (I). (H and I) n = 10–11 mice per group.(J and K) Analysis of intimal LGALS3+ area as percentage of plaque size in BCA sections at three consecutive locations (J) and in plaques from aortic valves (K) (n = 10–11 each).(L) Schematic illustration of media and intima processing from aortae of Ccl2SMC+/+ and Ccl2SMCΔ/Δ littermates after 14 weeks of western diet (left). Heatmap displaying expression of differentially regulated genes in bulk RNA-seq of Ccl2SMC+/+ mice (n = 3) and Ccl2SMCΔ/Δ mice (n = 4). Rows represent individual replicates, differentially expressed genes are illustrated in columns (right).(M) Volcano plots of intima/media RNA-seq showing differentially expressed genes in Ccl2SMC+/+ mice (n = 3) and Ccl2SMCΔ/Δ mice (n = 4), x-axis depicts Log2FC, y-axis depicts -Log10(adj. p-value). Data are shown as mean and SEM. (H and K), Student’s t test was used. (F, G, I, and J) Repeated measures two-way ANOVA or mixed-effects model was used. ∗p < 0.05; NS, not significant. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression.
Fig 2: Identification of MIF and MDK as immunosuppressive factors based on transcriptomic data. a, Schematic representation of analysis strategy of scRNAseq data, published earlier by our group, in order to determine significant correlations between the cytotoxicity score of immune cell subsets and predicted interactions of those immune cells with other cells in the tumor microenvironment. b, Dotplot representing the gene expression of 7 cytotoxicity genes to determine the cytotoxicity score of γδ-T cells, CD8 T cells and NK cells. c, Score of cytotoxicity genes in Fig. 1b in NK, CD8-T, and γδ-T cells. Tukey’s multiple comparisons test for one-way ANOVA. d, Heatmap representing the correlation between genes in specific cell subsets, which significantly interact with the indicated immune cell subset and significantly correlate with the cytotoxicity of the indicated immune cell subset. Genes with at least one significant correlation, with either NK, CD8-T, or γδ-T, were included. The black box indicates a negative sum of the correlations of the three immune cell subsets and represents the genes selected for further analysis. e, Correlation of 13 selected genes from Fig. 1d with cytotoxicity in the dataset with bulk-RNA data from 498 neuroblastoma tumors (r2.amc.nl/; Tumor Neuroblastoma - SEQC - 498 - RPM - seqcnb1; GSE497104243). f, Survival analysis using the 13 selected genes from Fig. 1d using the SEQC bulkRNA cohort. Left panel represents event-free survival (EFS) and right panel represents overall survival (OS). Survival when the expression of the gene is high is depicted on the x-axis, the Bonferroni p-value on the y-axis. g, Kaplan-Meier curve indicating event-free survival for high- or low expression of MIF (left panel, expression cutoff: 157.498) and MDK (right panel, expression cutoff: 216.718). Bonferroni p-value is depicted. h, Dotplot representing the expression of MIF and MDK in several cell subsets in scRNAseq dataset [21].
Fig 3: MIF PROTAC reduces MIF secretion, enabling more efficient activation of CAR-T cells. a, Schematic representation of PROTAC degraders, which causes ubiquitination and degradation of proteins of interest through the proteosome. Figure made with Biorender.com. b, MIF concentration after treatment with 0.1, 1, or 10 µM MD13 for 48 h. Left panel represents MIF concentration of tumoroid model AMC691B and right panel of tumoroid model AMC691T. Measured using Luminex. c, IFN-γ concentration of supernatant from co-culture of AMC691B without treatment (grey) or with 1uM MD13 treatment (blue) in combination with GPC2 CAR-T cell in a 1:5 Effector:Target ratio, as measured by ELISA. Left panel shows concentration at 24 h and right panel shows concentration at 48 h. Statistical analysis shows results for paired t-test. (n = 4 CAR T-cell donors). d, Left panel: Luminescence signal of luciferase transduced tumoroid model AMC691B after co-culture of 24 h. Normalized to untreated tumoroid only. Tumoroids were pre-treated with or without PROTAC for 48 h before co-culture. (n = 2 CD19-CAR T-cell donors, n = 4 GPC2-CAR T-cell donors). Right panel: Normalized GPC2 CAR-T cell killing. Data were normalized to the tumoroid only untreated or treated control, respectively. Statistical analysis shows results for paired t-test. e, IFN-γ concentration of supernatant from co-culture of AMC691T without treatment (grey) or with 1uM MD13 treatment (blue) in combination with GPC2 CAR-T cell in a 1:5 Effector:Target ratio, as measured by ELISA. Left panel shows concentration at 24 h and right panel shows concentration at 48 h. Statistical analysis shows results for paired t-test. (n = 4 CAR T-cell donors). f, Left panel: Luminescence signal of luciferase transduced tumoroid model AMC691T after co-culture of 24 h. Normalized to untreated tumoroid only. Tumoroids were pre-treated with or without PROTAC for 48 h before co-culture. (n = 2 CD19-CAR T-cell donors, n = 4 GPC2-CAR T-cell donors). Right panel: Normalized GPC2 CAR-T cell killing. Data were normalized to the tumoroid only untreated or treated control, respectively. Statistical analysis shows results for paired t-test. g, IFN-γ concentration of supernatant from co-culture of AMC691B without treatment (grey) or with 1µM MD13 treatment (blue) in combination with B7-H3 CAR-T cell in a 1:5 Effector:Target ratio, as measured by ELISA. (n = 3 B7-H3-CAR T-cell donors). h, IFN-γ concentration of supernatant from co-culture of AMC691T without treatment (grey) or with 1µM MD13 treatment (blue) in combination with B7-H3 CAR-T cell in a 1:5 Effector:Target ratio, as measured by ELISA. (n = 3 B7-H3-CAR T-cell donors).
Fig 4: Reducing MIF secretion by the tumor increases the cytotoxicity of CAR-T cells. a, IncuCyte S3 experiment measuring tumor growth during co-culture with CAR-T cells. SK-N-BE2C neuroblastoma cells with shCtrl (grey) or shMIF (blue) were cultured with a control CAR-T cell targeting CD19 (triangles) or tumor antigen GPC2 (diamonds). Effector:Target ratio of 1:1. b, Normalized residual tumor cells at endpoint of the experiment in Fig. 4a. One-way ANOVA statistical test with Holm-Šídák's multiple comparisons test for significance. c, Schematic overview of procedure to study the effect of MIF-knock down on efficacy of CAR-T cells in mice. Figure made with Biorender.com. d, Average SK-N-BE2C shCtrl or shMIF tumor growth. Measuring of tumor size started when CD19 or GPC2 CAR-T cells were injected (5 × 106, iv, at arrow indication). Experimental groups of n = 6–9. Statistics show two-way ANOVA using Tukey’s multiple comparisons test at t = 2.6 weeks, when mice in the control group were sacrificed. e, Progression free survival (PFS) of shCtrl or shMIF tumor bearing mice treated with CD19.CAR or GPC2.CAR. Statistical analysis shows p-values for a log-rank (Mantel-Cox) test.
Fig 5: The immunosuppressive effect of MIF and MDK on CAR-T cell activation. a, Flow cytometry analysis of healthy donor peripheral blood T cells after 4 day in vitro stimulation with anti-CD3/anti-CD28 beads, in the presence of rMIF or rMDK (10 ng/mL). Representative graphs of CellTrace Violet peaks in CD8+ population (left panel). Middle and right panel represent the normalized division index of the CD8+ and CD4+ population from three healthy donors, respectively. Division index was determined using FlowJo’s proliferation platform. Dunnett’s multiple comparisons test for one-way ANOVA (n = 3 healthy donors, blue indicates recombinant MIF, red indicates recombinant MDK). b, Granzyme B MFI (Median Fluorescence Intensity) analysis from same experiment as Fig. 3a. Representative graphs (left two panels) of granzyme B expression in CD8+ population. Combined data from three healthy donors (right panel). MFI was normalized to stimulated control. Dunnett’s multiple comparisons test for one-way ANOVA (n = 3 healthy donors, blue indicates recombinant MIF, red indicates recombinant MDK). c, Validation of MDK knock-down by western blot (intracellular; left) and ELISA (secreted; right) on SK-N-AS and SH-SY5Y. d, Validation of MIF knock-down by western blot (intracellular; left) and ELISA (secreted; right) on SK-N-BE2C and Kelly. e, Activation of GPC2 CAR T-cells after co-culture with shCtrl and shMDK SK-N-AS and SH-SY5Y models measured by IFN-γ ELISA using two effector:target ratios (E:T; 1:1 and 1:5) at 2 different timepoints (days 2 and 5). Two-way ANOVA with Šídák's multiple comparisons test. (n = 1 CAR donor with 3 technical replicates). f, Activation of GPC2 CAR T-cells after co-culture with shCtrl and shMIF SK-N-Be2C and Kelly models measured by IFN-γ ELISA using 1:1 and 1:5 E:T ratios at days 2 and 5. Two-way ANOVA with Šídák's multiple comparisons test. (n = 1 CAR donor with 3 technical replicates). g, Proliferation of GPC2 CAR T-cells in co-culture with SK-N-BE2C-shCTRL (grey) or SK-N-BE2C (blue) in 1:1 and 1:5 Effector:Target ratio. Left plot show proliferation (CFSE) plots of one representative assay. Two-way ANOVA with Šídák's multiple comparisons test. (n = 1 CAR donor with several technical replicates). h, % of CD25 + population in GPC2 CAR-T cells in co-culture with SK-N-BE2C-shCTRL (grey) or SK-N-BE2C (blue) in 1:5 Effector:Target ratio. Each CAR-T cell donor is connected by a line. Statistics represent a multiple paired t-test with False Discovery Rate correction. n = 3 CAR-T cell donors. i, % of CD107a+ population in GPC2 CAR-T cells in. Each CAR-T cell donor is connected by a line. Statistics represent a multiple paired t-test with False Discovery Rate correction. n = 3 CAR-T cell donors. j, Mean % of TIM3, LAG3, PD-1 and CD39 on GPC2 CAR-T cells day 5 of co-culture with SK-N-BE2C-shCTRL (grey) or SK-N-BE2C (blue). Statistics represent 2-way ANOVA with Šídák's multiple comparisons correction n = 3 CAR-T cell donors.
Supplier Page from BioLegend for Recombinant Human MIF (carrier-free)