IL-18-mediated IFN-γ secretion activates macrophages to promote elimination of antigen-low myeloma. (A) Z-score heat map of proinflammatory cytokines in the peripheral blood of CAR T-cell–treated mice on day 8 of experiment, measured with the BioLegend Legendplex mouse inflammation panel of 13 cytokines. (B) IFN-γ concentration in the peripheral blood from panel A, measured with the BioLegend Legendplex mouse inflammation kit. Statistics were performed with the 1-way ANOVA test. (C) Abundance of CD11b+ F4/80+ macrophages in the BM of CAR T-cell–treated mice. Data are combined from at least 2 independent experiments. (D) Volcano plot of selected upregulated genes in BM macrophages from APRIL-BB-1XX/IL-18 vs APRIL-BB-1XX T-cell–treated mice. Genes with P value = 1 were filtered out before plotting. (E) GSEA plots of upregulated pathways related to hallmark interferon-gamma response, antigen presentation/processing and glucose utilization in BM macrophages from APRIL-BB-1XX/IL-18 treated mice. (F) Dotplot of mRNA transcript expression of genes related to antigen presentation and interferon response (more M1-like), M2/immunosuppression, macrophage activation, and glucose utilization in BM macrophages from mice treated with APRIL-BB-1XX T cells ± IL-18. Columns represent 3 replicate mice in each group. (G-H) Expression of MHC-II in BM macrophages as measured by CITE-seq (G) and flow cytometry (H). Flow cytometry data are combined from at least 2 independent experiments. (I) Production of tumor necrosis factor α (TNF-α) in BM macrophages as measured with flow cytometry, taken from a BM aspirate on day 10 of experiment. (J) Experimental setup of macrophage culture and isolation, followed by coculture of macrophages with T cells and antigen-low MOPC315.BMlow. BM was cultured with 25 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then macrophages were harvested. Approximately 10 000 macrophages were cultured with 10 000 MOPC315.BMlow and 100 000 CAR T cells. (K-L) Incucyte cytotoxicity assay of APRIL-CAR T cells ± IL-18 expression and MOPC315.BMlow cocultured with macrophages. AUC was generated and statistical analysis was performed with the 1-way ANOVA test. Results are depicted as comparison of each T-cell group with macrophage coculture, or macrophages alone (K) and each T-cell product ± macrophages (L). Data are representative of at least 2 independent experiments. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

IL-18-mediated IFN-γ secretion activates macrophages to promote elimination of antigen-low myeloma. (A) Z-score heat map of proinflammatory cytokines in the peripheral blood of CAR T-cell–treated mice on day 8 of experiment, measured with the BioLegend Legendplex mouse inflammation panel of 13 cytokines. (B) IFN-γ concentration in the peripheral blood from panel A, measured with the BioLegend Legendplex mouse inflammation kit. Statistics were performed with the 1-way ANOVA test. (C) Abundance of CD11b+ F4/80+ macrophages in the BM of CAR T-cell–treated mice. Data are combined from at least 2 independent experiments. (D) Volcano plot of selected upregulated genes in BM macrophages from APRIL-BB-1XX/IL-18 vs APRIL-BB-1XX T-cell–treated mice. Genes with P value = 1 were filtered out before plotting. (E) GSEA plots of upregulated pathways related to hallmark interferon-gamma response, antigen presentation/processing and glucose utilization in BM macrophages from APRIL-BB-1XX/IL-18 treated mice. (F) Dotplot of mRNA transcript expression of genes related to antigen presentation and interferon response (more M1-like), M2/immunosuppression, macrophage activation, and glucose utilization in BM macrophages from mice treated with APRIL-BB-1XX T cells ± IL-18. Columns represent 3 replicate mice in each group. (G-H) Expression of MHC-II in BM macrophages as measured by CITE-seq (G) and flow cytometry (H). Flow cytometry data are combined from at least 2 independent experiments. (I) Production of tumor necrosis factor α (TNF-α) in BM macrophages as measured with flow cytometry, taken from a BM aspirate on day 10 of experiment. (J) Experimental setup of macrophage culture and isolation, followed by coculture of macrophages with T cells and antigen-low MOPC315.BMlow. BM was cultured with 25 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then macrophages were harvested. Approximately 10 000 macrophages were cultured with 10 000 MOPC315.BMlow and 100 000 CAR T cells. (K-L) Incucyte cytotoxicity assay of APRIL-CAR T cells ± IL-18 expression and MOPC315.BMlow cocultured with macrophages. AUC was generated and statistical analysis was performed with the 1-way ANOVA test. Results are depicted as comparison of each T-cell group with macrophage coculture, or macrophages alone (K) and each T-cell product ± macrophages (L). Data are representative of at least 2 independent experiments. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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