IL-18-secreting CAR T cells increase interferon signaling and drive effector-like differentiation of T cells in vivo. (A) Experimental setup describing in vivo antigen-low IL-18-CAR T-cell model and BM harvest for flow or single-cell sequencing. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 4E5 MOPC315.BM^low. 2E6 CAR T cells were injected 3 days later. Harvest data for single-cell sequence or flow cytometry were collected on day 7 of experiment unless otherwise specified. (B) UMAP projection of 34628 CD45⁺ cells post-QC processing, annotated by different cell types (left) and biological group (right). (C) GSEA plot depicting enrichment of the type-I/II interferon signaling pathways in T cells from APRIL-BB-1XX/IL-18 treated mice. (D-E) Dotplot of selected CITE-seq epitope expression (D) and RNA transcript expression (E) related to antigen presentation, interferon signaling/response, T-cell activation/effector function, T-cell stemness, and cell cycle. Columns represent 3 replicate mice in each group. (F) Abundance of total T cells, CD45.2⁺ host T cells, and CD45.1⁺ donor CAR T cells, as measured by flow cytometry. Data are combined from at least 2 independent experiments. Statistics performed with the 1-way ANOVA test. (G) Expression of stemness/naïve-like markers in CD45.1⁺ donor CAR T cells, as measured by flow cytometry. Data are combined from at least 2 independent experiments. (H) UMAP projection of T cells and annotation of CD45.2⁺ host T cells and CD45.2^– donor CAR T cells. Annotations were assigned using CD45.2 expression with CITE-seq. (I) Volcano plots of the Wilcoxon rank-sum scores from the differential gene expression analysis of APRIL-BB-1XX/IL-18 vs APRIL-BB-1XX CAR T-cell treatment in CD45.2⁺ host T cells and CD45.2^– donor CAR T cells, respectively. Genes with adjusted P value = 1 were filtered out before plotting. (J) GSEA plot displaying upregulation of NF-κB signaling pathway in CD45.2^– donor APRIL-BB-1XX/IL-18 T cells. UMAP: Uniform Manufold Approximation and Projection; GSEA: gene set enrichment analysis. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.