Extended Data Fig. 10: c-Jun overexpressing CAR TILs demonstrate increased activity in osteosarcoma xenograft tumours.
From: c-Jun overexpression in CAR T cells induces exhaustion resistance
Experimental design described in Fig. 6. a–c, Frequency (a), phenotype (b) and ex vivo functional activity (c) of CD4+ TILs from mice treated with Her2-BBz or JUN-Her2-BBz CAR T cells. a, Left, CD4+ levels as a proportion of total live tumour cells. Right, CAR+ as a frequency of total live CD4+. b, Percentage of PD-1+ (left) and PD-1 mean fluorescence intensity (MFI; middle) of total live CD4+ with representative flow histograms (right). Mock untransduced T cells were from spleens of tumour-bearing mice at the same time point. c, Frequency of indicated cytokine- or CD107a-producing cells after 5-h re-stimulation with Nalm6-Her2+ target cells. Gated on total, live CD4+ T cells (left) with representative contour plots (right). Data are mean ± s.e.m. of n = 6 mice per group. Each data point represents an individual mouse. P values determined by unpaired two-tailed t-tests. In d–g, dissociated tumour cell suspensions were labelled and sorted by FACS analysis to isolate live, human CD45+ TILs. Sorted cells from six mice per group were pooled and approximately 10,000 cells were processed for 3′ single-cell RNA-seq on the 10X Genomics platform. d, Volcano plot showing results of differential expression analysis comparing JUN-Her2-BBz CAR T cells with control Her2-BBz CAR T cells. Top three upregulated and downregulated genes are highlighted. e, Heat map of the top 20 most significantly upregulated and downregulated genes. f, Uniform manifold approximation and projection (UMAP) embedding analysis showing JUN-Her2-BBz and control Her2-BBz CAR T cells overlaid. g, Expression of indicated transcripts in JUN-Her2-BBz or control Her2-BBz CAR T cells showing localization of CD4+ and CD8+ subsets, activation marker (IL2RA), exhaustion marker (NR4A2), and maintenance of a small memory-like population (IL7RA, KLF2) in JUN-overexpressing Her2-BBz CAR T cells within the solid osteosarcoma tumour microenvironment.
