Fig. 4: NR4A2/IL-12 engineered T cells exhibit an enhanced proinflammatory phenotype and effectively promote host antitumour immunity.

a–f, Mock, NR4A2 knockout or NR4A2/IL-12 OT-I cells (CD45.2⁺) were adoptively transferred into CD45.1⁺ mice bearing AT-3-ova tumours, and ex vivo analyses were performed 8 days later. Data are t-SNE plots comparing protein expression in intratumoral OT-I cells (a); flow cytometry plots (left) and quantification (right) of IFNγ, TNF, Ki67, PD-1 and Tim3 expression in intratumoral (b,c) or splenic (d) OT-I cells; quantification of H-2K^b SIINFEKL tetramer⁺ host splenic CD8⁺ T cells (e); and flow cytometry plots (left) and quantification (right) of IFNγ and TNF expression in host intratumoral CD8⁺ T cells (f). g–m, 5 × 10⁶ mock, NR4A2 knockout or NR4A2/IL-12 murine anti-hHer2 CAR T cells were adoptively transferred into MC38-hHer2 tumour-bearing mice and ex vivo analyses were performed 9 days later. Data are quantification of intratumoral CD8⁺ T cells (g) and their Ki67 expression (h); immunofluorescence imaging of tumours showing CD4, CD8 and DAPI, representative of n = 3 mice per group (i); quantification of H-2K^b p15E tetramer⁺ splenic CD8⁺ T cells (j) and IFNγ⁺ TNF⁺ intratumoral CD8⁺ T cells (k); green data points indicate mice with tumour dLNs analysed by TCR sequencing (l–m), showing cumulative frequencies of the top five TCR clonotypes (indicated by five colours; black indicates all other TCR clonotypes) in tumour dLNs. a–c,f,k, Tumours were ex vivo stimulated with SIINFEKL (a–c,f) or MC38 neoantigen peptides (k) for 3 h. a–d,f, Plots concatenated from n = 5 mice per group. b–h,j–k, Data are mean ± s.e.m. from n = 8 (NR4A2/IL-12), n = 9 (mock) and n = 10 (NR4A2 knockout) mice per group (b–c,f) or n = 10 mice per group (d,e) pooled from n = 2 experiments; n = 6 (mock, NR4A2 knockout) and n = 3 (g,k) or n = 5 (h) or n = 12 (j) (NR4A2/IL-12) mice per group. One-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar in i, 50 μm.

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