Fig. 3. Intra-tumoral IL-12:Fc administration reinvigorates EGFRvIII-specific CAR-T cells.

A Experiment timeline. GL261 EGFRvIII⁺ cells were implanted in the right striatum at day 0. Mice received 5 Gy TBI on day 15 post implantation, while tumor engraftment was confirmed on day 17. On day 20, mice underwent surgery and received either PBS or 300 ng of IL-12:Fc at tumor site, followed by intravenous injection of 1 × 10⁶ CAR-T cells or non-transduced cells. On day 29 mice were euthanized and FACS analysis was performed on recovered lymphoid cells from brain and spleen. B Manual gating of flow cytometry data on CAR-T defined as CD45.1⁺TCR-β⁺CD34⁺CD8⁺ cells in a representative brain sample among glioma-bearing mice treated with CAR+IL-12:Fc. C Relative marker distributions, shown as scaled histograms of arcsinh-transformed marker expression, for all CAR-T cells (violet) and the selected population (red) by CellCNN analysis (left panel); boxplot showing the median frequency and 25^th and 75^th percentile of the selected population in CAR and CAR+IL-12:Fc group (right panel), n = 5 mice per condition, representative of n = 2 independent experiments. D t-SNE map showing the FlowSOM-guided metaclustering of live intratumoral CD8⁺CAR-T cells in the different treatment groups; each color represents a metacluster and is associated with a different immune population. Heatmap showing the median marker expression in each metacluster (value range: 0–1, black and white). E Frequency of CD8⁺CAR-T cells for each metacluster in each condition, n = 5 mice per condition, representative of n = 2 independent experiments. F Median expression of selected cell markers shown for LAG3^hiPD1^hi CAR-T cells and LAG3^lowPD1^low CAR-T cells in both CAR+PBS and CAR+IL12:Fc conditions, n = 5 mice per condition, representative of n = 2 independent experiments. Data are presented as mean values ± SEM. 2-tailed unpaired Mann–Whitney T test (C, E, F). Source data are provided as a Source Data file.