Extended Data Fig. 8: AC484 increases immune infiltration into tumors and increases STAT5 signaling and the metabolic fitness of T cells.

a, Bar plot showing quantification of total CD45⁺ cells from flow cytometry analysis of tumour-infiltrating lymphocytes from untreated B16 tumours, or B16 tumours treated with AC484 or anti-PD-1. b-e, CT26 tumour bearing animals were treated for 8 days with AC484 starting after tumour size-match at day 7. b, MFI of pSTAT5 in whole blood CD3⁺ T cells. c, Frequency of ICOS-expressing and GZMB-expressing CD8⁺ T cells in blood. d, Frequency of ICOS-expressing and GZMB-expressing CD8⁺ T cells as well as frequency of CD8⁺ T cells among CD45⁺ immune cells in tumours. e, Frequency of GZMB-expressing NK cells and CD86 and MHC I expression on macrophages in tumours. f-g, Murine splenic CD8⁺ T cells were stimulated with plate-bound anti-CD3 (5 µg mL⁻¹) and soluble anti-CD28 (2 µg mL⁻¹), expanded in IL-2, and then treated with IL-2 (100 ng mL⁻¹), AC484 (20 μM), or both for 1 or 20 h. f, Flow cytometry histograms of total STAT5 and pSTAT5 staining and relative pSTAT5 quantification of stimulated T cells. g, Western blot for and relative quantification (normalized to loading control) of pSTAT5 of stimulated T cells. h, Western blot for pSTAT5 ± IFNγ, ± IL-2, and increasing dose of AC484 (0.1 μM, 0.5 μM, 20 μM). i, Schematic representation of experimental design for the Seahorse assay on primary mouse T cells treated with the indicated cytokines ± AC484; figure created with BioRender.com. j, Oxygen consumption rate over time via the Seahorse assay using naïve T cells stimulated with plate-bound anti-CD3 (1 μg mL⁻¹), soluble anti-CD28 (2 μg mL⁻¹), and no cytokine (left) or IFNγ (right), treated with or without AC484 for 72 h. Oxidative phosphorylation profile was characterized by measuring OCR before and after injections of oligomycin (1.5 μM), FCCP (1 μM) and antimycin A and rotenone (0.5 μM). k, Quantification of mean OCR. l, Quantification of extracellular acidification rate (ECAR) over time (left) and mean ECAR (right) via the Seahorse assay using naïve T cells stimulated with plate-bound anti-CD3 (1 μg mL⁻¹), soluble anti-CD28 (2 μg mL⁻¹), IL-2 (100 ng mL⁻¹), and treated with or without AC484 for 72 h. ECAR was measured before and after injections of oligomycin (1.5 μM), FCCP (1 μM) and antimycin A and rotenone (0.5 μM). m, Bar plot quantifying Mitotracker dye staining of naïve T cells stimulated with plate-bound anti-CD3 (1 μg mL⁻¹), soluble anti-CD28 (2 μg mL⁻¹), and IL-2 (100 ng mL⁻¹) ± AC484 for 96 h.

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