Extended Data Fig. 6: o9R signalling in vitro and in vivo drives a T_SCM phenotype in pmel T cells.

a, Surface expression of CD62L, Fas (CD95) and CD44 as percentage of CD8⁺Thy1.1⁺ sorted o9R pmel T cells, o2R pmel T cells, or pmel T cells transduced with wild-type IL-9R (pmel-IL-9R) and treated with MSA-IL2 (0.05 μM), MSA-oIL2 (5 μM), or IL-9 (.05 μM) for 48 h in vitro. Shown are mean ±SD, n = 3 biological replicates/group. ns, not significant; ****, p < 0.0001; unpaired t-tests, two-sided. b, unsupervised transcriptomic analysis (RNA-seq) of sorted pmel-o2R (n = 3) and pmel-o9R (n = 3) T cells 48 h after exposure to MSA-oIL2 or MSA-IL2 in vitro. Heat map of genes differentially expressed between o2R and o9R pmel T cells treated with MSA-oIL2 (5 μM)(left panel). MSA-IL2 treated groups (50nM) also shown. On principal component analysis (PC1 v PC2, top right panel), samples separate by treatment group. Samples cluster by treatment group when arranged by sample-sample distances in a heat map (bottom right panel), with o9R pmel T cells treated with oIL-2 most distinct among the four groups. c, Frequency of CD62L⁺ o2R or o9R pmel T cells (Thy1.1⁺YFP⁺) from tumour draining lymph nodes (DLN), spleen or tumours of mice treated with MSA-oIL2 (without prior lymphodepletion). Tissues were collected one or five days after adoptive transfer. Shown are mean ±SD with individual data points (n = 3-5 mice per group). **, p < 0.01; ***, p < 0.001; ****, <0.0001; unpaired t-test, two-sided.