Extended Data Fig. 6. Ret tumor-infiltrating ILC2 express high levels of GM-CSF and high Csf2 expression in human melanoma tumors is associated with increased survival.

a, Representative flow cytometric full gating strategy used to identify GM-CSF-expressing ILC2 in Ret melanoma tumors 7 days after tumor inoculation. GM-CSF expression in other immune and non-immune cell subsets is also depicted. b, Frequency of GM-CSF producing cells in Ret melanoma tumors. Each circle represents one mouse and data and show mean ± s.e.m. Data are pooled from 2 independent experiments (n=12 mice) with 6 mice/experiment. c, Representative flow-cytometric gating strategy used to identify polyfunctional ILC2 (IL-5⁺IL-13⁺GM-CSF⁺). a-c, Single cell suspensions of digested tumor cells were stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin in the presence of GolgiStop™ for 4h before intracellular staining for IL-5, IL-13 and GM-CSF. b, Each circle represents one mouse and data show mean ± s.e.m. and are pooled from 2 independent experiments with 6 mice/experiments. a and c, Data show one of two independent experiments performed with 6 mice/experiment. d,e, Analysis of the publicly available TCGA database. d, Plot shows tumor CSF2 gene expression according to type 2 immune cell tumor enrichment probabilities in individual human metastatic melanoma samples. Tumor enrichment probabilities were determined as described in the Material and Methods using machine learning. Mean difference in CSF2 expression between TCGA metastatic samples predicted with type 2 immune cell infiltration (red) or no infiltration (blue). Data show statistical significance was determined by Student’s t test. e, Kaplan-Meier overall survival curves of metastatic melanomas (n=367) plotted against the likelihood of high and low tumor CSF2 expression. Kaplan-Meier survival statistical analysis was performed using a Log-rank test. p-value is indicated.