Fig. 4: scRNA-seq reveals transcriptional regulation and mechanisms involved in NK cell subsets’ response to IL-35.

a–c UMAP of human purified NK cells cultured in IL-2 with or without IL-35 for 2 or 4 days from two different healthy donors. Cells are colored according to a the unsupervised clustering, b the proliferation phase, and c the treatment received. d, e Pathway signatures enriched (Fisher’s test adjusted P-value < 0.01) in genes up-regulated in absence or presence of IL-35 in ConvNK, AdaptNK and ProlifNK. f, g Venn diagram of significantly f downregulated or g upregulated genes in IL-2 + IL-35-exposed cells compared to IL-2-exposed control cells, by comparing ConvNK, AdaptNK, and ProlifNK being respectively colored in light blue, dark blue and pink. h Representative raw histograms of GP130, IL-12Rβ2, IL-27Rα in NKG2C⁺ versus NKG2C^- NK cells that were cultured as indicated for 24 h (left). Cumulative histograms of % positive NK cells for each marker (right). Mean values ± S.D are shown (n = 3 individual donors). i, j Supernatants from sorted NKG2C⁺ and NKG2C^- NK cells cultured for 24 h in the presence of IL-12 and IL-18 with or without IL-35 were collected to quantify i IFN-γ and  j VEGF-A release by ECLIA assay. Results are expressed as pg/ml. Mean values ± S.D are shown (n = 3 individual donors). k UMAP of AdaptNK (top), ConvNK (middle), and ProlifNK (bottom). Cells are colored according to the unsupervised clustering. l Frequency of IL-2-treated or IL-2 + IL-35-treated cells in each Seurat cluster among AdaptNK (top), ConvNK (middle) and ProlifNK (bottom). m Violin plots showing the distribution across Seurat clusters of the ILC1-like signature score among AdaptNK (top), ConvNK (middle) and ProlifNK (bottom). n Venn diagram of the top50 most highly expressed genes among genes specifically expressed in the identified ILC1-like cluster versus other clusters from AdaptNK (dark blue) and ConvNK (light blue). Source data are provided as a Source Data file.