Fig 2: Human cutaneous Vδ1+ γδ T cells were maintained up to 14 days in xSCC.(A) Expression of indicated chemokine receptors by in vitro expanded, live gated CD3+ Vδ1+ γδ T cells. Blood-derived αβ T cells were used as staining control. (B) As (A), graphical summary of the percentage of Vδ1+ γδ T cell expressing the chemokine receptors (n = 5 skin donors). Error bars represent mean ± SD. (C) 10 × 106 human skin–derived T cells, containing approximately 7% of Vδ1+ γδ T cells, were injected intravenously (i.v.) into NSG mice carrying a xSCC of a volume ranging from 100 to 200 mm3 [reached approximately 60 to 80 days (d) post–i.d. injection of SCC-13 cells]. Each mouse was injected intraperitoneally (i.p.) with recombinant IL-2 and IL-15 daily until the harvest day. Figure created in BioRender. I. Gratz (2026) https://BioRender.com/zmnd28b. (D) Representative plot of the percentage of Vδ1+ γδ T cells engrafted in the spleen, blood, xSCC and murine skin 2, 7, or 14 days posttransfer. (E to G) Bar graphs show the absolute numbers of ingoing Vδ1+ γδ T cells, Vδ1+ γδ T cells engrafting spleen and blood normalized to mouse weight (grams), and xSCC and murine skin normalized to tissue weight (grams). (E) n = 7 mice per group; pool of two independent experiments. (F) n = 12 mice per group; pool of four independent experiments; (G) n = 12 mice per group; pool of two independent experiments. Each symbol represents one skin donor. Error bars represent mean ± SD. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons test. All data points, including extreme values, are shown. (H) Representative immunofluorescent staining of colocalized TCRδ/DAPI in HD skin and xSCC 7 days after γδ transfer. Scale bars, 100 μm. Staining controls are shown in fig. S4.

Fig 3: xSCC produced IL-1α and IL-18, which enhanced anti-CD3 induced-Vδ1+ γδ T cell proliferation in vitro.(A) Levels of cytokines (pg/mg tissue) produced by HD skin, huSCC, ES, and xSCC. Heatmap bars represent the mean of n = 5 HD skin and huSCC donors, and mean n = 5 of xenograft mice. (B) Representative gating strategy and bar graphs of the human skin–derived and ex vivo expanded Vδ1+ γδ T cells expressing IL-1RAcP and IL-18Rα. Peripheral blood αβ T cells and ex vivo expanded skin-derived αβ T cells were used as staining controls. Mean of n = 5 skin donors. (C) eFluor450-labeled γδ T cells were cultured under basal conditions [unstimulated or with IL-2 (100 IU/ml) and IL-15 (20 ng/ml)] or stimulated with anti-CD3 (1 μg/ml) and/or IL-1α and IL-18 (9 ng/ml) for 6 days. Proliferation was assessed by the median fluorescence intensity (MFI) of eFluor450 in Vδ1+ by flow cytometry. The representative histograms show the eFluor450 dilution in Vδ1+ T cells in the different conditions. Cell counts were normalized to unit area. (D) Bar graphs show the fold change of efluor450 MFI of Vδ1+ treated with anti-CD3, IL-1α, and IL-18 relative to IL-2 and IL-15. Mean of n = 6 skin donors. Statistical analysis was performed using a Friedman test followed by Dunn’s multiple comparisons test. Data in bar graphs (B) and (D) are shown as mean ± SD.