Fig 2: T cell epitope determination following stimulation with artificial APCs pulsed with overlapping peptides.(A–C) Identification of a new epitope B*18:01/MART125-33 in polyclonally expanded M87 TILs. (A) When stimulated with B*18:01-artificial APCs pulsed with MART1-derived overlapping peptides, the M87 TILs showed positive responses to two adjacent peptides with the shared sequence 21YTTAEEAAGIGILTV35. See also Supplementary file 2a. (B) M87 TILs were stained with the indicated multimers. B*18:01/unexchanged and HIV gag161-170 multimers were employed as controls. (C) IFN-γ production by M87 TILs in a B*18:01/MART125-33-specific manner. B*18:01-artificial APCs pulsed with MART125-33 or HIV gag161-170 control peptide were employed as stimulator cells. (D–F) Identification of a new epitope, B*40:01/NY-ESO-1125-133, recognized by polyclonally expanded M31 TILs. CD8+ T cells isolated from M31 TILs were stimulated with B*40:01-artificial APCs pulsed with 10 μg/ml NY-ESO-1-derived overlapping peptides and cultured with 10 IU/ml IL-2, 10 ng/ml IL-15, and 30 ng/ml IL-21 for 14 days. (D) Following peptide-specific stimulation, the M31 TILs showed positive responses to two adjacent peptides with the shared sequence 121VLLKEFTVSGNILTI135. See also Supplementary file 2b. (E) Data from B*40:01/NY-ESO-1125-133 or control B*40:01/HIV nef92-100 multimer staining before stimulation (day 0) and 14 days after stimulation (day 14) are shown. (F) IFN-γ production by M31 TILs in a B*40:01/NY-ESO-1125-133-specific manner following peptide-specific stimulation. CD8+ T cells stimulated with B*40:01-artificial APCs pulsed with the NY-ESO-1125-133 peptide were employed as responder cells. B*40:01-artificial APCs pulsed with the indicated peptide were used as stimulator cells. The HIV nef92-100 and NY-ESO-1125-132 peptides were employed as controls. The percentage of multimer+ cells in CD8+ T cells is shown in (B) and (E). In (A), (C), and (F), the data shown represent the mean ± SD of experiments performed in triplicate. All the results are representative of at least two independent experiments. *p<0.05, ***p<0.001 (two-tailed Welch’s t tests).

Fig 3: Human AML tumours express proximal tubule markers. a Representative immunofluorescence co-stainings for the melanocytic/AML marker HMB45/MART-1 and the proximal tubule epithelial markers NAPI2A, AQP1 and CD133 and distal tubule epithelial marker Tamm Horsfall glycoprotein (THP) in human renal AMLs (top row) and normal kidney (bottom row). Scale bars: 50 μm. b Relative mRNA abundance (renal AML vs. matched normal kidney) of three selected transcription factors (TAL1, HNF4A and TFEC) and three proximal tubule markers (FMO1, CUBN and VIL1) in seven human AML cases. The graph shows the values for each patient and the whisker plot depicts mean ± s.d. * denotes P < 0.05 and **P < 0.01 (Student’s t test) statistically significant different expression between AML and normal

Fig 4: An ex vivo genetically engineered model of renal AML. a Proliferation assay, western blot and soft-agar assay of Cdkn2a∆/∆ primary kidney cells following infection with lentiviruses expressing an empty shRNA (shRNA-Ctrl) or shRNAs against Tsc1 (shRNA-Tsc1) or Tsc2 (shRNA-Tsc2). b Proliferation assay, western blot and soft-agar assay of primary kidney cells infected with lentiviruses expressing shRNAs against Cdkn2a (shRNA-Cdkn2a) or Tsc2 and Cdkn2a (shRNA-Tsc2 + Cdkn2a). All graphs depict mean ± s.d. Student’s t test, n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. c Phase contrast images of spheres formed in suspension culture conditions from shRNA-Tsc2/Cdkn2a∆/∆ and shRNA-Tsc2 + Cdkn2a primary kidney cells. Scale bars: 50 μm. d Representative allografttumours derived from subcutaneous injections of respective sphere cells into SCID-beige mice.Scale bars: 1 cm. e Representative histology of allograft renal AML-like tumours. Characteristic histological features of AML are indicated by letters, where A shows adipocyte, S shows spindle cells and V shows an aberrant blood vessel. Epithelial structures are indicated by Ep. Scale bars: 50 μm. f Positive immunofluorescence or immunohistochemical staining in engineered renal AML-like allograft tumours for PMEL(HMB45), HMB45/MART-1, CATHEPSIN K, SMA, VIMENTIN, PDGFRβ, CD31 and S100. Scale bars: 50 μm

Fig 5: Differential gene expression highlight basal stem cell heterogeneity.a Heatmap showing top 300 differentially expressed genes per cluster. Dotted lines outline differentially expressed genes. b Violin plots of relative expression of marker genes split by cell cohorts and color-coded by cell community as in A. c Library 3 violin plots of relative expression of GJB2 and RHCG. d Feature plots showing expression of select basal and granular cell marker genes. e Immunostaining of KRT14 (red), KRT10 (green), and DAPI (blue) in human neonatal skin. Scale bar 100 µm. n = 3. Immunostaining of differentially expressed proteins (red) from the (f) BAS-III cluster (ASS1 [n = 6] and COL17A1 [n = 4]), (g) BAS-III/IV clusters (KRT19 [n = 3] and GJB2 [n = 4]), (h) BAS-I cluster (PTTG1 [n = 6] and CDC20 [n = 6]), (i) BAS-II cluster (RRM2 [n = 6] and PCLAF [n = 2]), (j) GRN cluster (SPINK5 [n = 3] and CALML5 [n = 2]), and (k) the MEL (MLANA [n = 2]) and LAN (CD74 [n = 2]) clusters in human neonatal skin. COL7A1 (green) and DAPI (blue) are costained. Dotted lines denote position of basement membrane where COL7A1 staining is absent. Scale bar 100 µm. l Quantification of ASS1 (n = 14 ridges) and KRT19 (n = 10 ridges) staining intensity at the top and bottom of Rete ridges. ***p < 0.0001. m Quantification of PTTG1 (n = 102 cells) and RRM2 (n = 104 cells) distance from the basement membrane, with KRT14 + basal cell (n = 167 cells) or KRT10+ suprabasal cell (n = 142 cells) distance shown as controls. Black stars represent significance compared with basal cell position whereas green stars represent significance compared with suprabasal position. ***p < 0.0001. Box represents 25th to 75th percentiles. Whiskers represent minimum and maximum data points. Bar represents mean. Significance was determined by unpaired two-tailed t test.