Skint-1 is a highly specific, unique selecting component for epidermal T cells

Abstract

αβ T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1(+) dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1(+) dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.

Fig. 1.

Ubiquitous Tg.NF-Skint1 expression restores Vγ5Vδ1⁺ DETC development. (A) Flow cytometry of thymocyte suspensions prepared from FVB.Jax, FVB.Tac, and FVB.Tac Tg.NF-Skint1^−/+ line 39 fetuses at E17. Data are gated to show only Vγ5Vδ1⁺ (17D1⁺) thymocytes. Similar results were observed in all Tg.NF-Skint1 founder lines. (B) Flow cytometry of epidermal suspensions prepared from body wall skin of 3-wk-old mice. The bottom panels show γδTCR⁺ cells only. (C) Staining of DETC in FVB.Jax and Tg.NF-Skint1 epidermal sheets. (Scale bar: 20 μm.) (D and E) Quantitative RT-PCR analysis of Skint-1 expression in pooled E17 thymi and in skin from individual animals from Tg.NF-Skint1 (D) and untagged Skint1 (E) Tg lines, calculated relative to GAPDH. (F and G) Flow cytometry analysis of Skint1 Tg animals versus B6xSJL NLCs, performed as in A and B.

Fig. 2.

Tg NF-Skint1 does not regulate γδTCR repertoires outside the skin. (A) Western blot of Tg.NF-Skint1 protein in tissues (T, thymus; S, spleen; G, gut) from founder line 39. Protein was immunoprecipitated from Tg.NF-Skint1⁻ and Tg.NF-Skint1⁺ tissue lysates with mouse anti-FLAG M2 antibody, and immunoblotted with rabbit anti-FLAG antibody. Putative monomer (◀) and dimer species (◁) are observed in all Tg tissues. (B–E) Flow cytometry of cell suspensions prepared from the thymus, spleen, gut, and uterus of Tg⁻ and Tg⁺ animals of Tg.NF-Skint1 line 39. In B and C, all cells shown are gated on γδTCR⁺ and numbers reflect the percentage of γδ T cells, whereas CD3⁺ cell and total live cells are shown in D and E, respectively. (D) 17D1 binds to γδTCR⁺ cells that are Vγ5⁻; this corresponds to a demonstrated GL3-dependent cross-reactivity with the Vγ6Vδ1⁺ TCR used by the uterine IEL compartment (33).

Fig. 3.

Skint-1 is expressed in thymic medullary epithelium in a TCR-independent fashion and effects function from a stromal cell context. (A) Quantitative RT-PCR analysis of Skint-1 expression in CD45⁻ thymic stroma (Left and Center) or FACS-purified TEC (Right). CD45⁻EpCam⁺ TEC from pooled FVB.Jax E15-E18 thymi were divided into Ly51⁺ cTEC and Ly51⁻ mTEC subsets, and compared with total E17 thymus. Skint-1 expression is relative to GAPDH. (B) Quantitative RT-PCR analysis of Skint-2 expression as in A. (C) In situ hybridization analysis of Skint-1 expression in adult FVB.Jax thymus. Right: Close-up of medullary area indicated (Left). (D) Analysis of Vγ5Vδ1⁺ DETC precursor maturation (17D1⁺CD45RB^hi) in mixed RTOC of purified CD45⁺ thymocyte progenitors and CD45⁻ stroma from FVB.Jax, FVB.Tac, or Tg.NF-Skint1 E15 thymi.

Fig. 4.

Biological and biochemical properties of Skint1. (A) Graphic representation of the Skint-1 constructs tested. (B) Surface flow cytometry of 293 cells transfected with the constructs in bicistronic vectors. All constructs include a FLAG tag at the N terminus and the KYVERTELL motif in the IgV domain; hCD2 is the marker of transduction efficiency. (C) Analysis of Vγ5Vδ1⁺ DETC precursor maturation in FVB.Tac RTOC 12 d after transduction with the constructs from A. (D) The multimerization of WT and Tac alleles is analyzed by Western blot analysis of 293 transfectants. Protein was immunoprecipitated with mouse anti-FLAG M2 or mouse anti-HA antibody, and immunoblotted with rabbit anti-FLAG antibody.

Fig. 5.

Proteolytic processing specified by IgC is required for function. (A and B) Western blot analysis of 293 cells transfected with N-terminal FLAG-tagged Skint-1/Skint-2 chimeric constructs. Protein was immunoprecipitated with mouse anti-FLAG M2 and immunoblotted with rabbit anti-FLAG antibody. In A, the recombination between Skint1 and Skint2 was shifted in 10-aa increments in the IgC, whereas in B, only the first 10 aa of the Skint1 IgC was replaced with the analogous sequence from Skint2. (C) Analysis of Vγ5Vδ1⁺ DETC precursor maturation in FVB.Tac RTOC 12 d after transduction with the indicated chimeric constructs.