Skint1, the prototype of a newly identified immunoglobulin superfamily gene cluster, positively selects epidermal gammadelta T cells

Abstract

B cells, alphabeta T cells and gammadelta T cells are conserved lymphocyte subtypes encoding their antigen receptors from somatically rearranged genes. alphabeta T cells undergo positive selection in the thymus by engagement of their T cell receptors (TCRs) with self-peptides presented by major histocompatibility complex molecules. The molecules that select gammadelta T cells are unknown. Vgamma5+Vdelta1+ cells comprise 90% of mouse epidermal gammadelta T cells. By mapping and genetic complementation using a strain showing loss of Vgamma5+Vdelta1+ cells due to a failure of thymic selection, we show that this defect is caused by mutation in Skint1, a newly identified gene expressed in thymus and skin that encodes a protein with immunoglobulin-like and transmembrane domains. Skint1 is the prototypic member of a rapidly evolving family of at least 11 genes in mouse, with greatest similarity to the butyrophilin genes. These findings define a new family of proteins mediating key epithelial-immune interactions.

Figure 1

Recessive transmission of Vγ5⁺Vδ1⁺ T cell deficiency in a B6 x FVB_Tac F₂ cross. Epidermal cells from mice were stained with antibodies to TCRγδ and the Vγ5Vδ1 idiotype and analyzed by flow cytometry. (a) Representative results from F₂ mice harboring high or low proportions of Vγ5⁺Vδ1⁺ T cells. Numbers in upper quadrants indicate the percentage of total epidermal cells within the quadrant; numbers in parentheses indicate percentage of γδ⁺ T cells that are Vγ5⁺Vδ1⁺. (b) Percentages of epidermal γδ T cells that are Vγ5⁺Vδ1⁺ in individual mice of the indicated groups. Brackets indicate values considered high and low for analysis of linkage

Figure 2

Linkage of Vγ5 Vδ1 T cell deficiency to chromosome 4. (a) Multipoint lod scores for linkage of Vγ5⁺Vδ1⁺ T cell deficiency across chromosome 4. 4cen, centromere of chromosome 4. The maximum lod score is 26.1, with a lod −3 interval of 3.3 Mb. (b) Haplotypes of mice with recombination in the interval between D4Mit146 and D4Mit12. The genetic (cM) and physical (Mb) map positions of markers are indicated. Mouse 551 had high (+) whereas mice 597 and 681 had low (−) Vg5⁺Vd1⁺ T cell levels. The B6 and FVB_Tac alleles are denoted 1 and 2, respectively. The genotypes showing co- segregation with the Vγ5⁺Vδ1⁺ level of each mouse are shown in gray boxes, and the minimum linked interval is indicated by a bracket. (c) Genes within the chromosome 4 linkage interval in B6. The 3.3-Mb interval between locus rs13477910 and nucleotide 113807721 is shown as a solid line, and the transcriptional orientations of genes are indicated by arrows. Skint gene depictions (red) are based on structural characterization described herein; prior aliases for predicted genes are shown in Supplementary Figure 1. The region absent in some strains is indicated with brackets.

Figure 3

Skint1 gene and protein structure. (a) Amplification of the full-length coding region of Skint1 from keratinocyte cDNA. The DNA sequence reveals a consensus Kozak sequence for translation initiation and a continuous open reading frame of 364 amino acids (Supplementary Fig. 2). (b) Deduced amino acid sequence of Skint1. The locations of the signal sequence, IgV, IgC and transmembrane domains (TMDs) are indicated. (c) Hydropathy plot from the TMHMM program predicts three TMDs with high probability, yielding an extracellular N terminus and cytoplasmic C terminus. (d) Alignment of cDNA with genomic DNA reveals that Skint1 is encoded in modular exons. Exon colors correspond to those of the encoded domains shown in b. The codon position of the last base of each exon is indicated. (e) Tissue distribution of Skint1 expression. Products of RT-PCR with Skint1-specific primers visualized on an agarose ge (Skint1); Actb amplification is shown as a control (b-Actin). Thymic epithelium and Vγ5⁺ T cell RNAs were obtained at E15–16; RNAs from all other tissues were obtained at adulthood. Skint1 expression is detected in whole thymus and thymic epithelium and in whole skin and keratinocyte.

Figure 4

Premature termination codon in Skint1 in FVB_Tac. (a) Genomic DNA sequence of Skint1 codons 322–326 from B6, FVB_Jax and FVB_Tac mice reveals a premature termination mutation in FVB_Tac (asterisk). (b) Schematic representation of the inferred protein structure of wild-type Skint1. Location of the FVB_Tac E324X mutation is indicated by ‘X’ and an arrow. The structure is inferred from domain predictions for Skint1 and its paralogs.

Figure 5

Rescue of Vγ5⁺Vδ1⁺ T cell deficiency by a wild-type Skint1 transgene. (a) Structure of the linearized Skint1 transgene. (b) E17 thymocytes prepared from individual transgene-injected mice were stained with antibodies to TCRγδ, the Vγ5Vδ1 idiotype and CD45RB and were analyzed by flow cytometry. Representative results from a transgene-negative embryo (left) and a transgenepositive, rescued embryo (right) are shown. In the unrescued embryo, 0.5% of Vγ5⁺Vδ1⁺ T cells are mature (that is, express high levels of CD45RB); in the rescued embryo, 84% of Vγ5⁺Vδ1⁺ T cells are mature. (c) The percentage of mature Vγ5⁺Vδ1⁺ T cells from mice of indicated groups. ‘Jax’ and ‘Tac’ represent the FVBJax and FVBTac lines; ‘tg(−)’ and ‘tg(+)’ represent transgene-negative and transgene-positive injected embryos. Phenotypic rescue is seen only among transgene positive embryos. (d) Expression of the Skint1 transgene in thymus of unrescued and rescued transgene-positive embryos shown in log scale. Rescued mice have high Skint1 transgene expression.

Figure 6

Skint1 haplotypes in diverse mouse strains. (a) Variants at 40 Skint1 codons, resulting in 35 missense substitutions, 4 silent substitutions and 1 premature termination (found only in FVBTac), are listed next to a schematic of the protein structure, with domains colored as in Figure 3, showing the domain location of the substitutions. The sequences present in B6, FVBJax and FVBTac, as well as 16 other laboratory strains and 5 strains derived from wild mice, are indicated with numbers corresponding to the codons listed at the left. Blue shading within the table highlights B6-like sequence (the reference sequence); Skint1 variation in other strains is shaded yellow (FVB-like), orange (found in I and NON but not FVB), lavender (found only in MOLF), green (found only in CAST) and pink (found only in SPRET). The stop mutation unique to FVBTac is shaded red. Four Skint1 haplotypes are observed among the 16 laboratory strains studied. (b) Presence (+) or absence (−) of an ~540 kb segment of mouse chromosome 4 containing Skint3, Skint4 and Skint9. The segment is present in B6 and 8 other distantly related mouse strains, but absent in FVB and 13 other strains. (c) Skint1 protein schematic, drawn to scale, indicating where missense substitutions occur relative to the protein structure; bar color denotes the strains harboring the substitutions, as shown in a. Missense substitutions are concentrated within the first two TMDs and the intervening loop.