A novel gene essential for the development of single positive thymocytes

Abstract

A critical step during intrathymic T-cell development is the transition of CD4(+) CD8(+) double-positive (DP) cells to the major histocompatibility complex class I (MHC-I)-restricted CD4(-) CD8(+) and MHC-II-restricted CD4(+) CD8(-) single-positive (SP) cell stage. Here, we identify a novel gene that is essential for this process. Through the T-cell phenotype-based screening of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we established a mouse line in which numbers of CD4 and CD8 SP thymocytes as well as peripheral CD4 and CD8 T cells were dramatically reduced. Using linkage analysis and DNA sequencing, we identified a missense point mutation in a gene, E430004N04Rik (also known as themis), that does not belong to any known gene family. This orphan gene is expressed specifically in DP and SP thymocytes and peripheral T cells, whereas in mutant thymocytes the levels of protein encoded by this gene were drastically reduced. We generated E430004N04Rik-deficient mice, and their phenotype was virtually identical to that of the ENU mutant mice, thereby confirming that this gene is essential for the development of SP thymocytes.

FIG. 1.

Peripheral T cells are reduced in ENU mutant mice. (A) CD4 versus CD8 profiles of peripheral blood mononuclear cells (PBMC) from a representative 12-week-old ENU-induced mutant and wild-type (WT) mouse. (B) Percentages of total CD3⁺ cells, CD4⁺ cells, and CD8⁺ cells in PBMC of wild-type (n = 23), heterozygous (n = 17), and mutant mice (n = 5) are indicated. The data from mice originally were tentatively grouped into normal, heterozygous (hetero), and homozygous groups according to phenotype, and in this figure the data have been regrouped based on themis genotyping. The asterisk indicates a P of <0.001 (t test).

FIG. 2.

Developmental arrest of SP thymocytes in ENU mutant mice. (A and B) Thymocytes from 8-week-old ENU mice and wild-type (WT) C57BL/6 mice were analyzed for the expression of CD4 and CD8. Representative flow-cytometric profile (A) and proportions of CD4/CD8 subsets (means ± standard errors of the means) (B) are shown. (C) The expression of TCRβ, CD5, CD24, and CD69 in double-negative (DN), DP, CD4SP, and CD8SP thymocyte fractions was analyzed for wild-type (blue) and mutant mice (red). (D) Histological analysis of thymi from 8-week-old ENU and B6 mice. Sections were stained with the medullary marker ER-TR5 (red) and DAPI (blue). (E) Representative flow-cytometric profiles of regulatory T-cell and NKT cells in thymi of 8-week-old mice. (F) The development of cells in major lineages other than T lineage in bone marrow was not affected in mutant mice. Bone marrow cells were analyzed with the indicated surface markers. In the bottom panels, profiles of cells gated on the lineage marker-negative (Lin⁻) fraction of bone marrow cells are shown.

FIG. 3.

Defect in thymocytes but not in thymic stromal cells is responsible for the phenotype of the SPOTR mice. (A) CD4 versus CD8 profiles of thymocytes from bone marrow chimeric mice. Lethally irradiated SPOTR mice (left) or B6Ly5.1 congenic mice (right) were reconstituted with 1 × 10⁶ bone marrow cells from B6Ly5.1 or SPOTR mice, respectively. Thymocytes were harvested 2 months after reconstitution. (B) Proliferative response of mutant and wild-type (WT) T cells. Sorted splenic T cells from mutant and wild-type mice were labeled with CFSE and stimulated with anti-CD3 and anti-CD28 monoclonal antibody. Cells were analyzed by flow cytometry 72 h after stimulation.

FIG. 4.

Genetic mapping of the SPOTR mutation by SNP analysis. (A) Half of the genetic mapping results, rearranged in accordance with CD3⁺ cell proportions in peripheral blood, are shown. With SNP analysis between the C57BL/6J and C3H/HeJ strains, a compatible homozygous C57BL/6J SNP pair with a phenotype for low levels of CD3⁺ cells were found at the D10SNP2 marker on chromosome (Chr.) 10, and the SNP genotype was changed to heterozygote or C3H/HeJ homozygote in accordance with the increase in CD3⁺ cell numbers. (B) Phenotype and SNP mapping are shown in detail.

FIG. 5.

Identification of a missense mutation in the SPOTR mouse genome. (A) A total of 254 mice were examined for genetic mapping, and the responsible loci were mapped to the 0.39 centimorgan (cM) region, including D10SNP2 (shown as a red arrow on the magnified chromosome bar). Calculating that 1 cM ≈ 2Mbp, a 0.78-Mbp region was predicted to contain the candidate loci (yellow on chromosome [Chr.] 10). Three genes in this region, ptprk, E430004N04Rik, and 2310057J18Rik, are indicated, with the direction of transcription indicated by the pointed end of the rectangular bars. Pink arrows indicate the approximate analogous region deleted in the LEC rat genome. (B) A missense mutation was found in exon 4 of the E430004N04Rik gene, A1799C, leading to the amino acid substitution T512P. The coding regions of the E430004N04Rik gene are indicated in light green, and the untranslated regions are indicated in black. (C) RT-PCR analysis of ptprκ and themis expression in total RNA prepared from sorted DP thymocytes. The amplification of β-actin cDNA is shown as a control. (D) Western blot analysis of Themis protein in DP thymocytes of wild-type (WT) and mutant mice. Erk1/2 expression is shown as a loading control. (E) Cytochemical analysis for the location of Themis protein. FLAG-tagged themis cDNA was introduced into 293T cells and stained with anti-FLAG monoclonal antibody (red) together with DAPI (blue).

FIG. 6.

themis protein orthologs in various Placentalia species. ClustalW protein alignment of themis protein orthologs between mouse, rat (91% identical to mouse), human (80%), chimpanzee (80%), horse (81%), and cow (75%) are shown. Amino acids conserved among all of these proteins are highlighted in dark blue. The location of the point mutation in the SPOTR mouse is indicated by a red rectangle.

FIG. 7.

themis-deficient mice showed a phenotype similar to that of SPOTR mice. (A) themis gene targeting strategy. The coding region of first exon was replaced by GFP and a neomycin (neo) cassette. The open box of exon 1 indicates the 5′ untranslated region. (B) Deletion was screened by PCR using primers shown in panel A. (C) The expression of themis was analyzed by using a GFP reporter mouse. GFP expression profiles in cells of CD4/CD8 thymic fractions from a 6-week old heterozygous mouse are shown. GFP expression profiles in CD3⁺ cells and B220⁺ cells from spleen also are shown. (D) CD4 versus CD8 profiles of thymocytes and peripheral blood mononuclear cells (PBMC) from a 4-week-old themis-deficient mouse (themis^gfp/gfp), a littermate (themis^+/gfp), and a wild-type mouse of the same age (themis^+/+) are shown. (E) Western blot analysis of Themis protein in thymocytes from wild-type, heterozygous, and themis-deficient mice.