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Contiguous Gene Deletion of Ptprk and Themis Causes T-Helper Immunodefi- Ciency (Thid) in the LEC Rat

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Contiguous Gene Deletion of Ptprk and Themis Causes T-Helper Immunodefi- Ciency (Thid) in the LEC Rat Biomedical Research 31 (1) 83-87, 2010 Contiguous gene deletion of Ptprk and Themis causes T-helper immunodefi- ciency (thid) in the LEC rat Ryohei IWATA, Nobuya SASAKI and Takashi AGUI Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hok- kaido University, Kita 18, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan (Received 6 January 2010; and accepted 15 January 2010) ABSTRACT The LEC rat has a spontaneous mutation of T-helper immunodeficiency (thid), which causes a marked defect in T cell maturation from double positive (DP) to CD4 single positive (SP) cells in the thymus. Previously, we identified the contiguous gene deletion of Ptprk and Themis genes in the thid locus that causes the simultaneous loss of expression of both genes, and the exogenous Ptprk expression partially rescued this phenotype. To determine whether the deletion of Themis influences thid phenotype, bone marrow (BM) cells were transduced with lentiviral vector express- ing Themis gene, and were transplanted into X-ray-irradiated LEC rats. Interestingly, the exoge- nous Themis expression rescued the development of CD4 SP cells as well as Ptprk. The result suggests that the deficiency of both genes is responsible for the thid mutation, and that both genes are indispensable for the development of SP cells from DP cells in the thymus. Differentiation of T cells from bone marrow (BM)- by neglect), whereas strong interaction of TCR and derived progenitor T cells in the thymus is depen- self peptide-MHC complex also induces apoptosis dent upon the complex interactions of signaling (negative selection) (6). pathways (15). Most immature T cells in the thymus The LEC rat displays an immunodeficiency, express neither T cell antigen receptor (TCR) nor known as T-helper immunodeficiency (thid). The the co-receptors of CD4 and CD8 (double-negative thid mutation causes a marked defect in T cell mat- or DN). DN cells that successfully rearrange and uration from DP to CD4 SP, but not to CD8 SP express a TCR β-chain, differentiate into CD4 and cells in the thymus, and in addition, the thid is a CD8 double-positive (DP) cells. The DP cells en- cell-autonomous mutation in T cells (1, 2, 18). Pre- gaged in appropriate interactions with self peptides viously, we have identified genomic deletion of ap- in the context of the major histocompatibility com- proximately 380 kb, that causes the deletion that plex (MHC) molecules, through their TCR and ma- encompasses the almost cording exons of Ptprk ture into CD4 or CD8 single-positive (SP) cells gene, and promoter region and the first exon of The- (positive selection), and this CD4 or CD8 linage mis (rat gene symbol: RGD1560849) gene in the commitment is dependent on both the strength and thid locus (Fig. 1A). The large deletion results in duration of TCR signaling (16). Cells that lack these loss of mRNA expression of both genes in thymus positive selection signals undergo apoptosis (death and BM (Fig. 1B). Furthermore, we have shown that the exogenous Ptprk re-expression partially rescues the thid phenotype (3). Recently, it has been report- Address correspondence to: Dr. Nobuya Sasaki ed that several mutations of Themis in mice causes Laboratory of Laboratory Animal Science and Medi- cine, Department of Disease Control, Graduate School the abnormality of T cell maturation from DP to of Veterinary Medicine, Hokkaido University, Kita-18, CD4 SP, which is quite similar to the thid pheno- Nishi-9, Kita-ku, Sapporo 060-0818, Japan type (5, 8–10, 12). Thus, we hypothesized that the Tel/Fax: +81-11-706-5107 deficiency of both Ptprk and Themis genes are in- E-mail: [email protected] (Nobuya Sasaki) volved in the thid mutation of LEC rat. 84 R. Iwata et al. Fig. 1 Delineation of deleted region and candidate genes in thid locus of LEC rat. (A) Genomic structure around the thid locus on rat chromosome (Chr. 1). The arrows indicate direction of transcription of both genes and exons are represented by blocks connected by horizontal lines representing introns. The exons of Ptprk and Themis are indicated in black boxes and open boxes, respectively. The large deletion causes the loss of exon 4–20 in Ptprk and promoter region and exon 1 in Themis, respectively. (B) Gene expression of both genes in the thymus (Th) and bone marrow (BM) from control BN rat and mutant LEC rat. Actb is the internal standard. In the present study, we investigated whether X-ray machine (Hitachi) was operated at 200 kV, Themis gene transfer would rescue thid phenotype. 20 mA, with a 2-mm-alminum filter, dose rate Furthermore, we compared the effects of Ptprk and 3.03 Gy/min in air, and focus distance of 600 mm. Themis, in a BM reconstitution experiment, in which BM cell suspension was isolated from LEC rats by LEC rat-derived BM cells were transduced with flushing the femurs with PBS. Single cell suspen- the Themis gene using a lentivirus vector, and sions were prepared by passing the cells through a were transplanted into X-irradiated recipient LEC cell strainer and viral transduction was performed rats. The expression vector was constructed as by incubating the BM cells in DMEM (Invitrogen) follows; the rat Ptprk cDNA (accession number, at 37°C. After 24 h, cells were washed with PBS; AB297790) and mouse Themis cDNA (A630021B05) lentivirus-infected BM cells (1 × 106 cells) were were inserted into multicloning site of FUGW lenti- transplanted into recipient LEC rats through tail virus vector (kind gift from Dr. David Baltimore, veins. After 4–5 weeks, recipient rats were sacri- California Institute of Technology). Recombinant ficed and the expression of these genes in the thy- lentiviruses were produced in human 293 FT cells mus and spleen was examined by RT-PCR. Fig. 2A (Invitrogen) by co-transfection with each vector shows that the reconstitution with BM cells trans- encoding viral accessory protein, pMD2.G and duced exogenous genes in recipient rats was suc- psPAX2 by Lipofectamine 2000 (Invitrogen) (11). cessful and well sustained. Thymocyte subsets were To generate BM-chimeric rats, recipient female LEC then examined by flow cytometry (Fig. 2B). Thymo- rats (6–8 weeks old) were treated with 6 Gy X-irra- cytes (1 × 106 cells) were stained with fluorescein diation, which was lethal dose for LEC rats (7). The isothiocyanate (FITC)-conjugated anti-rat CD4 and Both Ptprk and Themis genes are responsible for the thid mutation 85 Ptprk gene (Fig. 2B). In addition, we attempted further rescue of CD4 SP cells by co-transduction of both Ptprk and Themis genes. However, CD4 SP populations of the two genes transduction of BM-chimeric rats were at a similar level to that ob- served with the single Themis transduction (data not shown). It is difficult for lentivirus system to simul- taneously express both genes effectively on a single T cell, due to the insufficient virus titer and gene expression for non-optimal developmental stage, or silencing of lentiviral gene expression (13). Our re- sults indicated that Themis expression induced the development of CD4 SP cells more than that of Ptprk. These findings suggest that the deficiency of both genes is responsible for the thid mutation, and that both genes are indispensable for the develop- ment of SP cells from DP cells in the thymus. The Ptprk gene encodes a protein tyrosine phos- phatase, receptor κ (RPTPK) protein. RPTPK is composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and cyto- plasmic phosphatase domain. The extracellular re- gion of RPTPK plays a critical role in homophilic binding, which leads to cell-cell adhesion mediated by RPTPK (14, 19), while the cytoplasmic domains can dephosphorylate some important substrates such as epidermal growth factor receptor and β-catenin, RPTPK is also known to act as a tumor suppressor that induces transforming growth factor-β signaling Fig. 2 Complement study of thid phenotype using trans- downstream (17). RPTPK may play a role in the genic rescue. The transplantation of BM cells transduced regulation of adhesion, proliferation, and migration with Ptprk gene or Themis gene to X-irradiated LEC rats. of T-precursor cells in the thymus. However, there (A) RT-PCR analysis of expression of Ptprk and Themis in were no difference detected in the expression of the thymus (Th) and Spleen (Sp) of LEC rat in 4–5 weeks after bone marrow transplantation. Ptprk (+) and Themis (+) indi- Ptprk gene among DN, DP and CD4/CD8 SP cells cate that the lentiviruses encoding the Ptprk and Themis (Fig. 3B). These data suggest that the regulation of were infected in BM cells. Note that the Ptprk and Themis RPTPK function or expression of a RPTPK sub- genes were successfully expressed in both thymus and strate, but not Ptprk expression itself, is involved in spleen. BN indicates the natural expression of both genes from BN rat as a positive control. Data are representative the selective loss of CD4 SP cells. Recently, it was RT-PCR results of at least two independent experiments for reported that RPTPK positively regulates ERK1/2 each group of rats. (B) Flow cytometry profiles of CD4/CD8 phosphorylation, which influences CD4 SP T cell staining of thymocytes expressing transgenes. Numbers in development (4). Thus, a further analysis that uses plots indicate percent cells in each quadrant. Data are rep- Ptprk knockout (KO) mice is required for the eluci- resentative of two or three independent experiments for each group of rats. Mean number of CD4 SP cells was as dation of the mechanism of thymocyte differentia- follows: BN = 9.7 ± 0.6, LEC = 0.8 ± 0.3, Ptprk (+) = 2.6 ± 0.8, tion.
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