Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease.

The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So far, there have been no reports to our knowledge of a human deficiency in a tyrosine-specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other. The point mutation resulted in the alteration of intervening sequence 13 donor splice site. The patient presented at 2 months of age with severe combined immunodeficiency disease. The population of peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte dysfunction.

Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling.

The threshold at which antigen triggers lymphocyte activation is set by the enzymes that regulate tyrosine phosphorylation. Upon T cell activation, the protein tyrosine phosphatase SHP-1 was found to bind to the protein tyrosine kinase ZAP-70. This interaction resulted in an increase in SHP-1 phosphatase activity and a decrease in ZAP-70 kinase activity. Expression of a dominant negative mutant of SHP-1 in T cells increased the sensitivity of the antigen receptor. Thus, SHP-1 functions as a negative regulator of the T cell antigen receptor and in setting the threshold of activation.

CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli.

CD45 is a high-molecular-weight transmembrane protein tyrosine phosphatase expressed only by nucleated cells of hematopoietic origin. To examine function, mouse CD8+ cytolytic T-cell clones were derived that had a specific defect in the expression of CD45. Northern (RNA) blot analysis indicates that the CD45 deficiency is due to either a transcriptional defect or mRNA instability. The CD45-deficient cells were greatly diminished in their ability to respond to antigen. All functional parameters of T-cell receptor signalling analyzed (cytolysis of targets, proliferation, and cytokine production) were markedly diminished. A CD45+ revertant was isolated, and the ability to respond to antigen was restored. These results support a central and immediate role for this transmembrane protein tyrosine phosphatase in T-cell receptor signalling.

Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation.

The leukocyte-common antigen (L-CA) is a family of large molecular weight glycoproteins uniquely expressed on the surface of all nucleated cells of hematopoietic origin. The glycoprotein consists of a heavily glycosylated exterior domain, a single membrane spanning region, and a large cytoplasmic domain that contains tyrosine phosphatase activity. To investigate the function of this family, we generated T cell clones that lacked L-CA (L-CA-). The expression of the alpha beta T cell receptor, CD3, CD4, IL-2 receptor (p55), LFA-1, Thy-1, and Pgp-1 (CD44) was normal. The L-CA- T cell clones failed to proliferate in response to antigen or cross-linked CD3; however, they could still proliferate in response to IL-2. An L-CA+ revertant was obtained and the ability to proliferate in response to antigen and cross-linked CD3 was restored. These data indicate that L-CA is required for T cells to enter into cell cycle in response to antigen.

Activation of CD45-deficient T cell clones by lectin mitogens but not anti-Thy-1.

CD45, the leukocyte-common antigen, is a transmembrane protein tyrosine phosphatase uniquely expressed by cells of hematopoietic origin. We have developed CD4+ and CD8+ T cell clones that are deficient in the expression of CD45 and have previously shown that these cells fail to proliferate in response to antigen or cross-linked CD3. These studies have now been extended to show that stimulation with anti-Thy-1, a mitogenic signal for the CD4+CD45+ and CD8+CD45+ T cells, fails to induce proliferation in the CD45- T cells. Examination of the CD8+CD45- T cells correlates anti-Thy-1 unresponsiveness with a failure to increase in tyrosine phosphorylation. Furthermore, stimulation of CD8+CD45+ T cells with anti-Thy-1 results in an increase in p56lck activity but not in CD8+CD45- T cells. In contrast to the results with anti-Thy-1, both the CD4+CD45- and CD8+CD45- T cells respond to treatment with lectin mitogens, concanavalin A or phytohemagglutinin. Lectin-induced proliferation was inhibited by the addition of cyclosporin A. Treatment of CD45- T cells with PMA and ionomycin also results in proliferation indicating that activation of protein kinase C in conjunction with an increase in intracellular calcium rescues the defect caused by CD45 deficiency. The data suggest that CD45 is required for the activation of tyrosine kinase activity immediate or prior to transmembrane signaling.

Sequence conservation in potential regulatory regions of the mouse and human leukocyte common antigen gene.

The leukocyte common antigen is a family of glycoproteins uniquely expressed by cells of hemopoietic origin. The family is generated by alternative splicing of 3 exons, and individual members are expressed in a cell type-specific fashion. The glycoprotein consists of a heavily glycosylated exterior domain of approximately 100 kDa, a single membrane spanning region, and a cytoplasmic domain that has homology to a protein tyrosine phosphatase of 83 kDa. To further understand the regulation and structure of this family of molecules, genomic clones were isolated from mouse lambda and cosmid libraries. The genomic organization was determined from 20 genomic clones. Eighteen of the clones clustered within a 75-kilobase region, whereas the promoter region, the exons for the 5'-untranslated region, and the leader domain are located an unknown distance further 5' within in an unlinked clone. The gene is composed of 34 exons: two 5'-untranslated exons, 1a and 1b, and 32 exons that encode protein sequence. Exon 2 contains part of the 5'-untranslated region and encodes the 23 amino acids of the leader sequence. Exons 3-15 encode amino acid residues 1-538 of the external domain, exon 16 encodes residues 539-574, encompassing the membrane-spanning domain, and exons 17-33 encode residues 575-1268 of the cytoplasmic domain. Exon 33, the largest exon, contains the 1.1-kilobase 3'-untranslated region. A comparison between mouse and human (Hall, L.R., Streuli, M., Schlossman, S.F., and Saito, H. (1988) J. Immunol. 141, 2781-2787) promoter regions reveals significant homologies 5' to the transcription start site. The relationship between exon structure and glycoprotein structure is discussed.

Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor.

Stimulation of tyrosine phosphorylation is an early and important event in antigen-induced T-cell activation. T-cell clones deficient in expression of CD45, a transmembrane protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), are impaired in their ability to respond to either antigen or T-cell receptor cross-linking. Analysis of the CD45-deficient CD8+ T-cell clone L3M-93 demonstrates that the Src family members p56lck and p59fyn show increased immunoreactivity with anti-phosphotyrosine antibody and exhibit decreased kinase activity. The site of increased tyrosine phosphorylation in Src family members was identified by comparison of cyanogen bromide peptide maps. Phosphorylation of the C-terminal phosphopeptide, containing the negative regulatory site of tyrosine phosphorylation, from the CD45-deficient cells was increased 8-fold for p56lck and 2-fold for p59fyn. These data suggest that CD45 dephosphorylates the negative regulatory site of multiple Src family members in the cytotoxic T-lymphocyte clone L3 and show a correlation between the ability to respond efficiently to antigen and the dephosphorylation of Src family members by CD45.

Cloning and expression of the cDNA for the murine interferon gamma receptor.

A murine interferon gamma (IFN-gamma) receptor cDNA was isolated by screening a murine T-cell hybridoma library prepared in lambda gt10 with probes prepared from a human IFN-gamma receptor cDNA. The 2.1-kilobase (kb) cDNA encoded a serine-rich polypeptide of 477 amino acids that was 52% identical to the human protein. Southern and Northern (RNA) blot analyses indicated the presence of a single receptor gene and a single predominant 2.3-kb receptor transcript. Human embryonic kidney fibroblasts, stably transfected with the murine IFN-gamma receptor cDNA, expressed murine IFN-gamma receptors as detected by flow cytometry with either ligand or a receptor-specific monoclonal antibody. Nontransfected cells bound neither ligand nor antibody. Radioligand-binding analysis demonstrated that the transfectants expressed 530,000 murine IFN-gamma receptors per cell and bound murine IFN-gamma with a Ka of 1 x 10(9) M-1. However, despite high-level expression of murine IFN-gamma receptors, the transfected human cells responded only to human and not to murine IFN-gamma as detected by enhancement of major histocompatibility class I antigen expression and induction of antiviral activity. These results thus document the isolation and expression of a full-length murine IFN-gamma receptor cDNA and suggest that additional species-specific components may be necessary to form a biologically active IFN-gamma receptor.

CD45-associated protein is not essential for the regulation of antigen receptor-mediated signal transduction.

CD45 is a transmembrane protein tyrosine phosphatase required for signaling through the T-and B-cell antigen receptors. In lymphocytes, CD45 interacts with CD45-associated protein (CD45AP), a 32 000 Mr phosphoprotein, through their respective transmembrane domains. To determine whether CD45AP affects the ability of CD45 to regulate antigen receptor signaling, CD45AP-deficient mice were generated. Thymocyte development was grossly normal. Moreover, the cellularity of the thymus and spleens were normal. CD45 expression on thymocytes and splenocytes, ascertained by flow cytometry, was comparable between CD45AP-deficient mice and littermate controls. In contrast to a previous report (Matsuda et al., J. Exp. Med. 1998 187: 1863 - 1870). CD45AP-deficient and normal thymocytes and splenocytes proliferated similarly in response to various mitogens or antigen receptor cross-linking. Furthermore, thymocyte CD45-associated p56(lck) kinase activity was similar between CD45AP-deficient and normal cells. We conclude that CD45AP is not essential for the regulation of Src-family kinase activity by CD45.