A novel mutation in PTPRC interferes with splicing and alters the structure of the human CD45 molecule

CD45, encoded by the protein tyrosine phosphatase receptor type C ( PTPRC) gene, is essentially involved in maturation, activation, and migration of immune cells. Lack of CD45 results in severe immunodeficiency, and alterations of the receptor may result in autoimmunity. Here, we describe a novel mutation in PTPRCas a cause of variant CD45 expression in humans. Several members of a multiple sclerosis multiplex family showed expression of CD45RA on memory T cells and monocytes. The variant expression pattern was linked to the PTPRCgene by DNA microsatellite studies. DNA analysis identified a novel point mutation in exon 4 (position 59 C-->A) in all family members with variant CD45 expression, but not in donors with normal CD45 expression. The mutation interferes with alternative splicing and alters amino acid sequence (H-->Q), interfering with antibody binding to the CD45RA domain. Overall, we describe the first mutation in PTPRCthat interferes with splicing and results in surface expression of a structurally altered CD45 molecule in humans.     

TLR3-involved modulation of pregnancy tolerance in double-stranded RNA-stimulated NOD/SCID mice

This study aims to extend understanding of the relationship between TLR3-involved cell signaling and dsRNA-induced embryo resorption. Upon stimulation of dsRNA, the resorption rate of embryos was boosted dramatically in syngeneic mating BALB/c mice, but not significantly influenced in syngeneic mating NOD/SCID mice. Accordingly, there was an enhanced cell surface expression of TLR3 on placental CD45(+) cells derived from BALB/c mice, concomitant with both increased percentages of CD45(+)CD80(+) cells and CD8alpha(+)CD80(+) cells in flow cytometric analysis. In addition, both increased IL-2 and decreased IL-10 expression could be observed in CD45(+) cell group in the intracellular detection by flow cytometry. In contrast, no such trends were observed in NOD/SCID model, and its resorption rate of embryos was kept at a low level throughout pregnancy. Neutralizing Abs against TLR3 could abrogate the embryo rejection induced by dsRNA in BALB/c mice, and simultaneously could reduce the CD80(+) percentage in the CD45(+) cell group. These results indicate that the interaction between dsRNA and TLR3 may be involved in the mobilization of CD45(+)CD80(+) and CD8alpha(+)CD80(+) cells, followed by the up-regulation of IL-2 and down-regulation of IL-10 expression at the feto-maternal interface, and finally resulting in embryo rejection. The relatively low responsiveness of NOD/SCID mice may be one of the reasons why these mice appeared to be resistant to dsRNA-induced embryo resorption.     

Combinations of CD45 isoforms are crucial for immune function and disease

Expression of the CD45 Ag in hemopoietic cells is essential for normal development and function of lymphocytes, and both mice and humans lacking expression exhibit SCID. Human genetic variants of CD45, the exon 4 C77G and exon 6 A138G alleles, which alter the pattern of CD45 isoform expression, are associated with autoimmune and infectious diseases. We constructed transgenic mice expressing either an altered level or combination of CD45 isoforms. We show that the total level of CD45 expressed is crucial for normal TCR signaling, lymphocyte proliferation, and cytokine production. Most importantly, transgenic lines with a normal level, but altered combinations of CD45 isoforms, CD45(RABC/+) and CD45(RO/+) mice, which mimic variant CD45 expression in C77G and A138G humans, show more rapid onset and increased severity of experimental autoimmune encephalomyelitis. CD45(RO/+) cells produce more TNF-alpha and IFN-gamma. Thus, for the first time, we have shown experimentally that it is the combination of CD45 isoforms that affects immune function and disease.     

New insights into the transmembrane protein tyrosine phosphatase CD45

CD45 is expressed on all nucleated haematopoietic cells and was originally identified as the first and prototypic transmembrane protein tyrosine phosphatase. In CD45 mutant cell lines, CD45-deficient mice and CD45-deficient human SCID patients, CD45 is required for signal transduction through antigen receptors. CD45 can operate as a positive as well as a negative regulator of Src-family kinases. Moreover, CD45 was identified as the elusive JAK tyrosine phosphatase that negatively regulates cytokine receptor activation involved in the differentiation, proliferation and antiviral immunity of haematopoietic cells. Modulation of CD45 splice variants provides a unique opportunity to design drugs that turn off or turn on antigen and cytokine receptor signaling in cancer, transplantation or autoimmunity     

CD45 variant alleles: possibly increased frequency of a novel exon 4 CD45 polymorphism in HIV seropositive Ugandans

The CD45 (leucocyte common) antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor signalling in lymphocytes, and expression of different CD45 isoforms is associated with distinct functions. Here we describe a novel polymorphism in exon 4 (A54G) of the gene encoding CD45 (PTPRC) that results in an amino acid substitution of Thr-19 to Ala in exon 4. The 54G allele was identified in African Ugandan populations and was found with a suggestive but not statistically significant increase in frequency amongst HIV-seropositive Ugandans. This suggests that the 54G variant and CD45 splicing abnormalities might be associated with HIV infection.     

Restoration of T cell receptor-mediated signal transduction by transfection of CD45 cDNA into a CD45-deficient variant of the Jurkat T cell line.

The TCR is a multimeric structure comprised of distinct Ag recognition and signal transduction components. Although none of the molecules that make up the TCR possess intrinsic protein tyrosine kinase (PTK) activity, stimulation of T cells via the TCR results in the rapid appearance of newly tyrosine phosphorylated proteins in cell lysates. Evidence suggests ligation of the TCR induces activation of a PTK that may be a member of the src family. One early consequence of this TCR-mediated PTK activation is the phosphorylation of the gamma 1 isoform of phospholipase C. This phosphorylation event is associated with increased enzymatic activity resulting in the hydrolysis of phosphatidylinositol 4,5 bisphosphate into two second messengers, inositol 1,4,5 trisphosphate and diacylglycerol. Recently, our laboratory and others have isolated mutant T cells that lack surface expression of CD45, the major surface tyrosine phosphatase expressed on lymphoid cells. Stimulation of the TCR on these cells fails to result in the expected activation events. We demonstrate that reconstitution of surface expression of the 180-kDa isoform of CD45 by gene transfer into a CD45-deficient mutant of the Jurkat T cell leukemic line restores the ability of the TCR to couple fully to its signal transduction machinery. These results support the role of CD45 tyrosine phosphatase activity in regulating the TCR-activated PTK.     

Human activation-induced cytidine deaminase is induced by IL-4 and negatively regulated by CD45: implication of CD45 as a Janus kinase phosphatase in antibody diversification

Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and V(H) gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.     

Human recent thymic emigrants--identification, expansion, and survival characteristics

This study shows that, in humans at birth, circulating T cells represent recent thymic emigrants (RTEs) as reflected in their high level of expression of TCR excision circles. RTEs express "thymocyte-like" characteristics with regard to rapid rate of apoptosis. In the presence of common gamma-chain cytokines, in particular IL-7, they show enhanced potential to survive, entry into cell cycle, and proliferation. Although common gamma-chain cytokines were also potent antiapoptotic stimuli for mature adult-derived naive CD4+CD45RA+ T cells, these cells were refractory to IL-7-induced expansion in vitro. RTEs cultured with IL-7 could not reinduce recombination-activating gene-2 gene expression in vitro. These data suggest that postthymic naive T cells in the periphery during early life are at a unique stage in ontogeny as RTEs, during which they can undergo homeostatic regulation including expansion and survival in an Ag-independent manner while maintaining their preselected TCR repertoire.     

E-Selectin expression in a murine model of chronic colitis

The objective of this study was to quantify E-selectin surface expression in the colon as well as other tissues in a CD4(+) T-cell model of chronic colitis in mice using the newly developed dual radiolabel monoclonal antibody technique. Male SCID mice were reconstituted with either 5 x 10(5) CD4(+) CD45RB(low) or CD45RB(high) T-cells isolated from normal CB-17 donor mouse spleens and subsequently monitored for clinical signs of colitis. We found that animals injected with CD45RB(high) but not CD45RB(low) T-cells nor PBS developed colitis at 6-8 weeks following reconstitution as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of E-selectin compared to SCID mice injected with PBS or reconstituted with CD45RB(low) T-cells, both of which did not develop colitis. We also observed significant increases in E-selectin expression in cecum, small intestine, mesentery, and liver of colitic mice. Our data confirm that reconstitution of SCID mice with CD45RB(high) but not CD45RB(low) T-cells induces chronic colitis and demonstrate that this chronic colitis is associated with enhanced expression of an endothelial cell-specific adhesion molecule. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RB(high) T-cells enhances E-selectin expression in a variety of tissues distant from the site of active inflammation.     

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.     

Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype

We studied the induction, severity and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6-12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12-16 wk posttransfer. SCID mice transplanted with STAT-4-/- CD4+ T cells showed a late-onset IBD manifest > 20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-gamma and TNF-alpha. In SCID mice transplanted with IL-12-unresponsive STAT-4-/- CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-gamma but abundant levels of TNF-alpha. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency.     

Partial characterization of the CD45 phosphatase cDNA in the owl monkey (Aotus vociferans)

CD45 is a protein tyrosine phosphatase implicated in T and B cell activation, differentiation, and development. It dephosphorylates specific tyrosine residues on its substrates, principally on the Src-family of protein tyrosine kinases, thus regulating T cell or B cell activation during the immune response. In this study, we present the partial CD45 nucleotide and deduced amino-acid sequences for the owl monkey (Aotus vociferens). There is 97% identity in the nucleotide sequence and 96% in the amino acid sequence with the human counterpart. Aotus CD45 undergoes alternative splicing on the extracelular N-terminal tail, and has several conserved features characteristic of other species. This includes the two Tyr phosphatase domains and some residues and/or motifs involved in docking of signaling molecules, intramolecular interactions, and CD45 activity and activity regulation (YINAS, GXGXXG, WPD, and YWP motifs, and the Cys residues). This suggests that the Aotus CD45 molecule is a functional enzyme and that initial lymphocyte activation in Aotus monkeys and humans is very similar. Together with previous reports from our laboratory, this work supports the contention that immune responses in Aotus are similar to those of humans, and supports the strategy for using this experimental model for studies on activation of T lymphocytes in response to specific antigens.     

Expression of CD52 mRNA in the rat embryo

CD52 is a leukocyte differentiation antigen first discovered in humans as expressed on the surface of lymphocytes, monocytes and eosinophils. The human CD52 is found on chromosome 1, and two alleles are both known to be reasonably common. A closely homologous gene has been identified in the cynomologous monkey and related genes have been found in mouse, rat and dog. The role of CD52 in lymphocyte is still unclear but the anti-CD52 antibodies named CAMPATH-1 antibodies are largely used for therapy where depletion of lymphocytes is required. In the past expression of the antigen on progenitors of leukocytes in bone marrow had been excluded, but recent work indicates CD52 is highly expressed on cells with colony-forming and NOD/SCID (non-obese diabetic-severe combined immunodeficiency)-engrafting capacities, both at the mRNA and membrane protein level. We have investigated CD52 expression during development in rat embryos by in situ hybridization. We report here that the antigen is highly expressed in the liver that is the major organ where multipotent hematopietic stem cells differentiate but also in the splancnopleuric mesoderm, at early stages of embryo differentiation, where hematopietic stem cells are suggested to arise. CD52⁺ cells were found in areas active in vasculogenesis at early embryo stages and in the walls of the vessels in the liver at mid gestation. CD52⁺ cells were also found to emerge among c-Kit positive cells.     

Immune-Related Gene Expression Profile in Laboratory Common Marmosets Assessed by an Accurate Quantitative Real-Time PCR Using Selected Reference Genes

The common marmoset (Callithrix jacchus) is considered a novel experimental animal model of non-human primates. However, due to antibody unavailability, immunological and pathological studies have not been adequately conducted in various disease models of common marmoset. Quantitative real-time PCR (qPCR) is a powerful tool to examine gene expression levels. Recent reports have shown that selection of internal reference housekeeping genes are required for accurate normalization of gene expression. To develop a reliable qPCR method in common marmoset, we used geNorm applets to evaluate the expression stability of eight candidate reference genes (GAPDH, ACTB, rRNA, B2M, UBC, HPRT, SDHA and TBP) in various tissues from laboratory common marmosets. geNorm analysis showed that GAPDH, ACTB, SDHA and TBP were generally ranked high in stability followed by UBC. In contrast, HPRT, rRNA and B2M exhibited lower expression stability than other genes in most tissues analyzed. Furthermore, by using the improved qPCR with selected reference genes, we analyzed the expression levels of CD antigens (CD3ε, CD4, CD8α and CD20) and cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12β, IL-13, IFN-γ and TNF-α) in peripheral blood leukocytes and compared them between common marmosets and humans. The expression levels of CD4 and IL-4 were lower in common marmosets than in humans whereas those of IL-10, IL-12β and IFN-γ were higher in the common marmoset. The ratio of Th1-related gene expression level to that of Th2-related genes was inverted in common marmosets. We confirmed the inverted ratio of CD4 to CD8 in common marmosets by flow cytometric analysis. Therefore, the difference in Th1/Th2 balance between common marmosets and humans may affect host defense and/or disease susceptibility, which should be carefully considered when using common marmoset as an experimental model for biomedical research.     

Distinct T cell developmental consequences in humans and mice expressing identical mutations in the DLAARN motif of ZAP-70

The protein tyrosine kinase, ZAP-70, is pivotally involved in transduction of Ag-binding signals from the TCR required for T cell activation and development. Defects in ZAP-70 result in SCID in humans and mice. We describe an infant with SCID due to a novel ZAP-70 mutation, comparable with that which arose spontaneously in an inbred mouse colony. The patient inherited a homozygous missense mutation within the highly conserved DLAARN motif in the ZAP-70 kinase domain. Although the mutation only modestly affected protein stability, catalytic function was absent. Despite identical changes in the amino acid sequence of ZAP-70, the peripheral T cell phenotypes of our patient and affected mice are distinct. ZAP-70 deficiency in this patient, as in other humans, is characterized by abundant nonfunctional CD4(+) T cells and absent CD8(+) T cells. In contrast, ZAP-70-deficient mice lack both major T cell subsets. Although levels of the ZAP-70-related protein tyrosine kinase, Syk, may be sufficiently increased in human thymocytes to rescue CD4 development, survival of ZAP-70-deficient T cells in the periphery does not appear to be dependent on persistent up-regulation of Syk expression.     

Expression of Epstein-Barr Virus Nuclear Antigen 1 Is Associated with Enhanced Expression of CD25 in the Hodgkin Cell Line L428

Epstein-Barr virus is associated with several human malignancies including Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s disease (HD). To examine the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1) in the pathogenesis of HD, we transfected the gene into the HD cell line L428. EBNA-1 expression was associated with significantly enhanced CD25 expression (interleukin 2 [IL-2]-receptor α chain) in transient and stably transfected L428 cells but did not affect the expression of IL-2 receptor β and γ chains. There was no up-regulation of the B-cell activation molecules CD23, CD30, CD39, CD40, CD44, CD71, and CD54 (intercellular adhesion molecule 1) or enhanced production of IL-6, IL-10, lymphotoxin alpha, and the soluble form of CD25. Stable EBNA-1-expressing L428 cells were nontumorigenic in SCID mice but showed enhanced lymphoma development in nonobese diabetic-SCID mice compared to mock-transfected cells.     

Hck is a key regulator of gene expression in alternatively activated human monocytes

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.