Transmembrane Protein Tyrosine Phosphatase CD45 Research Articles

An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

The effects of a chimeric monoclonal antibody (chA6 mAb) that recognizes both the RO and RB isoforms of the transmembrane protein tyrosine phosphatase CD45 on human T cells were investigated. Chimeric A6 (chA6) mAb potently inhibited antigen-specific and polyclonal T cell responses. ChA6 mAb induced activation-independent apoptosis in CD4+CD45RO/RBhigh T cells but not in CD8+ T cells. In addition, CD4+ T cell lines specific for tetanus toxoid (TT) generated in the presence of chA6 mAb were anergic and suppressed the proliferation and interferon (IFN)-γ production by TT-specific effector T cells by an interleukin-10–dependent mechanism, indicating that these cells were equivalent to type 1 regulatory T cells. Similarly, CD8+ T cell lines specific for the influenza A matrix protein-derived peptide (MP.58-66) generated in the presence of chA6 mAb were anergic and suppressed IFN-γ production by MP.58-66–specific effector CD8+ T cells. Furthermore, chA6 mAb significantly prolonged human pancreatic islet allograft survival in nonobese diabetic/severe combined immunodeficiency mice injected with human peripheral blood lymphocytes (hu-PBL-NOD/SCID). Together, these results demonstrate that the chA6 mAb is a new immunomodulatory agent with multiple modes of action, including deletion of preexisting memory and recently activated T cells and induction of anergic CD4+ and CD8+ regulatory T cells.

Neuronal expression of CD22: novel mechanism for inhibiting microglial proinflammatory cytokine production.

Although considered an immunologically privileged site, the central nervous system (CNS) can display significant inflammatory responses, which may play a pathogenic role in a number of neurological diseases. Microglia appear to be particularly important for initiating and sustaining CNS inflammation. These cells exist in a quiescent form in the normal CNS, but acquire macrophage-like properties (including active phagocytosis, upregulation of proteins necessary for antigen presentation, and production of proinflammatory cytokines) after stimulation with inflammatory substances such as lipopolysaccharide (LPS). Recent studies have focused on elucidating the role of neurons in the regulation of microglial inflammatory responses. In the present study, we demonstrate, using neuron-microglial cocultures, that neurons are capable of inhibiting LPS-induced tumor necrosis factor-alpha (TNF-alpha) production by microglia. This inhibition appears to be dependent on secretion of substances at axon terminals, as treatment with the presynaptic calcium channel blocker omega-conotoxin abolishes this inhibitory effect. Moreover, we show that conditioned medium from neuronal cultures similarly inhibits microglial TNF-alpha production, which provides additional evidence that neurons secrete inhibitory substances. We previously demonstrated that the transmembrane protein-tyrosine phosphatase CD45 plays an important role in negatively regulating microglial activation. The recent characterization of CD22 as an endogenous ligand of this receptor led us to investigate whether neurons express this protein. Indeed, we were able to demonstrate CD22 mRNA and protein expression in cultured neurons and mouse brain, using reverse transcriptase-polymerase chain reaction and antibody-based techniques. Furthermore, we show that neurons secrete CD22, which functions as an inhibitor of microglial proinflammatory cytokine production.

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

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice.

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.

The Role of the Protein Tyrosine Phosphatase CD45 in Regulation of B Lymphocyte Activation

The transmembrane protein tyrosine phosphatase CD45 is expressed throughout B cell development and differentiation, with the exception of terminally differentiated plasma cells on which its expression is down regulated. Numerous studies using CD45-deficient B cell lines and CD45-deficient mice have clearly demonstrated that CD45 plays an important role in modulating the signal that is transduced via the B cell antigen receptor by regulating the phosphorylation state of Src family kinases. Spatial and temporal controls enable CD45 to promote B cell antigen receptor signal transduction by constitutively maintaining Src family kinases in a partially active state, such that the B cell is able to effectively respond to an antigenic challenge. Moreover, CD45 is required for optimal activation of Ca2+-dependent and MAP kinase-dependent signal transduction pathways in the B cell. The net result is that CD45 affects the B cell response by controlling the relative threshold of sensitivity to a given antigenic stimulus. Thus, CD45 expression and function is required for normal B cell development, tolerance induction, and responsiveness to antigen.

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.

Regulation of Integrin-Mediated T Cell Adhesion by the Transmembrane Protein Tyrosine Phosphatase CD45

The transmembrane protein tyrosine phosphatase CD45 is required for Ag receptor signal transduction in lymphocytes. Recently, a role for CD45 in the regulation of macrophage adhesion has been demonstrated as well. To investigate further the role of CD45 in the regulation of adhesion, we examined integrin-mediated adhesion to fibronectin of two T cell lines and their CD45-deficient variants. The absence of CD45 correlated with enhanced adhesion to fibronectin via integrin alpha5beta1 (VLA-5), but not alpha4beta1 (VLA-4) in both cell lines. Adhesion returned to normal levels upon transfection of wild-type CD45 into the CD45-deficient lines. Transfection of chimeric or mutant molecules expressing some, but not all, CD45 domains and activities demonstrated that both the transmembrane domain and the tyrosine phosphatase activity of CD45 were required for regulation of integrin-dependent adhesion, but the highly glycosylated extracellular domain was dispensable. In contrast, only a catalytically active CD45 cytoplasmic domain was required for TCR signaling. Transfectants that restored normal levels of adhesion to fibronectin coimmunoprecipitated with the transmembrane protein known as CD45-associated protein. These studies demonstrate a novel role for CD45 in adhesion regulation and suggest a possible function for its association with CD45-associated protein.

Expression of the p56(Lck) Y505F mutation in CD45-deficient mice rescues thymocyte development.

Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56(lck) (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.

Definition of amino acids sufficient for plasma membrane association of CD45 and CD45-associated protein.

The transmembrane protein tyrosine phosphatase CD45 functions to activate Src-family member kinase activity in T lymphocytes. The inability to activate p56(lck) in CD45-deficient cells results in a higher threshold of signaling through the T cell receptor. The lymphoid-specific CD45-associated protein, CD45AP, interacts with CD45 through transmembrane interactions. Cells lines and mice deficient in CD45 express CD45AP mRNA, yet the protein is poorly expressed, indicating that CD45 is required for efficient expression of CD45AP. Pulse-chase analysis indicates that CD45 associates with CD45AP within minutes of biosynthesis. Cell surface labeling and coimmunoprecipitation demonstrate that CD45AP associates with surface-expressed CD45. Therefore, CD45AP is localized to the plasma membrane. To further characterize this interaction, chimeric proteins containing mutations in CD45 transmembrane regions were expressed, and their ability to associate with CD45AP was determined. Alanine-scan mutations of the CD45 transmembrane region demonstrate that no single amino acid is essential for the interaction with CD45AP. However, the expression of chimeric transmembrane regions indicates that a minimum of three and a maximum of eight amino acids in this region are sufficient to allow interaction with CD45AP.

T cell development in mice expressing splice variants of the protein tyrosine phosphatase CD45.

The transmembrane protein tyrosine phosphatase CD45 is expressed in multiple isoforms as a result of alternative splicing of variable exons encoding the extracellular domain. CD45 expression is critical for T cell development, and thymocyte maturation is blocked at the immature CD4+ CD8+ double-positive stage in CD45 gene-deficient (CD45 -/-) mice. Moreover, splicing of variable CD45 exons changes during thymocyte selection. To test the role of CD45 extracellular splice variants in T cell selection and development, we introduced CD45RO (a low-m.w. splice variant lacking exons 4, 5, and 6) and CD45ABC (a high-m.w. isoform containing all exons) transgenes under the control of a thymocyte-specific promoter into a CD45 -/- background, generating CD45RO transgene-positive CD45 -/- (CD45RO) and CD45ABC transgene-positive CD45 -/- (CD45ABC) mice. We demonstrate that both CD45 splice isoforms can rescue development of CD4+ and CD8+ TCR-alphabeta+ thymocytes. Neither CD45 isoform rescued positive selection of H-Y TCR transgene thymocytes, and these cells were blocked at a HSA(high) CD69- CD5(low) stage of development. Peripheral T cells from CD45RO and CD45ABC mice proliferated in response to allogeneic stimulator cells and anti-CD3epsilon cross-linking. However, only CD45RO mice, not CD45ABC mice, generated cytotoxic T cell responses and neutralizing, Th cell-dependent IgG Abs after viral infections. In addition, we show that T cells from CD45RO and CD45ABC mice accumulate in lymph nodes but not in the spleen, liver, or skin, indicating that the CD45 phosphatase may control the homing behavior and trafficking of T cells.

The Transmembrane Protein-tyrosine Phosphatase CD45 Is Associated with Decreased Insulin Receptor Signaling

Overexpression of the transmembrane protein-tyrosine phosphatase (PTPase) CD45 in nonhematopoietic cells results in decreased signaling through growth factor receptor tyrosine kinases. Consistent with these data, insulin receptor signaling is increased when the CD45-related PTPase LAR is reduced by antisense suppression in a rat hepatoma cell line. To test whether the hematopoietic cell-specific PTPase CD45 functions in a manner similar to LAR by negatively modulating insulin receptor signaling in hematopoietic cells, the insulin-responsive human multiple myeloma cell line U266 was isolated into two subpopulations that differed in CD45 expression. In CD45 nonexpressing (CD45-) cells, insulin receptor autophosphorylation was increased by 3-fold after insulin treatment when compared to CD45 expressing (CD45+) cells. This increase in receptor autophosphorylation was associated with similar increases in insulin-dependent tyrosine kinase activation. These receptor level effects were paralleled by postreceptor responses. Insulin-dependent tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and Shc was 3-fold greater in CD45- cells. In addition, insulin-dependent IRS-1/phosphatidylinositol 3-kinase association and MAP kinase activation in CD45- cells were also 3-fold larger. While expression of CD45 was associated with a decrease in the responsiveness of early insulin receptor signaling, interleukin 6-dependent activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was equivalent between CD45- and CD45+ cells. These observations indicate that CD45 can function as a negative modulator of growth factor receptor tyrosine kinases in addition to its well-established role as an activator of src family tyrosine kinases.

Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases.

We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo.

Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases.

We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo.

The regulation of B- and T-lymphocyte activation by the transmembrane protein tyrosine phosphatase CD45

The expression of transmembrane protein tyrosine phosphatase CD45 is required for antigen-induced activation of T and B lymphocytes. During the past year, advances have been made in our understanding of CD45 regulation and its role in regulating Src-family members. The importance of CD45 in thymocyte maturation has been demonstrated through the generation of CD45-knockout mice.