Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction.

An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56(lck) tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0(+) than in the CD45RBC(+) sub-clones. Upon CD3-CD4 ligation, TCR-zeta phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-zeta phosphoisomers, ZAP-70 phosphorylation, as well as p56(lck), c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0(+) compared with CD45RBC(+) cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0(+) but not in CD45RBC(+) cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56(lck) kinase molecules. Upon CD3-CD4 co-ligation, the active p56(lck) increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-zeta chain phosphorylation and ZAP-70 recruitment.     

Homeostasis of the naive CD4+ T cell compartment during aging

Despite thymic involution, the number of naive CD4(+) T cells diminishes slowly during aging, suggesting considerable peripheral homeostatic expansion of these cells. To investigate the mechanisms behind, and consequences of, naive CD4+ T cell homeostasis, we evaluated the age-dependent dynamics of the naive CD4+ T cell subsets CD45RA+CD31+ and CD45RA+CD31-. Using both a cross-sectional and longitudinal study design, we measured the relative proportion of both subsets in individuals ranging from 22 to 73 years of age and quantified TCR excision circle content within those subsets as an indicator of proliferative history. Our findings demonstrate that waning thymic output results in a decrease in CD45RA+CD31+ naive CD4+ T cells over time, although we noted considerable individual variability in the kinetics of this change. In contrast, there was no significant decline in the CD45RA+CD31- naive CD4+ T cell subset due to extensive peripheral proliferation. Our longitudinal data are the first to demonstrate that the CD45RA+CD31+CD4+ subset also undergoes some in vivo proliferation without immediate loss of CD31, resulting in an accumulation of CD45RA+CD31+ proliferative offspring. Aging was associated with telomere shortening within both subsets, raising the possibility that accumulation of proliferative offspring contributes to senescence of the naive CD4+ T cell compartment in the elderly. In contrast, we observed retention of clonal TCR diversity despite peripheral expansion, although this analysis did not include individuals over 65 years of age. Our results provide insight into naive CD4+ T cell homeostasis during aging that can be used to better understand the mechanisms that may contribute to immunosenescence within this compartment.     

Nuclear factor of activated T cells is a driving force for preferential productive HIV-1 infection of CD45RO-expressing CD4+ T cells

Human immunodeficiency virus type-1 (HIV-1) preferentially replicates in CD4-expressing T cells bearing a "memory" (CD45RO+) rather than a "naive" (CD45RA+/CD62L+) phenotype. Yet the basis for the higher susceptibility of these cells to HIV-1 infection remains unclear. Because the nature of the CD45 isoform itself can affect biochemical events in T cells, we set out to determine whether these isoforms could differently modulate HIV-1 long terminal repeat (LTR) activity and thereby replication. Through the use of CD4+ Jurkat T cells specifically expressing distinct CD45 isoforms (i.e. CD45RABC or CD45RO), we demonstrated that a difference in CD45 isoform expression conferred preferential replication of HIV-1 to CD45RO-expressing T cell clones following a physiological CD3/CD28 stimulation. Closer analysis indicated that higher HIV-1 LTR activation levels were consistently observed in CD45RO-positive cells, which was paralleled by more pronounced nuclear factor of activated T cells (NFAT) activation in these same cells. Specific involvement of NFAT1 was revealed in studied Jurkat clones by mobility shift analyses. In addition, preferential activation of the LTR and viral replication in CD45RO T cells was FK506- and cyclosporin A-sensitive. These results underscore the importance of NFAT in HIV-1 regulation and for the first time identify the role of the CD45 isoform in limiting productive HIV-1 replication to the human CD4 memory T cell subset.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

In vitro differentiation from a pluripotent human CD4+CD8+ thymic cloned cell into four phenotypically distinct subsets

Human thymic cell differentiation is almost totally unknown. In the present study we developed an in vitro system using human thymic cloned cells to analyze precursor-progeny relationship. We obtained several CD4+CD8+ double positive thymic clones that could give rise after several weeks in culture only to either CD4 or CD8 single positive clones. By contrast we isolated a unique pluripotent thymic double positive clone, termed B12, which differentiated into four phenotypically distinct T cell clones, namely double-positive CD4+CD8+, double-negative CD4-CD8- or either single-positive phenotype. We derived stable subclones representative of each phenotype and we showed by molecular analysis that they expressed the same TCR. Utilization of either CD3 or anticlonotypic mAb revealed that this TCR expressed by the four subclones was functional.     

Evidence for increased T cell turnover and decreased thymic output in HIV infection.

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO-CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.     

Signal transduction via CD4, CD8, and CD28 in mature and immature thymocytes. Implications for thymic selection.

The positive and negative selection of immature thymocytes that shapes the mature T cell repertoire appears to occur at an intermediate stage of development when the cells express low levels of TCR/CD3. These cells are also CD4+CD8+ and CD28+ (dull), and signals delivered by these three accessory molecules have been implicated in the selection process. We have examined the regulatory function of these accessory molecules on responses of immature thymocytes stimulated through the TCR/CD3 complex. Cross-linking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosine phosphorylation and calcium mobilization. Subsequent cell proliferation could be induced by soluble anti-CD28 mAb, which was comitogenic for cells stimulated with CD3 x CD4 or CD3 x CD8 cross-linking, but was without effect on cells stimulated with CD3 x CD3 cross-linking. A potential role for CD28 signal transduction in thymic maturation is suggested by the demonstration that the BB-1 molecule, a natural ligand for CD28, is expressed on thymic stromal cells. Taken together, our data suggest a model of thymic development in which CD4 or CD8 may enhance TCR/CD3 signaling upon coligation by an MHC molecule. If the CD28 surface receptor is simultaneously stimulated by a BB-1 expressing stromal cell, this set of interactions could lead to proliferation and positive selection. In the absence of CD28 stimulation the enhanced TCR/CD3 signals might lead to apoptosis and negative selection.     

Human CD8+ T lymphocytes can be divided into CD45RA+ and CD45RO+ cells with different requirements for activation and differentiation.

The functional distinction between CD45RA+ and CD45RO+ cells within the human CD4+ T cell subset is well established. This study was undertaken to investigate whether a similar division can be made within the CD8+ T cell population. A quantitative comparison was made of the requirements for activation and differentiation of CD8+CD45RA+ and CD8+CD45RO+ cells. Stimulation of T lymphocytes with anti-CD3 mAb immobilized at high-density induced strong proliferation and CTL activity in both CD45RA+ and CD45RO+ cells. Suboptimal TCR/CD3 triggering, in contrast, induced substantially higher levels of proliferation and CTL activity in CD8+CD45RO+ cells compared with their CD45RA+ counterparts. Lymphokine secretion (i.e., Il-2 and TNF-alpha) was under any condition more readily induced in CD8+CD45RO+ cells. Markedly, proliferation of both CD8+CD45RA+ and CD8+CD45RO+ T cells initiated by anti-CD3 mAb immobilized at high densities was not inhibited by addition of anti-CD25 mAb, in contrast to proliferation induced by suboptimal anti-CD3 mAb concentrations. These findings show that a functional division between CD45RA+ and CD45RO+ T cells with distinct requirements for activation and differentiation may also be made in the CD8+ subset.     

Expression of CD45 lacking the catalytic protein tyrosine phosphatase domain modulates Lck phosphorylation and T cell activation

The function of the second protein tyrosine phosphatase domain (D2) in two-domain protein tyrosine phosphatases (PTP) is not well understood. In CD45, D2 can interact with the catalytic domain (D1) and stabilize its activity. Although D2 itself has no detectable catalytic activity, it can bind substrate and may influence the substrate specificity of CD45. To further explore the function of D2 in T cells, a full-length construct of CD45 lacking the D1 catalytic domain (CD45RABC-D2) was expressed in CD45+ and CD45- Jurkat T cells. In CD45- Jurkat T cells, CD45RABC-D2 associated with Lck but, unlike its active counterpart CD45RABC, did not restore the induction of tyrosine phosphorylation or CD69 expression upon T cell receptor (TCR) stimulation. Expression of CD45RABC-D2 in CD45+ Jurkat T cells resulted in its association with Lck, increased the phosphorylation state of Lck, and reduced T cell activation. TCR-induced tyrosine phosphorylation was delayed, and although MAPK phosphorylation and CD69 expression were not significantly affected, the calcium signal and IL2 production were severely reduced. This indicates that the non-catalytic domains of CD45 can interact with Lck in T cells. CD45RABC-D2 acts as a dominant negative resulting in an increase in Lck phosphorylation and a preferential loss of the calcium signaling pathway, but not the MAPK pathway, upon TCR signaling. This finding suggests that, in addition to their established roles in the initiation of TCR signaling, CD45 and Lck may also influence the type of TCR signal generated.     

Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.     

TSCOT+ thymic epithelial cell-mediated sensitive CD4 tolerance by direct presentation

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

CD45 tyrosine phosphatase-activated p59fyn couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx.

The role of the CD45 phosphotyrosine phosphatase in coupling the T cell antigen receptor complex (TCR) to intracellular signals was investigated. CD45- HPB-ALL T cells were transfected with cDNA encoding the CD45RA+B+C- isoform. The tyrosine kinase activity of p59fyn was found to be 65% less in CD45- cells than in CD45+ cells, whereas p56lck kinase activity was comparable in both sub-clones. In CD45- cells the TCR was uncoupled from protein tyrosine phosphorylation, phospholipase C gamma 1 regulation, inositol phosphate production, calcium signals, diacylglycerol production and protein kinase C activation. Restoration of TCR coupling to all these pathways correlated with the increased p59fyn activity observed in CD45-transfected cells. Co-aggregation of CD4- or CD8-p56lck kinase with the TCR in CD45- cells restored TCR-induced protein tyrosine phosphorylation, phospholipase C gamma 1 regulation and calcium signals. Receptor-mediated calcium signals were largely due (60-90%) to Ca2+ influx, and only a minor component (10-40%) was caused by Ca2+ release from intracellular stores. Maximal CD3-mediated Ca2+ influx occurred at CD3 mAb concentrations at which inositol phosphate production was non-detectable. These results indicate that CD45-regulated p59fyn plays a critical role in coupling the TCR to specific intracellular signalling pathways and that CD4- or CD8-p56lck can only restore signal transduction coupling in CD45- cells when brought into close association with the TCR.     

Analysis of expression of CD2, CD3, and T cell antigen receptor molecules during early human fetal thymic development

To define early stages of T cell maturation during human fetal thymic development, we have used mAb reactive with CD2, CD3, and TCR molecules in indirect immunofluorescence assays on a series of early human fetal thymic specimens. Using a technique of quantitating the relative proportions of fluorescent-positive cells present in tissue sections, we found at 8.5 wk of gestational age after arrival of CD7+ T cell precursors into the thymic rudiment, 60% of thymic CD7+ cells were CD2+, 4% were CD3+ and none was TCR-delta+ or TCR beta+. Moreover, cells reactive with anti-CD2 antibodies against T11(2) and T11(3) epitopes of CD2 as well as thymic stromal cells expressing the CD2 ligand, lymphocyte function associated Ag-3, were also present at 8.5 wk. From 9.5 wk to birth TCR beta+ cells increased to include greater than 90% of all CD7+ cells while TCR-delta+ cells fell from a peak of 11% of CD7+ cells at 9.5 wk to 1% of CD7+ cells at birth. These data suggest that epitopes of CD2 molecules are expressed early on during fetal thymic development. Moreover, these data suggest that CD7+, CD2+, cytoplasmic CD3+ T cell precursors in man give rise to both TCR-delta+ T cells as well as to T cells expressing TCR-alpha beta.     

The influence of age on T cell generation and TCR diversity

The ability to mount protective immune responses depends on the diversity of T cells. T cell diversity may be compromised by the declining thymic output of new T cells. The aging process imposes a threat to diversity, because thymic function deteriorates. In this study we have examined the relationship between thymic production, homeostatic T cell proliferation and TCR beta-chain diversity in young (approximately 25 years), middle-aged ( approximately 60 years), and elderly adults (approximately 75 years). TCR excision circles (TREC) as a marker of thymic output exponentially decreased by >95% between 25 and 60 years of age. The frequency of Ki67(+) cycling CD4 T cells remained steady, and surprisingly, the diversity of the naive CD4 T cell repertoire was maintained at approximately 2 x 10(7) different TCR beta-chains. After the age of 70 years, TRECs only slightly declined, but homeostatic proliferation doubled. The diversity of the T cell pool drastically contracted to 200,000 TCR beta-chains. Also, the phenotypic distinction between naive and memory CD4 T cells became fuzzy. The collapse in CD4 T cell diversity during the seventh and eighth decades indicates substantial T cell loss and implies that therapeutic measures to improve vaccine responses will have to include strategies for T cell replenishment.     

High levels of CD45 are coordinately expressed with CD4 and CD8 on avian thymocytes.

In this paper we describe the avian homolog of mammalian CD45. We show that this Ag is expressed on all leukocytes but not on erythroid cells or their immediate precursors. Immunoprecipitations demonstrated that B lineage cells from the bursa of Fabricius expressed a higher molecular mass variant (215 kDa) than did T lineage cells from the thymus (190 kDa), and crucially, these high molecular mass molecules had intrinsic phosphotyrosine phosphatase activity characteristic of mammalian CD45. We show that levels of CD45 expression as detected by mAb LT40 in the avian thymus are heterogeneous and further that mAb LT40 can deplete all phosphotyrosine phosphatase activity from thymocyte membrane preparations. Therefore total levels of CD45 are heterogeneous among avian thymocytes. Specifically, 87 to 89% of thymocytes expressed fourfold higher levels of surface CD45 (CD45hi) than the remaining 11 to 13% (CD45lo). The CD45lo population contained exclusively thymocytes with the phenotype CD3-4-8lo, characteristic of the immediate precursors to the CD3-4+8+ thymic population which are CD45hi. The shift from low to high levels of surface CD45 expression therefore occurred at the same stage as the transition from CD4-8lo to CD4+8+ and before the expression of CD3. The protein tyrosine kinase activity associated with CD4 and CD8 (p56lck) and the phosphatase activity of CD45 have been implicated elsewhere in jointly regulating peripheral T cell signal transduction and subsequent cellular responses. The coordinated expression of high levels of CD45 with both CD4 and CD8 in the avian thymus supports the possibility that these molecules may function together in regulating thymocyte growth and/or differentiation.     

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.     

Development of the CD4 and CD8 lineage of T cells: instruction versus selection

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8- and CD4-8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up-regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.     

The CD45 tyrosine phosphatase regulates specific pools of antigen receptor-associated p59fyn and CD4-associated p56lck tyrosine in human T-cells.

A newly isolated T-cell line (CB1) derived from a T-acute lymphoblastic leukaemia (T-ALL) patient contained cells (40% of total) which did not express the CD45 phosphotyrosine phosphatase. The cells were sorted into CD45- and CD45+ populations and shown to be clonal in origin. T-cell receptor (TCR) cross-linking or coligation of the TCR with its CD4/CD8 co-receptors induced tyrosine phosphorylation and calcium signals in CD45+ but not in CD45- cells. Unexpectedly, whole cell p56lck and p59fyn tyrosine kinase activities were not reduced in CD45- compared to CD45+ cells. A novel technique was therefore developed to isolated specific pools of aggregated receptors expressed at the cell surface, together with their associated tyrosine kinases. Using this technique it was shown that cell surface CD4-p56lck kinase activity was 78% lower in CD45- than in CD45+ cells. Phosphorylation of TCR zeta- and gamma-chains occurred in TCR immunocomplexes from CD45+ but not CD45- cells, despite comparable levels of p59fyn and TCR proteins. Furthermore, TCR-associated tyrosine kinase activity towards an exogenous substrate was 84% lower in CD45- than in CD45+ cells. Addition of recombinant p59fyn to TCR immunocomplexes isolated from CD45-cells restored the phosphorylation of the TCR zeta- and gamma-chains. Our results demonstrate that CD45 selectively regulates the pools of p59fyn and p56lck kinases which are associated with the TCR and CD4 at the cell surface. Activation by CD45 of these receptor-associated kinase pools correlates with the ability of the TCR and its coreceptors to couple to intracellular signalling pathways.