CD4+CD45RA+T cells from adults respond to recall antigens after CD28 ligation

The leukocyte common antigen isoforms CD45RA and CD45RO havelong been used to discriminate human naive and memory T cellsrespectively. This model was largely based on the observationthat CD45RO⁺ T cells respond preferentially to and show a higherfrequency of precursors specific for recall antigens. However,CD45RA⁺ T cells have more stringent requirements for stimulationand standard in vitro assays may favour CD45RO⁺ cells in thisrespect. We tested the hypothesis that CD45RAf T cells respondpoorly to in vitro stimulation with recall antigens becauseof inadequate stimulation rather than a lack of precursors.Limiting dilution analyses (LDA) for tetanus toxoid (lT)-specificT cells were performed in the presence or absence of exogenousantLCD28 antibody. Addition of antLCD28 yielded no proliferationin the absence of specific antigen. The precursor frequencyfor lT in the CD4⁺ CD45RO⁺ population was –1:4000, whilethe frequency of CD4⁺ CD45RA⁺ T cells specific for lT was 4-to >>20-fold lower. Addition of anti-CD28 antibody didnot significantly alter the apparent precursor frequency forCD45RA⁺ cells but yielded an enhancement of the value for CD45RA⁺cells by 3- to >>5-fold. No enhancement of antigen-specificproliferation by antLCD28 was observed with CD45RA⁺ T cellsderived from cord blood, although phytohemagglutinin responsesof these cells were amplified by CD28 antibody. These resultsindicate that conventional LDA underestimate the true precursorfrequency of antigen-specific cells within the adult CD45RA⁺population and support the possibility that a small number ofcells revert from a primed (CD45RO⁺) to an unprimed (CD45RA⁺)state. The majority of memory T cells, however, appear to residein the CD45RO⁺ population     

CD4-dependent and -independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56^lck, p59^fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56^lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolées, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4⁺ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4⁺ T lymphocytes. Our results show that, in normal CD4⁺ T lymphocytes, as in CD4⁺ T cell lines, there is a stable and readily detectable association between CD4: p56^lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4⁺ lymphocytes have detectable amounts of p56^lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59^fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56^fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4⁺ T cells and CD4⁺ T cell lines in kinase association to the TcR/CD3 complex.     

CD4+ lymphocytes from HIV-infected patients display impaired CD45-associated tyrosine phosphatase activity which is enhanced by anti-oxidants

It has been proposed that signal transduction defects may, at least partially, account for the functional impairment of CD4⁺ lymphocytes during HIV-1 infection. Recently, we have demonstrated that unresponsive CD4⁺ lymphocytes from these patients had reduced protein tyrosine phosphorylation after CD3 engagement, and that this defect was associated with constitutively altered levels of p56^lck and p59^fynkinases. Since CD45 is essential for T cell receptor (TCR) and CD2-mediated activation of protein tyrosine kinases, we study here CD45-associated tyrosine phosphatase activity in resting and activated CD4 T cells from HIV-infected patients. We found a significant decrease in the basal and post-activation phosphatase activity of CD45 which correlated well with impairment of proliferative responses. In addition, decreased levels of cellular thiols observed in resting CD4⁺ lymphocytes from these patients suggested a disturbed redox status. Although expression levels of CD45 were decreased in most patients, a significant recovery of phosphatase activity and proliferative responses was observed in most patients by preincubating cells with N-acetyl-l-cysteine and β₂-mercaptoethanol. In some patients, anti-oxidant treatment failed to significantly enhance phosphatase activity and proliferative responses. The low responses of purified CD4⁺ lymphocytes from these patients were associated with a high ratio of apoptotic cell death which did not appear to be influenced by anti-oxidant treatment.     

Defective protein tyrosine phosphorylation and altered levels of p59fyn and p56ick in CD4 T cells from HIV-1 infected patients

Early in HIV infection, CD4⁺ lymphocytes exhibit the propertiesof an anergic state characterized by unresponslveness to mltogensor to TCR stimulation and by defective IL-2 production. As tyrosinephosphorylation is the earliest of the biochemical events initiatedby stimulation of CD3-TCR, we studied protein tyrosine phosphorylationin purified CD4⁺ lymphocytes from 25 asymptomatic seropositlvepatients (CD4 T cells >350/mm³) previously stimulated invitro by immobilized antl-CD3 mAb or by co-lmmoblllzed antl-CD3and antl-CD28 mAbs. Purified CD4⁺ lymphocytes from HIV-lnfectedpatients exhibited defective early protein tyrosine phosphorylationin response to CD3 activation when compared with normal subjects.This defect was observed mainly in patients in whom prollferatlveresponses to immobilized antl-CD3 ranged from 2 to 50% of controlvalues obtained in healthy donors, and was frequently associatedwith increased cellular levels of p59^fyn and decreased cellularlevels of p56^ick. Interestingly, these defects appeared to correlatewith the degree of impairment in thymidine incorporation. SinceCD28 mAbs have been reported to enhance prollferatlve responsesto the CD3–TCR pathway in cloned murine or human anergicmodels and to induce tyrosine phosphorylation in human T cells,we studied the role of CD28 mAb as a co-signal. Although antl-CD28co-stlmulatlon augmented the prollferatlve responses in bothcontrols and HIV-lnfected patients, It failed to correct thetyrosine phosphorylation pattern in the latter. Our resultssuggest a relationship between defective early protein tyrosinephosphorylation and impairment of proliferatlve responses inCD4 T cells from HIV-lnfected patients, and evidence is providedthat associated altered cellular levels of the fyn and Ick tyrosinekinases might play an important role in the anergic responseobserved early during HIV infection.     

3H11, a unique cell surface molecule involved in the function of the CD45RA+subset of CD4+ cells

We have developed a mAb anti-3H11 by immunizing mice with aT cell line derived from the Callithrix Jacchus (common marmoset).anti-3H11 is reactive with 48% of unfractlonated T cells, 62%of CD4⁺ cells and 39% of CD8⁺ cells. Among CD4 cells, anti-3H11preferentially reacts with the CD45RA⁺ T cell subset. The majorityof helper activity for pokeweed mitogen (PWM)-driven B cellIgG synthesis and T cell response to recall antigen such astetanus toxold was found within the 3H11-CD4 cell population,whereas anti-3H11⁺CD4⁺ cells provided poor helper function forPWM-driven B cell IgG synthesis and were more responsive toconcanavalln A and autologous mixed lymphocyte reaction. Biochemicalcharacterization showed that anti-3H11 precipitated a singleprotein band with a relative molecular weight of 32,000 from¹²⁵l-surface labeled cell lysate. Biochemical, phenotyplc andfunctional studies revealed that the 3H11 molecule appearedto be different from previously established molecules on theT cell surface. Interestingly, addition of anti-3H11 to thecombination of CD4 and B cells in the presence of CDS cellsbut not to the combination of CD4 and B cells resulted in enhancementof the suppression of PWM-driven B cell IgG synthesis. Moreover,anti-3H11 had a co-mitogenic effect on T cells via the CD2 andCD3 pathways, and this co-mitogenic activity is restricted tothe CD45RA⁺ T cells. Taken together, our results show that the3H11 molecule is a novel antigen which may play an importantrole in the activation and function of the CD45RA⁺ subset ofT cells.     

CD45 up-regulation during lymphocyte maturation

CD4⁺CD8⁺ double-positive thymocytes differentiate into CD4⁺and CD8⁺ single-positive T cells during thymic positive selection.This process requires the interaction between the TCR and selfMHC molecules. In this context we have anaiyzed the expressionof CD45, an abundant transmembrane protein tyrosine phosphatase,and describe here its differential surface expression duringT cell maturation. Using four-color FACS analysis of thymocytesfrom normal as well as TCR-transgenic mice we demonstrate thatCD45 is up-regulated only during positive selection concomitantlywith the TCR-CD3 complex and the transient early activationmarker CD69, but that this up-regulation precedes heat stableantigen down-regulation. The tight linkage of the upregulationof the TCR-CD3 complex and CD45 may be required because theCD45 tyrosine phosphatase plays a role in modulating signaltransduction by the TCR-CD3 complex during positive selection.In addition, our findings argue for a regulation mechanism thatadapts the CD45 levels to increasing antigen receptor levelson mature T cells and B cells.     

Physical association of the cytoplasmic domain of CD2 with the tyrosine kinases p56lck and p59fyn

In T lymphocytes, CD2 forms part of a loosely associated membrane complex which includes the T cell receptor (TcR) for antigen, the CD3 subunits, CD4 or CD8, CD5 and the protein tyrosine kinases p56^lck and p59^fyn. The interaction of CD2 with tyrosine kinases in this complex provides a possible mechanism for transmembrane signal transduction by CD2. We have investigated whether the interaction of CD2 with the kinases is dependent on other known members of the complex, or whether an independent association can be observed. Using in vitro kinase assays with immune complexes precipitated from cell lysates, we demonstrate that CD2 can associate with p56^lck and p59^fyn in a rat thymoma line that does not express CD4 or CD8, and in a TcR-negative Jurkat cell line. In TcR-positive Jurkat cells that express rat CD2, interaction of CD2 with p56^lck and p59^fyn _Was clearly seen, but it was absent in cells where the cytoplasmic tail of CD2 is truncated, indicating that the interactions are mediated by the cytoplasmic region of CD2. Furthermore, using cells expressing CD2 molecules with partial truncations in the cytoplasmic domain, we show that the association of CD2 with p56^lck: is progressively lost as the cytoplasmic domain is shortened, and that the capacity of the mutants to associate with p56^lck correlates with their capacity to transduce transmembrane signals.     

Co-localization of Fyn with CD3 complex,CD45 or CD28 depends on different mechanisms

The Src family protein tyrosine kinase Fyn (p59^fyn) plays an important role in thymocyte development and T cell receptor (TCR) signal transduction. Fyn has been shown to associate with the TCR-CD3 complex, the protein tyrosine phosphatase CD45 and several co-receptors such as CD28 which are crucial for initiating T cell activation and proliferation. The molecular basis of how Fyn is associated with these transmembrane proteins is largely unknown. To investigate the Fyn association with the TCR-CD3 complex, CD45 and CD28 at the molecular level, various Fyn/β-galactosidase fusion proteins were constructed and expressed in Jurkat cells. Co-localization experiments applying antibody-induced co-capping and double immunofluorescence staining techniques were used to study the association of these fusion proteins with the TCR-CD3 complex, CD45 and CD28. Our results revealed that co-localization of Fyn with the TCR-CD3 complex requires the unique N terminus whereas co-localization with CD45 depends on the unique N terminus, the Src homology (SH)3- and a functional SH2 domain. CD28 co-localizes with Fyn molecules that contain the N terminus and a functional SH2 domain. These results suggest that Fyn association with the TCR-CD3 complex, CD45 and CD28 is mediated by different molecular mechanisms.     

Human CD45RA+ and CD45R0+ T cells exhibit similar CD3/T cell receptor-mediated transmembrane signaling capacities but differ in response to co-stimulatory signals

CD45RA⁺ cells have been described to be less responsive to CD3/T cell receptor (TcR)-mediated activation than CD45R0⁺ T cells. To analyze the underlying mechanism of the differential responses we compared CD3/TcR-triggered tyrosine phosphorylation in the two subsets and studied the role of co-stimulatory signals provided either by accessory cells or pharmacologic activation of protein kinase C by phorbol ester. Stimulation of purified CD45RA⁺ and CD45R0⁺ T cells with CD3/TcR antibodies induced similar patterns and intensities of tyrosine phosphorylation in the two subsets, but no proliferation. If accessory cells were used as the source of co-stimulatory signals, strong expression of the 55-kDa chain of the interleukin-2 (IL-2) receptor (CD25), significant IL-2 production and vigorous proliferation were observed in CD45R0⁺ cells, whereas CD45RA⁺ cells responded weakly. However, when CD3/TcR-mediated triggering was combined with activation of protein kinase C by phorbol ester, CD45RA⁺ cells responded strongly. These data indicate that the transmembrane signaling capacity of the T cell receptor expressed by CD45RA⁺ and CD45R0⁺ cells is similar and, therefore, is presumably not responsible for the differential reactivities of the two subsets. It is more likely that co-stimulatory signals determine whether CD3/TcR-initiated activation results in strong or weak responses.     

Role of CD4+CD45RA+ T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse

The non-obese diabetic (NOD) mouse spontaneously develops aT cell-mediated autoimmune disease, sharing many features withhuman insulin-dependent diabetes mellltus (IDDM), leading toinsulin-secreting ß cell destruction. The role ofCD4⁺ T cells has been evidenced at two levels. First, CD4⁺ Tcells from diabetic animals are required to transfer diabetesto non-diabetic recipients in conjunction with CD8⁺ effectorT cells. Second, suppressive CD4⁺ T cells have been characterizedin non-diabetic NOD mice. T cells with different functions canthus share the CD4⁺ phenotype. Since CD4⁺ T cells can be dividedinto at least two subgroups on the basis of CD45 isoform expression,we evaluated the distribution of CD4⁺ T cells expressing theCD45RA isoform on NOD mouse thymocytes and peripheral T cells.The percentage of CD45RA⁺ cells was dramatically increased amongthe most mature CD3^bright thymocytes and among CD4⁺ T cellsin lymph nodes of the NOD mouse as compared with control strains.This increase was related to the development of insulitls. Interestingly,the CD45RA isoform was expressed on most CD4⁺ T cells invadingthe islets. In vivo treatment with an antl-CD45RA mAb preventedthe development of insulitls and spontaneous diabetes in femaleanimals but not the transfer of diabetes by T cells collectedfrom diabetic NOD donors. These results indicate that anti-CD45RAmAb is only effective if given before the full commitment ofeffector T cells to the destruction of islet ß cells.ThusCD4⁺CD45RA⁺ T cells play a key role in early activation stepsof anti-islet immunity.     

Regulation of the p70zap tyrosine protein kinase in T cells by the CD45 phosphotyrosine phosphatase

Two classes of protein tyrosine kinases (PTK) are utilized by the T cell antigen receptor (TcR)/CD3 complex for initiation of the signaling cascade, the Src-family PTK p56^lck and p59^fyn, and the Syk-family PTK p70^zap and p72^syk. In addition, the CD45 phosphotyrosine phosphatase (PTPase) is required for the induction of tyrosine phosphorylation by the TcR/CD3, presumably by positively regulating Src-family PTK. Here we report that CD45 also regulates the Syk-family PTK p70^zap (or ZAP-70). In CD45-negative T cells, p70^zap was constitutively phosphorylated on tyrosine and co-immunoprecipitated with the TcR-ζ chain. In resting wild-type CD45-positive cells, p70^zap was mainly unphosphorylated, but it was rapidly phosphorylated on tyrosine upon treatment of the cells with anti-CD3 or PTPase inhibitors. Finally, p70^zap co-distributed with CD45 in intact T cells, and tyrosine phosphorylated p70^zap was dephosphorylated by CD45 in vitro. These findings suggest that CD45 plays an important role, direct or indirect, in the regulation of p70^zap and its function in TcR/CD3 signaling.     

The p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/CD3/ζ complex

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are thought to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56^lck SH2 domain with ζ-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/ζ complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8αβ and CD8αβ isoforms to the TCR. We demonstrate in vivo in association of CD8αα/p56^lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56^lck specifically impedes the association of ZAP-70 with CD8αα/p56^lck without affecting the ζ/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8αα or CD8αβ isoform with the TCR/CD3/ζ complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56^lck SH2 domain mediates recruitment of CD8/p56^lck to the activated TCR/CD3/ζ complex.     

HIV-1 glycoprotein gp120 disrupts CD4-p56lck/CD3-T cell receptor interactions and inhibits CD3 signaling

Using the CD4⁺ human T cell clone P28, we demonstrated that the HIV-1 glycoprotein gp120 inhibited CD3-induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56^lck from CD4 in CEM cells, with a concommittant inhibition of CD4-linked kinase activity. We have addressed the question whether disruption of CD4/p56^lck or CD4/CD3-T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti-CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4-associated p56^lck and CD4-linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti-CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3-induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide-specific phospholipase C-γ-1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3-induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4-p56^lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down-modulation of CD4-p56^lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3-linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.     

CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0⁺ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA⁺ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen-induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA⁺ and CD45R0⁺ human T lymphocyte subsets derived from peripheral blood. The mean CD3-induced rise in intracellular calcium was 80% greater in CD45R0⁺ than in CD45RA⁺ cells. Basal DAG levels in CD45R0⁺ cells were found to be, on average, 60% higher than in CD45RA⁺ cells (p = 0.002), but the CD3-induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3-induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0⁺ cells than in CD45RA⁺ cells (p = 0.015 and 0.023). The CD45R0⁺ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA⁺ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0⁺ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3-induced elevation in intracellular calcium observed in the CD45R0⁺ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0⁺ T cells to mitogenic stimuli compared to CD45RA⁺ cells.     

Comparison of lymphokine secretion and mRNA expression in the CD45RA+ and CD45RO+ subsets of human peripheral blood CD4+ and CD8+ lymphocytes

Flow cytometric analysis of human peripheral blood T lymphocytes demonstrated that the majority of the CD4⁺ cells were CD29⁺ or CD45RO⁺ “mature” cells while the CD8⁺ cells were primarily CD45RA⁺ “naive” cells. After an initial separation into CD4⁺ and CD8⁺ cells and a secondary separation into CD45 subsets, lymphokine secretion was assessed after phorbol 12-myristate 13-acetate and ionomycin or fixed anti-CD3 stimulation. Within the respective CD45 subsets, CD4⁺ cells produced more interleukin (IL)-2, IL-4, and IL-6; but the CD8⁺ cells secreted more interferon-γ and granulocyte/macrophage-colony-stimulating factor. Tumor necrosis factor-α secretion was similar in the matched CD45 subsets. Northern analysis revealed a parallel pattern of lymphokine mRNA expression in the four lymphocyte subsets. These results suggest that human CD8⁺ peripheral blood lymphocytes have a significant capacity to secrete lymphokines, and that the low lymphokine production observed in unseparated CD8⁺ cells reflects the higher percentage of less functional CD45RA⁺ cells.     

Increased Frequencies of the CD29 and CD57 Markers and Decreased Frequency of CD45RA Within CD4+ and CD8+ Subsets after Allogeneic Bone Marrow Transplantation in Man

Two monoclonal antibodies, anti-CD45RA and anti-CD29, reciprocally divide the CD4⁺ and CD8⁺ lymphocytes into CD4⁺ CD45RA⁺, CD4⁺ CD29⁺, CD8⁺ CD45RA⁺ and CD8⁺ CD29⁺ subsets. The CD4⁺ CD45RA⁺, CD4⁺ CD29⁺ and CD8⁺ CD45RA⁺ possess suppressor-inducer, helper-inducer and suppressor-effector functions respectively. Since the role of these subsets has not been established after allogeneic bone marrow transplantation we studied lymphocyte subpopulations in 12 patients 45- 227 days after the procedure. The fraction of CD4⁺ lymphocytes was significantly (P= 0.0005) decreased to 20±9% versus 43±3% in controls. Within the CD4⁺ compartment, we found an increase in the fraction of CD4⁺ cells that co-expressed CD29 (CD29⁺/CD4⁺) to 92±10% versus 48±15% (P=0.008) in controls and a concommittant decrease m CD45RA⁺/CD4⁺ to 16±12% versus 56±25% (P=0.008) Patients were also noted to have an increase in the percentage of CD8⁺ lymphocytes to 41±5% compared to 23±4% in controls (P=0.0004), Examination of the CD8⁺ subsets revealed a significant increase in the CD29⁺/CD8⁺ fraction to 97±3% versus 64±2% in controls (P= 0.008) and a decrease in the CD45RA⁺/CD8⁺ fraction to 36±11% versus 70±21% (P= 0.008). The number of cells co-expressing CD57 were also determined within the CD4⁺ and CD8⁺ subsets. In patients CD57⁺/CD4⁺ were increased to 29±7% versus 1±1% in controls (P=0.04), and CD57⁺/CD8⁺ to 49±12% versus 23±9% (P=0.02). Since CD29⁺ and CD57⁺ cells have a poor capability for IL-2 production and proliferation this shift in subset distribution may account for some of the defects in cellular immunity seen within the first year after allogeneic bone marrow transplantation.     

HIV envelope-directed signaling aberrancies and cell death of CD4+ T cells in the absence of TCR co-stimulation

HIV-1 infection in CD4₊ T cells initiates a viral cytopathiceffect (CPE) that is dependent on the activation of intracellularprotein tyrosine kinases (PTK). PTK in T cells are also activatedduring the course of TCR or CD4 receptor engagement and themanner of receptor engagement may generate signals leading eitherto cell proliferation, tolerance induction (anergy) or programmedcell death (PCD). We have identified PTK triggered during theinteraction of cells stably expressing surface HIV envelope(gp120/gp41; HIVenv) and CD4⁺ T cells, which leads to extensiveand rapid individual cell death. We have found that this killingis accompanied by tyrosine phosphorylation and activation ofthe CD4-associated p56^ick kinase, and by activation of a secondmember of the src family of PTK, p59^fyn kinase, normally associatedwith T cell stimulation through the TCR. Interestingly, in contrastwith normal T cell signaling, the subunit of the TCR failsto become tyrosine-phosphorylated during signaling accompanyingHIV-directed cell killing. Downstream activation of the ZAP-70PTK also does not occur. Unlike T cell apoptosis triggered bysoluble HIVenv glycoproteins, which requires co-stimulationof CD4 and the antigen-specific TCR, T cell killing by membrane-associatedHIVenv does not require TCR co-stimulation, because aberrantsignaling and cell death are triggered by CD4+ but TCR^–cell lines. These results are the first report where dual activationof the Lck and Fyn PTK does not result in normal downstreamsignaling through the ZAP PTK. We suggest by analogy to SCIDresulting from ZAP-70 mutations, that the dissociation of upstreamPTK activation from ZAP-70 signaling contributes to T cell depletionby HIV and to the development of AIDS.     

Alterations of CD2 association with T cell receptor signaling molecules in “CD2 unresponsive” human T lymphocytes

CD2-associated molecules were identified by means of in vitro kinase assays of CD2 immunoprecipitates obtained from nontransformed human T lymphocytes lysed in the detergent brij 58. Under these conditions CD2 was found to be associated with the protein tyrosine kinases p56^lck and p59^fyn as well as two low molecular weight phosphoproteins. The latter were identified as the ζ and ? chains, the major signaling components of the CD/7 TcR complex. Importantly, induction of T cell unresponsiveness towards CD2-mediated stimuli by means of CD3/Tcell receptor (TcR) modulation results in uncoupling of ζ and ? from the CD2 molecule, while its associations with p56^lck and p59^fyn remain unaffected. Moreover, despite the incapacity of T lymphocytes to undergo DNA synthesis in the CD3/TcR-modulated state, CD2 triggering still results in tyrosine phosphorylation of some unknown protein substrates. Thus, the same ζ and ? chains which are components of a functional TcR complex appear to also couple to the CD2 molecular complex. Moreover, dissociation of TcR and CD2 complexes in intact cells seems to block CD2-mediated Tcell growth but does not result in complete abolishment of the signal transducing capacity of the CD2 receptor.     

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

CD45, the leukocyte-common antigen, Is a transmembrane proteintyrosine phosphatase uniquely expressed by cells of hematopoletlcorigin. We have developed CD4⁺ and CD8⁺ T cell clones that aredeficient in the expression of CD45 and have previously shownthat these cells fall to proliferate in response to antigenor cross-linked CD3. These studies have now been extended toshow that stimulation with antl-Thy-1, a mltogenlc signal forthe CD4⁺CD45⁺ and CD8⁺CD45⁺ T cells, falls to induce proliferationin the CD45^– T cells. Examination of the CD8⁺CD45^–T cells correlates antl-Thy-1 unresponslveness with a failureto increase in tyrosine phosphorylatlon. Furthermore, stimulationof CD8⁺CD45⁺ T cells with antl-Thy-1 results in an increasein p56^ick activity but not in CD8⁺CD45^– T cells. In contrastto the results with antl-Thy-1, both the CD4^+– CD45 andCD8⁺CD45^– T cells respond to treatment with lectin mitogens,concanavalln A or phytohemagglutlnln. Lectin-lnduced proliferationwas inhibited by the addition of cyclosporln A. Treatment ofCD45^– T cells with PMA and lonomycln also results in proliferationindicating that activation of protein kinase C in conjunctionwith an increase in intracellular calcium rescues the defectcafsed by CD45 deficiency. The data suggest that CD45 Is requiredfor the activation of tyrosine kinase activity Immediate orprior to transmembrane signaling.