Involvement of p21ras activation in T cell CD69 expression

The involvement of p21^ras in the induction of the early activation antigen CD69 was investigated in T cells. Expression of a v-Ha-ras coding for a constitutively active ras protein in Jurkat cells resulted in CD69 induction on the cell surface. Transfected ras was shown to be constitutively activated and functionally efficient, since it could be immunoprecipitated in the guanosine triphosphate (GTP)-bound form and it induced transactivation of an AP-1 consensus-chloramphenicol acetyltransferase reporter gene. The requirement for ras activation in T cell receptor (TcR) CD3-mediated CD69 induction was also investigated. The expression of a dominant negative c-Ha-ras-N17 mutant markedly reduced the amount of GTP that could be immunoprecipitated from ras proteins after TcR/CD3 triggering in Jurkat cells, and concomitantly decreased TcR/CD3-mediated CD69 induction. These results suggest a central role for ras in TcR/CD3-mediated CD69 expression in T cells.     

CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection

CD69, an ‘activation marker’ that is rapidly inducedon mature T cells after stimulation through the T cell antigenreceptor (TCR) was found to be expressed on 10% of normal thymocytes.All of these CD69⁺ thymocytes express ß TCR, and theyinclude both TCR^lowCD4⁺CD8⁺ and TCR^highCD4⁺CD8^– or CD4^–CD8⁺thymocytes. The CD69⁺ cells can be further segregated into heat-stableantigen (HSA)⁺TCR^*HSA^–TCR^high and HSA⁺TCR^high thymocytepopulations. None of CD69⁺ cells express the mature T cell markerQa-2. Thus CD69⁺ cells present in vivo appear phenotyplcallyto represent transitional cell populations between immatureTCR^lowHSA⁺Qa-2^– double-positive cells and mature TCR^highHSA^–QA-2⁺single-positive cells. In addition, TCR engagement by MHC moleculesis required for CD69 expression in the thymus. Taken together,the CD69 ⁺ thymocytes appear to represent the cells auditioningin positive selection process or they are the cells that havebeen positively selected recently. Analysis of a TCR transgenicmouse model revealed an increased number of CD69⁺ thymocytesin a positively selecting thymus, whereas no CD69⁺ transgenicTCR⁺ thymocytes were observed in the non-selecting thymus. Basedon the results of this study, we suggest that the surface expressionof CD69 serves as a useful marker to identify and trace thosethymocytes that are engaged in the TCR-mediated positive selectionprocess in the thymus.     

Ligation of the CD5 or CD28 molecules on resting human T cells induces expression of the early activation antigen CD69 by a calcium- and tyrosine kinase-dependent mechanism.

The CD5 and CD28 molecules on T lymphocytes can each exert an accessory role in T-cell activation. Ligands for CD5 and CD28 have been identified as CD72 and B7/BB1 respectively. The function of, and the signal transduction pathways coupled to CD28 have been the subject of extensive studies. In contrast, it is still debated whether CD5 functions as a receptor which directly transduces an independent signal to the T cell. In this paper, it is reported that culture of purified T cells in the presence of either immobilized anti-CD5 monoclonal antibody (mAb) (OKT1, Leu-1 or 10.2) or cross-linked anti-CD28 (9.3) mAb (but not of anti-LFA-1 alpha, anti-LFA-1 beta, or anti-CD7) induces expression of CD69, an early activation marker, in the absence of other activating stimuli. CD69 expression was consistently detectable after 3-24 hr on 20-50% of T cells, within both the CD4 and CD8 subsets. CD45RO- CD45RA+ naive T cells were more responsive than CD45RO+ CD45RA- memory T cells. In the presence of recombinant (r) interleukin-2 (IL-2), anti-CD5- or anti-CD28- induced CD69 expression was further up-regulated, more sustained and, as previously shown, succeeded by IL-2 responsiveness. Simultaneous cross-linking of both CD5 and CD28 enhanced CD69 expression above the levels obtained with optimal amounts of both ligands separately. In the presence of a submitogenic dose of the protein kinase C (PKC) activating agent phorbol 12-myristate 13-acetate (PMA), co-stimulation with anti-CD5 or anti-CD28 increased CD69 expression above that induced by PMA alone. Cross-linking of CD5 or CD28 induces an early rise of cytoplasmic free calcium concentration ([Ca2+)]i) and both this rise and CD69 expression were inhibited by chelation of extracellular Ca2+ with ethyleneglycol-bis-(2-aminoethyl)-tetraacetate (EGTA). Pretreatment of the cells with the tyrosine kinase inhibitor herbimycin A also blocked CD69 expression. The data thus antigen-independent fashion. Moreover it is demonstrated that influx of Ca2+ and tyrosine kinase activity are involved in the signal transduction pathways of both receptors.     

Expression of early activation antigen (CD69) during human thymic development.

The novel early activation antigen, EA1, has been shown to be induced by mitogens, antigens and the tumour promoter, phorbol myristate acetate (PMA), on human lymphocytes. This antigen has been designated to be CD69. EA1 has also been shown to be expressed on thymocytes without exogenous activation stimuli. In order to characterize further the expression of EA1 on thymocytes, the ontogeny of its expression was studied. EA1 appeared between 7 and 9.5 weeks of gestation, after colonization of the thymic rudiment with CD7+ T cell precursors, but before the onset of compartmentalization of the thymus into cortical and medullary zones. After cortico-medullary differentiation, the majority of medullary thymocytes expressed EA1 while only a fraction of the cortical thymocytes expressed this antigen. In the fetal and post-natal cortex, EA1 expression appeared to cluster in the subcapsular cortex. EA1+ cells were also scattered throughout the inner cortex. By two-colour fluorocytometric analysis of post-natal thymocytes, it was shown that EA1 was expressed on 30 to 65% of thymocytes. EA1 was expressed on CD4+ CD8+ as well as on the more immature CD4- CD8- thymocytes. In contrast to circulating T cells, thymocytes were much less responsive to PMA stimulation for the expression of EA1. Molecular characterization showed that EA1 on thymocytes had the same structure as that of activated peripheral T cells. In addition, thymic EA1 was constitutively phosphorylated. Thus, EA1 expression is acquired early during thymic development after colonization of the thymic rudiment by CD7+ T cell precursors. However, the specific role that EA1 may play in the activation and function of developing thymocytes remains to be determined.     

A requirement for the CD5 antigen in T cell activation

Treatment of adult rats with a monoclonal antibody specific for the CD5 antigen led to a dramatic reduction in the number of CD5+ cells. However, a substantial number of T cells remained as assessed by other T cell-specific antibodies. These CD5- T cells did not proliferate in response to alloantigen or mitogenic stimulation, did not generate cytotoxic T lymphocytes in vitro, and did not induce graft-vs.-host disease when injected into susceptible recipients in vivo. Re-expression of the CD5 antigen occurred when CD5- T cells were placed in an environment devoid of the anti-CD5 antibody. Re-expression of the antigen was followed by return of the T cell proliferative responses. While CD5- T cells could not proliferate in response to alloantigen they could produce interleukin 2 following a short pulse with the T cell mitogen concanavalin A. However, T cell proliferative or cytotoxic responses could not be rescued by the addition of an exogenous source of interleukin 2. We conclude that the CD5 antigen appears to be required for proliferation of resting T cells.     

Molecular characterization of the early activation antigen CD69: A type II membrane glycoprotein related to a family of natural killer cell activation antigens

CD69 is a disulfide-linked homo-dimer expressed on the surface of activated T cells, B cells, natural killer cells, neutrophils and platelets. Antibody cross-linking of CD69 in the presence of phorbol ester results in cellular activation events including proliferation and the induction of specific genes. Using an expression cloning strategy we have isolated cDNA encoding human CD69 from a CD4⁺ T cell clone. Transfection of the cDNA clone in CV-1/EBNA cells results in the expression of a covalently linked homodimer. The cDNA insert hybridizes to a 1.7-kb mRNA in phorbol 12-myristate 13-acetate- or phytohemoagglutinin-stimulated human T cells. Using the human clone we have isolated cDNA encoding mouse CD69, which, when expressed in human T cells allowed those cells to respond to anti-mouse CD69 antibodies by secreting interleukin-2 and interferon-γ. Sequence analysis showed that both mouse and human CD69 are type II membrane glycoproteins related to the NKR-P1 and Ly-49 families of natural killer cell activation molecules.     

CD69 expression correlates with expression of other markers of Th1 T cell differentiation in peripheral T cell lymphomas

CD69, a marker of early T cell activation, is associated with Th1 T cell differentiation. Previously we found that peripheral T cell lymphomas could be subdivided based on the expression of markers of Th1 versus Th2 differentiation, including CXCR3, CD134/OX40, CCR4, and CD30. Here we report immunohistochemical staining for CD69 in frozen and paraffin sections of peripheral T cell lymphomas that exhibit immunoreactivity for markers of Th1 or Th2 differentiation. CD69 expression correlated with immunoreactivity for other Th1 differentiation markers in 18 of 19 frozen specimens of peripheral T cell lymphomas (P = 0.0005). In 10 of these cases in which paraffin-embedded tissue was available for study, CD69 immunohistochemical staining of paraffin sections correlated with frozen section expression. CD69 immunostaining was performed on paraffin sections from 53 additional cases of peripheral T cell lymphoma and correlated with immunoreactivity for other Th1 differentiation markers (P < 0.0001) and was associated with specific subtypes of peripheral T cell lymphoma, including angioimmunoblastic lymphoma, Lennert's lymphoma, and mycosis fungoides/Sezary syndrome, previously noted to express Th1 differentiation-associated markers. Anaplastic large cell lymphoma, both systemic and cutaneous, which typically exhibits immunoreactivity for markers of Th2 expression, was negative for CD69 immunostaining in 22 of 24 cases. CD69 immunostaining results support previous findings that a subset of T cell lymphomas exhibits immunophenotypic features of either Th1 or Th2 T cell differentiation. In addition, CD69 is a useful immunohistochemical marker for specific T cell lymphomas in frozen and paraffin-embedded tissue.     

Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes.

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.     

Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25

Rearrangements in the actin cytoskeleton play a pivotal role for costimulation-induced formation of the immunological synapse and T cell activation. Yet, little is known about the actin-binding proteins that link costimulation to rearrangements in the actin cytoskeleton. Here we demonstrate that phosphorylation of the actin bundling protein L-plastin in response to costimulation through TCR/CD3 plus CD2 or CD28, respectively, is important for the activation of human peripheral blood T lymphocytes (PBT). Mass spectrometry and site-directed mutagenesis revealed that Ser5 represents the only phospho-acceptor site of L-plastin in PBT. Wild-type L-plastin (wt-LPL) and a non-phosphorylatable 5A-L-plastin (5A-LPL) equally relocalized to the immunological synapse between PBT and APC. Yet importantly, cells expressing 5A-LPL showed a significantly lower expression of the T cell activation molecules CD25 and CD69 on the cell surface than cells expressing wt-LPL. This effect is due to a failure in the transport of CD25 and CD69 to the cell surface since the total amount of these proteins within the cells remained unchanged. In conclusion, phosphorylation of the actin bundling protein L-plastin represents a so-far-unknown mechanism by which costimulation controls the transport of activation receptors to the T cell surface.     

Alternative molecular form of human T cell-specific antigen CD27 expressed upon T cell activation

The CD27 membrane antigen is exclusively present on a large subset of human peripheral blood T lymphocytes and on mature thymocytes. Several observations point towards a specific role of this molecule on activated T cells. Upon T cell activation induced via the T cell receptor complex, CD27 expression greatly increases, while addition of anti-CD27 monoclonal antibodies amplifies the proliferative response. Within the CD4+ subset, only CD27+ T cells provide helper activity for B cell differentiation. Interestingly, CD27 expression differs not only quantitatively, but also qualitatively between resting and activated T cells. On resting cells, CD27 is a disulfide-linked homodimer with subunits of 55 kDa molecular mass. Upon activation, also a 55-kDa monomer seems to occur, while in addition a 32-kDa component is found. The relationship between these two proteins has been investigated. The 55-kDa and 32-kDa molecules do not seem to be physically associated. The two CD27 components are structurally highly homologous as determined by two-dimensional mapping of tryptic peptides. They both express the epitopes recognized by anti-CD27 antibodies, indicating that the 32-kDa molecule is also T cell specific. Both 55-kDa and 32-kDa molecules carry N-linked carbohydrate groups. However, they differ in other, not yet specified, post-translational modifications, and do not arise from a common precursor simply by alternative N-glycosylation. The 32-kDa form may be derived by proteolytic processing from the 55-kDa protein, but most likely not from a larger (common) precursor. Alternatively, both molecules may arise from a common gene by alternative mRNA splicing, or be derived from highly homologous genes. The dramatic change in molecular composition of CD27 suggests a newly acquired function for CD27 on activated T cells.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus , bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4⁺ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25⁺ and CD69⁺ lymphocytes were higher. Seventy-four percent of the CD25⁺ PBMC but only 55% of the CD69⁺ cells were CD3⁺ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69⁺ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

Expression of the CD69 activation antigen on lymphocytes of patients with hip prosthesis

The aim of the study was to evaluate the sensitization to metals in patients with Co-Cr hip prosthesis. Peripheral blood mononuclear cells (PBMC) were collected from 14 healthy donors and three groups of patients: 10 candidates for primary total joint replacements, 11 patients with well-fixed implant and 13 patients with aseptic loosening of the hip prosthesis. PBMCs were cultured with the metal ions employed for implant manufacturing and the expression of CD69 activation antigen on CD3/T lymphocytes was detected by flow cytometry. Chromium extract increased significantly the expression of CD3/CD69 phenotype in patients with loosening of hip prosthesis. The chromium-induced 'activation index' was higher in patients with loosening of hip prosthesis than in healthy donors and in pre-implant patients. The cobalt-stimulated PBMC of patients with either well-fixed or loosened prosthesis had an 'activation index' significantly higher than healthy donors. The activation index values were used to graduate the PBMC-response as 'normal' (> or = 0.9 and < 2), 'low' (< 0.9) and 'high' (> or = 2): an high-activation index was observed only in chromium-exposed PBMC of patients with prosthesis. Our data show that chromium released from orthopedic implants could be responsible for the lymphocyte sensitization and flow cytometry is an easy and reliable method for monitoring the hypersensitivity state in patients with metal prostheses. Activated lymphocytes in the peri-implant tissue are likely to elicit a localized immune response and contribute to maintain the inflammatory process evolving in the implant failure.     

Low-avidity CD8lo T cells induced by incomplete antigen stimulation in vivo regulate naive higher avidity CD8hi T cell responses to the same antigen

We have previously reported that multiple injections of soluble MHC class I tetramers assembled with wild-type HY peptide induces unresponsiveness to male skin grafts in naive female C57BL/6 (B6) mice. Induction of unresponsiveness is dependent on a population of unresponsive allospecific CD8(lo )T cells. Reduced expression of CD8 acts to limit a T cell response to HY peptide by limiting the avidity window of effective signal transduction. We and others have demonstrated that CD8(lo) T cells are an alternative stable phenotype of CD8alphabeta(+) T cells in vitro and in vivo after antigen stimulation. We show here that CD8(lo) T cells can suppress naive CD8(+) T cell responses to HY antigen in vitro and male skin graft rejection in vivo after adoptive transfer into female recipients. These novel regulatory T cells express surface TGF-beta1 and secrete T cytotoxic 2 cytokines after antigen-specific stimulation. Anti-TGF-beta antibody and latency-associated peptide inhibit the suppressive effects in vitro. We also show that HY-specific memory CD8(+) T cells overcome regulation by CD8(lo) T cells. These data define a novel peripheral regulatory CD8(+ )T cell population that arises after repeated antigen encounter in vivo. These cells have implications in the maintenance of tolerance and memory.     

Direct helper T cell-induced B cell differentiation involves interaction between T cell antigen CD28 and B cell activation antigen B7.

Cognate interactions between major histocompatibility complex class II antigen (Ag)-reactive CD4+ T helper (Th) and Ag-presenting B cells induce first the activation of B cells and their subsequent differentiation into Ig-secreting cells (IgSC). The Th cell-associated homodimeric glycoprotein CD28 has been implicated as an important regulator of Th activation. Recently, B cell-associated early activation Ag B7 has been identified as a ligand for the CD28 molecule. In this study, we have examined using monoclonal antibodies (mAb) the roles of CD28 and B7 molecules during the Th-B cell cognate interactions leading to the differentiation of B7+ B cells. Anti-CD28 mAb 9.3 specifically inhibited proliferative responses of CD4+ T cells to both allogeneic B cells and soluble Ag-presenting autologous non-T cells. In addition, anti-CD28 mAb 9.3 inhibited Th-induced differentiation of alloantigen-presenting B cells into ISC. Similar inhibition of both Ag-induced Th activation and B cell differentiation into ISC was observed using mAb BB1 which recognizes a B cell-associated molecule B7. In contrast, non-cognate Th-independent exogenous interleukin 6-induced differentiation of B7+ B cells into ISC was not inhibited by mAb to either molecule. These results clearly demonstrate the involvement of CD28 on Th and its ligand B7 on B cells during cognate Th-B interactions leading to the differentiation of B cells. Furthermore, these results also suggest the development of new mAb-based therapeutic approaches for exaggerated B cell activation associated with certain autoimmune diseases such as systemic lupus erythematosus.     

Regulation of D-3 phosphoinositides during T cell activation via the T cell antigen receptor/CD3 complex and CD2 antigens.

An immediate consequence of T cell activation via the T cell receptor (TcR)/CD3 complex and CD2 antigen is the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate and the generation of inositol-(1,4,5)-trisphosphate and diacylglycerol which then regulate intracellular calcium and protein kinase C. Changes in cellular levels of phosphoinositides phosphorylated on the D-4 and D-5 position during T cell activation have been well documented. Recently it has been proposed that phosphoinositides phosphorylated on the D-3 position of the inositol ring by a novel phosphoinositide (PI) 3 kinase may also be important in cell activation. In the present study we have examined the levels and regulation of D-3 phosphoinositides in T cells activated by the TcR/CD3 complex and CD2 antigens. The data show the existence of phosphatidylinositol-(3)-monophosphate [PtdIns(3)P], phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] in T cells. Activation of the TcR/CD3 complex or CD2 antigen results in modulation of PtdIns(3,4)P2 and a putative PtdIns(3,4,5)P3 in T cells but does not change levels of PtdIns(3)P. These data provide the first evidence that lipid products of a PI3 kinase exist in T cells.     

Functional expression of CD28 on T cell antigen receptor gamma/delta-bearing T lymphocytes

T lymphocytes express a T cell antigen receptor (TcR) complex composed of either an TcR alpha/beta or TcR gamma/delta heterodimer in noncovalent association with the CD3 glycoproteins. CD28, a 44-kDa disulfide-linked homodimer, is present on the surface of the majority of TcR alpha/beta-bearing T lymphocytes. Monoclonal antibodies (mAb) directed against CD28 potentiate activation signals delivered through the CD3/TcR alpha/beta complex. Herein, we demonstrate that CD28 is expressed on approximately 40%-60% of TcR gamma/delta-bearing T lymphocytes in most donors. Anti-CD28 mAb substantially augmented proliferative signals delivered through the TcR gamma/delta, demonstrating the presence of functional CD28 molecules on TcR gamma/delta-bearing T lymphocytes. The majority of TcR gamma/delta+ thymocytes also expressed CD28.     

The activation antigen CD69 is selectively expressed on CD8+ endomyocardium infiltrating T lymphocytes in human rejecting heart allografts.

We analyzed the presence of T-cell subsets (CD4/CD8) and the activation markers CD25 and CD69 in the cellular infiltrates of endomyocardium biopsies taken from transplanted human hearts. The results indicate that CD25 was present within specimens mainly infiltrated by CD4+ cells. In contrast, CD69 was found in infiltrated biopsies by CD8+ cells, as determined by single immunofluorescence. Double immunoenzymatic staining was used to investigate the cellular distribution of the activation markers studied in some representative cases. Thus, CD25 was found on both CD4+ and CD8+ cells while CD69 molecule was selectively expressed on CD8+ T-cell subset. These results suggest that CD69 is a surface molecule relevant to the CD8+ cell-mediated graft rejection events of allografted human hearts.     

CD69在细胞活化、凋亡中的双向免疫调节作用

分化抗原簇69(cluster of differentiation 69,CD69)于1981年发现,早期被称为活化诱导分子(activation inducer molecule,AIM),早期活化抗原-1(early activation antigen-l,EA-1)等,是C-型凝集素受体家族的成员,CD69基因属于自然杀伤细胞（natural killer cell,NK细胞)信号转导基因复合体家族,在细胞的生物功能调节中起着重要的信号转导作用.