CD3zeta and CD28 down-modulation on CD8 T cells during viral infection

Down-modulation of CD3zeta expression on CD8 T lymphocytes occurs, independently of other T-cell receptor (TCR)-CD3 components, in tumor-infiltrating lymphocytes, human immunodeficiency virus infection, and autoimmune disease. These associations suggest that it might be related to chronic antigenic stimulation. CD3zeta down-modulation was found, however, in CD8 T cells that proliferate in response to acute viral infections. In 3 otherwise healthy donors with acute gastroenteritis, infectious mononucleosis, and Epstein-Barr virus/cytomegalovirus/mononucleosis, 30% to 60% of circulating CD8 T cells had down-modulated CD3zeta to below the level of detection. The CD3zeta-T cells were also CD28- but expressed the activation markers HLA-DR and CD57. CD3zeta-CD28- T cells are effector CTL because they express perforin and produce IFN-gamma, but not IL-2, on activation and contain the viral-specific cytotoxic T lymphocyte (CTL). However, CD3zeta-CD28-T cells generally do not express CD25 after anti-CD3 and anti-CD28 stimulation and are not cytotoxic until they are cultured with IL-2 overnight. Cytotoxicity coincides with the re-expression of CD3zeta but not CD28. Down-modulation of CD3zeta and CD28 on effector CTL may control CTL triggering and proliferation to prevent immunopathogenesis.     

Decreased expression of CD28 coincides with the down-modulation of CD3zeta and augmentation of caspase-3 activity in T cells from hepatocellular carcinoma-bearing patients and hepatitis C virus-infected patients

BACKGROUND AND AIM: Hepatitis C virus (HCV) infection causes chronic inflammation and increases the risk of hepatocellular carcinoma (HCC). This immunosuppressive state may be one reason why HCV-infected patients often have multicentric cancers. Therefore, the purpose of the present study was to assess the cellular immune function in HCC-bearing and HCV-infected patients. METHODS: The expression of cluster of differentiation (CD)3zeta, CD28 and caspase-3 activity of peripheral blood T lymphocytes (PBL) from HCC-bearing patients, HCV-infected patients and normal subjects was measured by flow cytometric methods. Furthermore, intrahepatic T lymphocytes (IHL) and tumor-infiltrating T lymphocytes (TIL) from HCC patients were used. RESULTS: Decreased expressions of CD3zeta, CD28 and the augmentation of caspase-3 activity were recognized in PBL from HCC and HCV patients. These phenomena were more dominant in TIL and IHL than in PBL in HCC patients. Furthermore, the down-modulation of CD3zeta and increased caspase-3 activity occurred in CD28 down-modulated T cells. CONCLUSION: These results demonstrate impairment of the cellular immune system in HCC and HCV patients from the viewpoints of the down-modulation of CD3zeta and CD28 on T cells and T-cell apoptosis. In addition, the results imply that the down-modulation of CD3zeta and T-cell apoptosis take place in activated T cells.     

CD22-mediated stimulation of T cells regulates T-cell receptor/CD3-induced signaling.

Interaction between B lymphocytes and other cell types is mediated in part by the B-cell adhesion molecule CD22. Recent work has suggested one of the T-cell ligands of B cells to be CD45RO, an isoform of the receptor-linked phosphotyrosine phosphatase CD45. Here we demonstrate direct interaction between CD22 and several isoforms of CD45, including CD45RO, and propose that the interaction may participate in regulation of lymphocyte signaling. Cross-linking of CD3 and CD22 T-cell ligands with anti-CD3 antibody and soluble CD22 is shown to block anti-CD3-induced intracellular calcium increase and to inhibit tyrosine phosphorylation of phospholipase C gamma 1. These effects are consistent with those observed upon coligation of CD3 and CD45 with antibody, providing support to the possibility that ligand-mediated stimulation of CD45 may result in modulation of substrate phosphorylation and lymphocyte activation.     

Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function

Recent preclinical and clinical trials have demonstrated the therapeutic potential of T lymphocytes redirected with genetically engineered T-cell receptor (TCR) surrogates against infected, cancerous, or autoreactive cells. These surrogate TCRs link a ligand-recognition domain to signaling regions from the TCR. We previously compared the function of surrogate TCRs that include TCR or TCR and CD28 signaling regions. We found that primary murine T cells modified to specifically target Kb-restricted CD8+ T cells using either Kb-zeta or Kb-CD28-zeta receptors had similar functional activities, although the CD28-zeta receptor showed a 2-fold to 4-fold decreased expression. We have now identified a previously unrecognized dileucine motif in the murine CD28 signaling domain that accounts for this reduced expression. Inactivation of this motif increased chimeric receptor surface expression 2- to 5-fold. T cells expressing the dileucine-mutated CD28-zeta chimeric receptor demonstrated enhanced proliferation, cytokine production, and cytolytic activities. Further, cells expressing this dileucine-mutated receptor were highly effective in eliminating antigen-specific CD8+ T lymphocytes in vivo. These results therefore identify a critical motif limiting the function of receptor-modified T lymphocytes, demonstrate that inactivation of this motif enhances chimeric receptor function, and illustrate a potential novel application of receptor-modified T lymphocytes in the induction of immune tolerance.     

Alterations in the expression pattern of TCR zeta chain in T cells from patients with hematological diseases

The TCR zeta chain, a component of the T cell receptor (TCR)/CD3 complex, plays a significant role in the assembly of the receptor complex and in connecting antigen recognition to the intracellular signal transduction apparatus. Recently, studies have demonstrated altered expression and function of this signal transduction molecule in T cells from patients with hematological diseases. In this review, current knowledge concerning the biological feature and function of TCR zeta protein, splice variant and mutation of TCR zeta chain gene and alteration of expression pattern in hematological diseases and the related mechanism are summarized.     

Correlation between disease activity and T-cell CD3 chain expression in a B-cell lymphoma

Summary. The T-cell receptor (TCR)/CD3 complex is the surface molecule responsible for the highly specific response to foreign antigens, including tumour-associated antigens. T-cell responses are frequently defective in cancer patients, and correlations have been observed with disease status. Recently, low amounts of CD3γ and CD3 chains in the CD3/TCR complex have been reported in long-term tumour-bearing mice as a possible mechanism for the impaired T-cell responses. In this study we report a B-cell lymphoma in which T-cell CD3 expression correlated with disease activity.     

Deficiency of CD3gamma, delta, epsilon, and zeta expression in T cells from AML patients

In order to elucidate the feature of T-cell receptor (TCR) signal transduction in T-cells from acute myeloid leukemia (AML), the expression levels of CD3gamma, delta, epsilon and zeta chain genes in CD3+ T cells were analyzed using real-time PCR. CD3+ T cells sorted from peripheral blood of 10 AML patients and 10 healthy donors were used in the study. Significantly lower expression levels of all four CD3gamma, delta, epsilon, and zeta chain genes were found in the AML samples. The expression pattern of the four CD3 chains was epsilon>gamma>delta>zeta in CD3+ T cells from AML samples, which was different from the healthy control group. In conclusion, the results provide a global gene expression profile of CD3gamma, delta, epsilon, and zeta chains in AML patients. Deficiency of all four CD3 gene expression levels might represent the feature related to T-cell immunodeficiency.     

Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype

Thymic-derived natural T-regulatory cells (nTregs) are important for the induction of self-tolerance and the control of autoimmunity. Murine CD4+CD25(-)Foxp3(-) cells can be induced to express Foxp3 after T-cell receptor (TCR) activation in the presence of transforming growth factor beta (TGFbeta) and are phenotypically similar to nTregs. Some studies have suggested that TCR stimulation of human CD4+CD25(-) cells results in the induction of transient expression of FOXP3, but that the induced cells lack a regulatory phenotype. We demonstrate here that TCR stimulation alone was insufficient to induce FOXP3 expression in the absence of TGFbeta, whereas high levels of FOXP3 expression could be induced in the presence of TGFbeta. Although FOXP3 expression was stable, the TGFbeta-induced FOXP3+ T cells were neither anergic nor suppressive and produced high levels of effector cytokines. These results suggest that even high levels of FOXP3 expression are insufficient to define a human CD4+ T cell as a T-regulatory cell.     

Molecular cloning and chromosomal localization of the human T-cell receptor zeta chain: distinction from the molecular CD3 complex.

The T-cell antigen receptor (TCR) is a multisubunit receptor complex specific to T cells subserving both antigen recognition and signal transduction functions. The zeta chain of the TCR is a component of all surface receptor complexes. This chain was first identified in murine T cells by virtue of the fact that it coimmunoprecipitates with the TCR complex using antibodies directed against either the clone-specific subunits or invariant CD3 subunits of the receptor. Recently, we have isolated a cDNA encoding the murine zeta. Using this as a probe, we have now isolated cDNAs encoding the human zeta. Sequence analysis of cDNAs encoding human and murine zeta reveals that it is a highly conserved protein. In addition to amino acid homology, there is remarkable interspecies conservation in the nucleotide sequence of the 5' and 3' untranslated regions of the zeta mRNA. The previously characterized invariant delta, epsilon, and gamma chains of the TCR, referred to as the CD3 complex, share significant sequence and structural homology with each other and are all located within 300 kilobases of each other on human chromosome 11 (11q23). zeta has no sequence similarity to the CD3 chains and the localization of the human zeta gene to the centromeric region of chromosome 1 underscores the fact that it is a distinct genetic component of the TCR.     

The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein.

Stimulation of the T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including phospholipase C (PLC) gamma 1 and the TCR zeta chain. We describe here a 70-kDa tyrosine phosphoprotein (ZAP-70) that associates with zeta within 15 sec following TCR stimulation. The phosphorylation of ZAP-70 and its association with zeta is independent of the other TCR chains since stimulation of a functional CD8/zeta chimeric receptor in a TCR-negative T cell leads to coprecipitation of ZAP-70 with the chimeric protein. In a Jurkat cell expressing the TCR and the CD8/zeta chimeric protein, tyrosine phosphorylation and association of ZAP-70 occurs exclusively with the stimulated receptor complex. In addition, a tyrosine kinase that does not appear to be fyn associates with the cytoplasmic domain of zeta and phosphorylates zeta and ZAP-70 in vitro.     

Reconstitution of deficient T cell receptor zeta chain restores T cell signaling and augments T cell receptor/CD3-induced interleukin-2 production in patients with systemic lupus erythematosus

OBJECTIVE: T cells from a majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR zeta chain may reverse the known signaling abnormalities and defective interleukin-2 (IL-2) production in SLE T cells. METHODS: Freshly isolated SLE T cells were transfected with TCR zeta chain construct in a eukaryotic expression vector at high efficiency, by a recently developed nucleoporation technique. Restoration of TCR/CD3-mediated signaling was studied in the zeta chain-transfected cells. RESULTS: In SLE T cells transfected with TCR zeta chain, surface expression of TCR chain was increased and the TCR/CD3-induced increased free intracytoplasmic calcium concentration response was normalized, as was hyperphosphorylation of cellular substrates. Simultaneously, the previously noted increased expression of the Fc receptor gamma chain was diminished in SLE T cells transfected with the zeta chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR zeta chain replacement also augmented the expression of diminished TCR/CD3-mediated IL-2 production in SLE T cells, associated with increased expression of the p65 subunit of nuclear factor kappaB in the nuclear fractions of these T cells. CONCLUSION: These results suggest that reconstitution of deficient TCR zeta chain can reverse the TCR/CD3-mediated signaling abnormalities as well as the defective IL-2 production in T cells of patients with SLE.     

Coclustering of CD4 (L3T4) molecule with the T-cell receptor is induced by specific direct interaction of helper T cells and antigen-presenting cells

Blocking studies with monoclonal antibodies have suggested that helper T cell recognition and triggering involve the CD4 (L3T4) accessory molecule as well as the T-cell receptor (TCR) that is linked to the T3 complex. We have investigated the surface distribution of L3T4 and TCR during the direct interaction of a cloned murine helper T-cell line with an antigen-presenting B-cell line. Using immunofluorescence microscopy, we show that in 1:1 cell couples formed between the two cells, in which a specific interaction can be demonstrated, the L3T4 and the TCR become redistributed on the T-cell surface so that they are concentrated in the cell-cell contact region. This coclustering of L3T4 with TCR occurs only when the relevant antigen and appropriate major histocompatibility class II molecules are presented to the T cell, and it therefore requires the specific interaction of the TCR with its complex ligand on the antigen-presenting cell.     

Instability of assembled T-cell receptor complex that is associated with rapid degradation of zeta chains in immature CD4+CD8+ thymocytes.

The intracellular fate of newly synthesized T-cell receptor (TCR) chains was compared in CD4+CD8+ (double positive; DP) thymocytes and in CD4+CD8- or CD4-CD8+ (single positive; SP) thymocytes. Purified DP and SP thymocytes from normal adult mice were analyzed by pulse-chase metabolic labeling and immunoprecipitation with specific anti-TCR antibodies. Biosynthesis of invariant chains (CD3 gamma, -delta, -epsilon, and zeta) was comparable between DP and SP thymocytes, whereas DP thymocytes synthesized TCR alpha and TCR beta chains at lower and higher levels than SP thymocytes, respectively. These newly synthesized TCR chains were degraded at different rates in SP thymocytes based on their sensitivities for degradation as previously reported: TCR alpha, TCR beta, CD3 gamma, and CD3 delta chains were rapidly degraded and CD3 epsilon and zeta chains were stable. Although the degradation rates of clonotypic and invariant CD3 chains were similar in DP and SP thymocytes, the zeta subunit was rapidly degraded in DP thymocytes (t1/2, approximately 1.5 hr). Degradation of zeta was inhibited by NH4Cl, implicating lysosomes as the site of degradation. Comparison of TCR subunit assembly in DP and SP thymocytes demonstrated that, despite the same relative rate of formation of TCR complexes in a pulse period (30 min), complete complexes were unstable and degraded during the subsequent 6 hr of chase in DP thymocytes. This contrasted with the stability and a progressive increase in the levels of completely assembled complexes in SP thymocytes. Thus, these results demonstrate that a unique posttranslational regulation operates in the formation of TCR complexes in DP thymocytes and that lack of stability of complete TCR complexes is a crucial mechanism that may account for the limited surface TCR expression on this thymocyte subset.     

CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.     

Abnormal expression of various molecular forms and distribution of T cell receptor zeta chain in patients with systemic lupus erythematosus

OBJECTIVE: T cells from the majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain expression. The TCR zeta chain, a critical signaling molecule, exists in multiple molecular forms and membrane fractions with distinct functions in antigen-mediated signaling processes. This study was undertaken to investigate the complete spectrum of expression of the different forms and distribution of the TCR zeta chain in SLE T cells. METHODS: T cells were isolated from 48 SLE patients and 21 healthy subjects. The expression of various forms of the TCR zeta chain was investigated by immunoblotting with specific antibodies. The lipid raft-associated form of the zeta chain was determined by quantitating the solubilized zeta chain after disruption of the lipid rafts by cholesterol depletion using methyl-betacyclodextrin. The distribution of the zeta chain was investigated by fluorescence microscopy. RESULTS: The phosphorylated 21- and 23-kd forms and the detergent-insoluble membrane-associated form of the TCR zeta chain and alternatively spliced zeta chain were significantly decreased in SLE T cells. In contrast, major ubiquitinated forms of the zeta chain were increased in these cells. We also identified up-regulation of a novel 14-kd form of the zeta chain in SLE T cells. Resting SLE T cell membranes had an increased percentage of the residual membrane-bound zeta chain in the lipid rafts. Fluorescence microscopy findings indicated that the residual zeta chain is more clustered on the cell membranes of SLE T cells. CONCLUSION: These results suggest that, in addition to the 16-kd form, expression of other molecular forms and fractions of the TCR zeta chain as well as its membrane distribution are abnormal in SLE T cells. Increased lipid raft association and surface clustering of the zeta chain may explain the molecular mechanisms underlying the signaling abnormalities in these cells.     

Enhanced expression of interleukin-18 receptor alpha chain by CD4+ T cells in sarcoidosis

STUDY OBJECTIVES: To investigate the expression of interleukin-18 receptor alpha chain (IL-18Ralpha) in BAL and peripheral blood (PB) T cells in patients with sarcoidosis compared with control subjects, to evaluate the relationship between the expression and clinical manifestations, and to clarify the mechanisms of altered expression. SUBJECTS AND METHODS: The study subjects consisted of 21 patients with sarcoidosis and 8 normal control subjects. The expression of IL-18Ralpha was examined by flow cytometry. RESULTS: The proportions of BAL CD4+ and PB CD4+ T cells expressing IL-18Ralpha were significantly increased in patients with sarcoidosis compared to control subjects. BAL CD4+ T cells expressed IL-18Ralpha in a higher proportion than did paired CD8+ T cells in patients with sarcoidosis but not in control subjects. Greater proportions of BAL CD4+ T cells and BAL CD8+ T cells than of their PB counterparts expressed IL-18Ralpha in both patients and control subjects. CD4+ T cells were more sensitive to the induction of IL-18Ralpha by cytokines in vitro, such as interleukin (IL)-2, IL-12, and tumor necrosis factor-alpha than were CD8+ T cells. Increased expression of IL-18Ralpha by BAL T cells commonly observed in patients and control subjects was associated with the expansion of CD45RO+ cells in BAL T cells. However, there were no significant correlations between the expression of IL-18Ralpha by any cell populations and BAL findings, serum angiotensin-converting enzyme activities, radiograph stages, or clinical courses. CONCLUSION: The overexpression of IL-18Ralpha predominantly by CD4+ T cells in sarcoidosis emphasizes crucial roles played by T-helper type 1 cells in the IL-18/IL-18Ralpha system in sarcoidosis.     

T-cell expansions in patients with multiple myeloma have a phenotype of cytotoxic T cells

The presence of T-cell clones in peripheral blood has been previously shown to be associated with a survival advantage in patients with multiple myeloma and suggests that the expanded T-cell populations may be involved in an anti-tumour response. We studied the T-cell receptor (TCR) repertoire of 38 patients with myeloma to identify and characterize the expanded T-cell populations by flow cytometry. T-cell expansions were found in 79% of the patients. The expansions occurred randomly among the 21 variable regions of the TCR beta chain (Vbeta) studied, representing 62% of the V-beta repertoire, and were stable during an 18-month follow-up. The phenotype of the expanded V-beta populations was predominantly CD8+, CD57+, CD28- and perforin+, which differed significantly from the other non-expanded Vbeta populations. The expression of the apoptosis markers Fas (CD95) and bcl-2 were similar between the expanded and non-expanded Vbeta populations. In conclusion, expanded T-cell populations were frequent in patients with myeloma, they remained unchanged during follow-up and had phenotypic characteristics of cytotoxic T cells. These data add further support to the concept that the T-cell expansions may have an immunoregulatory role in myeloma.     

Altered T-cell receptor + CD28-mediated signaling and blocked cell cycle progression in interleukin 10 and transforming growth factor-beta-treated alloreactive T cells that do not induce graft-versus-host disease

The induction of anergy in T cells, although widely accepted as critical for the maintenance of tolerance, is still poorly understood at the molecular level. Recent evidence demonstrates that in addition to blockade of costimulation using monoclonal antibodies (mAbs) directed against cell surface determinants, treatment of mixed lymphocyte reaction (MLR) cultures with interleukin 10 (IL-10) and transforming growth factor-beta (TGF-beta) results in induction of tolerance, rendering alloreactive murine CD4(+) T cells incapable of inducing graft-versus-host disease (GVHD) after in vivo transfer to histoincompatible recipients. The present study, using these cells prior to adoptive transfer, determined that IL-10 + TGF-beta-tolerant CD4(+) T cells exhibit an altered pattern of T-cell receptor (TCR) + CD28-mediated signaling and are incapable of progressing out of the G(1) phase of the cell cycle during stimulation with HLA class II disparate antigen-presenting cells. TGFbeta + IL-10-tolerant cells were incapable of phosphorylating TCR-zeta, or activating ZAP-70, Ras, and MAPK, similarly to T-cell tolerized by blockade of B7/CD28 and CD40/CD40L pathways. Moreover, these cells were incapable of clonal expansion due to defective synthesis of cyclin D3 and cyclin A, and defective activation of cyclin-dependent kinase (cdk)4, cdk6, and cdk2. These cells also exhibited defective down-regulation of p27(kip1) cdk inhibitor and lack of cyclin D2-cdk4 activation, Rb hyperphosphorylation, and progression to the S phase of the cell cycle. These data link anergy-specific proximal biochemical alterations and the downstream nuclear pathways that control T-cell expansion and provide a biochemical profile of IL-10 + TGF-beta-tolerant alloreactive T cells that do not induce GVHD when transferred into MHC class II disparate recipients in vivo.