A splice variant of the TCR zeta mRNA lacking exon 7 leads to the down-regulation of TCR zeta, the TCR/CD3 complex, and IL-2 production in systemic lupus erythematosus T cells

The reduction or absence of TCR zeta-chain (zeta) expression in patients with systemic lupus erythematosus (SLE) is thought to be a factor in the pathogenesis of SLE. We previously reported a splice variant of zeta mRNA that lacks the 36-bp exon 7 (zeta mRNA/exon 7(-)) and is accompanied by the down-regulation of zeta protein in T cells from SLE patients. In this study, we show that EX7- mutants (MA5.8 cells deficient in zeta protein that have been transfected with zeta mRNA/exon 7(-)) exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab, compared with that in wild-type (WT) mutants (MA5.8 cells transfected with the WT zeta mRNA). Furthermore, real-time PCR analyses demonstrated that zeta mRNA/exon 7(-) in EX7- mutants was easily degraded compared with zeta mRNA by the WT mutants. Pulse-chase experiment showed zeta protein produced by this EX7- mutants was more rapidly decreased compared with the WT mutants. Thus, the lower stability of zeta mRNA/exon 7(-) might also be responsible for the reduced expression of the TCR/CD3 complex, including zeta protein, in SLE T cells.     

TCR zeta mRNA with an alternatively spliced 3'-untranslated region detected in systemic lupus erythematosus patients leads to the down-regulation of TCR zeta and TCR/CD3 complex

The reduction or absence of TCR zeta-chain (zeta) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of zeta mRNA containing an alternatively spliced 3'-untranslated region (3'UTR; zetamRNA/as-3'UTR) and a reduction in the expression of zeta mRNA containing the wild-type 3'UTR (zetamRNA/w-3'UTR) in T cells from SLE patients. Here we show that AS3'UTR mutants (MA5.8 cells deficient in zeta protein that have been transfected with zetamRNA/as-3'UTR) exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab compared with that in wild-type 3'UTR mutants (MA5.8 cells transfected with zetamRNA/w-3'UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of zetamRNA/as-3'UTR in AS3'UTR mutants (3 h) was much shorter than that of zetamRNA/w-3'UTR in wild-type 3'UTR mutants (15 h). Thus, the lower stability of zetamRNA/as-3'UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including zeta protein, in SLE T cells.     

Polymorphisms in the CD3Z gene influence TCRzeta expression in systemic lupus erythematosus patients and healthy controls

TCRzeta (CD247) functions as an amplification module in the TCR signaling cascade and is essential for assembly and surface expression of the TCR/CD3 complex. The TCRzeta-chain is down-regulated in many chronic infectious and inflammatory diseases, including systemic lupus erythematosus (SLE). It is unclear whether reduced TCRzeta expression is a cause or a consequence of chronic inflammatory responses. We have addressed this question by adopting a combined genetic and functional approach. We analyzed TCRzeta protein expression using a FACS-based expression index and documented considerable, but longitudinally stable, variation in TCRzeta expression in healthy individuals. The variation in TCRzeta expression was associated with polymorphisms in the CD3Z 3'-untranslated region (UTR) in SLE patients and healthy controls. Detailed mapping of the 3'-UTR revealed that the minor alleles of two single nucleotide polymorphisms (SNPs) in strong disequilibrium (rs1052230 and rs1052231) were the causal variants associated with low TCRzeta expression (p=0.015). Using allelic imbalance analysis, the minor alleles of these 3'-UTR SNPs were associated with one-third of the level of mRNA compared with the major allele. A family-based association analysis showed that the haplotype carrying the low-expression variants predisposes to SLE (p=0.033). This suggests that a genetically determined reduction in TCRzeta expression has functional consequences manifested by systemic autoimmunity.     

The CD3 zeta subunit contains a phosphoinositide-binding motif that is required for the stable accumulation of TCR-CD3 complex at the immunological synapse

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.     

CD3- T cells with cis- or trans-acting mutations affecting expression of T cell receptor beta-chain mRNA.

Mutants of an untransformed T cell clone that no longer respond to TCR/CD3 stimulation have been derived using a selection procedure based on the loss of functional response to Ag. This functional selection gives rise to clones of several different phenotypes. We have previously described mutants with a TCR/CD3+ cell surface phenotype whose TCR are uncoupled from cellular responses. We describe six additional mutants that do not express TCR/CD3 at the cell surface. One of the CD3- clones contains a deletion in the successfully rearranged TCR-alpha gene, whereas another carries a deletion in the successfully rearranged TCR-beta gene. TCR/CD3 expression in these deletion mutants can be restored by transfection of TCR-alpha or TCR-beta DNA. Four other clones do not express TCR-beta mRNA, yet contain no obvious deletions or rearrangements in the TCR-beta genes. One of these clones does not transcribe TCR-beta chain mRNA. The mutation in this clone does not reside in the TCR-beta gene itself, but may instead reside in a trans-acting regulatory element affecting TCR-beta gene expression, because the TCR-beta mRNA-phenotype is complemented by fusion with a TCR-alpha-beta- cell line. TCR-beta chain regulatory mutants will be valuable in contributing to our understanding of how TCR expression is regulated.     

Mutation in splicing consensus sequences causes lack of TCR membrane expression due to exon excision

 T-cell antigen receptor (TCR) membrane-negative T-cell mutants can be divided into two groups: 1) those which lack one of the six TCR polypeptides and 2) those which contain a mutated TCR chain. The present experiments reveal a new mechanism for the development of TCR membrane-negative T-cell variants: mutations in splicing consensus motifs causing excision or misreading of an entire exon (exon 3 of the TCRAC or TCRBC genes). C27.15 cells transcribe a TCR α chain consisting of TCRAVJCexon1Cexon2-encoded amino acids plus six new amino acids. The assembly defect seems to be that the truncated α chain does not interact with CD3 δ molecules; consequently, no TCR αβ/CD3 δεγε complexes are formed. E6.E12 cells transcribe a TCR β chain composed of TCRBVDJCexon1Cexon2-encoded amino acids plus twenty-seven new amino acids, which seem not to form a transmembrane region. The truncated β chain does associate with CD3 γε heterodimers, yet no TCR αβ/CD3 δεγε complexes are made. This may be due either to low assembly of TCR β/CD3 γε trimers or to lack of access of the mutated TCR β/CD3 γε trimers to the TCR α/CD3 δε compartment in the endoplasmic reticulum. Received: 25 September 1996 / Revised: 7 November 1996     

A single cell analysis of TCR AV24AJ18+ DN T cells.

The T-cell receptor (TCR) BV gene of human TCR AV24+ double-negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single-cell sorting and single-cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24+ DN T-cell clones utilized TCR BV8, three BV9, and one BV6. Six of seven TCR AV24/BV8+ DN T-cell clones had identical TCR beta and alpha chains, indicating that they were the same clone. All three TCR AV24/BV9+ DN T-cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR beta and alpha chains on TCR AV24+ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T-cell epitopes on antigens. All TCR AV24+ clones expressed the NKR-P1A mRNA, and so were true NK T cells. IL-2 and IL-4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0-type T cells. Six clones overexpressed Fas-ligand (Fas-L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24+ DN T cells might recognize restricted T-cell epitopes on antigens and function as Th0-type T cells, inducer cells to Th1- or Th2-type T cells (regulatory T cells), and as Fas-L-positive cytolytic T cells.     

Analysis of the individual role of the TCRzeta chain in transgenic mice after conditional activation with chemical inducers of dimerization

Signaling through the TCR/CD3 complex plays a critical role in T-cell development and activation. Gene-targeted mice lacking particular components of this complex show arrested T-cell development in the thymus. As all TCR/CD3 components are required for efficient surface expression of the complex, it is difficult to assess the specific signaling role of each receptor component. To overcome this problem, we designed a strategy to examine the specific role(s) of individual receptor chains. A chimeric protein, containing binding domains for chemical inducers of dimerization fused to the cytoplasmic tail of TCRzeta, was generated. Activation of the chimeric receptor after stimulation with chemical dimerizers in Jurkat cells showed tyrosine phosphorylation of the TCRzeta chain chimera, recruitment of phosphorylated Zap70, and generation of NFAT in a reporter assay. Analysis of thymocytes from transgenic mice expressing this chimeric receptor showed that intracytoplasmic crosslinking of the chimera induced tyrosine phosphorylation of the protein, as well as a slow and very weak calcium mobilization response. However, this signaling did not lead to increased expression of activation markers, T-cell proliferation, or apoptosis. In addition, stimulation of thymocytes in suspension or in fetal thymic organ cultures with chemical inducers of dimerization did not lead to alterations in positive or negative selection. We conclude that signaling through the TCRzeta chain alone is not sufficient to generate downstream events leading to full T-cell activation or thymocyte selection; instead, additional CD3 components must be required to induce a functional response in primary thymocytes and peripheral T cells.     

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis,but enhances DNA polymerase ζ – dependent spontaneous mutagenesis

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ.     

Circulating TCR gammadelta cells in the patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a disorder with a wide range of immunological abnormalities. The results of the studies undertaken in the last decade indicated that SLE pathogenesis was mainly connected with the breakdown of the activation control of B and T cells, generating humoral or cell-mediated responses against several self-antigens of affected cells. The last studies demonstrate that the role of gammadelta T lymphocytes in autoimmune diseases can be especially important. Flow cytometry techniques were used to investigate the number and percentage of TCR gammadelta T cells and their most frequent subtypes in peripheral blood of 32 patients with SLE and 16 healthy volunteers. We also correlated TCR gammadelta cells number with the level of T CD3+, T CD4+, T CD8+, and NK (CD16) cells (cytometric measurements) and SLE activity (on the basis of clinical investigations). Our studies were preliminary attempts to evaluate the role of that minor T cell subpopulation in SLE. Absolute numbers of cells expressing gammadelta TCR in most SLE blood specimens were significantly lower than in the control group (P<0.006). However, since the level of total T cell population was also decreased in the case of SLE, the mean values of the percentage gammadelta T cells of pan T lymphocytes were almost the same in both analysed populations (7.1% vs 6.3%, respectively). In contrast to Vdelta2+ and Vgamma9+ subtypes of pan gammadelta T cells, Vdelta3+ T cells number was higher in SLE patients (20 x 10 cells/microl) than in healthy control group (2 x 2 cells/microl) (P=0.001). However, we found no differences between the numbers of pan gammadelta T lymphocytes and studied their subtypes in the patients with active and inactive disease. These cell subpopulations were doubled in the treated patients with immunosuppressive agents in comparison with untreated ones; however, data were not statistically significant. Our study indicated that Vdelta3+ subtype of gammadelta T cells seems to be involved in SLE pathogenesis; however, we accept the idea that the autoimmunity does not develop from a single abnormality, but rather from a number of different events.     

The CysB motif of Rev3p involved in the formation of the four‐subunit DNA polymerase ζ is required for defective‐replisome‐induced mutagenesis

Eukaryotic DNA replication is performed by high‐fidelity multi‐subunit replicative B‐family DNA polymerases (Pols) α, δ and ?. Those complexes are composed of catalytic and accessory subunits and organized in multicomplex machinery: the replisome. The fourth B‐family member, DNA polymerase zeta (Pol ζ), is responsible for a large portion of mutagenesis in eukaryotic cells. Two forms of Pol ζ have been identified, a hetero‐dimeric (Pol ζ₂) and a hetero‐tetrameric (Pol ζ₄) ones and recent data have demonstrated that Pol ζ₄ is responsible for damage‐induced mutagenesis. Here, using yeast Pol ζ mutant defective in the assembly of the Pol ζ four‐subunit form, we show in vivo that [4Fe‐4S] cluster in Pol ζ catalytic subunit (Rev3p) is also required for spontaneous (wild‐type cells) and defective‐replisome‐induced mutagenesis – DRIM (pol3‐Y708A, pol2‐1 or psf1‐100 cells), when cells are not treated with any external damaging agents.     

Low frequency of CD8+CD25+FOXP3(BRIGHT) T cells and FOXP3 mRNA expression in the peripheral blood of allergic asthma patients

The aim of this study is to determine whether frequencies of CD8+CD25+ T cells and FoxP3 messenger RNA (mRNA) expression levels in CD8+ T cells isolated from peripheral blood are related to allergic asthma and disease severity. We enrolled 50 patients with allergic asthma (AA) and 25 healthy control subjects (NC) in our study. The frequencies of CD8+CD25+FoxP3 -/+ T cells were assessed with flow cytometry, and mRNA FoxP3 level in CD8+ T cells was determined with real time polymerase chain reaction (RT-PCR). Asthma patients had fewer CD8+CD25+FoxP3bright T cells [SA (median = 3.4%, IQR = 3.1) vs MA (median = 7.5%, IQR = 4.7)] than controls NC [median = 12.1 %, IQR = 8, P < 0.0001] but more CD8+CD25+FoxP3- T cells [SA (median = 96 %, IQR = 3.1) vs MA (median = 92.5%, IQR = 4.7)] than controls NC [median = 87.9%, IQR = 9.2, P < 0.0001]. FoxP3 mRNA level was significantly decreased in CD8+ T cells of severe asthma patients (median = 0.82, IQR = 0.54) than that of patients with mild to moderate asthma and controls [(median = 2.29, IQR = 4.40) vs (median = 2.11, IQR = 3.2)]. The percentage of FoxP3+ T cells was correlated positively with the percentage of forced expiratory volume in 1 second (FEV1) (r = 0.71, p< 0.01) in patients with severe asthma. The proportion of CD8+CD25+FoxP3bright T cells and the level of FOXP3 gene expression in CD8+ T cells are relevant to allergic asthma and disease severity. The manipulation of FoxP3+CD25+CD8+ T cells may prevent chronic allergic inflammation and improve lung function during an acute allergic asthma exacerbation.     

Virus-specific CD8+ T-cell responses in mice transgenic for a T-cell receptor beta chain selected at random.

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.     

Differential regulation of T-cell receptor processing and surface expression affected by CD3 theta, an alternatively spliced product of the CD3 zeta/eta gene locus.

The T-cell receptor (TCR) is a multisubunit complex consisting of the clonotypic Ti alpha and beta (or Ti gamma and delta) subunits and the invariant CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, and CD3 eta subunits. Herein, we describe an additional product from the CD3 zeta/eta gene locus which we have termed CD3 theta. The cDNA derives from the first seven exons common to CD3 zeta and CD3 eta, 94 base pairs (bp) of the CD3 eta-specific exon 9 and an additional exon 10 encoding the carboxyl-terminal 15 amino acids and the 3'-untranslated region. The expression of CD3 theta is equivalent to that of CD3 eta in tissue distribution and level of expression as judged by RNase protection analysis. Despite the identity of the amino-terminal 121 amino acids of CD3 zeta, CD3 eta, and CD3 theta and an additional 31 amino acids shared between CD3 eta and CD3 theta, transfection of CD3 theta into the CD3 zeta- eta- T-cell hybridoma, MA5.8, failed to restore detectable surface TCR expression in contrast to transfection with CD3 zeta or CD3 eta. Analysis of the CD3 theta protein in transfectants indicated that CD3 theta is associated with the TCR intracellularly. However, unlike with CD3 zeta, Ti alpha-beta chains remain endoglycosidase H sensitive, suggesting a role for the unique COOH-terminal segment of CD3 theta in mediating TCR retention and/or degradation in a pre-Golgi compartment.     

CD3ζ-Chain Expression of Human T Lymphocytes Is Regulated by TNF via Src-like Adaptor Protein-Dependent Proteasomal Degradation

Decreased expression of the TCR ζ-chain has been reported in several autoimmune, inflammatory, and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. Although ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is less clear. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data have reported that SLAP regulates immunoreceptor signaling. We have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation. TNF treatment of human T lymphocytes (15-40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose-dependent manner (p < 0.05) and decreases activation-induced IL-2 expression (p < 0.01). Although blocking of the lysosomal compartment fails to restore TNF-induced CD3 ζ-chain downregulation, inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalization of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p < 0.05 and p < 0.01, respectively), whereas TNF-induced ζ-chain downregulation was inhibited by gene silencing of SLAP with small interfering RNA. SLAP levels of the CD4(+) T lymphocytes isolated from patients with rheumatoid arthritis were more than 2-fold higher than that of the healthy donors' (p < 0.05); moreover, TNF treatment did not alter the SLAP expression of the CD4(+) cells of anti-TNF therapy-treated patients. Our present data suggest that TNF modulates T cell activation during inflammatory processes by regulating the amount of CD3 ζ-chain expression via a SLAP-dependent mechanism. These data provide evidence for SLAP-dependent regulation of CD3 ζ-chain in the fine control of TCR signaling.     

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.