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.     

Conservative sequences in 3'UTR of TCRzeta mRNA regulate TCRzeta in SLE T cells

We have demonstrated that T-cell receptor ζ (ζ) mRNA with a 562-bp deleted alternatively spliced 3′-untranslated region (3′UTR) observed in T cells of patients with systemic lupus erythematosus (SLE) can lead to a reduction in ζ and TCR/CD3 (J. Immunol., 2003 & 2005). To determine the region in ζ mRNA 3′UTR for the regulation of ζ, ζ mRNA with 3′UTR truncations ligated into pDON-AI was used to infect murine T-cell hybridoma MA5.8 cells, which do not contain ζ. As a Western blot analysis demonstrated the importance of the regions from +871 to +950, containing conservative sequence 1 (CS1), and +1070 to +1136, containing CS2, for the production of ζ, we constructed MA5.8 mutants carrying ζ mRNA 3′UTR with deletions of these regions (ΔCS1 and ΔCS2 mutants). Western blot and FACS analyses showed significant reduction in the cell surface ζ and TCR/CD3 in both these mutants, and IL-2 production was decreased, compared with MA5.8 cells transfected with wild-type ζ mRNA. Furthermore, real-time PCR demonstrated the instability of ζ mRNA with 3′UTR deletions in these MA5.8 mutants. In conclusion, CS1 and CS2 may be responsible for the regulation of ζ and TCR/CD3 through the stability of ζ mRNA in SLE T cells.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

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.     

TCR/CD3 down-modulation and zeta degradation are regulated by ZAP-70

TCR down-modulation following binding to MHC/peptide complexes is considered to be instrumental for T cell activation because it allows serial triggering of receptors and the desensitization of stimulated cells. We studied CD3/TCR down-modulation and zeta degradation in T cells from two ZAP-70-immunodeficient patients. We show that, at high occupancy of the TCR, down-modulation of the CD3/TCR is comparable whether T cells express or do not express ZAP-70. However, if TCR occupancy was low, we found that CD3/TCR was down-regulated to a lesser extent in ZAP-70-negative than in ZAP-70-positive T cells. We studied CD3/TCR down-modulation in P116 (a ZAP-70-negative Jurkat cell-derived clone) and in P116 transfected with genes encoding the wild-type or a kinase-dead form of ZAP-70. Down-modulation of the TCR at high occupancy did not require ZAP-70, whereas at low TCR occupancy down-modulation was markedly reduced in the absence of ZAP-70 and in cells expressing a dead kinase mutant of ZAP-70. Thus, the presence of ZAP-70 alone is not sufficient for down-modulation; the kinase activity of this molecule is also required. The degradation of zeta induced by TCR triggering is also severely impaired in T cells from ZAP-70-deficient patients, P116 cells, and P116 cells expressing a kinase-dead form of ZAP-70. This defect in TCR-induced zeta degradation is observed at low and high levels of TCR occupancy. Our results identify ZAP-70, a tyrosine kinase known to be crucial for T cell activation, as a key player in TCR down-modulation and zeta degradation.     

The isolation and characterization of the murine T cell antigen receptor zeta chain gene

The T cell antigen receptor (TCR) is a multisubunit complex which has a dual function of antigen recognition and signal transduction. One of its invariant subunits, the zeta chain, has been shown to have a significant role in the expression and function of the TCR on the cell surface. The mouse and human zeta cDNAs share significant homologies to each other but are distinct from all of the previously characterized TCR components. We now report the isolation and structural analysis of the complete murine zeta gene. This gene spans at least 31 kilobases and divides into eight exons. The first exon, which is located at least 20 kilobases upstream from the second exon, codes for the 5'-untranslated region and most of the signal peptide. The second exon codes for the remainder of the signal peptide, the extracellular domain, the transmembrane domain, and the first three amino acids of the intracytoplasmic domain. Exons 3-7 encode the majority of the intracytoplasmic domain. The eight exon encodes the carboxyl-terminal 21 amino acids and the 3'-untranslated region. Four groups of mRNA initiation sites have been identified at approximately 140 base pairs upstream to the AUG codon. No TATA-like box has been detected. The gene is localized to the distal part of chromosome 1 in a linkage group highly conserved between man and mouse.     

The T-cell receptor zeta chain contains two homologous domains with which simian immunodeficiency virus Nef interacts and mediates down-modulation

We have recently demonstrated that simian immunodeficiency virus (SIV) Nef binds to the zeta chain of the T-cell receptor (TCR), leading to its down-modulation from T-cell surfaces (I. Bell, C. Ashman, J. Maughan, E. Hooker, F. Cook, and T. A. Reinhart, J. Gen. Virol. 79:2717-2727, 1998). Using a panel of human as well as rhesus macaque TCR zeta cytoplasmic domain mutants, we have identified in this report two linear peptides in the cytoplasmic domain of TCR zeta which independently interact with SIV Nef. Each SIV Nef interaction domain was sufficient in the absence of the other for interaction with SIV Nef in a yeast two-hybrid assay. In parallel, we demonstrated that Nef down-modulation of CD8-TCR zeta fusion proteins containing full-length or truncated portions of the TCR zeta cytoplasmic domain occurs in transiently transfected 293T cells. Furthermore, using proteins expressed in Escherichia coli, a glutathione S-transferase-Nef fusion protein coprecipitated histidine-tagged portions of the TCR zeta cytoplasmic domain which contained SNID-1 or SNID-2. The peptides targeted by SIV Nef, YNELNL and YSEIGMKGERRR, are portions of the first and second of three immunoreceptor tyrosine-based activation motifs which are important in signal transduction, thymocyte development, and TCR biogenesis. These results demonstrate that SIV Nef binds to two distinct domains on TCR zeta in the absence of other T-cell-specific factors, and that interaction with either domain is sufficient to cause down-modulation of TCR zeta.     

TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex

We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50-100 nm, are delimited by a lipidic bilayer membrane, and bear TCR beta, CD3epsilon, and zeta. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated zeta in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.     

T cell antagonism is functionally uncoupled from the 21- and 23-kDa tyrosine-phosphorylated TCR zeta subunits

The functional effects of altered peptide ligands on T cells is proposed to involve differential intracellular signaling mediated by the 21- and 23-kDa tyrosine-phosphorylated derivatives of the TCR zeta subunit (p21 and p23). To understand the functional contribution of p21 and p23 to T cell development and T cell antagonism, we generated selected TCR zeta transgenic mice maintained on the P14 alphabeta TCR transgenic line such that p23 or both p21 and p23 were selectively eliminated. Importantly, one line (YF1,2) retains the constitutively tyrosine-phosphorylated p21 in the complete absence of inducible p23. We determined that T cell development was uncoupled from p21 and/or p23. Using a series of agonist, weak agonist, and antagonist peptides, we analyzed the role of each of the phosphorylated forms of TCR zeta on T cell activation and antagonism. In this study, we report that the proliferative responses of alphabeta P14 T cells to agonist peptides and the inhibition of proliferation resulting from antagonist peptide treatments was functionally uncoupled from p21 and/or p23. These results suggest that the mechanism of T cell antagonism is independent of the two phosphorylated TCR zeta derivatives.