L-Arginine modulates CD3zeta expression and T cell function in activated human T lymphocytes

Engagement of the T cell receptor (TCR) by antigen or anti-CD3 antibody results in a cycle of internalization and re-expression of the CD3zeta. Following internalization, CD3zeta is degraded and replaced by newly synthesized CD3zeta on the cell surface. Here, we provide evidence that availability of the amino acid L-arginine modulates the cycle of internalization and re-expression of CD3zeta and cause T cell dysfunction. T cells stimulated and cultured in presence of L-arginine, undergo the normal cycle of internalization and re-expression of CD3zeta. In contrast, T cells stimulated and cultured in absence of L-arginine, present a sustained down-regulation of CD3zeta preventing the normal expression of the TCR, exhibit a decreased proliferation, and a significantly diminished production of IFNgamma, IL5, and IL10, but not IL2. The replenishment of L-arginine recovers the expression of CD3zeta. The decreased expression of CD3zeta is not caused by a decreased CD3zeta mRNA, an increased CD3zeta degradation or T cell apoptosis.     

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.     

Helicobacter pylori vacuolating cytotoxin inhibits activation-induced proliferation of human T and B lymphocyte subsets

Helicobacter pylori are Gram-negative bacteria that persistently colonize the human gastric mucosa despite the recruitment of immune cells. The H. pylori vacuolating cytotoxin (VacA) recently has been shown to inhibit stimulation-induced proliferation of primary human CD4(+) T cells. In this study, we investigated effects of VacA on the proliferation of various other types of primary human immune cells. Intoxication of PBMC with VacA inhibited the stimulation-induced proliferation of CD4(+) T cells, CD8(+) T cells, and B cells. VacA also inhibited the proliferation of purified primary human CD4(+) T cells that were stimulated by dendritic cells. VacA inhibited both T cell-induced and PMA/anti-IgM-induced proliferation of purified B cells. Intoxication with VacA did not alter the magnitude of calcium flux that occurred upon stimulation of CD4(+) T cells or B cells, indicating that VacA does not alter early signaling events required for activation and proliferation. VacA reduced the mitochondrial membrane potential of CD4(+) T cells, but did not reduce the mitochondrial membrane potential of B cells. We propose that the immunomodulatory actions of VacA on T and B lymphocytes, the major effectors of the adaptive immune response, may contribute to the ability of H. pylori to establish a persistent infection in the human gastric mucosa.     

Cross-linking of T3 (CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes

Monoclonal antibodies reactive with defined T lymphocyte surface antigens were covalently coupled to protein A-Sepharose beads using the bifunctional imidoester, dimethyl pimelimidate. Sepharose-immobilized antibody reactive with T3 induced the proliferation of resting T lymphocytes in the presence of either recombinant interleukin 2 or phorbol myristate acetate. When monoclonal antibodies reactive with T3 and T4 were coupled to the same Sepharose bead (hereafter designated Sepharose (T3:T4)), proliferation was enhanced an average of three-fold. Similarly prepared Sepharose beads coupled to anti-T3 and anti-T8 also enhanced proliferation over that observed with anti-T3 alone. Sepharose (T3:T4) similarly increased the proliferation of T4+ lymphocytes and a T4+ clone but failed to enhance the proliferation of T8+ lymphocytes. The increased proliferation of T4+ lymphocytes resulted from a preferential activation of the T4+2H4- helper population over the T4+2H4+ suppressor-inducer population. The enhanced proliferation induced by Sepharose (T3:T4) could be completely inhibited by soluble anti-T4. These results suggest that perturbation of T3 may be a minimal signal for T cell activation and that the assembly of a multimeric complex including T3 and T4 may be required for optimal T cell activation.     

Tyrosine phosphorylation of the human T cell antigen receptor zeta-chain: activation via CD3 but not CD2

TCR stimulation by Ag or anti-receptor antibodies in murine T cells results in the activation of two independent protein kinases, protein kinase C (PKC) and a protein tyrosine kinase. Similarly, stimulation of murine Thy-1 or Ly-6 with mAb also results in activation of both of these kinase pathways. Tyrosine phosphorylation in all cases occurs on the TCR zeta-chain. It is known that Ag and anti-receptor antibodies activate PKC in human T cells. In this study we demonstrate that mitogen or anti-CD3 antibodies activate tyrosine phosphorylation of the human TCR-zeta-chain. PMA, which activates PKC, does not result in zeta-chain tyrosine phosphorylation. Stimulation of human T cells by antibodies that bind the CD2 molecule is an alternate mode of inducing T cell proliferation. These antibodies surprisingly do not induce tyrosine phosphorylation of the zeta-chain. Thus, different methods of cellular activation can result in distinguishable patterns of receptor-mediated biochemical signaling events.     

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.     

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

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.     

The FcR gamma subunit and Syk kinase replace the CD3 zeta-chain and ZAP-70 kinase in the TCR signaling complex of human effector CD4 T cells

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3zeta-related FcRgamma signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3epsilon, but not CD3zeta. The TCR/CD3/FcRgamma complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.     

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.     

Clonal competition inhibits the proliferation and differentiation of adoptively transferred TCR transgenic CD4 T cells in response to infection

CD4 and CD8 T cells have been shown to proliferate and differentiate to different extents following antigenic stimulation. CD4 T cells form a heterogenous pool of effector cells in various stages of division and differentiation, while nearly all responding CD8 T cells divide and differentiate to the same extent. We examined CD4 and CD8 T cell responses during bacterial infection by adoptive transfer of CFSE-labeled monoclonal and polyclonal T cells. Monoclonal and polyclonal CD8 T cells both divided extensively, whereas monoclonal CD4 T cells underwent limited division in comparison with polyclonal CD4 T cells. Titration studies revealed that the limited proliferation of transferred monoclonal CD4 T cells was due to inhibition by a high precursor frequency of clonal T cells. This unusually high precursor frequency of clonal CD4 T cells also inhibited the differentiation of these cells. These results suggest that the adoptive transfer of TCR transgenic CD4 T cells significantly underestimates the extent of proliferation and differentiation of CD4 T cells following infection.     

L-arginine consumption by macrophages modulates the expression of CD3 zeta chain in T lymphocytes

L-Arginine plays a central role in the normal function of several organs including the immune system. It is metabolized in macrophages by inducible nitric oxide synthase to produce nitric oxide, important in the cytotoxic mechanisms, and by arginase I (ASE I) and arginase II (ASE II) to synthesize L-ornithine and urea, the first being the precursor for the production of polyamines needed for cell proliferation. L-Arginine availability can modulate T cell function. Human T cells stimulated and cultured in the absence of L-arginine lose the expression of the TCR zeta-chain (CD3zeta) and have an impaired proliferation and a decreased cytokine production. The aim of this work was to test whether activated macrophages could modulate extracellular levels of L-arginine and alter T cell function, and to determine which metabolic pathway was responsible for this event. The results show that macrophages stimulated with IL-4 + IL-13 up-regulate ASE I and cationic amino acid transporter 2B, causing a rapid reduction of extracellular levels of L-arginine and inducing decreased expression of CD3zeta and diminished proliferation in normal T lymphocytes. Competitive inhibitors of ASE I or the addition of excess L-arginine lead to the re-expression of CD3zeta and recovery of T cell proliferation. In contrast, inducible nitric oxide synthase or ASE II failed to significantly reduce the extracellular levels of L-arginine and modulate CD3zeta expression. These results may provide new insights into the mechanisms leading to T cell dysfunction and the down-regulation of CD3zeta in cancer and chronic infectious diseases.     

Cytokine-independent Jak3 activation upon T cell receptor (TCR) stimulation through direct association of Jak3 and the TCR complex

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.