Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes.

It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.

RelA/NF-kappaB recruitment on the bax gene promoter antagonizes p73-dependent apoptosis in costimulated T cells.

The balance between antiapoptotic and proapoptotic proteins of the Bcl-2 family is critical in determining the fate of T cells in response to death stimuli. Proapoptotic genes, such as bax, are generally regulated by the p53 family of transcription factors, whereas NF-kappaB subunits can activate the transcription of antiapoptotic Bcl-2 members. Here, we show that CD28 activation protects memory T cells from irradiation-induced apoptosis by both upregulating bcl-xL and inhibiting bax gene expression. We found that p73, but not p53, binds to and trans-activates the bax gene promoter in irradiated T cells. The activation of RelA/NF-kappaB subunit in CD28 costimulated T cells and its binding onto the bax gene promoter results in suppression of bax transcription and decrease in both p73 and RNA polymerase II recruitment in vivo. RelA recruitment on the bax gene promoter is also accompanied by the lost of p300 binding and the parallel appearance of histone deacetylase-1-containing complexes. These findings identify RelA/NF-kappaB as a critical regulator of T-cell survival by affecting the balance of Bcl-2 family members.

Organization of plasma membrane functional rafts upon T cell activation.

Raft microdomains have been shown to play a key role in T cell activation. We found that in human T lymphocytes the formation of functional rafts at the plasma membrane was induced by T cell priming. In resting T cells from peripheral blood Lck and the raft glycosphingolipid GM1 resided in intracellular membranes. T cell activation induced synthesis of GM1 and effector cells showed very high levels of this lipid, which became predominantly plasma membrane associated. TCR triggering also induced targeting of the cytosolic Lck to the plasma membrane. Thus, effector cells acquire an improved signaling machinery by increasing the amount of rafts at the plasma membrane. The fact that, when compared with naive T cells, memory T cells showed higher GM1 levels suggests that raft lipid synthesis may be developmentally regulated and tune T cell responsiveness.

CD28 utilizes Vav-1 to enhance TCR-proximal signaling and NF-AT activation.

The mechanism through which CD28 costimulation potentiates TCR-driven gene expression is still not clearly defined. Vav-1, an exchange factor for Rho GTPases thought to regulate, mainly through Rac-1, various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. Here, we provide evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of staphylococcus enterotoxin E/MHC required for TCR-induced NF-AT activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76 but necessitated the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1, and SLP-76 phosphorylation, an event which was favored by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic cooperation between Vav-1 and SLP-76 and probably via cortical actin changes to facilitate the organization of a signaling zone.

Mitogen-activated kinase kinase kinase 1 regulates T cell receptor- and CD28-mediated signaling events which lead to NF-kappaB activation.

Optimal activation of Rel/NF-kappaB transcription factors in T lymphocytes requires a CD28-delivered co-stimulatory signal in addition to TCR engagement. Although, Rel/NF-kappaB transcription factors are critical regulators of many T cell functions, the mechanisms and molecules, which link the surface receptors to their activation, are poorly characterized. Using Jurkat T cells stimulated with superantigen presented on B7-positive APC, we showed that CD28- and TCR-stimulated NF-kappaB-dependent transcription is associated to the activation of IkappaB kinase beta (IKKbeta) and, to a lesser extent, of IkappaB kinase alpha (IKKalpha). A dominant negative mutant of the MAP3 kinase MEKK1, a kinase known to regulate the JNK pathway and to activate NF-kappaB-dependent transcription in many cell types, strongly inhibits CD28- and TCR-induced IKK activity, whereas the dominant negative mutants of the NF-kappaB-inducing kinase (NIK) did not exert any significant effects. In addition, TCR/CD28 stimulation results in the recruitment and autophosphorylation of endogenous MEKK1, whereas endogenous NIK was not detectably activated. Our data identify MEKK1 as a critical step in coupling signals initiated by TCR and CD28 to the downstream pathways which lead to both AP-1 and NF-kappaB activation in T lymphocytes.

Engagement of CD4 before TCR triggering regulates both Bax- and Fas (CD95)-mediated apoptosis.

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (DeltaPsim) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.

Hypervariable region 1 variants act as TCR antagonists for hepatitis C virus-specific CD4+ T cells.

In various human viral infections, the appearance of mutated epitopes displaying TCR antagonistic activity has been correlated with the severity and persistence of infection. In hepatitis C virus (HCV) infection, where the virus persistence has been associated with the rapid and substantial Ag modifications occurring during replication, TCR antagonism has been evidenced in CD8+ T cell responses. However, CD4+ T cell antagonism may be another important strategy by which HCV eludes a protective response, because sustained Th responses directed against several HCV Ags are associated with a self-limited course of infection. The data reported here represent the first evidence that variants of the hypervariable region (HVR1) of the putative Envelope 2 protein of HCV can act as powerful TCR antagonists for HVR1-specific CD4+ T cells isolated from HCV-infected individuals. Using classical antagonism assays, we observed strong inhibition of cellular proliferation and cytokine production when the agonist and the antagonist ligands were simultaneously presented by the same APCs. The presence in HVR1 of conserved residues, critical for binding to HLA-DR molecules, supports the function of HVR1 variants as TCR antagonists. In conclusion, our data evidence an antagonism phenomenon, which was achieved by naturally occurring class II-restricted T cell epitopes whose mechanism was addressed in terms of the antagonist capacity to inhibit agonist-mediated TCR down-regulation and early signal transduction.

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2.

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.

Fyn and ZAP-70 are required for Vav phosphorylation in T cells stimulated by antigen-presenting cells.

In T cells, triggering of the T cell antigen receptor or of the co-stimulatory receptor CD28 can direct tyrosine phosphorylation of the signaling protein Vav. We investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in these processes in a T cell hybridoma after physiological stimulation of the T cell receptor (TCR) and CD28. A dominant-negative mutant approach based on overexpression of catalytically inactive alleles of these kinases showed that CD28-induced Vav phosphorylation preferentially requires Fyn, whereas ZAP-70 had no role. Consistently, Vav was strongly phosphorylated in Lck-deficient JCAM-1 cells after CD28 ligation. In contrast, ZAP-70 appeared to control TCR-directed Vav phosphorylation. However, overexpression of ZAP-70 carrying a mutated Tyr315, contained within a motif previously suggested to be a Vav Src homology 2 domain binding site, had little or no effect. Immunoprecipitation assays showed that phosphorylated Vav associated with Fyn after CD28 triggering and that this interaction, likely to involve binding of Fyn Src homology 2 domain to Vav, was more strongly detectable after concomitant CD28 and TCR stimulation. These data suggest that Fyn plays a major role in controlling Vav phosphorylation upon T cell activation and that the mechanism implicating ZAP-70 in this process may be more complex than previously anticipated.

CD28 affects the earliest signaling events generated by TCR engagement.

The efficiency and magnitude of T cell responses are influenced by ligation of the co-stimulatory receptor CD28 by B7 molecules expressed on antigen-presenting cells (APC). In contrast to most previous studies in which agonistic anti-TCR/CD3 and anti-CD28 antibodies were employed, here we have investigated the contribution of CD28 to T cell activation under physiological conditions of antigen presentation. Jurkat T cells and primary T cells from TCR-transgenic mice stimulated with superantigen and antigen, respectively, presented by B7-expressing APC were utilized. In both systems we show that inhibiting CD28/B7 interaction resulted in impaired TCR-induced tyrosine phosphorylation of the signal-transducing zeta chain and ZAP-70. Consistent with a blockade of TCR-proximal signaling events, Jurkat cells stimulated in the absence of CD28 ligation were found to have strongly diminished tyrosine phosphorylation of cellular substrates and downstream signaling pathways such as Ca2+/calcineurin, ERK/MAPK and JNK. Our results provide evidence for a role of CD28 in enhancing TCR signaling capacity during the earliest stages of T cell:APC interaction.

Quantitative contribution of CD4 and CD8 to T cell antigen receptor serial triggering.

CD4 and CD8 are thought to function as coreceptors by binding to the cognate major histocompatibility complex (MHC) molecules recognized by the T cell antigen receptor (TCR) and initiating the signal transduction cascade. We report that during T cell-antigen-presenting cell interaction, triggered TCRs and coreceptors are downregulated and degraded with identical kinetics. This coordinated disappearance takes place whenever the TCR is triggered, even when the coreceptor does not engage the cognate MHC molecule and is the consequence of binding of the coreceptor-associated Lck to ZAP-70. The interaction of coreceptor and cognate MHC molecules is dispensable when T cells are stimulated by optimal ligands, but becomes crucial when suboptimal ligands are used. In the latter case the coreceptor increases the efficiency of TCR triggering without changing the activation threshold or the quality of the T cell response.

Mutation of tyrosines 492/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways.

The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56(lck), in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR zeta chain and was able to bind p56(lck). This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56(lck) with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56(lck), which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation.

p95vav associates with tyrosine-phosphorylated SLP-76 in antigen-stimulated T cells.

p95vav, the product of the vav protooncogene, has been implicated in the T cell receptor (TCR)-mediated signaling cascade p95vav is phosphorylated on tyrosine residues after TCR stimulation by anti-TCR/CD3 antibodies and possesses a number of landmark features of signaling molecules such as a putative guanine nucleotide exchange factor domain, a pleckstrin homology domain, and an Sre homology (SH) 2 and two SH3 domains, which provide the capacity to form multimeric signaling complexes. However, the precise role of p95vav in TCR signaling remains unclear. In this work we show that physiological stimulation of T cell hybridomas with antigen presented by major histocompatibility complex class II molecules leads to a strong tyrosine phosphorylation of p95vav and its association with tyrosine-phosphorylated SLP-76. SLP-76 is a newly described SH2-containing protein that has been previously found to bind to the adapter molecule Grb2. Moreover, we provide evidence that p95vav-SI P-76 association is SH2-mediated by demonstrating that this interaction can be inhibited by a phosphopeptide containing a putative p95vav-SH2-binding motif (pYESP) present in SLP-76. Furthermore, in vitro experiments show that after antigen stimulation, phosphorylated p95vav-SLP-76 can bind to Grb2 in a complex that contains pp36/38 and pp116 proteins. Our data provide a clue to explain recent independent observations that overexpression of p95vav or SLP-76 enhances TCR-mediated gene activation.

Natural killer clones recognize specific soluble HLA class I molecules.

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from natural killer (NK) cell recognition in several systems. MHC class I gene products are released from the cell surface and can be found in sera as soluble forms. To investigate the possible immunoregulatory role of soluble HLA (sHLA) in NK cell-target recognition, several sHLA antigens were studied for their ability to induce NK cell cytotoxicity modulation. NK cell-target recognition was inhibited by the addition of sHLA during the cytotoxicity assay. Our results indicate that sHLA molecules can down-regulate NK killing at the effector level. Moreover, different NK clones are able to specifically recognize different sHLA antigens. Kp43 molecules seem to be involved in the NK recognition of sHLA-B7.

Differential susceptibility to monomeric HIV gp120-mediated apoptosis in antigen-activated CD4+ T cell populations.

To support the hypothesis that indirect mechanisms mediated by viral products like the HIV envelope glycoprotein gp120 could be responsible for T lymphocyte depletion in HIV infection, we developed a system in which the impairment of T cell functions could be investigated in vitro. In particular, we characterized the conditions that allow T lymphocytes repeatedly stimulated with an antigen to be sensitive or resistant to gp120-mediated apoptotic signals. To achieve this goal, a panel of antigen-specific CD4+ T cell clones and primary CD4+ T lymphocytes were treated for 2 and 18 h with saturating amounts of monomeric gp120 (without cross-linking with specific antibodies) and antigen-driven T cell proliferation and apoptosis were analyzed. We show that monomeric gp120 induces apoptosis only in T lymphocytes repeatedly stimulated with the antigen, that primary T lymphocytes are resistant to programmed cell death mediated by monomeric gp120, but are sensitive to anti-CD4 antibodies, and that gp120-mediated apoptosis is dependent on the period of time between the binding of gp120 to CD4 and the encounter with antigen. To investigate the different susceptibility to gp120 induced apoptosis of primary CD4+ and T cell clones further, the number of membrane CD4 molecules and their affinity for gp120, together with Bcl-2 and Fas expression, were studied. Our data suggest that a down-modulation of membrane CD4 together with high expression of the Bcl-2 gene and protein characterizes the susceptibility to apoptosis of gp120-treated cells. In conclusion, our results define the phenotypic features of T cells susceptible to HIV gp120-induced apoptosis and demonstrate that the same clonotype, depending on the activation state, may present a differential sensitivity to apoptosis induction.

Ligation of either CD2 or CD28 rescues CD4+ T cells from HIV-gp120-induced apoptosis.

Temporal or quantitative imbalance in signals delivered to T cells via T cell antigen receptor (TCR), the CD4 co-receptor, and accessory molecules can lead to anergy, apoptosis, or both. This has been observed following ligation of CD4 by HIV gp120 prior to TCR occupancy. The ability of molecules such as CD2 and CD28, interacting with their ligands LFA-3 and B7, to provide signals that protect T cells from the induction of anergy, has been reported. Here, we demonstrate that ligation of CD2 and CD28 in conjunction with TCR occupancy rescue T cells that have been programmed for apoptotic death by prior CD4 ligation to gp120. This appears to be the result of augmented interleukin-2 and interleukin-4 release by the T cells following these molecular interactions. In conclusion, our results suggest that an impairment of antigen-presenting accessory cell functions could favor gp120-mediated apoptosis in HIV-uninfected cells.

Identification of a novel HLA-B27 subtype by restriction analysis of a cytotoxic gamma delta T cell clone.

Seven HLA-B27 alleles are known, which share the same allospecificity, but differ by one to six amino acid substitutions. Herein, we describe a novel HLA-B27 allele, provisionally named B27-ci, which is expressed by an individual from whom a B27-restricted gamma delta T cell clone has been derived. This clone recognizes B cell lines from the proband and all of the other B27-positive members of the family, but does not lyse B cell lines that express other HLA-B27 alleles. The amino acid sequence deduced from three B27-ci cDNA clones was found to differ from the B*2705 sequence by one amino acid substitution (Asp to His) in position 116 of the alpha 2 domain. This position has been shown to lie in the floor of the F pocket, where it plays a key role in determining the nature of the amino acid side chain that will fit into this pocket. Moreover, the fact that the clone described here possesses a TCR-gamma delta indicates that this subset of cells not only can be HLA-restricted, but also can finely discriminate among classical class I molecules.

Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes.

The contributions of individual amino acids in the polymorphic beta chain and the conserved alpha chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the beta 1 domain and 11 positions in the alpha 1 domain. The beta 1-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR alpha 1 domain were based on the sequences of other human and mouse class II alpha chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the beta 1-domain alpha-helix caused substantial reduction in the responses of all of the clones. Substitutions at multiple positions in the beta 1-domain floor and in the alpha 1 domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the beta 1 domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules.

Analysis of susceptibility of mature human T lymphocytes to dexamethasone-induced apoptosis.

We present evidence that dexamethasone (Dex), a synthetic glucocorticosteroid, causes apoptosis in mature human T cells, similarly to what has been reported for murine T lymphocytes. Human T cell clones and short-term activated T lymphocytes treated with Dex show the characteristic pattern of apoptotic cells, such as hypodiploid nuclei, chromatin condensation and DNA fragmentation into oligonucleosomal fragments. However, Dex susceptibility of T cells to apoptosis is cell cycle-dependent. The progression in the proliferative cell cycle (G1 versus S) rescues Dex-treated T cells from apoptosis. Moreover, occupancy of the T cell receptor reverses Dex-induced apoptotic phenomena. These observations suggest that glucocorticoids contribute to the regulation of the proliferative or the suicidal response of antigen-activated human T cells.

Mycobacterial antigen complex A60-specific T-cell repertoire during the course of pulmonary tuberculosis.

The Mycobacterium bovis antigen complex A60 is known to be immunodominant in tuberculosis and to have a protective effect against experimental infection in vitro and in vivo. To identify immunodominant and possibly protective antigens in pulmonary tuberculosis, the T-cell repertoire directed to nitrocellulose-bound fractions of A60 antigen was analyzed in active tuberculosis patients during the course of the infection and after recovery. The results show that patients infected with Mycobacterium tuberculosis acquired complete A60-T-cell reactivity only in the late phases of infection. At disease onset, patients with active tuberculosis were characterized by (i) T-cell unresponsiveness to most A60 fractions, (ii) high tumor necrosis factor alpha production, and (iii) low gamma interferon (IFN-gamma) release. Several weeks after chemotherapy, the unresponsive state disappeared and the following reverse situation was observed: (i) high blastogenic response to almost all A60 fractions, (ii) low tumor necrosis factor alpha release, and (iii) high IFN-gamma production. In addition, 60% of these patients significantly responded against seven A60 fractions (61 to 58, 56 to 53, 49 to 46, 46 to 44, 35 to 33, 33 to 30, and 30 to 28 kDa), indicating that they included immunodominant antigens. Furthermore, only the fractions within the molecular mass ranges of 56 to 44 and 35 to 28 kDa induced IFN-gamma synthesis. One year after complete recovery from infection, more than 60% of past-active tuberculosis subjects had memory T cells specific for the immunodominant fractions of 61 to 58, 56 to 53, 49 to 46, and 33 to 30 kDa. Since the same fractions induced the strongest IFN-gamma production, known to exhibit antimycobacterial effects, it is suggested that these may represent the inducers of a protective immune response.