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Unlike BCR and secreted Ig, TCR expression is not thought to occur in a bivalent form. The conventional monovalent model of TCR/CD3 is supported by published studies of complexes solubilized in the detergent digitonin, in which bivalency was not observed. We revisited the issue of TCR valency by examining complexes isolated from primary αβ T cells after solubilization in digitonin. Using immunoprecipitation followed by flow cytometry, we unexpectedly observed TCR/CD3 complexes that contained two TCRs per complex. Standard anti-TCR Abs, being bivalent themselves, tended to bind with double occupancy to bivalent TCRs; this property masked the presence of the second TCR per complex in certain Ab binding assays, which may partially explain why previous data did not reveal these bivalent complexes. We also found that the prevalence of bivalency among fully assembled, mature TCR/CD3 complexes was sufficient to impact the functional performance of immunoprecipitated TCRs in binding antigenic peptide/MHC-Ig fusion proteins. Both TCR positions per bivalent complex required an Ag-specific TCR to effect optimal binding to these soluble ligands. Therefore, we conclude that in primary T cells, TCR/CD3 complexes can be found that are physically and functionally bivalent. The expression of bivalent TCR/CD3 complexes has implications regarding potential mechanisms by which Ag may trigger signaling. It also suggests the possibility that the potential for bivalent expression could represent a general feature of Ag receptors.

Nef, an important pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), elevates virus replication in vivo. Among other activities, Nef affects T-cell receptor (TCR) signaling via several mechanisms. For HIV-1 Nef these include alteration of the organization and function of the immunological synapse (IS) such as relocalization of the Lck kinase, as well as early inhibition of TCR/CD3 complex (TCR-CD3)-mediated actin rearrangements and tyrosine phosphorylation. Although most SIV and HIV-2 Nef alleles (group 2) potently downregulate cell surface TCR-CD3, this activity was lost in the viral lineage that gave rise to HIV-1 and its SIV counterparts (group 1). To address the contribution of TCR-CD3 downregulation to Nef effects on TCR signal initiation, we compared the activities of 18 group 1 and group 2 Nef proteins, as well as SIV Nef mutants with defects in TCR-CD3 downmodulation. We found that alteration of Lck's subcellular localization is largely conserved and occurs independently of actin remodeling inhibition or TCR-CD3 downregulation. Surprisingly, Nef proteins of both groups also strongly reduced TCR-induced actin remodeling and tyrosine phosphorylation on TCR-stimulatory surfaces and TCR-CD3 downmodulation competence by group 2 Nef proteins only slightly elevated these effects. Furthermore, Nef proteins from HIV-1 and SIV reduced conjugation between infected primary human T lymphocytes and Raji B cells and potently prevented F-actin polarization at the IS independently of their ability to downmodulate TCR-CD3. These results establish alterations of early TCR signaling events at the IS, including F-actin remodeling and relocalization of Lck, as evolutionary conserved activities of highly divergent lentiviral Nef proteins.

The HIV and SIV Nef accessory proteins are potent enhancers of viral persistence and accelerate progression to AIDS in HIV-1-infected patients and non-human primate models. Although relatively small (27-35 kD), Nef can interact with a multitude of cellular factors and induce complex changes in trafficking, signal transduction, and gene expression that together converge to promote viral replication and immune evasion. In particular, Nef recruits several immunologically relevant cellular receptors to the endocytic machinery to reduce the recognition and elimination of virally infected cells by the host immune system, while simultaneously interacting with various kinases to promote T cell activation and viral replication. This review provides an overview on selected Nef interactions with host cell proteins, and discusses their possible relevance for viral spread and pathogenicity.

The success of vaccination is directly or indirectly based on the specificity of antigen recognition by T lymphocytes, their efficient activation and expansion, and the generation of vaccine-specific effectors and memory cells. These traits are largely dependent on the correct assembly and expression of sufficient number of functional TCR/CD3 complexes in the cell surface. In this review, some of the genetic and epigenetic factors that determine the correct assembly and structure of the TCR/CD3 complex are summarized. Those physiologic or pathologic factors leading to natural variations, or pathologic alterations of the standard that might lead to poor response to vaccination and that could give some possibilities to pharmacological intervention are emphasized.

The CD8 receptor plays a central role in the recognition and elimination of virally infected and malignant cells by cytolytic CD8(+) T cells. In conjunction with the TCR, the CD8 coreceptor binds Ag-specific class I MHC (MHC-I) molecules expressed by target cells, initiating signaling events that result in T cell activation. Whether CD8 can further function as an adhesion molecule for non-Ag MHC-I is currently unclear in humans. In this study, we show that in human CD8(+) T cells, TCR complex signaling activates CD8 adhesion molecule function, resulting in a CD8 interaction with MHC-I that is sufficient to maintain firm T cell adhesion under shear conditions. Secondly, we found that while CD8 adhesive function was triggered by TCR complex activation in differentiated cells, including in vitro generated CTL and ex vivo effector/memory phenotype CD8(+) T cells, naive CD8(+) T cells were incapable of activated CD8 adhesion. Lastly, we examine the kinetics of, and signaling for, activated CD8 adhesion in humans and identify notable differences from the equivalent CD8 function in mouse. Activated CD8 adhesion induced by TCR signaling may contribute to the more rapid and robust elimination of pathogen-infected cells by differentiated CD8(+) T cells.

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).

The de novo generation of Foxp3+ regulatory T (Treg) cells in the peripheral immune compartment and the differentiation of Th17 cells both require TGF-beta, and IL-6 and IL-21 are switch factors that drive the development of Th17 cells at the expense of Treg cell generation. The major vitamin A metabolite all-trans retinoic acid (RA) not only enforces the generation of Treg cells but also inhibits the differentiation of Th17 cells. Herein we show that RA enhances TGF-beta signaling by increasing the expression and phosphorylation of Smad3, and this results in increased Foxp3 expression even in the presence of IL-6 or IL-21. RA also inhibits the expression of IL-6Ralpha, IRF-4, and IL-23R and thus inhibits Th17 development. In vitro, RA significantly promotes Treg cell conversion, but in vivo during the development of experimental autoimmune encephalomyelitis it does not increase the frequency of Treg cells in the face of an ongoing inflammation. However, RA suppresses the disease very efficiently by inhibiting proinflammatory T cell responses, especially pathogenic Th17 responses. These data not only identify the signaling mechanisms by which RA can affect both Treg cell and Th17 differentiation, but they also highlight that in vivo during an autoimmune reaction, RA suppresses autoimmunity mainly by inhibiting the generation of effector Th17 cells.

Antigenic peptides initiate an immune response in T cells via the T cell receptor (TCR). The TCR itself is widely regarded as one of the most complex receptors in nature, as it is comprised of at least six different subunits, the antigen recognizing TCRalpha and beta chains, and the signal transmitting CD3deltavarepsilon, gammaepsilon, and zeta2 dimers. In order for a signal to be transmitted from the TCR to the cytoplasm, the CD3 chains must "sense" that an antigenic peptide has been presented to the TCRalpha and beta subunits. After accomplishing this, there are a total of 10 different immunoreceptor tyrosine activation motifs (ITAMs) present within the CD3 chains which effectively activate the T cell and hence the immune response. The importance of each CD3 chain and subsequently each ITAM has been the focus of intense research. However, the precise role(s) played by each CD3 chain has remained elusive. Using the immunomodulatory peptide termed core peptide (CP), which is proposed to inhibit TCR activation by disrupting TCR-CD3 interactions, a tri-modular signaling system for T cell activation is proposed. By contrast to the existing two distinct signaling model (zeta2, CD3epsilongamma/epsilondelta), in this model each of the three dimers, CD3gammaepsilon, deltaepsilon, and zeta2, are proposed to act as three separate and distinct signaling modules, performing both specific and redundant functions.

Adaptor proteins positively or negatively regulate the T cell receptor for antigen (TCR) signaling cascade. We report that after TCR stimulation, the inhibitory adaptor downstream of kinase (Dok)-2 and its homologue Dok-1 are involved in a multimolecular complex including the lipid phosphatase Src homology 2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP)-1 and Grb-2 which interacts with the membrane signaling scaffold linker for activation of T cells (LAT). Knockdown of LAT and SHIP-1 expression indicated that SHIP-1 favored recruitment of Dok-2 to LAT. Knockdown of Dok-2 and Dok-1 revealed their negative control on Akt and, unexpectedly, on Zap-70 activation. Our findings support the view that Dok-1 and -2 are critical elements of a LAT-dependent negative feedback loop that attenuates early TCR signal. Dok-1 and -2 may therefore exert a critical role in shaping the immune response and as gatekeepers for T cell tolerance.

Electrostatic amino acid interactions between receptor subunits within the T-cell antigen receptor (TCR) transmembrane domain are critical for the formation of the TCR-CD3 complex. Core peptide, a short peptide corresponding to the TCR-alpha transmembrane region, containing two positively charged amino acids, is known to inhibit T-cell function in vitro and in vivo. The aim of this study was to examine peptides corresponding to the syntactic transmembrane CD3 region binding to TCR-alpha for their ability to inhibit T-cell activation in vitro and in vivo. Three peptides matching the transmembrane sequence of CD3-delta, -epsilon and -gamma were synthesized and tested in different biological in vitro and in vivo systems for their effect on T-cell activity. The CD3-peptides had no impact on T-cell function in vitro, but surprisingly, decreased signs of inflammation in the adjuvant arthritis rat model in vivo. Preliminary evidence suggests that peptides with CD3 transmembrane-derived sequences can inhibit an immune response as assessed by adjuvant-induced arthritis. The lack of in vitro activity may lead to a wasteful disregard of active compounds in the process of drug discovery and development.

The T-cell antigen receptor (TCR x CD3) is a multi-subunit complex that is responsible for triggering an adaptive immune response. It shows high specificity and sensitivity, while having a low affinity for the ligand. Furthermore, T cells respond to antigen over a wide concentration range. The stoichiometry and architecture of TCR x CD3 in the membrane have been under intense scrutiny because they might be the key to explaining its paradoxical properties. This review highlights new evidence that TCR x CD3 is found on intact unstimulated T cells in a monovalent form (one ligand-binding site per receptor) as well as in several distinct multivalent forms. This is in contrast to the TCR x CD3 stoichiometries determined by several biochemical means; however, these data can be explained by the effects of different detergents on the integrity of the receptor. Here, we discuss a model in which the multivalent receptors are important for the detection of low concentrations of ligand and therefore confer sensitivity, whereas the co-expressed monovalent TCR x CD3s allow a wide dynamic range.

When T cells encounter antigens via the T cell antigen receptor (TCR), information about the quantity and quality of antigen engagement is relayed to the intracellular signal transduction machinery. This process is poorly understood. The TCR itself lacks a significant intracellular domain. Instead, it is associated with CD3 molecules that contain intracellular signaling domains that couple the TCR/CD3 complex to the downstream signaling machinery. The earliest events in TCR signaling must involve the transfer of information from the antigen binding TCR subunit to the CD3 signaling subunits of the TCR/CD3 complex. Elucidating the structural organization of the TCR with the associated CD3 signaling molecules is necessary for understanding the mechanism by which TCR engagement is coupled to activation. Here, we review the current state of our understanding of the structure and organization of the TCR/CD3 complex.

Termination of an immune response requires elimination of activated T lymphocytes by activation-induced cell death (AICD). In AICD, CD95 (Apo-1/Fas) ligand (L) triggers apoptosis of CD95-positive activated T lymphocytes. In AIDS patients, AICD is strongly enhanced and accelerated. We and others have previously shown that HIV-1 trans-activator of transcription (HIV-1 Tat) sensitizes T cells toward CD95-mediated apoptosis and up-regulates CD95L expression by affecting the cellular redox balance. In this study, we show that it is hydrogen peroxide (H(2)O(2)) that functions as an essential second messenger in TCR signaling. The H(2)O(2) signal combined with simultaneous calcium (Ca(2+)) influx into the cytosol constitutes the minimal requirement for induction of CD95L expression. Either signal alone is insufficient. We further show that HIV-1 Tat interferes with TCR signaling and induces a H(2)O(2) signal. H(2)O(2) generated by HIV-1 Tat combines with CD4-dependent calcium influx and causes massive T cell apoptosis. Thus, our data provide an explanation for CD4(+) T lymphocyte depletion during progression of AIDS.

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.

The Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood-derived HSCs cultured on Delta-like 1-expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 alpha beta-T-cell receptor (TCR)-bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro-differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.

PKCepsilon has been strongly linked to cell activation and proliferation in many cell types, including leukemic T-cell lines. In particularly, an essential role of PKCepsilon has been established in the IKK-beta/I-kappaB/NF-kappaB transactivation cascade. To study the physiological function of PKCepsilon in primary T-cells, we used our newly established PKCepsilon null mice. Unexpectedly, however, we did not reveal any defect in the development and function of CD3+ T-cells. Proliferative responses as well as IL-2 cytokine secretion of PKCepsilon-deficient T-cells induced by allogenic MHC, plate-bound anti-CD3 antibodies (with or without anti-CD28 costimulation), or mitogenic stimuli such as phorbol ester and Ca2+ ionophore were comparable with wild-type controls. Consistently, after CD3/CD28 engagement, deficiency of PKCepsilon did not impair NF-kappaB transactivation as well as CD25, CD44 and CD69 induction. Thus, PKCepsilon-deficient T-cells had similar physiological thresholds for activation in vitro. This finding suggests that PKCepsilon plays a redundant role in TCR-induced regulation of T-cell proliferation.

TCR/CD3 complex-mediated signals play critical roles in regulating CD4(+) Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule zeta-chain in regulating the differentiation of Th cells. We have studied T cells from zeta-chain-deficient mice (zetaKO mice), zeta-chain-bearing mice (zeta(+) mice), and from zetaKO mice expressing a FcRgamma chain transgene (FcRgammaTG, zetaKO mice). Our results demonstrated that, compared with those of control mice, CD4(+) T cells and not CD8(+) T cells from zetaKO mice were polarized into IFN-gamma-producing cells. Some of these IFN-gamma-producing cells could also secrete IL-10. Interestingly, zetaKO mouse T cells produced IFN-gamma even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-gamma production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from zetaKO mice. Further studies showed that noncultured zetaKO mice CD4(+) T cells and thymocytes bore a unique memory cell-like CD44(high), CD62L(low/neg) phenotype. Altogether, these results suggest that, in the absence of the zeta-chain, CD4(+) T cells develop as polarized IFN-gamma-producing cells that bear a memory cell-like phenotype. The zeta-chain-bearing T cells may produce a large amount of IFN-gamma only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of zeta-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.

The role of membrane cofactor protein (MCP, CD46) on human T cell activation has been analyzed. Coligation of CD3 and CD46 in the presence of PMA or CD28 costimuli enhanced IL-2, IFN-gamma, or IL-10 secretion by CD4+ T lymphocytes. The effect of CD46 on IL-10 secretion did not require additional costimuli like anti-CD28 antibodies or phorbol esters. CD46 also enhanced IL-2 or IFN-gamma secretion by CD4+ blasts. In contrast, IL-5 secretion was inhibited upon CD46-CD3 coligation, in all the cells analyzed. These effects were independent of IL-12 and suggest that CD46 costimulation promotes a Th1-biased response in human CD4+ T lymphocytes. CD46 enhanced TCR/CD3-induced tyrosine phosphorylation of CD3zeta and ZAP-70, as well as the activation of the ERK, JNK, and p38, but did not modify intracellular calcium. The effect of specific inhibitors shows that enhanced ERK activation contributes to augmented IFN-gamma and lower IL-5 secretion and, consequently, to the Th1 bias. Cross-linking CD46 alone induced weak tyrosine phosphorylation of CD3zeta and ZAP-70. However, CD46 cross-linking by itself did not induce cell proliferation or lymphokine secretion, and pretreatment of CD4+ T lymphocytes with anti-CD46 antibodies did not significantly alter TCR/CD3 activation., (Copyright 2004 Wiley-VCH Verlag GmbH & Co.)

The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position -236 and -93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position -117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.

Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors' native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.

Altered signaling through the TCR is currently showing promise for immunotherapy. However, the molecular mechanisms are not completely understood. Therefore, we investigated whether varying the strength of TCR engagement in various human T cells would yield different second messenger responses. The kinetics and duration of extracellular signal-regulated kinase (ERK) activation, central to multiple cellular responses, are distinctly dependent on the T cell activation state (naive vs effector), strength of TCR cross-linking, and input from the phosphatidylinositol-3 kinase pathway, which is regulated by cytokines and growth factors. Moreover, the duration of ERK activation affects c-Fos expression, a component of the AP-1 transcription complex. Thus, the character of ERK activation, transient or sustained, acts as a signal integrator to quantify the strength of TCR engagement and direct the cellular response.

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.

T cell activation leads to the induction of the transcription factors of the NFAT and NF-kappa B families, important regulators of T cell activation and function. In this study we demonstrate that TCR/CD3-stimulated T cells from mice expressing a constitutively active form of protein kinase B (myr PKB alpha) lack significant nuclear accumulation/shuttling of NFATc1 and NFATp as well as NF-kappa Bp65 and RelB proteins. Notably, despite this deficit in nuclear NFAT and NF-kappa B proteins, myr PKB T cells show lower activation threshold for proliferation, enhanced cell cycle progression and increased production of Th1 and Th2 cytokines similar to signals provided by CD28 costimulation. The enhanced T cell response correlates with increased expression of cyclins D3 and B1 and cytokine-induced Src homology 2 protein, and inactivation of the forkhead transcription factor FKHR. In addition, coimmunoprecipitation studies indicate a direct regulation of NFATc1 by active PKB. Together, our results demonstrate that the positive regulatory role of myr PKB on TCR responsiveness, subsequent cell division, and effector function is linked to a negative regulatory mechanism on the nuclear accumulation/shuttling of NFAT and NF-kappa B proteins.

T cell anergy has been demonstrated to play a role in maintaining peripheral tolerance to self Ags as well as a means by which tumors can evade immune destruction. Although the precise pathways involved in anergy induction have yet to be elucidated, it has been linked to TCR engagement in the setting of cell cycle arrest. Indeed, rapamycin, which inhibits T cell proliferation in G(1), has the ability to promote tolerance even in the presence of costimulation. To better define the role of the cell cycle in regulating anergy induction, we used the novel cyclophilin-binding ligand, sanglifehrin A (SFA). We demonstrate that SFA can inhibit TCR-induced cytokine and chemokine production without preventing TCR-induced anergy. Our data also indicate that despite its ability to induce G(1) arrest, SFA does not induce anergy in the presence of costimulation. Furthermore, although SFA blocks proliferation to exogenous IL-2, it does not prevent IL-2-induced reversal of anergy. When we examined the phosphorylation of 4EBP-1, a downstream substrate of the mammalian target of rapamycin, we found that rapamycin, but not SFA, inhibited the mammalian target of rapamycin activity. Based on these data, we propose that the decision as to whether TCR engagement will lead to productive activation or tolerance is dictated by a rapamycin -inhibitable pathway, independent of the G(1)-->S phase cell cycle progression.

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-gamma production.

Peripheral T lymphocytes undergo activation by antigenic stimulation and function in hypoxic areas of inflammation. We demonstrated in CD3-positive human T cells accumulating in inflammatory tissue expression of the hypoxia-inducible factor-1alpha (HIF-1alpha), indicating a role of hypoxia-mediated signals in regulation of T cell function. Surprisingly, accumulation of HIF-1alpha in human T cells required not only hypoxia but also TCR/CD3-mediated activation. Moreover, hypoxia repressed activation-induced cell death (AICD) by TCR/CD3 stimulation, resulting in an increased survival of the cells. Microarray analysis suggested the involvement of HIF-1 target gene product adrenomedullin (AM) in this process. Indeed, AM receptor antagonist abrogated hypoxia-mediated repression of AICD. Moreover, synthetic AM peptides repressed AICD even in normoxia. Taken together, we propose that hypoxia is a critical determinant of survival of the activated T cells via the HIF-1alpha-AM cascade, defining a previously unknown mode of regulation of peripheral immunity.

X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the gene encoding NEMO/IKKgamma, the regulatory subunit of the IkappaB kinase (IKK) complex. IKK normally phosphorylates the IkappaB-inhibitors of NF-kappaB at specific serine residues, thereby promoting their ubiquitination and degradation by the proteasome. This allows NF-kappaB complexes to translocate into the nucleus where they activate their target genes. Here, we describe an autosomal-dominant (AD) form of EDA-ID associated with a heterozygous missense mutation at serine 32 of IkappaBalpha. This mutation is gain-of-function, as it enhances the inhibitory capacity of IkappaBalpha by preventing its phosphorylation and degradation, and results in impaired NF-kappaB activation. The developmental, immunologic, and infectious phenotypes associated with hypomorphic NEMO and hypermorphic IKBA mutations largely overlap and include EDA, impaired cellular responses to ligands of TIR (TLR-ligands, IL-1beta, and IL-18), and TNFR (TNF-alpha, LTalpha1/beta2, and CD154) superfamily members and severe bacterial diseases. However, AD-EDA-ID but not XL-EDA-ID is associated with a severe and unique T cell immunodeficiency. Despite a marked blood lymphocytosis, there are no detectable memory T cells in vivo, and naive T cells do not respond to CD3-TCR activation in vitro. Our report highlights both the diversity of genotypes associated with EDA-ID and the diversity of immunologic phenotypes associated with mutations in different components of the NF-kappaB signaling pathway.

T cells have an amazing ability to discern and differentially respond to MHC-embedded peptides that can differ by only a single amino acid. This potential involves a combination of the precise ligand-binding specificities of the T-cell receptor (TCR) and the distinct intracellular signaling processes it transmits. Signaling processes are controlled by the ten immunoreceptor tyrosine-based activation motifs (ITAMs) present in the invariant chains of the TCR complex (TCR zeta and CD3-gamma, -delta and -epsilon ). Here, we discuss recent studies of the functions of TCR invariant chains and the contribution of the ten ITAMs to T-cell signal transmission. We incorporate these results into two non-exclusive models of TCR signal transduction: the ITAM multiplicity model, which describes a functional redundancy within the TCR zeta and CD3 ITAMs; and the differential signaling model, which proposes distinct functions for the CD3-gamma, -delta and -epsilon and TCR zeta modules.

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.

CD43 is an abundant cell surface sialoglycoprotein implicated in hemopoietic cell adhesion and activation. Cell stimulation through CD43 results in recruitment of different signaling proteins, including members of the Src family kinases, Syk, phospholipase Cgamma2, the adapter protein Shc, the guanine nucleotide exchange factor Vav, and activation of protein kinase C. In this study, we report that in human T lymphocytes, the zeta-chain is part of the CD43 signaling pathway. Upon CD43 engagement, the zeta-chain was tyrosine-phosphorylated, generating docking sites for tyrosine-phosphorylated zeta-associated protein of 70 kDa and Vav. In vitro kinase assays suggested that zeta-associated protein of 70 kDa could account for the kinase activity associated with the zeta-chain following CD43 engagement. Cross-linking CD43 on the surface of the Lck-deficient JCaM.1 cells failed to phosphorylate the zeta-chain and associated proteins, suggesting that Lck is a key element in the CD43 signaling pathway leading to zeta phosphorylation. CD43 engagement with beads coated with anti-CD43 mAb resulted in concentration of the zeta-chain toward the bead attachment site, but interestingly, the distribution of the T cell Ag receptor complex remained unaffected. Recruitment of the zeta-chain through CD43-mediated signals was not restricted to T lymphocytes because phosphorylation and redistribution of the zeta-chain was also observed in NK cells. Our results provide evidence that the zeta-chain functions as a scaffold molecule in the CD43 signaling pathway, favoring the recruitment and formation of downstream signaling complexes involved in the CD43-mediated cell activation of T lymphocytes and other leukocytes such as NK cells.

Protein kinase B (PKB), a serine threonine kinase is critically involved in cellular proliferation and survival. To characterize its role in T cell development in vivo, we have analyzed transgenic mice that express a membrane-targeted constitutively active version of PKB (myr PKB) in thymocytes and peripheral T cells. We report that myr PKB renders proliferative responses of thymocytes more sensitive to TCR signals by increased and sustained activation of Src kinase Lck and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In addition, the proliferative response of myr PKB T cells is relatively independent of calcium mobilization and calcineurin activity. We also find that myr PKB enhances phosphorylation of glycogen synthase kinase 3, a negative regulator of NFAT and T cell activation, and the recruitment of the adapter protein Cbl-c. Interestingly, we demonstrate that upon TCR/CD3 stimulation of wild-type T cells PKB is translocated into lipid rafts, adding a new role for PKB in TCR-initiated signalosome formation in T cell activation. Localization of transgenic PKB in lipid rafts could contribute to the higher TCR sensitivity of myr PKB thymocytes which is reflected in an increase in positive selection toward the CD4 lineage and variable effects on negative selection depending on the model system analyzed. Thus, our observations clearly indicate a cross-talk between PKB and important signaling molecules downstream of TCR that modulate the thresholds of thymocyte selection and T cell activation.

Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fcgamma receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.

The earliest biochemical events at the plasma membrane that lead to gene activation appear to depend not only on the local concentration of signaling molecules, but also on the mobility of these molecules at the site of signaling. To elucidate the process of signal transduction after receptor engagement in the immune system, it is important to analyze the mobility of signaling molecules in living lymphocytes. Current knowledge of the changes in intracellular localization and dynamic movements of signaling molecules during lymphocyte activation is limited. Here, we describe a method for known as fluorescence recovery after photobleaching, used to measure the diffusion mobility of a signaling molecule in a T cell line after T cell receptor stimulation. This method is a useful tool in studies of spatiotemporal regulation in immunoreceptor signaling.

The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.

The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The beta(2) leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758-760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the beta(2) integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, beta(7), has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused beta(7) integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the beta(7) subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in beta(2) phosphorylation.

High level expression of Fc epsilon RI gamma chain replaces the deficient TCR zeta-chain and contributes to altered TCR/CD3-mediated signaling abnormalities in T cells of patients with systemic lupus erythematosus. Increased responsiveness to Ag has been considered to lead to autoimmunity. To test this concept, we studied early signaling events and IL-2 production in fresh cells transfected with a eukaryotic expression vector encoding the Fc epsilon RI gamma gene. We found that the overexpressed Fc epsilon RI gamma chain colocalizes with the CD3 epsilon chain on the surface membrane of T cells and that cross-linking of the new TCR/CD3 complex leads to a dramatic increase of intracytoplasmic calcium concentration, protein tyrosine phosphorylation, and IL-2 production. We observed that overexpression of Fc epsilon RI gamma is associated with increased phosphorylation of Syk kinase, while the endogenous TCR zeta-chain is down-regulated. We propose that altered composition of the CD3 complex leads to increased T cell responsiveness to TCR/CD3 stimulation and sets the biochemical grounds for the development of autoimmunity.

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of I kappa B kinase (IKK) complex and, consequently, to NF-kappa B activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKK alpha in both Jurkat and primary CD4(+) T cells. Vav-1/IKK alpha association is mediated by their helix-loop-helix domains, does not involve IKK beta, and is functionally relevant in that Vav-1-associated IKK alpha kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-kappa B activation is augmented by both IKK alpha and Vav-1, but not IKK beta. Confocal microscopy showed that endogenous Vav-1 and IKK alpha, but not IKK beta, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-kappa B pathway by targeting IKK alpha in the T cell membrane and favoring its activation in response to CD28 stimulation.

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.

In Jurkat T cells, the type 3 ryanodine receptor (RyR) was knocked-down by stable integration of plasmid expressing type 3 ryanodine receptor antisense RNA. Stable integration of the antisense plasmid in individual clones was demonstrated by PCR of genomic DNA, expression of antisense RNA by reverse transcriptase PCR, and efficiently reduced expression of type 3 ryanodine receptor protein by Western blot. Selected clones were successfully used to analyze T cell receptor/CD3 complex-mediated Ca(2+) signaling. Reduced expression of the type 3 RyR resulted in (i) significantly decreased Ca(2+) signaling in the sustained phase and (ii) in permeabilized cells in a significantly impaired response toward cyclic ADP-ribose but not to d-myo-inositol 1,4,5-trisphosphate. For the first time, the role of the type 3 RyR in sustained Ca(2+) signaling was directly visualized by confocal Ca(2+) imaging as a significant contribution to the number and the magnitude of subcellular Ca(2+) signals. These data suggest that the type 3 ryanodine receptor is essential in the sustained Ca(2+) response in T cells.

Previous research has shown that many of the CD4 T cells from older mice do not form functional immune synapses after conjugation with peptide-pulsed APC. We now show that the defect lies at a very early stage in the cytoskeletal reorganization that precedes movement of protein kinases and their substrates to the TCR/APC interface. Antagonist peptides presented to T cells from young mice induce migration of talin (but not paxillin, vinculin, or F-actin) to the APC contact zone, but CD4 T cells from older donors typically fail to show the talin polarization response. A spreading assay in which contact with anti-CD3-coated slides induces CD4 T cells to assume a conical shape and develop lammelopodia also shows a decline with age in the proportion of T cells that can initiate cytoskeletal changes in response to this simplified stimulus. Finally, the transition from detergent-soluble to cytoskeletal forms of the p16, p21, and p23 isoforms of CD3zeta in response to CD3/CD4/CD28 cross-linking is much stronger in young than in old T cells. Thus, defects in cytoskeletal reorganization triggered by initial contact between TCR and peptide-bearing APC precede, and presumably contribute to, defective activation of protein kinase-mediated signals in the first few minutes of the activation cascade in T cells from aged mice.

A dual specific human CTL clone harboring one beta and two inframe alpha transcripts of TCR was previously reported to recognize an HIV Pol-derived nonapeptide (IPLTEEAEL) endogenously presented by both syngeneic HLA-B*3501 and HLA-B*5101. In the current study, a retrovirus-mediated TCR transfer of individual alpha- and beta-chains to TCR-negative hybridoma showed that Valpha12.1 TCR in complex with Vbeta5.6 were responsible for the peptide-specific response in the context of both HLA-B*3501 and HLA-B*5101, confirming single TCR-mediated dual specificity. The second TCR-alpha chain was not somehow expressed on the cell surface. Remarkably, the Valpha12.1/Vbeta5.6 TCR also recognized the same peptide presented by allogeneic HLA class I molecules that share the similar peptide-binding motifs, such as HLA-B*5301 and HLA-B*0702. The sensitivity of peptide recognition by the Valpha12/Vbeta5.6 TCR appeared to be comparable when the peptide was presented by syngeneic and allogeneic HLA class I molecules, with changes in T cell responsiveness caused largely by peptide-binding capacity. Moreover, the CTL clone bearing Valpha12.1/Vbeta5.6 TCR showed substantial cytolytic activity against the peptide-loaded cells expressing HLA-B*3501, HLA-B*5101, HLA-B*5301, or HLA-B*0702, providing further evidence that a single TCR complex can recognize the same peptide presented by a broad range of HLA class I molecules. A TCR with fine specificity for an HIV Ag but broad specificity to multiple HLA molecules may provide an advantage to the generation of allorestricted, peptide-specific T cells, and thus could be a potent candidate for immunotherapy against HIV infection.

Maturation arrest and interference with selection are two well-documented effects of cyclosporin-A (CsA) on the thymus. We recently hypothesized that these effects are related and owing to the reduced T-cell receptor (TCR)-CD3 complex-mediated signal transduction in thymocytes upon CsA treatment. In this hypothesis, the maturation arrest is the result of the additional depletion of thymocytes that normally survive by positive selection, whereas the impaired self-tolerance induction is caused by an increased survival of thymocytes that normally undergo negative selection. In this view, it is anticipated that CsA differentially affects thymocyte apoptosis during in vivo thymocyte maturation. Indeed, we report in this study a strong increase in apoptotic cells in the thymic cortex on in situ analysis. Simultaneously, the number of apoptotic cells had decreased at the cortico-medullary zone which is held to be the site for negative selection. Rapamycin (Rapa) also interferes with thymocyte maturation by inhibiting cytokine-driven proliferation. Hence, Rapa preferentially affects the early maturational stages of thymocyte development and is considered not to alter thymocyte selection and subsequent apoptotic events. Indeed, the number of apoptotic events appears not to be altered. However, possibly owing to the decrease in cortical macrophages, the apoptotic cells revealed an atypical enumeration around blood vessels. Taken together, our results favour the hypothesis that the dominant effect of CsA on the thymus is the reduction of the TCR-CD3 complex-mediated signal transduction in thymocytes upon interaction with stromal cells. Furthermore, the preferential localization of apoptotic cells next to blood vessels upon Rapa administration may indicate that endothelial cells are a back-up system for the removal of apoptotic cells.

NF-kappa B-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-beta receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-kappa B activity in both mature and immature T cells; the impaired NF-kappa B activity in mature T cells was also associated with the failure of maintenance of activated NF-kappa B. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-kappa B activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.

Peyer's patches (PP) are believed to be the principal sites for induction of tolerance to Ags from food and commensal flora, yet the phenotype of T cells activated within the PP is largely unexplored. We hypothesize that exposure to Ags within the PP promotes differentiation of T cells with immunoregulatory functions. Cytokine production and cell surface marker expression of murine PP mononuclear cells (MC) are compared with those from mesenteric lymph nodes and peripheral lymph nodes (PLN). In response to stimulation through the TCR/CD3 complex, PP MC exhibit vigorous proliferation, modest production of IL-2, and significantly elevated synthesis of IL-10. Exogenous IL-12 enhances both IL-10 and IFN-gamma secretion by activated PP MC. Cell surface marker analysis reveals that PP T cells consist of activated and memory subpopulations compared with the predominantly naive T cells identified in the PLN and mesenteric lymph nodes. Upon stimulation, only CD45RB(low)CD4(+) PP T cells produce IL-10, whereas secretion of IL-2, IL-4, and IFN-gamma was not detected. Furthermore, PP MC, but not PLN MC, stimulated through the TCR/CD3 complex suppress proliferation of purified PLN T cells in vitro, evidence for a regulatory function among PP lymphocytes. We conclude that PP favor differentiation of an IL-10-producing, regulatory CD45RB(low)CD4(+) T cell population and that inhibition of T cell proliferation by activated PP MC may reflect regulatory activity consistent with T regulatory cells.

In the present study, we aimed to demonstrate that CD4 may represent a critical turning point that governs the apoptotic and survival programs in T cells, without modifying the physical association with the TCR-CD3 complex. To address this issue, we have explored the possibility that the activation of CD4 may transduce apoptotic signals unless signaling effectors neutralize them. Our data show that in Jurkat T cells CD4 engagement by Leu3a mAb results in a rapid and strong increase of Lck kinase activity, subsequent alterations of mitochondrial membrane potential, and apoptosis. Critical parameters are coassociation of CD4/Lck with TCR/CD3 and up-regulation of the proapoptotic protein Bax. Indeed, Leu3a-mediated Lck activation failed to induce apoptotic features in Jurkat cells either defective for TCR/CD3 or overexpressing the antiapoptotic protein Bcl-2. Furthermore, we demonstrate that Leu3a treatment of Jurkat cells overexpressing Vav results in the inhibition of mitochondrial damage and apoptosis; this rescue effect is accompanied with a significant decrease of Bax expression observed in apoptotic cells. Our evidence that the activation of Lck activates in T cells apoptotic pathways which are counteracted by Vav, a signaling molecule that cooperates with CD28 to boost TCR signals, suggests a novel role for costimulation in protecting T cells from CD4-mediated cell death.

Adhesion of leukocytes to the vascular endothelium is an early event in inflammation. Since cell-cell signaling may be an important stimulus for endothelial activation, we focused in this study on the role of contact-mediated activation by T lymphocytes of endothelial cells (EC). T lymphocytes were cultured with anti-CD3 monoclonal antibody or in the presence of a combination of TNF-alpha, interleukin (IL)-6, and IL-2, prior to fixation and coculture with human umbilical vein EC. Fixed, activated (anti-CD3- or cytokine-stimulated), but not unstimulated T cells, induced release of monocyte chemotactic protein-1, IL-8, and IL-6 by EC in a contact-dependent manner. Moreover, expression of tissue-factor antigen and activity was also significantly increased. Addition of anti-CD40 ligand antibody abolished T cell-induced activation of EC. Our data suggest that contact-mediated activation of EC by T cells, involving ligand:counter ligand interactions such as CD40:CD40 ligand, may represent a novel pathogenic mechanism of progression in inflammatory diseases such as atherosclerosis or rheumatoid arthritis.

CD28 provides a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell activation, cytokine production, and clonal expansion. We have recently shown that CD28 directly regulates progression of T lymphocytes through the cell cycle. Although a number of signaling pathways have been linked to the TCR/CD3 and to CD28, it is not known how these two receptors cooperate to induce cell cycle progression. Here, using cell-permeable pharmacologic inhibitors of phosphatidylinositol 3-hydroxykinase (PI3K) and mitogen-activated protein kinase kinase (MEK1/2), we show that cell cycle progression of primary T lymphocytes requires simultaneous activation of PI3K- and MEK1/2-dependent pathways. Decreased abundance of cyclin-dependent kinase inhibitor p27(kip1), which requires simultaneous TCR/CD3 and CD28 ligation, was dependent upon both MEK and PI3K activity. Ligation of TCR/CD3, but not CD28 alone, resulted in activation of MEK targets extracellular signal-related kinase 1/2, whereas ligation of CD28 alone was sufficient for activation of PI3K target protein kinase B (PKB; c-Akt). CD28 ligation alone was also sufficient to mediate inactivating phosphorylation of PKB target glycogen synthase kinase-3 (GSK-3). Moreover, direct inactivation of GSK-3 by LiCl in the presence of anti-CD3, but not in the presence of anti-CD28, resulted in down-regulation of p27(kip1), hyperphosphorylation of retinoblastoma tumor suppressor gene product, and cellular proliferation. Thus, inactivation of the PI3K-PKB target GSK-3 could substitute for CD28 but not for CD3 signals. These results show that the PI3K-PKB pathway links CD28 to cell cycle progression and suggest that p27(kip1) integrates mitogenic MEK- and PI3K-dependent signals from TCR and CD28 in primary T lymphocytes.

The mechanisms by which immunological memory is maintained after infection or vaccination are still a matter of debate. Long-term survival of memory T cells does not require major histocompatibility complex (MHC) contact. We show here that compared with memory CD4+ T cells that maintain contact with MHC class II, memory CD4+ T cells deprived of MHC class II contact show distinct functional defects upon antigen re-encounter. Thus, in contrast to their survival, maintenance of the typical quality of memory T cells crucially depends on MHC-derived signals.

T lymphocyte activation by specific antigen requires prolonged TCR occupancy and sustained signaling. This is accomplished by the formation of a specialized signaling domain, the immunological synapse, at the T cell-antigen-presenting cell contact site. Surface receptors and signaling components are progressively recruited into this domain where they are organized in defined three-dimensional structures. To better understand how TCR are supplied to the signaling domain during the activation process, we measured (using confocal microscopy and photo-bleaching recovery techniques) lateral mobility of GFP-tagged TCR on living Jurkat cell surface. We show that: (i) surface-expressed TCR exhibit an intrinsic, actin cytoskeleton-independent, lateral mobility which allows them to passively diffuse over the entire T cell surface within approximately 60 min and (ii) non-stimulated TCR rapidly enter the signaling domain. Our results indicate that TCR lateral mobility per se is sufficient to ensure TCR supply to the immunological synapse in the course of sustained T cell activation.

CD95 plays a critical role in the homeostasis of the immune system, and has been reported to participate in T cell death during HIV infection. Here we report that the response to CD3-TCR stimulation of CD4(+) T cells from HIV-infected individuals and CD4(+) T cells from healthy donors incubated in vitro with HIV-1(Lai) depends on the manner the CD3-TCR complex is engaged. While stimulation by anti-CD3 antibodies in solution induced CD4 T cell apoptosis both in the absence or presence of anti-CD95 antibodies, stimulation by immobilized anti-CD3 antibodies rendered CD4(+) T cells resistant to CD95-mediated death and led to increased CD4 T cell proliferation in response to CD95 ligation. CD95 ligation of CD4(+) T cells led to the activation of caspases, while costimulation induced by anti-CD3 and anti-CD95 mAb prevented the full processing of caspase-3 and caspase-8. Proliferation of CD4(+) T cells induced by CD3-TCR and CD95 costimulation was decreased by treatments with a caspase-1 inhibitor or with neutralizing antibodies to IL-1ss, indicating a requirement for caspase-1-mediated IL-1beta processing and secretion. Our findings suggest a novel mechanism whereby in addition to its role in inducing T cell apoptosis, CD95 signaling during HIV infection may also provide a costimulatory signal leading to an enhancement of CD4 T cell proliferation in response to CD3-TCR complex engagement.