TCR beta chain that forms peptide-independent alloreactive TCR transfers reduced reactivity with irrelevant peptide/MHC complex

A major feature of the TCR repertoire is strong alloreactivity. Peptides presented by allogeneic MHC are irrelevant for recognition by a subset of alloreactive T cells. To characterize peptide-independent TCRs at the molecular level, we forced the expression of a TCRbeta chain isolated from a peptide-independent alloreactive CD8+ T cell line. The alloreactive TCR repertoire in the transgenic mouse was peptide dependent. However, analysis of essential TCR contacts formed during the recognition of self-MHC-restricted Ag showed that fewer contacts with peptide were established by the transgenic TCRbeta chain, and that this was compensated by additional contacts formed by endogenous TCRalpha chains. Thus, reduced interaction with the peptide appears to be a transferable feature of the peptide-independent TCRbeta chain. In addition, these findings demonstrate that reactivity to peptides is preferred over the reactivity to MHC during the formation of the TCR repertoire.     

TCR internalization induced by peptide/MHC ligands requires the transmembrane domains of alphabeta chains of TCR, but not the expression of CD8 and Thy-1 molecules

T-cell receptor (TCR) internalization occurs via TCR recognition of the peptide/MHC molecule complex on antigen presenting cell (APC). In this study, the requirements for inducing the internalization of TCR molecules on Ld major histocompatibility complex (MHC) class I-restricted T-cells were investigated with 2C cytotoxic T-lymphocyte (CTL) clones with defined peptides as the antigen. To evaluate the function of the transmembrane region of TCR alphabeta chains in TCR internalization, we generated T-cell transfectants expressing the wild type and glycosylphosphatidyl inositol (GPI)-linked form of 2C TCR. Among all peptides forming proper ligands to 2C TCR, only the Qp2Ca peptide induced TCR internalization, which was known to have the highest affinity to both Ld MHC class I molecules and TCR in association with Ld molecules. Such TCR internalization was not observed in cells expressing the GPI-linked form of 2C TCR. Furthermore, the expression of CD8 coreceptor and Thy-1 accessory molecules were both not required for Qp2Ca-induced TCR internalization, and these molecules did not accompany TCR internalization. Altogether, these results suggest that TCR internalization on CTL is not a prerequisite for CTL function.     

High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC

The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D(b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E. coli as inclusion bodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicate that human p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D(b). Yet, T cell binding and cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs that block CD8 association with MHC. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produces p56(lck) activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding. Taken together, these results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement.     

Plasticity and rigidity in adaptor protein-2-mediated internalization of the TCR:CD3 complex

Many cell surface proteins are internalized via dileucine- or tyrosine-based motifs within their cytoplasmic domains by the heterotetrameric adaptor protein complex, AP-2. In this study we have examined how AP-2 mediates internalization of large cell surface receptors, such as the eight-chain TCR:CD3 complex. Although most receptors have a single signal that drives internalization, the TCR complex has two (D/E)xxxL(L/I) motifs and 20 Yxx? motifs. Using 293T cells, we show that AP-2 is completely dependent on both signals to mediate TCR internalization, because deletion of either completely blocks this process. Significant plasticity and redundancy were observed in the use of the Yxx? motifs, with a clear hierarchy in their use (CD3delta > CD3gamma >or= CD3zeta > CD3epsilon). Remarkably, a single, membrane-distal Yxx? motif in CD3delta could mediate approximately 75% of receptor internalization, whereas its removal only reduced internalization by approximately 20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3gamma. This was due to an absolute requirement for the position of this signal in the context of the TCR complex and for a highly conserved lysine residue, K128, which is not present in CD3delta. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.     

Unidirectional, heterologous desensitization of the pertussis toxin receptor by the CD3/TCR complex.

Prolonged exposure of many types of receptors to their cognate agonists can lead to a progressive lack of responsiveness. When this occurs after stimulation by the primary agonist for a given receptor it is termed homologous desensitization, and heterologous desensitization when to an agonist binding to a different type of receptor. Pertussis toxin (PTx) is a potent mitogen for human T lymphocytes. We have previously identified the human T cell PTx receptor (PTx-R) as a 43-kDa plasma membrane protein that, when stimulated, leads to the production of the intracellular second messengers, inositol-1,4,5-triphosphate, 1,2-sn-diacylglycerol, and elevated cytosolic calcium. The PTx-R appears to require the co-expression of the CD3/TCR complex because mutant cells that lack the AgR, but express the PTx-R, fail to respond to PTx. In this report, we have investigated the relationship between these two receptor systems. Activation of the PTx-R with submaximal concentrations of PTx did not affect the ability of an anti-CD3 antibody combined with rabbit anti-mIg to stimulate increases in intracellular free calcium concentration [Ca2+]i or diacylglycerol in human peripheral blood T cells. However, treatment with soluble anti-CD3 mAb, which lead to only a modest increase in [Ca2+]i, completely inhibited the effect of PTx. The cells were not refractory to further stimulation of the AgR because cross-linking with rabbit anti-mIg resulted in the standard maximal stimulation. This effect could be observed within 1 min of treatment with anti-CD3 mAb, and persisted for at least 1 h. The effect was not caused by production of either diacylglycerol (leading to activation of PK-C) or an increase in [Ca2+]i by anti-CD3 mAb because the effect could not be mimicked by either phorbol esters or a calcium ionophore. Pretreatment of either resting T lymphoblasts or PBL with anti-CD3 mAb also prevented enhanced [3H]TdR incorporation stimulated by PTx. These observations suggest a model in which T cells can regulate amplification of a non-AgR stimulatory pathway by heterologous desensitization.     

Altered peptide ligand-mediated TCR antagonism can be modulated by a change in a single amino acid residue within the CDR3 beta of an MHC class I-restricted TCR

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

Thymic selection and peripheral activation of CD8 T cells by the same class I MHC/peptide complex

Thymic selection is controlled by the interaction between TCR and MHC/peptide. Strength and quality of the signal determine whether thymocytes are selected or deleted. The factors that contribute to this signal remain poorly defined. Here we show that fetal thymic organ cultures (FTOCs) derived from OT-I transgenic mice (the OT-I TCR is restricted by K(b)-SIINFEKL) on a K(b)D(b-/-) background support positive selection, but only when provided with soluble H-2K(b)-SIINFEKL complexes. Selection of CD8 T cells is independent of the valency of the ligand or its capability to coengage CD8 molecules. Both CD8alphaalpha and CD8alphabeta T cells are selected by H-2K(b)-SIINFEKL, but only CD8alphabeta cells are capable of releasing IFN-gamma in response to the same ligand. The alpha(4)beta(7) integrin is up-regulated on postselection thymocytes from FTOCs. After adoptive transfer, FTOC-derived OT-I CD8 T cells divide in response to the agonist peptide SIINFEKL. These results establish that CD8 T cells responsive to their nominal peptide-Ag can be generated in FTOC supplemented with soluble MHC class I molecules equipped with the same peptide.     

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.     

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.     

Stimulation through the TCR/CD3 complex up-regulates the CD2 surface expression on human T lymphocytes.

Despite the well known interrelationship between the CD2- and CD3-mediated signal transduction pathways, it is not well established whether the CD2 surface expression can be regulated by triggering of TCR/CD3 complex. In this study we show that the stimulation of human PBMC with the Cris-7 (CD3) mAb, both in soluble and particulate form, results in hyperexpression of the CD2 surface Ag, as assessed by immunofluorescence and semi-quantitative immunoprecipitation assays. Similar effects on CD2 surface expression were obtained when different CD3 mAb (OKT3, RW2-8C8 and Leu-4) were tested. The CD3-mediated CD2 up-regulation was suppressed by cycloheximide and actinomycin D, indicating that it requires de novo protein and RNA synthesis. In agreement with this, increased CD2 RNA levels were observed after 3 h of stimulation, reaching a plateau at 24 h that was maintained for 72 h. The CD2 up-regulation was concomitant to other CD3-induced activation-related events such as induction of surface CD25 and CD71 and high RNA levels for c-myc, IL-2R alpha- and beta-chains, CD71, and IFN-gamma. CD2 up-regulation appeared to be elicited by a protein kinase C-dependent mechanism because it was abrogated by staurosporine, a potent protein kinase C inhibitor. Moreover, IL-2-dependent events may also help in enhancing CD2 hyper-expression because it was only partially inhibitable by cyclosporine, dexamethasone, or Mar-108 (CD25) mAb. In conclusion, our data suggest that CD2 up-regulation can be a relevant event in T cell activation triggered by the physiologic engagement of the TCR/CD3 complex.     

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.     

Hapten addition to an MHC class I-binding peptide causes substantial adjustments of the TCR structure of the responding CD8(+) T cells

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR alpha- or beta-chain of a VSV8 (unmodified)/H-2K(b)-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3alpha and beta loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3beta residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jbeta region, while the Jalpha region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3alpha and beta loops to allow interaction with a haptenated peptide.     

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.     

Endocytosis of the TCR/CD3 complex and the class-I major histocompatibility complex in a human T cell line.

We investigated the expression of the T cell receptor (TCR)/CD3 complex on a CD4-positive human T cell lymphoma cell line treated with phorbol myristate acetate (PMA) and/or CA2+ ionophore using fluorescence flow cytometry and fluorescence microscopic analysis. PMA induced a significant decrease in the expression of the CD3 complex on the cell membranes. Fluorescence microscopy confirmed that the down regulation is due to internalization of the antigens. Ca2+ ionophore treatment had no effect on the internalization of the CD3 complex. Double staining revealed that the vesicles containing the internalized CD3 complex and those containing intra-cytoplasmic class I major histocompatibility complex antigen had similar distribution in the PMA-stimulated cells, implying coexistence of these two antigens in a cytoplasmic perinuclear distribution.     

Mature CD4+ T cells perceive a positively selecting class II MHC/peptide complex in the periphery

A repertoire of TCRs is selected in the thymus by interactions with MHC bound to self-derived peptides. Whether self peptides bound to MHC influence the survival of mature T cells in the periphery remains enigmatic. In this study, we show that the number of naive CD4+ T cells that developed in mice with class II MHC bound with endogenous peptides (Abwt) diminished when transferred into mice with Ab covalently bound with a single peptide (AbEp). Moreover, transfer of a mixture of naive CD4+ T cells derived from Abwt and from AbEp mice into AbEp mice resulted in the expansion of the latter and decline of the former. In contrast, when wild-type activated CD4+ T cells were transferred into AbEp or Abwt mice, these cells survived in both recipients for more than 4 wk, but further expanded in the Abwt host. We conclude that to survive, naive CD4+ T cells favor peripheral expression of the class II MHC/peptide complex(es) involved in their thymic selection, whereas some of activated CD4+ T cells may require them only for expansion.     

Opening a window on thymic positive selection: developmental changes in the influence of cosignaling by integrins and CD28 on selection events induced by TCR engagement

How TCR and non-TCR signals are integrated by thymocytes to generate a decision to undergo either positive or negative selection remains incompletely understood. Recent evidence suggests that TCR signal transduction changes its quality during thymocyte maturation, but whether the contributions of various cosignaling or costimulatory pathways to thymocyte selection also are modified during development is unclear. Questions also remain about the possible selective roles of specific costimulatory pathways in induction of differentiation vs death among thymocytes at any given stage of maturity. To address these issues, a quantitative in vitro analysis of initiation of CD4+CD8+ thymocyte differentiation as measured by CD69 up-regulation/coreceptor down-modulation was conducted in parallel with an analysis of induction of death. Using transfected cells varying in their surface display of ICAM-1 or B7.1 along with antibody blocking experiments, we demonstrate here that ICAM-1 provides a selective boost to signaling for differentiation without substantially affecting induction of death among CD4+CD8+ cells, a property that is lost as thymocytes mature further. In contrast, B7 engagement enhances both cell activation and death in parallel. Based on these data, we propose that the high level of ICAM-1 on cortical epithelial cells plays a special role in opening a window between TCR signaling for differentiation vs death, permitting efficient initiation of positive selection on epithelial ligands. In contrast, late CD28-dependent cosignaling on hemopoietic cells in the medulla would help enforce negative selection by augmenting the effects of TCR engagement by low levels of high affinity ligands.