The αβ T Cell Receptor Clustering Upon Superantigen/MHC Recognition

Antigen recognition by T cells is the key event for the antigen specific immune responses to be triggered. This recognition is initiated by the binding of the T cell receptor (TCR) to antigen peptide/major histocompatibility complex (MHC) on the surface of the antigen presenting cells. TCR on most of the T cells is a heterodimer composed of α and β chains which are associated with CD3 γδε as well as ζ chains, the signal transmission molecules. The dynamics of this TCR complex upon antigen/MHC recognition, however, has not been well understood. In this paper the authors analyse the configuration of TCR complex on T cells from a TCR β chain gene transgenic mouse (TGM) strain. Unlike many other TGM strains reported, a considerable proportion of T cells from this TGM expresses both transgene-encoded (Vβ3) and endogenous TCR β chains on their surface. By immunoprecipitation and immunoblotting analysis of T cells stimulated with a superantigen, staphylococcal enterotoxin B (SEB), the authors found that Vβ3 was coprecipitated with Vβ8, demonstrating the clustering of TCR αβ upon superantigen/MHC recognition.     

Human intraepithelial lymphocytes

The location of intraepithelial lymphocytes (IEL) between epithelial cells, their effector memory, cytolytic and inflammatory phenotype positions them to kill infected epithelial cells and protect the intestine against pathogens. Human TCRαβ⁺CD8αβ⁺ IEL have the dual capacity to recognize modified self via natural killer (NK) receptors (autoreactivity) as well as foreign antigen via the T cell receptor (TCR), which is accomplished in mouse by two cell subsets, the naturally occurring TCRαβ⁺CD8αα⁺ and adaptively induced TCRαβ⁺CD8αβ⁺ IEL subsets, respectively. The private/oligoclonal nature of the TCR repertoire of both human and mouse IEL suggests local environmental factors dictate the specificity of IEL responses. The line between sensing of foreign antigens and autoreactivity is blurred for IEL in celiac disease, where recognition of stress ligands by induced activating NK receptors in conjunction with inflammatory signals such as IL-15 can result in low-affinity TCR/non-cognate antigen and NK receptor/stress ligand interactions triggering destruction of intestinal epithelial cells.     

Changes in Arrangement and in Conformation of Molecular Components of Peripheral T-Cell Antigen Receptor Complex After Ligand Binding: Analyses by Co-Precipitation Profiles

Amounts of co-precipitating CD3 components by anti-T-cell receptor (TCR)Vβ or anti-CD4/8 monoclonal antibodies were compared between non-stimulated and stimulated splenic T cells. The amounts of co-precipitating CD3δ, ε and γ chains with TCRαβ and with CD4/8 were not significantly changed after TCR ligation. The apparent amount of CD3ζ chain co-precipitated with TCRαβ increased up to threefold, while the actual amount of co-precipitating CD3ζ with TCRαβ and the total amount of specifically precipitated CD3ζ are not changed after cross linking of TCR. The apparent amount of CD3ζ chain co-precipitated with CD4/8 also increased. Unlike co-precipitation with TCRαβ, the actual amount of CD3ζ co-precipitated with CD4/8 increased significantly. This observation suggests a conformational change as well as the relocation of CD3ζ molecules within the TCR complex after the signal delivery. After TCR ligation, CD3ζ chains relocate to the vicinity of either CD4 or CD8 molecules. In addition, when cross linking and binding signals are compared, CD3 chains undergo two distinct phases of conformational change. The early conformational change caused by ligand binding is positively related to the induction of proliferative responses, while the later conformational change caused by the cross linking of TCR does not induce but enhances the proliferative response.     

Involvement of inhibitory NKRs in the survival of a subset of memory-phenotype CD8+ T cells

Inhibitory natural killer receptors (NKRs) such as killer cell immunoglobulin-like receptors (KIRs) in humans and Ly49 molecules in mice are expressed on NK cells and recognize multiple major histocompatibility (MHC) class I proteins. In humans and mice, a subset of CD8+ T cells also expresses NKRs and harbors a memory phenotype. Using mice that are transgenic for KIR2DL3 and its cognate HLA-Cw3 ligand, we show that engagement of inhibitory NKRs selectively drives the in vivo accumulation of a subset of memory-phenotype CD8+ T cells that express the beta chain of the interleukin 2 receptor. In vitro, recognition of MHC class I molecules by inhibitory NKRs on T cells down-regulated activation-induced cell death. These results unveil an MHC class I-dependent pathway that promotes the survival of a subset of memory-phenotype CD8+ T cells and also reveal an unexpected biological function for inhibitory NKRs on T cells.     

The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR alpha/beta-mediated recognition.

Cytolytic T lymphocytes (CTL) are known to recognize antigen peptides in association with major histocompatibility complex (MHC) class I molecules expressed on target cells. However, a fraction of human CD8(+) CTL has been shown to lyse certain natural killer (NK)-susceptible target cells via still undefined mechanism(s). These CD8(+) T cells, hereafter referred to as NK-CTL, are frequently composed of cells expressing one single TCR Vbeta expansion (different in different individuals), display a memory phenotype and express HLA class I-specific inhibitory NK receptors. Here we show that cell populations or clones of NK-CTL isolated from three healthy donors homogeneously expressed Vbeta16, Vbeta9 and Vbeta3 TCR, respectively. Various clones isolated under limiting dilution conditions from Vbeta16(+) cells of donor 1 displayed identical TCR Vbeta and Valpha rearrangements, thus suggesting a substantial monoclonality of the NK-CTL subset analyzed. NK-CTL lysed a number of NK-susceptible tumor target cells with the exception of those characterized by beta2-microglobulin (beta2m) deficiency. However, the latter targets became susceptible to lysis upon beta2m transfection. Using monoclonal antibodies specific for the relevant TCR Vbeta or beta2m we provide evidence suggesting that target cell lysis by NK-CTL is mediated by the TCR itself upon recognition of beta2m-associated proteins. The cellular distribution of the potential beta2m-associated proteins in susceptible target cells suggested, as a likely candidate for TCR-mediated recognition, the non-classical HLA-E molecule. The use, as target cells, of the murine TAP2-deficient RMA-S cells, either untransfected or transfected with HLA-E, and loaded with an appropriate HLA-E-binding peptide, provided the direct demonstration that HLA-E represents a ligand recognized by the TCR expressed by NK-CTL. This is the first evidence that human TCR alpha/beta can recognize HLA-E molecules, thus revealing a novel type of TCR-mediated recognition, which may offer new insight in immune responses in both normal and disease conditions.     

Vδ1+ Gamma/Delta T Lymphocytes Infiltrating Human Lung Cancer Express the CD8α/α Homodimer

Murine γ/δ T lymphocytes localize to different epithelial tissues and are phenotypically distinct from peripheral γ/δ T cell-populations in that they show limited TCR diversity, express the CD8 α/α homodimer and lack the CD8β chain. In humans, a compartmentalization of γ/δ cells sharing similar phenotypic features has been documented to date only in the case of intestinal epithelium. In the present study we show that about half of Vδ1⁺ (as well as Vδ1⁻Vδ2⁻) γ/δ lymphocytes, which can be selectively expanded from human lung cancers, coexpress the CD8α/α homodimer. The accumulation of intraepithelial CD8⁺γ/δ⁺ lymphocytes might then be a more general phenomenon, possibly as a result of common mechanisms operating at those sites.     

TCR and Notch signaling in CD4 and CD8 T-cell development

Summary:  The generation of CD4 and CD8 αβ T-cell lineages from CD4⁺CD8⁺ double-positive (DP) thymocyte precursors is a complex process initiated by engagement of major histocompatibility complex (MHC) by T-cell receptor (TCR) and coreceptor. Quantitative differences in TCR signaling induced by this interaction impose an instructional bias on CD4/CD8 lineage commitment that must be reinforced by MHC recognition and TCR signaling over subsequent selection steps in order for the thymocyte to progress and mature in the adopted lineage. Our studies show that the transmembrane receptor Notch plays a role in this process by modifying TCR signal transduction in DP thymocytes. In this review, we consider the functional relationship of TCR and Notch signaling pathways in the selection and specification of CD4 and CD8 T-cell lineages.     

Interactions between CD8alphabeta and the TCRalphabeta/CD3-receptor complex

CD8+ T cells recognize antigenic peptides bound to major histocompatibility complex (MHC) class I molecules on normal antigen-presenting cells (APC), as well as on virus-infected cells or tumour cells (pMHC). At least two receptor types participate in recognition of these complexes: T-cell receptor (TCR) alphabeta heterodimers and CD8alphabeta molecules. The former molecules react with antigenic peptide and variable regions of MHC class I molecules, whereas the latter molecules react with constant alpha3 regions of MHC class I molecules. As the avidity of both receptor-MHC interactions is low, it is believed that TCRalphabeta and CD8alphabeta heterodimers collaborate in T-cell recognition. We have established a TCR/CD3-CD8 capture ELISA, which can measure the interaction of pMHC with CD8alphabeta molecules and with TCR/CD3 complexes. The major findings are: (1) TCR/CD3 complexes derived from in vitro activated T cells and captured by anti-CD3 MoAb, do bind specific pMHC and (2) CD8+ T cells express at least three forms of CD8alphabeta molecules: single CD8alphabeta, CD3-CD8 and TCR/CD3-CD8 complexes. Only the latter complexes are associated with CD3zeta homodimers, and the quantity of TCR/CD3-CD8 complexes relative to total CD8alphabeta molecules appears to increase and to be selected into sucrose-gradient microdomains as a function of TCRalphabeta-mediated T-cell activation.     

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR). whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-l can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.     

Late postnatal expansion of self-reactive CD8alphaalpha+ intestinal intraepithelial lymphocytes in mice

The intestinal epithelium is unique in that it harbors auto-reactive T cells largely absent from the peripheral TCR repertoire in normal mice. Intestinal intraepithelial lymphocytes (IEL) expressing self-reactive TCR are mostly CD8alphaalpha+ cells in adult H-Y TCR RAG(-/-) male mice homozygous for the restricting MHC I allele, H-2D(b). By contrast, in male mice heterozygous for the restricting and non-restricting MHC I allele, H-2D(d) (MHC F(1), H-2D(b/d)), IEL are composed of CD8alphabeta and CD8alphaalpha+ T cells. Here we demonstrate that IEL in the immediate postnatal period of MHC homozygous male mice were mostly CD8(-) T cells, while IEL in MHC F(1) male mice were CD8(-) and CD8alphabeta+ T cells. Regardless of the MHC I configuration and the ability to support positive selection of CD8alphabeta+ cells in the thymus, the expansion of CD8alphaalpha+ IEL was a late postnatal event that followed a reduction in CD8(-) IEL. Furthermore, although in vivo treatment with the specific peptide antigen resulted in an earlier accumulation of activated IEL, the expansion of CD8alphaalpha+ IEL remained inefficient until late in postnatal life. Finally, as CD8(-) IEL stimulated with TCR agonists in vitro, acquired expression of CD8alphaalpha, we propose that CD8alphaalpha+ IEL derive from CD8(-) IEL intermediates. Whether CD8(-) IEL are CD8alphabeta-lineage cells that escape deletion in the thymus or are T cells targeted to the intestine from the thymus because of the early and high level TCR transgene expression in this model, is not clear. The signals required for the expansion of CD8alphaalpha+ IEL are however, incomplete in the immediate postnatal intestine. Determining the factors required for the expansion or retention of CD8alphaalpha+ IEL bearing high affinity, self-specific TCR will further elucidate the in vivo role of these T cells in intestinal homeostasis and perhaps, autoimmunity.     

Intraepithelial lymphocytes in celiac disease immunopathology

Celiac disease is a T cell-mediated immune disorder induced by dietary gluten that is characterized by the development of an inflammatory anti-gluten CD4 T cell response, anti-gluten antibodies, and autoantibodies against tissue transglutaminase 2 and the activation of intraepithelial lymphocytes (IELs) leading to the destruction of the intestinal epithelium. Intraepithelial lymphocytes represent a heterogeneous population of T cells composed mainly of cytotoxic CD8 T cells residing within the epithelial layer, whose main role is to maintain the integrity of the epithelium by eliminating infected cells and promoting epithelial repair. Dysregulated activation of IELs is a hallmark of CD and is critically involved in epithelial cell destruction and the subsequent development of villous atrophy. In this review, we compare and contrast the phenotype and function of human and mouse small intestinal IELs under physiological conditions. Furthermore, we discuss how conditions of epithelial distress associated with overexpression of IL-15 and non-classical MHC class I molecules induce cytotoxic IELs to become licensed killer cells that upregulate activating NKG2D and CD94/NKG2C natural killer receptors, acquiring lymphokine killer activity. Pathways leading to dysregulated IEL activation could eventually be targeted to prevent villous atrophy and treat patients who respond poorly to gluten-free diet.     

Recent insights into the signals that control αβ/γδ-lineage fate

Summary:  During thymopoiesis, two major types of mature T cells are generated that can be distinguished by the clonotypic subunits contained within their T-cell receptor (TCR) complexes: αβ T cells and γδ T cells. Although there is no consensus as to the exact developmental stage where αβ and γδ T-cell lineages diverge, γδ T cells and precursors to the αβ T-cell lineage (bearing the pre-TCR) are thought to be derived from a common CD4⁻CD8⁻ double-negative precursor. The role of the TCR in αβ/γδ lineage commitment has been controversial, in particular whether different TCR isotypes intrinsically favor adoption of the corresponding lineage. Recent evidence supports a signal strength model of lineage commitment, whereby stronger signals promote γδ development and weaker signals promote adoption of the αβ fate, irrespective of the TCR isotype from which the signals originate. Moreover, differences in the amplitude of activation of the extracellular signal-regulated kinase- mitogen-activated protein kinase-early growth response pathway appear to play a critical role. These findings will be placed in context of previous analyses in an effort to more precisely define the signals that control T-lineage fate during thymocyte development.     

CD8+ T-cell clones in old mice

Summary: Most old mice and human beings contain large clones of CD8⁺αβ TCR⁺ T cells. In mice clones bearing Vβ7 appear more frequently in animals infected with mouse hepatitis virus than in uninfected animals. This property is controlled by some non MHC gene in the animals. The frequency of old mice containing such clones is affected by the origin of the animals. Although the clones are relatively anergic to acute stimuli in vitro, they can divide in vivo since in old animals they divide and turnover with about the same kinetics as other, non-clonally expanded CD8⁺T cells. Moreover the clones expand slowly but inexorably after transfer into recipient animals. These data suggest that the CDS⁺αβ TCR clones arise because they are specific for some exogenous or auto antigen to which the cells are continuously exposed in vivo.     

A flavonoid sulfate antigen activates human alphabeta CD8+ Th2 lymphocytes in pollen allergy

Cellular immune responses are initiated when T lymphocytes expressing alphabeta TCR recognize peptide antigens bound to MHC molecules or, less frequently, double-stranded glycolipid antigens bound to CD1 molecules. In the allergy to Parietaria judaica, human alphabeta CD8+ Th2 lymphocytes react to a non-peptide pollinic antigen presented by B cells. The environmental allergen was purified and identified as a new flavonoid pigment: 2'-O-sulfate, 6-O-betaD-glucuronopyranosyl, 2',5,6-trihydroxy-isoflavone. Its specific recognition by alphabeta CD8+ Th2 T cells (1) depends upon an MHC- and CD1-independent presentation mediated by B cells, (2) is determined by the flavonoid carbohydrate and sulfate groups and (3) leads to positive skin prick test in allergic patients. Hence, an unusual mode of aromatic sulfated antigen recognition by alphabeta CD8+ Th2 T lymphocytes might underlie the cellular mediation of human allergy to plant allergens.     

Staphylococcal enterotoxin-A directly stimulates signal transduction and interferon-γ production in psoriatic T-cell lines

Abstract: Bacterial superantigens such as staphylococcal enterotoxin-A (SEA) have been implicated in the pathogenesis of psoriasis vulgaris. Major histocompatibility complex (MHC) class II molecules are high affinity receptors for SEA, and T cells found in psoriatic skin lesions express high levels of MHC class II. Here we address the question of whether SEA can directly activate psoriatic T cells in the absence of professional antigen-presenting cells. We show that SEA induces i) tyrosine phosphorylation of several proteins, ii) downregulation of the T-cell receptor (TCR), and iii) production of interferon-γ (IFN-γ), but not autocrine mitogenesis in CD8-posi-tive T clones obtained from skin lesions of a patient with psoriasis vulgaris. Psoriatic T cells do not respond to SEA molecules if mutations are introduced in the TCRβ- or in both the two MHC class II α- and β-binding sites of SEA. Mutations in only one of the two MHC class II binding sites of SEA has different effects on T-cell activation. Thus, SEA molecules with a mutation in the MHC class LT β-binding site induce protein tyrosine phosphorylation, but not LFN-γ production or co-stimulation of cytokine-mediated proliferation. In contrast, SEA with a mutation in the MHC class II α-binding site induces IFN-γ and a qualitatively changed tyrosine phosphorylation profile. Both mutations delete the co-stimulatory effect on cytokine-mediated proliferation. This suggests that both MHC class II binding sites are involved in the autopresentation of SEA by psoriatic T cells. In conclusion, we provide evidence that SEA directly activates MHC class II-posi-tive psoriatic T-cell lines to produce IFN-γ, a key cytokine in the pathogenesis of psoriasis vulgaris.;     

HIV-Neutralizing Antibodies: Epitope Identification and Significance for Future Vaccine

Recent studies of the TCR α and β chains expressed by normal human IELs suggest that these intestinal lymphocytes are directed at a limited set of antigens, presumably on intestinal epithelial cells in view of their anatomic location. The direct sequence analysis of these cells has indicated that they are oligoclonal and cannot, therefore, be responding to the complex mixture of antigens which are present in the lumen. The abundant expression of the CD8 accessory molecule by the IELs, in addition, indicates that these putative intestinal epithelial cell antigens are presented by MHC class I or I-like molecules. The expression of CD8 also suggests that these cells function biologically in part as cytolytic T lymphocytes which is consistent with a variety of functional studies. Taken together with their expression of the CD45RO isoform, these phenotypic and functional observations suggest that iIELs are cytolytic, memory cells which are responsive to an extremely limited number of antigens bound to major histocompatibility complex (MHC) class I or class I-like molecules. Several non-polymorphic MHC class I-like molecules such as Qa, the thymus leukemia antigen (TL) and CD1 in the mouse and CD1 in humans represent important candidate ligands for these oligoclonal iIELs. TL and CD1 are expressed specifically by murine intestinal epithelial cells. In humans, CDld is constitutively expressed by intestinal epithelial cells. In addition, we have isolated iIEL T cell clones which specifically recognize members of the CD1 gene family when expressed on a transfected B cell line that lacks HLA-A and B and have shown that the proliferation of peripheral blood T cells to intestinal epithelial cells is CDld dependent. Thus, the evidence to date strongly implicate the nonpolymorphic, class lb molecules as novel restriction elements for unique populations of lymphocytes within the intestinal epithelium.     

Regulation of T cell function by NK cell receptors for classical MHC class I molecules

Inhibitory receptors for MHC class I molecules were initially characterised on NK cells. Human and mouse NK cell receptors (NKRs) are also expressed on T cells, predominantly on a subset of memory-phenotype CD8(+) T cells. This review focuses on the precise determination of interactions between NKRs and MHC class I, as well as on the unexpected in vivo function of NKRs on T cells.     

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.     

MHC Class II-Dependent Peptide Antigen Versus Superantigen Presentation to T Cells

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.     

A molecular basis for NKT cell recognition of CD1d-self-antigen

The antigen receptor for natural killer T?cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3???resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.