Antigen recognition by γδ T cells

Summary:  γδ T cells contribute to host immune competence uniquely. This is most likely because they have distinctive antigen-recognition properties. While the basic organization of γδ T-cell receptor (TCR) loci is similar to that of αβ TCR loci, there is a striking difference in how the diversity of γδ TCRs is generated. γδ and αβ T cells have different antigen-recognition requirements and almost certainly recognize a different set of antigens. While it is unclear what most γδ T cells recognize, the non-classical major histocompatibility complex class I molecules T10 and T22 were found to be the natural ligands for a sizable population (0.2–2%) of murine γδ T cells. The recognition of T10/T22 may be a way by which γδ T cells regulate cells of the immune system, and this system has been used to determine the antigen-recognition determinants of γδ T cells. T10/T22-specific γδ T cells have TCRs that are diverse in both V gene usage and CDR3 sequences. Their Vγ usage reflects their tissue origin, and their antigen specificity is conferred by a motif in the TCR δ chain that is encoded by V and D segments and by P-nucleotide addition. Sequence variations around this motif modulate affinities between TCRs and T10/T22. That this CDR3 motif is important in antigen recognition is confirmed by the crystal structure of a γδ TCR bound to its ligand. The significance of these observations is discussed in the context of γδ T-cell biology.     

Evolutionarily Conserved Function of CD28 in αβ T Cell Activation

The functional role of the chicken homologue of CD28 was studied. It is expressed on all thymocytes, and both Vβ1- and Vβ2-family expressing peripheral αβ T cells. Peripheral γβ T cells are CD28-negative. Monoclonal antibody against CD28 had a costimulatory effect on T cells stimulated by phorbol myristate acetate (PMA), concanavalin A or MoAb against TCR. Vβl and Vβ2 expressing cells responded equally well to stimulation with anti-CD28 in combination with PMA. These responses were resistant to cyclosporin A, but inhibited by herbimycin A, suggesting that CD28 employs a signalling pathway at least partly distinct from that triggered by TCR/CD3. These data indicate a striking conservation of the costimulatory function of CD28 and emphasize the importance of this costimulatory pathway.     

Retrovirally Induced Mouse Anti-TCR Monoclonals can Synergize the In Vitro Proliferative T Cell Response to Bacterial Superantigens

Antibodies directed against the β chain of the T-cell receptor (TCR) have been detected in animals and in humans in a number of distinct immune states that do not involve direct immunization with either T cells or TCR epitopes. When C57Bl/6 mice are infected experimentally with the LP-BM5 retrovirus mixture they produce increased titres of autoantibodies directed against TCR Vβ complementarity determining region 1 (CDR1) epitopes. Here, the authors utilized hybridoma technology to isolate monoclonal immunoglobulin (Ig)M antibodies (MoAbs) that arose at the peak of infection. The authors characterized the binding specificity tested using synthetic peptides modelling the CDR1 segments of 24 distinct Vβ gene products and determined the V_H gene usage by two such monoclonals. One binds to a restricted set of TCR Vβ CDR1 peptides, and the second reacts with approximately half of the CDR1 peptide homologues. These MoAbs are specific for T-cell receptor β chains and do not bind to immunoglobulin light chains or to unrelated protein molecules. Both MoAbs bind to intact T cells expressing the Vβ domain (human Vβ8 and mouse Vβ11) from which selection peptides were derived, and costimulate a Vβ specific in vitro T cell proliferative response induced by the staphylcoccal enterotoxin E (SEE) superantigen.     

Skin γδ T-cell functions in homeostasis and wound healing

Summary:  There is a resident population of T cells found in murine skin that expresses an invariant Vγ3Vδ1 T-cell receptor (TCR), and these cells are significantly different from lymphoid γδ T cells and αβ T cells in terms of ontogeny, tissue tropism, and antigen receptor diversity. These dendritic epidermal T cells are derived from fetal thymic precursor cells, are in constant contact with neighboring epidermal cells, and express a monoclonal γδTCR only found in the skin. Skin γδ T cells have been shown to play unique roles in tissue homeostasis and during tissue repair through local secretion of distinct growth factors including keratinocyte growth factors and insulin-like growth factor-1. In this review, we discuss evidence supporting a role for cross talk between skin γδ T cells and keratinocytes that contributes to the maintenance of normal skin and wound healing.     

Conformation of the T-Cell Antigen Receptor-β Chain C-Domain Contributes to Vβ3 Epitope Recognition by Monoclonal Antibody KJ25

The clonotypic T-cell antigen receptor (TCR)-β chain contains two extracellular intrachain disulfide bonds. It belongs to the immunoglobulin gene superfamily and is subdivided into variable (V), joining (J), diversity (D) and constant (C) region. Monoclonal antibody (MoAb) KJ25 is believed to recognize an epitope in the V-domain of TCR-β (Vβ₃) chain, but its epitope requirements are unknown. In this study of TCR-αβ chain interactions using chimeric recombinant TCR-β chains, the authors found that partial substitution of the Cβ-domain with that of interleukin-2 receptor α chain (Tac) sequences led to the loss of TCR-Vβ₃ epitope recognition by KJ25. These results suggest that epitope recognition of the TCR-Vβ₃ by KJ25 MoAb is dependent not only on the V-domain, but also on the close contact with the extracellular C-domain which influences the conformation and epitope recognition of the Vβ₃-region. This may not be unique to Vβ₃ and may be a general feature of TCR-β protein folding.     

Development and selection of γδ T cells

Summary:  Two main lineages of T cells develop in the thymus: those that express the αβ T-cell receptor (TCR) and those that express the γδ TCR. Whereas the development, selection, and peripheral localization of newly differentiated αβ T cells are understood in some detail, these processes are less well characterized in γδ T cells. This review describes research carried out in this laboratory and others, which addresses several key aspects of γδ T-cell development, including the decision of precursor cells to differentiate into the γδ versus αβ lineage, the ordered differentiation over the course of ontogeny of functional γδ T-cell subsets expressing distinct TCR structures, programming of ordered Vγ gene rearrangement in the thymus, including a molecular switch that ensures appropriate Vγ rearrangements at the appropriate stage of development, positive selection in the thymus of γδ T cells destined for the epidermis, and the acquisition by developing γδ T cells of cues that determine their correct localization in the periphery. This research suggests a coordination of molecularly programmed events and cellular selection, which enables specialization of the thymus for production of distinct T-cell subsets at different stages of development.     

Human CD8+ intraepithelial lymphocytes: a unique model to study the regulation of effector cytotoxic T lymphocytes in tissue

Summary:  The epithelium of the human small intestine contains a large population of intraepithelial cytolytic αβ T-cell receptor (TCR) CD8αβ T lymphocytes (IE-CTLs), whose main role is to sustain epithelial integrity by rapidly eliminating infected and damaged cells. In mouse, the recognition of inducible/modified self-molecules, i.e. non-classical major histocompatibility complex (MHC) class I molecules, is mediated by the TCR and natural killer receptors (NKRs) co-expressed on the cell surface of a non-conventional autoreactive CD8αααβTCR cell subset. In contrast, in humans, the recognition of non-classical MHC class I molecules induced by stress and inflammation on intestinal epithelial cells (IECs) is principally mediated by NKRs expressed on conventional CD8αβαβTCR cells. By sensing microenvironmental signals of inflammation and stress through NKRs, IE-CTLs fine tune their TCR activation threshold. Furthermore, IE-CTLs under particular conditions, involving interleukin-15 upregulation, acquire the capacity to kill distressed intestinal epithelial cells in an antigen non-specific manner. Adaptive IE-CTLs appear hence to have autoreactive properties and modulate their immune response based on innate signals, reflecting the fitness of the tissue.     

No Evidence for TCR Vβ Repertoire Changes Influencing Disease Protection in E-Transgenic NOD Mice

In order to study whether positive selection of T cells plays any role in the MHC-dependent protection from diabetes in the non-obese-diabetic (NOD) mouse, the T cell Vβ repertoire has been studied in NOD mice and in NOD mice either transgenic for the wildtype MHC class II Eα gene, or for ΔY, a promotor-mutagenized Eα gene with a restricted tissue expression. The Eα transgenic line is protected from both insulitis and diabetes. The ΔY transgenic line is neither protected from insulitis nor from diabetes, although it can perform both positive and negative E-mediated selection in the thymus. The Vβ repertoire was studied in the pancreatic lymph nodes as these drain the area which is the target for the autoimmune attack. We see no evidence for Ea TCR Vβ repertoire differing from both nontransgenic NOD mice and ΔY mice despite its striking difference in susceptibility to autoimmunity. We conclude that none of the differences in the TCR Vβ repertoire of Eα-transgenic NOD mice hitherto observed are likely to explain the protective effect of E molecule expression in NOD mice.     

Cytokine Induction by Mycoplasma arthritidis-Derived Superantigen (MAS), but not by TSST-1 or SEC-3, is Correlated to Certain HLA-DR Types

Superantigens bind to major histocompatibility complex (MHC) class II molecules on antigen presenting cells and T cells in a Vβ-restricted manner. Both cell types are activated resulting in cytokine production. Although the MHC-II binding site for superantigens has been well described, little is known as to whether this binding complex has an influence on cytokine induction. In order to assess superantigen induced cytokine production and its correlation to HLA-DR types, the authors stimulated peripheral blood from 40 subjects with superantigens toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin C-3 (SEC-3) and Mycoplasma arthritidis -derived superantigen (MAS), and measured cytokine levels thereafter. The HLA-DR type was determined in each subject. A statistical evaluation was carried out between the highest superantigen cytokine induction and the presence of certain HLA-DR types. Whereas MAS presented a statistical association between the highest cytokine production with HLA-DR4, DR7 and DR12, no such associations were observed for TSST-1 and SEC-3. These results demonstrate that T cell stimulation, and consequently its cytokine production by MAS but not by TSST-1 and SEC-3, depends on the presenting HLA-DR type. Because the diverse HLA-DR specificities are given according to the variability of the β chain of the HLA-DR molecule, the data suggest the participation of the human MHC-II β chain in the MAS/MHC-II binding.     

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.     

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.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

Accessibility control of variable region gene assembly during T-cell development

Summary: T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an αβ or γδ T-cell antigen receptor (TCR), The genes that encode the TCR β and y chains lie in distinct loci, whereas the genes that encode the TCR a and S chains he in a single locos (TCR α/δ locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR β, γ and δ variable region genes are assembled prior to TCR α variable region genes. Furthermore, assembly of TCR β variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement arid expression of genes within the TCR α/δ locus presents a complicated problem. TCR α and δ variable region genes are assembled at different stages of T-cell development, and fully assembled TCR α and δ variable region genes must be expressed in distinct hneages of T cells, αβ and γδ. respectively We have developed several experimental approaches lo assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR β and TCR α/δ foci during T-cell development.     

Role of the T cell receptor α-chain in superantigen recognition

Superantigens bind to antigen-presenting cells on the outside of the major histocompatibility complex (MHC) class II molecule and to T cells via the external face of the T cell receptor (TCR) Vβ element. As a consequence, superantigens stimulate populations of T cells in a Vβ-specific, non-MHC-restricted manner. However, accumulating evidence has shown an additional contribution of the TCR α-chain and polymorphic residues of the MHC molecule to superantigen recognition by some T cells. These data suggest that the TCR and MHC come into contact during superantigen engagement and indirectly modulate the superantigen reactivity. Thus, additional interactions between non-Vβ elements of the TCR and MHC play a role in the overall stability of the superantigen/MHC/TCR complex, explaining the influence of the TCR α-chain. It is likely that this additional interaction is of greater consequence for weakly reactive T cells. This modulation of superantigen reactivity in individual T cells may have physiological consequences, for example, in the induction of autoimmunity.     

Toll-Like Receptor Expression and Function in Subsets of Human γδ T Lymphocytes

Two subsets of human γδ T cells can be identified by T cell receptor (TCR) V gene usage. Vδ2Vγ9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vδ1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-γ (IFN-γ) production in peripheral blood γδ T cells and in Vδ2Vγ9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated γδ T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vδ1 and Vδ2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vδ1 and Vδ2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-γ and chemokine secretion in TCR-activated Vδ1 and Vδ2 subsets, although the levels of IFN-γ secreted by Vδ1 T cells were much lower than those produced by Vδ2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vδ1 and Vδ2 T cells and underscore the intrinsically different capacity for IFN-γ secretion of Vδ1 versus Vδ2 T cells.     

The role of the T-cell receptor (TCR) in αβ/γδ lineage commitment: clues from intracellular TCR staining

Summary: T cells belong to two mutually exclusive lineages expressing either αβ orγδ T-cell receptors (TCR). Although αβ and γδ cells are known to share a common precursor the role of TCR rearrangement and specificity in the lineage commitment process is controversial. Instructive lineage commitment models endow the αβ or γδ TCR with a deterministic role in lineage choice, whereas separate lineage models invoke TCR-independent lineage commitment followed by TCR-dependent selection and maturation of αβ and γδ cells. Here we review the published data pertaining to the role of the TCR in αβ/γδ lineage commitment and provide some additional information obtained from recent intracellular TCR staining studies. We conclude that a variant of the separate lineage model is best able to accommodate all of the available experimental results.     

Intravenous Immunoglobulin (IVIg) Modulates the Expansion of Vβ3+ and Vβ17+ T Cells Induced by Staphylococcal Enterotoxin B Superantigen In Vitro

The authors investigated the effect of IVIg on T-cell proliferation induced by the superantigen, staphylococcal enterotoxin B (SEB). The addition of IVIg to normal PBMC stimulated with SEB resulted in a threefold increase in the proportion of CD3⁺ blast cells expressing Vβ3 and Vβ17, two subsets of T cells that selectively expand in the presence of SEB. There was no increase in the proportion of T-cell blasts expressing Vβ2, Vβ8 and Vβ13.6 antigens that do not respond to SEB in the absence of IVIg. As described previously, IVIg inhibited T-cell proliferation independently of Vβ specificity. The effects of IVIg were mediated by variable regions of immunoglobulins since they were reproduced with F(ab')₂ fragments of IVIg but not with purified Fc fragments of IgG. The observation that SEB-activated T cells are rescued from inhibition of proliferation by IVIg indicates that IVIg modulates the effects of superantigen on T cells. These results may be of relevance for understanding the mechanisms underlying the effects of IVIg in patients with diseases in which T-cell superantigens are of pathophysiological significance.     

Repertoire of the αβ T-cell receptor in the intestine

Summary:  The majority of T cells in the human and mouse intestine express the T-cell receptor (TCR) as an αβ heterodimer on their cell surface. As the major recognition element of antigens in the context of major histocompatibility complex-derived proteins, an examination of the structure of the αβTCR in intestines has provided significant insights into the potential function of these cells and the major determinants that drive their selection. Studies in the human intestine have shown that the repertoires of intraepithelial lymphocytes (IELs), and likely lamina propria lymphocytes, are polyclonal before and shortly after birth, with the repertoire becoming oligoclonal in adults. Similarly, in adult mice the repertoire is oligoclonal, while in the newborn it is polyclonal. Investigations in mice have shown that some T cells may evade thymic selection. The population size and oligoclonality of IELs is influenced by the microbial content of the luminal microenvironment. This microenvironment probably directly determines the TCR repertoire. Studies in human inflammatory bowel disease (IBD) indicate that inflammation further skews the TCR repertoire. We speculate that dominant antigens associated with the pathogenesis of IBD are responsible for such skewing and that identifying the antigenic drivers may shed light on the environmental factors that trigger or potentiate human IBD.     

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.     

Strict Adherence to a Common Rank Order of T-Cell Receptor Vβ Usage in Human Leucocyte Antigen Disparate Individuals

In order to investigate the T-cell receptor (TCR) Vβ usage in different T-cell subsets, the authors performed flow cytometric analyses using a large panel of TCR Vβ-specific monoclonal antibodies on CD4⁺, CD8⁺ CD28⁺ and CD8⁺ CD28⁻ T cells from 15 random blood donors, six umbilical cords and seven human leucocyte antigen (HLA) identical non-twin sibling pairs. The authors found that the proportion of T cells expressing each Vβ gene product was similar within CD4⁺ and CD8⁺ CD28⁺ T cells from all samples studied. For these T-cell subsets a rank order of Vβ usage could be identified which was adhered to by all donors. In contrast, within CD8⁺ CD28⁻ T cells a wide variation of Vβ usage was found between individuals, and no rank order correlation could be detected. Members of HLA identical sibling pairs were found to be no more similar in their usage of Vβ gene products than pairs of HLA disparate random blood donors. Groups of individuals sharing HLA antigens were no different from the groups not sharing such antigens in their usage of Vβ segments. The results suggest that HLA polymorphisms play no more than a minor part in determining TCR Vβ usage.