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.     

A retrospective on the requirements for γδ T-cell development

Summary:  Since the discovery of γδ T cells two decades ago, considerable effort has been made to understand their developmental program, their antigen specificity, and their contribution to the immune response. In this review, we focus on what is known about γδ T-cell development and on the advances that have been made in determining which genes are required. In addition, we compare the genetic requirements for αβ and γδ T-cell development with the hope of gaining a better picture of the signaling pathways that govern the development of γδ lineage cells.     

γδ T cells: firefighters or fire boosters in the front lines of inflammatory responses

Summary:  Intradermal inoculation of cloned self-reactive αβ T cells into the footpads of mice induced cutaneous graft-versus-host disease (GVHD), and after recovery from GVHD, the epidermis became resistant to subsequent attempts to induce GVHD. Resistance to GVHD was not induced in the epidermis of T-cell receptor δ-deficient (TCRδ^−/−) mice that lacked γδ T cells bearing canonical Vγ5/Vδ1⁺γδTCRs, known as dendritic epidermal T cells (DETCs), and resistance was restored by reconstitution of these mutant mice with precursors of Vγ5⁺ DETCs. Pulmonary infection by Cryptococcus neoformans induced an increase of γδ T cells in the lung, and in comparison with wildtype mice, TCRδ^−/− mice eliminated C. neoformans more rapidly and synthesized more interferon-γ in the lung. In the mouse small intestine, the absence of γδ T cells is associated with a reduction in epithelial cell turnover and downregulation of the expression of major histocompatibility complex class II molecules. The protective role of γδ T cells was verified in a dextran sodium sulfate-induced inflammatory bowel disease (IBD) model, whereas in a spontaneous model of IBD, γδ T cells were involved in the exacerbation of colitis in TCRα^−/− mice. Taken together, in addition to the homeostatic regulation of epithelial tissues, γδ T cells appear to play a pivotal role in the modification of inflammatory responses induced in many organs containing epithelia.     

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.     

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 γδ T Cells Produce the Protease Inhibitor and Antimicrobial Peptide Elafin

Human γδ T cells rapidly secrete pro-inflammatory cytokines in response to T cell receptor-dependent recognition of pyrophosphates produced by many bacteria and parasites. In further support of an important role of γδ T cells in the immune defence against infection, human γδ T cells have been shown to produce the antimicrobial peptide LL37/cathelicidin. In this study, we have investigated whether γδ T cells can produce additional antimicrobial peptides. To this end, we have screened human γδ T cell clones by RT-PCR for mRNA expression of a broad range of antimicrobial peptides. While α-defensins were absent and β-defensins (HBD1) present only in rare γδ T cell clones, elafin mRNA was induced by supernatant of Pseudomonas aeruginosa grown under static conditions. Elafin is a protease inhibitor that also displays antimicrobial activity. Constitutive intracellular expression of elafin was demonstrated by flow cytometry and Western blot analysis. Furthermore, trappin-2 (pre-elafin) could be immunoprecipitated in cell lysates but also in the supernatant of γδ T cells stimulated by Ps. aeruginosa supernatant. Taken together, our studies reveal a novel effector function of γδ T cells which might be important for local immune defence.     

γδ T-cell receptors: functional correlations

Summary:  The γδ T-cell receptors (TCRs) are limited in their diversity, suggesting that their natural ligands may be few in number. Ligands for γδTCRs that have thus far been determined are predominantly of host rather than foreign origin. Correlations have been noted between the Vγ and/or Vδ genes a γδ T cell expresses and its functional role. The reason for these correlations is not yet known, but several different mechanisms are conceivable. One possibility is that interactions between particular TCR-V domains and ligands determine function or functional development. However, a recent study showed that at least for one ligand, receptor specificity is determined by the complementarity-determining region 3 (CDR3) component of the TCR-δ chain, regardless of the Vγ and/or Vδ. To determine what is required in the TCR for other specificities and to test whether recognition of certain ligands is connected to cell function, more γδTCR ligands must be defined. The use of recombinant soluble versions of γδTCRs appears to be a promising approach to finding new ligands, and recent results using this method are reviewed.     

An In Vitro Model for the Expansion of Vγ9δ2 T Lymphocytes During Development

The 7/γ/δTlymphocytes represent a minority of T lymphocytes in human peripheral blood. Although there have been reports of reactivity against (myco-) bacterial antigens and heat shock proteins, their function and antigen specificity remain ill defined. The biological role of γ/δ T cells has been related to functions within the 'first line of defense'. Similar to γ/δ T lymphocytes in the T-cell compartment, CD5 positive B cells represent a small subset of B lymphocytes, which is thought to be involved in the maintenance of natural immunity and autoimmunity. We provide evidence for the cooperation of γ/δ T cells and CD5 positive B cells in the proliferative response of γ/δ T cells to bacterial antigens. Our data indicate a strong proliferation of Vγ9δ2 T cells in response to gram-negative bacteria, which is dependent upon the presence of CD5 positive B-CLL or activated normal B lymphocytes. The selective stimulation of the Vγ9δ2 subpopulation by gram-negative bacteria is also confirmed by analysis of different γ/δ T-cell clones. The interaction of γ/δ T cells with activated B cells and gram-negative bacteria may prove to be a useful model similar to the expansion of the Vγ9δ2 subpopulation during development. In addition, our in vitro system should provide new insights in the interaction of CLL B cells with the immune system and the antigens recognized by γ/δ T cells.     

Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vγ2Vδ2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens

Summary:  Human Vγ2Vδ2 T cells play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. Vγ2Vδ2 T cells recognize the pyrophosphorylated isoprenoid intermediates ( E )-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the foreign 2- C -methyl- d -erythritol 4-phosphate (MEP) pathway, and isopentenyl pyrophosphate (IPP), an intermediate in the self-mevalonate pathway. Infection with bacteria and protozoa using the MEP pathway leads to the rapid expansion of Vγ2Vδ2 T cells to very high numbers through preferential recognition of HMBPP. Activated Vγ2Vδ2 T cells produce proinflammatory cytokines and chemokines, kill infected cells, secrete growth factors for epithelial cells, and present antigens to αβ T cells. Vγ2Vδ2 T cells can also recognize high levels of IPP in certain tumors and in cells treated with pharmacological agents, such as bisphosphonates and alkylamines, that block farnesyl pyrophosphate synthase. Activated Vγ2Vδ2 T cells are able to kill most tumor cells because of recognition by T-cell receptor and natural killer receptors. The ubiquitous nature of the antigens converts essentially all Vγ2Vδ2 T cells to memory cells at an early age. Thus, primary infections with HMBPP-producing bacteria are perceived by Vγ2Vδ2 T cells as a repeat infection. Extensive efforts are underway to harness these cells to treat a variety of cancers and to provide microbial immunity.     

Activated Human γδ T Lymphocytes Express Functional Lactoferrin Receptors

Lactoferrin (Lf), an iron-binding protein in milk, mucosal secretions and neutrophil granules has bactericidal properties and is a source of iron for breast-fed infants. In this paper the authors show that most in vivo activated lymphocytes, i.e. freshly isolated lymphocytes from first trimester human decidua, and most in vitro activated human blood lymphocytes, express lactoferrin receptors (Lf-R), while unstimulated blood lymphocytes do not. All major lymphocyte subsets, i.e. αβ T cells, γδ T cells, CD8⁺ T cells, CD4⁺ T cells, B cells and NK cells, express Lf-R after activation. The proportion of Lf-R expressing activated γδ T cells is significantly larger than that of activated αβ T cells. Lf-R and transferrin receptors (Tr-R/CD71) show the same kinetics of appearance on activated blood lymphocytes and are, to a large extent, expressed on the same cells. However, 35% of decidual lymphocytes and 15% of activated blood lymphocytes express Lf-R only. Addition of Lf to cultures containing an optimal concentration of Tr augments the proliferative response to polyclonal T cell activators and alloantigens, suggesting that presently used standard culture conditions for in vitro activation are suboptimal in particular for γδ T cells. Lf-R on decidual lymphocytes contain bound Lf, which probably is produced locally. The results suggest that Lf is a growth-supporting factor, especially important in local immune responses in the mucosa.     

γδ and αβ T Cells are Equally Susceptible to Apoptosis

Gamma/delta TCR bearing T lymphocytes represent a T-cell subset whose functional relevance remains unclear. Nevertheless these T cells may play a role in the early immune reponse against bacteria. Until now the regulatory mechanisms on this response have not been investigated. The study described here evaluated the immunoregulatory effects of Interleukin-10 on γ/δ and α/β TCR-positive T-cell clones and freshly isolated peripheral-blood mononuclear cells (PBMC). IL-10 has been shown previously to inhibit lectin and antigen-induced proliferation and cytokine production by α/β T cells. The results outlined below show that rhIL-10 strongly inhibits lectin-induced production of IFN-γ, TNF-α. IL-2, and to a lesser degree proliferation and IL-4 production of both T-cell subsets. As IL-10 did not inhibit proliferation but at the same time strongly suppressed cytokine production in various experiments, the hypothesis that it could function as a growth factor for human T cells as has been described for murine thymoeytes was tested. The data demonstrate that, although the γ/δ T-cell clones tested do not produce IL-10 they can use it as a growth factor in combination with IL-2, IL-4 or alone. Furthermore, IL-10 has the same properties on human α/β T-cell clones and PBMC. In summary, it is shown that IL-10 has pleiotropic effects on γ/δ and α/β TCR⁺ T cells by inhibiting lectin-induced cytokine production and by acting as a growth factor for these cells alone or in combination with IL-2 or IL-4.     

Self/non‐self discrimination by human γδ T cells: simple solutions for a complex issue?

Summary:  Although γδ T cells express clonally distributed T-cell receptors (TCRs), a hallmark of adaptive immunity, they are classically considered as innate-like effectors, owing to the high frequency of preactivated γδ T cells, with restricted antigen recognition repertoire in particular tissue locations. Actually, such features are shared only by a fraction of γδ T-cell subsets located in the skin and reproductive organ mucosa in rodents or in peripheral blood in humans. By contrast, other γδ subsets, e.g. those found in rodent and human spleen, show diverse antigenic reactivity patterns and mixed naive/memory phenotypes. Thus, γδ T cells are made of both 'primitive' subsets endowed with innate-like properties and 'evolved' subsets able to mount anamnestic responses like conventional major histocompatibility complex-restricted αβ T cells. In this article, we show that human γδ T cells, although heterogeneous, do share recurrent innate features that distinguish them from mainstream αβ T cells. In particular, most of them are activated on TCR- or natural killer receptor-mediated recognition of a restricted set of conserved yet poorly defined endogenous stress determinants. This rather simple recognition mechanism allows human γδ T cells to discriminate healthy cells from altered cells and to exert a variety of immunostimulatory or regulatory functions. The recent availability of synthetic γδ T-cell agonists mimicking these natural stress-induced ligands have fostered development of immunotherapeutic strategies, with broad indications against infectious and tumor diseases, which are briefly reviewed here.     

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.     

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.     

γδ T-Cell Precursor-Derived CD4− CD8−αβ T Cells Retain γδ Cell Function

We have previously shown that some of the DNαβ⁺ T cells arising in TcRα-chain transgenic mice are of γδ T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of αβ TcR expression on the functional programme of the mature γδ precursor-derived DNαβ⁺ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of γδ T cells. Furthermore, both transgenic DNαβ⁺ T cells and DNγδ⁺ T cells up-regulate CD8α expression after activation, but, in contrast to CD4⁺αβ T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.     

T Lymphocytes Bearing the γδ T Cell Receptor are Susceptible to Steroid-Induced Programmed Cell Death

The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined. In steroid-sensitive pathological conditions, an increase in γδ T cells can occur in certain untreated systemic autoimmune disorders and seems to be a persistent feature in most cases of systemic lupus erythematosus (SLE). Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms. To establish how corticosteroid treatment determines the disappearance of peripheral blood γδ T lymphocytes, circulating αβ and γδ T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10_-7M Dexamethasone (DEX). Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method. Conventional agarose-gel electrophoresis on the same T cell populations was carried out for comparison. Regular follow-ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood γδ T cells, and in vitro experiments seem to indicate that DEX-triggered apoptotic signals are confined to the double negative (CD4_- CD8_-) γδ T cell subpopulation which disappears after in vivo immunosuppressive therapy. Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids. Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses.     

Experimental Human Plasmodium Falciparum Infections: Longitudinal Analysis of Lymphocyte Responses with Particular Reference to γδ T Cells

The kinetics of the γδ T-cell response was analysed in the context of the overall haematological response in subjects experimentally infected with sporozoites of Plasmodium falciparum . Numbers of γδ and αβ T cells and NK cells declined markedly during infection to reach minimum values 12–13 days post-infection when the patients were ill. This decline commenced from the beginning of the erythrocytic cycle and well before parasites could be detected microscopically and clinical symptoms developed. Platelet numbers also declined. In vivo activation of γδ T cells was evident with sequential up-regulation of the activation markers CD69 and HLA-DR. γδ T cell numbers were highest after treatment with the majority being CD4⁻CD8⁻, HLA-DR⁺ and showing reduced CD45RA expression. Contrary to some published observations γδ T-cell percentages remained within the normal range. Little evidence of up-regulation of activation or memory markers was observed in the αβ T-cell population. In vitro proliferative responses to malaria antigen which involve γδ T cells were lost as the infection progressed and the lymphocyte count declined but these could be restored with the addition of exogenous IL-2 to cultures. The authors findings are consistent with a protective and/or immunomodulatory role for γδ T cells in malaria.     

Decreased peripheral blood γδ T cells in patients with bronchial asthma

Many cell populations are thought to be involved in the etiopathogenesis of bronchial asthma. We examined by flow cytometry the relative and absolute number of CD3*, CD4*, CD8*γδ TcR* T cells. CD19* B cells; and CD56* natural killer (NK) cells in the peripheral blood of 26 adult patients with difficult-to-control asthma (DCA) and 22 patients with minimally symptomatic asthma (MSA). Statistically higher relative and absolute numbers of NK cells (18.39±10.67% and 0.38±0.17×10⁹/l) in comparison with healthy controls (ll.77±8.06% and 0.25±0.19×10⁹/l) and significantly decreased relative and absolute numbers of γδ T cells (3.02±2.16% and 0.06±0.04×10⁹/l) in comparison with controls (5.65+2.90% and 0.13±0.08×10⁹/l) in the DCA patient group were found. After pooling of data from both MSA and DCA patients and dividing the patients according to the presence of allergy, the relative and absolute numbers of 78 T celts were found to be diminished in both the allergy (3.77±2.98 and O.O7±0.O5 ×10⁹/l) and nonallergy (3.06±1.78% and 0.06±0.03 ×10⁹/l) groups in comparison with healthy controls. The reason for the low number of 78 T cells in the peripheral blood of patients suffering from bronchial asthma is under investigation.