Intraepithelial γδ-T cells are present in all epithelial tissues, where they reside in close contact with neighboring epithelial cells. Our data support the idea that the role of these cells is to monitor neighboring cells for signs of damage or disease. Once a problem is detected, the intraepithelial γδ-T cells can lyse damaged or malignant epithelial cells, directly participate in tissue repair through production of epithelial growth factors, and play a unique role in the recruitment of inflammatory cells to the site of damage. Intraepithelial γδ-T cells play unique roles in homeostasis and disease.
- tissue repair
- γδ-T cells
althoughγδ-T cells are a relatively minor population in lymphoid organs and peripheral blood, there is a resident population of γδ-T cells in all epithelial tissues in all species that have been studied (2, 15). In some of these epithelial tissues, including murine skin, γδ-T cells are the only resident population of lymphocytes. In other tissues such as the murine and human intestine and lung, they coexist with αβ-T cells and other lymphocyte populations. In each of these epithelial sites, γδ-T cells reside in intimate contact with neighboring epithelial cells. Increasing evidence from many laboratories has shown that these intraepithelial γδ-T cells play unique roles in maintaining epithelial homeostasis and in response to tissue injury or malignancy. The mechanisms of γδ-T cell function include timely production of specific cytokines, chemokines, growth factors, and glycosoaminoglycans that have specialized effects on neighboring epithelial cells.
LOCALIZATION OF γδ-T CELLS TO EPITHELIAL SITES
A striking feature of T cell ontogeny is the ordered rearrangement and expression of individual T cell antigen receptor (TCR) genes in the developing thymus. We and others (2, 15) showed that γ- and δ-TCR genes are rearranged and expressed before expression of αβ-TCRs. Individual γδ-TCRs are expressed in a series of overlapping waves. As a consequence, specific γδ-T cell populations are exported to the periphery in an ordered manner where they then localize to specific epithelial sites (13, 16). γδ-T cells arising later in ontogeny and after birth, as well as αβ-T cells, circulate in the blood and populate lymphoid tissues. This regimented pattern of development results in the establishment of tissue-specific γδ-TCR-bearing cells (Fig. 1).
Mechanisms of gene recombination are exploited by αβ-TCRs to generate a large pool of receptors with the ability to recognize diverse antigens. Although γδ-TCRs have the potential to generate receptors with even more diversity, in general, this potential is not realized, resulting in populations of cells bearing γδ-TCRs with limited or, in some cases, invariant sequence diversity (2, 15). The reasons for this are not clear, although coupled with the restricted tissue distribution, it suggests that γδ-T cells may recognize very different kinds of antigens than αβ-T cells.
RECOGNITION OF UNKNOWN ANTIGENS ON NEIGHBORING EPITHELIAL CELLS
The distinctive tissue-specific localization of γδ-T cells raises the possibility that the intraepithelial γδ-T cells recognize tissue-specific antigens. Unfortunately, little is known about the rules for antigen recognition by intraepithelial γδ-T cells. A few antigens have been identified for lymphoid γδ-T cells including nonclassical major histocompatibility complex (MHC) molecules and nonpeptidic phosphoantigens (7). In general, it seems that, in contrast to αβ-T cells that see peptide antigens presented by self-MHC molecules, the rules for γδ-T cell antigen recognition are quite different. Although some γδ-TCRs do recognize nonclassical MHC molecules, including TL in mice and MICA/B in humans, this recognition is not peptide dependent (7). Recent resolution of the first crystal structure of a murine γδ-TCR bound to its ligand, a nonclassical MHC molecule, demonstrated an unexpected restriction of contact of the ligand to a particular region of the TCR δ-chain (1). Although this result is for a single γδ-TCR and ligand, it is in striking contrast to the multiple areas of contact seen between αβ-TCRs and their peptide/MHC ligands. This distinction between types of ligands recognized by αβ- and γδ-TCRs and means of engagement may be a reflection of specialized functions that differ for these T cell subsets. Further studies may also demonstrate differences in antigen recognition between lymphoid and epithelial γδ-T cells, because these populations appear to have distinct functions.
We have shown that mouse skin γδ-T cells recognize an unknown antigen that is expressed on neighboring keratinocytes after damage or disease. Recognition of distressed keratinocytes is mediated by the γδ-TCR because only cells bearing the skin-specific γδ-TCR (Vγ3Vδ1) respond to the keratinocytes, responses can be blocked with Fab fragments of the anti-Vγ3 TCR monoclonal antibody, and transfection of the skin γδ-TCR into human Jurkat T cells confers reactivity with the distressed keratinocytes (14). Work is in progress to identify the unknown antigen expressed by the distressed keratinocytes using biochemical isolation and fractionation techniques (5) and through binding and purification of ligands detected by use of soluble skin γδ-TCR molecules (3).
Human intestinal intraepithelial γδ-T cells have been shown to recognize the nonclassic MHC molecules MICA and MICB through the Vδ1 TCR (11). MICA/B are expressed by intestinal epithelial cells after damage or disease (10). These molecules are also ligands for the activating natural killer (NK) receptor NKG2D that is also expressed by intraepithelial γδ-T cells (22). As such, NKG2D has been shown to function as not only a TCR ligand but also to provide coreceptor or costimulatory signals important for both skin and intestinal intraepithelial γδ-T cell functions including recognition and lysis of malignant epithelial cells (10, 22). Work from our laboratory has identified additional costimulatory molecules for epithelial γδ-T cells that appear to provide important signals for epithelial γδ-T cell activation and function (24).
Although most epithelial γδ-T cell antigens remain unknown, the evidence supports that this population of cells recognize nonpeptidic self-antigens that are expressed on neighboring epithelial cells after damage or disease. In that way, these cells with limited TCR diversity are able to respond to a wide range of infectious organisms and diseases through recognition of a particular antigen that is expressed after any kind of distress rather than an antigen that is disease or infection specific. In addition, particular costimulatory molecules expressed by γδ-T cells may recognize ligands on epithelial cells that are differentially expressed under conditions of malignancy, damage, or infection. This would provide an additional level of specificity to the response. For example, signals delivered through NKG2D recognition of ligand on malignant keratinocytes lead to tumor cell lysis (9), whereas these interactions do not appear to function during tissue repair (17). Instead, signals generated through alternative costimulatory molecules may lead to functional responses such as are required for effective wound healing or responses to infection. As additional costimulatory molecules and ligands are identified, this possibility can be tested.
ROLES FOR INTRAEPITHELIAL γδ-T CELLS DURING HOMEOSTASIS
Epithelial tissues are in a constant state of regulated proliferation and apoptosis as epithelial cells are differentiated and renewed. A proper balance is required for effective tissue homeostasis. This requires both regulation of homeostatic proliferation of the intraepithelial γδ-T cells and of the neighboring epithelial cells.
Lymphocyte homeostasis is regulated to maintain a constant level of T and B cells throughout life. This may be of particular importance for populations of intraepithelial γδ-T cells such as those in the skin that are generated during a discrete window in development (24). Recent studies (4) have evaluated the mechanisms of control of homeostatic proliferation of lymphoid γδ-T cells in the mouse. The cytokines IL-7 and IL-15 are known to impact aspects of the biology of γδ-T cells including the initiation of γδ-TCR gene rearrangement and development of γδ-T cells as well as localization and survival of γδ-T cells (24). Results now also indicate a fundamental role for IL-7 and IL-15 in the process of homeostatic proliferation. IL-7 and IL-15 are also the primary modulators of αβ-T cell homeostasis, whereas IL-15 alone appears sufficient for NK cell homeostasis (4). In addition to cytokine-mediated signals, the γδ-T cells appear to require signals through the antigen receptor and can be inhibited by the presence of αβ-T cells. This suggests that requirements for γδ-T cell homeostasis are shared with other lymphoid populations but that additional signals through TCR subset-specific antigens are also necessary.
Because the presence of γδ-T cells is tightly regulated, this raises the possibility that interactions between γδ-T cells and epithelial neighbors may be necessary for effective epithelial homeostasis. Evidence from mouse skin supports this view. IGF-1 can stimulate keratinocyte proliferation and migration and may be an important factor for epidermal homeostasis. We demonstrated that skin γδ-T cells express both IGF-1 and upregulate IGF-1 receptor expression after stimulation through the TCR (23). IGF-1 can protect both the skin γδ-T cells and keratinocytes from apoptosis. In the absence of γδ-T cells, there is increased apoptosis in the skin. The addition of either skin γδ-T cells or IGF-1 reverses the epidermal apoptosis. Keratinocytes undergo apoptosis during the final stage of their terminal differentiation pathway. Epidermal apoptosis must be tightly regulated to ensure that death does not occur earlier in differentiation. These results indicate that skin γδ-T cells provide IGF-1 for the homeostatic maintenance of keratinocytes by regulation of apoptosis and survival. Aberrant homeostasis is a feature of a number of epithelial disorders. It will be interesting to determine whether intraepithelial γδ-T cells similarly regulate homeostasis in other epithelial tissues.
INTRAEPITHELIAL γδ-T CELL FUNCTIONS DURING SKIN WOUND REPAIR
The striking localization of the γδ-T cells to epithelial sites and the tissue-specific expression of γδ-TCRs raised the possibility of specialized functions for these intraepithelial T cells. This was confirmed in a mouse model of wound healing (17). In the absence of γδ-T cells, full-thickness punch biopsy wounds heal slower than wounds in normal mice. Effective wound healing requires a complex series of processes that are mediated by a variety of different cell types. We demonstrated that skin γδ-T cells are required for effective keratinocyte proliferation and wound reepithelialization (17). This is mediated by skin γδ-T cell production of the epithelial mitogens KGF-1 (FGF-7) and KGF-2 (FGF-10). In vitro organ culture experiments confirmed that the addition of either activated skin γδ-T cells or recombinant KGFs to wounds from mice lacking γδ-T cells would restore effective wound reepithelialization. IGF-1 has also been shown to play a role in wound reepithelialization and neovascularization (23). γδ-T cell-deficient mice have reduced IGF-1 receptor phosphorylation at wound sites, suggesting that production of IGF-1 by skin γδ-T cells can increase effective wound healing by rescuing cells from apoptosis (23).
Effective wound repair is also dependent on the recruitment of cells with unique functions to the wound site. Neutrophil migration is not affected by γδ-T cells. However, there is a significant delay in macrophage entry into the wound site in the absence of skin γδ-T cells (18). Hyaluronan is a glycosoaminoglycan that is produced in large amounts during wound repair and can play a key role in inflammatory cell migration associated with wound repair. We found that hyaluronan synthase and hyaluronan expression in wound sites is diminished in the absence of γδ-T cells. It was previously known that keratinocytes were capable of producing hyaluronan. We determined in addition that skin γδ-T cells express hyaluronan synthases and hyaluronan. In a coculture assay, soluble factors produced by activated skin γδ-T cells also induced keratinocyte expression of hyaluronan synthases. It was recently shown that KGF-1 could induce hyaluronan production by rat keratinocytes (19), raising the possibility that in addition to impacting keratinocyte proliferation, skin γδ-T cell production of KGFs might induce hyaluronan production by keratinocytes. Additional coculture experiments demonstrated that skin γδ-T cell production of KGFs was required for effective production of hyaluronan by keratinocytes following wounding and for timely migration of macrophages into wound sites (18). These results demonstrate unique roles for skin-resident intraepithelial γδ-T cells in multiple aspects of tissue repair.
Intraepithelial γδ-T cells in the skin can also produce factors such as thymosin-β4 splice variants that prevent neutrophil infiltration and inhibit inflammation in models of contact hypersensitivity (15). In certain strains of mice, spontaneous skin inflammation occurs in the absence of γδ-T cells, suggesting that γδ-T cells may normally inhibit leukocyte infiltration into the skin (8). Other important roles for skin γδ-T cells include tumor surveillance. Epithelial γδ-T cells effectively lyse skin tumor cells, and mice lacking γδ-T cells develop tumors at significantly higher rates in response to carcinogens and ultraviolet radiation (9). Effective tumor recognition and lysis requires NKG2D-mediated signals in addition to TCR recognition of antigen (9). Together, these results indicate that skin γδ-T cells employ multiple mechanisms to respond to epithelial damage or disease (Fig. 2). Further studies are necessary to determine how cells expressing a single invariant antigen receptor can produce differential functional responses. The roles of signals through coreceptors and costimulatory molecules need to be explored as well as the contributions of factors produced by neighboring epithelial cells in creating different function-influencing microenvironments.
INTRAEPITHELIAL γδ-T CELL FUNCTIONS IN THE INTESTINE
To determine whether intraepithelial γδ-T cells in tissues other than the skin could play a role in tissue repair, the dextran sulfate sodium (DSS)-induced murine colitis model was used to examine tissue repair in the colon. In this model, the DSS is thought to cause apoptosis of epithelial cells in the colon, resulting in the formation of discrete focal lesions, crypt drop-out, and inflammation (6). If the DSS treatment is discontinued, tissue repair occurs. In this model, there is an increase in the numbers of intraepithelial γδ- and αβ-T cells following DSS treatment. There is a striking localization of the γδ-T cells to sites of damaged crypts, whereas the αβ-T cells are found at the base of the damaged mucosa in clusters. In situ hybridization showed dramatically increased levels of KGF-1 around damaged crypts. Analysis of isolated cells demonstrated that γδ-intraepithelial lymphocytes (IEL), but not αβ-IEL, had upregulated expression of KGF-1 following DSS treatment. In the absence of γδ-T cells, but not αβ-T cells, the damage caused by DSS treatment is more severe, and there is delayed tissue repair once the DSS is discontinued. These results demonstrate that participation in tissue repair is a function shared by intraepithelial γδ-T cells in multiple tissues. In addition, this is a unique function of γδ-IELs because αβ-IELs do not produce KGF or appear to contribute to these aspects of tissue repair.
MICA/B are human MHC class I-related molecules that are recognized both by the human intestinal Vδ1 TCR and the NKG2D molecule. MICA/B are highly expressed by a variety of epithelial tumors in humans, whereas normal expression of these molecules is only seen on intestinal epithelial cells. In vitro studies demonstrated that human γδ-IEL can effectively lyse MICA/B-expressing epithelial tumor cells (10). Because mouse skin γδ-T cells similarly recognize epithelial tumors through NKG2D, these results indicate a conserved function for intraepithelial γδ-T cells in immunosurveillance and immune responses against malignant cells that is directed through recognition of antigen plus NKG2D ligands.
INTRAEPITHELIAL γδ-T CELL FUNCTIONS IN THE LUNG
Studies in the lung provide further evidence that intraepithelial γδ-T cells play unique functional roles in tissue repair. Both Nocardia asteroides infection and ozone inhalation have been evaluated as models of lung injury (20). In the absence of γδ-T cells, there is a more rapid onset of disease, and epithelial injury is more severe. In addition, there are defects in the clearance of damaged epithelial cells. Studies from other investigators demonstrate an additional role for γδ-T cells in inflammatory airway hyperresponsiveness (12, 21). Atopic asthma patients have increased numbers of γδ-T cells in the bronchoalveolar lavage fluid (25). These levels drop to normal following steroid treatment. Although the function of these γδ-T cells is not clear, they may be involved in regulation of inflammation and/or tissue repair as was shown in the animal models. Together, these studies demonstrate the importance of intraepithelial γδ-T cells in a variety of airway epithelial immune responses.
γδ-T cells are in intimate contact with neighboring epithelial cells in all of the barrier tissues. These two populations of cells depend on effective interactions for their own homeostasis and to effectively combat epithelial damage or disease. The expression of tissue-specific antigen receptors suggests that these intraepithelial γδ-T cells recognize antigens with restricted tissue distribution. In many tissues, the TCR use is restricted with limited or an absence of diversity. This would imply limited antigen recognition. Increasing evidence points to a role for costimulatory molecules in directing the functions of these cells. Differential expression of costimulatory molecule ligands on epithelial cells after damage, infection, or malignancy could regulate production of specific cytokines, chemokines, and other molecules that may have specialized roles in tissue repair, lysis of damaged epithelial cells, and modulation of neighboring cell functions. A more thorough understanding of the functions of intraepithelial γδ-T cells may allow the modulation of their responses for more effective epithelial defense.
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