Objetivos

The lEL, as discussed in Chapter 1, consist almost entirely of T cells. A further distinction from the lamina propria lymphocytes, is the striking predominance of CD8+ cells among the lEL, such that approximately 90% of the lEL carry this marker (Cerf-

y/ô T C ells in Gluten Sen sitive Enteropathy

Bensussan et al. 1987; Janossy et al. 1980; Selby et al. 1983b).. The remainder are either CD4+ or CD4-CD8-,"double negative" (Spencer et al. 1989b). The CD8+ lEL also express CD5 but at reduced levels (Malizia et al. 1985). Similarly, TEL demonstrate reduced expression of LFA-1 (Smart et al. 1991).

Mechanisms of TEL homing are generally poorly understood, but clearly they are of considerable importance to the selective accumulation of certain subgroups of mucosal lymphocytes in the epithelial compartment. The HML-1 antibody, which binds to an antigen highly expressed by lEL (Cerf-Bensussan et al. 1987), is now known to recognise the aEp7 integrin. This suggests that the aEp7 integrin may help mediate localisation of of lEL to the epithelium (Springer 1994). Data presented in the previous chapter has suggested that the ICAM-l-LFA-1 interaction is unlikely to function in the epithelial compartment. The state of activation of lymphocytes might affect their ability to preferentially migrate into the epithelial compartment; activated lymphocytes appear more mobile (Wilkinson 1986) and possess stronger adhesive qualities than resting T cells (Shimizu et al. 1990).

Activation and Proliferation

In order to respond to antigen, lymphocytes need to be activated via the CD3- TCR complex by antigen presented on MHC-encoded molecules. Classical antigen presenting cells, generally belong to the macrophage-mononuclear cell lineage which are not normally present within the intestinal epithelium. Since all enterocytes express HLA class I antigens and villous enterocytes also express HLA-DR, -DP and possibly DQ molecules, a role for the enterocyte in presenting antigen to lEL has been proposed (Bland et al. 1986; Mayer et al. 1987). These in vitro experiments however, used isolated rat or human enterocytes to present soluble protein antigens, such as ovalbumin, to previously sensitised peripheral T cells. The lack of ICAM-1 expression in the epithelial compartment argues further against activation of lEL by the classical route. Thus role of the enterocyte in vivo, in respect of antigen presentation remains to be defined.

Specific binding of the CD3-TCR complex leads to T cell proliferation. In vitro studies suggest that TEL have a relatively weak response to alloantigens, mitogens and anti-CD3 antibody (Ebert 1989). They do respond vigorously, however, to anti-CD2 antibodies, however, which activate T cells throught the CD2-LFA-3 pathway. Other studies have emphasised the role of accessory signals in the activation of T cells via the CD3-TCR pathway and suggest that the nature of such accessory signals determine whether triggering of this pathway results in activation or tolerance (Geppert et al. 1990). There is, for example, some evidence that the integrin oEp? may function as an alternative accessory molecule for lEL allowing activation via CD3-TCR pathways (Samacki et al. 1992). The nature of accessory signals available to lEL in vivo are not known but they may be of considerable importance in determining the response of lEL following binding of antigen.

Function of lEL

The functions of lEL are not clearly defined. Defence against foreign organisms at the epithelial barrier seems likely and could be mediated either through direct cytotoxic mechanisms or through the local secretion of cytokines.

Animal studies provide evidence that lEL are cytotoxic cells with the identification of granzymes and perforins in murine lEL (Guy-Grand et al. 1991). Cytotoxicity by both ap and y5 TCR+ lEL against target cells has been demonstrated in rodents (Goodman et al. 1989; Viney et al. 1990b). TEL are markedly increased in numbers during parasitic infestation with Giardia lamblia (Ferguson 1977) and spontaneous cytotoxicity has been observed against the parasite (Kanwar et al. 1986). Studies suggest that lEL are spontaneously cytotoxic against virally infected cells (Carman et al. 1986) and may therefore be able to protect the mucosa by eliminating infected epithelial cells. Knowledge of the cytotoxic properties of human lEL are limited by the relative paucity of relevant studies. They do not appear to be spontaneously cytotoxic against malignant cell lines (Cerf-Bensussan et al. 1985) but in vivo their cytotoxic activity may be increased under certain circumstances (Ruthlein et al. 1992). Lymphokine-activated

y/ô T C ells in Gluten S en sitiv e Enteropathy

killer and allogeneic cytolytic activity but not antibody-dependent cellular cytotoxicity has been observed (Roberts et al. 1993).

There is little direct evidence for the secretion of cytokines by lEL and such a role for these cells is not proven, certainly in humans. Secretion of a soluble factor with all the characteristics of IFN-y, by rat lEL has been observed (Cerf-Bensussan et al. 1984). It has been suggested that in situations where excessive antigenic stimulation leads to an accumulation of lEL (Amaud-Battandier et al. 1986; Ciclitira et al. 1986b; Guy-Grand et al. 1986), lEL synthesising large amounts of IFN-y, induce increased expression of HLA class II molecules. There is no direct evidence that the lEL are responsible for such IFN-y secretion which may be produced by the adjacent lamina propria lymphocytes. Evidence against lEL being major cytokine producers is provided by a study of experimental graft-versus host disease where cytokine secretion by CD8+ cells was poor (Guy-Grand et al. 1986). In addition, recent studies using radioactive in situ hybridisation to detect and localise mRNA expression of cytokines in human jejunal mucosa detected little or no expression of cytokine mRNA by lEL (Kontakou et al. 1995a; Kontakou et al. 1994). Immunohistochemical studies, however, have shown expression of IFN-y by lEL (Al-Dawoud et al. 1992). The differences in results may reflect the difficulties of localising a secreted product by immunohistochemistry.

The y/ô TCR and y/ô TCR POSITIVE INTRAEPITHELIAL LYMPHOCYTES

The a/p TCR had been well characterised before the isolation of the a third gene which, similarly to TCR a and p chains, was specifically rearranged in T cells and the biochemical characterisation of the corresponding cell surface glycoprotein together with another polymorphic chain, the Ô chain. The structural homologies between the a/p and y/S chains together with its obligatory association with the CD3 signal transduction complex suggested that y/5 chains formed another class of recognition structure (Lefranc et al. 1990). The lymphocytes that are y/ô TCR+ can be subdivided into several groups depending their stage of appearance during ontogeny, the sets of y and Ô genes they use and the extent of VDJ junctional diversity. Most y/6 T cells do not

express either CD4 or CDS, suggesting they may recognise antigen in its native form or presented by non-classical MHC molecules. The y/5 T cells vary in their tissue distribution and interest arose when evidence was presented suggesting that intestinal lEL of mice preferentially use the y/5 TCR (Bonneville et al. 1988; Goodman et al.

1988). Confusion occurred regarding the degree of y/5 TCR usage probably because of the dietary and microbiological status of the experimental animals, since in germ free or weanling mice, lEL were mostly y/5 TCR+, compared to normal mice where the frequency of ot/p TCR usage rose (Viney et al. 1990a).

The human differentiation pathway of y/5 TCR+ lEL has not been established, but in mice there is reasonable evidence that thymus independent differentiation may occur, possibly under the influence of the intestinal microenviroment. Human in vitro studies have demonstrated that immature T cell precursors can differentiate into y/5 TCR+ cells in the presence of various cytokines and y/5 TCR+ cells have been found in a patient with complete thymic aplasia (Cerf-Bensussan et al. 1991). Human y/5 TCR+IEL are usually double negative for CD4 and CD8, with approximately 30% expressing CD8, some of which only express the a-chain of the CD8 molecule. This phenotype is a characteristic of thymus-independent mouse lEL, as is lack of expression of CD5, which is also demonstrated by human y/5 TCR+IEL (Jarry et al.

1990).

The availablity of different monoclonal antibodies directed at epitopes encoded by certain Vy or V5 gene segments has allowed further analysis of the repertoire of human y/5 T cells. Monoclonal antibody BB3 (Ciccone et al. 1988) recognises an epitope encoded by the V52 gene and monoclonal 5TCS-1 (Wu et al. 1988) recognises an epitope dependent on the V51-J51 combination. The subset identified by the 5TCSi predominates in the thymus and peripheral blood at birth and then persists as a relatively constant proportion of peripheral T cells. The subset of y/5 TCR+ cells recognised by the BB3 antibody constitute a small proportion of y/5 TCR+ cells in the thymus and cord blood but rapidly expand after birth and accounts for the major population y/5 TCR+ cells in the peripheral blood of adults. The distribution of

Y/ô T C ells in Gluten S en sitiv e Enteropathy

intestinal y/Ô TCR+IEL subsets appears more variable than in the peripheral blood but there appears to be a preferential accumulation of cell using the V51 gene segment (Halstensen et al. 1990; Jarry et al. 1990; Spencer et al. 1989a). The mechanisms underlying the differential expansion of y/Ô TCR+ cells in different tissues is unknown.

The antigens recognised and functions of y/0 T cells remains unknown. Janeway and colleagues have suggested that they perform an “autologous surveillance” role by removing damaged epithelial cells possibly by recognition of heat shock or “stress” proteins, which are evolutionarily highly conserved (Janeway 1988). Evidence also exists of recognition of peptide presented by both MHC class I and II molecules, although the proportion of y/5 T cell clones able to perform this function appears to be relatively low (Spits et al. 1990). y/5 T cell clones also display high levels of in vitro cytolytic activity against various cell lines.

y/5 TCR Positive Intraepithélial Lym phocytes and C oeliac D is e a s e

Patients with coeliac disease have a significantly increased number of jejunal epithelial y/5+IEL (Halstensen et al. 1989; Savilhati et al. 1990; Spencer et al. 1989a). The role of these cells in the pathogenesis of coeliac disease was unclear, but removal of damaged intestinal epithelial cells or direct cytotoxic damage to the epithelium were possibilities. The increase appeared to be constant despite the dietary status of patient and therefore irrespective of the degree of enteropathy. This suggested that .y/6+IEL might have a fundamental role in the aetiology of coeliac disease