Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and

unequal T-cell repertoire? Leo Lefran ois

A significant number of intraepithelial intestinal T cells appear to mature extrathymically. In this paper, Leo Lefran¢ois discusses the implication of this for T-cell receptor selection and repertoire generation. He goes on to develop the concept that such cells may constitute discrete subsets of organ-specific lympho- cytes, with unique T-cell receptor repertoires that may be evolutionary

antecedents of thymus-derived T cells.

Intraepithelial lymphocytes (IEL) of the small intestine are anatomically positioned to be the first line of cellular defense against enteric pathogens. Like other peripheral T-cell populations, IEL contain both o~f3 T-cell receptor (TCR) and y8 TCR cells. In the mouse, IEL are distinct from other T-cell populations in that a significant per- centage (20-80%) express the y~ TCR 1,2. Both c43 and y8 mouse IEL are predominantly CD4-CD8 ÷, while a small number (less than 10%) are CD4÷CD8-cells. Major histocompatibility complex (MHC)-restricted anti- viraP -s and alloreactive cells 6 can be generated from the CD8 + (x13 + IEL population. Cytolytic activity can also be demonstrated in the y8 subset but specificity and MHC restriction patterns have not yet been defined 7. In ad- dition, y8 IEL have the capacity to reverse oral tolerance when adoptively transferred 8, but, again, direct antigenic reactivity of these y8 IEL has not been demonstrated. Thus, the functional role of y8 IEL remains unclear.

Extrathymic maturation of y8 IEL Mouse y8 IEL are phenotypically distinct from any

thymic or peripheral T-cell subpopulation so far de- scribed. First, CD8 expression distinguishes mouse y8 IEL from other y8 T-cell populations which are, for the most part, CD4-CD8-. Second, no ~/8 IEL express the CD 813 chain (Ly-3) whereas virtually all CD 8 + peripheral and thymic (including CD4+CD8 +, double-positive) T cells are CD86 + (Refs 9 and 10). Third, the y8 IEL population can be subdivided on the basis of Thy-1 expression: roughly half of the cells lack Thy-1. Fourth, few if any y8 IEL express CD5 (Ly-1), a molecule that is found on virtually all peripheral T cells and thymo- cytes n.

Early experiments with nude mice and with thymec- tomized, irradiated and bone marrow reconstituted (ATXBM) mice suggested that a proportion of IEL is gener- ated extrathymically 12qs. Recently, studies using mono- clonal antibodies specific for the murine y8 TCR have confirmed that reconstitution of irradiated, thymec- tomized mice with bone marrow or day 15 fetal liver results in the generation of y8 IEL 16-18, and that IEL from nude mice are predominantly y8 TCR + (Refs 9, 18-20). In our hands, both Thy-1 ÷ and Thy-l-subsets are gener-

ated in ATXBM mice, indicating that the expression of Thy-1 on murine T cells is not necessarily linked to thymic derivation. In some nude mice a large number of Thy-1 + cells are found, while in others Thy-1 expression is absent; this result is independent of the percentage of oL[3 IEL present (T. Goodman and Lefrancois, un- published). Other reports have suggested that only the Thy-1- subset is extrathymically derived zl. Since coloniz- ation of the gut by normal flora affects Thy-1 expression of IEL 22, these variable results may be due to the status of bacterial colonization, temporal differences in analysis or

strain differences. The fact that there is little difference in Vy5 and Vs4 expression (the predominant V regions expressed by y8 IEL) between the Thy-1 + and Thy-1- subsets 7 further suggests that these populations undergo common maturational pathways.

Additional evidence for the process of extrathymic development of y~ T cells comes from analysis ofTCR gene rearrangement in day 11 fetal gut and liver, prior to T-cell colonization of the thymus 23. In these experiments, Vv5 rearrangements were detected in both sites, again indi- cating that ~ TCR rearrangement can occur extra- thymically.

There is also evidence that y8 IEL undergo selection during maturation: 50-70% of y8 IEL in H-2 k mice express Vs4, whereas only about 30% express Va4 in H-2 d mice 17. Moreover, this selection process occurs in the absence of a thymus. Analysis of IEL from congenic and recombinant inbred mice has revealed that ex- pression of the MHC class II molecule I-E is necessary for selection of V~4 ÷ IEL 17. However, results from I-E trans- genic mice and other recombinant inbred sets indicate that other element(s) are involved in the selection process (Lefran~ois and Goodman, unpublished). Since selection of a~ TCRs by I-E is largely dependent on superantigen expression, perhaps similar events can occur for y8 IEL. It has also been suggested that nonpolymorphic MHC class I molecules are involved in y8 T-cell reactivity. In fact, CD1 and thymus leukemia (TL) antigens, both relatively nonpolymorphic class-I-like molecules, have been shown to be strongly expressed in the intestinal epithelium 24,2s. However, IEL reactivity to such mol- ecules has not so far been demonstrated.

© 1991, Elsevier Science Publishers Ltd, UK. 0167 -4919/9 I/S02.{)0

Immunology Today 436 Vol 12 No. 12 1991

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Extrathymic maturation of oq3 IEL As is the case with ~/8 IEL, oL[3 IEL can be subdivided on

the basis of expression of Thy-1, CD813 and CD5 (Refs 10,26). Three major populations of CD4-CD8 + (x13 IEL can be detected: Thy-1+CD5+CD813 + (approximately 50%); Thy-1+CD5-CD813 - (approximately 20%); and Thy- 1-CD5-CD 813- (approximately 20%)1°. In addition about 10% of cx[3 IEL are CD4+CD8+Thy-1 +, but CD8[3-, thus distinguishing them from their thymic counterparts 1°,27,28. The Thy-1- and Thy-1 + CD813- populations are phenotypically distinct from thymus- derived CD8 + T cells, while the Thy-1+CD813 + subset is phenotypically similar to thymus-derived T cells. The precursor-product relationships between the various oq3 IEL populations have yet to be determined.

Recent reports suggest that some cx[3 IEL mature extra- thymically16,26, 28. In our hands, reconstitution of this compartment in ATXBM mice is highly variable 28. Similarly, IEL from nude mice (even those more than six months old) are rarely (x13 +, although an occasional preparation can contain significant numbers of these cells (author's unpublished results). Several groups have been unable to detect (x13 IEL in nude mice 9,18-21, further indicating that the requirements for oq3 versus ~/8 IEL maturation and/or expansion are different, since the latter are readily detectable in nude mice. This dichotomy is further exemplified by the relative lack of a13, but not ~8, IEL in germ-free mice 29.

Rocha et al. 26 have shown that oLf3 TCR+CD813 - IEL express TCR V¢ regions that are deleted in peripheral T cells and in CD813 + IEL. These findings are a source of interest from a number of viewpoints. First and foremost, they provide circumstantial evidence for extrathymic maturation of o~13 IEL. Circumstantial because the exper- iments have not been performed using ATXBM mice, and because nude mice generally do not have ~13 IEL 9,18-21, so the possibility that these potentially self-reactive cells require intrathymic maturation yet fail to be deleted cannot be ruled out. This possibility is, however, at odds with the current understanding of thymic selection. An alternative explanation for their absence in nude mice is that the CD813- cells themselves mature extrathymically but require interaction with thymus-dependent T cells to do so.

It is also not clear at present whether local selection of ~13 IEL occurs. No instance of gut-specific deletion of cells bearing particular V~ regions has been reported. The existence of such a population could indicate true tissue specificity of selection, rather than lack of selection elsewhere. Nor is it known if these potentially autoreac- tive T cells are functional: they may be anergized follow- ing interaction with antigen in the gut or elsewhere, a form of nondeletional tolerance known to occur in per- ipheral tissues 3° and in the thymus 31.

Benefits of a multiple repertoire T-ceU system It is clear that, for ~/8 T cells, distinct TCR repertoires

exist in distinct anatomical sites, based not only on thymic influence but also on nonoverlapping V region usages. For example, dendritic epidermal cells (DEC) invariably use V~3 and V8t (Ref. 32), while IEL never use these V regions 33. A major distinction between epithelium-associated'y8 cells and the bulk of c~13 T cells is

that the former do not appear to recirculate between cellular compartments.

Barriers between compartments, perhaps in the form of tissue-specific homing receptors, may serve to optimize recognition of tissue-specific antigens. Distinct but lim- ited V region usage by regional populations may have evolved to combat tissue-specific pathogens or self pro- teins modified during stress, as has been proposed for heat shock proteins in the skin and elsewhere 32. In this way an efficient and beneficial system of tissue-specific effectors can be established. The identification of the antigens recognized by IEL and other ~/8 T-cell popu- lations should shed light on this hypothesis.

The existence of a major extrathymic pathway for o~13 T cells is hypothetical at this point. What is clear is that different selection pressures are applied to CD813- IEL on the one hand and to CD813 + IEL and other peripheral T cells 26 on the other. This is manifested in the presence of 'forbidden clones' of IEL. If these cells are functional, then they may be responsible for policing of tissue- specific abnormalities that are likely to be distinct from those recognized by ~/8 cells. Thus, very rapid responses could be mounted against foreign or induced antigens without the need for lymphocyte recirculation or chemo- tactic systems.

Dangers of multiple T-cell repertoires The dangers of differentially selected T-cell repertoires

become evident when the stringent control that would be required to maintain the integrity of the individual com- partments is considered. It could be argued that if extra- thymic ~8 and oq3 T cells resident in one organ cannot react with antigens present in other sites, owing to the strict selection of a highly tissue-specific repertoire, then disruption of the barriers between compartments would not be a problem. However, TCRs selected in one com- partment, such as the small intestine, would not necess- arily be tolerant to, or react with, the same array of MHC-antigen complexes (or other antigens) expressed in another compartment, such as the thymus. Indeed, TCRs reactive with cell-type-specific allogeneic ligands have been reported 34.

Disruption of compartmental barriers via physical, biochemical or genetic dysfunction could result in in- itiation of a detrimental immune response. In this regard, it is interesting to note that neonatal thymectomy in some mouse strains results in autoimmune disease, but it has not been established whether this is due to the lack of deletion of autoreactive T cells 35,36.

The gut as a lymphoid organ The concept of the intestinal epithelium as a primary

lymphoid organ was originally developed by Fichtelius 37 some 20 years ago. This theory was supported by the presence of 'theliolymphocytes' (IEL) in primitive ver- tebrates 38. Indeed, it appears that gut-associated lym- phoid tissue (GALT) arose prior to development of the thymus 39-41, and in fact may be the evolutionary anteced- ent of thymic tissue, although this theory is by no means universally accepted. Thus, an extrathymic pathway of T-cell development may have arisen early in phylogeny and evolved along a course that eventually became dis- tinct from that of thymus-dependent T-celt maturation,

Immunology Today 437 Vol. 12 No. 12 1991

but that retained certain similarities (for example the involvement of epithelium). If ontogeny recapitulates phylogeny in a functional as well as a developmental sense in this case, it might be expected that IEL, and other GALT components, would be important in immunologi- cal events early in life, such as the induction of oral tolerance and bacterial colonization of the neonatal gut.

Leo Lefran~ois is at the Dept of Cell Biology, The Upjohn Company, Kalamazoo, M149001, USA.

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Proc. Natl Acad. Sci. USA 88, 43-47 19 DeGeus, B., Van den Enden, M., Coolen, C. et al. (1990) Fur. J. ImmunoI. 20, 291-298 20 Viney, J.L., Kilshaw, P.J. and MacDonald, T.T. (1990) Fur. J. Immunol. 20, 1623-1626 21 Bonneville, M., Itohara, S., Krecko, E.G. et al. (1990) J. Exp. Med. 171, 1015-1026 22 Lefrangois, L. and Goodman, T. (1989) Science 243, 1716-1718 23 Carding, S.R., Kyes, S., Jenkinson, E.J. et al. (1990) Genes Dev. 4, 1304-1315 24 Bleicher, P.A., Balk, S.P., Hagen, S.J. et al. (1990) Science 250, 679-682 25 Hershberg, R., Eghtesady, P., Sydora, B. et al. (1990) Proc. Natl Acad. Sci. USA 87, 9727-9731 26 Rocha, B., Vassalli, P. and Guy-Grand, D. (1991) J. Exp. Med. 173,483-486 27 Mosley, R.L., Styre, D. and Klein, J.R. (1990) Int. lmmunol. 2, 361-365 28 Lefranqois, L., Mayo, J.M. and Goodman, T. (1990) in Cellular immunity and the Immunotherapy of Cancer (Lotze, M. and Finn, O.J., eds), pp. 31-40, Wiley-Liss, Inc. 29 Bandeira, A., Mota-Santos, T., Itohara, S. et al. (1990) J. Exp. Med. 172, 239-244 30 Jones, L.A., Chin, L.T., Merriam, G.R., Nelson, L.M. and Kruisbeek, A.M. (1990) J. Exp. Med. 172, 1277-1285 31 Ramsdell, F., Lantz, T. and Fowlkes, B.J. (1989) Science 246, 1038-1041 32 Asarnow, D.M., Kuziel, W.A., Bonyadi, M. et al. (1988) Cell 55,837-847 33 Asarnow, D.M., Goodman, T., Lefrangois, L. and Allison, J.P. (1989) Nature 341, 60-62 34 Marrack, P. and Kappler, J. (1988) Nature 332, 840-843 35 Yunis, E.J., Hong, R., Grewe, M.A. et al. (1967) J. Exp. Med. 125,947-966 36 Kojima, A. and Prehn, K.T. (1981) lmmunogenetics 14, 15-27 37 Fichtelius, K.E. (1968) Exp. Cell Res. 49, 87-104 38 Fichtelius, K.E., Finstad, J. and Good, R.A. (1969) Int. Arch. Allergy 35, 119-133 39 Du Pasquier, L. (1989) in Fundamental immunology (2nd edn) (Paul, W.E., ed.), pp. 139-165, Raven Press Ltd 40 McCumber, L.J., Sigel, M.M., Trauger, R.J. and Cuchens, M.A. (1982) in The ReticuloendotheliaI System (Vol. 3) (Cohen, N. and Sigel, M.M., eds), pp. 393-422, Plenum Press 41 Zapata, A. (1983) Bull. Inst. Pasteur 81, 165-186

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