T cell development within the intestinal mucosa: Clues to a novel immune-endocrine network?

Pergamon

Advances in Neuroimmunology Vol.6, pp. 397-405, 1996 © 1997Elsevier ScienceLtd. All rights reserved Printed in Great Britain 0960-5428/96 $32.00 PII: S0960-5428(97)00032-0

T cell development within the intestinal mucosa: clues to a novel immune-endocrine network? J o h n R. K l e i n Department of BiologicalScienceand the Mervin BovairdCenter for Studies in MolecularBiologyand Biotechnology, University of Tulsa, Tulsa. OK 74104, USA Keywords--Intraepithelial lymphocytes,extrathymic T cell development, immune-endocrineinteractions, intestine, thymopoiesis, endocrine-paracrine regulation.

Summary Small intestine intraepithelial lymphocytes (IELs) comprise a heterogeneous and phenotypically complex population of T cells that are part of the gut-associated lymphoid tissues (GALTs). Recent studies from a number of laboratories indicate that murine IELs are greatly enriched for extrathymic T cells, although many aspects of the IEL extrathymic developmental pathway remain controversial, and there is currently no consensus of opinion as to which IELs are extrathymic and which are thymus-derived. Those differences reflect variations in the IEL repertoire in athymic animals depending upon the specific model used to study IELs, and they correlate with the age at which mice became or were rendered athymic, implying that the thymus participates either directly or indirectly in the local extrathymic IEL developmental process. In this article, the basic findings regarding intestinal T cell development are discussed, and a hypothesis is provided which links neuroendocrine interactions targeted to the intestine epithelium to the striking relationship between animal developmental age and the thymopoietic potential of the intestine. © 1997 Elsevier Science Ltd. All rights reserved.

The intestinal immune system is distinguished by novel phenotypic and functional properties In addition to its role in digestion and absorption, the intestinal mucosa constitutes a major host barrier to foreign antigen entry. It is not surprising, therefore, that the intestinal immune system bears numerous characteristics which set it apart from lymphoid tissues located throughout the interior of the animal. Moreover, extensive phenotypic studies of intraepithelial lymphocytes (IELs) in mice now reveal a remarkably diverse and complex array of T cell populations associated with intestinal IELs, some with properties typical of T cells found elsewhere, others with properties unique to the intestine. Thus, murine IELs consist of both c~13and y8 T cells in about equal proportions, the majority of which are CD8+4- T cells (reviewed in Klein and Mosley, 1994). However, unlike CD8+ T cells in the thymus or the periphery, virtually all of which consist of a CD8 c ~ heterodimer complex, roughly three-quarters of the small intestine IELs express CD8 as an (xc~ homodimeric molecule (Rocha etal., 1991; Guy-Grand et al., 1991); the

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remaining CD8+ IELs are CD8~z~+ T cells. Although CD4+8- T cells are also normally present within the intestine epithelium, they rarely comprise more than about 5-10% of the total IELs. CD5 and Thy-1, both of which are markers of T cells in the periphery, are variably expressed on murine IELs (Croitoru et al., 1992; Wang and Klein, 1994). Curiously, the intestine epithelium frequently contains a subset of CD4+8+ (double-positive) T cells which bears little resemblance to double-positive thymocytes in that double-positive IELs appear to be mature CD4+ peripheral T cells which have migrated to the intestine and have acquired C D 8 c ~ expression (Rudolphi et al., 1994). Functionally, some murine IELs use unique signal transducing elements associated with the T cell receptor (TCR)/ CD3 complex, and/or with molecular components used in T cell activation (Guy-Grand, 1994).

Two autonomous peripheral immune systems with tightly regulated lymphocyte trafficking Studies using parabiotic mice demonstrate that lymphoid cell communication between the intestine and other peripheral immune compartments is very limited even though trafficking is extensive and occurs rapidly between the spleen, the Peyer's patches, and the intestinal lamina propria (Poussier et al., 1992). These findings have important implications for gastrointestinal immunity since they suggest that in the purlieu, particularly within the intestine, immunity is for the most part controlled locally and that it is not merely an extension of the peripheral immune system. This implies that lymphocyte circulation to mucosal tissues does not involve the haphazard migration of T cells into the intestine epithelium in the absence of antigenic stimulation. Likewise, the functional implications of such immunological restrictions can be seen by the fact that mature IELs in athymic mice do not protect against systemic tumor challenge, suggesting that there is little if any intestine/peripheral T cell trafficking. These findings, of course, by no means imply that T cell migration into mucosal sites never

occurs, but rather that lymphocyte trafficking into the intestinal epithelium is a well-regulated event involving the expression of specialized adhesion molecules and ligands on lymphocytes and mucosal endothelia (reviewed in Girard and Springer, 1995). Nonetheless, it is reasonable to envision two nearly independent immune systems in mice, with minimal or no overlap in the immunologic jurisdictions between peripheral and intestinal compartments under ordinary circumstances.

Extrathymic development of intestinal T cells The notion that the murine small intestine has thymopoietic potential was first suggested by Fichtelius nearly three decades ago (Fichtelius 1967). Using neonatally-thymectomized (NTX) mice, it was demonstrated that lymphocytes with general characteristics of T cells were reproducibly present in the intestinal mucosa of NTX mice (Fichtelius 1967; Fichtelius et al., 1968). This ultimately led to the prediction that the small intestine is itself a first-level lymphoid organ - a supposition which has gained ground in recent years, though it is by no means universally accepted. Other studies using fetal intestine tissues grafted subcutaneously or into the kidney capsule of mice (Ferguson and Parrott, 1972), or using athymic radiation chimeras in rats (Mayrhofer and Whatley, 1983), demonstrated that the numbers of IELs are equivalent between athymic and euthymic mice, implying that small intestine IELs arise from bone marrow-derived hematopoietic stem cells in a thymus-independent manner. Within the past decade far more definitive evidence for an extrathymic IEL developmental pathway has come from detailed studies using athymic bone marrow radiation chimeras and/or congenitally-athymic mice in conjunction with detailed phenotypic analyses employing markers of mature or developing T cells (Klein, 1986; Mosley etal., 1990; Lefrancois etal., 1990a; Guy Grand et al., 1991; Mosley and Klein, 1992a; Stickney et al., 1994). The reproducible findings

Developmental regulation of gut T cell maturation from these studies are that athymic chimeras have an abundance of phenotypically and functionally mature T cells in the small intestine despite a generalized lack of T cells (and T cell function) in extraintestinal lymphoid tissues, again leading to the ultimate conclusion that much of the intestinal T cell repertoire can, and most likely does, develop in the absence of the thymus. Although, taken together, the above studies point to a unique process of extrathymic T cell development, they also pose new challenging questions, not the least of which is where and how extrathymic IELs mature. Do IELs represent a small number of peripheral extrathymic T cells which have migrated to the intestine and proliferated? Or is the intestine itself vested with thymopoietic properties ? Whereas most studies favor the latter, until very recently these conclusions have been derived from indirect or circumstantial evidence, in particular from studies ofT cell selection patterns used by IELs (Hershberg etal., 1990; Lefrancois et al., 1990b; Poussier et al., 1992, 1993; Rocha et al., 1992a), as discussed further below. However, more direct evidence for a local extrathymic T cell development process now has come from the identification of a resident IEL progenitor cell population located within the small intestine lamina propria (Kanamori et al., 1996). Those cell clusters, termed cryptopatch cells, appear to be direct predecessors of mature IELs, and consist ofphenotypically heterogeneous cells which bear various characteristics of immature and/or developing T cells, including dependence upon interleukin 7 and stem cell factor.

Positive and negative selection of extrathymic intestinal T cells IfT cells develop within the intestinal epithelium, one might intuitively predict that alternative mechanisms exist for rendering such cells tolerant of normal host tissues despite the absence of intrathymic T cell maturation. In fact, empirical evidence for this comes from a variety of studies, in particular from analyses of VB6 or VBll in appropriate strains of athymic and euthymic mice, which demonstrate a process of negative selec-

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tion of potentially autoreactive T cell populations within the intestinal epithelium (Poussier et al., 1992, 1993; Rocha, 1992a). Interestingly though, the mechanisms by which autoreactive IELs are negatively selected vary according to the particular IEL subset in question. For example, CD4+CD8- and CD4-CD8c~I]+ IELs are primarily deleted during intestinal selection, whereas CD4CD8aa+ and CD4+CD8c~c~+ IELs are retained in the epithelium but rendered functionally anergic (Poussier et al., 1993). Similar selection patterns also have been observed in transgenic mice expressing TCR for the H-Y male histocompatibility antigen of mice in which both negative and positive extrathymic T cell selection occurs at the level of the intestinal epithelium (Poussier et al., 1993; Rocha et al., 1992a). Collectively, these findings strongly imply that the intestinal epithelium has an intricate and well-refined process for the elimination of autoreactive T cells in a thymus-independent manner.

Extrathymic intestinal T cells: the conundrum of the athymic animal model Despite the overall compelling evidence for an extrathymic developmental pathway for murine IELs, opinions are sharply drawn as to exactly which IELs develop extrathymically and which are thymus-derived (Lefrancois, 1991; Rocha et al., 1992b; Klein, 1996). The bases for such differences reflect, to a large extent, variations in experimental findings derived from the particular athymic animal system used to study IELs. Specifically, these differences are linked to whether mice were athymic at birth, as is the case for nude and NTX mice, or whether they were rendered athymic as adults, as with adult radiation chimeras. The salient features of findings from these experiments are described below and are delineated in Fig. 1. A t h y m i c radiation chimeras

IELs in adult athymic radiation chimeras following hematopoietic reconstitution with bone marrow or with fetal liver express all major T cells subsets found in euthymic mice, despite the virtual

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euthymic a ghymic

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Fig. 1. Differences in IEL repertoires in normal euthymic mice and athymic radiation chimeras, versus congenitally athymic nude mice and NTX mice. absence of mature T cells in extraintestinal peripheral tissues (Mosley etal., 1990; Lefrancois, 1990a; Poussier et al., 1992, 1992). Moreover, experiments using congenic mice convincingly establish IELs in athymic chimeras as descendents of donor hematopoietic stem cells, and effectively rule out the possibility that they are residual hostderived Tcells. Thus, IELs in athymic chimeras are composed of both TCR(z~ and TCR't'~5T cells, and include CD4+8-, CD4+8+, CD4-8+, and CD4-8subsets in numbers and proportions equivalent to those found in normal euthymic mice. CD8 IELs consist of either CDSo~+ or CD8c~13+ cells. Virtually all TCRyI5 IELs express CD8czcz, whereas TCRoq3 IELs consist of roughly equivalent proportions of CD8czcz and CD8o~ cells.

Congenital!y athymic nude (nu/nu) mice IELs in congenitally athymic nude mice differ notably from IELs in athymic chimeras in that although nude mice have approximately normal numbers of TCR78 and CD8c~c~ IELs, numbers of

TCR(x[3, CD8czl3, Thy- 1 and CD5 IELs are greatly reduced (Viney et al., 1989; DeGues et al., 1990; Klein and Mosley, 1994). Moreover, it is clear that the lack of these IELs in nude mice cannot be attributed to a stem cell defect since athymic chimeras constructed from nude mouse bone marrow develop a complete IEL repertoire which is indistinguishable from either normal mice or adult athymic chimeras. This inherently suggests that the IEL impairment in nude mice is an intrinsic defect of factors or conditions needed to facilitate extrathymic IEL maturation. With the identification of local IEL progenitors (Kanamori et al., 1996), detailed studies can now be undertaken to explore the point at which IELs in nude mice are arrested during development. Such information will undoubtedly be ofvahie for understanding the local developmental process of extrathymic IELs.

Neonatally thymectom&ed mice IELs in NTX mice bear some characteristics of both nude mice and athymic chimeras, although

Developmental regulation of gut T cell maturation the IEL repertoire, overall, is most closely similar to that found in nude mice (Lefrancois and Olson, 1994; Lin etal., 1993, 1994a,b; Wang and Klein, 1994, 1995). Thus, NTX mice have greatly reduced numbers of TCRc~[~ and C D 8 c ~ IELs, modestly reduced numbers of TCRT~i and CD8c~c~ cells, and few if any CD4+8+ IELs. In some NTX mice, there is an age-dependent increase in the numbers of TCRoq3 IELs (and occasionally CD4+8+ IELs). This, however, is by no means a consistent finding in all animals and, in fact, the extent to which these IELs result from incomplete or late thymectomy after birth has not been adequately determined.

Physiological basis for age-dependent differences in IEL development There are several potential ways to explain the differences noted in IEL populations between athymic animal systems. Undoubtedly, the sensitivity of the intestine epithelium to ionizing radiation used in the construction of radiation chimeras could lead to molecular or structural changes of intestinal cells (Klein, 1982). If such changes substantially alter the IEL repertoire in athymic chimeras, this would inherently imply that some IELs in athymic chimeras may constitute an experimentally-induced artifact rather than a population of specialized T cells which have originated via a true extrathymic developmental process. Obviously, nude and NTX mice not exposed to irradiation would be spared such effects. Several lines of evidence, nonetheless, argue against a radiation-induced effect, including the fact that IELs in athymic chimeras consist of all types of cells found in nonirradiated mice in terms of cell numbers, phenotypic characteristics, and the proportional distribution of IELs throughout the intestine. Or, viewed alternatively, the question can be considered as to why normal non-irradiated euthymic mice have IELs more similar to athymic chimeras (which have been exposed to radiation) than to nude or NTX mice (which have not been exposed to radiation)? In an attempt to directly understand the magnitude of the contribu-

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tion of ionizing irradiation on the development of IELs in athymic chimeras, we have examined the IEL repertoire of adult NTX mice following irradiation. These experiments convincingly demonstrate that although NTX mice exposed to irradiation occasionally have slight increases in CD4+ T cells, no changes occur in the numbers or proportions of TCRB+ and/or CD8B+ IELs compared to non-irradiated NTX mice (Wang and Klein, 1995), thus indicating that radiation alone does not account for differences in the IEL repertoires in athymic chimeras and nude or NTX mice. So what might account for variations in IEL subsets between the different athymic animal models? To understand this we need to look beyond classical factors associated with immune development and examine epi-immunologic processes which impinge upon the overall developmental process of the organism, in particular events associated with the time at which mice have been rendered or became athymic. Notice that the athymic alteration in nude and NTX mice, whether naturally or artificially introduced, occurs during a period of active biological development that involves multiple organ systems, not just immune development. In contrast, athymic chimeras thymectomized as adults have already proceeded through most maj or developmental phases prior to thymectomy. In this vein, it is known from earlier studies that mice that are athymic at birth have defects in hypothalamus-pituitary hormone responses which can be, at least partially, restored by reintroduction of thymus tissues (Pierpaoli and Sorkin, 1972; Pierpaoli and Besedovsky, 1975; Pierpaoli et al., 1977). Thus, a plausible explanation for the effect of early thymectomy on the immune system is that a thymus-initiated signal is delivered directly or indirectly to the hypothalamus or the pituitary. This could occur via one of several thymus hormones currently identified, including thymosin c~1, thymosin g4, thymopoietin, thymulin, etc. (Dardenne and Savino, 1994), or by as yet unknown thymus-derived molecules. This thymus-derived signal would in turn induce a

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hypothalamic/pituitary response, possibly mediated by thyrotropin-releasing hormone and/or thyroid-stimulating hormone, that would act directly on stromal elements ofT cell lymphopoietic tissues, including the intestine. The mode of action of the hypothalamus/pituitary response in this pathway does not appear to involve the thyroid gland, since blockers of thyroid biosynthesis such as 6-methyl-2-thiouracil do not interfere with the beneficial immunological outcomes of hormone treatment in NTX mice (Pierpaoli and Changxian, 1990). However, a central role for the pineal gland cannot be excluded in the overall pathway of T cell developmental activation (Maestroni et al., 1988). Obviously, immunological jeopardy from a failure to complete the thymus-endocrine loop would be greatest during fetal/neonatal life when the thymus itself is in an active phase of development. The thymus-neuroendocrine-IEL network is delineated in Fig. 2. Note that the principal effect of the pathway is to "prime" both the thymus

epithelium and the intestine epithelium for the eventual maturation of T cells. Most important, however, is that once the necessary bidirectional signals have been delivered, all subsequent IEL processes are thereafter thymus-independent. Conversely, failure to receive a thymus-endocrine signal in nude or NTX mice would result in a longterm defect in the thymopoietic capacity of the intestine epithelium, whereas mice rendered athymic as adults would have intestine epithelia suitable for IEL maturation in a thymus-independent fashion. This hypothesis has now been tested through a series of studies in our laboratory as discussed in the chapter by J. Wang of this volume.

Ontogenic and evolutionary factors linking immune-endocrine involvement to intestinal T cell development How might this system fit together developmentally and mechanistically? From a developmental point of view there are notable similarities

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Fig. 2. Proposed bidirectional immune-endocrine pathway linking thymopoietic function of thymus-derived and

intestine-derived T cell repertoires.

Developmental regulation of gut T cell maturation between the intestine and the thymus in that both tissues appear at about the same time of embryonic life, each being derived from the embryonic pharyngeal pouches (Rugh, 1990). From an evolutionary point of view, the gut-associated lymphoid tissues (GALTs) constitute the first distinct lymphoid tissues in vertebrates, appearing prior to the spleen, the thymus, the bone marrow, and the lymph nodes (du Pasquier, 1993). Thus, GALT-like tissues are present in primitive vertebrates such as sea lampreys and hagfish, whereas a well-developed thymus (and thymuslike Tcell functions) does not appear in vertebrates for at least another hundred million years of evolution. It is likely, therefore, that the thymus arose as an adaptation of the GALT, and that earlier immunologic functions of the GALT (possibly the use of y5 T cells) were retained in the intestine despite the subsequent acquisition of T cell thymopoiesis in the thymus. Predictably, across time differences would arise in the types of T cells (and T cell functions) that are associated with the thymus and the intestine as driven by the immunological needs of tissues served by these immune compartments. Similarly, the differential use of neuroendocrine hormones in the regulation of immunity by peripheral and mucosal immune systems may have resulted in different requirements for, or sensitivities to, hormones at each site. Clearly, much additional work is needed to understand the full impact of immuneendocrine interactions at the level of the gastrointestinal tract, and elsewhere.

Conclusions and implications The major points of this discussion can be summarized as follows: (i) the small intestine T cell repertoire in mice is distinguished by a high proportion of extrathymic T cells, which appear to develop locally within the intestinal mucosa; (ii) studies in athymic animal models yield contradictory findings regarding the types of T cells which are found in the intestine epithelium which correlate with the time of animal development during which the thymus was present or absent, i.e. during fetal, neonatal, or adult life;

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(iii) there is a well-documented role for the fetal/ neonatal thymus in the subsequent expression of competent endocrine and immune systems; and (iv) regulation of intestinal immunity, throughout the life of the animal, appears to be inextricably linked to endocrine function. From a practical point of view, the significance of this immuneendocrine loop with regard to intestinal immunity is that it provides a dynamic mechanism for continually adjusting the distribution of T cell subsets throughout the small intestine. This would provide a rapid and efficient means by which to alter the intestinal T cell repertoire so as to accommodate the diverse array of antigens and pathogens within the gastrointestinal tract.

Acknowledgement This work was supported in part by NIH grant DK35566.

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