- Email: [email protected]
Role of the gut as a primary lymphoid organ
Immunology Letters 140 (2011) 1–6
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Review
Role of the gut as a primary lymphoid organ Laetitia Peaudecerf ∗ , Benedita Rocha INSERM, U1020, Medical Faculty Descartes Paris V, 75015 Paris, France
a r t i c l e
i n f o
Article history: Received 17 January 2011 Received in revised form 11 May 2011 Accepted 23 May 2011 Available online 16 June 2011 Keywords: Gut CD8␣␣ intraepithelial T lymphocyte T cell differentiation Precursor export
s u m m a r y The TCR-␣/␥␦ CD8␣␣ intraepithelial T lymphocytes (T-IEL) located in the gut mucosa of the small intestine are an abundant population believed to have a major role in ensuring the integrity of the gut wall. Here, we describe their unique characteristics and the controversies regarding the origin and differentiation of these T-IELs. We show how accumulated experimental evidence has finally arrived at a unifying concept, which demonstrates that these cells originate from early thymus precursors that have not yet undergone TCR rearrangement and TCR-␣/␥␦ commitment. These precursors colonize the gut lamina propria during the perinatal period and complete rearrangements and TCR-␣/␥␦ commitment while migrating to the epithelium. Therefore, the gut epithelium, which shares the same embryonic origin as the thymus epithelium, behaves as a primary lymphoid organ responsible for the differentiation of a major local T cell set. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The gut mucosa is a major site of interaction between the almost sterile environment of the body and the outside world. The oral route is one of the pathways used most frequently by infectious agents to penetrate the body. Moreover, the gut harbors abundant microbiota, including the commensal bacteria that have a fundamental role in ensuring our survival through food processing, the production of some essential elements like vitamin K, and by keeping potentially pathological bacteria in check. The gut environment thus faces the unique challenge of preserving the subtle balance between immune responses and tolerance. While elsewhere in the body tolerance must be maintained against self antigens only, in the gut environment, tolerance must also be developed against abundant exogenous antigens—food antigens as well as the local microbiota. In addition, although microbiota are innocuous and useful when present in the lumen, they must not penetrate into the body, where they could cause septic shock and death. One of the major requirements of the local environment is thus the maintenance of epithelial barrier integrity. Local protection is ensured by the most abundant effector T cell compartment in the body: the gut intraepithelial T lymphocytes (T-IEL).
lumen, mucins, and the far more abundant epithelial cells, which rapidly die in vitro, forming clumps in which T-IEL are also trapped. Although we have attempted several methods to improve isolation procedures, we found that initial techniques involving passage through glass wool columns followed by separation in density gradients were optimal for obtaining T-IEL isolates; however, these techniques lead to variable and important losses. Therefore, the most reliable way to evaluate T-IEL recovery is to count individual T-IEL in tissue sections in adult mice bred in Specific Pathogen Free (SPF) conditions. Since the number, the size of villi, and the number of T-IEL vary in different portions of the small intestine, we examine the whole small intestine, divided into portions 1 cm in length. We determine the total number of villi present in each fragment from the number of villi in 1 cm long sections and the number of villi present at the perimeter of the gut in each location. We next determine the number of epithelial cells present in each villus by counting epithelial cells present along the length and at the perimeter of individual villi. Finally, we determine the T-IEL/epithelial cell ratio at each location. These results allow us to estimate the total number of T-IEL, which we found was comparable to the total number of T cells located in all the conventional peripheral lymphoid organs, i.e. lymph nodes plus the spleen [1].
2. Quantification of T-IEL
3. Different types of gut epithelium T-IEL
Recovery of T-IEL is hindered by multiple technical problems. Isolates are contaminated with bacteria, detritus located in the gut
Two main categories of T-IEL can be distinguished in adult mice. One population originates from peripheral T cells; after responding to antigens in the Peyer patches or the mesenteric lymph nodes, these cells up-regulate expression of ␣47 integrin and chemokine receptor CCR9 and home to the gut mucosa [2–4]. These conventional T-IEL have the phenotype of peripheral antigen-experienced
∗ Corresponding author. Tel.: +33 1 40 61 53 61. E-mail address: [email protected] (L. Peaudecerf). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.05.009
2
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
cells; i.e. TCR␣+ Thy1+ CD5+ LFA-1+ CD28+ CD44+ , and they are CD4+ or CD8␣+ . The other subpopulation is specific to the gut wall. In mice, but not in other species, they are commonly called CD8␣␣ T-IEL, because these cells are CD4− CD8− and frequently express the CD8␣␣ homodimeric form of the CD8 coreceptor [5]. Although CD8␣␣ expression is not specific to T-IEL, (CD8␣␣ is expressed by a subclass of dendritic cells [DC] and by highly activated peripheral T cells [6]), these unconventional cells have globally unique properties that distinguish them from all the other T cell types present elsewhere. They are TCR-␥␦ or TCR-␣ and CD5− CD28− LFA-1− , with only a fraction expressing Thy1 [7–9]. A major fraction expresses B220, and CD11c is also expressed, although elsewhere these markers are considered to be restricted to the B and DC lineages, respectively [10,11]. They are NK1.1− but can express several alternative NK receptors [12]. Their repertoire is quite pauciclonal; they result from the major expansion of a few clones [13,14]. These cells show no evidence of having ever expressed CD8. Indeed, TCR-␣+ cells that differentiate in the thymus go through a CD4+ CD8␣+ (DP) stage where they demethylate cytosines in the CD8 gene 5 regulatory region. This epigenetic modification is maintained in all conventional T cells; the CD8 locus remains demethylated even in peripheral CD4+ T cells. In contrast, the CD8 locus is methylated in both CD8␣␣ T-IEL and their precursors [15]. Finally, the CD3 complex of T-IEL has a peculiar composition, as it contains CD3- -FcRI␥ heterodimers or FcRI␥ -FcRI␥ homodimers instead of the CD3- -CD3- homodimers that characterize thymus-derived cells [16,17]. In adult mice in SPF conditions, CD8␣␣ constitute about half of the total T-IEL, and this proportion is maintained as the mice age. However, in situations where mice are kept in conventional animal houses, or in humans who undergo multiple infections, the proportion of nonconventional TIEL declines with age. It is not known if such decreases correspond to a reduction in their absolute numbers, or to a dilution due to the accumulation of conventional T-IEL.
4. CD8␣␣ T-IEL repertoires are generated by agonist selection Before CD8␣␣ T-IEL were studied, two mechanisms were described to determine the selection of T cell repertoires: negative selection, which purges the T cell repertoires of self-reactive cells, and positive selection, which ensures that T cells that differentiate in the thymus are restricted to self MHC class I or class II, and express CD8␣ or CD4, respectively. Negative selection was first identified in mice expressing endogenous Mls superantigens. These superantigens activate T cells expressing TCRs with particular V chains, like V6 or V11. Their expression as self antigens induces the deletion of V6- and V11-expressing cells in the thymus and in CD8␣+ and CD4+ T-IEL, but not in TCR-␣+ CD8␣␣ T-IEL [1]. These results demonstrated that CD8␣␣ T-IEL do not undergo negative selection. The subsequent study of mice expressing a TCR transgenic (Tg) specific to the male antigen revealed that while conventional T-IEL were deleted in male mice, TCR-␣+ CD8␣␣ T-IEL specific to the male antigen were highly enriched [18]. Conversely, TCR-␣+ CD8␣␣ T-IEL male-specific T cells were absent in female mice. This led to the surprising conclusion that instead of being deleted by the presence of self antigens, as is the case for CD8␣ and CD4T cells, CD8␣␣ T-IEL require the presence of self antigens to be generated. It was then proposed that this phenomenon could be due to the TCR/CD8/CD3 complex that is unique to TCR-␣+ CD8␣␣ T-IEL. The reduced number of ITAM motifs present in the FcRI␥, chain as well as the absence of CD8, would reduce the intensity of the TCR-mediated signals. Under these circumstances, rather than inducing cell death, signals induced by self antigens would function as survival signals. Decades later, this selection mechanism, called
agonist selection, was shown to control selection of the repertoire of TCR-␣ CD4+ CD25+ FOXP3+ regulatory T cells [19]. 5. Functions of CD8␣␣ T-IEL Because CD8␣␣ T-IEL are selected by self antigens, their function has been suggested to be in the transition between innate and adaptive responses, mainly directed toward control of the integrity of the gut wall. Thus, TCR-␥␦ – deficient mice are susceptible to mutagen – induced local carcinomas [20] and to localized inflammation by toxic chemicals [21]. TCR-␣+ CD8␣␣ T-IEL were shown to mimic the role of CD4+ CD25+ regulatory T cells in preventing the colitis induced by injection of CD4+ naïve T cells into T cell – deficient mice; this regulation was demonstrated to be mediated by IL-10 [22]. Moreover, these cells were also shown to promote the generation of CD4+ CD25+ regulatory T cells against orally presented autoantigens and were reported to have a major immunoregulatory role by preventing the development of autoimmune diabetes in NOD mice [23]. It must be noted, however, that the functional role of CD8␣␣ T-IEL has been studied mostly in mice; few studies have been performed in other species. However, human IEL were shown to kill carcinoma cell lines in vitro [24], and celiac disease is marked by a major accumulation of unconventional TIEL, which appears to contribute to reducing the local inflammation induced by antigen-specific conventional T cells [25]. 6. CD8␣␣ T-IEL development: evidence for an extrathymic origin The multiple peculiarities of CD8␣␣ T-IEL that distinguish them from peripheral thymus-derived cells suggest that they do not follow a classic T cell differentiation pathway. Several independent observations have led to the notion that these cells could be generated within the gut wall. The adaptative immune system appears in the gastrointestinal region of primitive vertebrates before the emergence of the thymus. In contrast with other epithelia, the gut epithelium shares a common endodermic origin with the liver and the thymus epithelia, and thus may express common factors that support hematopoiesis in general and T cell lymphopoiesis in particular. These cells are present in nude mice that have a mutation in the Foxn1 gene, which is essential for thymopoiesis [7]. Several genes specific to T lymphopoiesis, such as Rag1 (Rag, recombination-activating gene), CD3ε, and pT˛, were detected in lineage marker – negative (Lin− ) cells directly isolated from the gut wall of normal euthymic mice [16,26,27]. Finally, it was formally demonstrated that TCR-␥␦ T-IEL could rearrange their TCRs in situ. Indeed, in IL-7 – deficient mice, TCR-␥␦ T cells cannot develop in the thymus or elsewhere. However, the selective expression of IL-7 in the gut epithelium reconstitutes TCR-␥␦ T-IEL, since outside the gut, TCR-␥␦ T cells remain absent [28]. These results indicate that TCR-␥␦ rearrangements should occur directly within the gut wall. It must be noted that CD8␣␣ T-IEL were mostly studied in mice. However, Rag expression was also detected in Lin− cells isolated from the human intestine [29]. Research into extrathymic T cell generation in the gut changed markedly with the description of “thymus-like structures” that are scattered throughout the lamina propria (LP) at the base of some gut crypts. These small aggregates called cryptopatches (CPs) contain Lymphoid inducer cells (Lti) and further complex mixtures of Lin− populations expressing different levels of c-kit, IL-7R and ROR␥t, many of them not yet fully characterized, and were also proposed to be precursors of isolated lymphoid follicles [30]. However, CP structures also harbor T cell precursors since when Lin− cells were isolated directly from CP by micromanipulation, they showed evidence of ongoing TCR rearrangements and were able to generate
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
T-IEL after adoptive transfer [27,31,32]. However, the majority of studies on local differentiation were not performed after direct CP isolation; instead, they used Lin− cells isolated either from the LP or from the epithelium. The study of cell surface and molecular markers, TCR rearrangements, the kinetics of reconstitution, and the impact of different mutations affecting T cell development led to the establishment of a sequence of T cell development in the gut of euthymic mice, as described in [27] and reviewed in detail in [33]. Briefly, this sequence is initiated in Lin− precursors that are located in the LP and are Thy1+ IL-7R+ CD25int CD44+ c-kit+/− . Thus, they have the phenotype of triple negative ([TN] CD3− CD4− CD8␣− ) TN1-to-TN2 transition thymocytes [34]. This population expresses Rag and pTa; the Rag mutation blocks local differentiation at this stage. Injection of these precursors into irradiated nude mice reconstitutes the euthymic CD8␣␣ T-IEL compartment. These precursors migrate from the LP to the epithelium [35], where they give rise to an intermediate c-kit− Thy1+ B220+ CD8␣␣+ population. These cells show complete TCR- rearrangements and ␥5 rearrangements, but no TCR-␣ rearrangements. The TCR-␣ mutation blocks differentiation at this stage. Finally, these precursors generate Lin− B220+ CD8␣␣+ cells that lose Thy1 expression and complete the rearrangements of the TCR-␣ chain. The multiple characteristics of the gut differentiation process in euthymic mice could explain the differences between the T cell repertoires generated in the thymus and in the gut. Indeed, compared to the thymus, pT␣ is less expressed in the gut precursors [27], which may account for the relative local abundance of TCR-␥␦ cells. Rag1 expression frequency is also lower in the gut compared to the thymus, which could explain the pauciclonality of CD8␣␣ T-IEL repertoires. Indeed, because rearrangements would be relatively rare, repertoires in the gut would result from the expansion of relatively few clones [27]. 7. CD8␣␣ T-IEL development: differences between euthymic and athymic mice Although CD8␣␣ T-IEL can be generated in athymic mice, in the absence of a thymus, the pool of CD8␣␣ T-IEL is reduced 5–10-fold, TCR-␣ CD8␣␣ T-IEL are rare, and the majority of CD8␣␣ T cells do not express Thy1. Moreover, the molecular characteristics of Lin− precursors located in the gut in nude mice are rather different from those found in euthymic mice [36]. Although the phenotype and relative location (LP or epithelium) of these precursors is similar, Lin− cells isolated either from the LP from micromanipulated CPs or from the epithelium show less-advanced TCR rearrangements compared to the subpopulations with equivalent phenotypes recovered from euthymic mice [36,37]. Thus, in nude mice, complete TCR- rearrangements are not detected the Lin− LP cells, and the rearrangements of the TCR- and TCR-␣ chain are less extensive in Lin− subpopulations recovered from the nude epithelium. Moreover, although pT˛ is expressed [26], expression frequencies are quite low (Rocha, unpublished observations). All of these results indicate that the presence of the thymus has a major influence on the local differentiation process. 8. CD8␣␣ T-IEL development: evidence for a thymic origin It is well known that seeding of the CD8␣␣ T-IEL compartment is a postnatal event. At birth, these cells are virtually undetectable, but reach normal numbers by 3 wk of age. It is generally accepted that in euthymic mice, this wave of colonization has a thymic origin. Injection of bone marrow cells from normal mice into athymic mice leads to the generation of CD8␣␣ T-IEL in the same proportion as in nude mice [31]. By contrast, neonatal thymus transplants reconstitute the CD8␣␣ T-IEL compartment in the same proportion as in
3
normal mice; these cells originate from the thymus graft [38–40]. Migration from the thymus to the gut appears to be restricted to a short time period after birth. Thus, thymectomy only affects the CD8␣␣ T-IEL pool when performed in the perinatal period. From 3 wk after birth, thymectomy does not affect CD8␣␣ T-IEL numbers or composition [38]. Moreover, parabiosis experiments show an absence of CD8␣␣ T-IEL chimerism between adult partners [41]. Overall, these results demonstrate that the influence of the thymus on CD8␣␣ T-IEL generation is confined to a very narrow perinatal window. 9. CD8␣␣ T-IEL development: role of the thymus While it is generally accepted that CD8␣␣ T-IEL are thymus derived in euthymic mice, the differentiation stage at which these thymocytes exit the thymus and colonize the gut is the subject of debate. Two hypotheses have been proposed: (A) CD8␣␣ T-IEL originate from T cell – committed immature thymocyte precursors that have not yet rearranged their TCRs. These precursors colonize the gut LP and finish their differentiation in situ. This hypothesis reconciles a thymic origin with an extrathymic differentiation process. (B) CD8␣␣ T-IEL originate from more mature thymocytes that rearrange their TCRs in the thymus and acquire the particular phenotypes of CD8␣␣ T-IEL after colonization of the gut wall. These two theories are not mutually exclusive; both are explored below in further detail. 9.1. CD8˛˛ T-IEL originate from T cell–committed immature thymocyte precursors We studied the capacity of different thymocyte populations to generate CD8␣␣ T-IEL after adoptive transfer and found that only CD4− CD8− CD3− TN thymocyte sets had that potential. However, TN populations are mixtures of several precursor populations at different differentiation stages. It is well established that early precursors have the CD44+ CD25− phenotype (TN1) and have not yet rearranged their TCRs. These cells give rise to the CD44+ CD25+ TN2 population, which it is not yet fully committed to T cell differentiation, since it is still able to generate NK cells. These cells have incomplete TCR- DJ rearrangements and most are not yet committed to either TCR-␣ or TCR-␥␦ lineages, because these events occur at the more mature CD44− CD25+ TN3 stage of differentiation. Complementary evidence allowed us to determine at what stage of differentiation the TN precursors could leave the thymus and give rise to CD8␣␣ T-IEL. Only CD44+ TN cells fully reconstituted the CD8␣␣ T-IEL compartment with a Thy1+/− phenotype and the TCR-␣/TCR-␥␦ representation seen in euthymic mice [42]. After thymus transplant, we could find early precursors of thymic origin in the blood and in the gut LP [42]. Most of the Lin− precursors detected in the blood corresponded to a previously uncharacterized cell type in the transition between the CD44+ CD25− (TN1) and CD44+ CD25+ (TN2) thymocyte sets (CD44+ CD25int TN1–TN2 transitional cells). These precursors expressed a high frequency of Rag1 [42]. Direct studies of immature TN subsets isolated from the neonatal thymus also showed that the TN1–TN2 subset had the potential to leave the thymus and colonize the gut. We showed that during the TN1–TN2 transition, thymocytes strongly upregulate the expression of ␣47 integrin [42] and also express CCR9 (unpublished), both of which are required for homing to the gut. They also express a high frequency of S1p1, which codes for the receptor of sphingosine-1-phosphate, which is required for thymus egress [42]. Moreover, the location of TN1–TN2 transitional cells in the thymus also favors egress, which is described as taking place through capillaries located at the corticomedullary junction. Indeed, TN1 cells are present around this boundary and must cross
4
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
this zone to reach the inner cortex where TN2 cells reside [43]. Therefore, CD25int TN1–TN2 transition cells are likely present very close to the capillaries leaving the thymus. Thus, both the characterization of TN migrants in the blood after thymus transplantation and the direct study of thymocyte population properties indicate that the bulk of TN egress should occur during the TN1–TN2 transition. Finally, we also observed that transplants of Rag− thymus (which only harbor TN1–TN3 subsets) reconstituted CP-like structures in the gut, and all individual cells from these structures expressed CD3ε [40]. Although these experiments did not allow us to determine which TN subpopulation colonizes the gut, they did show that early precursors can leave the thymus before TCR rearrangements. They also create a direct link between gut colonization by thymus precursors and CP-like structures. It must be noted that within the CPs, thymus-derived precursors are only a minority population; several other cell types are also present (discussed below). The identification of the TN1–TN2 subset as responsible for gut colonization allows us to reconcile a thymic origin with a gut T cell-differentiation process. Single-cell cultures revealed that all TN1 cells and about 50% of TN2 cells have the capacity to generate both TCR-␣ and TCR-␥␦ cells [44]; thus, it is likely that at least the majority of these TN1–TN2 cells are bipotential. Moreover, these cells have probably not rearranged their TCRs, since TCR rearrangements are absent in the TN1 set and only rare and incomplete in the TN2 set [45,46]. Therefore, CP colonization precedes TCR-␣/TCR-␥␦ commitment and rearrangements that will occur in the gut. Finally, TN1 and TN2 cells have the potential to generate NK cells, and it was recently described that the CPs could be involved in the generation of a unique population of NK cells in the gut: the IL-22 – producing NKp46+ cells [47]. It remains to be determined if these cells may also derive from TN precursors. 9.2. CD8˛˛ T-IEL TCR-˛ˇ+ IEL originate from mature thymocytes that acquire particular phenotypes locally 9.2.1. Genetic labeling and genetic fate experiments The notion that TCR-␣+ CD8␣␣ T-IEL may originate from more mature thymocyte precursors arose from genetic labeling and genetic fate experiments. In mice expressing green fluorescent protein (GFP) under the promoter of the nuclear hormone receptor ROR␥t, both DP thymocytes and CP cells are GFP+ , but TCR-␣+ CD8␣␣ T-IEL are not. Elimination of ROR␥t expression in these mice induced the apoptosis of DP thymocytes, CP cells, and TCR-␣+ CD8␣␣ T-IEL. Although restoration of DP thymocyte survival by the anti-apoptotic factor Bcl-xl led to the reconstitution of the TCR-␣+ CD8␣␣ T-IEL pool, CP cells were not detected [48]. The authors claimed that Lin− c-kit+ IL-7R+ CP precursors were aggregates of lymphoid inducer cells (Lti) that were not required for TCR-␣+ CD8␣␣ T-IEL generation. However, in another study that used the same mice but directly isolated the CPs, ROR␥t deficiency appeared to eliminate just a fraction of Lin− c-kit+ IL-7R+ precursors and CP structures and had no effect on TCR-␣+ CD8␣␣ T-IEL generation [37]. The reasons for such discrepancies are unclear, since the same mice were used in both studies. Using ROR␥t promoter – driven Cre recombinase to induce yellow fluorescent protein (YFP) expression, it was shown that, in contrast to TCR-␥␦+ CD8␣␣ T-IEL, the majority of TCR-␣+ CD8␣␣ T-IEL expressed ROR␥t during their development [49]. Because YFP was detected in DP thymocytes, the authors also concluded that TCR-␣+ CD8␣␣ T-IEL had passed through the DP stage. These types of experiments are elegant and attractive, but may lead to serious overinterpretations (Fig. 1). In this particular case, the expression of YFP in both DP thymocytes and TCR-␣+ CD8␣␣ T-IEL does not prove that they are directly related: ROR␥t expression is part of the pre-TCR activation cascade during -selection [50]. Since Lin− cells isolated from the epithe-
Fig. 1. Possible relationships between two genetically labeled cells. Upper graphs: cells are directly related. Middle graphs: cells derived from a common precursor. Lower graphs: cells are not related. The gene is expressed independently in two different lineages. Genetic labeling and genetic fate experiments alone do not discriminate between these three possibilities. Relationships must be confirmed by complementary classic approaches to determining precursor/product relationships. Modified from Rocha [33].
lium express the pT␣ chain and undergo -selection [27], the YFP detected in TCR-␣+ CD8␣␣ T-IEL could be induced directly during T cell differentiation in the gut wall. The notion that CP cells are simply aggregates of Lti cells was reviewed extensively when a more-detailed analysis of CP composition was conducted. After direct isolation of CP aggregates by micromanipulation, these aggregates were found to contain multiple cell types that could be distinguished by different levels of c-kit, IL-7R, and ROR␥t expression. In fact, only 10% of the CP cells have the CD4+ c-kithi IL-7Rhi ROR␥thi phenotype typical of Lti [37]. CPs also harbor a population of NK cells with the unusual NKp46+ IL-7R+ ROR␥t+ phenotype. Moreover, while in early studies, ROR␥t expression was thought to be limited to Lti and immature thymocytes, it is now known that this gene is expressed by cells from other lineages (including certain TCR-␥␦ populations and IL-17 expressing cells), preventing the use of this marker to define possible lineage relationships with either Lti or other ROR␥t+ cells also present in the gut LP. Thus, the complexity of CP subpopulations and the lineage relationships between their subpopulations and the other cell types present in the gut LP and also expressing ROR␥t+ remains to be established. 9.2.2. Binding to thymus-leukemia antigen tetramers TCR-␣+ CD8␣␣ T-IEL have also been claimed to be derived from a DP population that, like IEL, expresses CD8␣␣. This minority of DP cells is identified by the specific ability of CD8␣␣ homodimers to bind tetramers of the thymus leukemia antigen (TL-tet) [51]. This cell set would be agonist selected in the thymus cortex, giving rise to agonist-selected NK1.1− TCR-␣+ DN cells that would reconstitute TCR-␣+ CD8␣␣ T-IEL in the gut. Thus, when TL-tet+ DP cells expressing the self-reactive anti-HY TCR-␣ transgene were stimulated in vitro with the HY peptide, they generated transgenic (Tg) TCR-␣+ DN or CD8␣␣+ cells, and intravenous injection of NK1.1− TCR-␣+ DN thymocytes into Rag-2−/− mice reconstituted the CD8␣␣ T-IEL compartment in the gut [51]. However, these experiments used mice in which the Tg TCR-␣ chain was expressed under the control of the TCR- promoter, resulting in a precocious and very abnormal expression of the transgenic TCR-␣ already at the TN2 stage [52]. By contrast, other experiments in which the Tg TCR-␣ chain was inserted in the TCR-␣ locus, resulting in normal expression of the TCR-␣ transgene in DP cells, suggested that self-reactive DP thymocytes did not give rise to CD8␣␣ T-IEL. Indeed, in these male mice, in spite of the vast opportunity to generate agonist-selected DP thymocytes, there is no more Tg TCR-␣+
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
5
Thus, when immature precursors are prevented from colonizing the gut, CD8␣␣ T-IEL generation is virtually abrogated [42]. These experiments demonstrate that in euthymic mice, reconstitution of the CD8␣␣ T-IEL compartment during the first 3 wk after birth depends upon the colonization of the gut by very immature thymocyte precursors.
11. Concluding remarks The origin of CD8␣␣ T-IEL has been the subject of much debate. The identification of early TN subsets as responsible for CD8␣␣ T-IEL generation allows us to reconcile a thymic origin with an extrathymic T cell – differentiation process, and to confer to the gut a major role as a primary lymphoid organ. Thus, the thymus is fundamental for a first imprinting of the gut precursors, since both the gut precursors and CD8␣␣ T-IEL are very different in athymic and euthymic mice. The precursors of the gut Lin− cells leave the thymus and colonize the gut during the perinatal period. These thymocytes have a restricted T and NK potential, TCR rearrangements are rare and incomplete, and TCR-␣/␥␦ lineage commitment has not yet occurred. Lin− c-kit+ IL-7R+ cells recovered from the gut LP of euthymic mice share the same molecular characteristics and finish TCR rearrangements and TCR-␣/␥␦ commitment after migration from the LP to the gut epithelium. However, CD8␣␣ T-IEL precursors are only a minority population within Lin− c-kit+ IL-7R+ cells in the LP. The complexity of these populations and the additional roles that various Lin− subsets may have in local physiology and pathology have yet to be further explored. Fig. 2. The relative role of TN and other thymocyte populations in CD8␣␣ T-IEL generation.
References CD8␣␣ T-IEL than in female mice [52]. Moreover, the expression of CD8␣␣ homodimers in a minority of DP cells was never directly demonstrated, and our recent data do not support this differentiation pathway. We showed that TL-tet binding is not CD8␣␣ specific, since these tetramers fail to bind many CD8␣␣ cells while adhering to several populations expressing CD8␣. Moreover, we demonstrated that the vast majority of the TCR-␣+ DN population is depleted of self-reactive cells and thus cannot give rise to the self-reactive, agonist-selected CD8␣␣ T-IEL pool [42]. 10. The relative roles of “immature” versus “mature” thymocytes in T-IEL generation To investigate the relative contribution of TN cells versus the more mature thymocytes in T-IEL generation, we transplanted neonatal thymi into different mice and followed the gut colonization by thymocytes originating from the transplant. Hosts were either Rag-2/␥c−/− or Rag-2−/− mice. The former mimic the conditions found in the postnatal period (both Lin− c-kit+ IL-7R+ precursors and the absence of T cells in the gut), while Rag-2−/− mice have an abundant Lin− c-kit+ IL-7R+ population but lack mature T cells. We reasoned that if CD8␣␣ T-IEL originate from immature precursors, the presence of a full niche of Lin− c-kit+ IL-7R+ cells in the gut of Rag-2−/− mice should hinder precursor entry and CD8␣␣ T-IEL generation. By contrast, if either TCR-␣ or TCR-␥␦ derive from more mature thymocyte sets, the reconstitution of the CD8␣␣ T-IEL pool should be similar in both host mice, since both lack more mature T cells [42] (Fig. 2). We found that the presence of local precursors in Rag-2−/− mice blocked CD8␣␣ T-IEL reconstitution. By contrast, CD4+ /CD8␣+ T-IEL (known to derive from mature thymocytes) colonized the gut of both mouse strains similarly, demonstrating the efficiency of the thymus transplantation.
[1] Rocha B, Vassalli P, Guy-Grand D. The V repertoire of mouse gut homodimeric ␣ CD8+ intraepithelial T cell receptor ␣/+ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J Exp Med 1991;173:483–6. [2] Koseki S, Miura S, Fujimori H, Hokari R, Komoto S, Hara Y, et al. In situ demonstration of intraepithelial lymphocyte adhesion to villus microvessels of the small intestine. Int Immunol 2001;13:1165–74. [3] Svensson M, Marsal J, Ericsson A, Carramolino L, Broden T, Marquez G, et al. CCL25 mediates the localization of recently activated CD8␣+ lymphocytes to the small-intestinal mucosa. J Clin Invest 2002;110:1113–21. [4] Guy-Grand D, Vassalli P. Gut intraepithelial lymphocyte development. Curr Opin Immunol 2002;14:255–9. [5] Rocha B, Guy-Grand D, Vassalli P. Extrathymic T cell differentiation. Curr Opin Immunol 1995;7:235–42. [6] Madakamutil LT, Christen U, Lena CJ, Wang-Zhu Y, Attinger A, Sundarrajan M, et al. CD8␣␣-mediated survival and differentiation of CD8 memory T cell precursors. Science (New York, NY) 2004;304:590–3. [7] Guy-Grand D, Cerf-Bensussan N, Malissen B, Malassis-Seris M, Briottet C, Vassalli P. Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med 1991;173:471–81. [8] Lefrancois L. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J Immunol 1991;147:1746–51. [9] Ohteki T, MacDonald HR. Expression of the CD28 costimulatory molecule on subsets of murine intestinal intraepithelial lymphocytes correlates with lineage and responsiveness. Eur J Immunol 1993;23:1251–5. [10] Barrett TA, Gajewski TF, Danielpour D, Chang EB, Beagley KW, Bluestone JA. Differential function of intestinal intraepithelial lymphocyte subsets. J Immunol 1992;149:1124–30. [11] Huleatt JW, Lefrancois L. Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8+ T cells in vivo. J Immunol 1995;154:5684–93. [12] Guy-Grand D, Cuenod-Jabri B, Malassis-Seris M, Selz F, Vassalli P. Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages. Eur J Immunol 1996;26:2248–56. [13] Regnault A, Cumano A, Vassalli P, Guy-Grand D, Kourilsky P. Oligoclonal repertoire of the CD8␣␣ and the CD8␣ TCR-␣/ murine intestinal intraepithelial T lymphocytes: evidence for the random emergence of T cells. J Exp Med 1994;180:1345–58. [14] Regnault A, Levraud JP, Lim A, Six A, Moreau C, Cumano A, et al. The expansion and selection of T cell receptor ␣ intestinal intraepithelial T cell clones. Eur J Immunol 1996;26:914–21. [15] Hamerman JA, Page ST, Pullen AM. Distinct methylation states of the CD8 gene in peripheral T cells and intraepithelial lymphocytes. J Immunol 1997;159:1240–6.
6
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
[16] Guy-Grand D, Rocha B, Mintz P, Malassis-Seris M, Selz F, Malissen B, et al. Different use of T cell receptor transducing modules in two populations of gut intraepithelial lymphocytes are related to distinct pathways of T cell differentiation. J Exp Med 1994;180:673–9. [17] Malissen M, Gillet A, Rocha B, Trucy J, Vivier E, Boyer C, et al. T cell development in mice lacking the CD3-/ gene. EMBO J 1993;12:4347–55. [18] Rocha B, von Boehmer H, Guy-Grand D. Selection of intraepithelial lymphocytes with CD8␣␣ co-receptors by self-antigen in the murine gut. Proc Natl Acad Sci USA 1992;89:5336–40. [19] Jordan MS, Boesteanu A, Reed AJ, Petrone AL, Holenbeck AE, Lerman MA, et al. Thymic selection of CD4+ CD25+ regulatory T cells induced by an agonist selfpeptide. Nat Immunol 2001;2:301–6. [20] Matsuda S, Kudoh S, Katayama S. Enhanced formation of azoxymethaneinduced colorectal adenocarcinoma in ␥␦ T lymphocyte-deficient mice. Jpn J Cancer Res 2001;92:880–5. [21] Inagaki-Ohara K, Chinen T, Matsuzaki G, Sasaki A, Sakamoto Y, Hiromatsu K, et al. Mucosal T cells bearing TCR␥␦ play a protective role in intestinal inflammation. J Immunol 2004;173:1390–8. [22] Poussier P, Ning T, Banerjee D, Julius M. A unique subset of self-specific intraintestinal T cells maintains gut integrity. J Exp Med 2002;195:1491–7. [23] Locke NR, Stankovic S, Funda DP, Harrison LC. TCR␥␦ intraepithelial lymphocytes are required for self-tolerance. J Immunol 2006;176:6553–9. [24] Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial ␥␦ T cells. Science 1998;279:1737–40. [25] Hayday A, Theodoridis E, Ramsburg E, Shires J. Intraepithelial lymphocytes: exploring the third way in immunology. Nat Immunol 2001;2:997–1003. [26] Bruno L, Rocha B, Rolink A, von Boehmer H, Rodewald HR. Intra- and extra-thymic expression of the pre-T cell receptor ␣ gene. Eur J Immunol 1995;25:1877–82. [27] Lambolez F, Azogui O, Joret AM, Garcia C, von Boehmer H, Di Santo J, et al. Characterization of T cell differentiation in the murine gut. J Exp Med 2002;195:437–49. [28] Laky K, Lefrancois L, Lingenheld EG, Ishikawa H, Lewis JM, Olson S, et al. Enterocyte expression of interleukin 7 induces development of ␥␦ T cells and Peyer’s patches. J Exp Med 2000;191:1569–80. [29] Bas A, Hammarstrom SG, Hammarstrom ML. Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression. J Immunol 2003;171:3359–71. [30] Eberl G, Sawa S. Opening the crypt: current facts and hypotheses on the function of cryptopatches. Trends Immunol 2010;31:50–5. [31] Suzuki K, Oida T, Hamada H, Hitotsumatsu O, Watanabe M, Hibi T, et al. Gut cryptopatches: direct evidence of extrathymic anatomical sites for intestinal T lymphopoiesis. Immunity 2000;13:691–702. [32] Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, Nariuchi H, et al. Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J Exp Med 1996;184:1449–59. [33] Rocha B. The extrathymic T-cell differentiation in the murine gut. Immunol Rev 2007;215:166–77. [34] Godfrey DI, Kennedy J, Suda T, Zlotnik A. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3− CD4− CD8− triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol 1993;150:4244–52.
[35] Saito H, Kanamori Y, Takemori T, Nariuchi H, Kubota E, Takahashi-Iwanaga H, et al. Generation of intestinal T cells from progenitors residing in gut cryptopatches. Science 1998;280:275–8. [36] Lambolez F, Rocha B. Molecular characterization of gut T cell precursors in euthymic and athymic mice. Adv Exp Med Biol 2001;495:15–24. [37] Naito T, Shiohara T, Hibi T, Suematsu M, Ishikawa H. ROR␥t is dispensable for the development of intestinal mucosal T cells. Mucosal Immunol 2008;1: 198–207. [38] Lin T, Matsuzaki G, Kenai H, Nakamura T, Nomoto K. Thymus influences the development of extrathymically derived intestinal intraepithelial lymphocytes. Eur J Immunol 1993;23:1968–74. [39] Lin T, Matsuzaki G, Yoshida H, Kobayashi N, Kenai H, Omoto K, et al. CD3− CD8+ intestinal intraepithelial lymphocytes (IEL) and the extrathymic development of IEL. Eur J Immunol 1994;24:1080–7. [40] Lambolez F, Arcangeli ML, Joret AM, Pasqualetto V, Cordier C, Di Santo JP, et al. The thymus exports long-lived fully committed T cell precursors that can colonize primary lymphoid organs. Nat Immunol 2006;7:76–82. [41] Sugahara S, Shimizu T, Yoshida Y, Aiba T, Yamagiwa S, Asakura H, et al. Extrathymic derivation of gut lymphocytes in parabiotic mice. Immunology 1999;96:57–65. [42] Peaudecerf L, Ribeiro Dos Santos P, Boudil A, Ezine S, Pardigon N, Rocha B. The role of the gut as a primary lymphoid organ: CD8␣␣ intraepithelial T lymphocytes in euthymic mice derive from very immature CD44+ thymocyte precursors. Mucosal Immunol 2010;4:93–101. [43] Petrie HT, Zuniga-Pflucker JC. Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annu Rev Immunol 2007;25:649–79. [44] Ciofani M, Knowles GC, Wiest DL, von Boehmer H, Zuniga-Pflucker JC. Stagespecific and differential notch dependency at the ␣ and ␥␦ T lineage bifurcation. Immunity 2006;25:105–16. [45] Livak F, Tourigny M, Schatz DG, Petrie HT. Characterization of TCR gene rearrangements during adult murine T cell development. J Immunol 1999;162:2575–80. [46] Capone M, Hockett Jr RD, Zlotnik A. Kinetics of T cell receptor  ␥, and ␦ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+ CD25+ Pro-T thymocytes. Proc Natl Acad Sci USA 1998;95:12522–7. [47] Luci C, Reynders A, Ivanov II, Cognet C, Chiche L, Chasson L, et al. Influence of the transcription factor ROR␥t on the development of NKp46+ cell populations in gut and skin. Nat Immunol 2009;10:75–82. [48] Eberl G, Littman DR. Thymic origin of intestinal ␣ T cells revealed by fate14 mapping of ROR␥t+ cells. Science (New York, NY) 2004;305:248–51. [49] Egawa T, Kreslavsky T, Littman DR, von Boehmer H. Lineage diversion of T cell receptor transgenic thymocytes revealed by lineage fate mapping. PLoS One 2008;3:e1512. [50] Villey I, de Chasseval R, de Villartay JP. ROR␥T, a thymus-specific isoform of the orphan nuclear receptor ROR␥/TOR, is up-regulated by signaling through the pre-T cell receptor and binds to the TEA promoter. Eur J Immunol 1999;29:4072–80. [51] Gangadharan D, Lambolez F, Attinger A, Wang-Zhu Y, Sullivan BA, Cheroutre H. Identification of pre- and postselection TCR␣+ intraepithelial lymphocyte precursors in the thymus. Immunity 2006;25:631–41. [52] Baldwin TA, Sandau MM, Jameson SC, Hogquist KA. The timing of TCR ␣ expression critically influences T cell development and selection. J Exp Med 2005;202:111–21.
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Review
Role of the gut as a primary lymphoid organ Laetitia Peaudecerf ∗ , Benedita Rocha INSERM, U1020, Medical Faculty Descartes Paris V, 75015 Paris, France
a r t i c l e
i n f o
Article history: Received 17 January 2011 Received in revised form 11 May 2011 Accepted 23 May 2011 Available online 16 June 2011 Keywords: Gut CD8␣␣ intraepithelial T lymphocyte T cell differentiation Precursor export
s u m m a r y The TCR-␣/␥␦ CD8␣␣ intraepithelial T lymphocytes (T-IEL) located in the gut mucosa of the small intestine are an abundant population believed to have a major role in ensuring the integrity of the gut wall. Here, we describe their unique characteristics and the controversies regarding the origin and differentiation of these T-IELs. We show how accumulated experimental evidence has finally arrived at a unifying concept, which demonstrates that these cells originate from early thymus precursors that have not yet undergone TCR rearrangement and TCR-␣/␥␦ commitment. These precursors colonize the gut lamina propria during the perinatal period and complete rearrangements and TCR-␣/␥␦ commitment while migrating to the epithelium. Therefore, the gut epithelium, which shares the same embryonic origin as the thymus epithelium, behaves as a primary lymphoid organ responsible for the differentiation of a major local T cell set. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The gut mucosa is a major site of interaction between the almost sterile environment of the body and the outside world. The oral route is one of the pathways used most frequently by infectious agents to penetrate the body. Moreover, the gut harbors abundant microbiota, including the commensal bacteria that have a fundamental role in ensuring our survival through food processing, the production of some essential elements like vitamin K, and by keeping potentially pathological bacteria in check. The gut environment thus faces the unique challenge of preserving the subtle balance between immune responses and tolerance. While elsewhere in the body tolerance must be maintained against self antigens only, in the gut environment, tolerance must also be developed against abundant exogenous antigens—food antigens as well as the local microbiota. In addition, although microbiota are innocuous and useful when present in the lumen, they must not penetrate into the body, where they could cause septic shock and death. One of the major requirements of the local environment is thus the maintenance of epithelial barrier integrity. Local protection is ensured by the most abundant effector T cell compartment in the body: the gut intraepithelial T lymphocytes (T-IEL).
lumen, mucins, and the far more abundant epithelial cells, which rapidly die in vitro, forming clumps in which T-IEL are also trapped. Although we have attempted several methods to improve isolation procedures, we found that initial techniques involving passage through glass wool columns followed by separation in density gradients were optimal for obtaining T-IEL isolates; however, these techniques lead to variable and important losses. Therefore, the most reliable way to evaluate T-IEL recovery is to count individual T-IEL in tissue sections in adult mice bred in Specific Pathogen Free (SPF) conditions. Since the number, the size of villi, and the number of T-IEL vary in different portions of the small intestine, we examine the whole small intestine, divided into portions 1 cm in length. We determine the total number of villi present in each fragment from the number of villi in 1 cm long sections and the number of villi present at the perimeter of the gut in each location. We next determine the number of epithelial cells present in each villus by counting epithelial cells present along the length and at the perimeter of individual villi. Finally, we determine the T-IEL/epithelial cell ratio at each location. These results allow us to estimate the total number of T-IEL, which we found was comparable to the total number of T cells located in all the conventional peripheral lymphoid organs, i.e. lymph nodes plus the spleen [1].
2. Quantification of T-IEL
3. Different types of gut epithelium T-IEL
Recovery of T-IEL is hindered by multiple technical problems. Isolates are contaminated with bacteria, detritus located in the gut
Two main categories of T-IEL can be distinguished in adult mice. One population originates from peripheral T cells; after responding to antigens in the Peyer patches or the mesenteric lymph nodes, these cells up-regulate expression of ␣47 integrin and chemokine receptor CCR9 and home to the gut mucosa [2–4]. These conventional T-IEL have the phenotype of peripheral antigen-experienced
∗ Corresponding author. Tel.: +33 1 40 61 53 61. E-mail address: [email protected] (L. Peaudecerf). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.05.009
2
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
cells; i.e. TCR␣+ Thy1+ CD5+ LFA-1+ CD28+ CD44+ , and they are CD4+ or CD8␣+ . The other subpopulation is specific to the gut wall. In mice, but not in other species, they are commonly called CD8␣␣ T-IEL, because these cells are CD4− CD8− and frequently express the CD8␣␣ homodimeric form of the CD8 coreceptor [5]. Although CD8␣␣ expression is not specific to T-IEL, (CD8␣␣ is expressed by a subclass of dendritic cells [DC] and by highly activated peripheral T cells [6]), these unconventional cells have globally unique properties that distinguish them from all the other T cell types present elsewhere. They are TCR-␥␦ or TCR-␣ and CD5− CD28− LFA-1− , with only a fraction expressing Thy1 [7–9]. A major fraction expresses B220, and CD11c is also expressed, although elsewhere these markers are considered to be restricted to the B and DC lineages, respectively [10,11]. They are NK1.1− but can express several alternative NK receptors [12]. Their repertoire is quite pauciclonal; they result from the major expansion of a few clones [13,14]. These cells show no evidence of having ever expressed CD8. Indeed, TCR-␣+ cells that differentiate in the thymus go through a CD4+ CD8␣+ (DP) stage where they demethylate cytosines in the CD8 gene 5 regulatory region. This epigenetic modification is maintained in all conventional T cells; the CD8 locus remains demethylated even in peripheral CD4+ T cells. In contrast, the CD8 locus is methylated in both CD8␣␣ T-IEL and their precursors [15]. Finally, the CD3 complex of T-IEL has a peculiar composition, as it contains CD3- -FcRI␥ heterodimers or FcRI␥ -FcRI␥ homodimers instead of the CD3- -CD3- homodimers that characterize thymus-derived cells [16,17]. In adult mice in SPF conditions, CD8␣␣ constitute about half of the total T-IEL, and this proportion is maintained as the mice age. However, in situations where mice are kept in conventional animal houses, or in humans who undergo multiple infections, the proportion of nonconventional TIEL declines with age. It is not known if such decreases correspond to a reduction in their absolute numbers, or to a dilution due to the accumulation of conventional T-IEL.
4. CD8␣␣ T-IEL repertoires are generated by agonist selection Before CD8␣␣ T-IEL were studied, two mechanisms were described to determine the selection of T cell repertoires: negative selection, which purges the T cell repertoires of self-reactive cells, and positive selection, which ensures that T cells that differentiate in the thymus are restricted to self MHC class I or class II, and express CD8␣ or CD4, respectively. Negative selection was first identified in mice expressing endogenous Mls superantigens. These superantigens activate T cells expressing TCRs with particular V chains, like V6 or V11. Their expression as self antigens induces the deletion of V6- and V11-expressing cells in the thymus and in CD8␣+ and CD4+ T-IEL, but not in TCR-␣+ CD8␣␣ T-IEL [1]. These results demonstrated that CD8␣␣ T-IEL do not undergo negative selection. The subsequent study of mice expressing a TCR transgenic (Tg) specific to the male antigen revealed that while conventional T-IEL were deleted in male mice, TCR-␣+ CD8␣␣ T-IEL specific to the male antigen were highly enriched [18]. Conversely, TCR-␣+ CD8␣␣ T-IEL male-specific T cells were absent in female mice. This led to the surprising conclusion that instead of being deleted by the presence of self antigens, as is the case for CD8␣ and CD4T cells, CD8␣␣ T-IEL require the presence of self antigens to be generated. It was then proposed that this phenomenon could be due to the TCR/CD8/CD3 complex that is unique to TCR-␣+ CD8␣␣ T-IEL. The reduced number of ITAM motifs present in the FcRI␥, chain as well as the absence of CD8, would reduce the intensity of the TCR-mediated signals. Under these circumstances, rather than inducing cell death, signals induced by self antigens would function as survival signals. Decades later, this selection mechanism, called
agonist selection, was shown to control selection of the repertoire of TCR-␣ CD4+ CD25+ FOXP3+ regulatory T cells [19]. 5. Functions of CD8␣␣ T-IEL Because CD8␣␣ T-IEL are selected by self antigens, their function has been suggested to be in the transition between innate and adaptive responses, mainly directed toward control of the integrity of the gut wall. Thus, TCR-␥␦ – deficient mice are susceptible to mutagen – induced local carcinomas [20] and to localized inflammation by toxic chemicals [21]. TCR-␣+ CD8␣␣ T-IEL were shown to mimic the role of CD4+ CD25+ regulatory T cells in preventing the colitis induced by injection of CD4+ naïve T cells into T cell – deficient mice; this regulation was demonstrated to be mediated by IL-10 [22]. Moreover, these cells were also shown to promote the generation of CD4+ CD25+ regulatory T cells against orally presented autoantigens and were reported to have a major immunoregulatory role by preventing the development of autoimmune diabetes in NOD mice [23]. It must be noted, however, that the functional role of CD8␣␣ T-IEL has been studied mostly in mice; few studies have been performed in other species. However, human IEL were shown to kill carcinoma cell lines in vitro [24], and celiac disease is marked by a major accumulation of unconventional TIEL, which appears to contribute to reducing the local inflammation induced by antigen-specific conventional T cells [25]. 6. CD8␣␣ T-IEL development: evidence for an extrathymic origin The multiple peculiarities of CD8␣␣ T-IEL that distinguish them from peripheral thymus-derived cells suggest that they do not follow a classic T cell differentiation pathway. Several independent observations have led to the notion that these cells could be generated within the gut wall. The adaptative immune system appears in the gastrointestinal region of primitive vertebrates before the emergence of the thymus. In contrast with other epithelia, the gut epithelium shares a common endodermic origin with the liver and the thymus epithelia, and thus may express common factors that support hematopoiesis in general and T cell lymphopoiesis in particular. These cells are present in nude mice that have a mutation in the Foxn1 gene, which is essential for thymopoiesis [7]. Several genes specific to T lymphopoiesis, such as Rag1 (Rag, recombination-activating gene), CD3ε, and pT˛, were detected in lineage marker – negative (Lin− ) cells directly isolated from the gut wall of normal euthymic mice [16,26,27]. Finally, it was formally demonstrated that TCR-␥␦ T-IEL could rearrange their TCRs in situ. Indeed, in IL-7 – deficient mice, TCR-␥␦ T cells cannot develop in the thymus or elsewhere. However, the selective expression of IL-7 in the gut epithelium reconstitutes TCR-␥␦ T-IEL, since outside the gut, TCR-␥␦ T cells remain absent [28]. These results indicate that TCR-␥␦ rearrangements should occur directly within the gut wall. It must be noted that CD8␣␣ T-IEL were mostly studied in mice. However, Rag expression was also detected in Lin− cells isolated from the human intestine [29]. Research into extrathymic T cell generation in the gut changed markedly with the description of “thymus-like structures” that are scattered throughout the lamina propria (LP) at the base of some gut crypts. These small aggregates called cryptopatches (CPs) contain Lymphoid inducer cells (Lti) and further complex mixtures of Lin− populations expressing different levels of c-kit, IL-7R and ROR␥t, many of them not yet fully characterized, and were also proposed to be precursors of isolated lymphoid follicles [30]. However, CP structures also harbor T cell precursors since when Lin− cells were isolated directly from CP by micromanipulation, they showed evidence of ongoing TCR rearrangements and were able to generate
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
T-IEL after adoptive transfer [27,31,32]. However, the majority of studies on local differentiation were not performed after direct CP isolation; instead, they used Lin− cells isolated either from the LP or from the epithelium. The study of cell surface and molecular markers, TCR rearrangements, the kinetics of reconstitution, and the impact of different mutations affecting T cell development led to the establishment of a sequence of T cell development in the gut of euthymic mice, as described in [27] and reviewed in detail in [33]. Briefly, this sequence is initiated in Lin− precursors that are located in the LP and are Thy1+ IL-7R+ CD25int CD44+ c-kit+/− . Thus, they have the phenotype of triple negative ([TN] CD3− CD4− CD8␣− ) TN1-to-TN2 transition thymocytes [34]. This population expresses Rag and pTa; the Rag mutation blocks local differentiation at this stage. Injection of these precursors into irradiated nude mice reconstitutes the euthymic CD8␣␣ T-IEL compartment. These precursors migrate from the LP to the epithelium [35], where they give rise to an intermediate c-kit− Thy1+ B220+ CD8␣␣+ population. These cells show complete TCR- rearrangements and ␥5 rearrangements, but no TCR-␣ rearrangements. The TCR-␣ mutation blocks differentiation at this stage. Finally, these precursors generate Lin− B220+ CD8␣␣+ cells that lose Thy1 expression and complete the rearrangements of the TCR-␣ chain. The multiple characteristics of the gut differentiation process in euthymic mice could explain the differences between the T cell repertoires generated in the thymus and in the gut. Indeed, compared to the thymus, pT␣ is less expressed in the gut precursors [27], which may account for the relative local abundance of TCR-␥␦ cells. Rag1 expression frequency is also lower in the gut compared to the thymus, which could explain the pauciclonality of CD8␣␣ T-IEL repertoires. Indeed, because rearrangements would be relatively rare, repertoires in the gut would result from the expansion of relatively few clones [27]. 7. CD8␣␣ T-IEL development: differences between euthymic and athymic mice Although CD8␣␣ T-IEL can be generated in athymic mice, in the absence of a thymus, the pool of CD8␣␣ T-IEL is reduced 5–10-fold, TCR-␣ CD8␣␣ T-IEL are rare, and the majority of CD8␣␣ T cells do not express Thy1. Moreover, the molecular characteristics of Lin− precursors located in the gut in nude mice are rather different from those found in euthymic mice [36]. Although the phenotype and relative location (LP or epithelium) of these precursors is similar, Lin− cells isolated either from the LP from micromanipulated CPs or from the epithelium show less-advanced TCR rearrangements compared to the subpopulations with equivalent phenotypes recovered from euthymic mice [36,37]. Thus, in nude mice, complete TCR- rearrangements are not detected the Lin− LP cells, and the rearrangements of the TCR- and TCR-␣ chain are less extensive in Lin− subpopulations recovered from the nude epithelium. Moreover, although pT˛ is expressed [26], expression frequencies are quite low (Rocha, unpublished observations). All of these results indicate that the presence of the thymus has a major influence on the local differentiation process. 8. CD8␣␣ T-IEL development: evidence for a thymic origin It is well known that seeding of the CD8␣␣ T-IEL compartment is a postnatal event. At birth, these cells are virtually undetectable, but reach normal numbers by 3 wk of age. It is generally accepted that in euthymic mice, this wave of colonization has a thymic origin. Injection of bone marrow cells from normal mice into athymic mice leads to the generation of CD8␣␣ T-IEL in the same proportion as in nude mice [31]. By contrast, neonatal thymus transplants reconstitute the CD8␣␣ T-IEL compartment in the same proportion as in
3
normal mice; these cells originate from the thymus graft [38–40]. Migration from the thymus to the gut appears to be restricted to a short time period after birth. Thus, thymectomy only affects the CD8␣␣ T-IEL pool when performed in the perinatal period. From 3 wk after birth, thymectomy does not affect CD8␣␣ T-IEL numbers or composition [38]. Moreover, parabiosis experiments show an absence of CD8␣␣ T-IEL chimerism between adult partners [41]. Overall, these results demonstrate that the influence of the thymus on CD8␣␣ T-IEL generation is confined to a very narrow perinatal window. 9. CD8␣␣ T-IEL development: role of the thymus While it is generally accepted that CD8␣␣ T-IEL are thymus derived in euthymic mice, the differentiation stage at which these thymocytes exit the thymus and colonize the gut is the subject of debate. Two hypotheses have been proposed: (A) CD8␣␣ T-IEL originate from T cell – committed immature thymocyte precursors that have not yet rearranged their TCRs. These precursors colonize the gut LP and finish their differentiation in situ. This hypothesis reconciles a thymic origin with an extrathymic differentiation process. (B) CD8␣␣ T-IEL originate from more mature thymocytes that rearrange their TCRs in the thymus and acquire the particular phenotypes of CD8␣␣ T-IEL after colonization of the gut wall. These two theories are not mutually exclusive; both are explored below in further detail. 9.1. CD8˛˛ T-IEL originate from T cell–committed immature thymocyte precursors We studied the capacity of different thymocyte populations to generate CD8␣␣ T-IEL after adoptive transfer and found that only CD4− CD8− CD3− TN thymocyte sets had that potential. However, TN populations are mixtures of several precursor populations at different differentiation stages. It is well established that early precursors have the CD44+ CD25− phenotype (TN1) and have not yet rearranged their TCRs. These cells give rise to the CD44+ CD25+ TN2 population, which it is not yet fully committed to T cell differentiation, since it is still able to generate NK cells. These cells have incomplete TCR- DJ rearrangements and most are not yet committed to either TCR-␣ or TCR-␥␦ lineages, because these events occur at the more mature CD44− CD25+ TN3 stage of differentiation. Complementary evidence allowed us to determine at what stage of differentiation the TN precursors could leave the thymus and give rise to CD8␣␣ T-IEL. Only CD44+ TN cells fully reconstituted the CD8␣␣ T-IEL compartment with a Thy1+/− phenotype and the TCR-␣/TCR-␥␦ representation seen in euthymic mice [42]. After thymus transplant, we could find early precursors of thymic origin in the blood and in the gut LP [42]. Most of the Lin− precursors detected in the blood corresponded to a previously uncharacterized cell type in the transition between the CD44+ CD25− (TN1) and CD44+ CD25+ (TN2) thymocyte sets (CD44+ CD25int TN1–TN2 transitional cells). These precursors expressed a high frequency of Rag1 [42]. Direct studies of immature TN subsets isolated from the neonatal thymus also showed that the TN1–TN2 subset had the potential to leave the thymus and colonize the gut. We showed that during the TN1–TN2 transition, thymocytes strongly upregulate the expression of ␣47 integrin [42] and also express CCR9 (unpublished), both of which are required for homing to the gut. They also express a high frequency of S1p1, which codes for the receptor of sphingosine-1-phosphate, which is required for thymus egress [42]. Moreover, the location of TN1–TN2 transitional cells in the thymus also favors egress, which is described as taking place through capillaries located at the corticomedullary junction. Indeed, TN1 cells are present around this boundary and must cross
4
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
this zone to reach the inner cortex where TN2 cells reside [43]. Therefore, CD25int TN1–TN2 transition cells are likely present very close to the capillaries leaving the thymus. Thus, both the characterization of TN migrants in the blood after thymus transplantation and the direct study of thymocyte population properties indicate that the bulk of TN egress should occur during the TN1–TN2 transition. Finally, we also observed that transplants of Rag− thymus (which only harbor TN1–TN3 subsets) reconstituted CP-like structures in the gut, and all individual cells from these structures expressed CD3ε [40]. Although these experiments did not allow us to determine which TN subpopulation colonizes the gut, they did show that early precursors can leave the thymus before TCR rearrangements. They also create a direct link between gut colonization by thymus precursors and CP-like structures. It must be noted that within the CPs, thymus-derived precursors are only a minority population; several other cell types are also present (discussed below). The identification of the TN1–TN2 subset as responsible for gut colonization allows us to reconcile a thymic origin with a gut T cell-differentiation process. Single-cell cultures revealed that all TN1 cells and about 50% of TN2 cells have the capacity to generate both TCR-␣ and TCR-␥␦ cells [44]; thus, it is likely that at least the majority of these TN1–TN2 cells are bipotential. Moreover, these cells have probably not rearranged their TCRs, since TCR rearrangements are absent in the TN1 set and only rare and incomplete in the TN2 set [45,46]. Therefore, CP colonization precedes TCR-␣/TCR-␥␦ commitment and rearrangements that will occur in the gut. Finally, TN1 and TN2 cells have the potential to generate NK cells, and it was recently described that the CPs could be involved in the generation of a unique population of NK cells in the gut: the IL-22 – producing NKp46+ cells [47]. It remains to be determined if these cells may also derive from TN precursors. 9.2. CD8˛˛ T-IEL TCR-˛ˇ+ IEL originate from mature thymocytes that acquire particular phenotypes locally 9.2.1. Genetic labeling and genetic fate experiments The notion that TCR-␣+ CD8␣␣ T-IEL may originate from more mature thymocyte precursors arose from genetic labeling and genetic fate experiments. In mice expressing green fluorescent protein (GFP) under the promoter of the nuclear hormone receptor ROR␥t, both DP thymocytes and CP cells are GFP+ , but TCR-␣+ CD8␣␣ T-IEL are not. Elimination of ROR␥t expression in these mice induced the apoptosis of DP thymocytes, CP cells, and TCR-␣+ CD8␣␣ T-IEL. Although restoration of DP thymocyte survival by the anti-apoptotic factor Bcl-xl led to the reconstitution of the TCR-␣+ CD8␣␣ T-IEL pool, CP cells were not detected [48]. The authors claimed that Lin− c-kit+ IL-7R+ CP precursors were aggregates of lymphoid inducer cells (Lti) that were not required for TCR-␣+ CD8␣␣ T-IEL generation. However, in another study that used the same mice but directly isolated the CPs, ROR␥t deficiency appeared to eliminate just a fraction of Lin− c-kit+ IL-7R+ precursors and CP structures and had no effect on TCR-␣+ CD8␣␣ T-IEL generation [37]. The reasons for such discrepancies are unclear, since the same mice were used in both studies. Using ROR␥t promoter – driven Cre recombinase to induce yellow fluorescent protein (YFP) expression, it was shown that, in contrast to TCR-␥␦+ CD8␣␣ T-IEL, the majority of TCR-␣+ CD8␣␣ T-IEL expressed ROR␥t during their development [49]. Because YFP was detected in DP thymocytes, the authors also concluded that TCR-␣+ CD8␣␣ T-IEL had passed through the DP stage. These types of experiments are elegant and attractive, but may lead to serious overinterpretations (Fig. 1). In this particular case, the expression of YFP in both DP thymocytes and TCR-␣+ CD8␣␣ T-IEL does not prove that they are directly related: ROR␥t expression is part of the pre-TCR activation cascade during -selection [50]. Since Lin− cells isolated from the epithe-
Fig. 1. Possible relationships between two genetically labeled cells. Upper graphs: cells are directly related. Middle graphs: cells derived from a common precursor. Lower graphs: cells are not related. The gene is expressed independently in two different lineages. Genetic labeling and genetic fate experiments alone do not discriminate between these three possibilities. Relationships must be confirmed by complementary classic approaches to determining precursor/product relationships. Modified from Rocha [33].
lium express the pT␣ chain and undergo -selection [27], the YFP detected in TCR-␣+ CD8␣␣ T-IEL could be induced directly during T cell differentiation in the gut wall. The notion that CP cells are simply aggregates of Lti cells was reviewed extensively when a more-detailed analysis of CP composition was conducted. After direct isolation of CP aggregates by micromanipulation, these aggregates were found to contain multiple cell types that could be distinguished by different levels of c-kit, IL-7R, and ROR␥t expression. In fact, only 10% of the CP cells have the CD4+ c-kithi IL-7Rhi ROR␥thi phenotype typical of Lti [37]. CPs also harbor a population of NK cells with the unusual NKp46+ IL-7R+ ROR␥t+ phenotype. Moreover, while in early studies, ROR␥t expression was thought to be limited to Lti and immature thymocytes, it is now known that this gene is expressed by cells from other lineages (including certain TCR-␥␦ populations and IL-17 expressing cells), preventing the use of this marker to define possible lineage relationships with either Lti or other ROR␥t+ cells also present in the gut LP. Thus, the complexity of CP subpopulations and the lineage relationships between their subpopulations and the other cell types present in the gut LP and also expressing ROR␥t+ remains to be established. 9.2.2. Binding to thymus-leukemia antigen tetramers TCR-␣+ CD8␣␣ T-IEL have also been claimed to be derived from a DP population that, like IEL, expresses CD8␣␣. This minority of DP cells is identified by the specific ability of CD8␣␣ homodimers to bind tetramers of the thymus leukemia antigen (TL-tet) [51]. This cell set would be agonist selected in the thymus cortex, giving rise to agonist-selected NK1.1− TCR-␣+ DN cells that would reconstitute TCR-␣+ CD8␣␣ T-IEL in the gut. Thus, when TL-tet+ DP cells expressing the self-reactive anti-HY TCR-␣ transgene were stimulated in vitro with the HY peptide, they generated transgenic (Tg) TCR-␣+ DN or CD8␣␣+ cells, and intravenous injection of NK1.1− TCR-␣+ DN thymocytes into Rag-2−/− mice reconstituted the CD8␣␣ T-IEL compartment in the gut [51]. However, these experiments used mice in which the Tg TCR-␣ chain was expressed under the control of the TCR- promoter, resulting in a precocious and very abnormal expression of the transgenic TCR-␣ already at the TN2 stage [52]. By contrast, other experiments in which the Tg TCR-␣ chain was inserted in the TCR-␣ locus, resulting in normal expression of the TCR-␣ transgene in DP cells, suggested that self-reactive DP thymocytes did not give rise to CD8␣␣ T-IEL. Indeed, in these male mice, in spite of the vast opportunity to generate agonist-selected DP thymocytes, there is no more Tg TCR-␣+
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
5
Thus, when immature precursors are prevented from colonizing the gut, CD8␣␣ T-IEL generation is virtually abrogated [42]. These experiments demonstrate that in euthymic mice, reconstitution of the CD8␣␣ T-IEL compartment during the first 3 wk after birth depends upon the colonization of the gut by very immature thymocyte precursors.
11. Concluding remarks The origin of CD8␣␣ T-IEL has been the subject of much debate. The identification of early TN subsets as responsible for CD8␣␣ T-IEL generation allows us to reconcile a thymic origin with an extrathymic T cell – differentiation process, and to confer to the gut a major role as a primary lymphoid organ. Thus, the thymus is fundamental for a first imprinting of the gut precursors, since both the gut precursors and CD8␣␣ T-IEL are very different in athymic and euthymic mice. The precursors of the gut Lin− cells leave the thymus and colonize the gut during the perinatal period. These thymocytes have a restricted T and NK potential, TCR rearrangements are rare and incomplete, and TCR-␣/␥␦ lineage commitment has not yet occurred. Lin− c-kit+ IL-7R+ cells recovered from the gut LP of euthymic mice share the same molecular characteristics and finish TCR rearrangements and TCR-␣/␥␦ commitment after migration from the LP to the gut epithelium. However, CD8␣␣ T-IEL precursors are only a minority population within Lin− c-kit+ IL-7R+ cells in the LP. The complexity of these populations and the additional roles that various Lin− subsets may have in local physiology and pathology have yet to be further explored. Fig. 2. The relative role of TN and other thymocyte populations in CD8␣␣ T-IEL generation.
References CD8␣␣ T-IEL than in female mice [52]. Moreover, the expression of CD8␣␣ homodimers in a minority of DP cells was never directly demonstrated, and our recent data do not support this differentiation pathway. We showed that TL-tet binding is not CD8␣␣ specific, since these tetramers fail to bind many CD8␣␣ cells while adhering to several populations expressing CD8␣. Moreover, we demonstrated that the vast majority of the TCR-␣+ DN population is depleted of self-reactive cells and thus cannot give rise to the self-reactive, agonist-selected CD8␣␣ T-IEL pool [42]. 10. The relative roles of “immature” versus “mature” thymocytes in T-IEL generation To investigate the relative contribution of TN cells versus the more mature thymocytes in T-IEL generation, we transplanted neonatal thymi into different mice and followed the gut colonization by thymocytes originating from the transplant. Hosts were either Rag-2/␥c−/− or Rag-2−/− mice. The former mimic the conditions found in the postnatal period (both Lin− c-kit+ IL-7R+ precursors and the absence of T cells in the gut), while Rag-2−/− mice have an abundant Lin− c-kit+ IL-7R+ population but lack mature T cells. We reasoned that if CD8␣␣ T-IEL originate from immature precursors, the presence of a full niche of Lin− c-kit+ IL-7R+ cells in the gut of Rag-2−/− mice should hinder precursor entry and CD8␣␣ T-IEL generation. By contrast, if either TCR-␣ or TCR-␥␦ derive from more mature thymocyte sets, the reconstitution of the CD8␣␣ T-IEL pool should be similar in both host mice, since both lack more mature T cells [42] (Fig. 2). We found that the presence of local precursors in Rag-2−/− mice blocked CD8␣␣ T-IEL reconstitution. By contrast, CD4+ /CD8␣+ T-IEL (known to derive from mature thymocytes) colonized the gut of both mouse strains similarly, demonstrating the efficiency of the thymus transplantation.
[1] Rocha B, Vassalli P, Guy-Grand D. The V repertoire of mouse gut homodimeric ␣ CD8+ intraepithelial T cell receptor ␣/+ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J Exp Med 1991;173:483–6. [2] Koseki S, Miura S, Fujimori H, Hokari R, Komoto S, Hara Y, et al. In situ demonstration of intraepithelial lymphocyte adhesion to villus microvessels of the small intestine. Int Immunol 2001;13:1165–74. [3] Svensson M, Marsal J, Ericsson A, Carramolino L, Broden T, Marquez G, et al. CCL25 mediates the localization of recently activated CD8␣+ lymphocytes to the small-intestinal mucosa. J Clin Invest 2002;110:1113–21. [4] Guy-Grand D, Vassalli P. Gut intraepithelial lymphocyte development. Curr Opin Immunol 2002;14:255–9. [5] Rocha B, Guy-Grand D, Vassalli P. Extrathymic T cell differentiation. Curr Opin Immunol 1995;7:235–42. [6] Madakamutil LT, Christen U, Lena CJ, Wang-Zhu Y, Attinger A, Sundarrajan M, et al. CD8␣␣-mediated survival and differentiation of CD8 memory T cell precursors. Science (New York, NY) 2004;304:590–3. [7] Guy-Grand D, Cerf-Bensussan N, Malissen B, Malassis-Seris M, Briottet C, Vassalli P. Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med 1991;173:471–81. [8] Lefrancois L. Phenotypic complexity of intraepithelial lymphocytes of the small intestine. J Immunol 1991;147:1746–51. [9] Ohteki T, MacDonald HR. Expression of the CD28 costimulatory molecule on subsets of murine intestinal intraepithelial lymphocytes correlates with lineage and responsiveness. Eur J Immunol 1993;23:1251–5. [10] Barrett TA, Gajewski TF, Danielpour D, Chang EB, Beagley KW, Bluestone JA. Differential function of intestinal intraepithelial lymphocyte subsets. J Immunol 1992;149:1124–30. [11] Huleatt JW, Lefrancois L. Antigen-driven induction of CD11c on intestinal intraepithelial lymphocytes and CD8+ T cells in vivo. J Immunol 1995;154:5684–93. [12] Guy-Grand D, Cuenod-Jabri B, Malassis-Seris M, Selz F, Vassalli P. Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages. Eur J Immunol 1996;26:2248–56. [13] Regnault A, Cumano A, Vassalli P, Guy-Grand D, Kourilsky P. Oligoclonal repertoire of the CD8␣␣ and the CD8␣ TCR-␣/ murine intestinal intraepithelial T lymphocytes: evidence for the random emergence of T cells. J Exp Med 1994;180:1345–58. [14] Regnault A, Levraud JP, Lim A, Six A, Moreau C, Cumano A, et al. The expansion and selection of T cell receptor ␣ intestinal intraepithelial T cell clones. Eur J Immunol 1996;26:914–21. [15] Hamerman JA, Page ST, Pullen AM. Distinct methylation states of the CD8 gene in peripheral T cells and intraepithelial lymphocytes. J Immunol 1997;159:1240–6.
6
L. Peaudecerf, B. Rocha / Immunology Letters 140 (2011) 1–6
[16] Guy-Grand D, Rocha B, Mintz P, Malassis-Seris M, Selz F, Malissen B, et al. Different use of T cell receptor transducing modules in two populations of gut intraepithelial lymphocytes are related to distinct pathways of T cell differentiation. J Exp Med 1994;180:673–9. [17] Malissen M, Gillet A, Rocha B, Trucy J, Vivier E, Boyer C, et al. T cell development in mice lacking the CD3-/ gene. EMBO J 1993;12:4347–55. [18] Rocha B, von Boehmer H, Guy-Grand D. Selection of intraepithelial lymphocytes with CD8␣␣ co-receptors by self-antigen in the murine gut. Proc Natl Acad Sci USA 1992;89:5336–40. [19] Jordan MS, Boesteanu A, Reed AJ, Petrone AL, Holenbeck AE, Lerman MA, et al. Thymic selection of CD4+ CD25+ regulatory T cells induced by an agonist selfpeptide. Nat Immunol 2001;2:301–6. [20] Matsuda S, Kudoh S, Katayama S. Enhanced formation of azoxymethaneinduced colorectal adenocarcinoma in ␥␦ T lymphocyte-deficient mice. Jpn J Cancer Res 2001;92:880–5. [21] Inagaki-Ohara K, Chinen T, Matsuzaki G, Sasaki A, Sakamoto Y, Hiromatsu K, et al. Mucosal T cells bearing TCR␥␦ play a protective role in intestinal inflammation. J Immunol 2004;173:1390–8. [22] Poussier P, Ning T, Banerjee D, Julius M. A unique subset of self-specific intraintestinal T cells maintains gut integrity. J Exp Med 2002;195:1491–7. [23] Locke NR, Stankovic S, Funda DP, Harrison LC. TCR␥␦ intraepithelial lymphocytes are required for self-tolerance. J Immunol 2006;176:6553–9. [24] Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial ␥␦ T cells. Science 1998;279:1737–40. [25] Hayday A, Theodoridis E, Ramsburg E, Shires J. Intraepithelial lymphocytes: exploring the third way in immunology. Nat Immunol 2001;2:997–1003. [26] Bruno L, Rocha B, Rolink A, von Boehmer H, Rodewald HR. Intra- and extra-thymic expression of the pre-T cell receptor ␣ gene. Eur J Immunol 1995;25:1877–82. [27] Lambolez F, Azogui O, Joret AM, Garcia C, von Boehmer H, Di Santo J, et al. Characterization of T cell differentiation in the murine gut. J Exp Med 2002;195:437–49. [28] Laky K, Lefrancois L, Lingenheld EG, Ishikawa H, Lewis JM, Olson S, et al. Enterocyte expression of interleukin 7 induces development of ␥␦ T cells and Peyer’s patches. J Exp Med 2000;191:1569–80. [29] Bas A, Hammarstrom SG, Hammarstrom ML. Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression. J Immunol 2003;171:3359–71. [30] Eberl G, Sawa S. Opening the crypt: current facts and hypotheses on the function of cryptopatches. Trends Immunol 2010;31:50–5. [31] Suzuki K, Oida T, Hamada H, Hitotsumatsu O, Watanabe M, Hibi T, et al. Gut cryptopatches: direct evidence of extrathymic anatomical sites for intestinal T lymphopoiesis. Immunity 2000;13:691–702. [32] Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, Nariuchi H, et al. Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J Exp Med 1996;184:1449–59. [33] Rocha B. The extrathymic T-cell differentiation in the murine gut. Immunol Rev 2007;215:166–77. [34] Godfrey DI, Kennedy J, Suda T, Zlotnik A. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3− CD4− CD8− triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol 1993;150:4244–52.
[35] Saito H, Kanamori Y, Takemori T, Nariuchi H, Kubota E, Takahashi-Iwanaga H, et al. Generation of intestinal T cells from progenitors residing in gut cryptopatches. Science 1998;280:275–8. [36] Lambolez F, Rocha B. Molecular characterization of gut T cell precursors in euthymic and athymic mice. Adv Exp Med Biol 2001;495:15–24. [37] Naito T, Shiohara T, Hibi T, Suematsu M, Ishikawa H. ROR␥t is dispensable for the development of intestinal mucosal T cells. Mucosal Immunol 2008;1: 198–207. [38] Lin T, Matsuzaki G, Kenai H, Nakamura T, Nomoto K. Thymus influences the development of extrathymically derived intestinal intraepithelial lymphocytes. Eur J Immunol 1993;23:1968–74. [39] Lin T, Matsuzaki G, Yoshida H, Kobayashi N, Kenai H, Omoto K, et al. CD3− CD8+ intestinal intraepithelial lymphocytes (IEL) and the extrathymic development of IEL. Eur J Immunol 1994;24:1080–7. [40] Lambolez F, Arcangeli ML, Joret AM, Pasqualetto V, Cordier C, Di Santo JP, et al. The thymus exports long-lived fully committed T cell precursors that can colonize primary lymphoid organs. Nat Immunol 2006;7:76–82. [41] Sugahara S, Shimizu T, Yoshida Y, Aiba T, Yamagiwa S, Asakura H, et al. Extrathymic derivation of gut lymphocytes in parabiotic mice. Immunology 1999;96:57–65. [42] Peaudecerf L, Ribeiro Dos Santos P, Boudil A, Ezine S, Pardigon N, Rocha B. The role of the gut as a primary lymphoid organ: CD8␣␣ intraepithelial T lymphocytes in euthymic mice derive from very immature CD44+ thymocyte precursors. Mucosal Immunol 2010;4:93–101. [43] Petrie HT, Zuniga-Pflucker JC. Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annu Rev Immunol 2007;25:649–79. [44] Ciofani M, Knowles GC, Wiest DL, von Boehmer H, Zuniga-Pflucker JC. Stagespecific and differential notch dependency at the ␣ and ␥␦ T lineage bifurcation. Immunity 2006;25:105–16. [45] Livak F, Tourigny M, Schatz DG, Petrie HT. Characterization of TCR gene rearrangements during adult murine T cell development. J Immunol 1999;162:2575–80. [46] Capone M, Hockett Jr RD, Zlotnik A. Kinetics of T cell receptor  ␥, and ␦ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+ CD25+ Pro-T thymocytes. Proc Natl Acad Sci USA 1998;95:12522–7. [47] Luci C, Reynders A, Ivanov II, Cognet C, Chiche L, Chasson L, et al. Influence of the transcription factor ROR␥t on the development of NKp46+ cell populations in gut and skin. Nat Immunol 2009;10:75–82. [48] Eberl G, Littman DR. Thymic origin of intestinal ␣ T cells revealed by fate14 mapping of ROR␥t+ cells. Science (New York, NY) 2004;305:248–51. [49] Egawa T, Kreslavsky T, Littman DR, von Boehmer H. Lineage diversion of T cell receptor transgenic thymocytes revealed by lineage fate mapping. PLoS One 2008;3:e1512. [50] Villey I, de Chasseval R, de Villartay JP. ROR␥T, a thymus-specific isoform of the orphan nuclear receptor ROR␥/TOR, is up-regulated by signaling through the pre-T cell receptor and binds to the TEA promoter. Eur J Immunol 1999;29:4072–80. [51] Gangadharan D, Lambolez F, Attinger A, Wang-Zhu Y, Sullivan BA, Cheroutre H. Identification of pre- and postselection TCR␣+ intraepithelial lymphocyte precursors in the thymus. Immunity 2006;25:631–41. [52] Baldwin TA, Sandau MM, Jameson SC, Hogquist KA. The timing of TCR ␣ expression critically influences T cell development and selection. J Exp Med 2005;202:111–21.