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Role of Gut Cryptopatches in Early Extrathymic Maturation of Intestinal Intraepithelial T Cells¹

Takatoku Oida^*,, Kenji Suzuki^*, Masanobu Nanno, Yutaka Kanamori^§, Hisashi Saito^¶, Eiro Kubota^¶, Shingo Kato^*, Mamoru Itoh^||, Shuichi Kaminogawa and Hiromichi Ishikawa^2,*

^* Department of Microbiology, Keio University School of Medicine, Tokyo, Japan; Department of Applied Biological Chemistry, University of Tokyo, Tokyo, Japan; Yakult Central Institute for Microbiological Research, Tokyo, Japan; ^§ Department of Pediatric Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan; ^¶ Second Department of Oral and Maxillofacial Surgery, Kanagawa Dental School, Kanagawa, Japan; and ^|| Central Institute for Experimental Animals, Kanagawa, Japan

	Abstract

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Lympho-hemopoietic progenitors residing in murine gut cryptopatches (CP) have been shown to generate intestinal intraepithelial T cells (IEL). To investigate the role of CP in progenitor maturation, we analyzed IEL in male mice with a truncated mutation of common cytokine receptor -chain (CR^-/Y) in which CP were undetectable. IEL-expressing TCR- (-IEL) were absent, and a drastically reduced number of Thy-1^highCD4⁺ and Thy-1^highCD8ß⁺ ß-IEL were present in CR^-/Y mice, whereas these ß-IEL disappeared from athymic CR^-/Y littermate mice. Athymic CR^-/Y mice possessed a small TCR- and _Eß₇ integrin-negative IEL population, characterized by the disappearance of the extrathymic CD8⁺ subset, that expressed pre-T, RAG-2, and TCR-Cß but not CD3 transcripts. These TCR^- IEL from athymic CR^-/Y mice did not undergo Dß-Jß and V-J joinings, despite normal rearrangements at the TCR-ß and - loci in thymocytes from euthymic CR^-/Y mice. In contrast, athymic severe combined immunodeficient mice in which CP developed normally possessed two major TCR^-_Eß₇⁺ CD8⁺ and CD8^- IEL populations that expressed pre-T, RAG-2, TCR-Cß, and CD3 transcripts. These findings underscore the role of gut CP in the early extrathymic maturation of CD8⁺ IEL, including cell-surface expression of _Eß₇ integrin, CD3 gene transcription, and TCR gene rearrangements.

	Introduction

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Numerous intraepithelial T cells (IEL)³ bearing either TCR-ß (ß-IEL) or TCR- (-IEL) are localized between columnar epithelial cells of the mouse small intestine and are believed to maintain the anatomical front of the intestine under constant immune surveillance. However, IEL have a number of cellular and behavioral characteristics that distinguish them from thymocytes and other peripheral T cells (1, 2, 3, 4, 5). For example, IEL are enriched with TCR- T cells (6, 7), and virtually all -IEL and about one-third of ß-IEL, unlike thymus-derived T cells that use -chain as part of their CD3 complex, express the unique CD8 homodimer (8, 9, 10, 11) instead of the CD8ß heterodimer and can use the Fc receptor -chain (12, 13, 14) in place of the -chain. Accumulating evidence indicates that these CD8⁺ IEL are potentially capable of developing somewhere in the intestinal mucosa without passing through the thymus (9, 15, 16, 17, 18, 19, 20). Moreover, the presence of lymphoid cells with properties of precursor T cells among IEL (1, 9, 19, 21, 22, 23, 24) and the expressions of recombination activating gene-1 (RAG-1) (9, 19, 21) and RAG-2 (25) by a subset of IEL support the notion that T lineage-committed precursors may enter the epithelium and undergo all steps of TCR gene rearrangement and subsequent differentiation into mature IEL in situ.

Recently, however, we identified multiple tiny clusters (1500) filled with 1000 c-kit⁺IL-7R⁺Thy-1⁺ lympho-hemopoietic progenitors in crypt lamina propria (LP) of the mouse small intestine (cryptopatches; CP) (26) and corroborated that c-kit⁺Lin^- (Lin; CD3, B220, Mac-1, Gr-1, and TER119) cells separated by flow cytometry from CP cells were capable of reconstituting ß- and -IEL in irradiated SCID mice (27). In contrast, cells from Peyer’s patches (PP) and mesenteric lymph nodes (MLN), which belong in the same intestinal immune compartment but lack c-kit⁺Lin^- cells, failed to do so. These findings indicate that CP are the key extrathymic anatomical sites in which precursor T cells develop to provide mature IEL and lead to the view that T lineage-committed precursors concentrating in gut CP and those residing in the IEL compartment represent at least two distinct intermediates along the extrathymic IEL lineage pathway, the more immature of which settles in CP.

Three athymic systems, namely congenitally athymic nude mice, neonatally thymectomized mice, and adult thymectomized, lethally irradiated, and hemopoietic stem cell-reconstituted mice, demonstrated that the generation of most peripheral T cells is wholly dependent on the thymus. Thus, mice that lack CP are extremely valuable not only for assessment of thymus-independent (TI) CD8⁺ IEL as the true descendants of progenitors residing in CP but also for dissection of precursor IEL maturation in CP. Because it is impossible to obtain experimentally manipulated mice lacking CP by surgical excision of every gut CP residing along the length of the intestine, we determined genetically manipulated mutant mice that lack CP.

Mice carrying null mutation at the common cytokine receptor -chain (CR) exhibit generalized lymphoid abnormality associated with a variety of immunological disorders (28, 29, 30). In the null mutant mice, development of IEL is severely diminished and PP are not detected (28, 29). We examined extensively tissue sections of small intestine prepared from male mice with a truncated mutant of the CR chain (CR^-/Y mice) that showed a phenotype similar to that of the null mutant mice in terms of development of the lymphocyte population (31, 32) and verified that CP were undetectable. Furthermore, not only - but also ß-IEL disappeared from the IEL compartment of athymic (nu/nu) CR^-/Y mice leaving a small population of TCR^- IEL that expressed c-kit, Thy-1, B220, CD4, and CD8ß molecules. Remarkably, these TCR^- IEL did not appear to contain the CD8⁺ subset. Thus, the absence of TCR^-CD8⁺ IEL in athymic CR^-/Y mice contrasts sharply with the phenotype of putative TCR^- IEL precursors present in young wild-type (WT) (19), athymic (nu/nu) nude (9), SCID (9, 33), RAG-1^-/- (23, 24), lck^-/- x fyn^-/- (23), CD3^-/- (24), and CD3^-/- (24) mice, a predominant fraction of which expresses the CD8 homodimer.

We (26) have previously demonstrated that the development of CP is unaltered in athymic nude, SCID, TCR-ß^-/- x -^-/-, and RAG-2^-/- mice and is comparable with that of normal B6 mice. In this study, we confirmed that athymic (nu/nu) SCID mice, which lacked ß- and -IEL but, unlike athymic CR^-/Y mice, in which CP developed normally, possessed the major TCR^-CD8⁺ subset in their IEL compartment. Further comparative analysis of TCR^- IEL from athymic CR^-/Y mice and those from athymic SCID mice with respect to the cellular and genetic levels of events associated with T cell development revealed other noteworthy distinctions between these two putative IEL precursors. Overall, the data are consistent with the view that maturation of precursor IEL in the small intestine proceeds sequentially in CP followed by intestinal epithelium and suggest an early and indispensable role of gut CP in the generation of an extrathymic subset of IEL-expressing CD8 homodimer.

	Materials and Methods

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Mice

C57BL/6J Jcl (B6), BALB/cA Jcl (B/c), athymic (nu/nu) nude, and C.B-17/Icr Jcl SCID (scid/scid) mice were purchased from the CLEA Japan (Tokyo, Japan). IL-7R -chain-deficient (7R^-/-) (34), RAG-2^-/- (35), and TCR-Cß^-/- (36) mice have been described previously (26). IL-2R ß-chain-deficient (2Rß^-/-) mice (37) that had been backcrossed seven times to B6 mice were a generous gift from Dr. H. Suzuki (Nagoya University School of Medicine, Nagoya, Japan), and heterozygous WT female mice carrying a truncated mutation of the CR (CR^-/X mice) (31) that had been backcrossed more than 20 times to B6 mice were kindly provided by Dr. K. Sugamura (Tohoku University School of Medicine, Sendai, Japan). Female CR^-/X mice were intercrossed with male B6 mice, and male CR^-/Y offspring were typed by PCR analysis of tail DNA with a set of primers to the neomysin-resistant gene described elsewhere (38). The CR^-/X mice were also crossed with athymic B/c mice and their heterozygous CR^-/X progeny were backcrossed to athymic B/c mice to obtain euthymic (nu/+) WT CR^+/Y, athymic (nu/nu) nude CR^+/Y, euthymic (nu/+) mutant CR^-/Y, and athymic (nu/nu) mutant CR^-/Y littermates. Although the genetic composition was different between individual littermate mice, it was confirmed that the difference was irrelevant to the distinctive cellular and phenotypic properties of lymphoid cells from the corresponding WT CR^+/Y, athymic CR^+/Y, euthymic CR^-/Y, and athymic CR^-/Y mice. We also obtained athymic SCID mice by intercrossing C.B-17/Icr Jcl SCID mice and athymic B/c mice. All mice used for experiments were between 8 and 20 wk of age, and the absence of the thymus in various athymic mice was checked at necropsy.

Antibodies

The following mAbs described elsewhere (26) were used for immunohistochemical staining: anti-c-kit mAb (ACK-2), anti-CD3 mAb (145-2C11), anti-CD4 mAb (GK1.5), and anti-CD8 mAb (53-6.7). Anti-CD103 (_Eß₇) mAb (2E7; PharMingen, San Diego, CA) and biotinylated anti-Ly5.2 mAb (104; PharMingen) were also employed in this study. The following FITC-conjugated and biotinylated mAbs were used for flow cytometric analysis: anti-CD3 mAb (145-2C11; PharMingen), anti-ß mAb (H57-597; PharMingen), anti- mAb (GL3; PharMingen), anti-Thy-1.2 mAb (30-H12; Becton Dickinson, San Jose, CA), anti-CD4 mAb (RM4-5; PharMingen), anti-CD8 mAb (53-6.7; Becton Dickinson), and anti-CD8ß mAb (53-5.8; PharMingen). We also used FITC-conjugated anti-B220 mAb (RA3-6B2; PharMingen), FITC-conjugated anti-E mAb (2E7; PharMingen), FITC-conjugated anti-CD19 mAb (1D3; PharMingen), FITC- conjugated anti-IgM mAb (II/41; PharMingen), and biotinylated anti-c-kit mAb (ACK-4; a gift from Dr. S. Nishikawa, Kyoto University, Kyoto, Japan).

Immunohistochemical procedure

Immunohistochemical staining was as described previously (26). In brief, longitudinally opened small intestine 10 mm in length was embedded in OCT compound (Tissue-Tek; Miles, Elkhart, IN) at -80°C. The tissue segments were sectioned with a cryostat at 6 µm, and sections were preincubated with Block-ace (Dainippon Pharmaceutical, Osaka, Japan) to block nonspecific binding of mAbs. The sections were then incubated with rat or hamster mAbs for 30 min at 37°C and rinsed three times with PBS, followed by incubation with biotin-conjugated goat anti-rat IgG Ab (Cedarlane Laboratories, Hornby, Ontario, Canada) or with biotin-conjugated goat anti-hamster IgG (Vector Laboratories, Burlingame, CA). In staining with biotinylated anti-Ly5.2 mAb, the second biotin-conjugated anti-IgG Ab was not used. Subsequently, the sections were washed three times with PBS and then incubated with avidin-biotin peroxidase complexes (Vectastatin ABC kit; Vector Laboratories). The histochemical color development was achieved by Vectastatin DAB (3,3'-diaminobenzidine) substrate kit (Vector Laboratories) according to the manufacturer’s instructions. Finally, the sections were counterstained with hematoxylin for microscopy. Endogenous peroxidase activity was blocked with 0.3% H₂O₂ and 0.1% NaN₃ in distilled water for 10 min at room temperature. Tissue sections incubated either with isotype-matched normal rat IgG or with nonimmune hamster serum showed only minimal background staining.

Flow cytometry and cell sorting

A single lymphoid cell suspension was prepared and nucleated cells were counted using a hemocytometer. IEL were isolated as described (39), and CP cells were isolated according to a newly devised method described elsewhere (27). In brief, with the aid of transillumination stereomicroscope, we isolated a tiny fragment of the small intestine containing one CP using an amputated and tapered 21-gauge needle. Lymphoid cells were incubated first with biotinylated mAb and then with streptavidin-PE (Becton Dickinson) and FITC-conjugated second mAb. Stained cells were suspended in staining medium (Hanks solution without phenol red, 0.02% NaN₃, and 2% heat-inactivated FBS) containing 0.5 µg/ml propidium iodide (PI) and analyzed using FACScan with LYSYSII software (Becton Dickinson). Dead cells were excluded by PI gating. Lymphoid cells were incubated with anti-Fc II/III receptor mAb (2.4G2; PharMingen) before staining to block nonspecific binding of labeled mAbs to FcR. CD8⁺ and CD8^- subpopulations of TCR^- IEL from athymic SCID mice were sorted by FACS Vantage (Becton Dickinson).

Semiquantitative RT-PCR analysis of mRNA levels

Total RNA was prepared from various lymphocytes with an RNeasy Mini Kit (Qiagen, Chatsworth, CA). RNA samples were treated with DNase (RT grade) (Nippon Gene, Toyama, Japan) to remove contaminating genomic DNA and repurified. Serial dilutions of each RNA sample were reverse transcribed with 5 µM random hexamers, 1 mM dNTP, 20 U of RNase inhibitor (Takara, Kyoto, Japan), and 100 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Rockville, MD) in a volume of 20 µl at 42°C for 30 min. PCR was conducted in a volume of 100 µl containing all reverse transcriptase products, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 1 µM of each primer, and 2.5 U of Taq DNA polymerase (Takara). Amplification for 35 cycles was performed for 15 s at 94°C, 30 s at 60°C, and 1 min at 72°C. The PCR products were run on 2% agarose gel and visualized by ethidium bromide staining. PCR primers and fragment length of PCR products were: pre-T, 5'-GTGTCAGGCTCTACCATCAGG-3 and 5'-GCAGAAGCAGTTTGAAGAGGA-3', 449 bp (40); CD3, 5'-ATGGCCAAGAGCTGCCT-3' and 5'-AGAATACAGGTCCCGCT-3', 383 bp (41); RAG-2, 5'-CACATCCACAAGCAGGAAGTACAC-3' and 5'-GGTTCAGGGACATCTCCTACTAAG-3', 472 bp (42); TCR-Cß, 5'-GAGCAATTATAGCTACTGCC-3' and 5'-TCAGAGTCAAGGTGTCAACG-3', 467 bp (40); and ß-actin, 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3', 349 bp (43).

Semiquantitative PCR analysis of TCR gene rearrangements

Genomic DNA was prepared from various lymphocytes with a QIAamp Blood Kit (Qiagen). For the analysis of TCR Dß-Jß gene rearrangement, we conducted a nested PCR to amplify exactly the rearranged DNA sequences. PCR primers were designed to be positioned 5' to the Dß2 gene segment and 3' to the Jß2.2 gene segment for both external (E) and internal (I) primers that are capable of amplifying the rearranged Dß2-Jß2.1 and Dß2-Jß2.2 sequences. Moreover, to minimize the amplification of germline sequence (1072-bp fragment), which competitively inhibits amplifications of rearranged Dß2-Jß2.1 (458-bp fragment) and Dß2-Jß2.2 (289-bp fragment) sequences, genomic DNA was first digested with EcoRI and EcoRV restriction enzymes (Takara), both of which have one recognition site in the germline DNA sequence extending from the Dß2 to Jß2 segment but have none in the rearranged Dß2-Jß2.1 and Dß2-Jß2.2 sequences. Serial dilutions of the digested DNA samples were subjected to the first PCR consisting of 20 cycles of 30 s at 94°C and 2 min at 68°C in a volume of 50 µl containing 1 µM of Dß2 (E) and Jß2.2 (E) primers, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, and 1.25 U of Taq polymerase. The PCR products were further digested with EcoRI and EcoRV and purified. Subsequently, one-tenth of the purified products were subjected to the second PCR with Dß2 (I) and Jß2.2 (I) primers for 20 cycles under the same conditions employed for the first round PCR. This method allowed us to determine nearly a two-order smaller amount of rearranged Dß2-Jß2.1 and Dß2-Jß2.2 DNA as compared with a standard PCR method in the detection of a small number of rearranged sequences in WT thymocyte DNA that had been serially diluted with unrearranged DNA from RAG-2^-/- thymocytes (data not shown). For the analysis of TCR V-D-J gene rearrangement, serial dilutions of DNA samples were amplified by PCR with V4 and J1 primers for 35 cycles. Each cycle consisted of 30 s at 94°C, 30 s at 61°C, and 1 min at 72°C. In this case, the unrearranged germline V4-J1 sequence was too long for PCR amplification. PCR primers used were: Dß2(E)-Jß2.2(E), 5'-CAGTCAGACAAACCTCTCTGCCAC-3' and 5'-GGCTCAGGACAAAAACTCAGTGCT-3', this study; Dß2(I)-Jß2.2(I), 5'-GTAGGCACCTGTGGGGAAGAAACT-3' and 5'-GCTCCTGCTGCTCCAACCCTGACT-3', K. Hozumi, Tokai University School of Medicine, unpublished observation; and V4-J1, 5'-CCGCTTCTCTGTGAACTTCC-3' and 5'-CAGTCACTTGGGTTCCTTGTCC-3', 168 bp (44).

	Results

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CR^-/Y mice lack CP, -IEL, CD8⁺ ß-IEL, and Thy-1^- ß-IEL

To explore the developmental events that proceed in CP toward IEL generation, we hoped to find mice that lack CP and to characterize IEL that emerge in the absence of CP. We (26) have previously reported that CP are undetectable in 7R^-/- mice. However, although -IEL are absent owing to the selective blockade of TCR- gene rearrangements (34), we have noticed only slightly decreased development of TI as well as thymus-dependent (TD) ß-IEL subsets in 7R^-/- mice (Fig. 1B; data not shown). With these observations in mind, we reinvestigated hundreds of cryosections prepared from the small intestines of 7R^-/- mice by immunohistochemistry and verified that conspicuously emaciated CP filled with c-kit⁺ cells and decreased by >16-fold in number were present in the mutant intestine (Fig. 1A). As it has been reported (45), 2Rß^-/- mice exhibited a dramatic reduction of TI CD8⁺ IEL (Fig. 1B). This finding raised the possibility that the development of CP might be hampered in the 2Rß^-/- condition. However, we found that 2Rß^-/- mice have barely decreased CP filled with c-kit⁺ cells (Fig. 1A). Severely diminished development of IEL in CR mutant mice has also been reported (28, 29). Surprisingly, we found that lymphoid cell aggregates filled with a meaningful number of c-kit⁺ cells, namely CP, were hardly detectable in the intestinal LP of CR^-/Y mice and that most c-kit⁺ cells were localized individually throughout the length of the small intestine, although two to three c-kit⁺ cells settled together in several locations (Fig. 1A). These results indicate that the CR-mediated signaling is needed for the generation of lymphoid and/or stromal CP cells. Concomitantly, a conspicuous decrease in the total number of IEL was also observed in CR^-/Y mice (Fig. 1B). As shown in Fig. 1C, flow cytometric analysis on IEL isolated from CR^-/Y revealed that mutant intestine retained Thy-1⁺ ß-IEL expressing either CD4 or CD8ß molecules but lacked -IEL and Thy-1^- as well as CD8⁺ ß-IEL. Because the recombination of TCR- genes is blocked in 7R^-/- mice (34) and the CR mutation also inhibits 7R-mediated signaling pathway (28, 29, 30, 31), CR^-/Y mice completely lacked -IEL, and all CD3⁺ IEL in this mutant animals were ß-IEL (Fig. 1, B and C). To compare WT and CR^-/Y B6 mice with respect to the phenotype of ß-IEL, the expression of Thy-1, CD4, CD8, and CD8ß molecules restricted to the ß-IEL from WT mice was also shown (Fig. 1C, bottom panel).

View larger version (71K): [in this window] [in a new window]   FIGURE 1. Immunohistochemical characterization of CP and flow cytometric analysis of IEL in B6, 7R-/-, 2Rß-/-, and CR-/Y mice. A, Representative immunohistochemical visualization of c-kit+ lymphocytes in the small intestinal CP (x400). Arbitrarily chosen duodenal, jejunal, and ileal tissue fragments from different mice (n = 4–6) were examined. We checked 50–60 fragments from each strain of mice, and 54 fragments from B6 mice equivalent to one and three-quarters of a small intestine gave 2745 CP. This number basically agreed with our previous CP enumeration of 1500 CP per small intestine in adult B6 mice (26 ). Based on the same calculation, the numbers of CP per intestine in the other mice are also shown. B, Absolute numbers of - and ß-IEL and the composition of Thy-1+ and CD8+ ß-IEL subsets from B6, 7R-/-, 2Rß-/-, and CR-/Y mice (n = 5 or 7) were determined by two-color flow cytometry. The composition (%) of CD8+ ß-IEL was calculated from (%CD8+ ß-IEL - %CD8ß+ ß-IEL). C, Two-color flow cytometric analysis was performed on IEL isolated from B6 and CR-/Y mice. Percentage of positive cells in the corresponding quadrants is shown.

Distinctive TCR^- IEL are localized in the intestinal epithelium of athymic CR^-/Y mice

Histogenesis of CP and generation of ß-IEL expressing CD8 homodimer are almost completely blocked in CR^-/Y mice, implying that CP are indispensable for the TI pathway of ß-IEL development. In an attempt to further explore this issue, we produced athymic CR^-/Y mice and analyzed IEL that migrated to epithelial destinations in the absence of the thymus and CP. As shown in Fig. 2A, euthymic WT CR^+/Y mice have ß- and -IEL and euthymic CR^-/Y mice have ß-IEL but lack -IEL. In contrast to these littermates, athymic CR^-/Y mice lacked both ß- and -IEL, leaving a small TCR^- IEL population, a substantial fraction of which expressed Thy-1, B220, CD4, and CD8ß (Fig. 2A). These results indicate: 1) Thy-1⁺ ß T cells that settle in the IEL compartment of euthymic CR^-/Y mice are most likely the thymic-derived progeny, and 2) as predicted from the near absence of CD8⁺ ß-IEL subset in CR^-/Y mice, most TCR^-CD8⁺ IEL isolated from athymic CR^-/Y mice bear CD8ß instead of CD8. It should also be pointed out that the B220⁺ IEL from athymic CR^-/Y mice did not express cell-surface IgM nor CD19, indicating that these TCR^- IEL are not B lineage cells (data not shown). Immunohistochemical analysis of tissue sections of intestinal villi confirmed that a small number of IEL detected by flow cytometry such as CD3⁺, CD4⁺, and CD8⁺ IEL in euthymic CR^-/Y mice and CD4⁺ and CD8⁺ IEL in athymic CR^-/Y mice were indeed localized in the intestinal epithelium rather than in the villous LP (Fig. 2B).

View larger version (98K): [in this window] [in a new window]   FIGURE 2. Flow cytometric and immunohistochemical analyses of IEL from WT (nu/+) CR+/Y, euthymic (nu/+) CR-/Y, and athymic (nu/nu) CR-/Y mice. A, Both ß- and -IEL are absent from the IEL compartment of athymic CR-/Y mice, leaving a significant number of TCR- IEL expressing either Thy-1, B220, CD4, and/or CD8ß but not those expressing CD8. Percentage of positive cells in the corresponding quadrants is shown. B, Representative immunohistochemical visualization of IEL expressing CD3, CD4, or CD8 (x400). Arrowheads indicate IEL. Although not shown by arrowheads, a considerable number of CD3+ and CD8+ IEL are evident in villous epithelia of the small intestine from WT (nu/+) CR+/Y mice.

View larger version (54K): [in this window] [in a new window]   FIGURE 3. IEL compartment contains TCR- lymphocytes expressing cell-surface molecules of mouse T lineage cells, and CD8+ IEL subset, irrespective of the TCR expression, are reduced drastically in mice that lack CP. A, Both IEL and MLN cells from athymic CR-/Y mice lack ß and T cells, whereas a significant fraction of IEL but not MLN cells expresses c-kit, Thy-1, CD4, and/or CD8. Percentage of positive cells in the corresponding quadrants is shown. B, Representative small intestinal CP of athymic SCID mice filled with c-kit+ lymphocytes, and the number of CP per intestine based on the calculation described in Fig. 1A is shown. C, Athymic (nu/nu) SCID mice in which CP develop normally possess a major TCR- IEL population expressing CD8, and athymic (nu/nu) nude mice in which CP develop normally (26 ) possess major TCR+ and TCR- populations expressing CD8. Percentage of positive cells in the corresponding quadrants is shown. D, Two-color (CD8 vs CD8ß) flow cytometric profiles of IEL from various mice carrying and not carrying CP. The profile of IEL from athymic (nu/nu) CR+/Y mice was essentially the same as that of IEL from B/c athymic nude mice depicted in the figure. Percentage of positive cells in the corresponding rectangular regions is shown.

Athymic CR^-/Y mice without CP lack but athymic SCID mice with CP possess the TCR^-CD8⁺ IEL subset

SCID mice in which CP develop normally (26) harbor a significant fraction of the TCR^- IEL population that includes cells expressing Thy-1^-CD8^- and three Thy-1⁺ and/or CD8⁺ subsets (33). The data presented in the preceding section show the compartmentalization of distinctive TCR^- IEL within the intestinal epithelium of athymic CR^-/Y mice in the absence of CP. To evaluate the role of CP in TCR^- IEL generation, we conducted a comparative analysis of these two TCR^- IEL populations with respect to the cellular and subcellular levels of events taking place in CP. For this purpose, we produced athymic SCID mice to eliminate any thymic emigrant IEL that might obscure the analysis (20, 50, 51, 52, 53) and verified that the development of CP populated with c-kit⁺IL-7R⁺Thy-1⁺ lymphocytes was maintained in these mice (Fig. 3B). As shown in Fig. 3C, athymic SCID mice lack ß- and -IEL, but athymic nude mice possess both ß- and -IEL although absolute numbers of ß-IEL are markedly reduced. A substantial fraction of TCR^- IEL from athymic SCID mice expressed c-kit, Thy-1, CD4, and CD8 molecules (Fig. 3C). Next, we examined the proportion of the TI CD8⁺ IEL subset to the whole CD8⁺ IEL population in the above-mentioned mice with and without CP. Major CD8⁺ IEL from euthymic WT CR^+/Y (68%), athymic SCID (96%), and athymic nude (99%) mice in which CP developed normally expressed CD8 homodimer (Fig. 3D). In sharp contrast, only a marginal fraction of CD8⁺ IEL from euthymic CR^-/Y (2.9%) and athymic CR^-/Y (2.0%) mice in which CP were undetectable expressed CD8 homodimer (Fig. 3D). These findings indicate that the generation of TI CD8⁺ IEL is dependent on CP. Absolute numbers of TCR⁺ (ß- and -IEL) and TCR^- IEL as well as the population size of CD4^-CD8^-, CD4⁺CD8^-, CD4⁺CD8⁺, CD8⁺, and CD8ß⁺ subsets in the IEL isolated from different mice (Fig. 3D) are presented in Table I. It is noteworthy that absolute numbers of TD CD4⁺CD8⁺ and CD8ß⁺ IEL subsets are drastically reduced while those of TD CD4⁺CD8^- IEL subset are maintained in nu/+ CR^-/Y mice (Table I). Because Cao et al. (28) have reported that the CD4:CD8 ratio was increased in the thymus and spleen of the CR mutant mice they produced, the increase in CD4:CD8 ratio is an inherent property of T cells irrespective of their location in mice lacking CR-mediated signaling pathway.

Major TCR^- IEL from athymic CR^-/Y mice fail to express but major TCR^- IEL from athymic SCID mice express _Eß₇ integrin

IEL are known to express _Eß₇ integrin (54, 55, 56, 57, 58, 59, 60), and it has been shown that _Eß₇ recognizes E-cadherin on gut epithelial cells (55, 59, 60). In fact, 90% IEL from euthymic WT CR^+/Y mice expressed _Eß₇ (Fig. 4, A and B), whereas 98% PP cells were _Eß₇ negative (Fig. 4A). Interestingly, a significant population (16%) of CP cells from these mice expressed _Eß₇ (Fig. 4A). However, this _Eß₇⁺ lymphocyte subset is most likely the unavoidable LP lymphocyte and/or IEL contaminants present in the CP cell preparation because c-kit⁺ CP cells were _Eß₇^- by immunohistochemistry (data not shown). Provided that CP are important lymphoid tissues in which early maturation of precursor IEL takes place, it can be assumed that the expression of _Eß₇ on IEL is also influenced by the absence of CP. Consistent with this proposition, most TCR^- common leukocyte alloantigen-positive (Ly5.2⁺) IEL (80%) were _Eß₇^- in athymic CR^-/Y mice that lacked CP (Fig. 4B). In contrast, most TCR^- Ly5.2⁺ IEL (80%) expressed high levels of _Eß₇ in athymic SCID mice that possessed CP (Fig. 4B). To confirm _Eß₇ expression in situ, we used anti-_Eß₇ and anti-Ly5.2 mAbs to stain frozen sections of the small intestine. As shown in the lower panel of Fig. 4B, IEL are identifiable by anti-Ly5.2 but not by anti-_Eß₇ mAbs in the small intestinal villi of athymic CR^-/Y mice, whereas they are identifiable by both mAbs in those of athymic SCID mice. Note that although the pictures are not enough for accurate quantitation of IEL, Ly5.2⁺ IEL of athymic CR^-/Y mice are about 10% of WT euthymic CR^+/Y mice, endorsing the results presented in Table I. Taken together, these results underline the role of CP in converting precursor IEL into the _Eß₇⁺ state.

View larger version (69K): [in this window] [in a new window]   FIGURE 4. Major TCR- IEL from athymic CR-/Y mice that lack CP fail to express Eß7 integrin. A, The expression of Eß7 on lymphoid cells isolated from PP, CP, and intestinal intraepithelial compartments of WT CR+/Y mice. B, The upper panel shows a representative histogram of Eß7 expression on the leukocyte common alloantigen (Ly5.2)-positive IEL from WT CR+/Y (with CP), athymic CR-/Y (without CP), and athymic SCID (with CP) mice, and the lower panel verifies immunohistochemically the flow cytometric profiles shown in the upper panel. Two-color flow cytometric analysis by using biotinylated anti-Ly5.2 mAb and FITC-conjugated anti-Eß7 mAb revealed that most of the IEL from WT CR+/Y mice (99%), athymic CR-/Y mice (95%), and athymic SCID mice (97%) expressed Ly5.2 molecules. Note that anti-Eß7 mAb but not anti-Ly5.2 mAb can hardly visualize IEL in the villous epithelia of the small intestine from athymic CR-/Y mice, whereas both mAbs visualize almost the same numbers of IEL in the villous epithelia of the small intestine from athymic SCID mice. Arrowheads indicate IEL. Although not shown by arrowheads, numerous Eß7+ and Ly5.2+ IEL are evident in the villous epithelia of the small intestine from WT CR+/Y mice.

Pre-T and CD3 gene transcripts in TCR^- IEL

Putative TCR^- IEL precursors in athymic CR^-/Y mice are distinct from those in athymic SCID mice in that the majority of former TCR^- IEL do not express CD8 and _Eß₇ molecules. However, the phenotype of these TCR^- IEL does not necessarily prove their T cell commitment but instead may represent cells with characteristics of NK, dendritic cell, and/or mast cell progenitors (1, 48, 53, 61). To determine whether these two TCR^- IEL populations include T cell precursors, we investigated whether these cells express pre-T- and CD3-specific mRNA. As internal standard for the mRNA and cDNA preparations, the intensities of the actin RT-PCR products, corresponded in all experiments to mRNA concentrations that were within the linear range of the template titration curve (Fig. 5).

View larger version (52K): [in this window] [in a new window]   FIGURE 5. Semiquantitative RT-PCR analysis of pre-T and CD3 mRNA levels in CP cells, IEL, MLN cells and thymocytes from WT (nu/+) CR+/Y, euthymic (nu/+) CR-/Y, athymic (nu/nu) CR-/Y, and athymic (nu/nu) SCID mice. Serial 5-fold dilutions of RNAs equivalent to RNAs extracted from the indicated numbers of cells were reverse transcribed, and the cDNA products were PCR amplified, electrophoresed in agarose gels, and visualized with ethidium bromide. CP cells and IEL from all four strains of mice express comparable levels of pre-T gene. In contrast, while the signal for CD3 transcripts is high in IEL from WT CR+/Y and euthymic CR-/Y mice that include TCR+ lymphocytes, and also in TCR- IEL from athymic SCID mice carrying CP, the same signal is almost undetectable in TCR- IEL from athymic CR-/Y mice not carrying CP. Note that ß-actin-specific mRNA levels are comparable in all RNA preparations.

CD3-specific mRNA-encoding TCR-associated molecules are expressed in the earliest T-committed mouse fetal thymocytes (41, 53, 64) and represent a marker to define whether immature lymphoid cells are committed to T cell lineages. RT-PCR analysis of mRNA in lymphocytes from athymic SCID mice revealed that CD3 transcripts were found in TCR^- IEL at high levels and in CP cells, albeit at >25-fold reduced levels, but were undetectable in MLN cells (Fig. 5). In contrast, the same CD3 transcripts were not detected even in mRNA extracted from a large number (6250 cells) of athymic CR^-/Y IEL (Fig. 5). Taken together, these data demonstrate that most TCR^- IEL from athymic CR^-/Y mice do not express CD8 (Fig. 3D) and include cells that express pre-T but not CD3 transcripts, whereas that TCR^- IEL from athymic SCID mice are comprised of two major CD8⁺ and CD8^- subpopulations (Fig. 3D) that express pre-T and CD3 transcripts.

TCR^- IEL and CP lymphocytes express RAG-2 transcripts

Proteins encoded by RAG-1 and -2 are essential in TCR and Ig gene rearrangements and are present in T and B lineage cells of the early stages but not in other lympho-hemopoietic cells. RT-PCR (9, 19) as well as in situ hybridization (21) analyses of IEL revealed the expression of RAG-1 mRNA by a small and confined subset of IEL. RAG-2 transcripts are also detectable by RT-PCR in IEL from the small intestine but not the large intestine (25). Thus, it is important to explore not only TCR^- IEL but also cells that reside in CP for the expression of RAG-1 and/or -2 genes because CP were shown to be responsible for generating IEL (27). For this purpose, we determined RAG-2 transcripts by semiquantitative RT-PCR analysis and compared mRNA of gut lymphocytes with those of thymocytes from WT and RAG-2^-/- mice, with the latter two mRNA serving as positive and negative templates, respectively.

mRNA from 50 WT thymocytes displayed a strong signal for RAG-2 transcripts, whereas mRNA from 6250 RAG-2^-/- thymocytes failed to display any detectable signals (Fig. 6). Under this condition, low levels of RAG-2 transcripts were constantly detected in an amount of mRNA equivalent to 6250 lymphocytes such as IEL from euthymic WT CR^+/Y, athymic CR^-/Y, and athymic SCID mice and CP cells from euthymic WT CR^+/Y and athymic SCID mice (Fig. 6). Because half of TCR^- IEL from athymic CR^-/Y mice (Fig. 2A) and about two-thirds of TCR^- IEL from athymic SCID mice (data not shown) expressed B220, a CD45R determinant that is generally considered a B cell-specific marker, it should be pointed out that the question of whether these TCR^- IEL also contain B cell progenitors expressing RAG-2 mRNA remains unanswered. However, on the basis of the following considerations, the possibility that these TCR^- IEL contain a meaningful fraction of B lineage cells appears to be remote. First, this anti-B220 mAb (clone; RA3-6B2) reacts with a large fraction of TCR⁺ IEL, whereas it does not react with other peripheral T cells (Ref. 65 and data not shown). Second, B220 has been reported to be expressed on lymphopoietic T progenitors (40) as well as on activated (66) and superantigen-induced apoptotic (67) T cells. Third, and importantly, almost all TCR^-B220⁺ IEL from athymic SCID mice expressed _Eß₇ (Fig. 4B and data not shown), an integrin found on most TCR⁺ IEL (56, 58, 59, 60) (Fig. 4) and on other mucosal T cells, dendritic cells, macrophages, and mast cells but not on mucosal B cells (59, 60). In any event, the results obtained by PCR analysis are sensitive to small contaminations by cells of other lineages and localizations. Thus, the possible pitfalls in the above interpretations remain to be formally excluded.

View larger version (45K): [in this window] [in a new window]   FIGURE 6. CP cells and IEL express low levels of RAG-2 gene. Amounts of RNAs equivalent to RNAs contained in 6250 CP cells and IEL but not MLN cells from WT CR+/Y, athymic CR-/Y, and athymic SCID mice exhibit low levels of RT-PCR signals for RAG-2 transcripts. RNAs equivalent to RNAs contained in 50 thymocytes from WT mice and those contained in 6250 thymocytes from RAG-2-/- mice were used for positive and negative control RNAs, respectively. Note that ß-actin-specific mRNA levels are comparable in all RNAs equivalent to RNAs contained in two cells.

To test further the proposition that TCR^- IEL and CP cells include lymphoid precursors committed to the T cell lineage, we used nested as well as standard DNA-PCR strategies to examine the status of TCR-ß and - gene rearrangements among these cells. The nested PCR analysis was capable of diminishing markedly the unrearranged germline Dß2-Jß2.2 band and thus allowed us to titrate much more accurately the template DNA containing a small number of rearranged Dß2-Jß2.1 and Dß2-Jß2.2 gene segments (see Materials and Methods). The rearranged Dß2-Jß2.1 and Dß2-Jß2.2 bands were not detected in TCR^- IEL from athymic CR^-/Y mice, unlike thymocytes and IEL from euthymic CR^-/Y mice and CP cells and IEL from athymic nude mice that exhibited Dß2-Jß2.1 and Dß2-Jß2.2 rearrangements (Fig. 7A). Likewise, a standard DNA-PCR analysis revealed that cells undergoing V4-J1 rearrangement were also not detected in TCR^- IEL from athymic CR^-/Y mice, whereas cells undergoing the rearrangement were detected not only in thymocytes (68, 69) but also in IEL that include thymus-derived ß T cells (Fig. 1, B and C, Fig. 2A, and Table I) from euthymic CR^-/Y mice as well as in CP cells and IEL from athymic nude mice (Fig. 7A). As depicted in Fig. 7A, neither IEL nor CP cells from athymic SCID mice exhibit Dß2-Jß2.1, Dß2-Jß2.2, and V4-J1 rearrangements (negative control).

View larger version (54K): [in this window] [in a new window]   FIGURE 7. Semiquantitative DNA-PCR analysis of TCR gene rearrangements and semiquantitative RT-PCR analysis of TCR-Cß mRNA levels in CP cells, IEL, and thymocytes from euthymic (nu/+) CR-/Y, athymic (nu/nu) CR-/Y, athymic (nu/nu) SCID, and athymic (nu/nu) nude mice. A, Serial 5-fold dilutions of genomic DNAs equivalent to DNAs extracted from the indicated numbers of cells were subjected to TCR gene rearrangement analysis using indicated PCR primers as described in Materials and Methods. Subsequently, PCR products were electrophoresed in agarose gels and visualized with ethidium bromide. For the amplification of rearranged Dß2-Jß2.1 (458-bp fragment) and Dß2-Jß2.2 (289-bp fragment) sequences, we employed a newly devised PCR strategy as described in Materials and Methods to minimize the amplification of 1072-bp signals corresponding to germline Dß2-Jß2.2 DNA, which competitively inhibited the amplification of a minute number of rearranged DNA sequences. Dß2-Jß2.1, Dß2-Jß2.2, and V4-J1 rearrangements were absent among IEL from athymic CR-/Y mice, whereas rearrangements were present in CP cells from athymic nude mice and also among IEL from euthymic CR-/Y and athymic nude mice. CP cell and IEL DNAs from athymic SCID mice were used as negative control DNAs. B, Serial 5-fold dilutions of RNAs equivalent to RNAs extracted from the indicated numbers of cells were reverse transcribed, and the cDNA products were PCR amplified, electrophoresed in agarose gels, and visualized with ethidium bromide. Not only CP cells and IEL from athymic SCID mice but also IEL from athymic CR-/Y mice express the transcript of the TCR-Cß gene, indicating that the signals are most likely the germline transcript because these cells do not undergo rearrangement of the TCR-ß gene. CP cell, IEL, and thymocyte RNAs from TCR-Cß-/- mice were used as negative control RNAs. Note that ß-actin-specific mRNA levels are comparable in all RNA preparations.

Both CD8⁺ and CD8^- IEL from athymic SCID mice express pre-T, CD3, and germline TCR-Cß transcripts

TCR^- IEL from athymic SCID mice contain pre-T, CD3, and germline TCR-Cß transcripts (Figs. 5 and 7B), and the generation of TI CD8⁺ IEL in these mice appears to be dependent on CP (Fig. 3D). However, because TCR^- IEL from athymic SCID mice include two major CD8⁺ and CD8^- subsets (Fig. 3C), it is possible that the CD8⁺ IEL are not T lineage-committed precursors. To determine whether T lineage-committed cells are present in TCR^-CD8⁺ IEL from athymic SCID mice, we purified CD8⁺ and CD8^- IEL by flow cytometry. As shown in Fig. 8, RT-PCR analysis of mRNA extracted from CD8⁺ IEL (purity, 99.4%) and CD8^- IEL (purity, 99.5%) revealed that pre-T, CD3, and germline TCR-Cß transcripts were expressed by these two sorted IEL to the same extent, indicating that both subpopulations include T lineage-committed precursors.

View larger version (63K): [in this window] [in a new window]   FIGURE 8. Semiquantitative RT-PCR analysis of pre-T, CD3, and TCR-Cß mRNA levels in sorted CD8+ and CD8- IEL from athymic SCID mice. RNAs were extracted from purified CD8+ IEL (99.4%) and CD8- IEL (99.5%) (upper panel). Serial 5-fold dilutions of RNAs equivalent to RNAs extracted from the indicated numbers of cells were reverse transcribed, and the cDNA products were PCR amplified, electrophoresed in agarose gels, and visualized with ethidium bromide. CD8+ and CD8- IEL from athymic SCID mice express comparable levels of pre-T, CD3, and TCR-Cß genes (lower panel). Note that ß-actin-specific mRNA levels are comparable in all RNA preparations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, we verified that the small intestine of CR^-/Y mice, in which signal tranductions from IL-2, IL-4, IL-7, IL-9, and IL-15 receptors are compromised (28, 29, 31), was devoid of CP and did not contain -IEL and TI CD8⁺ ß-IEL but did contain a small number of TD CD4⁺ and CD8ß⁺ ß-IEL-expressing Thy-1 molecules. In fact, these remaining TD ß-IEL subsets disappeared completely from the IEL compartment of athymic CR^-/Y mice. Regarding the gut microenvironment of CR^-/Y mice, we assume that deficiencies of IL-7R- and IL-15R-mediated signaling pathways reduce synergistically and/or additively the histogenesis of CP and, consequently, result in the disappearance of CP. Truncated CR expressed in these mutant mice could also abolish signal transductions from IL-2R, IL-4R, and IL-9R. However, these signalings might not be involved in the histogenesis of CP or in maturation of TI IEL (30, 72, 73), although these possibilities remain to be ruled out.

From the point of view of the correlation between CP development and generation of TI IEL in these three mutant mice, the present findings appear to be rather conflicting. Nonetheless, given that CP are indispensable for early TI IEL maturation, it is conceivable that CP in the 7R^-/- intestine are functionally intact even if histogenesis is markedly diminished, whereas CP in the 2Rß^-/- intestine might be functionally crippled even if histogenesis remains nearly the same in its numerical and immunohistochemical appearances (Fig. 1A). The role of thymus in the generation of peripheral CD8ß⁺ T cells has been well established to date. In this context, the following observations are noteworthy. First, MHC class I mutant mice do not have peripheral CD8ß⁺ T cells but their thymus is almost normal in terms of size and appearance. Thus, even though MHC class I mutant mice have a normal appearing thymus, it must not function normally because it does not produce CD8ß⁺ T cells. Second, IL-7R mutant mice have much reduced but still a significant number of peripheral CD8ß⁺ T cells, although their thymus displays a drastic reduction in its cellularity. Third, athymic nude mutant mice do not have thymus and peripheral CD8ß⁺ T cells. Based simply on these observations, we cannot conclude that the correlation is seen between thymus development and generation of peripheral CD8ß⁺ T cells. However, because T cell development in the thymus has been extensively studied by many investigators, we now know that the development of most peripheral CD8ß⁺ T cells is wholly dependent on the thymus. But in any case, much yet remains to be learned about the cellular mechanism of precursor IEL maturation in CP before we conclusively establish the correlation between CP development and generation of TI IEL. This issue, for instance, could be explored by analysis of various types of bone marrow chimeric animals produced between WT, 7R^-/-, 2Rß^-/-, and CR^-/Y mice.

TCR^- IEL from athymic CR^-/Y mice without CP and those from athymic SCID mice with CP exhibited abundant signals for pre-T and germline TCR-Cß transcripts, markers of T cell commitment (41, 53, 62) and a weaker but comparable level of signal for RAG-2 transcripts, and included cells that expressed c-kit, Thy-1, CD4, and CD8ß molecules. Outstanding differences between these two TCR^- IEL revealed in the present study concerned the development of the CD8⁺ subset, cell-surface expression of _Eß₇ integrin, and transcription of CD3-specific mRNA (Figs. 3D, 4B, and 5). On the basis of these parameters, athymic CR^-/Y and athymic SCID mice could be classified into incapable and capable mutant strains, respectively, indicating that at least these three events take place or are determined to take place in CP during the early stages of IEL maturation.

Although the in vivo function of _Eß₇ integrin remains to be determined, it has been reported that IEL receive signals for activation through _Eß₇ molecules triggered by E-cadherin on the epithelial cells rather than use this integrin as a homing receptor for the intestinal epithelium (55, 59, 60) and that a decrease of about 2-fold in the number of IEL is observed in E-deficient BALB/c mice (74). We revealed that major TCR^-Ly5.2⁺ IEL from athymic CR^-/Y mice without CP failed to express _Eß₇, whereas major TCR^-Ly5.2⁺ IEL from athymic SCID mice with CP expressed _Eß₇, indicating an important role of CP in the expression of _Eß₇ integrin on these putative TCR^- IEL precursors.

The fact that mRNA for CD3 molecules was hardly detectable in IEL and MLN cells of athymic CR^-/Y mice, whereas the same mRNA was detectable in cells from CP and IEL but not MLN compartments of athymic SCID mice (Fig. 5), favors a scheme approving the sequential in situ maturation of precursor IEL in CP followed by intraepithelium. Compartmentalization of T lineage committed precursors in gut CP was also verified in the present study by showing: 1) TCR^- CP cells and TCR^- IEL from athymic SCID mice contained a comparable amount of pre-T-specific (Fig. 5) and germline TCR-Cß-specific (Fig. 7B) mRNA; and 2) although our previous immunohistochemical study failed to demonstrate the presence of RAG-1-bearing cells in CP (26), a low level of RAG-2 transcripts relative to that seen for thymocytes from WT mice was detected by RT-PCR analysis in lymphoid CP cells from WT and athymic SCID mice (Fig. 6) as well as athymic nude mice (data not shown). Taken together, the results indicate that T cells mature in CP only in small numbers and/or at a slow rate. Moreover, both CD8⁺ and CD8^- IEL subsets from athymic SCID mice displayed a comparable level of signal for CD3 transcripts (Fig. 8), and most CP cells (>99%) from the same animals did not express CD8 molecules (data not shown). These findings support our contention that the commitment of putative IEL precursors to express CD8 is also achieved during early IEL maturation in CP, whereas actual cell-surface expression of CD8 starts after migration of such cells into the epithelium. Endorsing this scenario, our preliminary immunohistochemical analysis of the small intestines from irradiated and WT bone-marrow reconstituted athymic CR^-/Y mice revealed the emergence of donor-derived TCR^- IEL during an early and confined time period after reconstitution in the restricted epithelial areas beneath which histogenesis of CP filled with donor-derived lymphoid cell was detected (our unpublished observation).

It is also important that both Dß-Jß and V-D-J gene rearrangements were hardly detectable in TCR^- IEL of athymic CR^-/Y mice (Fig. 7A). Because thymocytes and TD IEL from euthymic CR^-/Y mice not only displayed abundant CD3 signals but also included cells that undergo Dß-Jß and V-D-J joining, the machinery necessary to carry out these genetic events is not crippled by the mutation and is retained by TCR^- IEL of athymic CR^-/Y mice, suggesting that the permissive gut microenvironment in which immature IEL drive such machinery is canceled in the CR^-/Y condition most likely due to the lack of CP. Consistent with the status of TCR-ß gene rearrangement in the athymic/euthymic CR^-/Y condition, it has recently been demonstrated that major CD3^-CD8⁺CD16⁺ IEL from CD3 gene-deficient mice fail to undergo Dß-Jß joining despite normal rearrangements at the TCR-ß locus in thymocytes from these animals (24). However, unlike TCR^- IEL of athymic CR^-/Y mice, it should be pointed out that V-D-J rearrangements were detected in sorted CD3^-CD8⁺CD16⁺ IEL from CD3-deficient mice (24). All in all, their data (24) in conjunction with our present findings illuminate an early role of CD3 in IEL maturation and provide another distinction between TD and TI IEL by establishing that TCR-ß gene rearrangement is controlled differentially in the thymus and intestine.

In conclusion, the majority of TCR^- IEL isolated from athymic CR^-/Y mice retain a similar distinguishing characteristic from the recently described B220⁺ fetal liver T lymphoid progenitors (40) and HSA^lowc-kit^lowThy-1⁺CD3^- T/NK progenitor cells present in the fetal blood and spleen (53) and perhaps represent the most primitive gut T lineage-committed precursors passaged directly in small numbers into the epithelium without prior differentiation in CP, i.e., their development is at a standstill before the onset of TCR gene rearrangements, CD3 gene transcription, and _Eß₇ expression but after expression of pre-T and germline TCR-Cß-specific mRNA.

	Acknowledgments

 
We thank Dr. L. Lefrancois for valuable discussions, Drs. C. M. Parker, J. R. Klein, and H. Kiyono for comments and encouragement, and K. Ishimaru for technical assistance.

	Footnotes

 
¹ This work was supported by a Grant-in-Aid for Scientific Research (A), Ministry of Education, Science, Sports, and Culture of Japan, by a Health Science Research Grants, The Ministry of Health and Welfare, and by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (to S. K.), and by a Grant-in-Aid for Scientific Research, Ministry of Education, Science, Sports, and Culture of Japan, by the Agency of Science and Technology, Japan, by the Japan Society for the Promotion of Science (JSPS-RFTF 97L00701), and by a Keio Gijuku Academic Development Funds (to H. I.).

² Address correspondence and reprint requests to Dr. Hiromichi Ishikawa, Department of Microbiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address:

³ Abbreviations used in this paper: IEL, intestinal intraepithelial T cells; 7R, IL-7R -chain; 2Rß, IL-2R ß-chain; CP, cryptopatches; CR, common cytokine receptor -chain; LP, lamina propria; MLN, mesenteric lymph nodes; nu/nu, athymic nude; PI, propidium iodide; PP, Peyer’s patches; RAG, recombination activating gene; TD, thymus-dependent; TI, thymus-independent; WT, wild type; B6, C57BL/6J Jcl; B/c, BALB/cA Jcl; E, external; I, internal.

Received for publication October 18, 1999. Accepted for publication January 14, 2000.

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