Brief Definitive Report |
Address correspondence to Philippe Poussier, Sunnybrook and Women's Health Sciences Centre, 2075 Bayview Ave., Room A3 38, Toronto, Ontario M4N 3M5, Canada. Phone: 416-480-6136; Fax: 416-480-4375; E-mail: ppoussie{at}sten.sunnybrook.utoronto.ca
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Abstract |
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Key Words: regulatory autoreactive TCRß+CD4-CD8
+ß- IEL IL-10 IBD
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Introduction |
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Evidence has accumulated over the last 10 yr which established the T lymphopoietic capacity of the murine intestinal mucosa (25). Indirect evidence supports this conclusion in humans as well (6). Analyses of intraintestinal mononuclear cells (MNCs) developing in athymic radiation chimeras has enabled the phenotypic characterization of the various subsets of gut-derived T cells.
The first important observation is that donor-derived T cells are found on both sides of the basement membrane of the intestinal epithelium in numbers comparable to those observed in unmanipulated animals (2, 4, 7). Among intraepithelial T lymphocytes (iIELs), 3050% express TCR. The remaining CD3+ iIELs express a high level of TCR
ß. 510% express CD4 only, while 530%, in an age- and strain-dependent fashion, coexpress high levels of CD4 and the
chain of CD8. The remaining 6080% of TCR
ß+ iIELs are divided into two subsets of roughly equal size, those expressing only the
chain of CD8, and those coexpressing CD8
and CD8ß. In humans, the latter two TCR
ß+ subsets account for virtually all iIELs (8).
Murine TCRß+ iIELs are also distinguished based on their differential expression of functional antigen receptor complexes. Specifically, the CD4+CD8- and CD4- CD8
+ß+ subsets express functional TCR/CD3 complexes (4, 7, 9), while CD4+CD8
+ß- and CD4-CD8
+ß- TCR
ß+ iIEL subsets are refractory to antigen, superantigen, anti-TCR
ß stimulation (4, 7, 9). However, these latter subsets do respond to lectin (4), and mAb specific for CD3 (4), in vitro.
There is evidence that the selection of CD4- CD8+ß-TCR
ß+ iIELs is unique (4, 10, 11). Specifically, this subset is comprised of a mixture of cells, some specific for antigen presented by classical MHC class I molecules (12), and others recognizing antigen presented by molecules encoded by the Qa-2 locus (13). What is remarkable is that the development of CD4-CD8
+ ß-TCR
ß+ iIELs restricted by classical MHC class I is predicated by the expression of high affinity, self-specific TCR (10).
The first indication that iIELs play a role in the maintenance of mucosal integrity was derived from the analysis of IL-2/IL-2Rdeficient mice, which are prone to IBD (14). The analysis of euthymic and athymic hemopoietic radiation chimeras demonstrated the lack of intraintestinal T lymphopoiesis in IL-2-/-, IL-2R-/-, and IL-2Rß-/- mice. The absence of IBD in athymic radiation chimeras, and the presence of intestinal inflammation in euthymic recipients of hemopoietic precursors derived from the above three strains demonstrated the central role of thymus-derived T cells resident in the intestinal mucosa as mediators of disease, and the importance of a normal complement of gut-derived T cells in the maintenance of intestinal integrity. The above results are consistent with previous results demonstrating that normal thymus-derived T cells can induce gut inflammation upon adoptive transfer to T celldeficient animals (15, 16). Specifically, when low numbers of CD4+CD45RBhi T cells of thymus origin cells are passively transferred into SCID mice, lacking both gut- and thymus-derived T cells, donor T cells migrate to the intestinal mucosa, expand, secrete proinflammatory cytokines, and induce an ulcerative colitis-like syndrome in the recipients (16). Furthermore, and in the context of this model system, it was demonstrated that TCR
ß+CD4+ CD45RBlow T cells of thymus origin prevented the development of this syndrome (16), and did so in an IL-10dependent fashion (17).
In this study, we directly assessed the capacity of specific iIEL subsets to protect the intestinal mucosa from chronic inflammation induced by TCRß+CD4+CD45RBhi T cells of thymic origin.
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Materials and Methods |
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Monoclonal Antibodies, Three-Color Immunofluorescence, FACS® Analysis, Cell Preparation, Cell Sorting, and Cell Transfer.
The preparation of iIELs and lamina propria (LP) lymphocytes, and the monoclonal antibodies used in this study have been described previously (4, 7, 14, 16). Cells were then analyzed by flow cytometry on a Becton Dickinson FACSCaliburTM or sorted using a Moflo (Cytomation, Inc.). At least 104 cells/sample were acquired for analysis. The absolute number of cells within an MNC subset was calculated by multiplying the total number of MNCs isolated by the proportion of cells accounted for by this subset. The total number of MNCs from the intestinal epithelium and lamina propria was determined before their purification by discontinuous Percoll gradient centrifugation.
For sorting, iIELs and splenocyte suspensions were prepared and stained in sterile conditions with the appropriate combination of fluorochrome-conjugated antibodies (4). The purity of sorted iIEL and splenocyte subsets was analyzed before their injection into SCID recipients and was always 99%.
For iIEL reconstitution, a total number of 5 x 106 cells from each of the studied iIEL subsets was administered intravenously to the SCID recipients. Adoptive transfer of TCRßCD4+ CD45RBhi splenic T cells (0.5 x 106/recipient) was performed intravenously 1 wk after the iIEL reconstitution.
Histology.
The gut of experimental animals was differentially processed for histological examination depending on whether suspensions of MNCs from the intestinal epithelium and lamina propria were also prepared for flow cytometry analysis. Prior to the preparation of suspensions of gut-derived MNCs, 3 proximal, median, and distal fragments (510 mm long) of both small and large bowel were collected for histological examination. For the other experimental animals, the entire length of small and large bowel from SCID recipients was wrapped to form a roll before fixation in 10% neutral buffered formaldehyde for 24 h. Specimens were then embedded in paraffin and three 6-µm sections were taken 250 µm apart through the entire thickness of the bowel loops. Each section was stained with hematoxylin and eosin. Each section was examined and scored in a blind fashion by one of us (D. Banerjee) as described (17). Briefly, grade 0 corresponded to the absence of histological abnormalities; grade 1 to the presence of minimal and scattered inflammatory cell infiltrates, with or without minimal epithelial hyperplasia; grade 2 to mild scattered to diffuse inflammatory cell infiltrates, sometimes invading the submucosa and associated with erosions, with minimal to mild epithelial hyperplasia, and minimal to mild loss of mucin from goblet cells; grade 3 to mild to moderate inflammatory cell infiltrates, sometimes transmural, often associated with ulceration, with moderate epithelial hyperplasia, and loss of mucin; grade 4 to severe inflammatory cell infiltrates, often transmural and associated with ulceration, with marked epithelial hyperplasia, and loss of mucin; grade 5 to severe, transmural inflammatory cell infiltrates, with severe ulceration and loss of intestinal crypts.
Statistical Analysis.
The Mann-Whitney test and log-rank analysis were applied to compare histological scores and Kaplan-Meier survival curves, respectively. Student's t test was applied to weight variations. Differences were considered as statistically significant with P < 0.05.
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Results and Discussion |
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The results presented herein demonstrate a heretofore unrecognized function of iIELs. Specifically, of the two major subsets of iIELs in rodents and humans (8, 11, 19), those expressing CD4-CD8+ß-TCR
ß+ and those expressing CD4-CD8
+ß+TCR
ß+, we demonstrate that the former but not the latter can function to protect gut integrity in the presence of thymus-derived inflammatory T cells. In this regard, it is of note that there is precedent for the role of iIELs in modulating systemic immune responses. Specifically, TCR
+, iIELs have been shown to modulate low dose oral tolerance induction in an IL-10dependent fashion (20).
Protective iIELs Regulate Numbers of CD4+ T Cells in the Colonic Lamina Propria.
The use of CD45 congenic C57Bl/6 lines enabled us to discriminate amongst recipient, donor-derived splenic T cells and iIELs, and hence to assess the fate of donor-derived T cell subsets.
Tracking of donor T cell subsets using this strain combination demonstrated that the inability of an iIEL subset to afford protection was not related to its capacity to reconstitute the iIEL compartment of the recipient. As illustrated in Table I, >95% of donor (Ly 5.2) iIELs were rescued from the iIEL compartment of recipient small bowel while <2% of these donor cells were rescued from secondary lymphoid organs. Further, the total number of donor-derived CD4-CD8+ß+TCR
ß+, CD4-CD8
+ß-TCR
ß+, and TCR
+ iIELs recovered was not significantly different, and hence differential reconstitution did not correlate with protection. Of note, the number of rescued cells approximated the number of cells injected, and their membrane CD4/CD8 phenotype remained unchanged.
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The lamina propria of the large bowel was the exclusive site where the number of cells derived from Ly 5.1 TCRß+CD4+CD45RBhi T cells was differentially and significantly affected by the subset of reconstituting iIELs. Specifically, 89-fold more CD4+ T cells were rescued from this site in animals that developed colitis compared with those healthy recipients of protective CD4-CD8
+ ß-TCR
ß+ iIELs (Table I).
It is striking that protective iIELs were rescued almost exclusively in the small bowel. While consistent with the location of this subset in conventional animals, this study reveals two remarkable facts. The cells derived from CD4+CD45RBhi TCRß+ T cells expanded 50100-fold in all animals, independent of iIEL reconstitution. However, the majority of the progeny were found in the intestinal mucosa of the small bowel, which was free of pathology. The influence of the reconstituting iIEL subsets was only observed in the lamina propria of the large bowel. We conclude that progeny of CD4+CD45RBhiTCR
ß+ T cells resident in the small bowel must not be pathogenic, and that the protective function of CD4-CD8
+ ß-TCR
ß+ iIELs does not involve direct interaction with inflammatory effector cells.
Protection Against Colitis Afforded by CD4-CD8+ß- TCR
ß+ iIELs Is IL-10dependent.
When adoptive transfer of Ly 5.1 TCRß+CD4+CD45RBhi splenic T cells was preceded by reconstitution of the recipients with 5 x 106 CD4-CD8
+ß-TCR
ß+ iIELs derived from IL-10-/- donors, mild to severe colitis developed in 9/10 animals, compared with 1/10 animals reconstituted with IL-10+/+ CD4-CD8
+ß-TCR
ß+ iIELs (Figs. 2 C, 3, and 4, and Table I). Importantly, this lack of protection was not related to an impaired iIEL reconstitution, as the number of donor-derived CD4-CD8
+ß-TCR
ß+ iIELs rescued approximated the number of injected cells independent of their IL-10 genotype (Table I). Notwithstanding, the number of cells derived from donor TCR
ß+CD4+CD45RBhi T cells within the lamina propria of the large bowel was significantly higher in recipients of IL-10-/- CD4-CD8
+ß- TCR
ß+ iIELs (Table I), and comparable to that observed in both unreconstituted animals and recipients of nonprotective, IL-10+/+ iIELs (Table I). Of note, reconstitution of SCID.B6 mice with CD4-CD8
+ß-TCR
ß+ iIELs derived from IL-10-/- donors did not result in the development of colitis in the absence of subsequent injection of TCR
ß+CD4+CD45RBhi splenic T cells (data not shown).
The IL-10dependent protection afforded by CD4- CD8+ß-TCR
ß+ iIELs is consistent with a previous report demonstrating that administration of exogenous IL-10 to recipients of CD4+CD45RBhiTCR
ß+ T cells prevents colitis (18). However, we have been unable to obtain evidence for the capacity of CD4-CD8
+ß- TCR
ß+ iIELs to secrete IL-10. Specifically, we have assessed the secretion of IL-10 by FACS®-purified (>99%) CD4-CD8
+ß-TCR
ß+ iIELs in vitro, without stimulation, as well as in response to a combination of plate bound anti-CD3 and anti-CD28 monoclonal antibodies. In both circumstances, CD4-CD8
+ß-TCR
ß+ iIELs failed to secrete IL-10 (data not shown). Further, we did not detect surface expression of IL-10R on CD4-CD8
+ß-TCR
ß+ iIELs flow cytometrically (data not shown). These results, which are consistent with those of a recent study showing very low levels of transcripts for both IL-10 and IL-10R in iIELs expressing TCR
ß (21), strongly suggest that the IL-10 dependency of protection conferred by this subset is indirect. Importantly, murine enterocytes which are in physical contact with iIELs, constitutively express IL-10R (22). It is not implausible that the differentiation of CD4- CD8
+ß-TCR
ß+ iIELs into effector regulatory T cells depends on signals generated by surrounding enterocytes induced by IL-10.
Self-specific CD4-CD8+ß-TCR
ß+ iIELs Protect Against Colitis.
One of the paradoxes of intraintestinal T cell development is the presence of CD4-CD8+ß- TCR
ß+ iIELs expressing potentially self-specific TCR. In conventional mice this subset, while oligoclonal, contains high proportions of cells expressing Vß specific for endogenous retroviral superantigens (4, 11, 23). Analysis of RAG-deficient animals transgenic for MHC class Irestricted TCR has demonstrated that CD4-CD8
+ß- TCR
ß+ iIELs expressing the transgenic TCR develop in animals that express both the restricting MHC and the specific antigen, exclusively (7, 10, 12). Thus, 100% of the CD4- CD8
+ß- iIEL subset in RAG-/- H-2Db male mice transgenic for a H-Y/H-2Db-specific TCR
ß, express high levels of the transgenic TCR. This enabled us to directly assess the protective capacity of self-specific CD4- CD8
+ß- iIEL against intestinal inflammation.
Toward this end, 5 x 106 CD4-CD8+ß- iIELs derived from RAG-/- H-2Db male mice transgenic for a H-Y/H-2Db-specific TCR
ß were used to reconstitute Ly 5.2 SCID.B6 male (n = 5) and female (n = 3) recipients. As illustrated in Fig. 5 , of the male recipients only one developed severe colitis within 150 d after the transfer of 0.5 x 106 splenic CD4+CD45RBhi T cells derived from male Ly 5.1 C57Bl/6 donors (Fig. 5). The female recipients, which provided a specificity control in this system, all developed severe colitis within the same time frame (Fig. 5).
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It is tempting to speculate that CD4-CD8+ß-TCR
ß+ iIEL, present in other species, including humans (8) fulfill a similar role in the maintenance of intestinal integrity. The observation that exogenous IL-10 is inefficient in ameliorating symptoms of inflammatory bowel disease in humans (25), would suggest that, if protective, human CD4-CD8
+ ß-TCR
ß+ iIELs, may not function in an IL-10dependent manner. However, results obtained in murine models of colitis underscore the importance of the methodology used to assess the role of IL-10 in regulating IBD. Specifically, the first attempt to demonstrate its role, involved the administration of neutralizing antibodies, and was unsuccessful. In contrast, subsequent studies established that either systemic administration of IL-10, or the forced expression of IL-10 in CD4+CD45RBhi TCR
ß+ T cells, prevented colitis (18, 26). Hence, resolution of the apparent discrepancy regarding the role of IL-10 in humans may require the assessment of various regimens of IL-10 administration, minimally the assessment of outcome in circumstances in which it is certain that IL-10 reaches the target organ. Furthermore, murine models of inflammatory bowel diseases have not established the capacity of IL-10 to reverse the course of ongoing colitis, rather, its effectiveness in preventing the development of chronic inflammation. It is therefore inappropriate to compare the preventive role of IL-10 in murine colitis with its curative role in humans with established inflammatory bowel disease.
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Acknowledgments |
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This work was supported by the Canadian Institutes of Health Research.
Submitted: October 24, 2001
Revised: February 15, 2002
Accepted: March 27, 2002
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References |
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