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Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-, IL-4-, and IL-5-secreting T cells were decreased in the Peyer’s patches, mesenteric lymph nodes, and intestinal mucosa of transgenic mice. Adoptive transfer of OVA-specific CD8+ (OT-I) T cells resulted in their preferential expansion in the Peyer’s patches and mesenteric lymph nodes and subsequently in the epithelia and lamina propria but failed to cause mucosal inflammation. Thus, CFSE-labeled OT-I cells greatly proliferated in these tissues by 5 days posttransfer. Strikingly, OT-I cell-transferred Fabpl4x at –132-OVA transgenic mice underwent a transient weight loss and developed a CD8+ T cell-mediated acute enterocolitis 5 days after oral L. monocytogenes-encoding OVA infection. These findings indicate that intestinal epithelium-derived "self-Ag" gains access to the mucosal immune system, leading to Ag-specific T cell activation and clonal deletion. However, when Ag is presented in the context of bacterial infection, the associated inflammatory signals drive Ag-specific CD8+ T cells to mediate intestinal immunopathology. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several mechanisms have been proposed to participate in peripheral T cell tolerance, including immunologic ignorance, deletion of autoreactive T cells, and suppression through CD25+CD4+ regulatory T cell activation and regulatory cytokines (5). However, this issue remains elusive in the gut mucosa, which represents unique anatomical and physiological features that affect the local immune system. Mucosal immune tolerance is the induction of nonresponsiveness to components of the intestinal flora, which plays a pivotal role in preventing hypersensitivity reactions to luminal Ags. Once breakdown of this immunoregulation occurs, an unregulated effector T cell response to enteric bacteria or food leads to mucosal inflammation. Previous work has shown that DC and CD4+ T cells in the PP preferentially secrete IL-10 in response to stimulation, which may mediate natural tolerance to luminal Ag (6, 7). Repeated oral administration of low-dose Ag leads to oral tolerance and the development of CD4+ T cells that secrete high levels of IL-4, IL-10, and TGF-
(8). CD25+CD4+ T cells also play an important role in inducing mucosal immune tolerance to luminal flora and prevention of murine colitis (9). They may mediate bystander suppression via the secretion of immunoregulatory cytokines such as TGF- and IL-10 or indirect inhibition via a cognate interaction between membrane-bound molecules such as CTLA-4 on T cells and B7 molecules on APC (9). They suppress both proliferation and IFN- production by CD8+ T cells induced by either polyclonal or Ag-specific stimulation (10), and also restrict memory CD8+ T cell responses in vivo (11). Ag-specific CD25+CD4+ regulatory T cells are also observed in vivo and show potently suppressive roles in down-regulating Ag-specific T cell activation (12, 13, 14). Interestingly, these Ag-specific regulatory T cells also prevent Helicobacter hepaticus-induced colitis in T cell-deficient mice (15). Although over the past few years our understanding of mucosal tolerance has markedly increased, a precise characterization of tolerance induction remains to be achieved with regard to relationships with the intestinal epithelial barrier, luminal Ag uptake and presentation, and the potential role of regulatory T cells and related cytokines in the gut.
A significant impediment to advances in understanding of intestinal mucosal T cell homeostasis and pathogenic responses is the lack of identified target Ag and their corresponding TCR specificities. In our laboratory, systems have recently been designed to address Ag-specific immune responses in the intestinal mucosa. This includes a transgenic (Tg) model system in which we have studied CD8+ T cell activation and tolerance induction in response to a nominal Ag exclusively expressed in small bowel IEC (16). We have used MHC class I tetramers and adoptive transfer of CD8+ T cells to allow direct quantification and functional analysis of mucosal Ag-specific CD8+ T cells. To further study the role of mucosal CD8+ T cells reactive to self-Ag in the induction of tissue- and Ag-specific damage of gut mucosa or the development of autoimmune bowel diseases, we established a novel (Fabpl4x at –132-OVA) Tg mouse, wherein a truncated chicken OVA gene was placed directly under control of a modified liver fatty acid-binding protein promoter (Fabpl4x at –132), allowing constitutive truncated OVA expression in IEC of the terminal ileum and colon (17). These mice were used to examine IEC-derived Ag presentation, and mucosal Ag-specific CD8+ T cell activation, tolerance induction, the polarization of immune reactivity to OVA in the terminal ileum and colon, and particularly the induction of autoimmune bowel disease. In this study, we show a critical role of IEC-derived Ag in the induction of mucosal CD8+ T cell activation and tolerance, and demonstrate that cross-reactive Ag-associated inflammatory signals promote Ag-specific CD8+ T cell activation, leading to acute enterocolitis.
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Materials and Methods |
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Fabpl4x at –132-OVA Tg mice were generated using a construct containing the transcriptional regulatory elements of the modified rat liver FABP promoter (nt –596 to +21, with four additional tandem repeats of nt –172 to –133 added at nt –132, namely, Fabpl4x at –132, generously provided by Dr. J. Gordon (Washington University, St. Louis, MO)) (17), which drives target gene expression restricted to IEC of the terminal ileum and colon, cytoplasmic OVA cDNA (encoding aa 138–386), and the human growth hormone gene (nt +3 to +2150). A SalI fragment (7.38 kb) containing these elements was microinjected into C57BL/6 (B6)-Ly5.1 fertilized eggs. Tg mice were identified by PCR analysis of tail DNA samples using the primers (5'-CCC CTT ATA AAA TAG CCA AC-3', 5'-TCA GGC AAC AGC ACC AAC AT-3'), and/or by mRNA dot blot hybridization using a 32P-labeled OVA cDNA probe. Four founders were obtained, and the corresponding lines were established and tested. One of them (2-10 line) characterized by restricted expression of OVA mRNA in the terminal ileum and large bowel, but not in the stomach, duodenum, and liver, was selected for further analysis.
Normal C57BL/6J (Ly5.1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). OT-I mice transgenic for V
2V5 TCR that recognizes an OVA257–264 epitope (SIINFEKL) in the context of MHC class I H-2Kb (provided by Dr. F. Carbone (University of Melbourne, Parkville, Australia)) were maintained on a C57BL/6- Ly5.2+Rag-1–/– background. Mice were housed in a specific pathogen-free facility in microisolator cages with free access to food and water, and were used at 7–12 wk of age.
Reagents and Abs
Cell cultures were performed in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 50 µM 2-ME (all purchased from Invitrogen Life Technologies, Carlsbad, CA). PE- or FITC- or PerCP-conjugated anti-CD8 (53-6.7), PE-conjugated anti-CD4 (RM4-5), FITC-conjugated anti-CD11a (2D7), FITC-conjugated anti-CD45RB (16A), FITC-conjugated anti-CD25 (7D4), FITC-conjugated anti-TCR V2 (B20.1), PE-conjugated anti-TCR-V5.1 (MR9-4), and paired matched mAbs for ELISPOT assay of mouse IFN- (R46A2, XMG1.2), IL-4 (TRFK5, TRFK4), and IL-5 (BVD4-1D11, BVD4-24G2) were purchased from BD Pharmingen (San Diego, CA). Purified anti-Ly5.2 mAb was conjugated to Cy5 (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions.
Listeria monocytogenes (LM) infection
Recombinant LM-OVA, which contains a chromosomally integrated Ag cassette encoding truncated OVA (OVA134–387), was described previously (18, 19). Mice were infected with 109 CFU of LM-OVA or wild-type LM in 200 µl of PBS via gavage, and monitored daily for activities, weight, and soft stool or diarrhea. Animals were sacrificed at different time points and lymphocytes were isolated from the indicated tissues for further analysis.
Adoptive transfer and CFSE labeling
Single-cell suspensions from lymph nodes (LN; cervical, axillary, inguinal, and mesenteric) of Ly5.2+Rag-1–/– OT-I mice were prepared in HBSS. CD8+ LN cells (1 x 106) were injected i.v. into wild-type (WT) or Fabpl4x at –132-OVA Tg mice in 200 µl of PBS. Mice were killed at the indicated time points, and lymphocytes were isolated from the indicated tissues and analyzed for the presence of donor CD8+ cells on a FACSCalibur (BD Biosciences, San Jose, CA). For analysis of short-term proliferation in vivo, CD8+ LN cells were resuspended in HBSS at a concentration of 1 x 107/ml and incubated with 10 µM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C. After two washes, CFSE-labeled OT-I cells (1 x 106) were adoptively transferred i.v. into mice in 200 µl of PBS. Recipients were sacrificed on days 2, 3, and 5. Lymphocytes were harvested from the indicated tissues, and cell division was analyzed on a FACSCalibur according to cellular CFSE content.
Flow cytometric analysis
Cells were resuspended in PBS supplemented with 0.2% BSA and 0.1% NaN3 at a concentration of 1–10 x 106 cells/ml and stained with optimal concentrations of fluorochrome-labeled mAbs and H-2Kb tetramers containing the vesicular stomatitis virus nucleoprotein immunodominant peptide epitope or the OVA-derived peptide SIINFEKL (16). The cells were then fixed in 3% paraformaldehyde buffer and were analyzed on a FACSCalibur using CellQuest software.
Preparation of lymphocytes
MLN and PP were harvested, and single-cell suspensions were prepared using a tissue homogenizer. The resulting cell preparation was filtered through a 70-µm cell strainer, and the filtrate was centrifuged to pellet the cells. Isolation of intraepithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), and lymphocytes from spleen, PP, and MLN was performed as previously described (16, 18).
ELISPOT assay
Lymphocytes secreting IFN-, IL-4, and IL-5 in an Ag-specific manner were detected by ELISPOT assay as described previously (18). Briefly, serial dilutions of lymphocytes (2–10 x 105) were seeded into cytokine-specific capture Ab-precoated 96-well filtration plates, and incubated with 4 x 105 irradiated syngeneic splenocytes for 24 h, with or without 2 µg/ml OVA257–264 peptide (SIINFEKL) or 20 µg/ml OVA265–280 peptide (TEWTSSNVMEERKIKV) or 10 µg/ml listeriolysin O (LLO)190–201 peptide (NEKYAQAYPNVS) (all peptides purchased from Research Genetics (Huntsville, AL)). Wells were sequentially washed and incubated with biotinylated anti-cytokine Abs for 20 h. After incubation with 2 µg/ml peroxidase-labeled anti-biotin Ab (Vector Laboratories, Burlingame, CA) for 20 h, spots were developed with 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO), and then counted using computer-assisted image analysis (KS-ELISPOT; Carl Zeiss, Jena, Germany). A control assay was also performed for each sample using lymphocytes cultured in medium alone or incubated with irradiated syngeneic spleen cells in the absence of peptides. The number of spots observed in the control assay was subtracted from the number of spots counted in each test sample. Data are the mean ± SD per 106 CD8+ or CD4+ T cells. Comparisons between two groups were analyzed using Student’s t test, and values of p < 0.05 were considered significant.
Histology
Intestinal tissues were fixed in PBS containing 10% neutral-buffered formalin and paraffin-embedded sections were stained with H&E. Sections were analyzed and graded using a scoring system according to the number of infiltrating cells and severity of lesions (20).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine whether constitutive expression of Ag in large bowel IEC resulted in T cell tolerance, we generated a new transgenic mouse line (namely, Fabpl4x at –132-OVA), wherein the transgene was placed under control of the modified promoter, Fabpl4x at –132 (17), allowing constitutive expression of truncated OVA within IEC of the terminal ileum, cecum, and colon regions of the gut, which are sites commonly affected in inflammatory bowel disease. Lymphocytes were isolated from various tissues at the day 9 peak of the response and Ag-specific CD8+ T cells were detected using H-2Kb/SIINFEKL tetramer and ELISPOT assay. As shown in Fig. 1, OVA-specific tetramer+CD8+ T cells were found to be highly increased in the spleen, MLN, PP, IEL, and LPL of WT mice, but relatively few, if any, OVA-tetramer+CD8+ T cells were detected in these tissues of Fabpl4x at –132-OVA Tg mice. No tetramer staining was observed in uninfected transgenic or control mice (data not shown). All mice appeared healthy, and histological analysis revealed undetectable epithelial inflammatory damage in small and large bowels. These data indicated that at least those clones with sufficient affinity to bind tetramer had been deleted in Fabpl4x at –132-OVA Tg mice.
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-, IL-4-, and IL-5-producing lymphocytes by ELISPOT assay when stimulated with either the OVA257–264 or the OVA265–280 or the LLO190–201 peptides in vitro. Table I shows that a large number of IFN--, IL-4-, and IL-5-secreting OVA-specific CD8 T cells were observed in the spleen, MLN, and intestinal mucosa of WT mice. These results were in agreement with OVA-tetramer analysis (Fig. 1), indicating that Ag-specific CD8+ T cells generated from LM-OVA-infected WT mice were capable of synthesizing effector cytokines. Interestingly, low numbers of OVA-specific cytokine-secreting CD8+ T cells were also detected in LM-OVA-infected Fabpl4x at –132-OVA Tg mice, indicating that the Ag-specific CD8 response could be detected by ELISPOT assay, despite minimal H-2Kb-tetramer reactivity (Fig. 1). Table II demonstrates a quantitative analysis of functional OVA-specific CD4+ T cells to LM-OVA infection. Substantial populations of OVA-specific CD4+ T cells secreting IFN-, IL-4, and IL-5 were present in WT spleen, MLN, and gut mucosa 9 days postinfection, yet the number was significantly decreased in the indicated tissues of Fabpl4x at –132-OVA Tg mice. We also observed that the frequency of IFN--, IL-4-, and IL-5-producing CD4+ T cells in response to the LLO190–201 peptide was similar in Fabpl4x at –132-OVA Tg and WT mice (Table III), indicating that the loss of OVA-specific CD4 T cells was Ag specific. Additionally, relatively low numbers of IL-10-secreting OVA-specific CD8 and CD4 T cells and LLO-specific CD4 T cells were also found in both groups, and few Ag-specific cells producing any cytokines were seen in uninfected mice. Collectively, these results implied that high-affinity tetramer-binding T cell clones were deleted in Fabpl4x at –132-OVA Tg mice, but that a small but detectable subset of low-affinity T cell clones remained.
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Evidence has demonstrated that material derived from apoptotic or dead IEC could be detected in DC in the MLN (2), suggesting that this may constitute a mechanism of tolerance induction to IEC Ag. To investigate whether IEC-derived OVA was functionally relevant to Ag-specific CD8 T cell activation in vivo, we transferred 106 Ly5.2+Rag–/– OT-I cells i.v. into either Fabpl4x at –132-OVA Tg or WT mice. Fig. 2 shows that, by day 4 after T cell transfer into Fabpl4x at –132-OVA Tg mice, low numbers of donor cells were observed in the spleen and gut mucosa, but were highly increased in the PP and MLN. Interestingly, the population of OT-I cells generated in the PP of the terminal ileum at 4 and 5 days after transfer was greater than that found in the duodenal PP (p < 0.05). By day 5 after cell transfer, the number of OT-I cells had declined in the MLN, whereas the peak number of OT-I cells was present in the PP, IEL, and LP at this time point. Moreover, the number of OT-I cells was substantially larger in the epithelium and LP of the ileum and large bowel compared with the duodenum. This result is in keeping with the predicted expression pattern of OVA in this transgenic line, where the modified Fabp promoter drives protein expression in the terminal ileum and large intestine. By day 6 after transfer, the percentage of OT-I cells had declined in all tissues examined. At later time points, up to 2 mo posttransfer, in some mice a small population of OT-I cells was present in IEL and LP, whereas donor cells could not be detected in the PP, MLN, or spleen. Thus, for the most part, Ag encounter in this system resulted in deletion of the reactive CD8 T cells, with the possibility that small numbers of autoreactive CD8 T cells preferentially remained in the Ag-bearing tissues. Comparative immunohistochemical analysis of cryosections also confirmed OT-I cell infiltrates in the PP, MLN, and mucosal areas, and histologic analysis revealed normal bowel structure (data not shown). These data illustrated that expansion of adoptively transferred OVA-specific OT-I CD8 cells directly correlated with the expected sites of Ag presentation. Thus, the data suggested that OVA Ag, released by apoptotic or dead IEC, was processed and presented by mucosal APC and/or IEC, and induced OVA-specific CD8 T cell activation and expansion. Whether intact protein percolates to the lymphoid tissues where it is processed by resident APC, or whether LP- or PP-resident APC acquire Ag and migrate to the MLN remains to be determined.
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The division of transferred CFSE-labeled alloreactive CD8+ T cells is a very sensitive measure of functional Ag presentation in host mice. To determine MHC class I-restricted cross-presentation of self-Ag leading to the proliferative response of Ag-specific CD8+ T cells, we used CFSE-labeled OT-I CD8+ T cells. When CFSE-labeled OT-I cells were adoptively transferred intoFabpl4x at –132-OVA Tg mice, some cells had divided three to four times in the PP and MLN, but not in the spleen by day 2 posttransfer (Fig. 3). Donor OT-I cells were not observed in the IEL and LPL compartments (data not shown). By day 3 posttransfer, the majority of transferred cells had divided six to eight times in the PP and MLN, as revealed by the sequential loss of CFSE intensity. Cells that had divided were also seen in the spleen at this time, indicating that donor cells were activated in the PP and MLN and subsequently migrated to the spleen, or, alternatively, that they were directly primed by Ag-pulsed DC migrating from the intestinal mucosa and MLN. Moreover, extensive proliferation of CFSE-labeled OT-I cells was seen in the gut, particularly in the LP, indicating the homing of activated CD8+ T cells into the intestinal mucosa. By day 5 posttransfer, all CFSE-labeled OT-I cells detected in gut mucosa and PP of Tg mice had lost detectable CFSE, indicating they had undergone more than eight divisions. OT-I cells in the MLN could be detected at various division steps, and the spleen contained few dividing cells at this time. In contrast, no cells undergoing division were observed in the spleen, PP, and MLN of WT mice, and donor cells were undetectable in the gut mucosa. These results suggested that adoptively transferred OT-I cells were preferentially activated and underwent vigorous proliferation in the PP and MLN in an Ag-driven manner and subsequently migrated to the gut mucosa where further proliferation may have occurred.
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It has been suggested that cross-reactive Ag-associated inflammatory signals can trigger Ag-specific T cell activation and induce autoimmune pathology (16, 21). To determine the potential role of oral infection of LM-OVA in the induction of mucosal autoimmunity, 106 OT-I cells were first adoptively transferred i.v. into Fabpl4x at –132-OVA Tg or WT mice, and 109 CFU of LM-OVA was orally administrated 1 day later. Fig. 4a demonstrates that OT-I cell-transferred Fabpl4x at –132-OVA Tg mice developed an overt disease 5 days after oral LM-OVA infection, displaying a transient weight loss, hunched posture, ruffled fur, and listlessness. Macroscopically, the terminal ileum, cecum, and colon were enlarged. Histological analysis of the terminal ileum and colon revealed acute ileitis and colitis, respectively (Fig. 5, e and f), characterized by a marked increase in the thickness of the mucosal layer and a large number of lymphocytes mixed with some monocytes and neutrophils in mucosa, submucosa, and, to some extent, in the muscular layers and serosa. Epithelial hyperplasia included loss of IEC covering the villi, loss of goblet cells, broadening of villi, and patchy necrosis. Intravillous abscesses were occasionally seen. Minimal inflammatory damage was also observed in the jejunum (Fig. 5d), characterized mainly by slight mucosal hyperplasia and some cellular infiltrates in the LP. Inflammatory changes were undetectable in the duodenum and stomach. Moreover, OT-I cells were found to have a robust expansion in the spleen, MLN, PP, IEL, and LPL in LM-OVA-infected Fabpl4x at –132-OVA Tg mice on day 5 postinfection compared with those in controls (p < 0.05; Fig. 4, c–f). In contrast, no disease was seen in control LM-infected Fabpl4x at –132-OVA Tg mice (Fig. 5, a–c) or LM-OVA-infected WT mice (data not shown), which showed a gradual increase of body weight and normal intestinal structure.
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2 wk after oral LM-OVA infection (Fig. 4b). In contrast, all animals in control groups appeared healthy. A kinetic analysis of transferred OT-I cells demonstrated that the number of harvested OT-I cells rapidly waned in the spleen, MLN, PP, IEL, and LPL in all groups 2 wk after LM-OVA infection (Fig. 4, c–f). Moreover, we also counted bacteria in the spleen and found that only small numbers of bacteria were present by day 6 postinfection (data not shown). These data suggested that inclusion of cross-reactive Ag-associated inflammatory signals (i.e., LM-OVA) facilitated Ag-specific CD8+ T cell activation in intestinal mucosa and was able to override mucosal immune tolerance, leading to the development of acute inflammatory damage at corresponding Ag-expressing sites. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cross-priming by DC is considered to play an important role in initiating MHC class I-restricted immune responses, including CD8 T cell activation and tolerance (22, 23). DC are found disseminated in the PP and LP (2, 3, 6), from which they can migrate to the MLN and perhaps elsewhere. Enterocytes located at the tips of villi are rapidly renewed by ascending enterocytes produced in the intestinal crypts. Thus, DC in the PP and LP could phagocytose either apoptotic or necrotic IEC and present Ag in the context of MHC class I or II. As a consequence, IEC-derived Ag presented by DC could gain access to the intestinal immune system via cross-presentation, thereby eliciting stimulatory or tolerogenic responses. Our studies demonstrated that cross-presentation of IEC-derived Ag by APC in the MLN and PP, led to robust expansion of transferred OT-I cells in the PP and MLN and subsequent migration into intestinal mucosal areas. Thus, these findings indicated that activation of transferred OT-I cells was dependent on TCR recognition of epithelium-derived Ag. It is likely that apoptotic or necrotic enterocytes could be taken up by intestinal DC in situ, after which OVA-loaded DC might migrate to the regional T cell areas of the PP and MLN, and then drive OVA-specific CD8+ T cell proliferation and differentiation followed by deletion, ultimately resulting in tolerance to self-Ag. Additionally, IEC might also act as nonprofessional APC and have the capacity to present OVA Ag in the context of MHC class I molecules to apposed CD8 T cells in the epithelium. This interaction could modulate Ag-specific T cell immune responses (1), and could also be involved in tolerance induction. Such a mechanism would be expected to occur primarily for effector or memory cells, because it is unlikely that many naive CD8 T cells recirculate through the intestinal epithelium.
Clonal deletion in the thymus and/or in the periphery has been considered as a major mechanism for the establishment of tolerance to self (5). Low-affinity interaction between T cells and peptide/MHC may enable autoreactive T cells to escape self-tolerance and thus to persist in the periphery (24), suggesting that the concentration of Ag and/or the affinity of TCR in the responding T cell population are likely to affect the efficacy of cross-presentation leading to deletion or anergy. In our studies, we have found that Ag-reactive CD8+ and CD4+ T cells could indeed be detected by the ELISPOT assay but, for CD8+ T cells, not by H-2Kb-tetramer reactivity in Fabpl4x at –132-OVA Tg mice after oral LM-OVA infection. The latter was likely due to deletion of high-affinity Ag-reactive CD8 T cell clones. These findings were consistent with an earlier report (25), in which CD8+ T cells with high avidity for self-peptide are deleted during tolerance induction, whereas low-avidity CD8 T cells are allowed to persist in the periphery. Moreover, mucosal CD8+ T cell tolerance to self-Ag may be also mediated through activation of inhibitory cytokines or Ag-specific CD4+CD25+ regulatory T cells. Evidence shows that DC and CD4+ T cells in the PP preferentially secrete IL-10 in response to stimulation, which may mediate natural tolerance to luminal Ag (6, 7). Oral tolerance induced by repeated oral administration of low-dose Ag is dependent on the development of regulatory CD4+ T cells that secrete high levels of IL-4, IL-10, and TGF- (8). CD4+CD25+ regulatory T cells also suppress CD8+ T cell immune responses (10, 11). Therefore, maintenance of a tolerogenic state to IEC-derived Ag is also likely to be mediated by the induction and activation of Ag-specific CD4+CD25+ regulatory T cells or inhibitory cytokines such as TGF-, IL-4, or IL-5.
Evidence has shown that LM can reside inside murine enterocytes of the epithelial lining as early as 12 h after bacterial ingestion and reach a maximum 3–4 days later (26). LM survive inside phagocytic cells of deeper intestinal tissues once across the IEC barrier and are rapidly eliminated thereafter (26). LM that are released from infected enterocytes/macrophages or that directly migrate from the intestinal lumen via a transcellular route can access the mucosal immune system, resulting in increased apoptotic death of enterocytes and macrophages, perhaps mediated by CTL activity (19). Thus, intestinal and mesenteric DC might not only simultaneously process bacteria and apoptotic enterocytes but may also sample luminal bacterial Ag directly by means of extending dendrites outside the epithelium (2, 3). Moreover, some proinflammatory cytokines (e.g., IFN-, IL-12, IL-1, TNF-) secreted by mucosal CD4+ and CD8+ T cells and APC during LM infection might also play an important role in triggering mucosal immunopathology (18, 27). Most importantly, cross-reactive Ag (i.e., OVA) released from LM-OVA but not from control LM could further facilitate Ag-specific CD8 T cell activation, including proliferation, cytokine secretion, and cytolysis of infected cells such as IEC. In agreement with this, a large number of OT-I cells were found in the spleen, MLN, PP, and gut mucosa 5 days after oral LM-OVA infection (Fig. 4, c–f), and mucosal inflammatory damage was also concomitantly present (Fig. 5, d–f). However, we also found that infection with LM lacking the cognate Ag was unable to trigger intestinal inflammation. This is in contrast to our previous results in which Ag was expressed in small bowel and where nonspecific inflammatory signals could trigger autoimmunity, although the effects of LM infection were not tested (28). It may also be possible that oral infection with LM in C57BL/6 mice does not result in a sufficiently robust infection to promote pathology. Nonetheless, different infectious agents, as well as the location of Ag expression, could conceivably regulate the induction of intestinal immunity. In agreement with this, data from others (29, 30) show that transfer of polarized Th1 or Th2 cells from Rag–/– DO11.10 mice results in the development of severe colitis in recipients colonized with OVA-expressing but not control Escherichia coli. Therefore, our findings support the possibility that mucosal self-Ag specific CD8+ T cells could be induced in vivo to become autoaggressive by cross-reactive Ag-associated inflammatory stimuli, leading to CD8+ T cell-mediated acute tissue damage at the sites where Ag was expressed.
Recent data have shown that Ag-specific regulatory T cells play an important role in down-regulating Ag-specific T cell activation (12, 13, 14). Interestingly, this subset of CD4+ T cells inhibits disease development of Leishmania major infection in SCID mice reconstituted with CD4+CD25– T cells by suppressing both Th1 and Th2 cell development in vivo (31), suggesting that regulatory T cells play a critical role in modulating responses to infections. In Fabpl4x at –132-OVA transgenic mice, OVA-specific regulatory T cells might be deficient as result of deletion of OVA-specific CD8+ and CD4+ T cells. Therefore, a lack of OVA-specific regulatory T cells could also be involved in the development of mucosal pathology in OT-I cell-transferred Tg mice after LM-OVA infection. The balance between survival and apoptosis of immune cells during an immune response is extremely important, because it may affect the generation of protective memory T cells and may eliminate potentially harmful activated T cells. Our data have shed some light on understanding the pathogenesis of inflammatory gastrointestinal disorders, including Crohn’s disease and ulcerative colitis, which are thought to be associated with a breakdown in tolerance (i.e., the loss of the down-regulation of T cell responses against luminal flora), leading to active immunity to food Ag or commensal bacteria (32). This work supports the concept that transient or perhaps chronic bacterial infection may lead to a breakdown of mucosal tolerance, facilitate mucosal Ag-specific T cell activation, and induce inflammatory damage. Targeting the mucosal lymphocyte immune response through the induction of Ag-specific T cell tolerance and/or Ag-specific regulatory T cells is a potential avenue for preventing intestinal autoimmune disease.
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Footnotes |
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1 This work was supported by U.S. Public Health Service Grant DK57932 (to L.L.). Z.L. is a recipient of a research fellowship award from the Crohn’s and Colitis Foundation of America. 
2 Address correspondence and reprint requests to Dr. Leo Lefrançois, Division of Immunology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: llefranc{at}neuron.uchc.edu
3 Abbreviations used in this paper: PP, Peyer’s patch; DC, dendritic cell; IEC, intestinal epithelial cell; LN, lymph node; MLN, mesenteric lymph node; Tg, transgenic; WT, wild type; LM, Listeria monocytogenes; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte; LLO, listeriolysin O.
Received for publication April 16, 2004. Accepted for publication July 26, 2004.
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W. Liu, D. P. Evanoff, X. Chen, and Y. Luo Urinary Bladder Epithelium Antigen Induces CD8+ T Cell Tolerance, Activation, and Autoimmune Response J. Immunol., January 1, 2007; 178(1): 539 - 546. [Abstract] [Full Text] [PDF]
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R. Chakraverty, D. Cote, J. Buchli, P. Cotter, R. Hsu, G. Zhao, T. Sachs, C. M. Pitsillides, R. Bronson, T. Means, et al. An inflammatory checkpoint regulates recruitment of graft-versus-host reactive T cells to peripheral tissues J. Exp. Med., August 7, 2006; 203(8): 2021 - 2031. [Abstract] [Full Text] [PDF]
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 Y. Chung, J.-H. Chang, M.-N. Kweon, P. D. Rennert, and C.-Y. Kang CD8{alpha}-11b+ dendritic cells but not CD8{alpha}+ dendritic cells mediate cross-tolerance toward intestinal antigens Blood, July 1, 2005; 106(1): 201 - 206. [Abstract] [Full Text] [PDF]
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