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CD8 Intraepithelial Lymphocytes Does Not Abolish Their Self-Tolerance in the Intestine1
* Institute of Pathology, Division of Immunopathology, University of Bern, Bern, Switzerland; and
Department of Pathology, Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
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Abstract |
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CD8 intestinal intraepithelial lymphocytes (IEL) represent an enigmatic subset of T cells, particularly, in regard to their potential functions and the apparent persistence of cells expressing self-specific TCR. We have used mice that are transgenic for the TCR specific for the lymphocytic choriomeningitis virus (LCMV)-derived peptide gp33, and TCR-transgenic mice that coexpress the gp33 Ag ubiquitously, to analyze the functional properties of TCR CD8 IEL in the presence, or absence, of their specific MHC-restricted Ag, and to assess the impact of molecular mimicry during a potent LCMV infection on potentially self-reactive TCR CD8 IEL. In this study, we show that the presence of the specific self-Ag results in reduced expression of IL-2, IFN-
, and IL-10 by resident TCR CD8 IEL while expression of mRNA for TGF is not affected. We further demonstrate that despite their secluded location in the epithelium, TCR CD8 IEL are activated after infection of the intestinal mucosa with LCMV. Importantly, LCMV-induced activation of self-specific TCR CD8 IEL does not reverse their tolerance as no cytotoxic activity or up-regulated expression of proinflammatory cytokines is detected and no overt signs of autoimmunity are seen. Taken together, these results are in support of an immunoregulatory role for self-specific TCR CD8 in the intestinal mucosa and clearly speak against an involvement of this cell subset in inflammatory reactions and tissue destruction. |
Introduction |
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TCR and a CD8 coreceptor; the rest, which expresses an TCR, can be subdivided into equal parts according to the expression of the heterodimeric (CD8) or homodimeric (CD8) form of the coreceptor (1). TCR CD8 IEL are subject to thymic selection (2) and can exert Ag-specific cytotoxicity against various pathogens (3, 4, 5, 6). Thus, they closely resemble their CD8+ counterparts in spleen and lymph nodes and may be derived from peripheral T cells that have entered the IEL compartment upon activation (7, 8). In contrast, TCR CD8 cells are restricted to the intestinal mucosa and bear features that are clearly distinct from peripheral T cells. Although it is now hypothesized that the thymus is involved in the selection process (9, 10, 11), at least some of the CD8 IEL may develop extrathymically (reviewed in Refs.12 and 13), and although all TCR CD8 IEL require 2-microglobulin expression, they are not exclusively restricted by the classical MHC class I molecules K and D (14, 15). Moreover, antigenic signals received by CD8 IEL are likely to be different from those received by peripheral T cells, since TCR CD8 IEL use different signaling molecules (16), and the CD8 coreceptor can modulate TCR transmitted signals by its semiautonomous interaction with the thymus leukemia (TL) Ag (17). Because of the unknown Ag specificity of CD8 IEL in wild-type mice, TCR CD8 IEL were mainly studied in TCR-transgenic (tg) models. These models collectively presented the intriguing finding that TCR CD8 IEL are not subject to negative selection as these cells persisted in TCR tg mice that coexpressed the specific MHC class I-restricted Ag (18, 19, 20, 21, 22). In normal mice, CD8 T cells are also enriched for cells expressing V segments reactive for endogenous superantigens (23, 24). Furthermore, it was recently claimed that the selection of CD8 T cells appears to involve a true positive selection process as opposed to an escape of negative selection (10). Thus, it is tempting to speculate that TCR CD8 IEL, which are specific for self-Ags, may fulfill particular functions in the intestinal mucosa. In contrast, these cells have a potential for autoreactivity and may cause chronic inflammation. With regard to the functional responses of self-specific TCR CD8 IEL, however, so far rather discrepant results have been obtained. Although some studies suggested that these cells are functionally active (10, 18, 21), others observed a hyporesponsiveness to antigenic stimulation (19, 20, 24). To directly investigate the potential functions exerted by TCR CD8 IEL in situ, we have used mice tg for the TCR specific for the lymphocytic choriomeningitis virus (LCMV) (3)-derived immunodominant epitope gp33 and TCR tg mice coexpressing the gp33 Ag. The gp33 transgene is under the control of a MHC class I promoter, resulting in a strong expression of gp33 also on intestinal epithelial cells. This model not only allowed us to reassess the effect of an ubiquitously expressed MHC class I-restricted Ag on selection and function of the TCR CD8 IEL subset, but also to look at the impact of a potent LCMV infection on potentially self-reactive TCR CD8 IEL. |
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C57BL/6 mice tg for the LCMV gp33-specific TCR (line 318) (25) and C57BL/6 mice tg for the LCMV gp33 (line H8) (26) were provided by H. Hengartner and R. M. Zinkernagel (University Hospital Zurich, Zurich, Switzerland). The tg mice were backcrossed to RAG2-/- mice, originally obtained from E. Wagner (Basel Institute for Immunology, Basel, Switzerland). 318 x RAG2-/- and H8 x RAG2-/- mice were then intercrossed to obtain double tg 318 x H8 x RAG2-/- mice. All mice were kept under specific pathogen-free conditions in the central animal facility of the Medical School, University of Bern (Bern, Switzerland). Experiments were performed in compliance with the regulations approved by the local committee on animal experimentation.
LCMV infections
A stock of LCMV strain WE was originally provided by S. Oehen (Zurich, Switzerland). Briefly, 2 x 104 PFU of LCMV-WE per mouse in a volume of 100 µl of MEM and 2% FCS were injected i.p.
Preparation of IEL
IEL were isolated from the small intestine as described previously (27). In brief, the entire small intestine was placed in Ca2+- and Mg2+-free HBSS, 10 mM HEPES, and 2% horse serum (HBSS-2). After longitudinally opening the gut, the tissue was cut into pieces of 1–2 cm each. Enterocytes and IEL were detached from the basement membrane by incubating pieces in HBSS-2 containing 2 mM DTT and 0.5 mM EDTA at 37°C with stirring. Isolated cells were sequentially passed through 70- and 40-µm pore size nylon mesh strainers (BD Biosciences, San Jose, CA). The IEL were then purified from enterocytes by 44/68% discontinuous Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation (15 min, 1000 x g, room temperature).
mAbs and gp33 tetramers
Fluorescence- or biotin-conjugated mAbs used in this study were either purchased from BD PharMingen (San Diego, CA; CD8 (53-6.7), CD8 (53-5.8), CD69 (H1.2F3), V2 (B20.1), V8 (MR5-2)) or purified by protein G columns from supernatants of hybridomas originally obtained from American Type Culture Collection (Manassas, VA) and subsequently labeled according to standard protocols (CD3 (145-2C11); TCR (GL3)). Soluble H-2Db tetramers complexed with biotinylated 2-microglobulin and gp33 peptide were prepared as described elsewhere (28, 29) and were kindly provided by R. M. Zinkernagel (Zurich, Switzerland).
FACS analysis
Cells were taken up in PBS-buffered 4% paraformaldehyde and acquired on a FACScan (BD Biosciences) by gating on the lymphocyte population in the forward and side angle scatter. Stainings were analyzed using CellQuest software (BD Biosciences).
Cell sorting
For cell sorting, cells were stained with mAb against V2, CD8, and CD8 and subsequently separated on a FACSVantage (BD Biosciences) into V2+CD8+CD8- and V2+CD8+CD8+ fractions (318 x RAG2-/- mice) and V2+CD8+CD8- and V2+CD8- fractions, respectively (318 x H8 x RAG2-/- mice). When cells were used for in vitro assays, staining for V2 was omitted and cells were sorted according to the expression of CD8 and CD8 as described above.
Proliferation assay
Briefly, 5 x 104 sorted cells were stimulated in triplicates in 96-well round-bottom microtiter plates (Costar, Cambridge, MA) with plate-bound anti-CD3 (10 µg/ml) or with 3 x 105 irradiated H8 x RAG2-/- spleen cells in a total volume of 200 µl of IMDM supplemented with 10% FCS. In some experiments, 50 U/ml IL-2 (Proleukin; Roche, Basel, Switzerland) was added. Cells were cultured for 48 h, pulsed with 1 µCi/well [3H]thymidine, and harvested 18 h later.
Analysis of cytokine expression
Briefly, 1 x 105 sorted cells were stimulated with plate-bound anti-CD3 (10 µg/ml) in 200 µl of IMDM/10% FCS. For the IL-2 ELISA, supernatants were harvested from triplicate wells after 48 h, and IL-2 was detected using the anti-IL-2 mAb JES6-1A12 as a coating Ab and the mAb JES6-5H4 as a detecting Ab (BD PharMingen). The standard curve was constructed using recombinant mouse IL-2 (PeproTech, London, U.K.). For analysis of IL-2 mRNA expression, cells were harvested after 6 h and resuspended in 1 ml of Tri-Reagent (Molecular Research Center, Cincinnati, OH). For analysis of ex vivo mRNA expression, cells (1–2 x 105) were immediately taken up in 1 ml of Tri-Reagent after cell sorting.
RNA isolation and cDNA synthesis
RNA was isolated by phenol-chloroform extraction and Tri-Reagent according to the manufacturer’s (Molecular Research Center) directions. RNA was DNase digested and reverse transcribed with random primers using a commercial cDNA kit (Promega, Madison, WI) and following the manufacturer’s suggested conditions.
Real-time PCR
Serial dilutions of cDNA were made to determine the linear range for amplification. Real-time PCR was performed with appropriate dilutions of cDNA using the Applied Biosystems PRISM 7700 sequence detector and either Assays-on-Demand (IFN-, TNF-, IL-10, and TGF) or the dsDNA binding dye SYBR Green (IL-2 and IL-4; Applied Biosystems, Foster City, CA). PCR amplifications were performed in a total volume of 25 µl according to the manufacturer’s instructions (Assays-on-Demand) or using SYBR Green PCR Master Mix and 300 nM of the appropriate primers (IL-2: 5'- TGGAGCAGCTGTTGATGGACCTAC-3', 5'-AGATGATGCTTTGACAGAAGGCTATC-3'; and IL-4: 5'-AAACGTCCTCACAGCAACGA-3', 5'-GGCTTTCCAGGAAGTCTTTCAG-3').
The real-time PCR was evaluated using the Sequence Detection System software version 1.7 (Applied Biosystems). Expression of specific cytokine genes was normalized based on levels of mRNA for 18S rRNA.
Cytotoxicity assay
The cytotoxicity assay was performed as described previously (30). Briefly, sorted effector cells were placed in a V-bottom microtiter plate (Costar) and serially diluted. A total of 3 x 104 RMA target cells, labeled with 75 µCi Na51Cr and pulsed with 1 µg/ml gp33 or adn5, was added per well of the assay plate. After incubating the plate for 5 h at 37°C in 5% CO2, 40 µl of supernatant from each well was harvested and measured for released 51Cr on a Top Count liquid scintillation counter (Canberra Packard, Meriden, CT). Specific lysis was calculated as follows: (experimental counts - spontaneous counts)/(maximum counts - spontaneous counts) x 100
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CD8 IEL in TCR tg mice expressing the specific Ag
Previous studies of H-Y and 2C TCR tg mouse models (18, 19, 20, 21) have demonstrated that in contrast to the CD8 IEL subset, TCR CD8 IEL do not appear to be negatively selected in the presence of their specific autoantigen. As shown in Fig. 1, this also applies to mice tg for the LCMV-derived immunodominant peptide gp33 and tg for the TCR specific for the gp33. In this model, TCR tg cells cannot only be identified by the mAbs V2 and V8 that recognize the tg TCR, but also by gp33-loaded MHC class I tetramers that bind to gp33-specific T cells. No gp33 tetramer-positive cells could be detected in the TCR CD8 IEL populations of C57BL/6 wild-type mice and of mice tg for the gp33 (H8), respectively (Fig. 1). However, in mice expressing the tg TCR (318), 3% of the TCR CD8 IEL stained positive for the gp33 tetramers and, in 318 x H8 mice that express both the gp33 Ag and the specific TCR, even 10% of the TCR CD8 IEL were gp33 tetramer positive (Fig. 1). Thus, in the presence of their cognate Ag, TCR CD8 IEL with self-specific TCR are indeed not deleted, but persistent and may even be enriched.
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CD8 IEL population
For a better characterization of these potentially self-reactive TCR CD8 IEL, 318 x H8 mice were crossed onto a RAG2-/- background to thus obtain a monospecific TCR CD8 IEL population and increased total TCR CD8 cell numbers in the absence of TCR and TCR CD8 T cells. Analysis of 318 x H8 x RAG2-/- mice showed that all IEL now only expressed the tg TCR, as determined by a staining with the mAbs V2 and V8 (Fig. 2a). The relative frequency of TCR tg IEL in the IEL compartment of 318 x H8 x RAG2-/- (Ag+) mice was the same as in 318 x RAG2-/- mice (Ag-) that did not express the specific Ag (Fig. 2a), and there was no difference in actual numbers of IEL recovered from Ag+ and Ag- mice, respectively (data not shown). However, whereas Ag- mice had equal numbers of TCR CD8 and TCR CD8 IEL, the latter population was almost completely absent from the Ag+ mice (Fig. 2b). In addition to the TCR CD8 IEL, which remained present in the 318 x H8 x RAG2-/- mice, a major population of TCR CD8- IEL was present among the TCR tg cells in the IEL compartment of these mice (Fig. 2b). The appearance of CD8- IEL has also been reported in other TCR tg mouse models in which the specific Ag was coexpressed (20, 21, 31). Although this may suggest that these cells arise from TCR CD8 or CD8 IEL that have down-regulated the CD8 coreceptor in response to constant antigenic stimulation, we found no evidence for intermediate forms of CD8 expression or down-modulation of the coreceptor upon in vitro stimulation of TCR CD8 IEL (data not shown).
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CD8 IEL from Ag+ and Ag- mice to stimulation in vitro
In 318 x H8 x RAG2-/- (Ag+) mice, the tg expression of the gp33 Ag is under the control of a MHC class I promoter. Hence, gp33 is expressed ubiquitously in the gut by a variety of cell types, including intestinal epithelial cells, and is thus likely to directly influence the functional behavior of resident TCR CD8 IEL. As shown in Fig. 3a, sorted TCR CD8 IEL from Ag+ mice indeed failed to proliferate in response to plate-bound anti-CD3 and Ag-bearing irradiated stimulator cells, as compared with TCR CD8 IEL from Ag- (318 x RAG2-/-) mice. To determine whether the absence of a proliferative response could be attributed to an impaired IL-2 production, IL-2 mRNA expression was assessed in anti-CD3-stimulated TCR CD8 IEL from Ag+ and Ag- mice, respectively, by means of real-time RT-PCR. Short-term stimulation (6 h) of TCR CD8 IEL from Ag- mice resulted in a considerable induction of IL-2 mRNA expression, whereas TCR CD8 IEL from Ag+ mice expressed only basal levels of mRNA for IL-2, even after stimulation with plate-bound anti-CD3 (Fig. 3b). Very similar results were obtained on a protein level with almost no IL-2 detectable in supernatants of stimulated TCR CD8 IEL from Ag+ mice while IL-2 was readily detected in the supernatants of TCR CD8 IEL from Ag- mice (Fig. 3c). Addition of exogenous IL-2 to cultures, however, only partially rescued proliferation of TCR CD8 IEL from Ag+ mice (data not shown).
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CD8 IEL from Ag+ and Ag- mice
To further assess the impact of the specific self-Ag on the functional behavior of TCR CD8 IEL, we compared the expression of additional proinflammatory and immunomodulatory cytokine genes in TCR CD8 IEL upon isolation from Ag+ and Ag- mice, respectively. In the absence of their specific Ag (318 x RAG2-/- mice), TCR CD8 IEL constitutively expressed comparable amounts of mRNA for IFN-, IL-10, and TGF (Fig. 4). The presence of the specific self-Ag in the 318 x H8 x RAG2-/- mice substantially reduced the expression of mRNA for IFN- and IL-10 by TCR CD8 IEL, whereas expression of mRNA for TGF remained unaffected and even was slightly increased (Fig. 4). In both Ag+ and Ag- mice, little or no expression of mRNA for TNF- or IL-4, respectively, could be detected. Thus, tolerance induction in self-specific TCR CD8 IEL not only affected proliferation and IL-2 production but also involved reduced expression of certain immunomodulatory cytokine genes.
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CD8 IEL in Ag+ mice
It has repeatedly been shown that viral infections can interfere with or even break tolerance of T cells to self-Ags (26, 32, 33, 34). LCMV not only produces the target self-Ag of our tg model, but when administered systemically also infects CD11c+ dendritic cells in the intestinal mucosa (6). Hence, systemic infection of 318 x H8 x RAG2-/- (Ag+) mice with LCMV seemed an ideal model system to determine whether virus-induced activation of self-specific TCR CD8 IEL leads to a breakdown in tolerance and results in the generation of autoaggressive effector T cells and subsequent intestinal pathologies.
FACS analysis of IEL isolated from Ag+ mice on different time points after LCMV infection (days 4, 6, and 8) indicated that despite their secluded location TCR CD8 IEL indeed responded to the ongoing virus infection. Thus, although no increased expression of CD25 was noted, there was a very distinct up-regulation of the early activation marker CD69 on day 6 after infection, concomitant with an apparent infection of the intestinal mucosa at this time point (Fig. 5a). LCMV-induced activation of self-specific TCR CD8 IEL, however, did not provoke increased expression of mRNA for the proinflammatory cytokines IFN- or TNF- (Fig. 6) while mean expression levels of TGF mRNA increased 
2-fold following LCMV infection. No cytotoxic activity could be detected in TCR CD8 IEL on day 8 after infection, i.e., at the time when maximum cytotoxic activity is observed for the CD8 IEL subset, isolated as a positive control from IEL-reconstituted and LCMV-infected RAG2-/- mice (Fig. 7). Most importantly, analysis of H&E-stained tissue sections revealed no signs of obvious histopathological alterations in the intestinal mucosa such as epithelial erosions or increased proliferative activity of enterocytes (data not shown), and all of the LCMV-infected 318 x H8 x RAG2-/- mice remained healthy. These results indicate that tolerance of self-specific TCR CD8 IEL is not readily abolished and that in consequence these cells are unlikely to initiate and mediate inflammatory reactions in the intestine.
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CD8 IEL expressing self-specific TCR (18, 19, 20, 21, 22). This finding also holds true for the TCR 318 tg RAG2-/- mice which, in the presence of the ubiquitously expressed LCMV-gp33 transgene, almost completely delete the TCR CD8 T cells while TCR CD8 T cells remain present in the IEL compartment.
Induction of functional unresponsiveness or anergy has been described as a mechanism to maintain peripheral tolerance in potentially autoreactive T cells that have escaped thymic negative selection (reviewed in Ref.37). The lack of a proliferative response and the defect in activation-induced IL-2 expression observed in TCR CD8 IEL from 318 x H8 x RAG2-/- (Ag+) mice apparently supports a role for such a mechanism in our model, in particular, since we also found no evidence for ex vivo gp33-specific cytotoxic activity mediated by self-specific TCR CD8 IEL in a short-term 51Cr release assay (data not shown).
It indeed makes sense that tolerance induction should extend to potentially autoreactive CD8 T cells in the IEL compartment since uncontrolled proliferation or cytotoxic activity would be particularly deleterious in the intestinal mucosa. In fact, despite the high precursor frequency of potentially self-reactive TCR CD8 IEL, we observed no signs of histopathological alterations in the intestinal mucosa of 318 x H8 x RAG2-/- mice. Why should, however, the murine intestine preserve self-specific T cells in the limited niches of the IEL compartment, if these cells are to be unfunctional? Guehler et al. (20) suggested that in the 2C TCR tg model, TCR CD8 IEL from Ag+ mice had undergone an immune deviation and differentiation into a TH2-like phenotype capable of mediating humoral immune responses, as these cells expressed mRNA for IL-4 upon in vitro restimulation. That T cells with an anergic phenotype need not necessarily be unfunctional is further illustrated by the CD4+CD25+ subset of T cells which proliferates very poorly upon TCR stimulation in vitro, yet has the potent capacity of suppressing immune responses in vivo by release of immunomodulatory cytokines (reviewed in Ref.38).
In support of a possible regulatory role for TCR CD8 IEL, we found that these cells express moderate levels of mRNA for IFN-, TGF, and IL-10 in the complete absence of their specific MHC-restricted Ag, indicating that TCR CD8 IEL may constitutively express mRNA for immunoregulatory cytokines as part of their genetic program or that other than TCR-mediated signals may induce and sustain cytokine gene expression. In fact, it has recently been shown for in vitro-stimulated CD8+ IEL that the interaction of the TL ligand with the CD8 homodimer enhances IL-2 and IFN- production independently of the TCR MHC specificity (17). Although it was beyond the scope of the present study to also assess the relative role of TL-CD8 and additional non-MHC-restricted cell-cell interactions for modulating the effector functions of TCR CD8 IEL, our results clearly demonstrate that Ag-specific, TCR-mediated signaling profoundly influences cytokine gene expression since TCR CD8 IEL from Ag+ mice consistently exhibit reduced levels of mRNA for IFN- and IL-10, but similar or even increased levels of mRNA for TGF.
The consistent expression of TGF by TCR CD8 IEL is intriguing. TGF is the major switch factor for IgA-producing plasma cells in the intestinal mucosa and appears to play a critical protective role in experimental models of intestinal inflammation (39, 40), possibly by inhibiting T cell proliferation and APC functions or by preventing the production of proinflammatory cytokines and chemokines by enterocytes (39, 41, 42). With their preferential expression of this important immunomodulatory cytokine and their reduced proliferative capacity, self-specific TCR CD8 IEL seem well adapted to an environment in which space for clonal expansion is lacking and preservation of tissue integrity is vital. Based on the results of Leishman et al. (10), which suggest a true positive selection process of self-specific TCR CD8 IEL as opposed to an escape of negative selection (10), it is therefore tempting to speculate that the presence of self-specific TCR CD8 IEL may indeed be of relevance to the maintenance of intestinal tissue homeostasis.
Evidence for a cytokine-dependent regulatory role of self-specific TCR CD8 IEL has very recently been provided by a study that demonstrated an IL-10-mediated protective capacity of adoptively transferred TCR CD8 IEL in an experimental model of colitis (43). This may be in apparent contrast to our finding that IL-10 mRNA expression was consistently reduced in TCR CD8 IEL from 318 x H8 x RAG2-/- mice in favor of the expression of TGF. Moreover, our tg system also failed to confirm a role for the expression of IL-4 by self-specific TCR CD8 IEL as previously observed for the 2C TCR tg model (20). These discrepancies between different models may be attributed to different methods used for the analysis of cytokine expression. A conclusive analysis of cytokine expression on a protein level is indeed difficult to accomplish for IEL as these cells seem to depend on survival signals provided by the epithelial cells and tend to rapidly undergo apoptosis in ex vivo culture (44). In fact, similar to Poussier et al. (43) who were unable to detect secretion of IL-10 in disease-preventing TCR CD8 IEL (43), we were not successful in detecting TGF in supernatants of in vitro-stimulated TCR CD8 IEL from 318 x H8 x RAG2-/- mice (data not shown). Given these circumstances, we believe that by assessing the effect of the presence or absence of the specific self-Ag on the expression of immunomodulatory cytokine genes directly in ex vivo isolated cells, thereby circumventing the problems associated with an in vitro culture of IEL, we have chosen an approach that is most likely to reflect the actual in vivo situation.
The seemingly conflicting results obtained so far for immunomodulatory cytokines preferentially expressed by self-specific TCR CD8 IEL may be best reconciled by a concept that also considers the interrelated roles of these factors in immunoregulation. With the general role of IL-4 in the intestinal mucosa remaining controversial, this may be particularly true for TGF and IL-10, both of which have been ascribed vital roles in the prevention of intestinal pathologies. In this respect, it has recently been suggested that TGF may serve as a primary mechanism for the counterregulation of Th1 T cell-mediated mucosal inflammation while IL-10 is necessary as a secondary factor that facilitates TGF production (45).
Although TCR CD8 IEL specific for self-Ags may be involved in the maintenance of intestinal tissue homeostasis, it is important to bear in mind that these cells have a potential for autoreactivity and could themselves initiate and contribute to intestinal inflammation. This in particular has to be considered with the constant and high exposure of the intestine to infectious and proinflammatory agents and the fact that also antigenically unrelated infections can prime for autoaggressiveness (26, 46). Although it has previously been shown in vitro that cytolytic responses of self-specific TCR CD8 IEL can be retrieved in the presence of increasing levels of exogenous self-peptide (47), no investigation has so far addressed the important issue of whether autoaggressive TCR CD8 effector T cells can be generated in vivo. In this study, we have been able to directly assess the consequences of molecular mimicry on self-specific TCR CD8 IEL in situ by the administration of a potent LCMV infection to the 318 x H8 x RAG2-/- (Ag+) mice.
It may be argued that due to their secluded location in the intestinal epithelium, TCR CD8 IEL may not be in contact with LCMV-infected, professional APCs. However, dendritic cells have been shown to penetrate the gut epithelium with their dendrites (48, 49), and the observed up-regulation of the early activation marker CD69 concomitant with an apparent infection of the intestinal mucosa on day 6 after infection clearly argues for a virus-induced activation of the TCR CD8 IEL. Activation appears to be Ag specific, since after LCMV infection of OT-I x RAG2-/- mice, which are TCR tg for an OVA-derived peptide, no up-regulation of CD69 could be detected on TCR CD8 IEL (data not shown).
Thus, we for the first time have been able to demonstrate an Ag-specific response of TCR CD8 IEL in situ, while previous attempts to detect an immune reaction by these cells after an in vivo priming have been hampered by unknown Ag specificities or low precursor frequencies (30). However, this potent activation of TCR CD8 IEL in situ does not lead to the generation of autoaggressive T cells. This is in clear contrast to CD8 T cells, where tolerance against the gp33 Ag expressed in the pancreatic cells was readily abolished following systemic LCMV infection, resulting in CD8 T cell-mediated cell destruction and the onset of overt diabetes (32, 50). Hence, it is intriguing that in our system all LCMV-infected mice remained healthy and no signs of intestinal pathologies such as epithelial erosion or crypt hyperplasia were observed. The absence of any overt signs of autoimmunity is supported by our observation that LCMV infection does not induce cytotoxic activity or even provoke the expression of proinflammatory cytokines in self-specific TCR CD8 IEL. Although these findings may be accounted for by the consistent and even increased expression of TGF by self-specific IEL throughout the course of LCMV infection, the exact molecular mechanism underlying the attenuation of immune effector functions in this IEL subset are still elusive.
The clearance from LCMV in a virus-infected immunocompetent host critically depends on the presence of cytotoxic TCR CD8 T cells (51). Hence, in Ag+ mice where TCR tg CD8 T cells are centrally deleted, the absence of cytotoxic activity in TCR CD8 T cells may contribute to the observed persistence of virus beyond day 8, i.e., at a time point where virus is generally cleared in an immunocompetent mouse. Because of these differences in the distribution, frequency, and persistence of virus-infected cells in Ag+ and Ag- mice, however, a conclusive comparative analysis of TCR CD8 IEL in the presence or absence of the autoantigen was not possible.
In summary, our results emphasize the unique nature of TCR CD8 IEL and imply that this cell subset is unlikely to initiate autoreactive responses or contribute to chronic inflammatory reactions in the intestine. Moreover, along with our finding that in the presence of their specific self-Ag TCR CD8 IEL preferentially express mRNA for TGF, these findings further underline the potential contribution of self-specific T cells in modulating local immune responses and in maintaining local tissue homeostasis.
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Acknowledgments |
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2 Current address: Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, U.K.
3 Address correspondence and reprint requests to Dr. Christoph Mueller, Division of Immunopathology, Institute of Pathology, University of Bern, Murtenstrasse 31, CH-3010 Bern, Switzerland. E-mail address: christoph.mueller{at}pathology.unibe.ch
4 Abbreviations used in this paper: IEL, intraepithelial lymphocyte; LCMV, lymphocytic choriomeningitis virus; TL, thymus leukemia; tg, transgenic.
Received for publication May 29, 2003. Accepted for publication January 16, 2004.
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promotes immune deviation by altering accessory signals of antigen-presenting cells. J. Immunol. 160:1589. and IL-10 in the regulation of experimental colitis. J. Immunol. 168:900.This article has been cited by other articles:
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  P. D. Holler, T. Yamagata, W. Jiang, M. Feuerer, C. Benoist, and D. Mathis The same genomic region conditions clonal deletion and clonal deviation to the CD8{alpha}{alpha} and regulatory T cell lineages in NOD versus C57BL/6 mice PNAS, April 24, 2007; 104(17): 7187 - 7192. [Abstract] [Full Text] [PDF]
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M. Schenk, A. Bouchon, S. Birrer, M. Colonna, and C. Mueller Macrophages Expressing Triggering Receptor Expressed on Myeloid Cells-1 Are Underrepresented in the Human Intestine J. Immunol., January 1, 2005; 174(1): 517 - 524. [Abstract] [Full Text] [PDF]
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). For a positive control, cytotoxic activity was assessed in CD8
). Unspecific cytotoxicity was measured using target cells pulsed with the irrelevant adenovirus-derived peptide adn5 instead of gp33 was <5% (data not shown).