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The Journal of Immunology, 1998, 160: 754-762.
Copyright © 1998 by The American Association of Immunologists

Lack of Local Suppression in Orally Tolerant CD8-Deficient Mice Reveals a Critical Regulatory Role of CD8+ T Cells in the Normal Gut Mucosa1

Dubravka Grdic, Elisabeth Hörnquist, Martin Kjerrulf and Nils Y. Lycke2

Department of Medical Microbiology and Immunology, University of Goteborg, Goteborg, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We found that feeding keyhole limpet hemocyanin (KLH) to CD8-deficient (CD8-/-) mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wild-type (wt) mice. The tolerance was dose dependent, and only higher doses of KLH caused significant reduction in specific Ab and T cell responses. Both Th1 and Th2 CD4+ T cell functions were affected. Feeding KLH together with cholera toxin (CT) adjuvant, however, abrogated the induction of oral tolerance equally well in CD8-/- and wt mice. On the contrary, CT adjuvant was unable to abrogate already established oral tolerance in both CD8-/- and wt mice. Most importantly, whereas Ag feeding induced hyporesponsiveness in systemic as well as in local gut IgA responses in wt mice, a lack of local suppression was evident in orally tolerant CD8-/- mice following oral immunizations. Thus, contrary to the situation in wt mice, Ag feeding induces systemic, but not local, gut IgA hyporesponsiveness in CD8-/- mice, suggesting that CD8+ T cells in the normal gut mucosa exert an important down-regulatory function. In wt mice the local suppression extended to an unrelated Ag, OVA, given together with KLH and CT adjuvant, i.e., bystander suppression. Based on these results we propose that tolerance induced by feeding Ag is highly compartmentalized, requiring CD8+ T cells for local suppression of IgA responses, whereas systemic tolerance may affect CD4+ T cells of both Th1 and Th2 types independently of CD8+ T cells. Finally, the adjuvant effect of CT abrogates induction, but not established, oral tolerance through a mechanism that does not require CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Two types of responses may be induced by feeding protein Ag: oral tolerance or local productive immunity involving Ag-specific IgA production (1, 2). Since most soluble protein Ags stimulate oral tolerance rather than productive immunity, it is thought that the former serves to protect the host against unwanted, potentially harmful, reactions, e.g., against food Ags (1, 2). A few strongly immunomodulatory molecules, such as cholera toxin (CT),3 have been found to act as powerful mucosal immunogens and adjuvants (3, 4). Interestingly, these agents, including CT, are known to abrogate oral tolerance while promoting strong intestinal IgA and CD4+ T cell responses to unrelated Ags (5, 6, 7, 8). Whether it is necessary to break oral tolerance to achieve mucosal immunity is, therefore, a debated issue, but with some exceptions (9, 10) most studies suggest that gut mucosal immunity and oral tolerance are two mutually exclusive phenomena (1, 2).

The mechanism by which the gut immune system selects the tolerance pathway rather than the productive pathway is largely unknown. At least three primary mechanisms for oral tolerance have been described: active suppression (11, 12, 13, 14, 15), clonal anergy (16, 17), and clonal deletion (18). These mechanisms can probably be operative simultaneously, although high doses of Ag seem to favor anergy and clonal deletion, while low doses of Ag may promote active suppression (19, 20, 21). Because oral tolerance has been found to affect CD4+ as well as CD8+ T cell functions and can be adoptively transferred by both these subsets (11, 13, 22, 23), it remains unclear whether feeding of Ag concomitantly affects both populations and, if so, whether CD4+ and CD8+ T cells are induced to exert control in the same or different compartments. The latter point is especially interesting, since the gut immune system is highly organized, with a majority of CD8+ T cells located to the intraepithelial (IE) compartment while CD4+ T cells predominate in the lamina propria (LP) of the gut mucosa as well as in the Peyer’s patches (PP) and lymphoid follicles (24, 25). Thus, tolerance following feeding of Ag may be a highly compartmentalized phenomenon, with CD4+ and CD8+ T cells having separate and unique regulatory functions.

The CD8+ T cells are abundant in the mucosal immune system, and although their presence is seen in LP and in PP, most of these cells are found in the IE compartment (25). Roughly 80 to 90% of the small intestinal IE lymphocytes are CD8+ T cells, of which half express the TCR{gamma}{delta} and half express the TCR{alpha}ß, whereas in LP and PP most CD8+ T cells are TCR{alpha}ß+ (25). Despite much recent interest in the intestinal CD8+ T cells, the immunobiologic function of these cells largely remains to be determined. Earlier studies have associated CD8+ cells with a regulatory function on epithelial cell growth and mucosal immune responses as well as with host resistance against some intracellular pathogens (25). Also, the potent immunomodulating action of CT on mucosal immune responses has been ascribed a selective inhibitory effect on suppressive gut CD8+ T cells (26).

Few studies have examined the induction of oral tolerance in the absence of CD8+ T cells using subset-specific mAbs for in vivo depletion (27, 28). However, as we have shown previously, in vivo T cell depletion may not be successful in eliminating cells from the intestinal immune system, albeit systemic lymphoid tissues demonstrate complete loss of T cells (29). Therefore, to better understand the complexity of mechanisms regulating induction and maintenance of oral tolerance at local and systemic levels, we undertook studies in CD8 gene-targeted (CD8-/-) mice (30). In an earlier study we reported that the CD8-/- mice have normal gut CD3+ T cell numbers, but whereas CD8+ cells are replaced by CD4+ T cells in organized lymphoid tissues (mesenteric lymph nodes and PP) and in the LP, by contrast, TCR{gamma}{delta}+ CD3+ CD4-CD8- (double-negative) cells greatly predominate among the IE T cells of CD8-/- mice (31). The current study addressed the role of CD8+ T cells in oral tolerance and whether evidence for compartmentalization of control of the tolerant state could be observed. In these studies CT adjuvant was used as a unique means to study oral tolerance and productive gut IgA immunity simultaneously.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

For these studies we used mice deficient for CD8 (CD8-/-; H-2b) by gene targeting the CD8 {alpha}-chain (129.Sv x C57Bl/6; provided by Prof. Tak Mak, Toronto, Canada) (30). The CD8-/- mice were bred for at least four generations onto the C57Bl/6 strain. Therefore, for comparisons we used both wild-type (wt) C57Bl/6 (H-2b) and F1 mice (129.Sv x C57Bl/6; H-2b) as wt controls. The mice were kept under pathogen-free conditions at the Department of Medical Microbiology and Immunology, University of Goteborg (Goteborg, Sweden). All experiments were conducted with 6- to 12-wk-old mice.

Immunizations and induction of oral tolerance

Please see the detailed outline of experiments in Figure 1Go. Briefly, age- and sex-matched mice were fed four oral doses of PBS or 2.5 mg of keyhole limpet hemocyanin (KLH; Sigma Chemical Co., St. Louis, MO), given alone or together with 10 µg of CT (List Biologic Laboratories, Campell, CA) in 0.5 ml of PBS containing 3% (w/v) NaHCO3. To investigate whether CT was able to abrogate already established oral tolerance, mice were immunized by one or three oral doses of 2.5 mg of KLH plus 10 µg of CT adjuvant as indicated in Figure 1Go. Feedings and immunizations were performed with a baby feeding tube under light ether anesthesia as previously described (8). Seven days after the last feeding or oral immunization, animals were immunized i.p. with 100 µg of KLH (Sigma) in RIBI adjuvant (TriChem ApS, Copenhagen, Denmark) and were boosted i.p. 14 days later with 100 µg of KLH alone. In experiments examining the effects of oral tolerance on subsequent mucosal immunity, tolerance was first induced as described above, and then mice were immunized with three oral doses of 2.5 mg of KLH admixed with 10 µg of CT adjuvant. Local intestinal suppression and bystander suppression were also evaluated after oral immunizations of KLH-tolerant mice with 10 mg of chicken egg albumin (OVA, grade V; Sigma Chemical Co.) plus 10 µg of CT adjuvant given with or without KLH at 2.5 mg/dose. Five to seven mice were included in each group throughout the study.



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  		FIGURE 1. Feeding and immunization protocols. Oral tolerance was induced by four consecutive feedings of 2.5 mg of KLH or PBS to CD8-/- or wt mice. Tolerance was defined as the systemic hyporesponsiveness observed after a priming and a booster immunization i.p. with 100 µg of KLH (Figs. 2Go and 3Go). In experiments aimed at testing the immunomodulating effect of CT on the induction of oral tolerance, CD8-/- or wt mice were given four oral doses of PBS or 2.5 mg of KLH with or without 10 µg of CT, and the systemic hyporesponsiveness was evaluated as described above (Fig. 4Go). Finally, mice that had developed oral tolerance after feeding Ag alone were challenged orally by KLH plus CT adjuvant on one or three occasions, and the ability of CT to abrogate established oral tolerance following i.p. priming and booster (Figs. 5Go and 6Go) or stimulate local gut mucosal anti-KLH, anti-OVA, or anti-CT IgA responses (Fig. 7Go) was evaluated.

 
Preparation of lymphocytes

After passing the spleens (SP) through a nylon net and lysis of RBC with Tris-ammonium chloride, lymphocytes in single cell suspensions were washed in PBS and resuspended to appropriate cell density. The isolation procedure for intestinal LP lymphocytes has been described in detail previously (32). Briefly, after removing the PP, the small intestines were cut into small pieces and washed in Ca2+- and Mg2+-free HBSS (Life Technologies, Paisley, Scotland). Thereafter, the tissue pieces were incubated in Ca2+- and Mg2+-free HBSS containing 5 mM EDTA (Merck, Darmstadt, Germany) to remove epithelial cells and IE lymphocytes. Finally, to extract the LP lymphocytes, the tissue was digested by repeated incubations in RPMI 1640 (Flow Laboratories, Irvine, Scotland) containing 100 U/ml of collagenase (type C2913, Sigma). SP cells were analyzed individually from five to seven mice, whereas LP cells were analyzed in pairs from six mice.

Serum Ab titers

The mice were bled at sacrifice, and the serum samples were analyzed individually in flat-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) coated with 100 µg/ml of KLH (Sigma) in PBS at 4°C overnight. After washing the plates in PBS and blocking with 0.1% BSA/PBS for 30 min at 37°C, sera at 1/1000 dilution were serially diluted 1/3 in 0.1% BSA/PBS in corresponding subwells. The plates were kept at 4°C overnight, and following washing in PBS/0.05% Tween-20, the plates were incubated with alkaline phosphatase (AP)-conjugated, isotype-specific, goat anti-mouse Abs (Southern Biotechnology, Birmingham, AL) at 1/500 dilution. After 2 h at room temperature, plates were washed, and the phosphatase substrate, p-nitrophenyl phosphatase (Sigma) was added to each well. The reaction was read at 405 nm using a Titer-Tek spectophotometer (Flow Laboratories). The Ab titers were defined as the interpolated dilutions of the samples giving rise to an absorbance on the linear part of the curve of 0.4 above background.

Enzyme-linked immunospot assay

The Ag-specific spot-forming cell (SFC) assay was performed as previously described (33). Briefly, analysis of KLH-specific SP SFC activity in tolerant and nontolerant mice was performed using 96-well Millipore HA plates (Millipore, Boston, MA) with a nitrocellulose (NC) membrane bottom. The NC membrane was coated with 100 µg/ml of KLH (Sigma) in PBS at 4°C overnight. After careful washing in PBS, the NC plates were blocked with Iscove’s medium containing 10% heat-inactivated FCS (Life Technologies), 5 x 10-5 M 2-ME (Sigma), 1 mM L-glutamine, and 50 µg/ml gentamicin (hereafter referred to as Iscove‘s total medium) for 30 min at 37°C in 8% CO2. Thereafter, 100 µl of splenocytes at 2 x 106 cells/ml were added to each well and serially diluted 1/2 in corresponding subwells. The plates were incubated for 4 h at 37°C in 8% CO2. For detection of Ag-specific LP SFC activity, plastic petri dishes (Nunc A/S, Roskilde, Denmark) were coated with OVA (Sigma) or KLH (Sigma) at 0.1 mg/ml in PBS. For detection of CT-specific SFC activity, the dishes were coated with GM1 ganglioside (Sigma) at 3 nmol/ml followed by CT (List) at 3 µg/ml in PBS. After blocking with 0.1% BSA/PBS, duplicate dishes were incubated with 400 µl of lymphocytes at 1 to 2 x 106 cells/ml, and the cells were allowed to grow for 2 to 4 h at 37°C. After thorough washing in PBS/0.05% Tween-20, developing Abs at 1/200 dilution were added sequentially as follows: 1) for all SP SFC activity, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig (Dako, Glostrup, Denmark) followed by HRP-conjugated swine anti-rabbit Ig (Dako); or 2) for all LP SFC activity, goat anti-mouse IgA (Cappel, Organon, PA) followed by HRP-conjugated rabbit anti-goat Ig (Dako). The bound Abs marking single Ab-producing cells were visualized by adding to Millipore HA plates 1) 100 µl/well of 10 mg/ml of 3-amino-9-ethyl-carbazole (Sigma) substrate in dimethylformamide diluted 1/30 in citrate buffer (pH 4.5) and 0.05% H2O2, or to petri dishes 2) 0.5 mg/ml of p-phenylene-diamine substrate (Sigma) with 0.01% H2O2 substrate in a 1% agar/PBS solution. Spots were counted under low magnification and SFC numbers were expressed as the mean per 107 cells ± SE of five to seven individual mice for SP SFC or three pairs of mice for LP SFC activity. Importantly, preparations of LP lymphocytes from CD8-/- and wt mice were carefully screened and compared for total numbers and distribution of phenotypes by FACS. Consistently, we found that these preparations were comparable in CD8-/- and wt mice with regard to the frequency of T, B, and other cells, such that 58 ± 9 and 54 ± 13% (mean percentage observed in 10 different experiments) were CD3+ T cells and 34 ± 10 and 35 ± 3% were Ig+ in lamina propria lymphocytes from CD8-/- and wt mice, respectively. Moreover, the total IgA SFC activity in LP lymphocytes ranged from 450,000 to 500,000 SFC/107 isolated cells in both strains, which corresponded well with our previous observations (31).

Cytokine assays

For analysis of cytokine production by tolerant and nontolerant SP T cells, cultures were set up in triplicate in round-bottom 96-well plates (Nunc A/S) with 2 x 106 cells/ml in complete Iscove’s medium. After 96 h at 37°C in 8% CO2 in the presence or the absence of KLH at 100 µg/ml, supernatants were harvested, and detection of IFN-{gamma}, IL-4, IL-5, and IL-10 was performed by ELISA as described in detail previously (31, 34): Microtiter ELISA plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 µl of 1.0 to 5 µg/ml of rat anti-mouse IFN-{gamma} (R4–6A2), IL-4 (BVD4–1D11), IL-5 (TRFK5), or IL-10 (JES5–2A5, PharMingen, San Diego, CA) in PBS at 4°C overnight. After washing in PBS and blocking with 0.1% BSA/PBS for 30 min at 37°C, serial 1/2 dilutions of supernatants and recombinant standards at appropriate dilutions (mouse IL-4 and IFN-{gamma} (Genzyme, Cambridge, MA); and mouse IL-5 and IL-10 (R&D, Minneapolis, MN)) were added in duplicate at 100 µl/well, and the plates were incubated at 4°C overnight. After washing in PBS/0.05% Tween-20, the plates were incubated with polyclonal rabbit anti-mouse IFN-{gamma} serum at 1/500 dilution for 2 h at room temperature, followed by an AP-conjugated goat anti-rabbit Ig (Southern Biotechnology) at 1/300 dilution or biotin-conjugated anti-IL-4, anti-IL-5, or anti-IL-10 (PharMingen) at 1 to 5 µg/ml in 0.1% BSA/PBS. Finally, the plates were incubated with extravidin-HRP (Sigma) at 2 µg/ml for an additional 2 h. As an enhancing step to the IL-4, IL-5, and IL-10 ELISAs, the wells were further incubated with 1.5 µg/ml biotinyl-tyramide (ELAST-kit, DuPont, Boston, MA) and 0.4 µl/ml H2O2 in Tris-HCl buffer for 15 min at room temperature, followed by an additional 30-min incubation with extravidin-HRP (Sigma). The IL-4, IL-5, or IL-10 bound in each well was visualized by using o-phenylenediamine (1 mg/ml)/0.04% H2O2 substrate in citrate buffer, pH 4.5, and the IFN-{gamma} reaction was assessed after the addition of p-nitrophenyl phosphatase substrate (Sigma) to each well at 1 mg/ml in ethanol amine buffer. The immunoenzymatic reactions were determined as absorbance in each well using a Titer-Tek Multiscan spectrophotometer (Flow Laboratories) at 450 (HRP) or 405 (AP) nm. The concentrations of cytokines in the supernatants were expressed in picograms per milliliter, as calculated from the plotted standard curves of serial dilutions of the recombinant cytokines. The sensitivity of detection for respective cytokine was 1.0 ng/ml for IFN-{gamma}, 40 pg/ml for IL-4, 50 pg/ml for IL-5, and 500 pg/ml for IL-10.

Adoptive transfer of splenocytes

Donor mice were fed four oral doses of 2.5 mg of KLH (Sigma) or PBS as outlined in Figure 1Go. Seven days after the last feeding, animals were killed, single cell suspensions of splenocytes were prepared, and recipient naive CD8-/- mice were injected i.v. with 20 x 106 splenocytes in 0.2 ml of PBS. On the day after transfer the mice were given a single immunization i.p. with 100 µg of KLH in RIBI adjuvant. Seven days later the mice were killed, and the SP anti-KLH SFC activity was determined.

Statistical analysis

We used Student’s t test for independent samples for analysis of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Feeding soluble protein Ags induces oral tolerance in CD8-/- mice

Since both CD4+ and CD8+ T cells have been implicated in the development and the maintenance of oral tolerance, we wanted to examine whether tolerance also would be induced in mice lacking CD8+T cells (2). To this end CD8-/- or wt mice were fed four doses of KLH or PBS. Systemic responsiveness was evaluated following priming and booster immunizations (i.p) with KLH, as outlined in Figure 1Go. Because the CD8-/- mice (C57Bl/6 x 129Sv) were derived from litters backcrossed for at least four generations onto C57Bl/6 mice, the experiment was performed with both C57Bl/6 and F1 (C57Bl/6 x 129Sv) mice as wt control mice. SP cells from tolerized or PBS-fed mice were analyzed using enzyme-linked immunospot assay technique for anti-KLH SFC activity. We found that CD8-/- mice developed systemic tolerance similar to or more pronounced than that found in wt mice (Fig. 2Go). The tolerized mice exhibited 70 to 80% reduced splenic SFC responses compared with PBS-fed control mice (p < 0.05; Fig. 2Go). Moreover, the induction of oral tolerance in CD8-/- mice was dose dependent, with higher doses giving stronger suppression (Fig. 2Go).



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  		FIGURE 2. Development of oral tolerance in CD8-/- mice. Four oral feedings with PBS (black bars) or KLH (open bars) were performed before parenteral immunizations as described in Figure 1Go. A, Seven days after the i.p. booster immunization with Ag, the anti-KLH SFC activity in the SP of CD8-/-, C57Bl/6, and F1 (C57Bl/6 x 129.Sv) mice was determined. The SFC values per 107 splenic lymphocytes are given as the mean ± SE of five to seven mice in each group. Two experiments are shown in which CD8-/- mice were compared with C57Bl/6 (I) or F1 (II) mice, respectively. B, The effect of varying the dose of KLH on the induction of oral tolerance in CD8-/- mice. Mice were fed four doses of KLH (open bars) or PBS (black bars) before i.p. priming and booster injection. These experiments are representative of at least four similar experiments using C57Bl/6 or F1 as wt controls. * denotes values that are significantly different from the controls (p < 0.05).

 
Stimulation of splenic T cells with recall Ag in vitro revealed that CD4+ T cell-mediated immunity was also impaired in orally tolerant CD8-/- mice; while T cell proliferation ranged from 19.0 ± 2.0 (mean stimulation index ± SD of two separate experiments) in naive mice to 9.0 ± 2.5 in KLH-fed wt mice, the corresponding figures in CD8-/- mice were 22.4 ± 3.5 to 12.4 ± 2.0 in KLH-fed mice. Moreover, the production of IFN-{gamma} was significantly (p < 0.05) reduced in KLH-fed mice compared with that in PBS control mice (Fig. 3Go). The degree of suppression was, again, comparable in orally tolerant CD8-/- and wt mice, i.e., roughly 80% reduction. Thus, we conclude that in CD8-/- mice, feeding of protein Ag results in systemic tolerance comparable in magnitude to that seen in wt mice. As oral tolerance developed identically in the F1 and C57Bl/6 wt mice, we used only C57Bl/6 mice as wt controls for the rest of the study.



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  		FIGURE 3. Impaired IFN-{gamma} production in orally tolerant CD8-/- mice. Mice were fed four doses of PBS (black bars) or KLH (open bars) followed by priming and booster immunizations i.p. with KLH. Seven days after the last immunization splenocytes were cultured in the absence or the presence of KLH. Culture supernatants were harvested after 96 h, and the content of IFN-{gamma} was determined. Results are given in nanograms per milliliter of IFN-{gamma} and are expressed as the mean ± SE of five to seven mice in each group. The values represent KLH-specific responses after subtraction of spontaneous production of IFN-{gamma} in the absence of Ag. This experiment is representative of at least four experiments with similar results using C57Bl/6 and F1 (C57Bl/6 x 129 Sv) mice as wt controls. * denotes values that are significantly different from the controls (p < 0.05).

 
Oral tolerance in CD8-/- mice affects both Th1 and Th2 CD4+ T cell functions

Conflicting findings have been reported as to the relative effect of feeding Ag on specific Th1 and Th2 CD4+ T cell functions (27, 35, 36). Since we noted a strong reduction of Th1 functions, i.e., IFN-{gamma} production, in the tolerized CD8-/- mice, we went on to evaluate the susceptibility of Th2 cells to oral tolerance. Ag-stimulated splenic T cells were analyzed for production of IL-4, IL-5, and IL-10. As shown in Table IGo, significant and equivalent decreases (p < 0.05) in IL-4, IL-5, and IL-10 production were found in tolerized CD8-/- and wt animals. In most experiments the reduction in Th2 cytokine production was less pronounced, however, than the reduction in IFN-{gamma} production (Fig. 3Go and Table IGo).


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  		Table I. Effect of Ag feeding on Th2-lymphokine production in CD8-/- and wt mice

 
In five consecutive experiments we attempted to adoptively transfer oral tolerance by splenocytes from KLH-fed mice to naive recipient CD8-/- mice. While in three experiments we observed roughly a 40% reduction in the SP SFC response following a single i.p. priming immunization with KLH plus RIBI, we failed to detect hyporesponsiveness in the remaining two experiments (not shown).

Taken together, oral tolerance in CD8-/- mice appeared to affect both Th1 and Th2 CD4+ T cell subsets.

CT adjuvant prevents induction of oral tolerance in CD8-/- mice

An alternative way to study regulatory mechanisms in oral tolerance is to use an adjuvant with strong immunomodulatory action on the intestinal immune system. CT is well known for its powerful mucosal adjuvant effects, which include strong enhancement of specific IgA and CD4+ T cell responses to unrelated Ags (5, 7, 8). The toxin also abrogates oral tolerance in normal mice, a mechanism thought to require the presence of CD8+ T cells (5, 26). However, we found that the ability of CT to prevent induction of oral tolerance was similar in CD8-/- and wt mice (Fig. 4Go). The relative effect of CT on anti-KLH SFC responsiveness to systemic challenge immunizations was comparable or stronger in CD8-/- compared with that in wt mice (Fig. 4Go). Furthermore, T cell responses, as determined by IFN-{gamma} production in response to recall Ag in vitro, were restored or even augmented by CT adjuvant in both CD8-/- and wt mice (p < 0.05; Fig. 4Go). Thus, CT efficiently abrogated the induction of oral tolerance independently of CD8+ T cells.



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  		FIGURE 4. CT prevents induction of oral tolerance in CD8-/- mice. Mice were fed four doses of PBS (black bars), KLH (open bars), or KLH plus CT (gray bars) followed by i.p. priming and booster immunizations (Fig. 1Go). Seven days after the final immunization splenocytes were isolated from CD8-/- or wt mice. A, Anti-KLH SFC were analyzed, and the results are given as mean SFC per 107 cells ± SE of five to seven mice per group. B, Splenocytes were cultured in the absence or the presence of KLH, and culture supernatants were harvested after 96 h. IFN-{gamma} production was assessed, and the results are given in nanograms per milliliter of IFN-{gamma} ± SE of five to seven mice per group. Values represent KLH-specific responses after subtraction of spontaneous production of IFN-{gamma} in the absence of Ag. This experiment is representative of three separate experiments producing similar results. * denotes values that are significantly different from the controls (p < 0.05).

 
By analyzing isotype distribution of serum anti-KLH Abs we indirectly sought to determine whether the abrogating action of CT adjuvant on CD4+ T cell functions may have influenced the Th1 and Th2 subsets differently in CD8-/- or wt mice. As illustrated in Table IIGo, we found significant (p < 0.05) enhancement of IgG1, IgG2a, IgG2b, and IgA anti-KLH responses in both CD8-/- and wt mice, suggesting that CT prevented the development of oral tolerance in both Th1 and Th2 CD4+T cell subsets (Table IIGo).


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  		Table II. Isotype distribution of serum anti-KLH antibodies in CD8-/- and wt mice after feeding Ag in the presence or absence of CT adjuvant

 
CT adjuvant cannot break already established oral tolerance

CT adjuvant abrogated the induction of oral tolerance independently of CD8+ T cells, probably by promoting the priming of productive rather than suppressive CD4+ T cells (6). However, earlier studies have suggested that CT may directly impair gut CD8+ T cell functions (5, 26), and such a mechanism could affect oral tolerance differently in CD8-/- and wt mice. Hence, we asked whether CT could break already established oral tolerance. Mice were fed Ag to achieve a tolerant state before single or repeated systemic or oral challenge immunizations with Ag plus CT adjuvant (see Fig. 1Go). We found that oral exposures to KLH plus CT adjuvant did not alter the level of tolerance established by feeding native Ag (Fig. 5Go). Thus, similar results were found regardless of whether one or even three oral doses of CT adjuvant were administered over a longer period of time, as evidenced by the strongly reduced anti-KLH SFC and IFN-{gamma}, responses (Fig. 5Go). Moreover, IL-4, IL-5, and IL-10 were significantly reduced (p < 0.05) in these mice, illustrating that established oral tolerance of Th2 CD4+ T cell functions largely remained resistant to treatment with Ag plus CT adjuvant (Fig. 6Go). Thus, CT adjuvant was unable to break already established oral tolerance in both CD8-/- and wt mice, indicating that T cell-mediated immune regulation following oral Ag exposure is resistant to immunomodulation by CT adjuvant. Furthermore, in mice fed KLH, the i.p. administration of CT instead of RIBI did not affect the level of suppression observed, i.e., in KLH-fed CD8-/- mice the splenic SFC response to KLH plus CT priming was 9,611 ± 4,308 (mean anti-KLH SFC value ± SE of two separate experiments) compared with that in unfed mice, which was 19,484 ± 5,558 anti-KLH SFC/107 cells.



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  		FIGURE 5. CT cannot abrogate already established oral tolerance. Oral tolerance was first induced by feeding KLH to CD8-/- or wt mice as described in Figure 1Go; then the animals were challenged orally once or three times with KLH in the presence of CT adjuvant to assess whether CT could abrogate established oral tolerance. The effects of CT administration were analyzed subsequent to i.p. priming and booster immunizations with KLH. A, Mice were fed PBS (black bars) or KLH (open bars) and challenged once with KLH plus CT (gray bars). For control purposes, PBS control mice were challenged by a single oral dose of KLH and CT before i.p. immunizations (striped bars). The anti-KLH SFC values were expressed as mean SFC per 107 cells ± SE of five to seven mice per group. B, IFN-{gamma} production by SP T lymphocytes in response to recall Ag in vitro was determined. Lymphocyte cultures from KLH-fed mice (open bars), PBS-fed mice (black bars), or mice fed KLH and then given one (gray bars, I) or three (gray bars, III) oral doses of KLH plus CT adjuvant are shown. The results are given in nanograms per milliliter and are expressed as the mean ± SE of five to seven mice per group. These are representative experiments of three that produced similar results. * denotes values that are significantly different from those in KLH-fed mice (p < 0.05).

 


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  		FIGURE 6. CT is unable to abrogate impaired Th2-cytokine production in orally tolerant CD8-/- mice. Oral tolerance was first induced by feeding KLH to CD8-/- (A) or wt (B) mice as described in Figure 1Go, and then the animals were challenged orally once or three times with KLH in the presence of CT adjuvant to assess whether CT could abrogate established oral tolerance in the Th2 subset. SP lymphocytes were prepared from KLH-fed tolerant mice (open bars), PBS-fed mice (black bars), or KLH-fed mice given a single oral immunization with KLH + CT (gray bars) before i.p. immunizations with KLH. The cells were cultured in the presence or the absence of KLH. Supernatants were collected after 96 h, and the content of IL-4, IL-5, or IL-10 was determined. The results are given in picograms per milliliter and are expressed as the mean ± SE of five to seven mice per group. Values represent KLH-specific responses. No background (spontaneous) production of IL-4, IL-5, or IL-10 was observed in the absence of Ag. This is one representative experiment of two that produced similar results. * denotes values that are significantly different from the controls (p < 0.05).

 
Lack of local suppression of gut immune responses in the absence of CD8+ T cells

Having the advantage of a potent mucosal adjuvant, we next explored whether local IgA immunity could be induced by oral immunizations even if the mice had been made tolerant by prior feeding of Ag (see Fig. 1Go). As demonstrated previously, feeding KLH alone gave little or no anti-KLH IgA SFC responses in the gut LP (Fig. 7Go) (6). After repeated oral immunizations with KLH plus CT adjuvant, we found that KLH-tolerant wt mice demonstrated significant reduction (p < 0.05), roughly 60%, in their local gut IgA anti-KLH SFC responses compared with nontolerant control mice (Fig. 7Go). By contrast, the local IgA response of tolerized CD8-/- mice after mucosal challenge with KLH plus CT adjuvant was of similar magnitude to that in nontolerant control mice (Fig. 7Go). Thus, whereas oral feeding of KLH affected both systemic and local responsiveness to the Ag in wt mice, only systemic, but not local, responses were reduced in the CD8-/- mice. In addition, significantly reduced serum anti-KLH IgA titers following oral immunizations were observed only in tolerant wt, but not in tolerant CD8-/-, mice (Fig. 7Go). These findings suggested that CD8+ T cells may play a critical down-regulatory role in the normal gut mucosal immune system. Also in support of such an idea, we consistently observed two- to threefold higher gut IgA anti-KLH SFC responses in PBS-fed CD8-/- compared with wt mice following oral immunizations with KLH plus CT (Fig. 7Go).



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  		FIGURE 7. Lack of local suppression of gut IgA responses in tolerant CD8-/- mice. The wt and CD8-/- mice were fed KLH to establish oral tolerance according to the protocol in Figure 1Go. Thereafter they were given three oral immunizations with KLH plus CT adjuvant and analyzed for specific responses in the LP (A) or serum (B). LP anti-KLH SFC or serum IgA responses are depicted for mice fed PBS (black bars) or KLH (gray bars) after oral immunizations with KLH plus CT adjuvant. In A, anti-KLH LP SFC responses in mice orally immunized with KLH alone (open bars) are shown. Of note, serum anti-KLH IgA levels directly reflected intestinal IgA responses to oral immunizations, because systemic administration of KLH plus CT adjuvant did not stimulate detectable serum anti-KLH IgA titers. LP cells from two mice were pooled, and the results are expressed as mean SFC per 107 cells ± SE of three pairs of mice in each group. Serum anti-KLH IgA titers were analyzed in individual mice. This is one representative experiment of three that produced similar results. The response to KLH alone was <150 SFC/107 cells. * denotes values that are significantly different from the controls (p < 0.05).

 
Finally, we asked whether CD8-/- or wt mice, fed KLH, also exhibited impaired mucosal responsiveness to an unrelated Ag given together with KLH plus CT adjuvant. Whereas there was no difference in the anti-OVA SFC response between KLH-tolerant and nontolerant CD8-/- mice, we found that despite no prior exposure to OVA, the intestinal response to OVA was reduced (p < 0.05) to roughly the same level as that seen for KLH in wt mice (Table IIIGo). By contrast, when OVA was administered together with CT in the absence of KLH in wt mice, no significant reduction of anti-OVA SFC was observed (Table IIIGo). Our data in wt mice, therefore, suggested that feeding of conventional Ags to normal mice may stimulate CD8+ T cells that exert bystander suppression on mucosal IgA responses. On the other hand, gut anti-CT SFC responses were not affected by prior feeding with KLH, demonstrating that the overall ability to respond with gut intestinal IgA SFC was not altered in mice made tolerant by feeding Ag compared with that in PBS-fed control mice (Table IIIGo). We conclude that whereas CD8+ T cells mediate suppression (KLH) and bystander suppression (OVA) of mucosal responses to conventional Ags given orally to wt mice, CT seemed to escape this regulatory control.


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  		Table III. Local suppression of gut IgA responses in orally tolerant wt mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first study to address regulatory mechanisms in oral tolerance using CD8-/- mice. By employing the CT adjuvant we were in the unique position to investigate oral tolerance and local productive IgA immunity simultaneously. Several new and unexpected findings were made. We initially established that feeding KLH to CD8-/- mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wt mice. Both Th1 and Th2 CD4+ T cell functions were reduced in orally tolerant CD8-/- and wt mice. Feeding KLH together with CT adjuvant, however, abrogated the induction of oral tolerance equally well in CD8-/- and wt mice, but did not break already established oral tolerance. Finally, contrary to that in wt mice, we found a lack of local suppression in orally tolerant CD8-/- mice following oral immunization with Ag plus CT adjuvant, suggesting that CD8+ T cells in the normal gut mucosa may exert an important down-regulatory function on intestinal immune responses.

The normal appearance of oral tolerance in CD8-/- mice is consistent with the results of several earlier studies showing that feeding Ag induces systemic hyporesponsiveness independently of CD8+ T cells but requiring CD4+ T cells (13, 23, 28, 37). Thus, although previous studies have documented the importance of CD8+ T suppressor cells, these cells do not appear to be critical for establishing oral tolerance to conventional protein Ags (11, 14, 15, 22, 38). Nevertheless, the reduced intestinal and serum IgA responses that we observed in tolerized wt, but not in CD8-/-, mice indicated that local suppression in the normal gut mucosa most likely relies on suppressive CD8+ T cells. This down-regulatory effect of gut CD8+ T cells also extended to an unrelated Ag when given orally together with KLH and CT adjuvant, indicating that regulatory gut CD8+ T cells can confer bystander suppression (39). The lack of a bystander effect in CD8-/- mice further strongly supports a role for CD8+ T cells in the local suppression of mucosal immunity. Moreover, the tolerant mice were not simply immunosuppressed because the response to OVA in the absence of KLH or to CT itself was unaffected by prior feeding with KLH. Therefore, we propose that tolerance following feeding of conventional soluble protein Ags appears to be Ag specific, highly compartmentalized, and require CD8+ T cells for local intestinal suppression, while CD4+ T cells are responsible for systemic hyporesponsiveness. Also, feeding of Ag coordinately promoted hyporesponsiveness in both the systemic and local intestinal immune systems, thus favoring the theory that systemic tolerance and local productive IgA immunity are mutually exclusive phenomena. Although few studies have simultaneously addressed the issue of tolerance and productive IgA immunity, this idea is further corroborated by Stok et al. and Elson et al., who also found a suppressed local IgA response in conjunction with oral tolerance (26, 40). Since local intestinal and serum IgA responses were unaffected in CD8-/- mice despite strong systemic hyporesponsiveness, it is likely that gut CD4+ T cells at local inductive sites, i.e., the PP and small lymphoid aggregates, may be more difficult to tolerize than SP by feeding Ag. Such an assumption finds support in a recent study by Marth et al. that showed increased IFN-{gamma} production in PP after feeding Ag, while SP demonstrated decreased IFN-{gamma} formation (41).

The MHC restriction for activation of CD4+ and CD8+ suppressive T cells argue for the existence of at least two pathways or sites for induction of oral tolerance. Most in vivo data identify the PP as an inductive site for both oral tolerance and productive IgA immunity (9, 42, 43, 44). However, epithelial cells express both MHC class I and II surface molecules, and in vitro studies have provided compelling evidence that the gut epithelium can preferentially stimulate suppressive CD8+ T cells, even though CD4+ T cells may also be generated (37, 45, 46). In addition, recent elegant studies have demonstrated that epithelial cells may present Ag to CD8+ T cells in the context of the CD1 molecule, a nonclassical MHC that is only distantly related to the MHC class I molecules (47, 48). Furthermore, the newly described membrane protein, gp180, on the epithelial cell apparently can act as a cofactor together with CD1 in stimulating CD8+ T cell activation (49). Thus, the surface expression of the CD8 molecule is critical for these interactions between epithelial cells and gut T cells, in particular for the activation of regulatory CD8+ T cells that are TCR{alpha}ß+ (47, 49). Of note, it is the CD8 {alpha}-chain, deleted in the CD8-/- mice, that has been shown to play important signaling functions (50). Whether the TCR{gamma}{delta}+ CD8+ cells have a regulatory role in the gut of normal mice is at present poorly understood. Several recent studies have provided strong evidence that TCR{gamma}{delta}+ CD8+ T cells may fulfill a down-regulatory role on immune responses by inhibiting cell-mediated immunity (51, 52, 53). Because of the lack of CD8-expression and the dominance of TCR{gamma}{delta}+CD4-CD8- cells, it is, however, likely that the IE T cells are nonfunctional in CD8-/- mice.

Previous studies have indicated that the quality of tolerance affecting CD4+ T cells after feeding Ag may be influenced by the dose of Ag used (20, 21, 54). Thus, a high dose of Ag, e.g., a single dose >20 mg of OVA or repeated milligram doses of other proteins (22, 35, 36, 41, 54), has been associated with T cell anergy or deletion (17, 18), which affected both Th1 and Th2 cytokine production. On the contrary, microgram doses of Ag were found to induce active suppression in which increased TGF-ß production, most often found in conjunction with increased production of Th2 cytokines, is thought to play a critical role (1, 12, 13). We found that oral tolerance in CD8-/- mice was strictly dose dependent, and only the higher doses of KLH produced significant suppression of Ab and Th1 and Th2 responses. Therefore, the tolerant state in the CD8-/- and wt mice fed KLH four times resembled that found with high doses of other Ags (35, 41, 54). It is interesting to note that recent studies of oral tolerance in IL-4- or IFN-{gamma}R-deficient mice, performed by our group and that of Garside et al., have indicated that the systemic hyporesponsive state achieved by feeding Ag does not appear to require Th1 or Th2 CD4+ T cells to develop (34, 35, 55).

Whether high and low dose oral tolerance represent uniquely different mechanisms for the control of immune reactivity against ingested proteins is not completely clear (1, 2). For example, such an idea does not explain why a low dose that generates active suppression mediated by CD4+ and perhaps CD8+ T cells secreting TGF-ß would be overtaken by a process leading to enhanced anergy and deletion among T cells when a higher dose is used. The alternative interpretation would be that these protocols exhibit variable degrees of the same feature, i.e., the generation of regulatory CD4+ and CD8+ T cells that exert immune suppression, leading to anergy and/or deletion. Whether such cells are short- or long-lived is currently unknown, but the duration of the oral tolerant state as observed in animal models would certainly favor the latter (1, 2). Moreover, this hypothesis would offer a more attractive theory because it would allow suppressive T cells to coexist with processes leading to anergy and deletion. In support of the idea that actively suppressive CD4+ T cells may be induced in CD8-/- mice, we were partly successful in adoptively transferring hyporesponsiveness by splenocytes from Ag-fed donor mice. Nevertheless, additional experiments will be required to clarify whether such regulatory cells can be found in Ag-fed CD8-/- mice.

It is clear from this study and our previous work using CD8-/- mice that the adjuvant affect of CT promotes both Th1 and Th2 CD4+ T cells and does not require the presence of CD8+ T cells (4, 31). In addition, one important message born out of the present study is that the CT adjuvant system may be used safely for enhancing the effectiveness of oral vaccines. We found that CT abrogated the induction of oral tolerance but was unable to break already established tolerance. This finding is of significant importance for mucosal vaccine development, and the result agrees well with a study by Nedrud et al. demonstrating that CT did not augment serum Ab levels against some common food Ags (56). If the hypothesis is correct that oral tolerance and productive IgA immunity are reciprocally regulated, then we require adjuvants in oral vaccines to circumvent the tolerance pathway to achieve productive IgA immunity. It is, therefore, reassuring that one of the most powerful mucosal adjuvants yet described, CT, does not cause loss of established tolerance, e.g., against food Ags. Although CT is unlikely to be used clinically because of its toxicity, this understanding should apply to adjuvant systems derived from CT, i.e., nontoxic mutants of the holotoxin or our newly developed nontoxic adjuvant, the CTA1-DD gene fusion protein (57). Whether it also applies to other mucosal adjuvant systems, such as iscoms, awaits to be investigated.


   Acknowledgments
 
We thank Karin Schön and Lena Ekman for skillful technical assistance, and Fredrik von Knoop for computer assistance.


   Footnotes
 
1 This work was supported by the World Health Organization GPV-Transdisease Program, the Swedish Medical Research Council, the National Institutes of Health (Grant 1R01AI40701–01), The Swedish Cancer Foundation, and The Martin Bergvalls and the Nanna Svartz Foundations. Back

2 Address correspondence and reprint requests to Dr. Nils Lycke, Department of Medical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg, Sweden. E-mail address: Back

3 Abbreviations used in this paper: CT, cholera toxin; IE, intraepithelial; LP, lamina propria; PP, Peyer’s patches; CD8-/-, CD8-deficient mice; wt, wild type; KLH, keyhole limpet hemocyanin; SP, spleen; AP, alkaline phosphatase; SFC, spot-forming cells; NC, nitrocellulose; HRP, horseradish peroxidase. Back

Received for publication April 17, 1997. Accepted for publication October 3, 1997.


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