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Induction of Oral Tolerance in Splenocyte-Reconstituted SCID Mice
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
Vol. 87, No. 3, June, pp. 282–291, 1998 Article No. II984538
Induction of Oral Tolerance in Splenocyte-Reconstituted SCID Mice Hiroko Yoshida,1 Satoshi Hachimura, Kazuki Hirahara,* Tatsuhiro Hisatsune, Ken-ichi Nishijima,2 Akio Shiraishi,* and Shuichi Kaminogawa Department of Applied Biological Chemistry, University of Tokyo, Tokyo, Japan; and *Biological Research Laboratories, Sankyo Co., Tokyo, Japan
The participation of each lymphocyte compartment in the induction of oral tolerance for antibody response was investigated by means of a new cell-transfer experimental system, using severe combined immunodeficiency (SCID) mice. Various lymphocyte compartments from BALB/c mice were transferred into SCID mice and these mice were evaluated for oral tolerance induction. First, whole splenocytes from BALB/c mice were transferred into SCID mice and these mice were orally administered bovine as1-casein. The specific antibody response in these mice after subsequent immunization with antigen was greatly reduced compared to controls which were not fed the antigen, and it was demonstrated that oral tolerance was induced in SCID mice bearing donor splenocytes. Oral tolerance was induced in SCID mice that were reconstituted with only T cells, revealing that B cells were not required for the induction of oral tolerance. Further, oral tolerance was induced in SCID mice reconstituted with CD8-depleted splenocytes but not in mice reconstituted with only CD8/ T cells. These results demonstrate that oral tolerance could be induced in SCID mice bearing normal splenocytes and that interaction of CD4/ T cells with antigen-presenting cells other than B cells are responsible for the induction of oral tolerance. Our experimental system may be useful for investigations with human lymphocytes. q 1998 Academic Press
Key Words: oral tolerance; T cells; B cells; SCID mice.
INTRODUCTION
Oral tolerance is known as the systemic immunological unresponsiveness induced by oral administration of exogenous antigen (1, 2). It is considered that the reduction of cellular and humoral immunological responses in the state of oral tolerance is mainly due to the reduced response of antigen-specific T cells. 1 Present address: Biomedical Research Laboratories, Sankyo Co., Tokyo, Japan. 2 Present address: Department of Biotechnology, School of Engineering, Nagoya University, Nagoya, Japan.
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0090-1229/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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Several mechanisms for the reduction of T cell response have been proposed (1, 2), such as clonal anergy (3 – 5), active suppression (6 – 11), and clonal deletion (12). It seems likely that an intricate combination of these mechanisms may be involved in oral tolerance. It is also possible that the responses of antigen-specific B cells are reduced to some extent in the state of oral tolerance (13). We have previously investigated the roles of each lymphocyte compartment (CD4/ T cells/CD8/ T cells/B cells) in the maintenance of oral tolerance for antibody response by means of cell-transfer experiments, using severe combined immunodeficiency (SCID) and BALB/ c nu/nu (nude) mice (13). SCID mice, which lack functional T cells and B cells (14), and nude mice, which lack functional T cells, are effective recipients for reconstitution and analysis of lymphocytes. Lymphocyte populations from tolerant or nontolerant mice were transferred in combination to SCID or nude mice. The specific antibody response was reduced dramatically in SCID or nude mice that were recipients of CD4/ T cells from tolerant mice (13). These results suggested that CD4/ T cells were hyporesponsive in the state of oral tolerance. However, which lymphocyte compartment induces CD4/ T cell unresponsiveness has not yet been examined. In this study, we show that oral tolerance was induced in SCID mice reconstituted with normal splenocytes. This system differs from the one used in the previous study, in that the recipient SCID mice were fed the antigen. We further investigated the role of each lymphocyte subset in the process of inducing oral tolerance by means of this newly established cell-transfer experimental system. There is much controversy as to whether CD8/ T cells actively participate in the induction of oral tolerance (15–17), and the role of B cells in oral tolerance has been poorly described. Therefore, we especially focused on the role of CD8/ T cells and B cells. Various lymphocyte compartments, i.e., whole splenocytes, T cells, CD8/ T-cell-depleted splenocytes, or CD8/ T cells only, were transferred into SCID mice. These mice were fed as1-casein, and subsequently immunized with this antigen, to evaluate whether oral
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tolerance was induced. We found that oral tolerance was induced in SCID mice that were recipients of only T cells or CD8-depleted cells. These findings indicate that neither B cells nor CD8/ T cells were necessary for the induction of oral tolerance, and suggest that interaction of CD4/ T cells with antigen-presenting cells (APC) other than B cells is responsible for the induction of oral tolerance. MATERIALS AND METHODS
Animals Seven- to 8-week-old female BALB/c mice were purchased from Japan Clea (Tokyo, Japan). Nine- to 10week-old female SCID mice were purchased from Charles River Japan (Tokyo, Japan). Antigen Bovine as1-casein was prepared from fresh raw skim milk by the method of Zittle et al. (18) and was purified by anion-exchange chromatography on DEAE-Sephacel (Pharmacia LKB Biotechnology, Uppsala, Sweden). A diet containing bovine whole casein was prepared as previously described (13). Adoptive Transfer Splenocytes were harvested from donor BALB/c mice and single-cell suspensions were prepared in RPMI medium and resuspended in PBS immediately before cell transfer. SCID mice were injected intraperitoneally (ip) with 2 1 108 splenocytes in 0.5 ml of PBS. T cells were isolated from BALB/c splenocytes by passing the splenocytes through a nylon wool column (Nylon Fiber; Wako Tokyo, Japan). The extent of contamination of B cells obtained in this manner was less than 4%. In cell-transfer experiments, 8 1 107 T cells resuspended in 0.5 ml of PBS were transferred ip into the SCID mice. BALB/c splenocytes were depleted of CD8/ T cells by treatment with anti-CD8 monoclonal antibody 83-12-5 (mouse IgM, Refs. 19, 20) and complement. The percentage of CD8/ T cells was reduced to less than 1% by this antibody-complement treatment. Positive selection of CD8/ T cells was performed using anti-CD8 antibody-coupled magnetic beads (20). The purity of the CD8/ T cells obtained in this manner was more than 93%. SCID mice were injected ip with 1.8 1 108 CD8-depleted splenocytes or 2.5 1 107 CD8/ T cells in 0.5 ml of PBS. In some experiments, on the day following the feeding period, additional cells required for antibody production were transferred into the SCID mice. That is, the mice which received T cells before the period of oral administration of the casein diet were given an injection containing 1 1 108 B cells. The mice that were
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recipients of CD8/ T cells were given an injection containing 2 1 108 whole splenocytes. The purity of the transferred cell population was confirmed by flow cytometric analysis using FACSort (Becton Dickinson, Mountain View, CA). The cells were stained with phycoerythrin (PE)-conjugated Thy1.2 monoclonal antibody 30-H12 (rat IgG2b , PharMingen, San Diego, CA) to measure the percentage of T cells, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse surface immunoglobulin antibody (Organon Teknika Corp., Durham, NC) to measure the percentage of B cells. The percentage of the CD8/ cell population was determined by staining with anti-CD8 antibody (83-12-5) followed by Thy1–PE and FITC– anti-mouse surface immunoglobulin antibody. The CD8/ cells were identified as cells positive for both colors. The gd-positive cells were measured by staining with FITC-conjugated anti-gd T cell receptor antibody GL-3 (Cedarlane, Hornby, Ontario, Canada). Migration of Transferred Splenocytes SCID mice received 2 1 108 of whole splenocytes of BALB/c mice. One week after the adoptive transfer, cells of various tissues, peritoneum, spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal epithelial layer were harvested from the recipient mice. The collected cells were counted and stained with antimouse Thy1.2–PE or goat anti-mouse surface immunoglobulin antibody–FITC, and the percentage of the stained cells was determined by FACSort. Induction of Oral Tolerance After the adoptive transfer, the SCID mice were fed a casein diet freely for 7 days as the means of oral administration of the antigen. The total amount of as1casein supplied by the casein diet per mouse during this period of feeding was estimated to be approximately 1.4 g. Control mice were fed a commercial diet (Oriental Yeast, Tokyo, Japan) not containing casein. Immunization SCID mice were immunized ip with 50 mg of as1casein in complete Freund’s adjuvant (CFA; Difco, Detroit, MI) on the day following the feeding period or in some experiments on the day following the additional cell transfer. One week after the immunization, they were boosted ip with 50 mg of as1-casein in incomplete Freund’s adjuvant (IFA; Difco). Each animal was bled from the tail vein 2 or 3 weeks after receiving the booster, and the serum was tested for anti-as1-casein antibody. Enzyme-Linked Immunosorbent Assay (ELISA) Anti-as1-casein antibody titers were measured by ELISA as described previously (13). Briefly, microtiter
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plates (MaxiSorp F96; Nunc, Roskilde, Denmark) were coated with 100 mg/ml of as1-casein. In some experiments, the coating concentration of 10 or 1 mg/ml was used. After the plates were washed and blocked with 3% bovine serum albumin, each sample of serum was added. The plates were washed and incubated with alkaline phosphatase-labeled goat anti-mouse (IgG, IgA, IgM) (Cappel; Organon Teknika), rabbit antimouse IgG1 , rabbit anti-mouse IgG2a , rabbit antimouse IgM, or rabbit anti-mouse IgA (Zymed, San Francisco, CA). The plates were washed and finally a p-nitrophenyl phosphate substrate solution was added and the absorbance was measured at 405 nm. Standard curves for anti-as1-casein antibody were generated by using pooled serum from BALB/c mice immunized with the antigen. The titers of anti-as1-casein antibody (IgG / IgM / IgA) were calculated with reference to a preparation of affinity-purified anti-as1-casein antibody. The titers of anti-as1-casein IgM, IgG1 , IgG2a were expressed as the relative concentration to the titer of the BALB/c anti-as1-casein antiserum. The percentage of suppression was calculated as 100 1 (1 0 (geometric mean value of the specific antibody concentration in the test group)/(mean value in the control group)). Statistical Analysis
as1-Casein-specific antibody titers were analyzed by analysis of variance by using data presented as the logarithmic antibody titers. RESULTS
Oral Tolerance Was Induced in SCID Mice That Were Recipients of Whole Splenocytes First, we examined whether oral tolerance could be induced by oral administration of a casein diet in SCID mice bearing whole splenocytes from donor BALB/c mice. SCID mice were injected ip with a suspension of 2 1 108 splenocytes, and thereafter the mice were fed a casein diet freely for 7 days. The mice were immunized with 50 mg of as1-casein in CFA and boosted with 50 mg of as1-casein in IFA. The mice were bled and the concentration of anti-as1-casein Abs in the serum was assayed by ELISA. Figure 1 shows the results for individual mice and the geometric mean value 3 weeks after boosting. The as1-casein-specific antibody concentration was profoundly decreased in the casein-fed group compared with the control group (80% suppression as indicated by comparison of geometric mean values, P õ 0.01). This result shows that oral tolerance to as1-casein was induced in SCID mice that were recipients of whole splenocytes. This is the first report of the induction of immunological tolerance to an oral antigen in SCID mice bearing donor lymphocytes. The antibody titers for individual isotypes (IgM, IgG1 , IgG2a , and
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FIG. 1. Oral tolerance to as1-casein was successfully induced in SCID mice that were recipients of whole splenocytes from donor BALB/c mice. SCID mice were injected ip with a suspension of whole splenocytes and fed a casein diet or control diet for 1 week. After the feeding period, the mice were immunized and boosted with as1-casein and the anti-as1-casein antibody titer was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values. Data from two experiments are presented.
IgA) were measured. The results are expressed as relative concentration to the standard antisera from as1casein-immunized BALB/c mice in Fig. 2. There was a difference in the degree of inhibition between individual isotypes. The production of IgG1 antibody was significantly suppressed (60% suppression, P Å 0.01), while the production of IgG2a isotypes was only slightly suppressed (34% suppression) in the casein-fed mice. There was considerable variation in IgM titers within each group. IgA titers were very low or IgA was undetected in both groups (data not shown). We also tested lower concentrations of antigen for coating (1 or 10 mg/ ml) in the ELISA, to examine the levels of antibodies with high affinity for the antigen in these mice. The degree of suppression evident under these conditions was similar to that observed at high antigen concentrations (79% suppression when 10 mg/ml was used, 77% suppression when 1 mg/ml was used, P õ 0.05 for both cases, data not shown). Splenocytes of Donor BALB/c Mice Migrated to Systemic and Intestinal Lymphoid Tissues in SCID Mice SCID mice received BALB/c splenocytes, and 1 week after the injection, cells of various tissues, peritoneum,
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cells before the adoptive transfer. As shown in Table 1, splenocytes of donor BALB/c mice migrated mainly to the spleen, and also migrated to intestinal lymphoid tissues such as mesenteric lymph nodes and the intestinal epithelial layer. Oral Tolerance Was Successfully Induced in SCID Mice Bearing Donor T Cells After confirming that oral administration of as1-casein could induce oral tolerance in SCID mice bearing whole splenocytes from BALB/c mice, we investigated whether B cells were required for the induction phase of oral tolerance. T cells were purified from splenocytes from donor BALB/c mice and were transferred into SCID mice (8 1 107 T cells). In these experiments, the recipients had only T cells as their lymphocytes during the feeding period. The number of T and B cells found 1 week after injection was examined. There were 1.6 1 107 cells found in the spleen, less than 5% of which were immunoglobulin-positive, demonstrating that B cells had not recovered during this period. After the feeding period, the missing B cell population was transferred and the mice were immunized with as1-casein. The concentrations of anti-as1-casein antibody observed in the sera of these mice are indicated in Fig. 3. As in the case of SCID mice that received whole splenocytes, the antibody titer was greatly decreased in the casein-fed group compared to the controls (96% suppression as indicated by comparison of geometric mean values, P õ 0.001). The titers of as1-casein-specific IgG1 and IgG2a antibodies in the casein-fed mice were reduced (Fig. 4, 83% suppression for IgG1 and 75% suppression for IgG2a , P õ 0.05 for both cases). The production of IgM antibodies was not inhibited in
TABLE 1 FIG. 2. The anti-as1-casein antibody response of individual isotypes in oral tolerance induced in splenocyte-reconstituted SCID mice. SCID mice were injected ip with a suspension of whole splenocytes and fed a casein diet or control diet for 1 week. After the feeding period, the mice were immunized and boosted with as1-casein and the anti-as1-casein antibody titer for each isotype was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. The anti-as1-casein antibody concentration for individual mice is indicated as relative values to a pooled serum from as1-casein-immunized BALB/c mice. The crossbars show the geometric mean values. Data from two experiments are presented.
spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal epithelial layer were harvested. The number of T cells or B cells in these harvested cells was calculated based on flow cytometric analysis. We had confirmed that SCID mice had no T cells and B
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Transferred Splenocytes Migrated to Systemic and Intestinal Lymphoid Tissues in Recipient SCID Mice Cell sourcea
T cellsb (1105 cells)
B cellsb (1105 cells)
Peritoneum Spleen Inguinal lymph nodes Mesenteric lymph nodes Intestinal epithelial layer
12 46 3 7 6
29 20 õ1 2 õ1
a SCID mice received 2 1 108 splenocytes of donor BALB/c mice. One week after the adoptive transfer, cells of various tissues such as peritoneum, spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal epithelial layer were collected. b The harvested cells were counted and stained with anti-Thy1.2 antibody or anti-surface immunoglobulin antibody and the percentage of T cells and B cells was measured by flow cytometric analysis. The number of T cells and B cells was calculated based on these data.
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observed in the SCID mice that were recipients of whole splenocytes, the specific antibody response in the SCID mice bearing CD8/ T-cell-depleted splenocytes and fed a casein diet was greatly decreased in comparison with the control recipient mice 3 weeks after boosting (84% suppression as indicated by comparison of geometric mean values, P õ 0.05). These results dem-
FIG. 3. Oral tolerance was induced in SCID mice bearing donor T cells. SCID mice were injected ip with a suspension of T cells from BALB/c mice and fed a casein diet or control diet for 1 week. After the feeding period, B cells were transferred into these SCID mice, and the mice were immunized with as1-casein. After being boosted, the specific antibody titer was measured by ELISA. Results obtained from sera collected 2 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values of the anti-as1-casein antibody titer. The result is representative of two experiments.
the casein-fed mice (Fig. 4). IgA antibody was low or IgA was undetected (data not shown). When lower concentrations of antigen were used for coating in the ELISA, the degree of suppression observed was slightly lower, but nevertheless significant (74% suppression when 10 mg/ml was used, and 66% suppression when 1 mg/ml was used, P õ 0.05 for both cases, data not shown). These results indicate that oral tolerance was successfully induced in SCID mice bearing donor T cells and that B cells were not required in the induction of oral tolerance. Oral Tolerance Was Induced in SCID Mice Reconstituted with CD8-Depleted Splenocytes but Not in Mice Reconstituted with Only CD8/ T Cells We next investigated the role of CD8/ T cells in the induction of oral tolerance by depleting the donor splenocytes of CD8/ T cells. BALB/c splenocytes were depleted of CD8/ T cells by treatment of the cells with anti-CD8 antibody and complement. SCID mice were injected ip with 1.8 1 108 CD8/ T-cell-depleted splenocytes and fed a casein diet. We confirmed that the depleted population had not recovered during this time (less than 5%). The concentration of anti-as1-casein antibody in the serum was measured after the mice had been immunized and boosted with as1-casein. The results are shown in Fig. 5. In a manner similar to that
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FIG. 4. The anti-as1-casein antibody response of individual isotypes in oral tolerance induced in SCID mice bearing donor T cells. SCID mice were injected ip with a suspension of T cells from BALB/ c mice and fed a casein diet or control diet for 1 week. After the feeding period, B cells were transferred into these SCID mice, and the mice were immunized with as1-casein. After being boosted, the specific antibody titer for each isotype was measured by ELISA. Results obtained from sera collected 2 weeks after booster immunization are shown. The anti-as1-casein antibody concentration for individual mice is indicated as relative values and the crossbars show the geometric mean values.
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T cells was not influenced by oral administration of as1-casein. DISCUSSION
In this study, we have shown that oral tolerance could be induced in SCID mice bearing normal spleno-
FIG. 5. Oral tolerance could be induced in SCID mice without CD8/ T cells. SCID mice were injected ip with a suspension of splenocytes depleted of CD8/ T cells and fed a casein diet or control diet for 1 week. Mice were immunized and boosted ip with as1-casein, and their subsequent antibody response was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values of the anti-as1-casein antibody titer.
onstrate that CD8/ T cells are not necessary for the induction of oral tolerance. Titers of as1-casein-specific IgG1 and IgG2a antibodies in the casein-fed mice were reduced (Fig. 6, 81% suppression for IgG1 and 80% suppression for IgG2a , P õ 0.05 for IgG2a). Although IgM titers varied within each group, the titers in the caseinfed mice were suppressed to some extent (Fig. 6). IgA titer was low or IgA was undetected (data not shown). When lower concentrations of antigen were used for coating in the ELISA, the degree of suppression observed was slightly lower (83% suppression when 10 mg/ml was used, and 74% suppression when 1 mg/ml was used; P õ 0.05 and P Å 0.06, respectively, data not shown). We next investigated the participation of CD8/ T cells in the induction of oral tolerance by transferring only CD8/ T cells into SCID mice. CD8/ T cells were separated from BALB/c splenocytes using anti-CD8 antibody-coupled magnetic beads and 2.5 1 107 cells per mouse were transferred into the recipient mice. The mice were additionally injected with a suspension of whole splenocytes after the feeding period. In this experiment, the SCID mice had only CD8/ T cells as lymphocytes during the feeding period. The concentration of as1-casein-specific antibody was measured after immunization and boosting, and compared with that of control mice. As shown in Fig. 7, specific antibody production in the SCID mice that were recipients of CD8/
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FIG. 6. The anti-as1-casein antibody response of individual isotypes in oral tolerance induced in SCID mice without CD8/ T cells. SCID mice were injected ip with a suspension of splenocytes depleted of CD8/ T cells and fed a casein diet or control diet for 1 week. Mice were immunized and boosted ip with as1-casein, and their subsequent antibody response for each isotype was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. The anti-as1-casein antibody concentration for individual mice is indicated as relative values and the crossbars show the geometric mean values.
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FIG. 7. Oral tolerance was not induced in SCID mice bearing donor CD8/ T cells. SCID mice were injected ip with a suspension of CD8/ T cells from BALB/c mice and fed a casein diet or control diet for 1 week. After the feeding period, whole splenocytes were transferred into SCID mice and these mice were immunized with as1-casein. After being boosted, the antibody titer was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values of the anti-as1casein antibody titer.
cytes, and we have investigated which lymphocyte compartment is primarily responsible for the induction of oral tolerance by means of this cell-transfer experimental system. This is the first report of the induction of immunological tolerance to oral antigen in SCID mice bearing donor lymphocytes. In preliminary experiments SCID mice were injected a suspension of various number of donor splenocytes and fed a casein diet for various periods. We found that transferring 2 1 108 splenocytes to SCID mice and feeding these mice for 1 week were the best conditions to observe the induction of oral tolerance. The results in Fig. 1 show that oral tolerance for antibody response was induced in SCID mice that had received donor splenocytes and that were fed the casein diet according to these conditions. In the following experiments the number of cells transferred was determined based on the number of cells of each kind estimated to be present in the population of 2 1 108 splenocytes. Our results demonstrate that splenocytes comprise a sufficient lymphocyte population for oral tolerance induction. We confirmed by flow cytometry that the splenocytes that were transferred migrated to various lymphoid tissues such as the spleen, mesenteric lymph nodes, and the intestinal epithelial layer in recipient mice 1 week after the injection (Table 1). It is possible
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that tolerance mediated by these splenocytes was induced at systemic sites and/or gut-associated lymphoid tissue (GALT). The Peyer’s patches (PP) are thought to play an important role in the induction of oral tolerance (6–8); however, we could not find PP in SCID mice either before and after cell transfer and it seemed that PP may not be essential for induction of oral tolerance in our study. However, we cannot rule out the existence of PP or PP-like structures that were too small to be visible. In a previous study, we showed that T cells play a principal role in the maintenance of oral tolerance as indicated by the results of cell-transfer experiments (13). That is, the state of oral tolerance was successfully reconstituted in SCID mice that were recipients of T cells from tolerant mice and B cells from normal controls remixed together. The tolerant state was partially but not completely reconstituted in those mice that were recipients of T cells from normal mice and B cells from tolerant mice. Based on these results, we examined whether B cells were required for the induction of oral tolerance. T cells from donor mice were transferred into SCID mice and these mice were fed a casein diet. After the feeding period, B cells were injected into these mice. In this experiment, the SCID mice had only T cells as their lymphocytes during the feeding period. As a consequence, the anti-as1-casein antibody titer was profoundly decreased compared with the control group (Fig. 3), indicating that oral tolerance could be induced in SCID mice with only T cells. Our result demonstrates that B cells are not required in the induction of oral tolerance. The percentage of contaminating B cells after nylon wool column passage was less than 4%, and we confirmed that after depletion the B cells were not restored during the feeding period in SCID mice. Furthermore, as1-casein-specific antibody production was reduced to a greater extent in mice without B cells than in mice that were recipients of whole splenocytes. Therefore, we consider that the effect of contaminating B cells is negligible. The results argue with the reports of antigen presentation by small resting B cells inducing tolerance to T cells (21, 22). On the other hand, it has been recently shown that T cell tolerance was induced in B-cell-deficient mice by injection of a soluble peptide, or deaggregated protein, or superantigen (23, 24). Our results are consistent with these results and further demonstrate that B cells are not required in induction phase of tolerance induction for antibody response. The results suggest that APC populations other than B cells are responsible for the induction of oral tolerance. Probable candidates are cell populations in the GALT (25). We are currently trying to identify these populations. The role of CD8/ T cells in the induction of oral tolerance has been controversial. Earlier experiments have demonstrated the immunoregulatory functions of T
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cells in oral tolerance by adoptive transfer experiments (6–8). More recently, it has been shown that such functions may be mediated by the secretion of TGF-b by CD4/ as well as CD8/ T cells in a series of experiments in experimental autoimmune encephalomyelitis (9, 10, 16, 26). In contrast, it has been shown that oral tolerance could be induced in the absence of such regulatory T cells (3, 4). It has been shown that regulatory cells are preferentially induced when ‘‘low-dose’’ antigen is administered (1, 11, 27). In our system, the daily intake of antigen was approximately 200 mg/mouse, which may be considered a high dose. High-dose feeding induces an unresponsive state of T cells termed anergy (1, 27). When splenocytes from mice tolerized by this regimen were cultured with IL-2, their responsiveness was restored, suggesting the presence of anergized T cells. Nevertheless, our previous study suggests that CD8/ T cells from mice rendered tolerant by this feeding regimen exhibit some immunosuppressive function (13). This may be because the antigen is not ingested at once, which may in some aspects resemble low-dose feeding. We next investigated whether CD8/ T cells were required in the induction of oral tolerance. In the course of the present study, Garside et al. (15) and Barone et al. (17) have reported that oral tolerance specific to OVA could be induced in mice that had been depleted of CD8/ T cells by treatment with anti-CD8 antibody. These studies suggest that CD8/ T cells were not necessary for the induction of oral tolerance. More recently, Vistica et al. demonstrated that oral tolerance could be induced in b2 (0/0) mice in which the development of CD8/ cells is defective (28). On the other hand, Weiner et al. (1, 2) consistently insist that CD8/ T cells have a suppressive role in oral tolerance mediated by TGF-b. They could induce oral tolerance to myelin basic protein in CD8/ T-cell-depleted mice as well as CD4/ T-cell-depleted mice, and have interpreted these results as indicating that the ability of both CD4/ and CD8/ T cells to induce oral tolerance is mediated by TGF-b, not that CD8/ T cells are unnecessary in the induction of oral tolerance (16). In our experimental system we examined the role of CD8/ T cells in the induction of oral tolerance, by depleting donor splenocytes of CD8/ T cells. In this experiment, the influence of administered antibody in vivo and the possibility of abnormal lymphocyte development could be excluded, which might have affected the results in the previous experiments (15–17, 28). SCID mice were injected with a suspension of CD8/ T-cell-depleted splenocytes and fed a casein diet. The percentage of contaminating CD8/ T cells after antibody-complement treatment was less than 1%. As in the case of B cell depletion, we have confirmed that the depleted cells were not restored during the feeding period. The subsequent as1-casein-specific antibody response was markedly reduced in a manner similar to that seen in SCID mice bearing
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whole spleen cells, demonstrating that oral tolerance could be induced without CD8/ T cells (Fig. 5). It was demonstrated that oral tolerance could be induced without CD8/ T cells. However, the results do not deny the role of CD8/ T cells as regulatory cells in oral tolerance. Our previous study suggest that CD8/ T cells from mice rendered tolerant by this feeding regimen exhibit some immunosuppressive function (13). In addition, we have found that CD8/ T cells remain responsive in mice tolerized by feeding the casein diet, and that CD8/ T cell clones established from these mice secrete lymphokines such as IFN-g and TGF-b, which are capable of inhibiting antibody responses (29). To investigate whether regulatory CD8/ T cells were induced by oral administration of antigen in the absence of other lymphocyte populations, only CD8/ T cells were transferred into SCID mice, and these mice were assessed for oral tolerance induction. In this way, mice that have only CD8/ T cells as lymphocytes can be prepared. The extent of anti-as1-casein antibody production in SCID mice that were recipients of CD8/ T cells was not affected by the oral administration of antigen (Fig. 7), and this result shows that oral tolerance was not induced in SCID mice having only CD8/ T cells. It is possible that CD8/ T cells obtain the capacity to function as regulatory T cells during the induction phase of oral tolerance by interacting with CD4/ T cells. Several recent studies have shown that minimal numbers of gd T cells are capable of mediating tolerance (30–32). The proportion of gd T cells in the transferred cells was very low, in the range of 0.5–1.5% in the case of whole splenocytes and purified T cells, and less than 0.5% in the case of CD8/ cells as determined by flow cytometry. (In the same analysis more than 20% of the intestinal intraepithelial lymphocytes from the BALB/c mice were stained positive for gd T cell receptor, indicating that the results were not due to defects in staining procedures.) Nevertheless, it has been reported that as few as 2 1 105 gd T cells can mediate tolerance (32). In our system, large numbers of cells (8 1 107 to 2 1 108 cells) were transferred to the recipients. Thus, there remains the possibility that the small numbers of gd T cells present in the population of transferred cells were mediating the induction of tolerance. Our study demonstrates some other interesting aspects of oral tolerance induction for antibody response. First, there was a difference in the degree of inhibition between different isotypes. The production of as1-casein-specific IgG1 antibody was reduced in the caseinfed mice whether tolerance was induced in mice with whole splenocytes, T cells, or CD80 cells. Similarly, profound inhibition of IgG2a antibody production was observed when tolerance was induced in mice with T cells or CD80 cells. On the other hand, the degree of inhibi-
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tion for IgG2a antibody production was relatively smaller in the case of mice reconstituted with whole splenocytes. At present, the reason for the difference in the degree of inhibition in the case of IgG2a antibody is unclear. We have previously reported that as1-caseinspecific CD8/ T cells secrete IFN-g (20) and these cells remain responsive in mice tolerized by feeding the casein diet (29). IFN-g supports production of antibodies of the IgG2a isotype (33). It is tempting to speculate that IFN-g-secreting as1-casein-specific CD8/ T cells were activated in the SCID mice reconstituted with whole splenocytes, and that B cells were required for the activation, although at present there is no direct evidence to support this idea. Our results also imply that production of antibodies of low affinity are more susceptible to tolerance induction. When a lower concentration of antigen was used to coat the plates in the ELISA, the apparent degree of reduction of antibody titers in the casein-fed mice was slightly lower in the case of mice that were recipients of T cells or CD80 cells. In summary, we have demonstrated that oral tolerance could be induced in SCID mice bearing donor splenocytes. Oral tolerance was induced in SCID mice that were recipients of T cells or CD8-depleted cells. These results indicate that interaction of CD4/ T cells with APC populations other than B cells is primarily responsible for the induction of oral tolerance. The potential of oral tolerance as therapy for autoimmune disease has been demonstrated (1, 2, 34, 35). However, knowledge concerning oral tolerance in humans remain limited. It has been shown that SCID mice can be employed as recipients of human lymphocytes to reproduce human immune responses (36, 37). We suggest that this cell transfer system may be useful to investigate the role of cell populations in oral tolerance not only for murine cells, but also for human lymphocytes.
4.
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14. ACKNOWLEDGMENTS We thank Mr. Yoshihiro Ueda for technical assistance. We also thank the faculty and staff of the Biotechnology Research Center, University of Tokyo, for use of the FACSort flow cytometer.
15.
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III. Evidence for clonal anergy. J. Immunol. 147, 2155–2163, 1991. Melamed, D., and Friedman, A., Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin. Eur. J. Immunol. 23, 935–942, 1993. Van Houten, N., and Blake, S. F., Direct measurement of anergy of antigen-specific T cells following oral tolerance induction. J. Immunol. 157, 1337–1341, 1996. Ngan, J., and Kind, L. S., Suppressor T cells for IgE and IgG in Peyer’s patches of mice made tolerant by the oral administration of ovalbumin. J. Immunol. 120, 861–865, 1978. Mattingly, J. A., and Waksman, B. H., Immunologic suppression after oral administration of antigen. I. Specific suppressor cells formed in rat Peyer’s patches after oral administration of sheep erythrocytes and their systemic migration. J. Immunol. 121, 1878–1883, 1978. Richman, L. K., Graeff, A. S., Yarchoan, R., and Strober, W., Simultaneous induction of antigen-specific IgA helper T cells and IgG suppressor T cells in the murine Peyer’s patch after protein feeding. J. Immunol. 126, 2079–2083, 1981. Miller, A., Lider, O., Roberts, A. B., Sporn, M. B., and Weiner, H. L., Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering. Proc. Natl. Acad. Sci. USA 89, 421– 425, 1992. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A., and Weiner, H. L., Regulatory T cell clones induced by oral tolerance: Suppression of autoimmune encephalomyelitis. Science 265, 1237– 1240, 1994. Chen, Y., Inobe, J., Kuchroo, V. K., Baron, J. L., Janeway, C. A., Jr., and Weiner, H. L., Oral tolerance in myelin basic protein Tcell receptor transgenic mice: Suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells. Proc. Natl. Acad. Sci. USA 93, 388–391, 1996. Chen, Y., Inobe, J., Marks, R., Gonnella, P., Kuchroo, V. K., and Weiner, H. L., Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 376, 177–180, 1995. Hirahara, K., Hisatsune, T., Nishijima, K., Kato, H., Shiho, O., and Kaminogawa, S., CD4/ T cells anergized by high dose feeding establish oral tolerance to antibody responses when transferred in SCID and nude mice. J. Immunol. 154, 6238–6245, 1995. Bosma, G. C., Custer, R. P., and Bosma, M. J., A severe combined immunodeficiency mutation in the mouse. Nature 301, 527–530, 1983. Garside, P., Steel, M., Liew, F. Y., and Mowat, A. M., CD4/ but not CD8/ T cells are required for the induction of oral tolerance. Int. Immunol. 7, 501–504, 1995. Chen, Y., Inobe, J., and Weiner, H. L., Induction of oral tolerance to myelin basic protein in CD8-depleted mice. Both CD4/ and CD8/ cells mediate active suppression. J. Immunol. 155, 910– 916, 1995. Barone, K. S., Jain, S. L., and Michael, J. G., Effect of in vivo depletion of CD4/ and CD8/ cells on the induction and maintenance of oral tolerance. Cell. Immunol. 163, 19–29, 1995. Zittle, C. A., Cerbulis, J., Pepper, L., and Della Monia, E. S., Preparation of calcium-sensitive a-casein. J. Dairy Sci. 42, 1897–1902, 1959. Leo, O., Foo, M., Segal, D. M., Shevach, E., and Bluestone, J. A., Activation of murine T lymphocytes with monoclonal antibodies: Detection on Lyt-2/ cells of an antigen not associated with the T cell receptor complex but involved in T cell activation. J. Immunol. 139, 1214–1222, 1987.
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22. Fuchs, E. J., and Matzinger, P., B cells turn off virgin but not memory T cells. Science 258, 1156–1159, 1992.
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23. Phillips, J. A., Romball, C. G., Hobbs, M. V., Ernst, D. N., Shultz, L., and Weigle, W. O., CD4/ T cell activation and tolerance induction in B cell knockout mice. J. Exp. Med. 183, 1339–1344, 1996. 24. Vella, A. T., Scherer, M. T., Schultz, L., Kappler, J. W., and Marrack, P., B cells are not essential for peripheral T-cell tolerance. Proc. Natl. Acad. Sci. USA 93, 951–955, 1996. 25. Harper, H. M., Cochrane, L., and Williams, N. A., The role of small intestinal antigen-presenting cells in the induction of Tcell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance. Immunology 89, 449–456, 1996. 26. Lider, O., Santos, L. M. B., Lee, C. S. Y., Higgins, P. J., and Weiner, H. L., Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein. II. Suppression of disease and in vitro immune responses is mediated by antigen-specific CD8/ T lymphocytes. J. Immunol. 142, 748–752, 1989.
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27. Friedman, A., and Weiner, H. L., Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc. Natl. Acad. Sci. USA 91, 6688–6692, 1994. 28. Vistica, B. P., Chanaud, N. P., III, Felix, N., Caspi, R. R., Rizzo, L. V., Nusenblatt, R. B., and Gery, I., CD8 T-cells are not essential for the induction of ‘‘low-dose’’ oral tolerance. Clin. Immunol. Immunopathol. 78, 196–202, 1996. 29. Nishijima, K., Hisatsune, T., Kato, K., Kohyama, M., Kakehi,
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M., Hachimura, S., and Kaminogawa, S., Oral administration of antigen does not influence the proliferation and IFN-g production of responsive CD8/ T cells, but enables us to establish T cell clones with different lymphokine production profile. Cytotechnology, in press. Ke, Y., Pearce, K., Lake, J. P., Ziegler, H. K., and Kapp, J. A., gd T lymphocytes regulate the induction and maintenance of oral tolerance. J. Immunol. 158, 3610–3618, 1997. Mengel, J., Cardillo, F., Aroeira, L. S., Williams, O., Russo, M., and Vaz, N. M., Anti-gd T cell antibody blocks the induction and maintenance of oral tolerance to ovalbumin in mice. Immunol. Lett. 48, 97–102, 1995. Harrison, L. C., Dempsey-Collier, M., Kramer, D. R., and Takahashi, K., Aerosol insulin induces regulatory CD8 gd T cells that precent murine insulin-dependent diabetes. J. Exp. Med., 184, 2167–2174, 1996. Snapper, C. M., and Paul, W. E., Interferon-g and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236, 944–947, 1987. Weiner, H. L., Mackin, G. A., Matsui, M., Orav, E. J., Khoury, S. J., Dawson, D. M., and Hafler, D. A., Double-blind pilot trial of oral tolerization with myelin antigens in multiple sclerosis. Science 259, 1321–1324, 1993. Trentham, D. E., Dynesius, T. R., Orav, E. J., Combitchi, D., Lorenzo, C., Sewell, K. L., Hafler, D. A., and Weiner, H. L., Effects of oral administration of type II collagen on rheumatoid arthritis. Science 261, 1727–1730, 1993. Duchosal, M. A., Eming, S. A., Fischer, P., Leturcq, D., Barbas, C. F., McConahey, P. J., Caothien, R. H., Thornton, G. B., Dixon, F. J., and Burton, D. R., Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries. Nature 355, 258–262, 1992. Pestel, J., Jeannin, P., Delneste, Y., Dessaint, J. P., Cesbron, J. Y., Capron, A., Tsicopoulos, A., and Tonnel, A. B., Human IgE in SCID mice reconstituted with peripheral blood mononuclear cells from Dermatophagoides pteronyssinus-sensitive patients. J. Immunol. 153, 3804–3810, 1994.
Received October 27, 1997; accepted February 19, 1998
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Induction of Oral Tolerance in Splenocyte-Reconstituted SCID Mice Hiroko Yoshida,1 Satoshi Hachimura, Kazuki Hirahara,* Tatsuhiro Hisatsune, Ken-ichi Nishijima,2 Akio Shiraishi,* and Shuichi Kaminogawa Department of Applied Biological Chemistry, University of Tokyo, Tokyo, Japan; and *Biological Research Laboratories, Sankyo Co., Tokyo, Japan
The participation of each lymphocyte compartment in the induction of oral tolerance for antibody response was investigated by means of a new cell-transfer experimental system, using severe combined immunodeficiency (SCID) mice. Various lymphocyte compartments from BALB/c mice were transferred into SCID mice and these mice were evaluated for oral tolerance induction. First, whole splenocytes from BALB/c mice were transferred into SCID mice and these mice were orally administered bovine as1-casein. The specific antibody response in these mice after subsequent immunization with antigen was greatly reduced compared to controls which were not fed the antigen, and it was demonstrated that oral tolerance was induced in SCID mice bearing donor splenocytes. Oral tolerance was induced in SCID mice that were reconstituted with only T cells, revealing that B cells were not required for the induction of oral tolerance. Further, oral tolerance was induced in SCID mice reconstituted with CD8-depleted splenocytes but not in mice reconstituted with only CD8/ T cells. These results demonstrate that oral tolerance could be induced in SCID mice bearing normal splenocytes and that interaction of CD4/ T cells with antigen-presenting cells other than B cells are responsible for the induction of oral tolerance. Our experimental system may be useful for investigations with human lymphocytes. q 1998 Academic Press
Key Words: oral tolerance; T cells; B cells; SCID mice.
INTRODUCTION
Oral tolerance is known as the systemic immunological unresponsiveness induced by oral administration of exogenous antigen (1, 2). It is considered that the reduction of cellular and humoral immunological responses in the state of oral tolerance is mainly due to the reduced response of antigen-specific T cells. 1 Present address: Biomedical Research Laboratories, Sankyo Co., Tokyo, Japan. 2 Present address: Department of Biotechnology, School of Engineering, Nagoya University, Nagoya, Japan.
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Several mechanisms for the reduction of T cell response have been proposed (1, 2), such as clonal anergy (3 – 5), active suppression (6 – 11), and clonal deletion (12). It seems likely that an intricate combination of these mechanisms may be involved in oral tolerance. It is also possible that the responses of antigen-specific B cells are reduced to some extent in the state of oral tolerance (13). We have previously investigated the roles of each lymphocyte compartment (CD4/ T cells/CD8/ T cells/B cells) in the maintenance of oral tolerance for antibody response by means of cell-transfer experiments, using severe combined immunodeficiency (SCID) and BALB/ c nu/nu (nude) mice (13). SCID mice, which lack functional T cells and B cells (14), and nude mice, which lack functional T cells, are effective recipients for reconstitution and analysis of lymphocytes. Lymphocyte populations from tolerant or nontolerant mice were transferred in combination to SCID or nude mice. The specific antibody response was reduced dramatically in SCID or nude mice that were recipients of CD4/ T cells from tolerant mice (13). These results suggested that CD4/ T cells were hyporesponsive in the state of oral tolerance. However, which lymphocyte compartment induces CD4/ T cell unresponsiveness has not yet been examined. In this study, we show that oral tolerance was induced in SCID mice reconstituted with normal splenocytes. This system differs from the one used in the previous study, in that the recipient SCID mice were fed the antigen. We further investigated the role of each lymphocyte subset in the process of inducing oral tolerance by means of this newly established cell-transfer experimental system. There is much controversy as to whether CD8/ T cells actively participate in the induction of oral tolerance (15–17), and the role of B cells in oral tolerance has been poorly described. Therefore, we especially focused on the role of CD8/ T cells and B cells. Various lymphocyte compartments, i.e., whole splenocytes, T cells, CD8/ T-cell-depleted splenocytes, or CD8/ T cells only, were transferred into SCID mice. These mice were fed as1-casein, and subsequently immunized with this antigen, to evaluate whether oral
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tolerance was induced. We found that oral tolerance was induced in SCID mice that were recipients of only T cells or CD8-depleted cells. These findings indicate that neither B cells nor CD8/ T cells were necessary for the induction of oral tolerance, and suggest that interaction of CD4/ T cells with antigen-presenting cells (APC) other than B cells is responsible for the induction of oral tolerance. MATERIALS AND METHODS
Animals Seven- to 8-week-old female BALB/c mice were purchased from Japan Clea (Tokyo, Japan). Nine- to 10week-old female SCID mice were purchased from Charles River Japan (Tokyo, Japan). Antigen Bovine as1-casein was prepared from fresh raw skim milk by the method of Zittle et al. (18) and was purified by anion-exchange chromatography on DEAE-Sephacel (Pharmacia LKB Biotechnology, Uppsala, Sweden). A diet containing bovine whole casein was prepared as previously described (13). Adoptive Transfer Splenocytes were harvested from donor BALB/c mice and single-cell suspensions were prepared in RPMI medium and resuspended in PBS immediately before cell transfer. SCID mice were injected intraperitoneally (ip) with 2 1 108 splenocytes in 0.5 ml of PBS. T cells were isolated from BALB/c splenocytes by passing the splenocytes through a nylon wool column (Nylon Fiber; Wako Tokyo, Japan). The extent of contamination of B cells obtained in this manner was less than 4%. In cell-transfer experiments, 8 1 107 T cells resuspended in 0.5 ml of PBS were transferred ip into the SCID mice. BALB/c splenocytes were depleted of CD8/ T cells by treatment with anti-CD8 monoclonal antibody 83-12-5 (mouse IgM, Refs. 19, 20) and complement. The percentage of CD8/ T cells was reduced to less than 1% by this antibody-complement treatment. Positive selection of CD8/ T cells was performed using anti-CD8 antibody-coupled magnetic beads (20). The purity of the CD8/ T cells obtained in this manner was more than 93%. SCID mice were injected ip with 1.8 1 108 CD8-depleted splenocytes or 2.5 1 107 CD8/ T cells in 0.5 ml of PBS. In some experiments, on the day following the feeding period, additional cells required for antibody production were transferred into the SCID mice. That is, the mice which received T cells before the period of oral administration of the casein diet were given an injection containing 1 1 108 B cells. The mice that were
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recipients of CD8/ T cells were given an injection containing 2 1 108 whole splenocytes. The purity of the transferred cell population was confirmed by flow cytometric analysis using FACSort (Becton Dickinson, Mountain View, CA). The cells were stained with phycoerythrin (PE)-conjugated Thy1.2 monoclonal antibody 30-H12 (rat IgG2b , PharMingen, San Diego, CA) to measure the percentage of T cells, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse surface immunoglobulin antibody (Organon Teknika Corp., Durham, NC) to measure the percentage of B cells. The percentage of the CD8/ cell population was determined by staining with anti-CD8 antibody (83-12-5) followed by Thy1–PE and FITC– anti-mouse surface immunoglobulin antibody. The CD8/ cells were identified as cells positive for both colors. The gd-positive cells were measured by staining with FITC-conjugated anti-gd T cell receptor antibody GL-3 (Cedarlane, Hornby, Ontario, Canada). Migration of Transferred Splenocytes SCID mice received 2 1 108 of whole splenocytes of BALB/c mice. One week after the adoptive transfer, cells of various tissues, peritoneum, spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal epithelial layer were harvested from the recipient mice. The collected cells were counted and stained with antimouse Thy1.2–PE or goat anti-mouse surface immunoglobulin antibody–FITC, and the percentage of the stained cells was determined by FACSort. Induction of Oral Tolerance After the adoptive transfer, the SCID mice were fed a casein diet freely for 7 days as the means of oral administration of the antigen. The total amount of as1casein supplied by the casein diet per mouse during this period of feeding was estimated to be approximately 1.4 g. Control mice were fed a commercial diet (Oriental Yeast, Tokyo, Japan) not containing casein. Immunization SCID mice were immunized ip with 50 mg of as1casein in complete Freund’s adjuvant (CFA; Difco, Detroit, MI) on the day following the feeding period or in some experiments on the day following the additional cell transfer. One week after the immunization, they were boosted ip with 50 mg of as1-casein in incomplete Freund’s adjuvant (IFA; Difco). Each animal was bled from the tail vein 2 or 3 weeks after receiving the booster, and the serum was tested for anti-as1-casein antibody. Enzyme-Linked Immunosorbent Assay (ELISA) Anti-as1-casein antibody titers were measured by ELISA as described previously (13). Briefly, microtiter
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plates (MaxiSorp F96; Nunc, Roskilde, Denmark) were coated with 100 mg/ml of as1-casein. In some experiments, the coating concentration of 10 or 1 mg/ml was used. After the plates were washed and blocked with 3% bovine serum albumin, each sample of serum was added. The plates were washed and incubated with alkaline phosphatase-labeled goat anti-mouse (IgG, IgA, IgM) (Cappel; Organon Teknika), rabbit antimouse IgG1 , rabbit anti-mouse IgG2a , rabbit antimouse IgM, or rabbit anti-mouse IgA (Zymed, San Francisco, CA). The plates were washed and finally a p-nitrophenyl phosphate substrate solution was added and the absorbance was measured at 405 nm. Standard curves for anti-as1-casein antibody were generated by using pooled serum from BALB/c mice immunized with the antigen. The titers of anti-as1-casein antibody (IgG / IgM / IgA) were calculated with reference to a preparation of affinity-purified anti-as1-casein antibody. The titers of anti-as1-casein IgM, IgG1 , IgG2a were expressed as the relative concentration to the titer of the BALB/c anti-as1-casein antiserum. The percentage of suppression was calculated as 100 1 (1 0 (geometric mean value of the specific antibody concentration in the test group)/(mean value in the control group)). Statistical Analysis
as1-Casein-specific antibody titers were analyzed by analysis of variance by using data presented as the logarithmic antibody titers. RESULTS
Oral Tolerance Was Induced in SCID Mice That Were Recipients of Whole Splenocytes First, we examined whether oral tolerance could be induced by oral administration of a casein diet in SCID mice bearing whole splenocytes from donor BALB/c mice. SCID mice were injected ip with a suspension of 2 1 108 splenocytes, and thereafter the mice were fed a casein diet freely for 7 days. The mice were immunized with 50 mg of as1-casein in CFA and boosted with 50 mg of as1-casein in IFA. The mice were bled and the concentration of anti-as1-casein Abs in the serum was assayed by ELISA. Figure 1 shows the results for individual mice and the geometric mean value 3 weeks after boosting. The as1-casein-specific antibody concentration was profoundly decreased in the casein-fed group compared with the control group (80% suppression as indicated by comparison of geometric mean values, P õ 0.01). This result shows that oral tolerance to as1-casein was induced in SCID mice that were recipients of whole splenocytes. This is the first report of the induction of immunological tolerance to an oral antigen in SCID mice bearing donor lymphocytes. The antibody titers for individual isotypes (IgM, IgG1 , IgG2a , and
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FIG. 1. Oral tolerance to as1-casein was successfully induced in SCID mice that were recipients of whole splenocytes from donor BALB/c mice. SCID mice were injected ip with a suspension of whole splenocytes and fed a casein diet or control diet for 1 week. After the feeding period, the mice were immunized and boosted with as1-casein and the anti-as1-casein antibody titer was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values. Data from two experiments are presented.
IgA) were measured. The results are expressed as relative concentration to the standard antisera from as1casein-immunized BALB/c mice in Fig. 2. There was a difference in the degree of inhibition between individual isotypes. The production of IgG1 antibody was significantly suppressed (60% suppression, P Å 0.01), while the production of IgG2a isotypes was only slightly suppressed (34% suppression) in the casein-fed mice. There was considerable variation in IgM titers within each group. IgA titers were very low or IgA was undetected in both groups (data not shown). We also tested lower concentrations of antigen for coating (1 or 10 mg/ ml) in the ELISA, to examine the levels of antibodies with high affinity for the antigen in these mice. The degree of suppression evident under these conditions was similar to that observed at high antigen concentrations (79% suppression when 10 mg/ml was used, 77% suppression when 1 mg/ml was used, P õ 0.05 for both cases, data not shown). Splenocytes of Donor BALB/c Mice Migrated to Systemic and Intestinal Lymphoid Tissues in SCID Mice SCID mice received BALB/c splenocytes, and 1 week after the injection, cells of various tissues, peritoneum,
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cells before the adoptive transfer. As shown in Table 1, splenocytes of donor BALB/c mice migrated mainly to the spleen, and also migrated to intestinal lymphoid tissues such as mesenteric lymph nodes and the intestinal epithelial layer. Oral Tolerance Was Successfully Induced in SCID Mice Bearing Donor T Cells After confirming that oral administration of as1-casein could induce oral tolerance in SCID mice bearing whole splenocytes from BALB/c mice, we investigated whether B cells were required for the induction phase of oral tolerance. T cells were purified from splenocytes from donor BALB/c mice and were transferred into SCID mice (8 1 107 T cells). In these experiments, the recipients had only T cells as their lymphocytes during the feeding period. The number of T and B cells found 1 week after injection was examined. There were 1.6 1 107 cells found in the spleen, less than 5% of which were immunoglobulin-positive, demonstrating that B cells had not recovered during this period. After the feeding period, the missing B cell population was transferred and the mice were immunized with as1-casein. The concentrations of anti-as1-casein antibody observed in the sera of these mice are indicated in Fig. 3. As in the case of SCID mice that received whole splenocytes, the antibody titer was greatly decreased in the casein-fed group compared to the controls (96% suppression as indicated by comparison of geometric mean values, P õ 0.001). The titers of as1-casein-specific IgG1 and IgG2a antibodies in the casein-fed mice were reduced (Fig. 4, 83% suppression for IgG1 and 75% suppression for IgG2a , P õ 0.05 for both cases). The production of IgM antibodies was not inhibited in
TABLE 1 FIG. 2. The anti-as1-casein antibody response of individual isotypes in oral tolerance induced in splenocyte-reconstituted SCID mice. SCID mice were injected ip with a suspension of whole splenocytes and fed a casein diet or control diet for 1 week. After the feeding period, the mice were immunized and boosted with as1-casein and the anti-as1-casein antibody titer for each isotype was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. The anti-as1-casein antibody concentration for individual mice is indicated as relative values to a pooled serum from as1-casein-immunized BALB/c mice. The crossbars show the geometric mean values. Data from two experiments are presented.
spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal epithelial layer were harvested. The number of T cells or B cells in these harvested cells was calculated based on flow cytometric analysis. We had confirmed that SCID mice had no T cells and B
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Transferred Splenocytes Migrated to Systemic and Intestinal Lymphoid Tissues in Recipient SCID Mice Cell sourcea
T cellsb (1105 cells)
B cellsb (1105 cells)
Peritoneum Spleen Inguinal lymph nodes Mesenteric lymph nodes Intestinal epithelial layer
12 46 3 7 6
29 20 õ1 2 õ1
a SCID mice received 2 1 108 splenocytes of donor BALB/c mice. One week after the adoptive transfer, cells of various tissues such as peritoneum, spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal epithelial layer were collected. b The harvested cells were counted and stained with anti-Thy1.2 antibody or anti-surface immunoglobulin antibody and the percentage of T cells and B cells was measured by flow cytometric analysis. The number of T cells and B cells was calculated based on these data.
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observed in the SCID mice that were recipients of whole splenocytes, the specific antibody response in the SCID mice bearing CD8/ T-cell-depleted splenocytes and fed a casein diet was greatly decreased in comparison with the control recipient mice 3 weeks after boosting (84% suppression as indicated by comparison of geometric mean values, P õ 0.05). These results dem-
FIG. 3. Oral tolerance was induced in SCID mice bearing donor T cells. SCID mice were injected ip with a suspension of T cells from BALB/c mice and fed a casein diet or control diet for 1 week. After the feeding period, B cells were transferred into these SCID mice, and the mice were immunized with as1-casein. After being boosted, the specific antibody titer was measured by ELISA. Results obtained from sera collected 2 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values of the anti-as1-casein antibody titer. The result is representative of two experiments.
the casein-fed mice (Fig. 4). IgA antibody was low or IgA was undetected (data not shown). When lower concentrations of antigen were used for coating in the ELISA, the degree of suppression observed was slightly lower, but nevertheless significant (74% suppression when 10 mg/ml was used, and 66% suppression when 1 mg/ml was used, P õ 0.05 for both cases, data not shown). These results indicate that oral tolerance was successfully induced in SCID mice bearing donor T cells and that B cells were not required in the induction of oral tolerance. Oral Tolerance Was Induced in SCID Mice Reconstituted with CD8-Depleted Splenocytes but Not in Mice Reconstituted with Only CD8/ T Cells We next investigated the role of CD8/ T cells in the induction of oral tolerance by depleting the donor splenocytes of CD8/ T cells. BALB/c splenocytes were depleted of CD8/ T cells by treatment of the cells with anti-CD8 antibody and complement. SCID mice were injected ip with 1.8 1 108 CD8/ T-cell-depleted splenocytes and fed a casein diet. We confirmed that the depleted population had not recovered during this time (less than 5%). The concentration of anti-as1-casein antibody in the serum was measured after the mice had been immunized and boosted with as1-casein. The results are shown in Fig. 5. In a manner similar to that
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FIG. 4. The anti-as1-casein antibody response of individual isotypes in oral tolerance induced in SCID mice bearing donor T cells. SCID mice were injected ip with a suspension of T cells from BALB/ c mice and fed a casein diet or control diet for 1 week. After the feeding period, B cells were transferred into these SCID mice, and the mice were immunized with as1-casein. After being boosted, the specific antibody titer for each isotype was measured by ELISA. Results obtained from sera collected 2 weeks after booster immunization are shown. The anti-as1-casein antibody concentration for individual mice is indicated as relative values and the crossbars show the geometric mean values.
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T cells was not influenced by oral administration of as1-casein. DISCUSSION
In this study, we have shown that oral tolerance could be induced in SCID mice bearing normal spleno-
FIG. 5. Oral tolerance could be induced in SCID mice without CD8/ T cells. SCID mice were injected ip with a suspension of splenocytes depleted of CD8/ T cells and fed a casein diet or control diet for 1 week. Mice were immunized and boosted ip with as1-casein, and their subsequent antibody response was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values of the anti-as1-casein antibody titer.
onstrate that CD8/ T cells are not necessary for the induction of oral tolerance. Titers of as1-casein-specific IgG1 and IgG2a antibodies in the casein-fed mice were reduced (Fig. 6, 81% suppression for IgG1 and 80% suppression for IgG2a , P õ 0.05 for IgG2a). Although IgM titers varied within each group, the titers in the caseinfed mice were suppressed to some extent (Fig. 6). IgA titer was low or IgA was undetected (data not shown). When lower concentrations of antigen were used for coating in the ELISA, the degree of suppression observed was slightly lower (83% suppression when 10 mg/ml was used, and 74% suppression when 1 mg/ml was used; P õ 0.05 and P Å 0.06, respectively, data not shown). We next investigated the participation of CD8/ T cells in the induction of oral tolerance by transferring only CD8/ T cells into SCID mice. CD8/ T cells were separated from BALB/c splenocytes using anti-CD8 antibody-coupled magnetic beads and 2.5 1 107 cells per mouse were transferred into the recipient mice. The mice were additionally injected with a suspension of whole splenocytes after the feeding period. In this experiment, the SCID mice had only CD8/ T cells as lymphocytes during the feeding period. The concentration of as1-casein-specific antibody was measured after immunization and boosting, and compared with that of control mice. As shown in Fig. 7, specific antibody production in the SCID mice that were recipients of CD8/
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FIG. 6. The anti-as1-casein antibody response of individual isotypes in oral tolerance induced in SCID mice without CD8/ T cells. SCID mice were injected ip with a suspension of splenocytes depleted of CD8/ T cells and fed a casein diet or control diet for 1 week. Mice were immunized and boosted ip with as1-casein, and their subsequent antibody response for each isotype was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. The anti-as1-casein antibody concentration for individual mice is indicated as relative values and the crossbars show the geometric mean values.
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FIG. 7. Oral tolerance was not induced in SCID mice bearing donor CD8/ T cells. SCID mice were injected ip with a suspension of CD8/ T cells from BALB/c mice and fed a casein diet or control diet for 1 week. After the feeding period, whole splenocytes were transferred into SCID mice and these mice were immunized with as1-casein. After being boosted, the antibody titer was measured by ELISA. Results obtained from sera collected 3 weeks after booster immunization are shown. Values for individual mice are indicated and the crossbars show the geometric mean values of the anti-as1casein antibody titer.
cytes, and we have investigated which lymphocyte compartment is primarily responsible for the induction of oral tolerance by means of this cell-transfer experimental system. This is the first report of the induction of immunological tolerance to oral antigen in SCID mice bearing donor lymphocytes. In preliminary experiments SCID mice were injected a suspension of various number of donor splenocytes and fed a casein diet for various periods. We found that transferring 2 1 108 splenocytes to SCID mice and feeding these mice for 1 week were the best conditions to observe the induction of oral tolerance. The results in Fig. 1 show that oral tolerance for antibody response was induced in SCID mice that had received donor splenocytes and that were fed the casein diet according to these conditions. In the following experiments the number of cells transferred was determined based on the number of cells of each kind estimated to be present in the population of 2 1 108 splenocytes. Our results demonstrate that splenocytes comprise a sufficient lymphocyte population for oral tolerance induction. We confirmed by flow cytometry that the splenocytes that were transferred migrated to various lymphoid tissues such as the spleen, mesenteric lymph nodes, and the intestinal epithelial layer in recipient mice 1 week after the injection (Table 1). It is possible
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that tolerance mediated by these splenocytes was induced at systemic sites and/or gut-associated lymphoid tissue (GALT). The Peyer’s patches (PP) are thought to play an important role in the induction of oral tolerance (6–8); however, we could not find PP in SCID mice either before and after cell transfer and it seemed that PP may not be essential for induction of oral tolerance in our study. However, we cannot rule out the existence of PP or PP-like structures that were too small to be visible. In a previous study, we showed that T cells play a principal role in the maintenance of oral tolerance as indicated by the results of cell-transfer experiments (13). That is, the state of oral tolerance was successfully reconstituted in SCID mice that were recipients of T cells from tolerant mice and B cells from normal controls remixed together. The tolerant state was partially but not completely reconstituted in those mice that were recipients of T cells from normal mice and B cells from tolerant mice. Based on these results, we examined whether B cells were required for the induction of oral tolerance. T cells from donor mice were transferred into SCID mice and these mice were fed a casein diet. After the feeding period, B cells were injected into these mice. In this experiment, the SCID mice had only T cells as their lymphocytes during the feeding period. As a consequence, the anti-as1-casein antibody titer was profoundly decreased compared with the control group (Fig. 3), indicating that oral tolerance could be induced in SCID mice with only T cells. Our result demonstrates that B cells are not required in the induction of oral tolerance. The percentage of contaminating B cells after nylon wool column passage was less than 4%, and we confirmed that after depletion the B cells were not restored during the feeding period in SCID mice. Furthermore, as1-casein-specific antibody production was reduced to a greater extent in mice without B cells than in mice that were recipients of whole splenocytes. Therefore, we consider that the effect of contaminating B cells is negligible. The results argue with the reports of antigen presentation by small resting B cells inducing tolerance to T cells (21, 22). On the other hand, it has been recently shown that T cell tolerance was induced in B-cell-deficient mice by injection of a soluble peptide, or deaggregated protein, or superantigen (23, 24). Our results are consistent with these results and further demonstrate that B cells are not required in induction phase of tolerance induction for antibody response. The results suggest that APC populations other than B cells are responsible for the induction of oral tolerance. Probable candidates are cell populations in the GALT (25). We are currently trying to identify these populations. The role of CD8/ T cells in the induction of oral tolerance has been controversial. Earlier experiments have demonstrated the immunoregulatory functions of T
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cells in oral tolerance by adoptive transfer experiments (6–8). More recently, it has been shown that such functions may be mediated by the secretion of TGF-b by CD4/ as well as CD8/ T cells in a series of experiments in experimental autoimmune encephalomyelitis (9, 10, 16, 26). In contrast, it has been shown that oral tolerance could be induced in the absence of such regulatory T cells (3, 4). It has been shown that regulatory cells are preferentially induced when ‘‘low-dose’’ antigen is administered (1, 11, 27). In our system, the daily intake of antigen was approximately 200 mg/mouse, which may be considered a high dose. High-dose feeding induces an unresponsive state of T cells termed anergy (1, 27). When splenocytes from mice tolerized by this regimen were cultured with IL-2, their responsiveness was restored, suggesting the presence of anergized T cells. Nevertheless, our previous study suggests that CD8/ T cells from mice rendered tolerant by this feeding regimen exhibit some immunosuppressive function (13). This may be because the antigen is not ingested at once, which may in some aspects resemble low-dose feeding. We next investigated whether CD8/ T cells were required in the induction of oral tolerance. In the course of the present study, Garside et al. (15) and Barone et al. (17) have reported that oral tolerance specific to OVA could be induced in mice that had been depleted of CD8/ T cells by treatment with anti-CD8 antibody. These studies suggest that CD8/ T cells were not necessary for the induction of oral tolerance. More recently, Vistica et al. demonstrated that oral tolerance could be induced in b2 (0/0) mice in which the development of CD8/ cells is defective (28). On the other hand, Weiner et al. (1, 2) consistently insist that CD8/ T cells have a suppressive role in oral tolerance mediated by TGF-b. They could induce oral tolerance to myelin basic protein in CD8/ T-cell-depleted mice as well as CD4/ T-cell-depleted mice, and have interpreted these results as indicating that the ability of both CD4/ and CD8/ T cells to induce oral tolerance is mediated by TGF-b, not that CD8/ T cells are unnecessary in the induction of oral tolerance (16). In our experimental system we examined the role of CD8/ T cells in the induction of oral tolerance, by depleting donor splenocytes of CD8/ T cells. In this experiment, the influence of administered antibody in vivo and the possibility of abnormal lymphocyte development could be excluded, which might have affected the results in the previous experiments (15–17, 28). SCID mice were injected with a suspension of CD8/ T-cell-depleted splenocytes and fed a casein diet. The percentage of contaminating CD8/ T cells after antibody-complement treatment was less than 1%. As in the case of B cell depletion, we have confirmed that the depleted cells were not restored during the feeding period. The subsequent as1-casein-specific antibody response was markedly reduced in a manner similar to that seen in SCID mice bearing
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whole spleen cells, demonstrating that oral tolerance could be induced without CD8/ T cells (Fig. 5). It was demonstrated that oral tolerance could be induced without CD8/ T cells. However, the results do not deny the role of CD8/ T cells as regulatory cells in oral tolerance. Our previous study suggest that CD8/ T cells from mice rendered tolerant by this feeding regimen exhibit some immunosuppressive function (13). In addition, we have found that CD8/ T cells remain responsive in mice tolerized by feeding the casein diet, and that CD8/ T cell clones established from these mice secrete lymphokines such as IFN-g and TGF-b, which are capable of inhibiting antibody responses (29). To investigate whether regulatory CD8/ T cells were induced by oral administration of antigen in the absence of other lymphocyte populations, only CD8/ T cells were transferred into SCID mice, and these mice were assessed for oral tolerance induction. In this way, mice that have only CD8/ T cells as lymphocytes can be prepared. The extent of anti-as1-casein antibody production in SCID mice that were recipients of CD8/ T cells was not affected by the oral administration of antigen (Fig. 7), and this result shows that oral tolerance was not induced in SCID mice having only CD8/ T cells. It is possible that CD8/ T cells obtain the capacity to function as regulatory T cells during the induction phase of oral tolerance by interacting with CD4/ T cells. Several recent studies have shown that minimal numbers of gd T cells are capable of mediating tolerance (30–32). The proportion of gd T cells in the transferred cells was very low, in the range of 0.5–1.5% in the case of whole splenocytes and purified T cells, and less than 0.5% in the case of CD8/ cells as determined by flow cytometry. (In the same analysis more than 20% of the intestinal intraepithelial lymphocytes from the BALB/c mice were stained positive for gd T cell receptor, indicating that the results were not due to defects in staining procedures.) Nevertheless, it has been reported that as few as 2 1 105 gd T cells can mediate tolerance (32). In our system, large numbers of cells (8 1 107 to 2 1 108 cells) were transferred to the recipients. Thus, there remains the possibility that the small numbers of gd T cells present in the population of transferred cells were mediating the induction of tolerance. Our study demonstrates some other interesting aspects of oral tolerance induction for antibody response. First, there was a difference in the degree of inhibition between different isotypes. The production of as1-casein-specific IgG1 antibody was reduced in the caseinfed mice whether tolerance was induced in mice with whole splenocytes, T cells, or CD80 cells. Similarly, profound inhibition of IgG2a antibody production was observed when tolerance was induced in mice with T cells or CD80 cells. On the other hand, the degree of inhibi-
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tion for IgG2a antibody production was relatively smaller in the case of mice reconstituted with whole splenocytes. At present, the reason for the difference in the degree of inhibition in the case of IgG2a antibody is unclear. We have previously reported that as1-caseinspecific CD8/ T cells secrete IFN-g (20) and these cells remain responsive in mice tolerized by feeding the casein diet (29). IFN-g supports production of antibodies of the IgG2a isotype (33). It is tempting to speculate that IFN-g-secreting as1-casein-specific CD8/ T cells were activated in the SCID mice reconstituted with whole splenocytes, and that B cells were required for the activation, although at present there is no direct evidence to support this idea. Our results also imply that production of antibodies of low affinity are more susceptible to tolerance induction. When a lower concentration of antigen was used to coat the plates in the ELISA, the apparent degree of reduction of antibody titers in the casein-fed mice was slightly lower in the case of mice that were recipients of T cells or CD80 cells. In summary, we have demonstrated that oral tolerance could be induced in SCID mice bearing donor splenocytes. Oral tolerance was induced in SCID mice that were recipients of T cells or CD8-depleted cells. These results indicate that interaction of CD4/ T cells with APC populations other than B cells is primarily responsible for the induction of oral tolerance. The potential of oral tolerance as therapy for autoimmune disease has been demonstrated (1, 2, 34, 35). However, knowledge concerning oral tolerance in humans remain limited. It has been shown that SCID mice can be employed as recipients of human lymphocytes to reproduce human immune responses (36, 37). We suggest that this cell transfer system may be useful to investigate the role of cell populations in oral tolerance not only for murine cells, but also for human lymphocytes.
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14. ACKNOWLEDGMENTS We thank Mr. Yoshihiro Ueda for technical assistance. We also thank the faculty and staff of the Biotechnology Research Center, University of Tokyo, for use of the FACSort flow cytometer.
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Received October 27, 1997; accepted February 19, 1998
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