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Department of Pathology and Microbiology, University of Bristol Medical School, Bristol, United Kingdom
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Introduction |
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There is now clear evidence that Ag-specific regulatory cells can be induced by appropriate administration of Ags or their fragments. Mucosal administration has been a favored route largely because the immune system has evolved mechanisms to tolerate the presence of Ags encountered at these surfaces. Mucosal administration of intact proteins tends to induce TGF-
secreting T (Th3) cells that are thought to have evolved to help IgA production (12). Th3 cells specific for myelin Ags have been shown to suppress autoimmunity in models of autoimmune encephalomyelitis. In addition, the repeated administration of superantigens in suitable transgenic mice leads to the generation of IL-10-secreting cells that have a regulatory function in vitro (13, 14). These cells may be analogous to T regulatory-1 cells that may be generated by repeated Ag stimulation in the presence of excess IL-10 (15) or vitamin D3 and dexamethasone (16) in vitro.
Previous work has shown that intranasal (i.n.) administration of peptides recognized by CD4+ T cells can induce peripheral tolerance to self Ags (17, 18, 19, 20). Intranasal administration of peptides could even induce tolerance in mice transgenic for an Ag-specific TCR (21). In this study, we characterize the cells arising from tolerance induction in the Tg4 transgenic model and demonstrate that peptide-induced regulatory cells suppress the response of naive T cells in vitro and in vivo. The suppressive properties of these cells are dependent on IL-10 for the suppression of T cell responses in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAbs directed to murine CD25, CD28, CD38, CD45RB, and CTLA-4 were purchased from BD PharMingen (San Diego, CA). The polyclonal antiserum specific for mouse glucocorticoid-induced TNFR (GITR) and the inducible costimulator protein (ICOS)-Fc fusion protein were purchased from R&D Systems (Abingdon, U.K.). The mAb specific for ICOS-1 was kindly provided by Dr. A. Coyle (Millenium, Boston, MA). The anti-IL-10R Ab (1B1.3a, rat IgG1) was kindly provided by Dr. F. Powrie with permission from DNAX (Palo Alto, CA). For in vivo experiments, this Ab was purified into endotoxin-free PBS (Life Technologies, Paisley, U.K.), filter sterilized, and stored at -20°C before its use. Control rat IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). CFSE was purchased from Molecular Probes (Leiden, Holland).
Peptides
The acetylated N-terminal peptide of murine MBP (Ac1-9, AcASQKRPSQR) and the high MHC-affinity analog with a tyrosine substituting the wild-type lysine in position 4 (Ac1-9[4Y]) were synthesized using standard fluorenyl methoxycarbonyl chemistry on an AMS 422 multiple peptide synthesiser (Abimed Analyes-Technik, Langenfeld, Germany). For in vivo experiments, peptides were purified into endotoxin-free PBS (Life Technologies), filter sterilized, and stored at -20°C before use.
Animals and treatments
The Tg4 transgenic mouse expresses TCR 
- and -chains derived from the 1934.4 T cell hybridoma specific for the N-terminal, acetylated CD4-T cell epitope of MBP (Ac1-9). This mouse strain was established as previously described (22) and back-crossed onto the B10.PL (H-2u) background. Mice were primed by a single i.n. dose of 100 µg Ac1-9[4Y]. Tolerance was induced by giving 10 doses of Ac1-9[4Y] at regular intervals over a period of 5 wk.
Cell separation
Spleens were taken from mice treated i.n. with Ac1-9[4Y] at different times before analysis. Purified CD4+ T cells (>95% CD4+ as determined by FACS analysis) were obtained by positive selection using magnetic beads coated with anti-CD4 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
CD4 T cell proliferation assays
Three days after the last Ag treatment, spleens were harvested and CD4 T cells were purified by positive selection as described. CD4+ T cells were cultured in IMDM medium supplemented with 5 x 10-5 M 2-ME, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Life Technologies), and 5% FCS (Sigma-Aldrich, Poole, U.K.) at 5 x 104 cells/well at 37°C in the presence of different concentrations of Ac1-9. Irradiated CD4-depleted splenocytes at 1 x 105 cells/well were used as APCs. After indicated times, cells were pulsed with 0.5 µCi [3H]thymidine overnight and the incorporated radioactivity was measured on a liquid scintillation beta-counter (1450 Microbeta; Wallac, Milton Keynes, U.K.).
Transwell experiments
CD4+ T cells were cultured in IMDM medium at 5 x 105 cells/well in 24-well plates in the presence of 10 µg/ml of Ac1-9. Irradiated CD4-depleted splenocytes at 5 x 105 cells/well were used as APC. In some of the cultures, the different CD4+ populations were separated by a semipermeable membrane (Falcon Transwell; BD Biosciences, Bedford, MA). When Transwells were used, APC and peptide were added to both chambers.
Cytokine protein levels
Blood samples, drawn 2 h after i.n. treatment of mice with Ac1-9[4Y], or cell culture supernatants were tested for cytokine content by specific ELISAs (BD PharMingen) according to instructions from the manufacturer.
FACS analysis (flow cytometry)
Cells were stained by direct immunofluorescence for two-color flow cytometry. Data were analyzed using CellQuest software (BD PharMingen).
CFSE staining and cell transfer
Labeling of naive and tolerant splenocytes with CFSE was performed as described previously (23). A total of 5 x 107 CFSE-labeled splenocytes were transferred i.v. into the tail vein of naive or tolerant syngeneic recipients. One day after the transfer, mice received one i.n. treatment of Ac1-9[4Y] or PBS, and T cell proliferation was monitored 2 days later in the spleen. Some mice received 0.5 mg anti-IL-10R Ab or isotype control IgG by i.p. injection 2 h before the peptide. Chimerism of CFSE+ cells in the spleen of recipient mice varied between 1 and 3% in all instances.
Tolerant cell transfer
Splenocytes (5 x 107 cells) from naive or tolerant mice were transferred by i.p. injection into recipient Tg4 mice. Some mice received 0.5 mg anti-IL-10R Ab by i.p. injection after 24 h. All mice were challenged with Ac1-9[4Y] (100 µg) i.n., and serum samples were obtained under terminal ether anesthesia after a further 2 h.
Statistical analysis
For statistical analysis, a Mann-Whitney U test was used for nonparametric data.
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Results |
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Splenocytes from the Tg4 TCR transgenic mouse respond spontaneously to challenge with the N-terminal Ac1-9 peptide of MBP in vitro (22). Previous work has shown that repeated administration of the high-affinity, MHC-binding analog of Ac1-9 (Ac1-9[4Y]) leads to profound unresponsiveness among Tg4 T cells (21). This was confirmed by the experiments shown in Fig. 1. Fig. 1, A and B, compares the proliferation of splenocytes from either naive mice, mice primed by a single i.n. dose of Ac1-9[4Y], or mice rendered tolerant by 10 i.n. doses of Ac1-9[4Y]. Primed Tg4 cells responded more rapidly (Fig. 1A) and at a lower dose of Ag (Fig. 1B) when compared with naive cells. However, peptide-induced tolerant (PItol) Tg4 cells failed to proliferate in response to Ac1-9.
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, and only produced marginal levels of IFN-
(data not shown). Cell surface phenotype of PItol cells
The cell surface phenotype of naive Tg4 cells was compared with that of primed and PItol cells. The expression of various markers was measured 3 (CD25, CD38, CD45RB) or 9 days (CTLA-4, ICOS-1) after administration of Ag. Priming of Tg4 cells by administration of Ac1-9[4Y] did not significantly increase the proportion of CD25+ cells within the CD4 subset (Table I). However, the proportion of CD25+ cells doubled in PItol Tg4 mice, although still at <10% of the total CD4+ population. A similar doubling of the proportion of CD38+ cells was noted in PItol mice. The proportion of CD45RBlow cells did not increase significantly in PItol when compared with naive mice. By contrast, both priming and tolerance induction led to a marked increase in the proportion of both CTLA-4- and ICOS-1-positive cells. Expression of these markers was measured at a time point when any transient changes following T cell activation would have subsided (24). The proportion of CTLA-4+ cells increased 10-fold after a single administration of peptide and increased further to reach 63% in PItol mice.
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Naive Tg4 cells were stimulated in vitro either alone or in the presence of an equal number of PItol cells. As shown in Fig. 2A, naive Tg4 cells proliferated well in response to Ac1-9, whereas PItol cells failed to respond. Addition of an equal number of PItol cells led to a substantial (93%) reduction in the proliferative response of naive Tg4 cells at the optimal dose of Ac1-9 (10 µg/ml). Because PItol cells secrete high levels of IL-10, it might be predicted that this cytokine would be responsible for this suppression. However, addition of excess anti-IL-10R Ab failed to significantly reverse suppression. A similar limited effect on suppression was seen by addition of an anti-TGF- Ab, while the two Abs together had a marginally additive effect. Thus, although both IL-10 and TGF- may contribute to the in vitro suppression of naive cell proliferation by PItol cells, they are certainly not sufficient. Other mechanisms must be involved in this process.
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Addition of IL-2 alone induced a low level of proliferation in both naive (
cpm = 13,184, stimulation index = 21.0) and tolerant (cpm = 16,204, stimulation index = 27.7) CD4+ T cells. However, simultaneous addition of Ag led to a significant reversal of the unresponsive state of PItol cells in the presence of IL-2 (Fig. 2B). Thus, addition of Ac1-9 to cultures stimulated a dose-dependent proliferation such that at 10 µg/ml of Ag the response of both naive and tolerant cells to Ag was equivalent. Similarly, suppression of the response of naive cells by PItol cells was lost by the addition of IL-2 even at a limiting dose of Ag.
Cell-cell contact is required for effective suppression of naive Tg4 cells by PItol cells in vitro
Transwell culture chambers were used to test whether cell-cell contact would be necessary for effective suppression of naive Tg4 cells by PItol cells in vitro. Secretion of IL-2 was used as the readout for activation of naive T cells. As shown in Table II, addition of Ag to the lower chamber of a transwell led to the secretion of high levels of IL-2 from naive Tg4 cells. However, this was totally suppressed by addition of an equal number of PItol cells to the lower chamber of the culture. Despite secretion of high levels of IL-10 by the tolerant cells (Fig. 1D), the PItol cells, alhough optimally stimulated with peptide, were not capable of effective suppression when placed in the upper chamber of a transwell, thus separating the cells from naive effectors by a semipermeable membrane. We believe that the reduced level of IL-2 measured in the lower chamber of the naive + tolerant transwell culture (1562 pg/ml) when compared with naive cells alone (2386 pg/ml) is the result of IL-2 consumption by the tolerant cells.
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Various cell surface molecules have been implicated in regulation of CD4 T cell growth and some have been shown to play a role in the function of regulatory T cells. Thus, various costimulatory pathways could contribute to the suppressive properties of PItol cells in vitro. Either Abs or fusion proteins specific for costimulatory pathways were introduced into cocultures of naive Tg4 and PItol cells in vitro. As shown in Fig. 3, Abs to GITR did not have a significant effect on either naive Tg4, PItol cells, or their cocultures. This is in contrast to the functional importance of the GITR receptor in suppression mediated by the CD4+CD25+ T cell subset (25, 26). Parallel experiments in this laboratory have shown that this reagent will abrogate the suppressive properties of IL-10-producing Tg4 cells grown in the presence of vitamin D3 and dexamethasone (M. Harber, unpublished observations) according to the method of Barrat and coworkers (16). The ICOS-Fc fusion protein did not affect IL-2 production by either the PItol cells or their cocultures with naive Tg4 cells. If anything, the fusion protein reduced IL-2 production by naive Tg4 cells. Abs to CTLA-4 had no effect on the response of PItol cells, but significantly increased the response of naive cells. Therefore, the minor reversal of suppression achieved by addition of anti-CTLA-4 to the cocultures may be accounted for by the effect of this Ab on the Ag-specific response of naive cells rather than a direct effect on PItol cells. Anti-CD28 Abs significantly increased the response of naive but not tolerant Tg4 cells to Ag in vitro. The level of suppression of naive T cell activation mediated by the PItol cells was also significantly reduced by addition of anti-CD28 Abs even though the additional costimulation had no effect on tolerant cells alone. Therefore, this significant reversal of suppression in cocultures by anti-CD28 could be accounted for by the direct effect of anti-CD28 on the naive T cell population. Alternatively, it could be that anti-CD28 affects tolerant cells so that they lose their suppressive capacity without restoring their ability to produce IL-2. Either way these results demonstrate that the suppressive effect of PItol Tg4 cells on naive cell activation, mediated by cell-cell contact, may be overcome by a dominant costimulatory signal through CD28.
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We wished to establish models of T cell-mediated suppression in vivo that would avoid any manipulation of cells in vitro. The first of these involved the transfer of CFSE-labeled Tg4 cells into either naive or tolerant Tg4 mice. Two populations of splenocytes obtained from either naive or PItol Tg4 mice were labeled with CFSE and transferred into either naive or PItol recipients by i.v. injection with each mouse receiving 5 x 107 cells. One day after cell transfer, the recipient mice were challenged with a single dose of Ac1-9[4Y] by the i.n. route. A single mouse in each group was left untreated to determine baseline CFSE levels. The spleens from these recipient mice were harvested on day 3 after transfer, disaggregated to yield a single-cell suspension, and counterstained with anti-CD4-tricolor Ab. FACS analysis of these splenocyte populations was performed gating on the CD4-tricolor, CFSE double-positive cells. Fig. 4A shows the response of naive cells transferred into naive recipients; the cells divided readily in response to Ag with only 14% remaining undivided. Fig. 4B shows that the same cells transferred into PItol recipients also divided; however, a greater proportion of these cells remained undivided (50%) or underwent lower numbers of divisions. Fig. 4, C and D shows the results of equivalent transfers of CFSE-labeled PItol cells. There was no measurable evidence of cell division when tolerant cells were transferred into tolerant recipients (Fig. 4D). However, this same population of tolerant cells did divide to some extent when transferred into naive recipients (Fig. 4C). However, a large proportion of the cells (66%) remained undivided.
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The CFSE-labeled cell transfer model was used to investigate the role of IL-10 in mediating suppression in vivo. Splenocytes from naive Tg4 mice were labeled with CFSE and then transferred into either naive or PItol recipients by i.v. injection (5 x 107 cells/mouse). One day after cell transfer, the recipient mice were challenged with a single dose of Ac1-9[4Y] by the i.n. route and splenocytes analyzed by FACS after a further 48 h. Fig. 5A shows the CFSE staining of naive cells transferred into naive recipients. In this experiment, only 13% of cells remained in the undivided fraction after 48 h. The CFSE staining of naive cells transferred into PItol recipients (Fig. 5B) shows that transfer of naive cells into a PItol recipient-suppressed proliferation of the CFSE-labeled naive cells with 59% of transferred cells remaining in the undivided fraction. Fig. 5C shows the CFSE staining of naive cells transferred into tolerant recipients then treated with anti-IL-10R Ab 2 h before antigenic challenge. The suppression of proliferation was significantly reversed when IL-10 was neutralized (Fig. 5C). PItol mice treated with control IgG showed a comparable level of suppression as the PItol recipients albeit slightly reduced (Fig. 5D). Taken together these observations emphasize the role of IL-10 in the maintenance of tolerance in PItol mice. The fact that an anti-IL-10R Ab can reverse suppression in vivo differs significantly from the lack of effect of the same Ab on suppression measured in vitro.
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We have investigated the use of a second in vivo measure of T cell activation. A time course of cytokine secretion was established in naive Tg4 mice treated with an i.n. dose of Ac1-9[4Y] and this demonstrated that high serum levels of IL-2 could be measured peaking at the 2 h time point (data not shown). This assay was used to confirm the observation that PItol cells do not secrete IL-2 and to test whether suppression of IL-2 production could be observed following transfer of PItol cells into naive Tg4 mice. Splenocytes were transferred on day 0 by i.p. injection from either naive (control transfer) or PItol (tolerant transfer) mice into two groups of naive Tg4 recipients. These recipient mice were then challenged with peptide, delivered by the i.n. route on day 2, and serum samples were obtained 2 h later. Two additional groups were included as controls: a group of naive mice that were primed with only a single dose of peptide and a PItol group that received 10 doses of peptide and were given the final dose 2 h before the collection of serum. The results (Fig. 6A) demonstrate that the suppression of IL-2 production among PItol cells in vitro is also maintained in vivo. Furthermore, the transfer experiment demonstrated that PItol cells were capable of transferring suppression of IL-2 production by naive Tg4 cells in vivo. The results of the control and tolerant cell transfer groups were compared using the Mann-Whitney U test demonstrating that the medians of these two groups were significantly different (p = 0.0079).
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The establishment of an assay for transfer of tolerance allowed us to investigate the role of IL-10 in vivo. Three groups of naive Tg4 recipients were prepared. Splenocytes from PItol mice were then transferred on day 0 by i.p. injection into two of the three groups of recipient Tg4 mice. The final control group received no cells. On day 2, anti-IL-10R Ab was administered to the mice in one of the cell transfer groups. All groups of mice were then challenged with peptide by the i.n. route and serum samples were obtained 2 h later. The results shown (Fig. 6B) are the mean serum IL-2 levels for each group with error bars depicting the SEM. The administration of anti-IL-10R Ab to tolerant cell-recipient mice resulted in reversal of suppression. This observation emphasizes the role of IL-10 in tolerance transfer. This result again differs significantly from the lack of effect of anti-IL-10R Ab on suppression measured in vitro.
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Discussion |
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rather than the cytokines normally associated with Th2 cells (28). Evidence that IL-10 can also play a role in protecting against autoimmune disease first arose through the work of Powrie and colleagues (32). They showed that protection from inflammatory bowel disease, following transfer of CD45RBlow cells, could be inhibited with anti-IL-10R Abs in lymphopenic recipients of CD45RBhigh cells. This observation is consistent with the fact that IL-10-/- mice are predisposed to inflammatory bowel disease.
Recent observations have revealed that IL-10-secreting cells may be induced both in vitro and in vivo. Culture of naive T cells in the presence of Ag and IL-10 leads to the generation of IL-10-secreting T cells (T regulatory-1 cells) capable of inhibiting inflammatory conditions when transferred back into the intact animal (15). These cells appear to be distinct from the IL-10/IFN--producing subset in that they produce IL-10 and IL-5 but only low levels of IFN-. Furthermore, there is evidence that cells with a similar cytokine profile can be induced in vivo. Thus, repeated administration of the superantigen staphylococcal enterotoxin A in TCR V3 transgenic mice led to the generation of CD4+ cells that secreted IL-10 but failed to produce significant amounts of IL-2, IFN-, or TNF- (13). Cells rendered tolerant by in vivo administration of superantigen were profoundly unresponsive and did not respond to Ag in the presence of IL-2 (14), a normal characteristic of anergic CD4+ T cell clones (33). Similarly, oral administration of IL-10 and Ag generated lymphocytes producing decreased amounts of IL-2 and IFN- and increased amounts of IL-10 and TGF- (34).
Previous studies from this laboratory showed that i.n. administration of peptide Ags could induce a state of tolerance in nontransgenic mice (17, 35). A similar state of tolerance could be induced in the Tg4 TCR transgenic mouse and the tolerant cells failed to secrete IL-2 but produced high levels of IL-10 (21). Recent studies have emphasized the role of CD4+CD25+ cells in immune regulation (36, 37), but questions have arisen as to whether these cells function by secreting cytokines such as IL-10 and TGF- (9). Therefore, to further characterize the PItol cells, we have analyzed their cell surface phenotype. CD4+ T cells from the tolerant Tg4 mouse were predominantly CD25-. This alone distinguishes these cells from the "natural", thymus-derived CD25+ population of regulatory/suppressor T cells. The most notable feature of the PItol cells was their expression of CTLA-4. Previous work from this laboratory showed that i.n. administration of the Ac1-9[4Y] peptide in Tg4 mice led to a rapid up-regulation of both CD28 and CTLA-4 (24). Although the cell surface expression of CD28 returned to baseline within 6 days, a significant proportion of CD4+ (10–20%) expressed CTLA-4 10 days after peptide administration. The fact that 
63% of PItol cells expressed CTLA-4 at stable levels in this study indicates that tolerance induction correlates with an increase in the proportion of CTLA-4+ cells. This peptide-induced tolerance model is a further example of various models of peripheral tolerance in which tolerance correlates with expression of CTLA-4 (38, 39, 40). Previous studies had shown that anti-CTLA-4 Abs would interfere with tolerance induction in vivo (41, 42). More recent studies have revealed that CTLA-4-/-CD4+ T cells were resistant to the induction of tolerance by high dose i.v. peptide (43). CTLA-4 plays a role in controlling cell cycle progression in these tolerant T cells and it is likely that this molecule contributes to the anergic state of PItol cells.
Recent experiments have revealed a role for ICOS in regulating EAE (44, 45, 46). Ab blockade of ICOS molecules during disease induction led to enhanced EAE and disease was exacerbated in ICOS knockout mice (44). We were particularly interested in studying the expression of ICOS because ICOS costimulation has been shown to induce IL-10 expression (47). Whereas 63% of PItol cells expressed CTLA-4 constitutively, only 16% of these cells expressed ICOS. This would indicate that ICOS expression is not required for the function of PItol cells. Indeed, blockade of the interaction between ICOS and its ligand in vitro did not reverse suppression mediated by the PItol cells. However, one cannot exclude the possibility that this molecule might be involved in the generation of such cells.
Previous in vitro experiments with the Tg4 model were limited to the demonstration of anergy and IL-10 production. We wished to know whether the tolerant cells might be capable of suppression. These cells were found to be potent suppressors of naive T cell proliferation and IL-2 production. However, interestingly, the suppressive effect of PItol cells could not be reversed by blockade of the IL-10R. Previous studies had shown that supernatants from cells rendered tolerant with superantigen were capable of suppressing the response of naive cells to Ag (14). The suppression mediated by supernatants could be blocked by addition of Abs to IL-10 and TGF-. By contrast, we noted that supernatants from tolerant cells had no significant effect on APCs. Thus, supernatants from PItol cells failed to block the up-regulation of MHC class II, CD80, and CD86 costimulatory molecules on purified immature dendritic cells and had no effect on T cell proliferation (data not shown). This intriguing difference between regulatory cells induced with peptide vs superantigen cannot be explained by the amount of IL-10 produced since the levels were similar in both studies. The apparent requirement for cell-cell contact was further confirmed using transwell cultures, indicating that PItol cells were totally dependent on cell-cell contact for suppression of naive cells in vitro. Therefore, despite the high level of IL-10 secreted by PItol cells in response to Ag, this cytokine does not account for the suppression of naive cells observed in vitro.
A further difference between the PItol cells and TCR transgenic cells rendered tolerant by repeated stimulation with superantigen lies in their response to Ag and IL-2. PItol cells appear to be conventionally anergic. In other words, the unresponsive state of these cells and their capacity to suppress naive cells in vitro could be reversed by the addition of exogenous IL-2 to cultures. However, previous studies by Schwartz and colleagues (14) showed that superantigen-treated cells were profoundly unresponsive and the addition of IL-2 at 10 U/ml did not increase their proliferative response, suggesting that responsiveness to IL-2 was impaired. Indeed, anergic cells were shown to display altered signaling via the common -chain of the IL-2R, which consequently resulted in diminished phosphorylation of several downstream proteins (48). This suggests to us that different degrees of unresponsiveness can be induced in T cells despite the fact that the two populations of regulatory cells both secrete IL-10 in response to Ag. It seems likely that this depends on the nature and strength of the tolerogen. It may well be that superantigens induce a "deeper state" of unresponsiveness involving impairment of more signaling pathways when compared with that induced by peptide Ags. However, most importantly, the IL-10 signaling pathway appears to be resistant to Ag-induced anergy.
We have established two separate models to test the function of PItol cells in vivo. The first transfer model involved transfer of either naive or tolerant cells that had been labeled with CFSE. The in vivo proliferation of naive cells was suppressed when these cells were transferred into tolerant mice, thus confirming the ability of tolerant cells to suppress the proliferation of naive cells in vitro. It was noticeable that tolerant cells transferred into naive mice underwent some rounds of division, whereas their proliferation was totally blocked in tolerant recipients. It seems likely that the proliferation of tolerant cells in naive mice resulted from a partial reversal of tolerance due to the secretion of IL-2 by naive T cells. This would be consistent with the reversal of tolerance by addition of IL-2 to tolerant cells in culture. Furthermore, the anergic state of PItol cells when transferred into tolerant mice was not reversed by injection of anti-IL-10R Abs (data not shown). This further emphasizes the requirement for IL-2 in reversal of anergy. However, most notably, the suppression observed when naive cells were transferred into tolerant mice was reversed by coinjection of an Ab directed to the IL-10R. This shows that IL-10 plays a significant role in the tolerance mediated by PItol cells in vivo and is in marked contrast to the results arising from experiments in vitro.
Naive Tg4 cells secrete IL-2 in response to peptide Ag and serum levels of the cytokine peak within 2 h. This observation was used as a read-out for transfer of tolerance. The results shown in Fig. 6 indicate that the transfer of PItol cells leads to the suppression of IL-2 production in vivo. However, most significantly, this suppression was reversed by coinjection of an Ab directed to the IL-10R. Therefore, we have used two independent methods to confirm the role of IL-10 in peripheral tolerance mediated by soluble peptide administration.
The results arising from this study reveal the limitations of in vitro models of suppression. A number of publications have demonstrated that the suppressive function of CD4+CD25+ natural regulatory T cells is cytokine-independent but requires cell-cell contact (37, 49, 50). These studies were based on assays conducted in vitro and the results clearly need to be carefully considered in light of the current findings. In other words, the fact that Abs to IL-10 or TGF- do not inhibit the suppressive properties of regulatory cells in vitro does not necessarily mean that these cytokines are not involved in vivo. Various groups have clearly shown a role for cytokines in the function of regulatory cells in vivo (32, 51, 52). This depended on the type of model and often involved the cotransfer of naive and tolerant cells into lymphopenic mice. Interestingly, the dependence on IL-10 depended on the disease model with suppression of autoimmune colitis being IL-10-dependent and autoimmune gastritis being IL-10-independent. A clue as to why cytokines are required in some but not other models arises from a study by Annacker et al. (53). These authors showed that both CD25+ and CD25-CD45RBlow populations contain regulatory cells. The CD25+CD45RBlow cells were able to prevent disease arising from expansion of CD45RBhigh cells in lymphopenic mice and the suppression of cell expansion was dependent on IL-10. As it stands there is evidence that both CD25+ and PItol cells (predominantly CD25-) can suppress the response of neighboring T cells in vitro in a cell-cell contact-dependent and cytokine-independent fashion. The need for IL-10 production in vivo varies depending on the nature of the model, but we can conclude from the results of this study that PItol cells are dependent on IL-10 for suppression in vivo. Further work from this laboratory has shown that PItol cells are distinct both in origin and function to the natural CD4+CD25+ cells that make up 5% of the T cell repertoire in the RAG+/+ Tg4 mouse (A. Sundstedt, E. O’Neill, K. Nicolson, S. Parry, and D. Wraith, manuscript in preparation).
PItol cells arise through repeated Ag encounter in an environment where there is little inflammation; hence, the majority of APCs are in a resting state. A number of recent studies have shown that Ags presented to T cells by dendritic cells that are either immature or intermediate in maturity drive the generation of IL-10-secreting regulatory T cells (54, 55, 56). It is likely that peptides delivered in a highly soluble form bind to such dendritic cells and that this form of Ag presentation in vivo leads to the generation of IL-10-secreting PItol cells. Similarly, addition of IL-10 or the combination of vitamin D3 and dexamethasone (57, 58, 59) would block the spontaneous maturation that occurs when immature dendritic cells are cultured in vitro. This could, in part, explain why repeated exposure to Ag in the presence of such agents leads to the generation of IL-10-secreting regulatory cells. There is now evidence that soluble peptide administration leads to the generation of IL-10-secreting regulatory cells in humans, induction of these cells correlating with effective desensitization of the immune response to allergens (60, 61, 62, 63). Therefore, cells with the properties of PItol cells can be induced in both mouse and man and these cells can moderate the immune response to both autoantigens and allergens. We believe that these cells function in the suppression of hypersensitivity conditions in general. Therefore, the findings of this paper are of central importance to the safe and effective use of peptides for immunotherapy of both allergic and autoimmune conditions.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Small Animal Clinical Studies, Veterinary College, University College Dublin, Dublin, Ireland.
3 Address correspondence and reprint requests to Dr. Anette Sundstedt at the current address: Active Biotech Research, P.O. Box 724, 220 07 Lund, Sweden. E-mail address: anette.sundstedt{at}activebiotech.com
4 Abbreviations used in this paper: MBP, myelin basic protein; EAE, experimental autoimmune encephalomyelitis; i.n., intranasal; PItol, peptide-induced tolerant cell; ICOS, inducible costimulator protein; GITR, glucocorticoid-induced TNFR; RAG, recombination-activating gene.
Received for publication July 30, 2002. Accepted for publication November 22, 2002.
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References |
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  L. Gabrysova, K. S. Nicolson, H. B. Streeter, J. Verhagen, C. A. Sabatos-Peyton, D. J. Morgan, and D. C. Wraith Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10-secreting Th1 cells J. Exp. Med., August 3, 2009; 206(8): 1755 - 1767. [Abstract] [Full Text] [PDF]
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 Y.-H. Huang, A. L. Zozulya, C. Weidenfeller, N. Schwab, and H. Wiendl T cell suppression by naturally occurring HLA-G-expressing regulatory CD4+ T cells is IL-10-dependent and reversible J. Leukoc. Biol., August 1, 2009; 86(2): 273 - 281. [Abstract] [Full Text] [PDF]
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J. D. Campbell, K. F. Buckland, S. J. McMillan, J. Kearley, W. L.G. Oldfield, L. J. Stern, H. Gronlund, M. van Hage, C. J. Reynolds, R. J. Boyton, et al. Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression J. Exp. Med., July 6, 2009; 206(7): 1535 - 1547. [Abstract] [Full Text] [PDF]
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 H. R. Kim, R. Rajaiah, Q.-L. Wu, S. R. Satpute, M. T. Tan, J. E. Simon, B. M. Berman, and K. D. Moudgil Green Tea Protects Rats against Autoimmune Arthritis by Modulating Disease-Related Immune Events J. Nutr., November 1, 2008; 138(11): 2111 - 2116. [Abstract] [Full Text] [PDF]
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 Z. Zhao, S. Yu, D. C. Fitzgerald, M. Elbehi, B. Ciric, A. M. Rostami, and G.-X. Zhang IL-12R{beta}2 Promotes the Development of CD4+CD25+ Regulatory T Cells J. Immunol., September 15, 2008; 181(6): 3870 - 3876. [Abstract] [Full Text] [PDF]
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A. D'Ambrosio, M. Colucci, O. Pugliese, F. Quintieri, and M. Boirivant Cholera toxin B subunit promotes the induction of regulatory T cells by preventing human dendritic cell maturation J. Leukoc. Biol., September 1, 2008; 84(3): 661 - 668. [Abstract] [Full Text] [PDF]
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 H. Ait-Oufella, B. Horvat, Y. Kerdiles, O. Herbin, P. Gourdy, J. Khallou-Laschet, R. Merval;, B. Esposito;, A. Tedgui, and Z. Mallat Measles Virus Nucleoprotein Induces a Regulatory Immune Response and Reduces Atherosclerosis in Mice Circulation, October 9, 2007; 116(15): 1707 - 1713. [Abstract] [Full Text] [PDF]
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 T. V. Johnson, C. B. Camras, and J. Kipnis Bacterial DNA Confers Neuroprotection after Optic Nerve Injury by Suppressing CD4+CD25+ Regulatory T-Cell Activity Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3441 - 3449. [Abstract] [Full Text] [PDF]
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I. R. Humphreys, C. de Trez, A. Kinkade, C. A. Benedict, M. Croft, and C. F. Ware Cytomegalovirus exploits IL-10-mediated immune regulation in the salivary glands J. Exp. Med., May 14, 2007; 204(5): 1217 - 1225. [Abstract] [Full Text] [PDF]
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S. Nylen, R. Maurya, L. Eidsmo, K. D. Manandhar, S. Sundar, and D. Sacks Splenic accumulation of IL-10 mRNA in T cells distinct from CD4+CD25+ (Foxp3) regulatory T cells in human visceral leishmaniasis J. Exp. Med., April 16, 2007; 204(4): 805 - 817. [Abstract] [Full Text] [PDF]
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A. Joetham, K. Takada, C. Taube, N. Miyahara, S. Matsubara, T. Koya, Y.-H. Rha, A. Dakhama, and E. W. Gelfand Naturally Occurring Lung CD4+CD25+ T Cell Regulation of Airway Allergic Responses Depends on IL-10 Induction of TGF-beta J. Immunol., February 1, 2007; 178(3): 1433 - 1442. [Abstract] [Full Text] [PDF]
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K. M. Spach, F. E. Nashold, B. N. Dittel, and C. E. Hayes IL-10 Signaling Is Essential for 1,25-Dihydroxyvitamin D3-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 2006; 177(9): 6030 - 6037. [Abstract] [Full Text] [PDF]
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M. Veldhoen, H. Moncrieffe, R. J. Hocking, C. J. Atkins, and B. Stockinger Modulation of Dendritic Cell Function by Naive and Regulatory CD4+ T Cells J. Immunol., May 15, 2006; 176(10): 6202 - 6210. [Abstract] [Full Text] [PDF]
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K. S. Nicolson, E. J. O'Neill, A. Sundstedt, H. B. Streeter, S. Minaee, and D. C. Wraith Antigen-Induced IL-10+ Regulatory T Cells Are Independent of CD25+ Regulatory Cells for Their Growth, Differentiation, and Function J. Immunol., May 1, 2006; 176(9): 5329 - 5337. [Abstract] [Full Text] [PDF]
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I. Kinjyo, H. Inoue, S. Hamano, S. Fukuyama, T. Yoshimura, K. Koga, H. Takaki, K. Himeno, G. Takaesu, T. Kobayashi, et al. Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor-{beta}1 J. Exp. Med., April 17, 2006; 203(4): 1021 - 1031. [Abstract] [Full Text] [PDF]
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 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
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G.-X. Zhang, S. Yu, D. Calida, Z. Zhao, B. Gran, M. Kamoun, and A. Rostami Loss of the Surface Antigen 3G11 Characterizes a Distinct Population of Anergic/Regulatory T Cells in Experimental Autoimmune Encephalomyelitis J. Immunol., March 15, 2006; 176(6): 3366 - 3373. [Abstract] [Full Text] [PDF]
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M. Elrefaei, B. Barugahare, F. Ssali, P. Mugyenyi, and H. Cao HIV-Specific IL-10-Positive CD8+ T Cells Are Increased in Advanced Disease and Are Associated with Decreased HIV-Specific Cytolysis J. Immunol., January 15, 2006; 176(2): 1274 - 1280. [Abstract] [Full Text] [PDF]
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I. P. Lewkowich, N. S. Herman, K. W. Schleifer, M. P. Dance, B. L. Chen, K. M. Dienger, A. A. Sproles, J. S. Shah, J. Kohl, Y. Belkaid, et al. CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function J. Exp. Med., December 5, 2005; 202(11): 1549 - 1561. [Abstract] [Full Text] [PDF]
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D. K. Sojka, A. Hughson, T. L. Sukiennicki, and D. J. Fowell Early Kinetic Window of Target T Cell Susceptibility to CD25+ Regulatory T Cell Activity J. Immunol., December 1, 2005; 175(11): 7274 - 7280. [Abstract] [Full Text] [PDF]
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T. Higuchi, T. Maruyama, A. Jaramillo, and T. Mohanakumar Induction of Obliterative Airway Disease in Murine Tracheal Allografts by CD8+ CTLs Recognizing a Single Minor Histocompatibility Antigen J. Immunol., February 15, 2005; 174(4): 1871 - 1878. [Abstract] [Full Text] [PDF]
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 H. Ben-David, M. Sela, and E. Mozes Down-regulation of myasthenogenic T cell responses by a dual altered peptide ligand via CD4+CD25+-regulated events leading to apoptosis PNAS, February 8, 2005; 102(6): 2028 - 2033. [Abstract] [Full Text] [PDF]
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R. K. Gregg, J. J. Bell, H.-H. Lee, R. Jain, S. J. Schoenleber, R. Divekar, and H. Zaghouani IL-10 Diminishes CTLA-4 Expression on Islet-Resident T Cells and Sustains Their Activation Rather Than Tolerance J. Immunol., January 15, 2005; 174(2): 662 - 670. [Abstract] [Full Text] [PDF]
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P. O. Anderson, A. Sundstedt, Z. Yazici, S. Minaee, R. Woolf, K. Nicolson, N. Whitley, L. Li, S. Li, D. C. Wraith, et al. IL-2 Overcomes the Unresponsiveness but Fails to Reverse the Regulatory Function of Antigen-Induced T Regulatory Cells J. Immunol., January 1, 2005; 174(1): 310 - 319. [Abstract] [Full Text] [PDF]
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J. Kipnis, H. Avidan, R. R. Caspi, and M. Schwartz Dual effect of CD4+CD25+ regulatory T cells in neurodegeneration: A dialogue with microglia PNAS, October 5, 2004; 101(suppl_2): 14663 - 14669. [Abstract] [Full Text] [PDF]
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 D S Robinson Regulation: the art of control? Regulatory T cells and asthma and allergy Thorax, August 1, 2004; 59(8): 640 - 643. [Full Text] [PDF]
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 J. Kipnis, M. Cardon, H. Avidan, G. M. Lewitus, S. Mordechay, A. Rolls, Y. Shani, and M. Schwartz Dopamine, through the Extracellular Signal-Regulated Kinase Pathway, Downregulates CD4+CD25+ Regulatory T-Cell Activity: Implications for Neurodegeneration J. Neurosci., July 7, 2004; 24(27): 6133 - 6143. [Abstract] [Full Text] [PDF]
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I. Apostolou and H. von Boehmer In Vivo Instruction of Suppressor Commitment in Naive T Cells J. Exp. Med., May 17, 2004; 199(10): 1401 - 1408. [Abstract] [Full Text] [PDF]
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P. L. Vieira, J. R. Christensen, S. Minaee, E. J. O'Neill, F. J. Barrat, A. Boonstra, T. Barthlott, B. Stockinger, D. C. Wraith, and A. O'Garra IL-10-Secreting Regulatory T Cells Do Not Express Foxp3 but Have Comparable Regulatory Function to Naturally Occurring CD4+CD25+ Regulatory T Cells J. Immunol., May 15, 2004; 172(10): 5986 - 5993. [Abstract] [Full Text] [PDF]
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M. Kohyama, D. Sugahara, S. Sugiyama, H. Yagita, K. Okumura, and N. Hozumi Inducible costimulator-dependent IL-10 production by regulatory T cells specific for self-antigen PNAS, March 23, 2004; 101(12): 4192 - 4197. [Abstract] [Full Text] [PDF]
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 C.-R. Shen, A.-R. Youssef, A. Devine, L. Bowie, A. M. Hall, D. C. Wraith, C. J. Elson, and R. N. Barker Peptides containing a dominant T-cell epitope from red cell band 3 have in vivo immunomodulatory properties in NZB mice with autoimmune hemolytic anemia Blood, November 15, 2003; 102(10): 3800 - 3806. [Abstract] [Full Text] [PDF]
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S. Grundstrom, L. Cederbom, A. Sundstedt, P. Scheipers, and F. Ivars Superantigen-Induced Regulatory T Cells Display Different Suppressive Functions in the Presence or Absence of Natural CD4+CD25+ Regulatory T Cells In Vivo J. Immunol., May 15, 2003; 170(10): 5008 - 5017. [Abstract] [Full Text] [PDF]
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