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* Institut National de la Santé et de la Recherche Médicale, Unité 343, Hopital de l’Archet, Nice, France; and
TxCell, Nice, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(Th3) have also been described in in vivo models. In the lymphocyte transfer model of inflammatory bowel disease, we show here that the control of inflammation during the first weeks is not due to a complete inhibition of differentiation of aggressive proinflammatory T cells, but is the result of a balance between proinflammatory and Tr cells. We also show that in the first weeks continuous IL-10 secretion was required to actively control inflammation. Indeed, treatment with anti-IL-10R Abs 3 wk after the start of the experiment completely reversed the protective effect of Tr cells. IL-10 secretion and control of inflammation could be provided by late injection of Tr1 cells that efficiently cure ongoing inflammatory responses in two different models of inflammation. In contrast, inflammation was not controlled when high numbers of CD4+CD45RBlow or CD4+CD25+ T cells were injected as early as 1 wk after the start of the experiment. These results confirm in vitro studies showing that CD4+CD45RBlow do not contain high IL-10-producing cells and suggest that CD4+CD45RBlow Tr cells maintain tolerance in vivo, in part indirectly, through the differentiation of IL-10-secreting Tr1 cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have focused on a population of CD4+ T cells that constitutively express the IL-2R
(CD25). CD4+CD25+ T cells comprise 
5–10% of the peripheral T cell pool and exhibit immunosuppressive abilities both in vitro and in vivo. Taken together, all in vitro studies of murine and human CD25+ T cells support a cell contact-dependent, cytokine-independent mechanism of suppression. Since suppression requires activation of CD25+ T cells, it has been hypothesized that activation of these cells via their TCR induces a cell surface molecule(s) that mediates suppression by binding to a counter-receptor on the responder cells (10, 11, 12). However, it is not known how CD4+ CD25+ T cells execute their function in vivo, if they constitute only a small population of peripheral CD4+ T cells (average, 6%) that needs direct cell contact as well as stimulation via the TCR to suppress unwanted T cell activation. In vitro studies usually employ high ratios of CD4+CD25-/CD4+CD25+ T cells, a situation that is hard to imagine in vivo. Moreover, in autoimmune diseases, IL-10, IL-4, and TGF- have been implicated in the suppression mechanism of CD4+CD25+, in contrast to in vitro experiments (10, 13, 14, 15, 16, 17). This suggests that the regulation induced by CD4+ CD25+ T cells might be indirect and would require the differentiation of T cells secreting anti-inflammatory cytokines, as recently suggested (18).
We and others have reported that repetitive Ag-specific stimulations of CD4+ T cells in the presence of IL-10 lead to the generation of another population of Tr cells (Tr1) predominantly producing IL-10 (19). We showed that these Tr1 cells have a regulatory function in vivo in a model of chronic inflammatory bowel disease (IBD) (7). In this model, injection of purified naive CD4+ CD45RBhigh T cells from BALB/c mice into immunodeficient C.B-17 scid mice results in chronic inflammation in the colon. Colitis could be equally prevented by coinjection of CD4+CD45RBlow T cells, CD4+CD25+ T cells, or Tr1 cells (20, 21). Although the regulatory function observed with CD4+CD45RBlow T cells could be restricted to the CD4+CD25+ T cell population (22, 23), the population of CD4+CD25-CD45RBlow T cells always displayed some regulatory effects (22, 23). We therefore performed most of the experiments with the complete CD4+CD45RBlow T cell population and controlled that the effects observed were similar to the effects obtained with the CD4+CD25+ T cell population.
In the present study using the same model of IBD we compared the mechanism of action of CD4+CD45RBlow T cells and Tr1 cells. We showed that although the protective effect of both CD4+CD45RBlow and Tr1 cells is prevented by blocking IL-10 function in vivo, only Tr1 cells display a curative effect when injected several weeks after the initiation of inflammation. These results support the hypothesis that the regulatory function of CD4+CD25+ T cells is mediated by the induction of anti-inflammatory Tr1 cell differentiation in vivo. Moreover, we confirmed the anti-inflammatory function of Tr1 cells in another model in which inflammation was induced in normal mice by repetitive applications of an irritant hapten.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free BALB/c and C.B-17 scid mice were obtained from CERJ (Le Genest Saint Isle, France). Homozygous DO11-10 mice were a gift from Dr. S. D. Hurst (DNAX Research Institute, Palo Alto, CA). Mice were maintained in our animal facility. C.B-17 scid mice were housed in microisolator cages with sterile filtered air (Rec Biozone, Margate, U.K.). Female mice were used at 8–12 wk of age.
Abs, media, and reagents
The medium used for T cell cultures was Yssel medium (24) supplemented with 10% FCS (Roche, Meylan, France) and 2 x 10-5 M 2-ME (Invitrogen, San Diego, CA). Recombinant mouse IL-10 and IL-4 were gifts from Dr. R. L. Coffman (DNAX Research Institute, Palo Alto, CA). Recombinant mouse IFN-
and IL-12 were purchased from R&D Systems (Minneapolis, MN). Purified anti-IL-4 (11B11), anti-IL-10 (2A5), anti-IFN- (XGM1.2), and biotin-anti-IL-4 (24G2), -anti-IL-10 (SXC1), and -anti-IFN- (R4-6A2; all from BD PharMingen, Le Pont de Claix, France) were used for cytokine assays. The following mAbs were used for mouse cell detection and purification: anti-I-Ad (AMS-32.1) anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-B220 (RA36B2), FITC-conjugated anti-mouse CD45RB (16A), Tricolor- or PE-conjugated anti-CD4 (GK1.5), PE-conjugated anti-CD62L (Mel-14), FITC-conjugated anti-CD25 (7D4), FITC- or biotynylated-KJ-1.26 mAb revealed by PE-labeled streptavidin, and FITC- and PE-conjugated isotype control Abs (BD PharMingen). The following Abs were used in vivo: GL113 (isotype control, rat IgG1) and 1B1.2 (anti-mIL-10R; provided by Dr. K. Moore, DNAX Research Institute). For in vivo use, mAb were purified by column chromatography from tissue culture supernatants. The resulting Abs were >98% pure and contained <3 endotoxin units of endotoxins/mg of protein. Lysis buffer, OVA323–339 peptide, OVA, and oxazolone were from Sigma-Chemie (Saint Quentin Fallavier, France). OVA323–339 lipopeptide was purchased from Bachem (Voisin-le-Bretonneux, France).
Cell purification and cytofluorometry
CD4+ T cell subsets were purified from the spleens of mice as previously described (7). Briefly, cells were depleted of B220+, Mac-1+, I-Ad+, and CD8+ cells by negative selection using sheep anti-rat Ab-coated Dynabeads (Dynal, Oslo, Norway). The remaining cells were labeled with FITC-conjugated anti-CD45RB (25 µg/ml) and PE-conjugated anti-CD4 (10 µg/ml) and separated into CD4+ CD45RBhigh and CD4+ CD45RBlow fractions by two-color sorting on a FACStar SE (BD Biosciences, Le Pont de Claix, France). All populations were >98% pure on reanalysis.
T cell populations and T cell clones
The mouse T cell clones were obtained from DO11-10 mice after in vitro differentiation as previously described (7). Naive (MEL-14bright) CD4+, KJ-1.26+ cells were stimulated for 3 wk repeatedly with OVA323–339 peptide in the presence of IL-4 and anti-IL-12, IL-12 and anti-IL-4, or IL-10 for Th2, Th1, or Tr1 cells, respectively. The populations obtained were either used in vivo or cloned at one cell per well by cytofluorometry (FACSVantage SE; BD Biosciences) and stimulated with irradiated splenocytes (4500 rad) and OVA peptide. Clones were then expanded and analyzed for cytokine secretion after activation with APCs and OVA peptide (Table I). Selected clones were then expanded by stimulation with irradiated splenocytes and OVA peptide every 2 wk and were further expanded with IL-2 (R&D Systems; 10 ng/ml). T cell clones were used at least 10 days after the last stimulation.
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C.B-17 scid mice were injected i.p. with 100 µl of PBS containing sorted CD4+ T cell subpopulations and different numbers of T cell clones as indicated. Anti-cytokine and control Abs were injected i.p. in PBS.
Microscopic examination of colons
Colons were removed from mice and fixed in PBS containing 10% formalin. Paraffin-embedded sections (6 µm) were cut and stained with H&E. Tissues were graded semiquantitatively from 0 to 5 in a blinded fashion. A grade of 0 was given when there were no changes observed. Changes typically associated with other grades are as follows: grade 1, minimal scattered mucosal inflammatory cell infiltrates, with or without minimal epithelial hyperplasia; grade 2, mild scattered to diffuse inflammatory cell infiltrates, sometimes extending into the submucosa and associated with erosions, with minimal to mild epithelial hyperplasia and minimal to mild mucin depletion from goblet cells; grade 3, mild to moderate inflammatory cell infiltrates that were sometimes transmural, often associated with ulceration, with moderate epithelial hyperplasia and mucin depletion; grade 4, marked inflammatory cell infiltrates that were often transmural and associated with ulceration, with marked epithelial hyperplasia and mucin depletion; and grade 5, marked transmural inflammation with severe ulceration and loss of intestinal glands.
Immunohistochemistry
Portions of colons were snap-frozen in liquid nitrogen and stored at -70°C. Frozen sections (5 µm) were cut and mounted on glass slides. They were thoroughly dried at room temperature for 1 h and fixed in acetone at 4°C for 15 min. Sections were stained by an immunoenzyme technique using the biotin-avidin-peroxidase system. Briefly, sections were washed in PBS for 5 min. Then sections were saturated with biotin and avidin (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions and incubated with KJ-1.26-biotin or the control isotype. After washing for 5 min in PBS, the sections were incubated with streptavidin-peroxidase. After a final wash, peroxidase was developed with a diaminobenzidene vector staining kit (Vectastain; Vector Laboratories), which gives a brown color.
Contact sensitivity to oxazolone
Contact sensitivity to oxazolone was performed by applying 20 µl of a 50 mg/ml oxazolone solution in acetone/olive oil (4/1, v/v) epicutaneously on the right ear once a day for 3 days. The left ear received the vehicle only. Ear thickness was monitored every day. OVA323–339 lipopeptide was diluted at 50 µM in olive oil. Mice were treated for 6 days by applying daily 20 µl of 50 µM OVA323–339 lipopeptide or olive oil directly on the inflamed ear.
Cytokine assays
A sandwich ELISA was used to measure IL-4, IL-10, and IFN-. In brief, ELISA plates (Polylabo, France) were coated with the appropriate coating anti-cytokine mAbs in carbonate buffer and incubated at 4°C overnight. Plates were blocked for 30 min at room temperature with 150 µl of 20% FCS/PBS added to each well. Fifty microliters of diluted supernatants from in vitro stimulated CD4+ T cells were then added to the plates and incubated overnight at 4°C. After a washing step, 50 µl/well of the biotinylated second-step Ab was added. Plates were incubated for 1 h at room temperature and washed. The enzyme conjugate (streptavidin-peroxidase) was then added to each well. Plates were incubated at room temperature for 1 h and washed, and 100 µl/well of substrate was added (1 mg/ml ABTS). Plates were read on an ELISA reader at a wavelength of 405 nm after color development (iEMS reader; Labsystems, Helsinki, Finland).
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Results |
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In the IBD model of lymphocyte transfer into immunodeficient mice, it has been shown that the induction of inflammation in the colon is correlated with the presence of Th1 cells (25), whereas protection is induced by regulatory cells secreting IL-10 and TGF-. However, the respective roles of Th1, Tr1, and Th2 cells in the regulation of inflammation has not been analyzed. To compare the in vivo function of Tr1 cells to Th1 and Th2 cells with the same antigenic specificity, SCID mice reconstituted with CD4+CD45RBhigh T cells were treated with different amounts of OVA-specific T cell clones of the different subtypes (Table I) and fed with OVA (100 ng/ml) in their drinking water (Fig. 1) as previously described (7). As few as 2 x 104 cells were required for Tr1 cells to display protection, whereas no inhibition of colitis was observed with Th2 cells even at 2 x 106 cells/mouse, suggesting that Th2 cells are not involved in the negative regulation of inflammation in this model. As expected, a slight enhancement in disease score was observed with high doses of Th1 cells. To analyze whether the protective effect of Tr1 cells, compared with that of Th2 cells, was due to higher survival or expansion, we analyzed the number of cells recovered after the transfer into SCID mice reconstituted with CD45RBhigh T cells. All mice were fed OVA. Analysis of the number of CD4+KJ1–26+ cells on day 2 after the transfer revealed that few cells survived (<1% in the different populations). As expected, in reconstituted mice a marked expansion of Th1 cells was observed 4 and 8 wk after the transfer. Th2 cells also proliferated in vivo, but to a lesser extent. In contrast, for Tr1 cells, an expansion was observed 4 wk after the transfer, but these cells were not recovered after 8 wk, suggesting that their expansion is only transient and correlates with the intensity of inflammation. As a control, treatment of mice with the different clones in the absence of CD4+CD4RBhigh T cells did not lead to colitis or significant expansion (not shown). These results suggest that Tr1 cells have a functional specificity in the control of inflammation.
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We have previously shown in vitro that IL-10 was required for both the function and the differentiation of Tr1 cells (7). Moreover, protection of IBD by regulatory CD4+CD45RBlow T cells has been shown to depend on the presence of IL-10 (15). To test whether IL-10 influences the function of in vitro-differentiated Tr1 cells in vivo, we used anti-IL-10R Abs to block the action of IL-10 in mice reconstituted with CD4+CD45RBhigh T cells. Mice were treated weekly with anti-IL-10R or the isotype control mAb and, as shown in Fig. 2, A and B, treatment with anti-IL-10R abrogated protection from colitis induced by Tr1 cells or the CD4+CD45RBlow population, as all mice in these groups treated with anti-IL-10R developed colitis. Ab treatment alone did not enhance IBD induced by CD4+CD45RBhigh cells (Fig. 2) or induced immune pathology in the absence of T cells, as unreconstituted recipients treated with anti-IL-10R did not develop colitis (data not shown). These results suggest that the secretion of IL-10 by regulatory T cells is crucial for the control of IBD.
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Differences between CD4+CD45RBlow T cells and Tr1 cells in the control of IBD
In this IBD model it has been clearly shown that the protective effect of CD4+CD45RBlow as well as CD4+CD25+ T cells is dependent on IL-10 secretion (15, 22). However, when activated in vitro with potent stimuli, these cells do not secrete IL-10 (10) (data not shown). Moreover, their inhibitory function in vitro is mediated by cell contact and is independent of IL-10 secretion. One could therefore hypothesize that CD4+CD45RBlow T cells do not have a direct inhibitory effect in vivo in this reconstitution model, but play their regulatory role indirectly by inducing the differentiation of IL-10-secreting Tr cells. To test this hypothesis, SCID mice reconstituted with CD4+CD45RBhigh T cells were treated with a population of CD4+CD45RBlow T cells, a population of CD4+CD25+ T cells, or a population of Tr1 cells on day 0, simultaneously with CD4+CD45RBhigh T cells or 1 wk after the onset of the experiment. In contrast to treatment with Tr1 cells, which remains effective after a delay of 1 wk (Fig. 3), treatment of SCID mice with CD4+CD45RBlow or CD4+CD25+ T cells is efficient only for the prevention of IBD, as injection of cells 1 wk after reconstitution failed to inhibit the development of colitis (Fig. 3C). We excluded the possibility that this absence of a protective effect of CD4+CD45RBlow or CD4+CD25+ T cells was due to low cell numbers in experiments performed with high doses (100 times the amount of cells required to show protection on day 0) of regulatory CD4+CD45RBlow or CD4+CD25+ T cells (Fig. 3), which again failed to display any inhibitory effect on colitis induction. We also excluded the possibility that the differences between Tr1 and CD25+ T cells in the inhibition of ongoing inflammation was due to a preferential expansion of Tr1 cells compared with CD4+CD25+ T cells. It has been shown (22) that CD4+CD25+ T cells expand in vivo and that 20–100% of the cells injected could be recovered 12–14 wk after their coinjection with CD45RBhigh T cells. In contrast, the expansion of Tr1 cells was less effective, as <5% of the injected cells were recovered 4 wk after their transfer (Table II), and no cells could be found 8 wk after the transfer. Taken together, the absence of a curative effect, the absence of IL-10 secretion in vitro, and the requirement for continuous IL-10 secretion to control inflammation support the hypothesis that the CD4+CD45RBlow or CD4+CD25+ T cell population regulates colitis indirectly by inducing the differentiation of T cells that actively secrete immunoregulatory cytokines such as IL-10.
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To further analyze the ability of Tr1 cells to cure an ongoing inflammatory response, C.B-17 scid mice restored on day 0 with CD4+CD45RBhigh T cells were transferred with Tr1 cells several weeks after the start of the experiment (Fig. 4, A and B). Even 6 wk after the transfer of pathogenic CD4+ CD45RBhigh T cells, treatment of mice with Tr1 cells and OVA administration was followed by a remission of all inflammatory signs in the colon (Fig. 4B) associated with the recovery of the initial weight (Fig. 4A). In other experiments Tr1 cells were injected 4 wk after the reconstitution of mice with CD4+CD45RBhigh T cells, and we analyzed the amount of Tr1 cells that infiltrated the colon as well as the extent of inflammation by immunohistochemistry. Analysis performed on colons 4 wk after the injection of CD4+ CD45RBhigh T cells revealed an ongoing inflammatory response (Fig. 4C, panel 1). One week after the injection of Tr1 cell clones, several KJ-1.26+ cells were detected within the inflamed colon (Fig. 4C, panel 6), and some signs of decreased inflammation were observed (Fig. 4C, panel 2). The inflammation quickly decreased within 3 wk (Fig. 4C, panels 3 and 4), and the mice were completely cured 3 wk after the injection of Tr1 cell clones. In some experiments treatment with OVA was interrupted 4 wk after injection of Tr1 cells. In that case mice remained healthy for several months and displayed no sign of inflammation at the time of sacrifice several weeks or even months later (Fig. 4B).
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To analyze the potential curative effect of Tr1 cells in a different inflammatory model, we set up skin inflammation experiments using the hapten oxazolone in three daily applications (Fig. 5A). Because Tr1 cells need to be activated at the site of inflammation, we first tested the ability of cutaneous application of lipo-OVA peptide to stimulate T cells. BALB/c mice were injected with naive OVA-specific DO11-10 T cells, and mice were treated for 6 days by applying daily 20 µl of 50 µM OVA323–339 lipopeptide or olive oil directly to the ear. Analysis of T cells in the draining lymph nodes revealed an accumulation of activated (CD25+) OVA-specific (KJ1-26+) only in the ear draining lymph node treated with the lipopeptide (Fig. 5A). In that model, 3 days after the induction of ear inflammation with oxazolone, the mice were treated with OVA-specific Th1, Th2, or Tr1 T cell populations or a Tr1 cell clone (Fig. 5B), and the lipo-OVA peptide was applied for 6 days. In mice treated with Tr1 cells a marked decrease in inflammatory signs was observed, whereas treatment with Th1 or Th2 cells enhanced inflammation and edema (Fig. 5B). These results show that the specific regulatory function of Tr1 cells is not restricted to the colon, but is also efficient in different tissues and different types of inflammation, as previously reported by others (9, 26).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, with or without TGF-, but with little or no IL-2 and IL-4 production, and proliferate poorly following TCR-mediated activation. Functional studies of Tr1 cells directed against different Ags have shown that Tr1 cells have potent immunosuppressive properties and have been shown to prevent the development of Th1-mediated autoimmune diseases (7, 19, 27) and Th2 responses (28). Recently, in vitro manipulation with immunosuppressive drugs (26) or complementation of IL-10 with addition of IFN- (29) or TGF- (30) has facilitated the expansion of Tr1 cells. Using defined polarized T cells sharing the same Ag specificity we showed in this manuscript that Tr1 cells specifically cure inflammatory responses. As previously shown in in vitro experiments (7), the protective effect of Tr1 cells in vivo is mediated by IL-10 secretion and is completely abrogated by the addition of anti-IL-10R Abs (Fig. 2). However, IL-10 secretion by itself is not sufficient to explain the protective effect of Tr1 cells, as Th2 cells, which also secrete IL-10, did not display any protective effect in the two different inflammatory models, IBD and skin inflammation. This lack of regulatory function of IL-10-secreting Th2 cells was not due to a preferential survival or expansion rate of Tr1 cells in vivo (Table II). However, the absence of inhibitory effects of Th2 in these models of inflammation could be due to the concomitant secretion of IL-4, a cytokine known to stimulate T cell functions, but other factors, such as different migration behavior and tissue localization in vivo, might also explain the specific function of Tr1 cells compared with Th2 cells.
It has been thoroughly demonstrated that CD4+ CD25+ T cells have important regulatory functions in vivo in rodents in the IBD model induced in immunosuppressed mice by the transfer of CD4+CD45RBhigh T cells, it has been shown that the injection of CD4+CD45RBlow T cells prevents colitis (20), and that the regulatory function could be restricted to the CD4+CD45RBlowCD25+ population (22, 23). However, the CD4+CD45RBlowCD25- population was also shown to display both a proinflammatory function when transferred alone and a regulatory function when coinjected with CD4+CD45RBhigh T cells (22, 23). These results suggest that this cell population contains a mixture of proinflammatory cells as well as Tr cells that do not express the CD25 molecule. It was also demonstrated that CD4+CD25+ T cells inhibited the proliferative response of CD4+ CD25- T cells in vitro (10). Moreover, a similar population of suppressor T cells with similar in vitro functions has been observed in humans (11, 12, 31). Numerous characteristics of CD4+ CD25+ T cells still need to be explained. One important question is how CD4+ CD25+ T cells execute their regulatory function in vivo, as they constitute only a small population of peripheral CD4+ T cells (average, 6%) that need direct cell contact as well as stimulation via the TCR to suppress unwanted T cell activation. Moreover, in contrast to the lack of involvement of cytokines in CD25+-mediated suppression in vitro (10, 13), IL-10, IL-4, and TGF- have been implicated in mediating suppression in autoimmune diseases controlled by Tr cells in vivo (14, 15, 16, 17). Recently, in vitro experiments using CD4+ CD25+ human T cells have suggested that the regulatory function of these cells might be supported in part by the differentiation of cytokine-secreting regulatory cells such as Tr1 or Th3 (18, 32). The data presented in this manuscript support that hypothesis. Here we showed that the control of inflammation is due not to a complete inhibition of proinflammatory cells in the first days after the transfer of Tr cells, but to a fine balance between proinflammatory and IL-10-secreting Tr cells. Indeed, some signs of inflammation were observed in the first weeks after the transfer of both proinflammatory CD4+CD45RBhighT cells and regulatory CD4+CD45RBlow T cells, suggesting that the regulatory cells actively control the inflammation. The importance of this balance, which is still active several weeks after the transfer of T cells, is demonstrated by the dramatic induction of colitis when IL-10 function is blocked 3 wk after the onset of the experiments. Therefore, the absence of a protective effect of the injection of high numbers of CD4+CD45RBlow (2 million cells) or CD4+CD25+ (1 million cells) T cells 1 wk after the reconstitution of SCID mice with CD4+CD45RBhigh T cells suggests that the Tr cells secreting IL-10 are not contained in that population. This has been confirmed in a recent manuscript (33) in which the authors show that IL-10 is mandatory for the control of IBD, but when they injected CD4+CD25+ T cells isolated from IL-10-/- mice these cells still retained their ability to inhibit colitis induced by CD4+CD45RBhigh T cells, demonstrating that IL-10 secretion by CD4+CD25+ T cells is not necessary to control IBD. Taken together, these results suggest that, at least in this model, the regulatory function of CD4+CD45RBlow or CD4+CD25+ T cells is in part indirect, by enhancing the differentiation of IL-10-secreting T cells. Recently, Mottet et al. (34) have reported that injection of CD4+CD25+ T cells inhibited ongoing colitis. This report is not discrepant with our results, as in both reports analysis of the colitis score 4–6 wk after the injection of CD4+CD25+ T cells did not reveal any marked beneficial effect, in contrast with the injection of Tr1 cells, which resulted in a rapid remission of inflammatory signs (Fig. 4). The curative effect of high number (106 cells) of CD4+CD25+ T cells was fully effective only after several weeks (10 wk), which again supports the hypothesis of an indirect mechanism in the control of inflammation mediated by CD4+CD25+ T cells.
The differentiation of IL-10-secreting regulatory cells (Tr1) by CD4+CD25+ T cells could be mediated by direct cell contact, as suggested recently by in vitro studies using human CD4+CD25+ T cells (18) or by the action of CD4+CD25+ T cells on APCs. Indeed, it has been shown that the populations of dendritic cells in immunodeficient mice are completely different from those in normal mice and that the transfer of T cells restores the populations of dendritic cells (35). Further studies will be required to analyze whether the transfer of CD4+CD25+ T cells modifies dendritic cell populations to induce the differentiation of Tr1 cells.
In contrast to CD4+CD25+ T cells, we showed here that the injection of in vitro differentiated Tr1 cells, even 6 wk after the transfer of aggressive naive CD4+ T cells, at the peak of the disease completely cures an ongoing inflammation. The regression of inflammation after the transfer of Tr1 cells was rapid and resulted in the complete absence of infiltrated leukocytes. Moreover, there was a surprising complete restoration of the damaged colon, which may be due to the combined action of TGF- secreted by Tr1 cells and the rapid turnover of epithelial cells. The protective effect was not restricted to the colon, as Tr1 clones could effectively down-regulate inflammation in a skin model in which inflammation was mediated by an irritant hapten. Moreover, our results emphasize in both models the importance of local delivery of the specific Tr1 Ag to ensure its anti-inflammatory function. This requirement has also been shown by others in an experimental autoimmune encephalomyelitis model (26).
Taken together, our results support the idea that Tr1 and CD4+CD25+ regulatory T cells are two separate and specialized subsets of Tr cells; the latter have a central homeostatic function to regulate T cell proliferation through direct cell-cell contact mechanisms, and the former, endowed with anti-inflammatory capacity, infiltrate injured tissues to locally control inflammation through the release of IL-10 and TGF-.
An important issue would be to isolate the natural counterparts of Tr1 cells differentiated in vitro. Preliminary studies using specific markers of these cells have shown that a decreased number of these natural Tr1 cells correlate with IBD in human (our manuscript in preparation).
Studies of Tr1 cells will soon offer some attractive avenues for potential therapeutic intervention. Indeed, they display several key advantages. They act through bystander suppression; thus, the pathogenic Ag that stimulates the aggressive T cells does not necessarily have to be known. Secondly, they preferentially migrate to inflamed organs and not to the sites of primary immune responses, thus preventing adverse side effects (our manuscript in preparation). Finally they require the presence of their specific Ag to be activated, providing a way to target them to a specific organ and to control their activation in vivo.
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Footnotes |
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2 F.C. and A.F. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Hervé Groux, Institut National de la Santé et de la Recherche Médicale, Unité 343, Route de St. Antoine de Ginestière, 06200 Nice, France. E-mail address: groux{at}unice.fr
4 Abbreviations used in this paper: Tr, T regulatory; IBD, inflammatory bowel disease.
Received for publication January 9, 2003. Accepted for publication September 4, 2003.
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References |
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1 production and responsiveness. J. Exp. Med. 196:237. and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4+CD45RC- cells and CD4+CD8- thymocytes. J. Exp. Med. 189:279. and IL-10 induce the differentiation of human type 1 T regulatory cells. J. Immunol. 166:5530.. J. Immunol. 163:3684.This article has been cited by other articles:
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  W. Ptak, M. Majewska, K. Bryniarski, M. Ptak, F. M. Lobo, K. Zajac, P. W. Askenase, and M. Szczepanik Epicutaneous Immunization with Protein Antigen in the Presence of TLR4 Ligand Induces TCR{alpha}{beta}+CD4+ T Contrasuppressor Cells That Reverse Skin-Induced Suppression of Th1-Mediated Contact Sensitivity J. Immunol., January 15, 2009; 182(2): 837 - 850. [Abstract] [Full Text] [PDF]
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H. Nagase, K. M. Jones, C. F. Anderson, and N. Noben-Trauth Despite Increased CD4+Foxp3+ Cells within the Infection Site, BALB/c IL-4 Receptor-Deficient Mice Reveal CD4+Foxp3-Negative T Cells as a Source of IL-10 in Leishmania major Susceptibility J. Immunol., August 15, 2007; 179(4): 2435 - 2444. [Abstract] [Full Text] [PDF]
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E. Marinova, S. Han, and B. Zheng Germinal Center Helper T Cells Are Dual Functional Regulatory Cells with Suppressive Activity to Conventional CD4+ T Cells J. Immunol., April 15, 2007; 178(8): 5010 - 5017. [Abstract] [Full Text] [PDF]
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 M. H. Maillard, V. Cotta-de-Almeida, F. Takeshima, D. D. Nguyen, P. Michetti, C. Nagler, A. K. Bhan, and S. B. Snapper The Wiskott-Aldrich syndrome protein is required for the function of CD4+CD25+Foxp3+ regulatory T cells J. Exp. Med., February 19, 2007; 204(2): 381 - 391. [Abstract] [Full Text] [PDF]
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H. H. Uhlig, J. Coombes, C. Mottet, A. Izcue, C. Thompson, A. Fanger, A. Tannapfel, J. D. Fontenot, F. Ramsdell, and F. Powrie Characterization of Foxp3+CD4+CD25+ and IL-10-Secreting CD4+CD25+ T Cells during Cure of Colitis J. Immunol., November 1, 2006; 177(9): 5852 - 5860. [Abstract] [Full Text] [PDF]
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Q. Ding, L. Lu, B. Wang, Y. Zhou, Y. Jiang, X. Zhou, L. Xin, Z. Jiao, and K.-Y. Chou B7H1-Ig Fusion Protein Activates the CD4+ IFN-{gamma} Receptor+ Type 1 T Regulatory Subset through IFN-{gamma}-Secreting Th1 Cells J. Immunol., September 15, 2006; 177(6): 3606 - 3614. [Abstract] [Full Text] [PDF]
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 A. Toda and C. A. Piccirillo Development and function of naturally occurring CD4+CD25+ regulatory T cells J. Leukoc. Biol., September 1, 2006; 80(3): 458 - 470. [Abstract] [Full Text] [PDF]
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N. N. Choileain, M. MacConmara, Y. Zang, T. J. Murphy, J. A. Mannick, and J. A. Lederer Enhanced Regulatory T Cell Activity Is an Element of the Host Response to Injury J. Immunol., January 1, 2006; 176(1): 225 - 236. [Abstract] [Full Text] [PDF]
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 B. Ruiz-Perez, D. R. Chung, A. H. Sharpe, H. Yagita, W. M. Kalka-Moll, M. H. Sayegh, D. L. Kasper, and A. O. Tzianabos Modulation of surgical fibrosis by microbial zwitterionic polysaccharides PNAS, November 15, 2005; 102(46): 16753 - 16758. [Abstract] [Full Text] [PDF]
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 A. J. M. van Oosterhout and N. Bloksma Regulatory T-lymphocytes in asthma Eur. Respir. J., November 1, 2005; 26(5): 918 - 932. [Abstract] [Full Text] [PDF]
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 D. Wolf, K. Hochegger, A. M. Wolf, H. F. Rumpold, G. Gastl, H. Tilg, G. Mayer, E. Gunsilius, and A. R. Rosenkranz CD4+CD25+ Regulatory T Cells Inhibit Experimental Anti-Glomerular Basement Membrane Glomerulonephritis in Mice J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1360 - 1370. [Abstract] [Full Text] [PDF]
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J. S. Satoguina, E. Weyand, J. Larbi, and A. Hoerauf T Regulatory-1 Cells Induce IgG4 Production by B Cells: Role of IL-10 J. Immunol., April 15, 2005; 174(8): 4718 - 4726. [Abstract] [Full Text] [PDF]
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 C Veltkamp, R B Sartor, T Giese, F Autschbach, I Kaden, R Veltkamp, D Rost, B Kallinowski, and W Stremmel Regulatory CD4+CD25+ cells reverse imbalances in the T cell pool of bone marrow transplanted TG{varepsilon}26 mice leading to the prevention of colitis Gut, February 1, 2005; 54(2): 207 - 214. [Abstract] [Full Text] [PDF]
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 M. K. Levings, S. Gregori, E. Tresoldi, S. Cazzaniga, C. Bonini, and M. G. Roncarolo Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells Blood, February 1, 2005; 105(3): 1162 - 1169. [Abstract] [Full Text] [PDF]
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B. E. Anderson, J. M. McNiff, C. Matte, I. Athanasiadis, W. D. Shlomchik, and M. J. Shlomchik Recipient CD4+ T cells that survive irradiation regulate chronic graft-versus-host disease Blood, September 1, 2004; 104(5): 1565 - 1573. [Abstract] [Full Text] [PDF]
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C. Vasu, B. S. Prabhakar, and M. J. Holterman Targeted CTLA-4 Engagement Induces CD4+CD25+CTLA-4high T Regulatory Cells with Target (Allo)antigen Specificity J. Immunol., August 15, 2004; 173(4): 2866 - 2876. [Abstract] [Full Text] [PDF]
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) or vehicle (
) directly to the inflamed ear. Results are shown as the mean ± SD thickness of inflamed ears for one representative experiment of two performed.