Transforming Growth Factor 1 Production by CD4+ CD25+ Regulatory T Cells in Peripheral Blood Mononuclear Cells from Healthy Subjects Stimula
http://www.100md.com
感染与免疫杂志 2005年第9期
Immunologie des Leishmanioses, Institut Pasteur de la Guyane, 97306 Cayenne
Institut d'Enseignement Superieur de la Guyane, Universite Antilles-Guyane, Cayenne
Institut Guyanais de Dermatologie Tropicale, E.A. 2188, Centre Hospitalier Andree Rosemon, 97300, Cayenne, French Guyana
WHO-IRTC, Department of Biochemistry, University of Lausanne, Epalinges, Switzerland
ABSTRACT
Transforming growth factor (TGF-) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF- production in response to Leishmania stimulation in healthy subjects. In this study, TGF-1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-1-producing cells were described as a distinct subpopulation of CD4+ CD25+ regulatory T cells. The suppressive properties of CD4+ CD25+ T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4+ CD25– T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-1 did not reverse the suppressive activity of CD4+ CD25+ T cells activated by anti-CD3, it reversed the suppressive activity of CD4+ CD25+ T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-1 is involved in the suppressive activity of L. guyanensis-stimulated CD4+ CD25+ T cells from healthy controls.
INTRODUCTION
Human infections with Leishmania species induce diseases ranging from cutaneous leishmaniasis to mucosal and visceral leishmaniasis, and the clinical outcomes depend largely on the specific immune responses to Leishmania antigens and particularly on cytokine production. In the murine model of infection with Leishmania major, the involvement of the development of CD4+ Th1 cells producing interleukin 2 (IL-2) and gamma interferon (IFN-) and of CD4+ Th2 cells producing IL-4, IL-10, and IL-13 in resistance and susceptibility, respectively, has been clearly demonstrated (28). However, in humans, the roles of Th1 and Th2 CD4+ T cells and the cytokines they produce are not yet well understood. IL-10 production has been demonstrated in human cutaneous leishmaniasis due to L. braziliensis, and its down-regulation of IFN- production has been proposed to explain disease (29). Transforming growth factor (TGF-) is also a well-characterized antiinflammatory cytokine with antiproliferative and antigen-presenting cell (APC)-deactivating properties which has been implicated in human leishmaniasis (5). Recent data have demonstrated, however, that in vitro TGF- could poorly inhibit IFN- production by peripheral blood mononuclear cells (PBMC) from leishmaniasis patients, suggesting that exogenous TGF- was not able to reverse IFN- antiparasite effector mechanisms (2). Therefore, the exact role of TGF- in disease susceptibility is still unclear.
Both IL-10 and TGF- are secreted by mouse and human regulatory T cells (Treg cells) (30), which represent 5 to 10% of the peripheral CD4+ T cells. The suppressive activity of CD4+ CD25+ Treg cells has been reported both in vitro and in vivo. Suppression was shown to depend on cell contact in vitro, while the cytokines IL-10 and TGF- appeared critical in their suppressive activity in vivo. CD4+ CD25+ Treg cells are restricted by self-antigens, but their role in the control of immune responses against several pathogens has been described recently (26). An important role for CD4+ CD25+ Treg cells has been reported in the control of L. major-specific immune responses in both susceptible BALB/c and resistant C57BL/6 mice (1, 7, 34). In these studies, CD4+ CD25+ Treg cells were shown to control both CD4+ Th1 and Th2 cellular immune responses. The role of CD4+ CD25+ Treg cells during infection with Leishmania in humans has not been investigated directly; investigations have relied on the measurement of cytokines secreted by CD4+ CD25+ Treg cells.
Most of our knowledge about cytokine interaction in humans has come from the studies on different clinical forms of leishmaniasis, and there is limited information about the development of cytokine responses in healthy individuals. In this regard, Leishmania-specific CD8+ and CD4+ T cells were isolated from PBMC of subjects who had never been previously exposed to Leishmania (9, 23), but the exact role of these cells in resistance versus susceptibility to infection is not yet established. Although IL-10- and IFN--producing T cells, which recognize the Leishmania homologue of receptors of activated C kinases (LACK), have been detected in naive subjects (9, 23), only a limited amount of IL-10 was detected in response to live L. guyanensis, which might be due to the presence of a suppressive cytokine. In the present study, we demonstrated that TGF- is produced by PBMC from healthy subjects in response to live Leishmania stimulation and that CD4+ CD25+ regulatory T cells are the TGF-1-secreting cells.
MATERIALS AND METHODS
Subjects. Blood samples were obtained by venipuncture from 13 healthy controls and 15 patients suffering from leishmaniasis due to L. guyanensis (duration of lesion development, <3 months) and collected into sterile tubes (Veinoject; Teruven, Leuven, Belgium). All subjects were seronegative for human immunodeficiency virus. Absence of prior exposure to Leishmania was assessed based on (i) the absence of scars due to leishmaniasis upon clinical examination, (ii) no history of any stay in a country in which leishmaniasis is endemic, and (iii) no reactivity against soluble Leishmania antigen from L. guyanensis based on the inability of these healthy subjects to develop IFN- reactivity and specific antibodies against soluble Leishmania antigens (10).
Informed consent was obtained from the subjects, and the human guidelines of the "Comite Consultatif de Protection des Personnes dans la Recherche Medicale" (CCPRB) of Guadeloupe were followed (project 99-3).
Antigens. Leishmania guyanensis (M4147) and Leishmania major (MRHO/SU/59/P strain) promastigotes were cultured in biphasic rabbit blood agar (22). L. major LV 39 amastigotes were obtained as previously described (15). Extracellular proteins were removed from the parasite pellets by three washes with phosphate-buffered saline. Parasites were used at 106/ml. In some experiments, L. major promastigotes were rendered unable to replicate by a 5-min irradiation with UV radiation (UVC; , 253 nm; 200 mW s/cm2) (27).
Reagents. The reagents for magnetic cell separation with anti-CD4-, anti-CD8-, and anti-mouse immunoglobulin G (IgG)-coated magnetic beads were obtained from Dynal (Compiegne, France). Mouse anti-human CD3 (UTCHT1; IgG1), CD25 (M-1251; IgG1), CD45RO (UCHL-1; IgG2a), DR (Tü36; IgG2b), CCR4 (1C1; IgG1,), CD62L (Dreg56; IgG1), CCR7 (2H4; IgM), CD29, or 1 integrin (HUTS-21; IgG2a), and CD49d, or 4 integrin (9F10; IgG1) antibodies, and rat anti-human cutaneous lymphocyte antigen (CLA) (HECA-452; IgM) and anti-human 7 integrin (FIB504; IgG2a) antibodies, were obtained from Pharmingen (San Diego, CA).
Neutralizing mouse anti-human TGF-1 (TB21; IgG1) and anti-IL-10 (JES3-9D7; IgG1) monoclonal antibodies (MAbs) were obtained from Biosource International (Carmarillo, CA) and Pharmingen, respectively. The purified mouse IgG1 isotype control (107.3, IgG1; anti-trinitrophenol) was obtained from Pharmingen.
Mouse anti-CD4 (RPA-T4; IgG1) and anti-CD8 (RPA-T8; IgG1) conjugated to phycoerythrin for flow cytometric experiments were obtained from Pharmingen.
For T-cell activation, the stimulatory anti-CD3 (UTCHT1; IgG1) antibody was used at 2.5 μg/ml.
Cell isolation and activation. PBMC were isolated after venipuncture on a Ficoll-Hypaque gradient (d = 1.077), and CD3+ and CD3– T cells were purified with a magnetic activated cell sorter from Dynal. Briefly, cells conjugated with an anti-CD3 MAb were suspended with anti-mouse IgG-coated magnetic microbeads and isolated after exposure to a magnetic field. The purity was 96%, as determined by fluorescence-activated cell sorter (FACS) analysis. CD4+ and CD8+ cells were purified and depleted from PBMC with CD4 and CD8 magnetic beads, as described by the manufacturer (Dynal). This resulted in 92% pure CD8+ T cells and 95% pure CD4+ T cells, as determined by FACS analysis. CD8 and CD4 MAbs were released with Detachabeads as described by the manufacturer (Dynal). The purified CD8+ and CD4+ T cells were then incubated with various mouse anti-human MAbs and isolated with magnetic beads conjugated with anti-mouse IgG or IgM MAbs. As anti-human CLA and anti-human 7 integrin were produced as an IgM and IgG from rat, respectively, an intermediate step with mouse anti-rat IgM or an anti-rat IgG was added before separation. For the CD4+ CD25+ T-cell positive selection, isolated CD4+ T cells were incubated with anti-CD25 MAb and anti-mouse IgG magnetic beads. The purity in all cases was >92%.
T cells purified positively and negatively (104 cells) were stimulated in the presence of autologous PBMC treated with mitomycin C (106 cells) used as antigen-presenting cells with or without antigens in RPMI medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 1 mg/ml streptomycin (all from Sigma), and 5% heat-inactivated human AB serum. In some experiments mouse anti-TGF-1, anti-IL-10, and the isotype control were added at 5 μg/ml. CD4+ CD25+ T cells or CD4+ CD25– T cells (5 x 105 cells) in the presence of 106 mitomycin C-treated autologous PBMC were stimulated with either anti-CD3 or L. guyanensis to analyze the cytokine profile of these cells.
Suppression assay and cytokine analysis. Cocultures of CD4+ CD25+ T cells and CD4+ CD25– T cells were activated with either anti-CD3 or L. guyanensis to analyze the suppressive activity of CD4+ CD25+ T cells on cytokine production. Briefly, a constant number of CD4+ CD25– T cells (5 x 105 cells) was cultured with CD4+ CD25+ T cells (2 x 105), which gave optimal suppression in preliminary experiments in the presence of mitomycin C-treated PBMC (data not shown). The supernatants for all the experiments were removed after 3 or 7 days of culture with anti-CD3 and L. guyanensis, respectively, and stored at –20°C. We analyzed cytokine production by specific IL-4, IL-10, IL-13, IFN-, and TGF-1 enzyme-linked immunosorbent assays (ELISA) (Pharmingen) with a sensitivity of 10 pg/ml (except for TGF-1, for which the assay sensitivity was 62.5 pg/ml).
Statistical analysis. The data were subjected to statistical analysis by the nonparametric Kruskal-Wallis test.
RESULTS
PBMC from naive subjects produce large amounts of TGF-1 in response to Leishmania promastigotes. We examined cytokine production by PBMC from 13 healthy subjects in the presence of live L. guyanensis promastigotes. PBMC from 15 patients suffering from leishmaniasis due to infection with L. guyanenis, the Leishmania species most frequently isolated in French Guyana, were also analyzed. Figure 1A clearly shows that TGF-1 is produced only by PBMC from healthy subjects stimulated with L. guyanensis. Production of TGF-1 required live L. guyanensis, since PBMC stimulated with UV-irradiated L. guyanensis or with frozen and thawed L. guyanensis promastigotes did not induce TGF-1 in PBMC from controls or localized cutaneous leishmaniasis (LCL) patients (data not shown). Furthermore, stimulation of PBMC with a 10-fold increase in UV-irradiated L. guyanensis (107) was not able to induce TGF-1 production in PBMC (data not shown).
To determine whether TGF-1 production by PBMC from healthy subjects is limited to stimulation with leishmaniae from the New World, we analyzed TGF-1 production after stimulation with live promastigotes of L. major, a representative parasite from the Old World. Figure 1B shows that TGF-1 is also produced by PBMC from healthy subjects stimulated with live L. major promastigotes.
Furthermore, the developmental stages—promastigotes versus amastigotes—involved in TGF-1 production by PBMC from healthy subjects were analyzed. Since we are unable to obtain amastigotes from L. guyanensis, however, we compared TGF-1 production after stimulation with either promastigotes or amastigotes from L. major. Interestingly, the levels of TGF-1 produced in response to L. major amastigotes were lower than those obtained with the promastigote forms (Fig. 1B).
As previously described (9, 11), live L. guyanensis induced IFN- in PBMC in either controls or LCL patients, but no IL-4 was detectable in response to live L. guyanensis (data not shown). However, some IL-2 (18 ± 7 pg/ml) and IL-10 (35 ± 12 pg/ml) are detected in supernatants of PBMC from LCL patients stimulated with live L. guyanensis.
CD4+ C25+ T cells are the cells producing TGF-1 in response to live L. guyanensis. To determine which cells produced TGF-1 in response to live L. guyanensis in PBMC from healthy subjects stimulated with live L. guyanensis, we analyzed cytokine production in the supernatants of unfractionated cells and of cells purified for CD3, CD4, and CD8. TGF-1-producing cells were found to be CD3+ CD4+ T cells (Fig. 2A). Since a subset of TGF--producing CD4+ T cells has been characterized by expression of the IL-2 receptor chain (CD25), we analyzed TGF-1 production by CD25+ and CD25– CD4+ T cells in response to live L. guyanensis. As shown in Fig. 2B, the TGF-1-producing cells are CD25+. Further characterization of the TGF--producing cells demonstrated that the TGF-1 producers were found among the CD45RO+ DR+ CD4+ T cells (Fig. 2B), which are phenotypically identical to the previously described CD4+ Treg cell population (30).
Recently, two distinct subsets of human Treg cells have been identified in peripheral blood of healthy subjects based on the expression of / integrins: the 47+ Treg cells produced large amounts of IL-10, whereas 41+ Treg cells produced TGF- (31). Thus, we characterized the expression of these integrins on the Treg cells producing TGF-1 in response to L. guyanensis. Figure 2C clearly demonstrates that these cells expressed the 41 integrin.
We then assessed the expression on TGF-1-secreting CD4+ T cells of CLA and CC chemokine receptor 4 (CCR4), which drive the migration of T cells to the skin (14, 17). In addition, expression of CCR7 and CD62L, both necessary for migration to lymph nodes (4, 12), was evaluated. Results presented in Fig. 2D indicate that the TGF-1-producing CD4+ T cells are CLA+ and CCR4+, implying that these cells are able to recirculate into the skin. TGF-1-secreting cells did not express CD62L or CCR7 molecules, since all the TGF-1 produced in response to live L. guyanensis was detected in the supernatants of CD62L– and CCR7– CD4+ T cells.
CD4+ CD25+ T cells produced TGF-1 but not IL-10 in response to L. guyanensis. Since CD4+ CD25+ T cells that produced TGF-1 are phenotypically similar to the previously described Treg cells, we analyzed the cytokine profile of these cells in comparison to that of CD4+ CD25– T cells. As shown in Fig. 3, CD4+ CD25+ T cells produced IL-10 and TGF-1 but not IL-2 and IFN- in response to anti-CD3 stimulation, corresponding to the cytokine profile described for human Treg cells. Interestingly, the fact that CD4+ CD25+ T cells produced TGF-1 but not IL-10 in response to L. guyanensis clearly reinforces results showing that this parasite stimulates a distinct subpopulation of CD4+ CD25+ Treg cells expressing the 41 integrin (Fig. 1).
As expected, CD4+ CD25– T cells produced IL-2, IL-10, and IFN- but not TGF-1 in response to anti-CD3 stimulation (Fig. 3). If IFN- production by CD4+ T cells in response to L. guyanensis was not surprising in subjects never exposed to Leishmania, since we and others have already demonstrated such cytokine production (9, 23), CD4+ CD25– T cells produced IL-2 and IL-10 but not TGF-1 in response to L. guyanensis stimulation. Since IL-2 and IL-10 were either undetectable or produced at very low levels in total PBMC stimulated with L. guyanensis (Fig. 1), these results suggest that IL-2 and IL-10 production in response to the parasite may be down regulated by the CD4+ CD25+ T cells.
CD4+ CD25+ T cells suppress IL-2 and IL-10 production by CD4+ CD25– T cells through TGF-1 production. To test the suppressive activity of CD4+ CD25+ T cells, we performed classical in vitro suppression assays by coculture of CD4+ CD25+ and CD4+ CD25– T cells in the presence of anti-CD3 or L. guyanensis. First, levels of cytokines were under the limit of detection in mitomycin C-treated APCs stimulated with L. guyanensis or anti-CD3. As shown in Fig. 4, CD4+ CD25+ T cells are able to inhibit the secretion of IL-2 and IL-10 by CD4+ CD25– T cells in the presence of either anti-CD3 or L. guyanensis. However, CD4+ CD25+ T cells were able to suppress IFN- production by CD4+ CD25– T cells in response to anti-CD3 (Fig. 4B) but were unable to inhibit the IFN- production of CD4+ CD25– T cells in response to L. guyanensis (Fig. 4A).
Since the role of cytokines in mediating the suppressive effect of CD4+ CD25+ T cells is not clear, we performed coculture assays in the presence of anti-TGF-1 and anti-IL-10 MAbs to analyze the mechanisms of suppression. Addition of anti-TGF-1, anti-IL-10, or isotype control MAbs did not reverse suppression induced by CD4+ CD25+ T cells in the presence of anti-CD3 (Fig. 4B). However, addition of anti-TGF-1, but not anti-IL-10 or isotype control MAbs, reversed the suppressive activity of CD4+ CD25+ T cells activated with L. guyanensis (Fig. 4A).
DISCUSSION
In the present study, we first demonstrated TGF-1 production by PBMC from healthy subjects in response to live L. guyanensis and further demonstrated that the TGF-1-producing cells are phenotypically and functionally identical to the CD4+ CD25+ T regulatory cells. TGF- is recognized as a central immunoregulator used by the parasite to escape protective mechanisms in human and murine infection with Leishmania. Indeed, previous reports demonstrated a central role of endogenous TGF- in the susceptibility of mice to L. major. TGF- also increased the susceptibility of BALB/c mice to infection with L. amazonensis (6) and reverted the resistance of C57BL/6 mice to L. chagasi infection (33). Furthermore, administration of anti-TGF- MAbs blocked the development of lesions in mice susceptible to infection with L. major (21). In human infections, TGF- has been detected in either active or chronic human cutaneous leishmaniasis (25), and macrophages from LCL patients were shown to produce TGF- after infection with different Leishmania species (5). Since the amount of TGF-1 produced by macrophages upon infection with Leishmania correlates with both the strain virulence and the multiplication of parasites within the macrophages, TGF- production was associated with a virulence mechanism during infection with Leishmania (5). Little is known about TGF- production in response to stimulation with Leishmania in healthy subjects. Our present results demonstrated that TGF-1 is also produced in PBMC of healthy subjects.
Among the populations of cells which are able to produce TGF--1, the recently described Treg cells have been analyzed extensively, mainly in the context of tolerance or autoimmunity (30). Several subsets of Treg cells which express CD25 have been described (24). However, since no specific surface antigens have been identified, characterization of Treg cells was done based on the pattern of secreted cytokines and the mechanism of suppression in vitro, although the transcription factor Foxp3 was reported to be a specific marker for CD4+ CD25+ T cells (13, 16, 18). Results presented in this paper clearly demonstrate that TGF-1-producing cells generated in response to L. guyanensis are phenotipically and functionally similar to the human CD4+ CD25+ Treg cells, since (i) they express CD25, CD45RO, and DR surface markers and (ii) they exert suppressive activity on CD4+ CD25– T cells in vitro. We further show that CD4+ CD25+ T cells stimulated by L. guyanensis produced TGF-1 but not IL-10. CD4+ CD25+ Treg cells stimulated with anti-CD3, however, produced both TGF and IL-10, suggesting that the former population of CD4+ CD25+ T cells might be a particular subpopulation of Treg cells.
The exact mechanisms by which CD4+ CD25+ T cells exert their suppressive activity are still controversial. Indeed, soluble suppressor cytokines such as IL-10 and TGF- have been reported to be involved in this process in vivo, but cell contact between suppressor cells and the responder cells has been shown to be required in vitro (3, 8). Our results show that addition of neutralizing MAbs to TGF-1 can reverse suppression of IL-2 and IL-10 production in response to L. guyanensis but not to anti-CD3 stimulation of CD4+ CD25+ T cells, demonstrating that TGF-1 is involved in the suppressive activity of L. guyanensis-stimulated CD4+ CD25+ T cells in vitro (30). These results reinforce the hypothesis that L. guyanensis could activate a subpopulation of Treg cells. In this context, CD4+ C25+ T-cell clones producing high levels of TGF-1 have been identified recently in humans, and their suppressive activity was reported to be partially dependent on TGF- (20). Thus, L. guyanensis could be able to stimulate such a subpopulation of CD4+ CD25+ T cells. This result is reinforced by the fact that TGF-1-producing cells expressed 41 integrin in response to live L. guyanensis. The 41 integrin has been recently described on a subpopulation of human Treg cells (31). Since 41 integrin binds to vascular cell adhesion molecule-1 (CD106; VCAM-1), which is induced on the endothelium of inflamed tissues, we could hypothesize that the TGF-1-producing cells migrate to the inflamed tissues to inhibit the T-cell response to parasites.
The CD4+ C25+ T cells generated in response to L. guyanensis have been identified in PBMC, and they were shown to express CLA and CCR4; therefore, they should be able to migrate into the inflamed skin where Leishmania parasites exert their functions. The lack of detection of TGF-1 production in supernatants of L. guyanensis-stimulated PBMC from infected patients might be explained by a specific recruitment of this subpopulation of CD4+ CD25+ T cells into the lesions. Analysis of lesions might give a response; however, the finding that murine CD4+ C25+ T cells preferentially accumulate in the skin during the chronic phases of infection with L. major is in accordance with this hypothesis (7). We cannot rule out the possibility that the suppressor function of human CD4+ CD25+ regulatory T cells in peripheral blood of patients with leishmaniasis is defective, as was recently reported for patients suffering from autoimmune diseases (19, 32).
In conclusion, TGF-1 production by a subpopulation of CD4+ C25+ T cells showing a regulatory phenotype was identified in PBMC from healthy subjects stimulated with Leishmania, but the role of these cells in infection and disease is not yet fully understood. The immune responses detected in healthy subjects could, however, reflect the propensity of Leishmania to infect (either clinically or subclinically) most exposed individuals. The fact that TGF-1 is known to down-regulate a number of immune responses associated with protective mechanisms (e.g., macrophage activation) is in accordance with the second hypothesis.
ACKNOWLEDGMENTS
We are especially grateful to J. A. Louis (Department of Parasitology, Institute Pasteur in Paris) for helpful comments during this project.
This study was supported by grants from the Institute Pasteur and the French Ministry of Research (Programme de Recherche Fondamentale, Microbiologie Maladies Infectieuses et Parasitaires du Ministere de l'Enseignement Superieur et de la Technologie).
Present address: Pasteur Institute of Iran, P.O. Box 11365-6699, Tehran, Iran.
REFERENCES
1. Aseffa, A., A. Gumy, P. Launois, H. R. MacDonald, J. A. Louis, and F. Tacchini-Cottier. 2002. The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+ CD25+ T cells. J. Immunol. 169:3232-3241.
2. Bacellar, O., H. Lessa, A. Schriefer, P. Machado, A. Ribeiro de Jesus, W. O. Dutra, K. J. Gollob, and E. M. Carvahlo. 2002. Up-regulation of Th1 responses in mucosal leishmaniasis patients. Infect. Immun. 70:6734-6740.
3. Bach, J.-F., and L. Chatenoud. 2001. Tolerance to islet autoantigens and type I diabetes. Annu. Rev. Immunol. 19:131-161.
4. Banchereau, J., and R. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-252.
5. Barral, A., M. Teixeira, P. Reis, V. Vinhas, J. Costa, H. Lessa, A. L. Bittencourt, S. Reed, E. M. Carvahlo, and M. Barral-Netto. 1995. Transforming growth factor- in human cutaneous leishmanisis. Am. J. Pathol. 147:947-954.
6. Barral-Netto, M., A. Barral, C. E. Brownell, Y. A. W. Skeiky, L. R. Ellingsworth, D. R. Twardzik, and S. G. Reed. 1992. Transforming growth factor- in leishmanial infection: a parasite escape mechanism. Science 257:545-548.
7. Belkaid, Y., C. A. Picirillo, S. Mendez, E. M. Shevach, and D. L. Sacks. 2002. CD4+ CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502-507.
8. Bluestone, J. A., and A. K. Abbas. 2003. Natural versus adaptive regulatory T cells. Nat. Rev. Immunol. 3:253-257.
9. Bourreau, E., M. Collet, G. Prevot, G. Milon, D. Ashimoff, H. Hasagewa, C. Parra-Lopez, and P. Launois. 2002. IFN- producing CD45RA+ CD8+ and IL-10 producing CD45RA– CD4+ T cells generated in response to LACK in naive subjects never exposed to Leishmania. Eur. J. Immunol. 32:510-520.
10. Bourreau, E., H. Pascalis, G. Prevot, A. Kariminia, N. Jolly, G. Milon, P. Buffet, R. Michel, J.-B. Meynard, J.-P. Boutin, D. Aschimoff, and P. Launois. 2003. Increased production of interferon- by Leishmania homologue of the mammalian receptor for activated C kinase-reactive CD4+ T cells among human blood mononuclear cells: an early marker of exposure to Leishmania Scand. J. Immunol. 58:201-210.
11. Bourreau, E., G. Prevot, J. Gardon, R. Pradinaud, H. Hasagewa, G. Milon, and P. Launois. 2002. LACK-specific CD4+ T cells that induce gamma interferon production in patients with localized cutaneous leishmaniasis during the early stage of infection. Infect. Immun. 70:3122-3129.
12. Butcher, E., and L. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60-66.
13. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat. Immunol. 4:330-336.
14. Fuhlbrigge, R., J. Kieffer, D. Armerding, and T. Kupper. 1997. Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells. Nature 389:978-981.
15. Green, S. J., R. M. Crawford, J. T. Hockmeyer, M. S. Meltzer, and C. A. Nacy. 1990. Leishmania major amastigotes initiate the L-arginine dependent killing mechanism in IFN--stimulated macrophages by induction of TNF-. J. Immunol. 145:4290-4297.
16. Hori, S., T. Nomura, and S. Sakagushi. 2003. Control of regulatory cell development by the transcription factor Foxp3. Science 229:1057-1061.
17. Hwang, S. T. 2001. Mechanisms of T-cell homing to skin. Adv. Dermatol. 17:211-241.
18. Khattri, R., T. Cox, S. A. Yasayko, and F. Ramsdell. 2003. An essential role for scurfin in CD4+ CD25+ T regulatory cells. Nat. Immunol. 4:337-342.
19. Kriegel, M. A., T. Lohmann, C. Gabler, N. Blank, J. R. Kalden, and H. M. Lorenz. 2004. Defective suppressor function of human CD4+ CD25+ regulatory T cells in autoimmune polyglandular syndrome type II. J. Exp. Med. 199:1285-1291.
20. Levings, M. K., R. Sangregorio, C. Sartirana, A. L. Moschin, M. Battaglia, P. C. Orban, and M.-G. Roncarolo. 2002. Human CD25+ CD4+ T suppressor cell clones produce transforming growth factor , but not interleukin 10, and are distinct from type 1 T regulatory cells. J. Exp. Med. 196:1335-1346.
21. Li, J., C. A. Hunter, and J. P. Farell. 1999. Anti-TGF- treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production. J. Immunol. 162:974-979.
22. Louis, J. A., E. Moedder, R. Behin, and H. D. Engers. 1979. Recognition of protozoan parasite antigens by murine T lymphocytes. Eur. J. Immunol. 9:841-847.
23. Maasho, K., I. Satti, S. Nyken, G. Guzman, F. Koning, and A. Akuffo. 2000. A Leishmania homologue of receptors for activated C-kinase (LACK) induces both interferon- and interleukin-10 in natural killer cells of healthy blood donors. J. Infect. Dis. 182:570-578.
24. McGuirk, P., and K. H. G. Mills. 2002. Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases. Trends Immunol. 23:450-455.
25. Melby, P. C., F. J. Andrare-Narvaez, B. J. Darnell, G. Valencia-Pacheco, V. V. Tryon, and A. Palomo-Ceita. 1994. Increased expression of proinflammatory cytokines in chronic lesions of human cutaneous leishmaniasis. Infect. Immun. 62:937-942.
26. Mittrücker, H.-W., and S. H. E. Kaufmann. 2004. Regulatory T cells and infection: suppression revisited. Eur. J. Immunol. 34:306-312.
27. Muller, I., P. Kropf, J. A. Louis, and G. Milon. 1994. Expansion of gamma interferon-producing CD8+ T cells following secondary infection of mice immune to Leishmania major. Infect. Immun. 62:2575-2581.
28. Reiner, S. L., and R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151-177.
29. Rocha, P. N., R. P. Almeida, O. Bacellar, A. R. de Jesus, D. C. Fihlo, A. C. Filho, A. Barral, R. L. Coffman, and E. M. Carvalho. 1999. Down-regulation of Th1 type of response in early human American cutaneous leishmaniasis. J. Infect. Dis. 180:1731-1734.
30. Shevach, E. M. 2002. CD4+ CD25+ suppressor T cells: more question than answers. Nat. Rev. Immunol. 2:389-400.
31. Stassen, M., S. Fondel, T. Bopp, C. Richter, C. Müller, J. Kubach, C. Becker, J. Knop, A. H. Enk, S. Schmitt, E. Schmitt, and H. Jonuleit. 2004. Human CD25+ regulatory T cells: two subsets defined by the integrins 47 or 41 confer distinct suppressive properties upon CD4+ T helper cells. Eur. J. Immunol. 34:1303-1311.
32. Viglietta, V., C. Baecher-Allan, H. L. Weiner, and D. A. Hafler. 2004. Loss of functional suppression by CD4+ CD25+ regulatory T cells in patients with multiple sclerosis. J. Exp. Med. 199:971-979.
33. Wilson, M. E., T. J. Recker, N. E. Rodriguez, B. M. Young, K. K. Burnelle, J. A. Streit, and J. N. Kline. 2002. The TGF- response to Leishmania chagasi in the absence of IL-12. Eur. J. Immunol. 32:3556-3565.
34. Xu, D., H. Liu, M. Komai-Koma, C. Campbell, C. McSharry, J. Alexander, and F. Y. Liew. 2003. CD4+ CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection and colitis in mice. J. Immunol. 170:394-399.(A. Kariminia, E. Bourreau)
Institut d'Enseignement Superieur de la Guyane, Universite Antilles-Guyane, Cayenne
Institut Guyanais de Dermatologie Tropicale, E.A. 2188, Centre Hospitalier Andree Rosemon, 97300, Cayenne, French Guyana
WHO-IRTC, Department of Biochemistry, University of Lausanne, Epalinges, Switzerland
ABSTRACT
Transforming growth factor (TGF-) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF- production in response to Leishmania stimulation in healthy subjects. In this study, TGF-1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-1-producing cells were described as a distinct subpopulation of CD4+ CD25+ regulatory T cells. The suppressive properties of CD4+ CD25+ T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4+ CD25– T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-1 did not reverse the suppressive activity of CD4+ CD25+ T cells activated by anti-CD3, it reversed the suppressive activity of CD4+ CD25+ T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-1 is involved in the suppressive activity of L. guyanensis-stimulated CD4+ CD25+ T cells from healthy controls.
INTRODUCTION
Human infections with Leishmania species induce diseases ranging from cutaneous leishmaniasis to mucosal and visceral leishmaniasis, and the clinical outcomes depend largely on the specific immune responses to Leishmania antigens and particularly on cytokine production. In the murine model of infection with Leishmania major, the involvement of the development of CD4+ Th1 cells producing interleukin 2 (IL-2) and gamma interferon (IFN-) and of CD4+ Th2 cells producing IL-4, IL-10, and IL-13 in resistance and susceptibility, respectively, has been clearly demonstrated (28). However, in humans, the roles of Th1 and Th2 CD4+ T cells and the cytokines they produce are not yet well understood. IL-10 production has been demonstrated in human cutaneous leishmaniasis due to L. braziliensis, and its down-regulation of IFN- production has been proposed to explain disease (29). Transforming growth factor (TGF-) is also a well-characterized antiinflammatory cytokine with antiproliferative and antigen-presenting cell (APC)-deactivating properties which has been implicated in human leishmaniasis (5). Recent data have demonstrated, however, that in vitro TGF- could poorly inhibit IFN- production by peripheral blood mononuclear cells (PBMC) from leishmaniasis patients, suggesting that exogenous TGF- was not able to reverse IFN- antiparasite effector mechanisms (2). Therefore, the exact role of TGF- in disease susceptibility is still unclear.
Both IL-10 and TGF- are secreted by mouse and human regulatory T cells (Treg cells) (30), which represent 5 to 10% of the peripheral CD4+ T cells. The suppressive activity of CD4+ CD25+ Treg cells has been reported both in vitro and in vivo. Suppression was shown to depend on cell contact in vitro, while the cytokines IL-10 and TGF- appeared critical in their suppressive activity in vivo. CD4+ CD25+ Treg cells are restricted by self-antigens, but their role in the control of immune responses against several pathogens has been described recently (26). An important role for CD4+ CD25+ Treg cells has been reported in the control of L. major-specific immune responses in both susceptible BALB/c and resistant C57BL/6 mice (1, 7, 34). In these studies, CD4+ CD25+ Treg cells were shown to control both CD4+ Th1 and Th2 cellular immune responses. The role of CD4+ CD25+ Treg cells during infection with Leishmania in humans has not been investigated directly; investigations have relied on the measurement of cytokines secreted by CD4+ CD25+ Treg cells.
Most of our knowledge about cytokine interaction in humans has come from the studies on different clinical forms of leishmaniasis, and there is limited information about the development of cytokine responses in healthy individuals. In this regard, Leishmania-specific CD8+ and CD4+ T cells were isolated from PBMC of subjects who had never been previously exposed to Leishmania (9, 23), but the exact role of these cells in resistance versus susceptibility to infection is not yet established. Although IL-10- and IFN--producing T cells, which recognize the Leishmania homologue of receptors of activated C kinases (LACK), have been detected in naive subjects (9, 23), only a limited amount of IL-10 was detected in response to live L. guyanensis, which might be due to the presence of a suppressive cytokine. In the present study, we demonstrated that TGF- is produced by PBMC from healthy subjects in response to live Leishmania stimulation and that CD4+ CD25+ regulatory T cells are the TGF-1-secreting cells.
MATERIALS AND METHODS
Subjects. Blood samples were obtained by venipuncture from 13 healthy controls and 15 patients suffering from leishmaniasis due to L. guyanensis (duration of lesion development, <3 months) and collected into sterile tubes (Veinoject; Teruven, Leuven, Belgium). All subjects were seronegative for human immunodeficiency virus. Absence of prior exposure to Leishmania was assessed based on (i) the absence of scars due to leishmaniasis upon clinical examination, (ii) no history of any stay in a country in which leishmaniasis is endemic, and (iii) no reactivity against soluble Leishmania antigen from L. guyanensis based on the inability of these healthy subjects to develop IFN- reactivity and specific antibodies against soluble Leishmania antigens (10).
Informed consent was obtained from the subjects, and the human guidelines of the "Comite Consultatif de Protection des Personnes dans la Recherche Medicale" (CCPRB) of Guadeloupe were followed (project 99-3).
Antigens. Leishmania guyanensis (M4147) and Leishmania major (MRHO/SU/59/P strain) promastigotes were cultured in biphasic rabbit blood agar (22). L. major LV 39 amastigotes were obtained as previously described (15). Extracellular proteins were removed from the parasite pellets by three washes with phosphate-buffered saline. Parasites were used at 106/ml. In some experiments, L. major promastigotes were rendered unable to replicate by a 5-min irradiation with UV radiation (UVC; , 253 nm; 200 mW s/cm2) (27).
Reagents. The reagents for magnetic cell separation with anti-CD4-, anti-CD8-, and anti-mouse immunoglobulin G (IgG)-coated magnetic beads were obtained from Dynal (Compiegne, France). Mouse anti-human CD3 (UTCHT1; IgG1), CD25 (M-1251; IgG1), CD45RO (UCHL-1; IgG2a), DR (Tü36; IgG2b), CCR4 (1C1; IgG1,), CD62L (Dreg56; IgG1), CCR7 (2H4; IgM), CD29, or 1 integrin (HUTS-21; IgG2a), and CD49d, or 4 integrin (9F10; IgG1) antibodies, and rat anti-human cutaneous lymphocyte antigen (CLA) (HECA-452; IgM) and anti-human 7 integrin (FIB504; IgG2a) antibodies, were obtained from Pharmingen (San Diego, CA).
Neutralizing mouse anti-human TGF-1 (TB21; IgG1) and anti-IL-10 (JES3-9D7; IgG1) monoclonal antibodies (MAbs) were obtained from Biosource International (Carmarillo, CA) and Pharmingen, respectively. The purified mouse IgG1 isotype control (107.3, IgG1; anti-trinitrophenol) was obtained from Pharmingen.
Mouse anti-CD4 (RPA-T4; IgG1) and anti-CD8 (RPA-T8; IgG1) conjugated to phycoerythrin for flow cytometric experiments were obtained from Pharmingen.
For T-cell activation, the stimulatory anti-CD3 (UTCHT1; IgG1) antibody was used at 2.5 μg/ml.
Cell isolation and activation. PBMC were isolated after venipuncture on a Ficoll-Hypaque gradient (d = 1.077), and CD3+ and CD3– T cells were purified with a magnetic activated cell sorter from Dynal. Briefly, cells conjugated with an anti-CD3 MAb were suspended with anti-mouse IgG-coated magnetic microbeads and isolated after exposure to a magnetic field. The purity was 96%, as determined by fluorescence-activated cell sorter (FACS) analysis. CD4+ and CD8+ cells were purified and depleted from PBMC with CD4 and CD8 magnetic beads, as described by the manufacturer (Dynal). This resulted in 92% pure CD8+ T cells and 95% pure CD4+ T cells, as determined by FACS analysis. CD8 and CD4 MAbs were released with Detachabeads as described by the manufacturer (Dynal). The purified CD8+ and CD4+ T cells were then incubated with various mouse anti-human MAbs and isolated with magnetic beads conjugated with anti-mouse IgG or IgM MAbs. As anti-human CLA and anti-human 7 integrin were produced as an IgM and IgG from rat, respectively, an intermediate step with mouse anti-rat IgM or an anti-rat IgG was added before separation. For the CD4+ CD25+ T-cell positive selection, isolated CD4+ T cells were incubated with anti-CD25 MAb and anti-mouse IgG magnetic beads. The purity in all cases was >92%.
T cells purified positively and negatively (104 cells) were stimulated in the presence of autologous PBMC treated with mitomycin C (106 cells) used as antigen-presenting cells with or without antigens in RPMI medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 1 mg/ml streptomycin (all from Sigma), and 5% heat-inactivated human AB serum. In some experiments mouse anti-TGF-1, anti-IL-10, and the isotype control were added at 5 μg/ml. CD4+ CD25+ T cells or CD4+ CD25– T cells (5 x 105 cells) in the presence of 106 mitomycin C-treated autologous PBMC were stimulated with either anti-CD3 or L. guyanensis to analyze the cytokine profile of these cells.
Suppression assay and cytokine analysis. Cocultures of CD4+ CD25+ T cells and CD4+ CD25– T cells were activated with either anti-CD3 or L. guyanensis to analyze the suppressive activity of CD4+ CD25+ T cells on cytokine production. Briefly, a constant number of CD4+ CD25– T cells (5 x 105 cells) was cultured with CD4+ CD25+ T cells (2 x 105), which gave optimal suppression in preliminary experiments in the presence of mitomycin C-treated PBMC (data not shown). The supernatants for all the experiments were removed after 3 or 7 days of culture with anti-CD3 and L. guyanensis, respectively, and stored at –20°C. We analyzed cytokine production by specific IL-4, IL-10, IL-13, IFN-, and TGF-1 enzyme-linked immunosorbent assays (ELISA) (Pharmingen) with a sensitivity of 10 pg/ml (except for TGF-1, for which the assay sensitivity was 62.5 pg/ml).
Statistical analysis. The data were subjected to statistical analysis by the nonparametric Kruskal-Wallis test.
RESULTS
PBMC from naive subjects produce large amounts of TGF-1 in response to Leishmania promastigotes. We examined cytokine production by PBMC from 13 healthy subjects in the presence of live L. guyanensis promastigotes. PBMC from 15 patients suffering from leishmaniasis due to infection with L. guyanenis, the Leishmania species most frequently isolated in French Guyana, were also analyzed. Figure 1A clearly shows that TGF-1 is produced only by PBMC from healthy subjects stimulated with L. guyanensis. Production of TGF-1 required live L. guyanensis, since PBMC stimulated with UV-irradiated L. guyanensis or with frozen and thawed L. guyanensis promastigotes did not induce TGF-1 in PBMC from controls or localized cutaneous leishmaniasis (LCL) patients (data not shown). Furthermore, stimulation of PBMC with a 10-fold increase in UV-irradiated L. guyanensis (107) was not able to induce TGF-1 production in PBMC (data not shown).
To determine whether TGF-1 production by PBMC from healthy subjects is limited to stimulation with leishmaniae from the New World, we analyzed TGF-1 production after stimulation with live promastigotes of L. major, a representative parasite from the Old World. Figure 1B shows that TGF-1 is also produced by PBMC from healthy subjects stimulated with live L. major promastigotes.
Furthermore, the developmental stages—promastigotes versus amastigotes—involved in TGF-1 production by PBMC from healthy subjects were analyzed. Since we are unable to obtain amastigotes from L. guyanensis, however, we compared TGF-1 production after stimulation with either promastigotes or amastigotes from L. major. Interestingly, the levels of TGF-1 produced in response to L. major amastigotes were lower than those obtained with the promastigote forms (Fig. 1B).
As previously described (9, 11), live L. guyanensis induced IFN- in PBMC in either controls or LCL patients, but no IL-4 was detectable in response to live L. guyanensis (data not shown). However, some IL-2 (18 ± 7 pg/ml) and IL-10 (35 ± 12 pg/ml) are detected in supernatants of PBMC from LCL patients stimulated with live L. guyanensis.
CD4+ C25+ T cells are the cells producing TGF-1 in response to live L. guyanensis. To determine which cells produced TGF-1 in response to live L. guyanensis in PBMC from healthy subjects stimulated with live L. guyanensis, we analyzed cytokine production in the supernatants of unfractionated cells and of cells purified for CD3, CD4, and CD8. TGF-1-producing cells were found to be CD3+ CD4+ T cells (Fig. 2A). Since a subset of TGF--producing CD4+ T cells has been characterized by expression of the IL-2 receptor chain (CD25), we analyzed TGF-1 production by CD25+ and CD25– CD4+ T cells in response to live L. guyanensis. As shown in Fig. 2B, the TGF-1-producing cells are CD25+. Further characterization of the TGF--producing cells demonstrated that the TGF-1 producers were found among the CD45RO+ DR+ CD4+ T cells (Fig. 2B), which are phenotypically identical to the previously described CD4+ Treg cell population (30).
Recently, two distinct subsets of human Treg cells have been identified in peripheral blood of healthy subjects based on the expression of / integrins: the 47+ Treg cells produced large amounts of IL-10, whereas 41+ Treg cells produced TGF- (31). Thus, we characterized the expression of these integrins on the Treg cells producing TGF-1 in response to L. guyanensis. Figure 2C clearly demonstrates that these cells expressed the 41 integrin.
We then assessed the expression on TGF-1-secreting CD4+ T cells of CLA and CC chemokine receptor 4 (CCR4), which drive the migration of T cells to the skin (14, 17). In addition, expression of CCR7 and CD62L, both necessary for migration to lymph nodes (4, 12), was evaluated. Results presented in Fig. 2D indicate that the TGF-1-producing CD4+ T cells are CLA+ and CCR4+, implying that these cells are able to recirculate into the skin. TGF-1-secreting cells did not express CD62L or CCR7 molecules, since all the TGF-1 produced in response to live L. guyanensis was detected in the supernatants of CD62L– and CCR7– CD4+ T cells.
CD4+ CD25+ T cells produced TGF-1 but not IL-10 in response to L. guyanensis. Since CD4+ CD25+ T cells that produced TGF-1 are phenotypically similar to the previously described Treg cells, we analyzed the cytokine profile of these cells in comparison to that of CD4+ CD25– T cells. As shown in Fig. 3, CD4+ CD25+ T cells produced IL-10 and TGF-1 but not IL-2 and IFN- in response to anti-CD3 stimulation, corresponding to the cytokine profile described for human Treg cells. Interestingly, the fact that CD4+ CD25+ T cells produced TGF-1 but not IL-10 in response to L. guyanensis clearly reinforces results showing that this parasite stimulates a distinct subpopulation of CD4+ CD25+ Treg cells expressing the 41 integrin (Fig. 1).
As expected, CD4+ CD25– T cells produced IL-2, IL-10, and IFN- but not TGF-1 in response to anti-CD3 stimulation (Fig. 3). If IFN- production by CD4+ T cells in response to L. guyanensis was not surprising in subjects never exposed to Leishmania, since we and others have already demonstrated such cytokine production (9, 23), CD4+ CD25– T cells produced IL-2 and IL-10 but not TGF-1 in response to L. guyanensis stimulation. Since IL-2 and IL-10 were either undetectable or produced at very low levels in total PBMC stimulated with L. guyanensis (Fig. 1), these results suggest that IL-2 and IL-10 production in response to the parasite may be down regulated by the CD4+ CD25+ T cells.
CD4+ CD25+ T cells suppress IL-2 and IL-10 production by CD4+ CD25– T cells through TGF-1 production. To test the suppressive activity of CD4+ CD25+ T cells, we performed classical in vitro suppression assays by coculture of CD4+ CD25+ and CD4+ CD25– T cells in the presence of anti-CD3 or L. guyanensis. First, levels of cytokines were under the limit of detection in mitomycin C-treated APCs stimulated with L. guyanensis or anti-CD3. As shown in Fig. 4, CD4+ CD25+ T cells are able to inhibit the secretion of IL-2 and IL-10 by CD4+ CD25– T cells in the presence of either anti-CD3 or L. guyanensis. However, CD4+ CD25+ T cells were able to suppress IFN- production by CD4+ CD25– T cells in response to anti-CD3 (Fig. 4B) but were unable to inhibit the IFN- production of CD4+ CD25– T cells in response to L. guyanensis (Fig. 4A).
Since the role of cytokines in mediating the suppressive effect of CD4+ CD25+ T cells is not clear, we performed coculture assays in the presence of anti-TGF-1 and anti-IL-10 MAbs to analyze the mechanisms of suppression. Addition of anti-TGF-1, anti-IL-10, or isotype control MAbs did not reverse suppression induced by CD4+ CD25+ T cells in the presence of anti-CD3 (Fig. 4B). However, addition of anti-TGF-1, but not anti-IL-10 or isotype control MAbs, reversed the suppressive activity of CD4+ CD25+ T cells activated with L. guyanensis (Fig. 4A).
DISCUSSION
In the present study, we first demonstrated TGF-1 production by PBMC from healthy subjects in response to live L. guyanensis and further demonstrated that the TGF-1-producing cells are phenotypically and functionally identical to the CD4+ CD25+ T regulatory cells. TGF- is recognized as a central immunoregulator used by the parasite to escape protective mechanisms in human and murine infection with Leishmania. Indeed, previous reports demonstrated a central role of endogenous TGF- in the susceptibility of mice to L. major. TGF- also increased the susceptibility of BALB/c mice to infection with L. amazonensis (6) and reverted the resistance of C57BL/6 mice to L. chagasi infection (33). Furthermore, administration of anti-TGF- MAbs blocked the development of lesions in mice susceptible to infection with L. major (21). In human infections, TGF- has been detected in either active or chronic human cutaneous leishmaniasis (25), and macrophages from LCL patients were shown to produce TGF- after infection with different Leishmania species (5). Since the amount of TGF-1 produced by macrophages upon infection with Leishmania correlates with both the strain virulence and the multiplication of parasites within the macrophages, TGF- production was associated with a virulence mechanism during infection with Leishmania (5). Little is known about TGF- production in response to stimulation with Leishmania in healthy subjects. Our present results demonstrated that TGF-1 is also produced in PBMC of healthy subjects.
Among the populations of cells which are able to produce TGF--1, the recently described Treg cells have been analyzed extensively, mainly in the context of tolerance or autoimmunity (30). Several subsets of Treg cells which express CD25 have been described (24). However, since no specific surface antigens have been identified, characterization of Treg cells was done based on the pattern of secreted cytokines and the mechanism of suppression in vitro, although the transcription factor Foxp3 was reported to be a specific marker for CD4+ CD25+ T cells (13, 16, 18). Results presented in this paper clearly demonstrate that TGF-1-producing cells generated in response to L. guyanensis are phenotipically and functionally similar to the human CD4+ CD25+ Treg cells, since (i) they express CD25, CD45RO, and DR surface markers and (ii) they exert suppressive activity on CD4+ CD25– T cells in vitro. We further show that CD4+ CD25+ T cells stimulated by L. guyanensis produced TGF-1 but not IL-10. CD4+ CD25+ Treg cells stimulated with anti-CD3, however, produced both TGF and IL-10, suggesting that the former population of CD4+ CD25+ T cells might be a particular subpopulation of Treg cells.
The exact mechanisms by which CD4+ CD25+ T cells exert their suppressive activity are still controversial. Indeed, soluble suppressor cytokines such as IL-10 and TGF- have been reported to be involved in this process in vivo, but cell contact between suppressor cells and the responder cells has been shown to be required in vitro (3, 8). Our results show that addition of neutralizing MAbs to TGF-1 can reverse suppression of IL-2 and IL-10 production in response to L. guyanensis but not to anti-CD3 stimulation of CD4+ CD25+ T cells, demonstrating that TGF-1 is involved in the suppressive activity of L. guyanensis-stimulated CD4+ CD25+ T cells in vitro (30). These results reinforce the hypothesis that L. guyanensis could activate a subpopulation of Treg cells. In this context, CD4+ C25+ T-cell clones producing high levels of TGF-1 have been identified recently in humans, and their suppressive activity was reported to be partially dependent on TGF- (20). Thus, L. guyanensis could be able to stimulate such a subpopulation of CD4+ CD25+ T cells. This result is reinforced by the fact that TGF-1-producing cells expressed 41 integrin in response to live L. guyanensis. The 41 integrin has been recently described on a subpopulation of human Treg cells (31). Since 41 integrin binds to vascular cell adhesion molecule-1 (CD106; VCAM-1), which is induced on the endothelium of inflamed tissues, we could hypothesize that the TGF-1-producing cells migrate to the inflamed tissues to inhibit the T-cell response to parasites.
The CD4+ C25+ T cells generated in response to L. guyanensis have been identified in PBMC, and they were shown to express CLA and CCR4; therefore, they should be able to migrate into the inflamed skin where Leishmania parasites exert their functions. The lack of detection of TGF-1 production in supernatants of L. guyanensis-stimulated PBMC from infected patients might be explained by a specific recruitment of this subpopulation of CD4+ CD25+ T cells into the lesions. Analysis of lesions might give a response; however, the finding that murine CD4+ C25+ T cells preferentially accumulate in the skin during the chronic phases of infection with L. major is in accordance with this hypothesis (7). We cannot rule out the possibility that the suppressor function of human CD4+ CD25+ regulatory T cells in peripheral blood of patients with leishmaniasis is defective, as was recently reported for patients suffering from autoimmune diseases (19, 32).
In conclusion, TGF-1 production by a subpopulation of CD4+ C25+ T cells showing a regulatory phenotype was identified in PBMC from healthy subjects stimulated with Leishmania, but the role of these cells in infection and disease is not yet fully understood. The immune responses detected in healthy subjects could, however, reflect the propensity of Leishmania to infect (either clinically or subclinically) most exposed individuals. The fact that TGF-1 is known to down-regulate a number of immune responses associated with protective mechanisms (e.g., macrophage activation) is in accordance with the second hypothesis.
ACKNOWLEDGMENTS
We are especially grateful to J. A. Louis (Department of Parasitology, Institute Pasteur in Paris) for helpful comments during this project.
This study was supported by grants from the Institute Pasteur and the French Ministry of Research (Programme de Recherche Fondamentale, Microbiologie Maladies Infectieuses et Parasitaires du Ministere de l'Enseignement Superieur et de la Technologie).
Present address: Pasteur Institute of Iran, P.O. Box 11365-6699, Tehran, Iran.
REFERENCES
1. Aseffa, A., A. Gumy, P. Launois, H. R. MacDonald, J. A. Louis, and F. Tacchini-Cottier. 2002. The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+ CD25+ T cells. J. Immunol. 169:3232-3241.
2. Bacellar, O., H. Lessa, A. Schriefer, P. Machado, A. Ribeiro de Jesus, W. O. Dutra, K. J. Gollob, and E. M. Carvahlo. 2002. Up-regulation of Th1 responses in mucosal leishmaniasis patients. Infect. Immun. 70:6734-6740.
3. Bach, J.-F., and L. Chatenoud. 2001. Tolerance to islet autoantigens and type I diabetes. Annu. Rev. Immunol. 19:131-161.
4. Banchereau, J., and R. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-252.
5. Barral, A., M. Teixeira, P. Reis, V. Vinhas, J. Costa, H. Lessa, A. L. Bittencourt, S. Reed, E. M. Carvahlo, and M. Barral-Netto. 1995. Transforming growth factor- in human cutaneous leishmanisis. Am. J. Pathol. 147:947-954.
6. Barral-Netto, M., A. Barral, C. E. Brownell, Y. A. W. Skeiky, L. R. Ellingsworth, D. R. Twardzik, and S. G. Reed. 1992. Transforming growth factor- in leishmanial infection: a parasite escape mechanism. Science 257:545-548.
7. Belkaid, Y., C. A. Picirillo, S. Mendez, E. M. Shevach, and D. L. Sacks. 2002. CD4+ CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502-507.
8. Bluestone, J. A., and A. K. Abbas. 2003. Natural versus adaptive regulatory T cells. Nat. Rev. Immunol. 3:253-257.
9. Bourreau, E., M. Collet, G. Prevot, G. Milon, D. Ashimoff, H. Hasagewa, C. Parra-Lopez, and P. Launois. 2002. IFN- producing CD45RA+ CD8+ and IL-10 producing CD45RA– CD4+ T cells generated in response to LACK in naive subjects never exposed to Leishmania. Eur. J. Immunol. 32:510-520.
10. Bourreau, E., H. Pascalis, G. Prevot, A. Kariminia, N. Jolly, G. Milon, P. Buffet, R. Michel, J.-B. Meynard, J.-P. Boutin, D. Aschimoff, and P. Launois. 2003. Increased production of interferon- by Leishmania homologue of the mammalian receptor for activated C kinase-reactive CD4+ T cells among human blood mononuclear cells: an early marker of exposure to Leishmania Scand. J. Immunol. 58:201-210.
11. Bourreau, E., G. Prevot, J. Gardon, R. Pradinaud, H. Hasagewa, G. Milon, and P. Launois. 2002. LACK-specific CD4+ T cells that induce gamma interferon production in patients with localized cutaneous leishmaniasis during the early stage of infection. Infect. Immun. 70:3122-3129.
12. Butcher, E., and L. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60-66.
13. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat. Immunol. 4:330-336.
14. Fuhlbrigge, R., J. Kieffer, D. Armerding, and T. Kupper. 1997. Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells. Nature 389:978-981.
15. Green, S. J., R. M. Crawford, J. T. Hockmeyer, M. S. Meltzer, and C. A. Nacy. 1990. Leishmania major amastigotes initiate the L-arginine dependent killing mechanism in IFN--stimulated macrophages by induction of TNF-. J. Immunol. 145:4290-4297.
16. Hori, S., T. Nomura, and S. Sakagushi. 2003. Control of regulatory cell development by the transcription factor Foxp3. Science 229:1057-1061.
17. Hwang, S. T. 2001. Mechanisms of T-cell homing to skin. Adv. Dermatol. 17:211-241.
18. Khattri, R., T. Cox, S. A. Yasayko, and F. Ramsdell. 2003. An essential role for scurfin in CD4+ CD25+ T regulatory cells. Nat. Immunol. 4:337-342.
19. Kriegel, M. A., T. Lohmann, C. Gabler, N. Blank, J. R. Kalden, and H. M. Lorenz. 2004. Defective suppressor function of human CD4+ CD25+ regulatory T cells in autoimmune polyglandular syndrome type II. J. Exp. Med. 199:1285-1291.
20. Levings, M. K., R. Sangregorio, C. Sartirana, A. L. Moschin, M. Battaglia, P. C. Orban, and M.-G. Roncarolo. 2002. Human CD25+ CD4+ T suppressor cell clones produce transforming growth factor , but not interleukin 10, and are distinct from type 1 T regulatory cells. J. Exp. Med. 196:1335-1346.
21. Li, J., C. A. Hunter, and J. P. Farell. 1999. Anti-TGF- treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production. J. Immunol. 162:974-979.
22. Louis, J. A., E. Moedder, R. Behin, and H. D. Engers. 1979. Recognition of protozoan parasite antigens by murine T lymphocytes. Eur. J. Immunol. 9:841-847.
23. Maasho, K., I. Satti, S. Nyken, G. Guzman, F. Koning, and A. Akuffo. 2000. A Leishmania homologue of receptors for activated C-kinase (LACK) induces both interferon- and interleukin-10 in natural killer cells of healthy blood donors. J. Infect. Dis. 182:570-578.
24. McGuirk, P., and K. H. G. Mills. 2002. Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases. Trends Immunol. 23:450-455.
25. Melby, P. C., F. J. Andrare-Narvaez, B. J. Darnell, G. Valencia-Pacheco, V. V. Tryon, and A. Palomo-Ceita. 1994. Increased expression of proinflammatory cytokines in chronic lesions of human cutaneous leishmaniasis. Infect. Immun. 62:937-942.
26. Mittrücker, H.-W., and S. H. E. Kaufmann. 2004. Regulatory T cells and infection: suppression revisited. Eur. J. Immunol. 34:306-312.
27. Muller, I., P. Kropf, J. A. Louis, and G. Milon. 1994. Expansion of gamma interferon-producing CD8+ T cells following secondary infection of mice immune to Leishmania major. Infect. Immun. 62:2575-2581.
28. Reiner, S. L., and R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151-177.
29. Rocha, P. N., R. P. Almeida, O. Bacellar, A. R. de Jesus, D. C. Fihlo, A. C. Filho, A. Barral, R. L. Coffman, and E. M. Carvalho. 1999. Down-regulation of Th1 type of response in early human American cutaneous leishmaniasis. J. Infect. Dis. 180:1731-1734.
30. Shevach, E. M. 2002. CD4+ CD25+ suppressor T cells: more question than answers. Nat. Rev. Immunol. 2:389-400.
31. Stassen, M., S. Fondel, T. Bopp, C. Richter, C. Müller, J. Kubach, C. Becker, J. Knop, A. H. Enk, S. Schmitt, E. Schmitt, and H. Jonuleit. 2004. Human CD25+ regulatory T cells: two subsets defined by the integrins 47 or 41 confer distinct suppressive properties upon CD4+ T helper cells. Eur. J. Immunol. 34:1303-1311.
32. Viglietta, V., C. Baecher-Allan, H. L. Weiner, and D. A. Hafler. 2004. Loss of functional suppression by CD4+ CD25+ regulatory T cells in patients with multiple sclerosis. J. Exp. Med. 199:971-979.
33. Wilson, M. E., T. J. Recker, N. E. Rodriguez, B. M. Young, K. K. Burnelle, J. A. Streit, and J. N. Kline. 2002. The TGF- response to Leishmania chagasi in the absence of IL-12. Eur. J. Immunol. 32:3556-3565.
34. Xu, D., H. Liu, M. Komai-Koma, C. Campbell, C. McSharry, J. Alexander, and F. Y. Liew. 2003. CD4+ CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection and colitis in mice. J. Immunol. 170:394-399.(A. Kariminia, E. Bourreau)