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Interleukin-10-Treated Dendritic Cells Modulate Immune Responses of Naive and Sensitized T Cells In Vivo

2002; Elsevier BV; Volume: 119; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.2002.00496.x

1523-1747

Gabriele Müller, Anke Müller, Thomas Tüting, Kerstin Steinbrink, Joachim Saloga, Claudia Szalma, Jürgen Knop, Alexander Enk,

Immune Cell Function and Interaction

Interleukin-10 is a pleiotropic cytokine known to have inhibitory effects on the accessory functions of dendritic cells. In vitro, interleukin-10 converts immature dendritic cells into tolerizing antigen- presenting cells. To assess whether interleukin-10-treated dendritic cells exert tolerizing effects in vivo, CD4+ T cells from DO11.10 ovalbumin-T cell receptor transgenic mice were transferred to syngeneic BALB/c recipients. Recipient animals were treated with ovalbumin-pulsed/unpulsed, interleukin-10-treated/untreated CD11c+ dendritic cells thereafter and ovalbumin-specific proliferation of lymph node cells was assessed by restimulation with the peptide in vitro. In prophylactic experiments, recipients received naive CD4+ DO11.10 T cells and were immunized with ovalbumin323-339 peptide in incomplete Freund's adjuvant after treatment with various subtypes of dendritic cells. Strong ovalbumin-specific proliferation was observed in animals immunized with control ovalbumin-dendritic cells. Minimal proliferation was found in mice treated with ovalbumin-pulsed, interleukin-10-treated dendritic cells. In therapeutic experiments, preactivated CD4+ DO11.10 T cells were transferred, and recipients were treated with dendritic cells as described. Ovalbumin-specific proliferation was strong in recipients treated with ovalbumin-dendritic cells. CD4+ T cell proliferation from ovalbumin-interleukin-10-dendritic cell treated animals was below background. When delayed type hypersensitivity reactions in the footpads of prophylactically or therapeutically vaccinated animals were tested, mice treated with ovalbumin-interleukin-10-dendritic cells showed no footpad swelling compared with controls. Rechallenge with the antigen in vitro and in vivo did not alter the inhibitory effect of interleukin-10-treated dendritic cells. Thus, interleukin-10-treated dendritic cells inhibit ovalbumin-specific immune responses in naive and sensitized mice. Interleukin-10 is a pleiotropic cytokine known to have inhibitory effects on the accessory functions of dendritic cells. In vitro, interleukin-10 converts immature dendritic cells into tolerizing antigen- presenting cells. To assess whether interleukin-10-treated dendritic cells exert tolerizing effects in vivo, CD4+ T cells from DO11.10 ovalbumin-T cell receptor transgenic mice were transferred to syngeneic BALB/c recipients. Recipient animals were treated with ovalbumin-pulsed/unpulsed, interleukin-10-treated/untreated CD11c+ dendritic cells thereafter and ovalbumin-specific proliferation of lymph node cells was assessed by restimulation with the peptide in vitro. In prophylactic experiments, recipients received naive CD4+ DO11.10 T cells and were immunized with ovalbumin323-339 peptide in incomplete Freund's adjuvant after treatment with various subtypes of dendritic cells. Strong ovalbumin-specific proliferation was observed in animals immunized with control ovalbumin-dendritic cells. Minimal proliferation was found in mice treated with ovalbumin-pulsed, interleukin-10-treated dendritic cells. In therapeutic experiments, preactivated CD4+ DO11.10 T cells were transferred, and recipients were treated with dendritic cells as described. Ovalbumin-specific proliferation was strong in recipients treated with ovalbumin-dendritic cells. CD4+ T cell proliferation from ovalbumin-interleukin-10-dendritic cell treated animals was below background. When delayed type hypersensitivity reactions in the footpads of prophylactically or therapeutically vaccinated animals were tested, mice treated with ovalbumin-interleukin-10-dendritic cells showed no footpad swelling compared with controls. Rechallenge with the antigen in vitro and in vivo did not alter the inhibitory effect of interleukin-10-treated dendritic cells. Thus, interleukin-10-treated dendritic cells inhibit ovalbumin-specific immune responses in naive and sensitized mice. antigen-presenting cell bone-marrow-derived dendritic cell dendritic cell ovalbumin recombinant mouse granulocyte-macrophage colony-stimulating factor recombinant mouse interleukin Dendritic cells (DC) are potent immunostimulatory cells whose capabilities to process and present antigen are unmatched in the immune system (Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-248Crossref PubMed Scopus (11805) Google Scholar). Besides serving as potent immunostimulatory cells, DC can be converted to tolerance-inducing cells under certain conditions (Schuler and Steinman, 1985Schuler G. Steinman R.M. Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro.J Exp Med. 1985; 161: 526-536Crossref PubMed Scopus (847) Google Scholar;Steinman and Young, 1991Steinman R.M. Young J.W. Signals arising from antigen-presenting cells.Curr Opin Immunol. 1991; 3: 361-371Crossref PubMed Scopus (84) Google Scholar;Young et al., 1992Young J.W. Koulova L. Soergel S.A. Clark E.A. Steinman R.M. Dupont B. The B7/BB1 antigen provides one of several costimulatory signals for the activation of CD4+ T lymphocytes by human blood dendritic cells in vitro.J Clin Invest. 1992; 90 ([published erratum appears in J Clin Invest 91(4):1853, 1993]): 229-235Crossref PubMed Scopus (252) Google Scholar). One of the factors known to affect DC in such a way that they become tolerizing antigen-presenting cells (APC) is interleukin-10 (IL-10) (de Waal et al., 1992ade Waal M. Yssel H. Roncarolo M.G. Spits H. de Vries J.E. Interleukin-10.Curr Opin Immunol. 1992; 4: 314-331Crossref PubMed Scopus (347) Google Scholar). It was shown by us and others previously that IL-10 is able to affect the expression of major histocompatibility complex (MHC) and costimulatory molecules such as CD86 on DC (Steinbrink et al., 1997Steinbrink K. Wolfl M. Jonuleit H. Knop J. Enk A.H. Induction of tolerance by IL-10-treated dendritic cells.J Immunol. 1997; 159: 4772-6684PubMed Google Scholar). Although the original work was performed in murine Langerhans cells (Enk et al., 1993Enk A.H. Angeloni V.L. Udey M.C. Katz S.I. Inhibition of Langerhans cell antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance.J Immunol. 1993; 151: 2390-2396PubMed Google Scholar;Enk et al., 1997Enk A.H. Jonuleit H. Saloga J. Knop J. Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma.Int J Cancer. 1997; 73: 309-314Crossref PubMed Scopus (339) Google Scholar;Dummer et al., 1995Dummer W. Becker J.C. Schwaaf A. Leverkus M. Moll T. Brocker E.B. Elevated serum levels of interleukin-10 in patients with metastatic malignant melanoma.Melanoma Res. 1995; 5: 67-78Crossref PubMed Scopus (123) Google Scholar;Dummer et al., 1996Dummer W. Bastian B.C. Ernst N. Schanzle C. Schwaaf A. Brocker E.B. Interleukin-10 production in malignant melanoma: preferential detection of IL-10-secreting tumor cells in metastatic lesions.Int J Cancer. 1996; 66: 607-614Crossref PubMed Scopus (97) Google Scholar), similar results were obtained in various subtypes of murine and human DC. As an example, IL-10-treated human-blood-derived DC induced anergy in CD4+ and CD8+ clonal or naive T cells. Induction of anergy in melanoma-antigen-specific T cell lines also resulted in a failure of these cytotoxic T lymphocytes to lyze tumor cells (Pisa et al., 1992Pisa P. Halapi E. Pisa E.K. et al.Selective expression of interleukin 10, interferon gamma, and granulocyte-macrophage colony-stimulating factor in ovarian cancer biopsies.Proc Natl Acad Sci USA. 1992; 89: 7708-7713Crossref PubMed Scopus (228) Google Scholar;Gastl et al., 1993Gastl G.A. Abrams J.S. Nanus D.M. et al.Interleukin-10 production by human carcinoma cell lines and its relationship to interleukin-6 expression.Int J Cancer. 1993; 55: 96-103Crossref PubMed Scopus (229) Google Scholar;Merlo et al., 1993Merlo A. Juretic A. Zuber M. et al.Cytokine gene expression in primary brain tumours, metastases and meningiomas suggests specific transcription patterns.Eur J Cancer. 1993; 29A: 2118-2121Abstract Full Text PDF PubMed Scopus (45) Google Scholar;Smith et al., 1994Smith D.R. Kunkel S.L. Burdick M.D. Wilke C.A. Orringer M.B. Whyte R.I. Strieter R.M. Production of interleukin-10 by human bronchogenic carcinoma.Am J Pathol. 1994; 145: 18-29PubMed Google Scholar;Kim et al., 1995Kim J. Modlin R.L. Moy R.L. Dubinett S.M. McHugh T. Nickoloff B.J. Uyemura K. IL-10 production in cutaneous basal and squamous cell carcinomas. A mechanism for evading the local T cell immune response.J Immunol. 1995; 155: 2240-2248PubMed Google Scholar;Steinbrink et al., 1999Steinbrink K. Jonuleit H. Muller G. Schuler G. Knop J. Enk A.H. Interleukin-10-treated human dendritic cells induce a melanoma-antigen- specific anergy in CD8(+) T cells resulting in a failure to lyse tumor cells.Blood. 1999; 93: 1634-1646PubMed Google Scholar). Additionally, data generated in patients with malignant melanoma showed that progressing metastases in contrast to regressing metastases contained high amounts of IL-10 (Enk et al., 1997Enk A.H. Jonuleit H. Saloga J. Knop J. Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma.Int J Cancer. 1997; 73: 309-314Crossref PubMed Scopus (339) Google Scholar). This IL-10 was derived from melanoma cells and inhibited the expression of CD86 on local DC. Therefore these DC were not only inhibited with regard to their potency to induce T cell proliferation, but also induced tolerance in the patients' T cells. In aggregate these data indicate that IL-10 serves as a factor that alters DC functions in such a way that these DC induce T cell tolerance. As tolerance induction might be a useful way to treat murine or human autoimmune diseases, we wondered whether IL-10-treated DC (DC-IL10) might also subserve tolerizing functions in vivo. To address the question of downregulation of immune responses by DC in vivo, we used a system of ovalbumin (OVA)-T cell receptor (TCR) transgenic animals (DO11.10 mice) where 50%-70% of all naive peripheral CD4+ T cells express an OVA-specific TCR (Murphy et al., 1990Murphy K.M. Heimberger A.B. Loh D.Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.Science. 1990; 250: 1720-1723Crossref PubMed Scopus (1627) Google Scholar). This system provides the advantage that a large number of peptide-specific T cells can be readily stimulated with the appropriate antigen. To exclude an influence of newly developing transgenic T cells that were not affected by the DC injected and to test transgenic T cells in a “normal” nontransgenic environment, CD4+ T cells from transgenic animals were transferred ex vivo into syngeneic BALB/c mice (Kearney et al., 1994Kearney E.R. Pape K.A. Loh D.Y. Jenkins M.K. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo.Immunity. 1994; 1: 327-333Abstract Full Text PDF PubMed Scopus (862) Google Scholar). We demonstrate that pretreatment of DC with IL-10 alters the APC functions of these cells in such a way that DC-IL10 are able to prevent and downmodulate activation of transgenic CD4+ T cells in vivo. Therefore, DC-IL10 might provide a potent tool for the prevention and treatment of murine or human autoimmune diseases. BALB/c mice (H-2d) and OVA-TCR DO11.10 mice (kindly provided by Dr. Dennis Loh, H-2d, OVA323-339 peptide) were bred in the animal facility of the Institute of Immunology in Mainz and used at 8–12 wk of age. For in vivo experiments five mice were used in each group (Murphy et al., 1990Murphy K.M. Heimberger A.B. Loh D.Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.Science. 1990; 250: 1720-1723Crossref PubMed Scopus (1627) Google Scholar). RPMI 1640 was supplemented with 100 IU per ml penicillin, 100 µg per ml streptomycin, 5 × 10-5 M 2′-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, and 1.5% heat-inactivated pooled BALB/c mouse serum. All cytokines used were recombinant mouse proteins. Final concentrations used in the cultures were 10 ng per ml for granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng per ml for IL-4, and 30 ng per ml IL-10 (Schering-Plough). The following antibodies were used anti-CD4 (clone GK1.5, ATCC, Rockville, MD), rIgG2b, RB6-8C5 (clone Ly-6G/Gr-1, ATCC), rIgG2a, class II I-Ab,d,q, I-Ed,k (clone M5/114.15.2, ATCC), rIgG2b, anti-CD80 (clone 16-10A1, PharMingen), anti-CD86 (clone GL1, PharMingen), anti-CD62L (clone MEL14, PharMingen). For magnetic separation we used the following beads: CD4-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Murine BM-DC were grown according to published protocols in RPMI medium supplemented with 10 ng per ml recombinant murine GM-CSF (rmGM-CSF), 10 ng per ml recombinant murine IL-4 (rmIL-4), and 1.5% mouse serum in a final volume of 25 ml. At day 2 cells were treated with 30 µg per ml rmIL-10 or left untreated. On day 5 of culture, nonadherent cells were pulsed with 50 µg per ml OVA or left unpulsed. After 2 d cells were assessed by fluorescence-activated cell sorter (FACS) for expression of CD11c, MHC class II, and costimulatory molecules (CD80, CD86). Regular yields were > 70% CD11c+ DC. Mice were sacrificed and ears removed. After disinfection with 70% ethanol ears were split into dorsal and ventral halves using fine tweezers. Subcutaneous fat and cartilage were removed by scraping with the tweezers. Ear halves were digested in 0.5% trypsin/0.2 mM ethylenediamine tetraacetic acid for 30 min. The epidermis was removed easily and epidermal cells were washed out of the tissue. After incubation of the cells in RPMI medium supplemented with 100 IU per ml penicillin, 100 µg per ml streptomycin, 5 × 10-5 M 2′-mercaptoethanol, 2 mM glutamine, 10% fetal bovine serum, and 30 µg per ml OVA overnight Langerhans cells were enriched by density centrifugation. CD4+ T cells of naive DO11.10 mice were purified with magnetic beads (Miltenyi, 98% purity) and injected intravenously into naive BALB/c mice (5 × 106 cells per mouse). Mice were injected intravenously with 5 × 105 DC treated with IL-10 or left untreated and pulsed with OVA or left unpulsed 48 h later. After a rest period of 8 d mice were sensitized by subcutaneous injection with 30 µg OVA323-339 peptide in incomplete Freund's adjuvant (IFA). Ten days later mice were either sacrificed to obtain lymph node cells for in vitro analysis or injected into the footpad with 30 µg OVA323-339 peptide in IFA for analysis of delayed-type hypersensitivity (DTH) reactions in vivo. The ratio of OVA-TCR transgenic CD4+ T cells contained in lymph node cells was analyzed by FACS and equal amounts were used for restimulation. Proliferation of OVA-specific T cells restimulated with 5 µg per ml OVA323-339 peptide was measured by 3H-thymidine incorporation. As tolerance control mice were injected once with 300 µg OVA323-339 peptide intraperitoneally and lymph node cells were processed as described. DO11.10 mice were sensitized three times every other day with 5 mg OVA per mouse subcutaneously in IFA. CD4+ T cells of these sensitized DO11.10 mice were isolated from lymph node cells (98% purity) and injected intravenously into naive BALB/c mice (5 × 106 cells per mouse). Mice were injected intravenously with 5 × 105 DC treated with IL-10 or left untreated and pulsed with OVA or left unpulsed 48 h later. After a rest period of 8 d mice were either sacrificed to obtain lymph node cells for in vitro analysis or injected into the footpad with 30 µg OVA323-339 peptide in IFA for analysis of DTH reactions in vivo. The ratio of OVA-TCR transgenic CD4+ T cells contained in lymph node cells was analyzed by FACS and equal amounts were used for restimulation. Proliferation of OVA-specific T cells restimulated with 5 µg per ml OVA323-339 peptide was measured by 3H-thymidine incorporation. For ex vivo restimulation analysis lymph node and spleen cells were prepared from all animals injected with DO11.10 cells and DC. CD4+ T cells were isolated and stimulated with OVA-pulsed epidermal cells. Three days later the medium was supplemented with 2 U per ml rhIL-2. After 10 d CD4+ cells were harvested and equal numbers of OVA-specific T cells were restimulated with OVA-pulsed epidermal cells. Proliferation was measured by 3H-thymidine incorporation. For in vivo restimulation mice reconstituted with CD4+ DO11.10 T cells and injected with DC as described were restimulated by subcutaneuos injection with 30 µg OVA323-339 peptide in IFA. Ten days later mice were injected into the footpad with 30 µg OVA323-339 peptide in IFA or IFA alone into the contralateral footpad for analysis of DTH reactions. CD4+ T cells from naive DO11.10 mice were injected into syngeneic BALB/c mice. Two days later, animals transplanted with transgenic T cells were injected intravenously with 5 × 105 BM-DC from normal BALB/c mice that were either pulsed with OVA (DC-OVA) or pulsed with OVA and pretreated with IL-10 (DC-OVA-IL10). Control mice received DC that were pretreated with IL-10 (DC-IL10) or left untreated (DC). Prior to injection DC were analyzed by FACS staining. IL-10 treatment reduced the expression of both stimulatory (MHC II) and costimulatory molecules (CD80, CD86, CD40). Expression of the DC marker CD11c was not affected (data not shown). After a period of 8 d animals were stimulated with the immunogenic OVA323-339 peptide by subcutaneous injection. Ten days later, total lymph node cells (containing APC) from these animals were prepared and restimulated with the peptide in vitro. The number of OVA-TCR transgenic T cells was determined by FACS and equal amounts were used for restimulation. Proliferation was measured after 3 d by thymidine incorporation. Whereas lymph node cells from mice treated with OVA-pulsed DC (DC-OVA) in vivo proliferated well to antigenic restimulation in vitro, there was only limited proliferation in the lymph node cells derived from animals treated with IL-10-treated OVA-pulsed DC (DC-OVA-IL10). This result is in concordance with the effects of DC-OVA-IL10 seen after stimulation in vitro (data not shown). The amount of proliferation detectable in this group was comparable to the amount of proliferation visible in the tolerance control group (lymph node cells from animals tolerized by intraperitoneal injection of immunogenic peptide in IFA) (Figure 2). Omission of antigen in the in vivo treatment resulted in proliferation rates similar to that observed with lymph node cells from naive animals. To analyze whether the inhibitory effects seen remained after antigen restimulation, CD4+ T cells were isolated from mice injected with DC-OVA and DC-OVA-IL10, respectively, and were restimulated with OVA-pulsed epidermal cells. After 3 d the medium was supplemented with 2 U per ml IL-2. After a rest period equal numbers of OVA-TCR transgenic T cells were restimulated with OVA-pulsed epidermal cells. CD4+ T cells from mice injected with DC-OVA-IL10 after reconstitution with naive CD4+ DO11.10 T cells remained impaired in their capacity to proliferate (Figure 3). CD4+ T cells isolated from mice injected with DC or DC-IL10 were driven into cell death by ex vivo restimulation.Figure 3OVA-pulsed IL-10-treated DC induce antigen-specific tolerance in naive T cells in vivo. CD4+ T cells from naive DO11.10 mice were transferred to syngeneic BALB/c mice. Two days later recipients were treated with either IL-10-treated or untreated, OVA-pulsed as described. Eight days later mice were immunized with OVA323-339 in IFA subcutaneously and CD4+ T cells were isolated from lymph node and spleen cells 10 d later. CD4+ T cells were restimulated in vitro with OVA-pulsed epidermal cells. After a rest period and addition of IL-2 to cultures (2 U per ml) CD4+ T cells were restimulated with OVA-pulsed epidermal cells and thymidine incorporation was determined after 3 d. Results shown are representative of five experiments with five mice per group. Groups are mice receiving naive CD4+ T cells and DC-OVA and mice receiving naive CD4+ T cells and DC-OVA-IL10. Error bars represent SEM.View Large Image Figure ViewerDownload (PPT) These data indicate that IL-10-treated DC inhibit antigen-specific immune responses in naive CD4+ transgenic T cells in vivo. This inhibition persists after multiple rounds of restimulation. DO11.10 mice were sensitized by subcutaneous injection of OVA in IFA. CD4+ T cells from these animals were prepared as described and injected intravenously into naive syngeneic BALB/c recipients. Two days later recipients were treated with 5 × 105 BM-DC from BALB/c mice that had been pulsed with OVA or were left unpulsed, and that were either pretreated with IL-10 or left untreated. Eight days later lymph node cells from all groups of recipients were prepared and restimulated with the immunogenic peptide in vitro. The number of OVA-TCR transgenic CD4+ T cells was determined by FACS and equal amounts were used for restimulation. Proliferation was measured by thymidine incorporation. As shown in Figure 4, lymph node cells from recipients treated with DC-OVA showed a strong proliferation upon stimulation with OVA peptide in vitro whereas lymph node cells from recipients treated with DC-OVA-IL-10 displayed 70% lower proliferation. In fact, proliferation of lymph node cells derived from DC-OVA-IL-10 injected mice was even lower than the proliferation of naive CD4+ transgenic T cells (from untreated mice) that first encountered antigen presentation in vitro (Figure 4). To analyze whether the inhibitory effects persist after restimulation with antigen, CD4+ T cells were isolated from mice injected with DC-OVA and DC-OVA-IL-10, respectively, and restimulated with OVA-pulsed epidermal cells. After 3 d the medium was supplemented with 2 U per ml IL-2. After a rest period of 8 d equal numbers of OVA-TCR transgenic CD4+ T cells were restimulated with OVA-pulsed epidermal cells. CD4+ T cells from mice injected with DC-OVA-IL-10 after reconstitution with sensitized CD4+ DO11.10 T cells remained impaired in their capacity to proliferate (Figure 5). CD4+ T cells of mice injected with DC or DC-IL-10 were driven into cell death by ex vivo restimulation. In summary these data indicate not only that IL-10-treated DC are able to suppress the function of preactivated CD4+ transgenic T cells but that these effects persist after repeated antigen restimulation.Figure 5OVA-pulsed IL-10-treated DC induce antigen-specific tolerance in sensitized T cells in vivo. CD4+ T cells from DO11.10 mice that had been immunized with OVA peptide in IFA in vivo as described were transferred to syngeneic BALB/c mice. Two days later recipients were treated with either IL-10-treated or untreated, OVA-pulsed or unpulsed DC as described. CD4+ T cells were isolated from lymph node and spleen cells from recipients after 8 d and were restimulated in vitro with OVA-pulsed epidermal cells. After a rest period and addition of IL-2 to the cultures (2 U per ml) CD4+ T cells were restimulated with OVA-pulsed epidermal cells and thymidine incorporation was determined after 3 d. Results shown are representative of five experiments with five mice per group. Groups are mice receiving primed CD4+ T cells and DC-OVA and mice receiving primed CD4+ T cells and DC-OVA-IL10. Error bars represent SEM.View Large Image Figure ViewerDownload (PPT) To look for an in vivo correlate of our in vitro findings, we performed classical DTH experiments with OVA peptide in IFA. Animals that had been either prophylactically or therapeutically treated as outlined above or mice that had been control-treated were injected intradermally with the immunogenic OVA peptide in IFA. Contralateral limbs just received IFA to determine background swelling responses. Forty-eight hours later footpad swelling reactions were measured. In mice that had been prophylactically treated with DC-OVA-IL-10, footpad swelling to OVA peptide was significantly suppressed compared to the control group of mice that had received DC or DC-IL-10 (Figure 6). Omission of antigen in the in vivo treatment resulted in swelling reactions similar to those observed in animals reconstituted with naive T cells. Mice that had received DC-OVA showed strong swelling responses. Similar results were obtained when DTH reactions were performed in a therapeutic setting (Figure 7). Analysis of DTH reactions in mice injected with DC-OVA-IL-10 showed impaired immune responses after repeated in vivo restimulation compared to mice injected with DC-OVA (Figure 8). In lymphocyte and splenocyte populations of mice injected with DC or DC-IL-10 no OVA-TCR transgenic T cells could be detected after repeated antigen restimulation in vivo. This might account for the small or absent swelling response seen in these groups after antigen inocculation into the footpad (Figure 8).Figure 7OVA-pulsed IL-10 DC downmodulate OVA-specific footpad swelling in mice injected with presensitized T cells in vivo. CD4+ T cells from DO11.10 mice that had been immunized with OVA peptide in vivo as described were transferred to syngeneic BALB/c mice. Two days later recipients were treated with either IL-10-treated or untreated, OVA-pulsed or unpulsed DC as described. Eight days later, mice were injected into the footpad with 30 µg OVA323-339 in IFA intradermally, or just IFA on the contralateral foot. Footpad swelling was determined 48 h later. Results shown are representative of three sets of experiments with five mice per group. Groups are mice receiving primed CD4+ T cells and DC-OVA (primed T cells/DC-OVA), mice receiving primed CD4+ T cells and DC-OVA-IL10 (primed T cells/DC-OVA-IL10), mice receiving primed CD4+ T cells and DC-IL10 (primed T cells/DC-IL10), mice receiving primed CD4+ T cells and unpulsed DC (primed T cells/DC), and mice receiving just primed CD4+ T cells (primed T cells). Error bars indicate SEM. *p <0.05 versus DC control. Statistical significance was determined by t test.View Large Image Figure ViewerDownload (PPT)Figure 8IL-10-treated DC inhibit footpad swelling after antigen restimulation in vivo. CD4+ T cells from DO11.10 mice that had been immunized with OVA peptide in IFA in vivo as described were transferred to syngeneic BALB/c mice. Two days later recipients were treated with either IL-10-treated or untreated, OVA-pulsed or unpulsed DC as described. Mice were injected with OVA323-339 in IFA subcutaneously after 8 d. Ten days later mice were injected into the footpad with 30 µg OVA323-339 in IFA intradermally, or just IFA on the contralateral foot. Footpad swelling was determined 48 h later. Results shown are representative of five experiments with five mice per group. Groups are mice receiving unpulsed DC, mice receiving DC-IL10, mice receiving DC-OVA, and mice receiving DC-OVA-IL10.View Large Image Figure ViewerDownload (PPT) In summary, these data indicate that application of IL-10-treated DC to OVA transgenic mice abrogates DTH reactions to OVA in vivo. IL-10 has been characterized as a modulator of APC function in many different systems. It was shown that IL-10 downmodulates the accessory functions of immature DC and monocytes, but not B cells. The inhibitory effects of IL-10 were due to a downregulation of MHC class II molecules and costimulatory molecules such as CD80, CD86, or ICAM-1 (de Waal et al., 1991de Waal M. Haanen J. Spits H. et al.Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression.J Exp Med. 1991; 174: 915-923Crossref PubMed Scopus (1701) Google Scholar;de Waal et al., 1992ade Waal M. Yssel H. Roncarolo M.G. Spits H. de Vries J.E. Interleukin-10.Curr Opin Immunol. 1992; 4: 314-331Crossref PubMed Scopus (347) Google Scholar;Hsu et al., 1992Hsu D.H. Moore K.W. Spits H. Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.Int Immunol. 1992; 4: 563-571Crossref PubMed Scopus (196) Google Scholar;Willems et al., 1994Willems F. Marchant A. Delville J.P. et al.Interleukin-10 inhibits B7 and intercellular adhesion molecule-1 expression on human monocytes.Eur J Immunol. 1994; 24: 1007-1014Crossref PubMed Scopus (345) Google Scholar). The effects of IL-10 were distinct for different subtypes of APC and also showed species dependency, however (Enk et al., 1993Enk A.H. Angeloni V.L. Udey M.C. Katz S.I. Inhibition of Langerhans cell antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance.J Immunol. 1993; 151: 2390-2396PubMed Google Scholar;Steinbrink et al., 1997Steinbrink K. Wolfl M. Jonuleit H. Knop J. Enk A.H. Induction of tolerance by IL-10-treated dendritic cells.J Immunol. 1997; 159: 4772-6684PubMed Google Scholar). Whereas the expression of MHC class II molecules and costimulatory signals was inhibited on both murine and human monocytes, various subtypes of DC were differentially affected by IL-10. Human-blood-derived DC showed reduced expression of MHC class II and CD80/CD86, whereas no such markers were affected on murine Langerhans cells (Enk et al., 1993Enk A.H. Angeloni V.L. Udey M.C. Katz S.I. Inhibition of Langerhans cell antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance.J Immunol. 1993; 151: 2390-2396PubMed Google Scholar;Steinbrink et al., 1997Steinbrink K. Wolfl M. Jonuleit H. Knop J. Enk A.H. Induction of tolerance by IL-10-treated dendritic cells.J Immunol. 1997; 159: 4772-6684PubMed Google Scholar). In general mature DC were resistant to IL-10 (Steinbrink et al., 1997Steinbrink K. Wolfl M. Jonuleit H. Knop J. Enk A.H. Induction of tolerance by IL-10-treated dendritic cells.J Immunol. 1997; 159: 4772-6684PubMed Google Scholar). Studies with murine Langerhans cells and human-blood-derived DC have shown that pretreatment with IL-10 converts the accessory functions from the induction of proliferation to anergy induction. This was demonstrated for CD4+ and CD8+ T cells (Steinbrink et al., 1997Steinbrink K. Wolfl M. Jonuleit H. Knop J. Enk A.H. Induction of tolerance by IL-10-treated dendritic cells.J Immunol. 1997; 159: 4772-6684PubMed Google Scholar). In this study we analyzed the effect of IL-10 treatment on the expression of MHC class II and costimulatory molecules on murine BM-DC and established a system to verify the in vitro results in vivo. We demonstrated in an ex vivo transfer system using CD4+ T cells from OVA-TCR transgenic mice in syngeneic BALB/c recipients that IL-10-treated DC are able to prevent the induction of sensitization in transgenic CD4+ T cells. Following sensitization with immunogenic peptide in IFA, lymph node cells from recipients treated with IL-10-treated, OVA-pulsed DC showed a proliferation similar to that of a tolerance control group that just received the immunogenic peptide in IFA intraperitoneally. In contrast, lymph node cells from recipients treated with normal OVA-pulsed DC mounted a strong proliferative response upon stimulation of the cells with immunogenic peptide in vitro. More importantly, IL-10-treated DC were also able to suppress ongoing immune responses in presensitized T cells. For this, CD4+ T cells from in vivo sensitized transgenic animals were tranferred into syngeneic recipients. When recipients were injected with IL-10-treated OVA-pulsed DC, lymph node T cells that were prepared and restimulated with immunogenic peptide in vitro showed a significantly lower T cell proliferation compared with the control group. Even after repeated antigen restimulation in vivo and in vitro full proliferative capacity of DC-OVA-IL10-treated cells cannot be restored. Our results are in agreement with earlier work byGroux et al., 1996Groux H. Bigler M. de Vries J.E. Roncarolo M.G. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells [see comments].J Exp Med. 1996; 184: 19-28Crossref PubMed Scopus (678) Google Scholar). Besides describing a direct tolerizing effect of IL-10 on CD4+ T cells in their earlier work, these authors have recently developed a transgenic mouse system where the effect is achieved by the expression of human IL-10 under the control of the murine MHC class II Ea promoter (Groux et al., 1999Groux H. Cottrez F. Rouleau M. et al.A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells.J Immunol. 1999; 162: 1723-1734PubMed Google Scholar). In these animals, IL-10 is secreted by APC such as macrophages or DC. Although no gross abnormalities in serum Ig levels or peripheral lymphocyte populations were detectable in these mice, transgenic animals failed to mount detectable T or B cell immune responses to OVA. In addition, these animals were highly susceptible to infection with intracellular pathogens like Listeria monocytogenes or Leishmania major. Although the exact reason for the poor immune response in IL-10 transgenic mice remained undetermined, T cell anergy and the induction of regulatory (Tr1-type) T cells were discussed as possible mechanisms. In human disease, there is also ample evidence to support our findings that IL-10 inhibits DC function in vivo.Mitra et al., 1995Mitra R.S. Judge T.A. Nestle F.O. Turka L.A. Nickoloff B.J. Psoriatic skin-derived dendritic cell function is inhibited by exogenous IL-10. Differential modulation of B7-1 (CD80) and B7-2 (CD86) expression.J Immunol. 1995; 154: 2668-2674PubMed Google Scholar) have shown that the secretion of IL-10 in human psoriatic plaques inhibits the APC function of human dermal DC. The effect was mediated by an inhibition of the expression of costimulatory molecules such as CD86, CD80, as well as HLA-DR molecules. For patients with malignant melanoma it has been demonstrated that high amounts of IL-10 in patients' sera is associated with a poor prognosis (Dummer et al., 1995Dummer W. Becker J.C. Schwaaf A. Leverkus M. Moll T. Brocker E.B. Elevated serum levels of interleukin-10 in patients with metastatic malignant melanoma.Melanoma Res. 1995; 5: 67-78Crossref PubMed Scopus (123) Google Scholar). Also, secretion of IL-10 by melanoma cells was shown to convert local DC function from induction of tumor immunity to the induction of T cell anergy (Dummer et al., 1996Dummer W. Bastian B.C. Ernst N. Schanzle C. Schwaaf A. Brocker E.B. Interleukin-10 production in malignant melanoma: preferential detection of IL-10-secreting tumor cells in metastatic lesions.Int J Cancer. 1996; 66: 607-614Crossref PubMed Scopus (97) Google Scholar). Similar results were obtained for lung cancer, where T-cell-derived IL-10 suppressed both APC and T cell functions (Smith et al., 1994Smith D.R. Kunkel S.L. Burdick M.D. Wilke C.A. Orringer M.B. Whyte R.I. Strieter R.M. Production of interleukin-10 by human bronchogenic carcinoma.Am J Pathol. 1994; 145: 18-29PubMed Google Scholar). On the other hand, the inhibitory effect of IL-10-treated DC in vivo makes these cells an attractive tool for future studies in experimental murine and hopefully human autoimmune diseases. The exact nature of the immune-modulatory effect exerted by IL-10-treated DC still needs to be analyzed (e.g., suppression versus anergy induction). Nevertheless there is a clear difference between OVA-pulsed IL-10-treated DC and control DC. In mice injected with control DC (DC or DC-IL10) no OVA-TCR transgenic T cells can be detected after repeated restimulation with OVA peptide in vivo, whereas CD4+ transgenic T cells can be isolated following antigen restimulation in mice injected with OVA-pulsed IL-10-treated DC. In addition, CD4+ T cells of mice injected with control DC were driven into cell death by secondary stimulation in vitro. Thus OVA-pulsed IL-10-treated DC seem to induce T cell survival without T cell activation. The inhibitory effect of DC-IL10 most likely is not a direct effect of IL-10 on CD4+ T cells, as no IL-10 was released by IL-10-treated DC (not shown). Studies to better define the nature of the tolerizing T cell response induced by IL-10-treated APC and the molecules involved in this reaction are currently in progress in our laboratory. Supported by Deutsche Forschungsgemeinschaft SFB 548-A3.