International Immunology Advance Access published online on August 13, 2007
International Immunology, doi:10.1093/intimm/dxm078
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Anti-thymocyte globulin (ATG) prevents autoimmune encephalomyelitis by expanding myelin antigen-specific Foxp3+ regulatory T cells
1 Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, HIM 780, Boston, MA 02115, USA
2 U.S. Scientific Development, Genzyme Corporation, Framingham, MA 01701, USA
3 Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
Correspondence to: Correspondence to: M. Oukka; E-mail: moukka{at}rics.bwh.harvard.edu
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Abstract |
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The T cell-depleting polyclonal antibody, anti-thymocyte globulin (ATG) has long been used in organ transplantation to treat acute rejection episodes. More recently, it is also being used as part of an induction regimen to protect allografts. It has been proposed that ATG might deplete effector T cells (T-effs) while sparing regulatory T cells (T-regs). In order to test whether ATG is effective in autoimmune disease, we used Foxp3gfp ‘knock-in’ mice in combination with a myelin oligodendrocyte glycoprotein (MOG)35–55/IAb tetramer to study more closely the effect of ATG treatment on antigen-specific T cell responses in vivo during MOG-induced experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple Sclerosis. ATG treatment enhanced the expansion of MOG-specific T-regs (CD4+Foxp3+) in MOG-immunized mice. T-effs were depleted, but on a single-cell basis, the effector function of residual T-effs was not compromised by ATG. Thus, ATG tipped the balance of T-effs and T-regs and skewed an auto-antigen-specific immune reaction from a pathogenic T cell response to a potentially protective T-reg response. In both acute and relapsing remitting disease models, ATG treatment resulted in the attenuation from EAE, both in a preventive and early therapeutic setting. We conclude that ATG treatment enforces the development of a dominant immunoregulatory environment which may be advantageous for the treatment of T cell-driven autoimmune diseases.
Keywords: anti-thymocyte globulin, T-reg cell, antigen-specific, EAE, multiple sclerosis
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Introduction |
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The ability of CD4+CD25+ regulatory T cells (T-regs) to actively suppress activation and expansion of self-reactive T cells is critical for the protection from autoimmunity (1, 2). In the clinical setting, it has been regarded as a potential therapeutic approach to manipulate the balance between T-regs and effector T cells (T-effs) in order to treat active autoimmune diseases in humans. T-reg-based treatment strategies have been tested in animal models for transplantation (3–5) and human autoimmune diseases including type I diabetes (T1D) (6), multiple sclerosis (MS) (7) and rheumatoid arthritis (8). Antibody treatments that have been used to shift the balance between T-effs and T-regs in vivo include therapy of T1D (9–11) and experimental autoimmune encephalomyelitis (EAE) (12) with anti-CD3. The exact mode of action of non-mitogenic anti-CD3 antibody remains elusive but it has been suggested that it may induce anergy in recently activated T cells (13), alter the balance between T-effs and T-regs by differential depletion of T-effs or induce de novo generation of T-regs (14).
The polyclonal antibodies, anti-lymphocyte serum (ALS) and anti-thymocyte globulin (ATG), that induce broad T cell depletion have also been used to treat graft versus host disease (15), acute rejection in organ transplantation (16), and autoimmune diabetes (17) and are FDA approved for the treatment of renal transplant patients. However, at this point, the exact mechanism of action is still unclear and has mostly been regarded as generalized immunosuppression by T cell depletion via complement-dependent lysis and Fas/Fas ligand-mediated activation-induced cell death (18, 19). Only recently, it has also been suggested that ATG might differentially deplete T-effs but spare T-regs (20) or even induce the generation of CD4+CD25+Foxp3+ T-regs de novo (21). However, functional analyses in these studies have been marred by using CD25 as a marker for T-regs since CD25 is also expressed on activated T-effs. Moreover, since activated T cells appear to be more susceptible to ATG-induced depletion (18, 19), the question arises whether ATG depletes the T cell repertoire in a skewed manner. Thus, it is essential to study antigen-specific responses of both T-regs and T-effs in order to address this issue.
In this report, we directly approach these questions by the combination of technologies that we recently developed in our laboratory. First, we used a Foxp3gfp ‘knock-in’ (Foxp3gfp.KI) mouse that allowed us to faithfully track Foxp3-expressing T cells in vivo. Second, we applied a myelin oligodendrocyte glycoprotein (MOG)35–55/IAb tetramer in order to visualize antigen-specific responses in vivo during EAE. Overall, our data show that ATG treatment drives the expansion of MOG-specific T-regs after preferential depletion of T-effs, resulting in reduced EAE.
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Mice
C57Bl/6 and SJL/J mice (6–8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Foxp3gfp KI mice have been described previously (22, 23). Briefly, a bicistronic eGFP reporter gene was introduced after the stop codon of the endogenous Foxp3 locus. This strategy enabled us to track Foxp3-expressing regulatory cells in vivo. The introduction of the eGFP reporter did not alter Foxp3 gene expression. Foxp3gfp. KI mice developed normally and were not phenotypically different from their wild-type littermates. All animals were kept in specific pathogen-free conditions. All experiments and protocols were performed in accordance to Institutional Animal Care and Use Committee IACUC guidelines. All experiments with SJL/J mice were performed in Genzyme Corporation (Framingham, MA, USA).
Induction of EAE and administration of ATG
Age-matched C57Bl/6 mice were immunized subcutaneously in the flanks with 100 µg of MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA supplemented with 5 mg ml–1 Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI, USA). Pertussis toxin (List Biological Laboratories, Campbell, CA, USA) was given intra-peritoneally (i.p.) on days 0 and 2 in a dose of 200 ng per mouse.
Age-matched SJL/J mice were immunized subcutaneously in the flanks with 60 µg of PLP139–151 (HSLGKWLGHPDKF) emulsified in CFA supplemented with 3 mg ml–1 M. tuberculosis H37Ra. Pertussis toxin (List Biological Laboratories) was given intravenously on days 0 and 2 in a dose of 100 ng per mouse.
The severity of clinical disease was assessed as follows: 0 = no signs of disease, 1 = limp tail, 2 = abnormal gait, 2.5 = one hindlimb paralyzed, 3 = complete hindlimb paralysis, 3.5 = complete hindlimb paralysis and one forelimb paralyzed, 4 = tetraplegia and 5 = moribund or death. Mice were monitored for a period of 40 days.
In the B6 model, 100 µg ATG or rabbit IgG was given i.p. on days –7 and –3 prior to immunization with MOG peptide for the preventive setting. In the therapeutic setting 100 µg of ATG or rabbit IgG was given i.p. when the mice developed a disease score of 1 followed by another 100 µg 2 days later.
In the SJL/J model, 450 µg ATG or PBS was given i.p. on days –7 and –3 prior to immunization with PLP peptide for the preventive setting. In the therapeutic setting, 450 µg ATG or PBS was given i.p. when the mice developed a disease score of 1 followed by another 450 µg 4 days later.
In vitro proliferation assay and cytokine ELISA
After immunization with MOG35–55/CFA, mice were given 100 µg ATG or rabbit IgG i.p. on days 1 and 3, following immunization. Spleen and lymph node cells were isolated on day 8 after immunization. CD4+ T cells were purified using CD4 MACS beads (Miltenyi Biotech, Auburn, CA, USA) following the manufacturer's instructions.
CD4+ T cells (1 x 105) and 4 x 105 irradiated (3300 rad) syngeneic splenic antigen-presenting cells (APCs) were seeded onto 96-well round-bottom plates in DMEM/10% FCS supplemented with 5 x 10–5 M ß-mercaptoethanol, 1 mM sodium pyruvate, non-essential amino acids, L-glutamine and 100 U penicillin/100 µg streptomycin per ml and stimulated with different concentrations of anti-CD3 (clone 145.2C11, Bioexpress, West Lebanon, NH, USA) antibody or MOG35–55 peptide for 72 h. Supernatants were collected after 48 h and analyzed for IL-4, IL-10, IFN-
and IL-17 by ELISA. Cells were pulsed with 1 µCi of [3H]thymidine ([3H]TdR) for 18 h and harvested on glass fiber filters. Incorporated [3H]TdR was determined using a beta plate scintillation counter.
Suppression assay
One hundred micrograms of ATG or rabbit IgG was injected i.p. into MOG35–55 immunized Foxp3gfp.KI mice on days 0 and 3 after MOG immunization. Mice were sacrificed on day 8. Single-cell suspensions were prepared from the spleen and lymph nodes. CD4+GFP+ T cells from both treatment groups were FACS sorted and used as regulatory cells. Naive cells (CD4+CD62LhighCD25–) from regular 2D2 (24) mice were used as responder T cells. Twenty thousand responder cells and regulatory cells from either treatment group were cultured in different ratios with 80 000 irradiated (3300 rad) syngeneic splenic APCs per well in the presence of 10 µg ml–1 MOG35–55 peptide or 1 µg ml–1 anti-mouse CD3 for 72 h. Cells were pulsed with 1 µCi of [3H]TdR for the last 18 h of culture and incorporated [3H]TdR was determined using a beta plate scintillation counter.
Isolation of T cells from the Central nervous system
Mice were perfused with cold PBS through the left cardiac ventricle. The brain was dissected and the spinal cord was flushed out by hydrostatic pressure. Central nervous system (CNS) tissue was cut into pieces and digested with 2.5 mg ml–1 of Collagenase D (Roche Diagnostics, Indianapolis, IN, USA) and 1 mg ml–1 of DNAse I (Sigma–Aldrich, St Louis, MO, USA) in DMEM medium at 37°C for 40 min. Single-cell suspensions were prepared using a 70-µm cell strainer followed by percoll gradient centrifugation (70/37%). Mononuclear cells were removed from the interphase, washed and re-suspended in culture medium.
MOG tetramer staining
T cells (5 x 105) from whole spleen and lymph nodes of immunized Foxp3gfp.KI mice were isolated on day 8 and stimulated with 10 µg ml–1 of MOG peptide for 4 days in medium containing IL-2. Mononuclear cells from the CNS were purified and isolated at the peak of disease as described above.
MOG35–55/IAb tetramers and control tetramers (TMEV 70-86/IAs) were generated as described (23, 25). Prior to ex vivo tetramer staining, 1 x 107 mononuclear cells per ml from the CNS were treated with 0.7 U ml–1 of neuraminidase (Sigma–Aldrich) in DMEM for 30 min at 37°C. Cells were incubated with the tetramers (30 µg ml–1) for 2.5 h in the dark at room temperature in DMEM containing 5 µM IL-2 and 2% FCS (pH 8.0). Cells were stained with anti-CD4–APC and 7-amino-actinomycin D (7-AAD) (PharMingen, San Diego, CA, USA). The gate was set on live (7-AAD–) CD4+ cells and the percentages of MOG-specific cells in the Foxp3/GFP+ (T-regs) and Foxp3/GFP– (T-effs) compartment were determined.
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Administration of ATG in vivo preferentially depleted CD4+Foxp3/GFP– T cells
We wished to test the effect of ATG on T cell populations in vivo. Therefore, we administered 100 µg of ATG i.p. to Foxp3gfp.KI mice on day 0 and day 3 and sacrificed the mice on day 8. We observed that the CD4+ population in the spleen was decreased by
75% following ATG treatment. However, the percentage of T-regs (GFP+Foxp3+) within the CD4+ population increased from 12 to 20% resulting in an increased T-reg to T-eff ratio (Fig. 1). Similar results were obtained when analyzing the lymph nodes of ATG-treated mice. Here, the percentage of CD4 cells within the T cell population decreased by 20% with the percentage of T-regs (GFP+Foxp3+) within the CD4+ population increasing from 7 to 32% after ATG treatment (data not shown). Thus, administration of ATG results in the preferential depletion of T-effs in vivo increasing the ratio of T-regs versus T-effs.
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ATG treatment inhibited proliferation and cytokine production of CD4+ T cells
In order to investigate whether ATG treatment affected antigen-specific recall responses to a class II restricted antigen, we isolated CD4+ T cells from the spleen and lymph nodes of MOG-immunized mice treated with ATG or rabbit IgG followed by re-stimulation in vitro in the presence of syngeneic APCs. CD4+ T cells from ATG-treated mice proliferated less when stimulated with anti-CD3 or MOG peptide (Fig. 2A) compared with the control groups. This also resulted in a significantly reduced production of IFN-
and IL-17 from the ATG-treated group while splenocytes from the rabbit IgG control group produced huge amounts of these two cytokines. The altered cytokine pattern resulted in markedly decreased IFN- to IL-10 and IL-17 to IL-10 ratios in splenocytes derived from ATG-treated mice as compared with controls (Fig. 2B). Collectively, these data suggested that the altered balance of T-effs/T-regs in the ATG-treated animals was functionally relevant for the response to auto-antigens.
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ATG did not alter the function of residual CD4+Foxp3/GFP– T-effs and CD4+Foxp3/GFP+ T-regs
Next, we asked whether ATG treatment affected the function of remaining CD4+Foxp3/GFP– T-effs. To test this, we FACS sorted CD4+Foxp3/GFP– T cells from MOG-immunized mice treated with either ATG or rabbit IgG and re-stimulated them with different concentrations of MOG peptide in the presence of syngeneic APCs. When plating identical numbers of responder cells, no difference in cell proliferation and cytokine production was observed (data not shown). These results suggested that ATG treatment did not alter the function of residual effector cells.
We also tested whether ATG treatment changed the ability of T-regs to suppress polyclonal and antigen-specific T cell responses. We performed suppression assays using responder cells from 2D2 MOG TCR transgenic mice and added FACS-sorted CD4+Foxp3/GFP+ T-regs from either ATG- or rabbit IgG-treated MOG-immunized Foxp3gfp. KI mice in different ratios. There was no difference in the ability of T-regs from ATG-treated and rabbit IgG-treated mice in suppressing 2D2 responder cells upon anti-CD3 or MOG35–55 stimulation (Fig. 3A and B). These data emphasize that ATG treatment did not impair the ability of T-regs to suppress polyclonal and antigen-specific responses of naive T cells.
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ATG treatment conferred protection from EAE in the B6–MOG model and SJL–PLP model
Given the preferential depletion of T-effs by ATG, we wondered whether administration of ATG conferred protection from EAE. We found that when ATG was administered prior to immunization with MOG peptide (preventive treatment setting), mice were relatively protected from disease. Although some ATG-treated mice still developed EAE, the disease onset was delayed and the disease was significantly milder (Fig. 4A). More importantly, when ATG was administered in a therapeutic setting (i.e. after the onset of clinical signs of disease), the disease did not progress and remained stable throughout the observation period (Fig. 4B). To further confirm this beneficial effect on EAE in the B6 model, we also tested ATG treatment in a remitting relapsing mouse model of EAE, the PLP139–151-induced EAE in SJL mice. Similar to the results observed in the B6 model, pre-treatment with ATG effectively decreased the incidence and resulted in a milder disease course (Fig. 4C). Furthermore, therapeutic administration of ATG also reduced the severity of the first disease episode in this model (Fig. 4D). These data suggest that ATG is effective in the treatment of EAE. However, the timing for the administration of ATG in a therapeutic setting appeared to be critical. When ATG was given at the peak of disease when the inflammatory infiltrate in the target tissue was already established, a beneficial effect of ATG was no longer observed (data not shown).
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ATG treatment resulted in the generation/expansion of antigen-specific T-regs
In order to investigate the basis for the reduction of clinical disease of ATG-treated animals, we determined the frequency of antigen-specific T-regs and T-effs using MOG35–55/IAb tetramers.
Spleen and lymph node cells from immunized Foxp3gfp KI mice were stimulated in vitro with MOG peptide and IL-2 for 4 days prior to MOG tetramer staining. In this antigen-specific recall culture of lymphocytes from rabbit IgG-treated control mice, MOG tetramers detected a distinct population of antigen-specific T-effs and T-regs. The expansion of T-effs was always dominant over the expansion of T-regs (Fig. 5A). In contrast, in ATG-treated mice, the expansion of antigen-specific T-regs largely prevailed over T-effs resulting in T-eff versus T-reg ratios of 1:15 in favor of T-regs (Fig. 5A). Similar results were also observed in recall cultures of splenocytes (data not shown). This suggested that ATG treatment changed the balance between T-effs and T-regs in favor of T-regs, allowing for a remarkable antigen-driven expansion of T-regs thus attenuating disease.
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In order to confirm this observation, we wished to investigate whether ATG induced a similar expansion of antigen-specific T-regs in vivo without further in vitro manipulation of antigen-primed T cells. Therefore, mononuclear cells were isolated from the CNS of MOG-immunized and IgG- or ATG-treated mice when the animals had reached the individual peak disease score, and stained with MOG35–55/IAb tetramers directly ex vivo. It has to be noted that it was uncommon for a mouse treated with ATG to develop severe EAE. The ATG-treated mice also recovered very quickly. So, the timing for MOG tetramer analysis was critical. We found that the ratio of MOG-specific T-effs to T-regs in the CNS of rabbit IgG-treated mice was 30:1 as opposed to 1:1 in the ATG-treated group (Fig. 5B). It is likely that this favorable ratio of antigen-specific T-effs/T-regs in the CNS of ATG-treated mice is responsible for inhibiting the establishment of a severe inflammatory infiltrate in the CNS of ATG-treated mice. It has recently been shown that the ratio of antigen-specific T-effs/T-regs in the CNS of EAE mice was 1:13 at the peak of disease and increased to 1:4 during remission, but also that the highly inflammatory cytokine milieu in an established autoimmune infiltrate in the CNS might impair T-reg function at the peak of disease (25). Thus, by manipulating the T-eff/T-reg ratio through administration of ATG before the onset of massive inflammation, autoimmune tissue destruction might be prevented.
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Discussion |
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In the present study, we tested a T cell-depleting polyclonal antibody (ATG) for its potency to treat organ-specific autoimmunity. Combining the use of Foxp3gfp.KI mice with the technology of tetramer staining, we created a model system that allowed us to track auto-antigen-specific T-effs and T-regs during EAE and thus determine differential effects of ATG on these T cell subsets. ATG, which is already used in humans to prevent transplant rejection, was very efficient in attenuating autoimmune encephalomyelitis both in a preventive and early therapeutic treatment regimen. We provide evidence, that ATG preferentially depletes T-effs while T-regs are resistant to depletion in vivo. Thus, upon immunization with MOG in CFA, the expansion of antigen-specific T-regs is favored over the expansion of T-effs resulting in robust protection from clinical disease. Furthermore, even if the ATG treatment is given only at the onset of clinical signs, the differential susceptibility of T-effs is still sufficient to reduce the severity of disease.
Peritransplantational depletion of T cells has proven to be a valuable way to prevent transplant rejection. A series of studies on kinetics and mechanism of ATG and ALS implied that these reagents induced a window for graft acceptance simply by inducing generalized suppression of T cell responses. However, recent studies indicated that various CD4+ subsets might have different susceptibilities to ATG-induced depletion (20, 21). Here, T-regs might be particularly resistant to ATG. We have previously determined the kinetics of antigen-specific T-effs and T-regs in autoimmunity and established that in the widely used model of MOG35–55/CFA-induced EAE, MOG-specific T-regs are generated by expansion of naturally occurring Foxp3-expressing T-regs in the peripheral immune compartment (23). These T-regs then traffic to the target tissue. The differential kinetics of accumulation of myelin-specific T-effs and T-regs in the CNS reflect the clinical course of the disease in that the T-reg/T-eff ratio in the CNS is 1:13 at the peak of disease, but 1:4 during recovery. It was also evident that T-reg-mediated regulation was more efficient in suppressing primary activation of responder cells, but not antigen-specific recall responses of MOG-specific T-effs isolated from the CNS (23). As a result, in order for T-reg-based therapeutic approaches to be successful, it is required to achieve a favorable ratio of antigen-specific T-regs and T-effs early during the activation of T-effs and before the onset of massive inflammation in the target tissue. This was the rationale for the application of ATG in EAE.
ATG treatment led to the depletion of the peripheral CD4+ T cell compartment by 75%. Since T-effs (CD4+Foxp3–) were preferentially depleted, T-regs (CD4+Foxp3+) were relatively enriched in the remaining CD4+ population. Near-complete T cell depletion involves the issue of homeostatic proliferation of the remaining T cell population. It has been shown that T-effs that proliferate homeostatically (due to lymphopenia) behave no longer as naive T cells, but as recently activated T cells or—after cessation of homeostatic proliferation—as memory-like T cells (26). This makes them resistant to tolerizing treatment approaches, which rely on the induction of anergy by blocking of co-stimulatory pathways. This is a severe conceptual setback for the clinically desirable combination of T cell depletion and immunomodulation. However, in vivo, T-regs are known to proliferate homeostatically to the same extent as T-effs (27). Thus, immunomodulation that is based on T-regs should not be impaired, especially in the case of ATG treatment, as the T-reg fraction within the CD4+ population is increased. In line with this concept, the unsorted CD4-positive T cell population isolated from lymph nodes and spleen of ATG-depleted animals that were immunized with MOG35–55/CFA showed reduced recall responses to MOG in terms of proliferation and production of inflammatory cytokines as compared with CD4+ cells from control-treated animals. When equal numbers of FACS-sorted T-effs (CD4+Foxp3–) were tested separately from the accompanying T-reg fraction, the recall responses to MOG were identical between the ATG and the control groups. Therefore, it can be ruled out that priming of antigen-specific T-effs on a single-cell basis is defective in ATG-treated animals. Nevertheless, ATG-treated mice were protected from disease induction by immunization with MOG35–55/CFA. This clearly indicates that the ATG-induced shift in balance toward a more favorable T-reg/T-eff ratio was clinically relevant in a setting of pending autoimmunity proving the hypothesis that T-reg-mediated suppression of autoaggressive T cell responses is well maintained after T cell depletion by ATG. It is very likely that the enrichment of T-regs in the CD4 population by ATG is the prerequisite for the massive expansion of MOG-specific T-regs after immunization with MOG35–55/CFA. Thus, MOG immunization in ATG-treated animals favors the expansion of fully functional MOG-specific T-regs in the peripheral immune compartment and the CNS. Although we have not formally ruled out in this study that the increase in frequency of MOG-specific T-regs upon ATG treatment followed by MOG sensitization is due to the conversion of Foxp3– into Foxp3+ T cells, we consider this unlikely. In fact, we have previously shown that there is no conversion of Foxp3– into Foxp3+ T cells under inflammatory conditions as generated by immunization with CFA as an adjuvant (23).
In the present study, we determined that ATG treatment is also efficient in an early therapeutic setting, i.e. after onset of clinical signs of disease, which is particularly relevant for the treatment of human autoimmune diseases. We propose that in this condition, the effect of ATG is due to the depletion of T cells that have infiltrated the target tissue or are in the process of trafficking to the CNS before the firm establishment of a severe autoimmune infiltrate. Based on our kinetic analysis of MOG-specific T cells in the natural course of EAE, both T-effs and T-regs are already present in the CNS at the onset of clinical signs. More importantly, the peak of IL-17 production by T-effs in the CNS is early after the onset of clinical signs of disease and it is believed that IL-17 initiates an inflammatory cascade that impairs T-reg function in situ (22, 23). Thus, in a therapeutic setting there is only a limited time frame for the depletion of T-effs. Indeed, when ATG was not administered at the onset, but at the peak of disease, the severity and duration of disease remained unchanged.
In theory, T-reg-based treatments of autoimmune diseases have an appealing elegance. However, much remains to be learned about the sites and modes of action of T-regs in organ-specific autoimmune diseases. It is particularly unclear whether transfer of ex vivo generated bona fide T-regs into inflamed tissue would be beneficial for controlling autoimmune tissue damage. So far, therapeutic approaches have therefore focused on shifting the balance between endogenous T-effs and T-regs. Recently, a superagonistic CD28 antibody that was shown to preferentially promote the expansion of CD4+CD25+ T-regs as opposed to T-effs in a series of pre-clinical models for autoimmune disease (28, 29) and transplantation (29) went into phase I clinical trial. Unfortunately, catastrophic side effects were observed that were most likely related to the mitogenic and activating properties of this reagent. The most promising results both in pre-clinical models and human trials were achieved with non-mitogenic antibodies like CD3 or depleting reagents like ATG or ALS. In this study, we provide evidence that ATG treatment is not only valid in transplantation but might also be extended to autoimmune diseases. We confirmed that ATG leads to a preferential depletion of T-effs and established that the protection from organ-specific autoimmunity is due to the enhanced expansion of auto-antigen-specific T-regs during the priming phase of the disease. In later disease phases, ATG treatment is still efficient as long as massive inflammation in the target organ is not yet established. Thus, in conclusion, ATG endorses the development and maintenance of dominant tolerance in autoimmunity, which might be relevant for the treatment of human autoimmune disease such as rheumatoid arthritis and MS.
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Deutsche Forschungsgemeinschaft (KO 2964/1-1) to T.K.
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Abbreviations |
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| ALS, anti-lymphocyte serum |
| APC, antigen-presenting cell |
| ATG, anti-thymocyte globulin |
| 7-AAD, 7-amino-actinomycin D |
| EAE, experimental autoimmune encephalomyelitis |
| Foxp3gfp.KI, Foxp3gfp knock-in |
| [3H]TdR, [3H]thymidine |
| i.p., intra-peritoneally |
| MOG, myelin oligodendrocyte glycoprotein |
| MS, multiple sclerosis |
| T-eff, effector T cell |
| T-reg, regulatory T cell |
| T1D, type I diabetes |
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Notes |
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* These authors contributed equally to this study.
Received 20 March 2007, accepted 30 May 2007.
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