In vitro-generated regulatory T cells induced by Foxp3 ... - Nature

Gene Therapy (2005) 12, 1294–1304 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt

RESEARCH ARTICLE

In vitro-generated regulatory T cells induced by Foxp3-retrovirus infection control murine contact allergy and systemic autoimmunity K Loser1, W Hansen2, J Apelt1, S Balkow1, J Buer2,3 and S Beissert1 Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany; 2Department of Mucosal Immunity, German Research Center for Biotechnology, Braunschweig, Germany; and 3Institute of Medical Microbiology, Hannover Medical School, Hannover, Germany 1

Regulatory T cells are promising candidates for the modulation of inflammation and autoimmunity. To generate regulatory T cells in vitro, we have infected naı¨ve CD4+CD25 T cells with a retrovirus encoding the transcription factor Foxp3. Foxp3-infected T cells are similar to naturally occurring regulatory T cells as evidenced by surface marker expression and function. To investigate the effects of Foxp3infected T cells on contact hypersensitivity (CHS) responses, sensitized mice were injected with Foxp3- or control virusinfected T cells. Only injection of Foxp3-infected T cells into sensitized mice significantly inhibited CHS compared to controls, indicating that Foxp3-infected T cells are suppressive in vivo. These findings prompted treatment of autoimmune-prone CD40L transgenic (tg) mice, which develop a

severe systemic autoimmune disease including autoreactive T cells and autoantibodies, with Foxp3-infected T cells. Interestingly, injections of Foxp3-infected T cells into CD40L tg mice inhibited the ongoing development of autoimmune dermatitis and activation of cytotoxic CD8+ T cells. Strikingly, treatment with Foxp3-infected T cells reduced serum concentrations of antinuclear antibodies in CD40L tg mice, which was paralleled with reduced renal immunoglobulin depositions and increased kidney function. Together, these findings indicate that newly in vitro-generated regulatory T cells can be successfully used to treat inflammatory and ongoing autoimmune disorders. Gene Therapy (2005) 12, 1294–1304. doi:10.1038/ sj.gt.3302567; published online 16 June 2005

Keywords: T cells; immunotherapy; contact allergy; autoimmunity; transcription factor

Introduction Immunological unresponsiveness is maintained by the elimination or inactivation of potentially hazardous selfreactive lymphocytes. Within the periphery, activation and expansion of autoreactive T cells that have escaped thymic deletion is actively suppressed by naturally occurring CD4+ regulatory T cells, the majority of which constitutively express CD25.1,2 In immunodeficient mice, transfer of CD4+CD25 T cells readily induced autoimmune disease and cotransfer of CD4+CD25+ T cells abrogated the development of disease.3 CD4+CD25+ T cells are able to inhibit the activation of both autoreactive Th1 and Th2 clones as well as CD8+ T-cell responses.3,4 From these studies, it was concluded that regulatory T cells inhibit primarily the induction of autoimmune responses. A classical phenotype of CD4+ regulatory T cells is that they are naturally anergic upon T-cell receptor (TCR) stimulation. Suppressor activity has been shown in vitro to be dependent on cell–cell contact between naı¨ve CD4+CD25 and CD4+CD25+ T cells. Thereby, CD4+CD25+ T cells inhibit the production of Correspondence: Professor S Beissert, Department of Dermatology, University of Mu¨nster, Von-Esmarch-Strasse 58, D-48149 Mu¨nster, Germany Received 6 December 2004; accepted 16 April 2005; published online 16 June 2005

interleukin (IL)-2 by CD4+CD25 T cells.5 However, several experimental in vivo models argue that suppressor activity might also be mediated by the release of immunosuppressive cytokines, such as IL-10 or transforming growth factor b (TGF-b).6,7 Regulatory CD4+ T cells are defined by a variety of surface molecules, which include cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), CD62L, CD45RBlow, integrin aeb7, glucocorticoid-induced TNF receptor (GITR), chemokine receptor (CCR) 4, CCR8 and neuropilin-1 (Nrp-1).8–13 Regulatory CD4+CD25+ T cells develop in the thymus. Hence, BALB/c mice that have been thymectomized on day three of life lack CD4+CD25+ T cells and suffer from a multiorgan autoimmune syndrome.3 Injection of CD4+CD25+ T cells into thymectomized recipients or thymus grafting prevents the induction of autoimmunity presumably via re-established regulatory effector cell functions. Within the periphery, several molecular mechanisms have been identified, which maintain regulatory T-cell numbers. CD4+CD25+ T cells are critically dependent on IL-2.14 Accordingly, IL-2/ or IL-2 receptor beta chain/ mice have almost no detectable numbers of CD4+CD25+ T cells and develop a lethal autoimmune disease.15 Addition of IL-2 to CD4+CD25+ T cells reversed the anergic state and induced proliferation of these cells indicating that, indeed, IL-2 is an essential growth factor of CD4+CD25+

In vitro generation of regulatory T cells K Loser et al

Results In vitro generation of regulatory T cells from naı¨ve CD4+ T cells by retroviral transduction of Foxp3 Since only 5–10% of all CD4+ T cells show regulatory functions, the availability of regulatory T cells for therapeutic interventions is greatly reduced. We therefore were interested to determine if regulatory T cells can be generated from naı¨ve T cells in vitro in numbers sufficient for immunotherapy by introduction of the Foxp3 gene. To this end, bicistronic retroviral vectors expressing Foxp3 and eGFP (Foxp3/eGFP) or eGFP alone were generated (Figure 1a). Splenic and lymph node CD4+CD25 T cells from naı¨ve mice were stimulated

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T cells. In addition to cytokines, molecules of the B7 family and the TNF superfamily have also been shown to be involved in the peripheral homeostasis of CD4+CD25+ T cells.16–19 More recent investigations suggest that regulatory CD4+CD25+ T cells may develop as a unique T helper (h) cell lineage and the transcription factor that appears to control lineage commitment is Foxp3.20–22 Foxp3 seems to be specifically expressed in regulatory T cells and, indeed, high levels of Foxp3 were detectable in sorted CD4+CD25+ T cells in contrast to CD4+CD25 T cells as evidenced by quantitative RT-PCR. Mice with spontaneous Foxp3 mutations (scurfy; sf) or with a genetargeted disruption of Foxp3 lack CD4+CD25+ T cells and develop a lethal autoimmune disorder.20,22 Hence, Foxp3 is a key factor involved in the development of regulatory CD4+CD25+ T cells. Therefore, modulation of Foxp3 expression appears to be an interesting alternative to manipulate regulatory T-cell numbers or even function. Impaired suppressor function of CD4+CD25+ T cells has been documented in patients suffering from autoimmune disorders such as multiple sclerosis.23 On the other hand, activation of suppressor function by injection of humanized anti-CD3 antibodies (Ab) might mediate in part the beneficial therapeutic effects of this regime in patients with early-onset type I diabetes.24 These findings point to regulatory T cells as promising candidates not only to prevent the induction but also to inhibit ongoing autoimmune disorders. Such efforts are, however, greatly impaired by the low available number of CD4+CD25+ T cells, which make up only 5–10% of T helper cells.25,26 To circumvent this problem, we have generated a retroviral transfection system that allows expression of the murine transcription factor Foxp3 in large numbers of naı¨ve T cells. Murine CD4+CD25 T cells were infected with a Foxp3-encoding retrovirus. Foxp3 overexpression was detectable after transfection and induced CD4+ T cells, which were similar to naturally occurring regulatory CD4+CD25+ T cells. Hence, Foxp3-infected T cells were anergic and suppressive in vitro. In the following, we show that by utilizing Foxp3 transfection, regulatory T cells can be in vitro generated from naı¨ve T cells in numbers sufficient enough for the in vivo treatment of T-cell-mediated allergic contact hypersensitivity (CHS) responses. Furthermore, we demonstrate that injection of Foxp3-infected T cells can be successfully used to control the progression of CD40L-induced ongoing severe systemic autoimmunity in mice.

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Figure 1 (a) Bicistronic retroviral vectors expressing Foxp3/eGFP or eGFP alone for transfection of naı¨ve CD4+CD25 T cells. (b) Transfection efficiency of CD4+CD25 cells was determined by FACS analysis (left), and 7 days after transfection, eGFP-expressing cells were purified by FACS-sorting (right). (c) Quantification of relative Foxp3 mRNA levels in retrovirus-infected CD4+CD25 T cells. cDNA samples of Foxp3 virus- or control virus-infected cells were subjected to quantitative real-time RT-PCR using primers and an internal fluorescent probe specific for Foxp3 or b-actin. The relative quantity of Foxp3 was normalized to the relative quantity of b-actin in each sample. One representative out of three independent experiments is shown.

with anti-CD3 and IL-2 and infected with either retrovirus. About 40–50% of CD4+ T cells expressed GFP after transfection; eGFP+ cells were isolated by FACS-sorting 7 days after transfection (Figure 1b), and proliferation to TCR stimulation, expression of cell surface markers and cytokine production of GFP+ T cells were analyzed. Importantly, enhanced levels of Foxp3 were detectable in Foxp3 virus-transfected compared to nontransfected or control virus-transfected T cells (Figure 1c). Classical characteristics of natural CD4+CD25+ regulatory T cells are the expression of CD25, which is also upregulated during T-cell activation, as well as CTLA-4 and Nrp-1 among others.8,12 Activation from retroviral infection induced in eGFP+ cells from both controland Foxp3-infected cultures the expression of CD25 (not shown). However, Foxp3-infected cells expressed CTLA4 and Nrp-1 at higher levels compared to eGFP+ cells from control virus-infected cells (Figure 2a). These findings indicate that Foxp3 transduction induced surface marker expression closely associated with naturally arising CD4+CD25+ regulatory T cells. CD4+CD25+ regulatory T cells are normally anergic to TCR stimulation with mitogenic antibodies.26 To test this, CD4+CD25 T cells transfected with either retrovirus Gene Therapy


and nontransfected CD4+CD25 T cells were stimulated with CD3/CD28 mAb. Upon stimulation, a vigorous proliferation was induced in eGFP+ cells from controltransfected and nontransfected T cells (Figure 2b). Interestingly, Foxp3/eGFP-transfected T cells showed a significantly reduced proliferation to TCR stimulation. In addition, eGFP+ cells from Foxp3/eGFP-infected cultures produced high concentrations of IL-10 and low concentrations of IL-2, IL-4 and IFN-g (Figure 2c and data not shown). Foxp3-infected T cells also produced more TGF-b than control virus-infected T cells as evidenced

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by quantitative RT-PCR (data not shown). These results suggest that Foxp3 transduction renders T cells hyporesponsive to TCR stimulation and instructs these cells to produce IL-10, a cytokine, which is associated with regulatory T-cell function.7

Suppressor activity of Foxp3-infected T cells in vitro CD4+CD25+ regulatory T cells can suppress the proliferation of CD4+CD25 T cells.3 Therefore, we assessed whether Foxp3/eGFP-transfected cells were able to demonstrate in vitro suppressor activity. eGFP+ cells from cultures transfected with either control or Foxp3/ eGFP retrovirus were added to freshly prepared CD4+CD25 T cells. The data depicted in Figure 3a show that eGFP+ cells from control virus-transfected cultures were unable to inhibit the proliferation of CD4+CD25 T cells after activation with CD3/CD28 mAb. In contrast, Foxp3-infected cells strongly suppressed CD4+CD25 T cells indicating that Foxp3 transduction was able to induce a suppressor phenotype indistinguishable from naturally occurring CD4+CD25+ regulatory T cells (Figure 3a).

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Figure 2 Retroviral transduction of Foxp3 into naı¨ve CD4+CD25 T cells results in a regulatory phenotype. (a) Foxp3 virus- or control virusinfected CD4+CD25 T cells were stained for CTLA-4, Nrp-1 and CD4, analyzed by flow cytometry and gated for GFP expression. CTLA-4 staining was performed after cell permeabilzation. Foxp3 virus-infected cells show surface marker expression normally associated with regulatory T cells. (b) Foxp3 virus-infected T cells are anergic in vitro. Proliferation assays were performed by stimulating 2 105 Foxp3 virus- or control virus-infected cells with anti-CD3/CD28 for 5 days. [3H]thymidine incorporation was measured as an indicator of cell proliferation. Data are mean7s.d. from three different experiments. * indicates statistical significance (Student’s t-test; Po0.05). (c) Foxp3 virus-infected T cells secrete IL-10. Naı¨ve CD4+CD25 as well as Foxp3 virus- and control virus-infected T cells (2 105) were stimulated with anti-CD3/CD28, and IL-10 levels were analyzed using the CBA inflammation kit. One representative out of three independent experiments is shown. Gene Therapy

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Figure 3 (a) Foxp3 virus-infected CD4 CD25 T cells are suppressive in vitro. CD4+CD25 T cells were infected with Foxp3 or control virus, and 7 days after infection, proliferation assays were performed by stimulating freshly isolated naı¨ve CD4+CD25 T cells (2 105) with anti-CD3 and anti-CD28 in the absence or presence of Foxp3 virus- and control virusinfected cells. Data are mean7s.d. from three independent experiments. * indicates statistical significance (Student’s t-test; Po0.05). (b) In vitro suppression of Foxp3 virus-infected cells is not IL-10-mediated but contact-dependent. Proliferation assays were performed in transwell plates by stimulating naı¨ve CD4+CD25 T cells (2 105) with anti-CD3/CD28 in the absence or presence of Foxp3- and control virus-infected CD4+CD25 T cells. To block IL-10, 50 mg of anti-IL-10 was added to the indicated assays. Data are mean7s.d. from three independent experiments. * indicates statistical significance (Student’s t-test; Po0.05).


Next we were interested to investigate whether suppressor activity of Foxp3-infected cells was dependent on cell–cell contact and/or IL-10 production. Therefore, eGFP+ cells from control virus- or Foxp3/ eGFP-infected cultures were either directly added to or separated from CD4+CD25 T cells by a semipermeable membrane. The findings shown in Figure 3b clearly demonstrate that suppressor activity of Foxp3-infected cells was lost in the absence of cell–cell contact whereas the inhibitory function was observed when cell–cell contacts were allowed. In addition, neutralization of IL10 by adding anti-IL-10 Ab to the cultures failed to block suppression of Foxp3-infected cells (Figure 3b). From these data, we concluded that in vitro suppressor activity of Foxp3-infected T cells requires cell contact and may not be mediated by IL-10.

Foxp3-infected T cells can inhibit allergic contact hypersensitivity responses in mice CHS is an antigen-specific T-cell-mediated disorder.27 Therefore, we were interested to examine whether Foxp3/eGFP-infected cells could modulate CHS responses in vivo. Groups of naı¨ve C57BL/6 mice were epicutaneously sensitized to dinitrofluorobenzene (DNFB). Negative controls were not sensitized. Experimental groups were intravenously (i.v.) injected with 2 106 CD4+CD25 T cells, eGFP+ control virus-transfected or eGFP+ Foxp3-infected cells. Subsequently, all groups of mice were ear challenged with DNFB and ear swelling was assessed as a measure of CHS responses. Mice that had been treated with CD4+CD25 T cells or control virus-infected cells showed a normal CHS response upon challenge (Figure 4). Interestingly, mice that were injected with Foxp3-infected cells developed a markedly reduced CHS response indicating that the injected Foxp3-infected cells were able to inhibit the

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36 h after challenge Figure 4 Reduced CHS responses in wild-type mice treated with Foxp3infected CD4+CD25 T cells. Animals were sensitized with DNFB, i.v. injected with 2 106 untreated, Foxp3 virus-infected or control virusinfected CD4+CD25 T cells and ear challenged. Ear swelling was evaluated 36 h after challenge and is expressed as difference between the challenged right ear and the unchallenged left ear. Data are mean7s.d. and are representative of 12 mice in three independent experiments. * indicates statistical significance (Student’s t-test; Po0.05).

elicitation phase of allergic CHS. Furthermore, these findings suggest that in vitro-generated regulatory T cells can be used to effectively control contact allergy.

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Foxp3-transfected T cells suppress the progression of ongoing systemic autoimmunity in CD40L transgenic mice Taking the data from Figure 4 into account, Foxp3infected cells appear to be intriguing candidates for the in vivo suppression of inflammation and systemic autoimmunity. CD40L transgenic (tg) mice overexpress CD40L in the basal keratinocytes of the epidermis, which leads to the spontaneous development of severe systemic autoimmunity as evidenced by the presence of autoantibodies and autoreactive T cells.28 To investigate the effects of Foxp3-infected cells on the progression of autoimmune dermatitis in CD40L tg mice, groups of tg mice were treated i.v. with 5 106 control virus-infected, Foxp3-infected cells or CD4+CD25 naı¨ve T cells once per week for 6 weeks. CD40L tg mice that were treated with GFP+ cells from control virus-infected cultures continued to develop severe autoimmune dermatitis similar to tg mice that were injected with CD4+CD25 T cells (Figure 5a and data not shown). Strikingly, CD40L tg animals that were injected with Foxp3-transfected cells showed a significant amelioration of dermatitis (Figure 5a). Also, histologic evaluation of ear skin from these differentially treated groups of tg mice showed significantly lower numbers of inflammatory infiltrates in mice treated with Foxp3-infected cells compared to controls (data not shown). Moreover, lymph node weight was significantly lower in mice injected with Foxp3-infected T cells for 6 weeks, suggesting that Foxp3-infected T cells reduced lymphadenopathy (Figure 5b). CD40L tg mice have detectable levels of autoantibodies within the serum.28 To examine the presence of serum autoantibodies in CD40L tg mice after cellular immunotherapy, serum was collected after 6 weeks of treatment and analyzed using HEp-2 cells. CD40L tg mice that received control virus-infected cells had detectable concentrations of autoantibodies within the serum that stained HEp-2 cells in a perinucleolar and nucleolar fashion similar to tg mice that were injected with CD4+CD25 T cells (Figure 5c and data not shown). Interestingly, CD40L tg mice that were injected with Foxp3-infected cells showed no presence of systemic autoantibodies (Figure 5c). Autoantibodies can participate in the involvement of internal organs such as nephritis during systemic autoimmune diseases. Nephritis usually leads to loss of renal function resulting in proteinuria. We have, therefore, evaluated the kidneys to analyze the effects of Foxp3-infected T-cell injections on internal organ function in autoimmune-prone CD40L tg mice. CD40L tg mice that were treated with control virus-infected cells for 6 weeks showed immunoglobulin depositions at the glomeruli of kidney tissue (Figure 5d). These findings were paralleled by enhanced protein contents in the urine of control virus-treated tg mice (Figure 5e). Interestingly, CD40L tg mice that received injections of GFP+ cells from Foxp3-infected cultures displayed markedly less renal immunoglobulin depositions after 6 weeks of treatment (Figure 5d). Importantly, the renal function was not compromised in CD40L tg mice as Gene Therapy


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Figure 5 (a) Treatment with Foxp3-infected CD4 CD25 T cells suppresses autoimmune dermatitis in CD40L tg mice. CD40L tg mice (n ¼ 6) were injected i.v. with 5 106 Foxp3 virus-infected or control virus-infected T cells six times once per week and disease development was evaluated weekly. Data are mean7s.d. * indicates statistical significance (Student’s t-test; Po0.05). Representative skin lesions after a 6 weeks treatment with Foxp3 virusinfected or control virus-infected cells are shown. (b) Weight of pooled cervical/submandibular lymph nodes from CD40L tg mice injected with either Foxp3transfected or control virus-transfected T cells for 6 weeks. *Po0.05. (c) Treatment of CD40L tg mice with Foxp3 virus-infected CD4+CD25 T cells for 6 weeks suppressed the development of antinuclear antibodies. Also shown is indirect fluorescence staining of HEp2 cells incubated with serum from CD40L tg mice treated with either Foxp3 virus-infected or control virus-infected T cells (magnification 200, serum dilution 1:80). (d, e) Reduced proteinuria and rescued renal function by 6 weeks treatment of CD40L tg mice with Foxp3 virus-infected cells. (d) Immunofluorescence staining of renal IgG/IgM deposits (magnification 400). (i+iii) IgG and IgM depositions before treatment with Foxp3-infected T cells; (i+iv) IgG and IgM depositions after treatment. (e) After a 6 weeks treatment period with Foxp3 virus-infected or control virus-infected (black column) T cells, urine was collected from CD40L tg mice. Protein concentration in urine was quantified using Bradford reagent. * indicates statistical significance (Student’s t-test; Po0.05).

evidenced by the reduced proteinuria (Figure 5e). These findings strongly suggest that Foxp3/eGFP-infected T cells can be successfully used to ameliorate ongoing CD40L-induced autoimmune dermatitis, autoantibody Gene Therapy

secretion and nephritis in CD40L tg mice. Moreover, these data support the concept of treating ongoing autoimmune diseases with newly in vitro-generated regulatory cells.


Reduced development of activated/cytotoxic CD8+ T cells in autoimmune-prone CD40L transgenic mice after injection of Foxp3-infected T cells Autoimmune dermatitis can be induced in naı¨ve recipient mice upon transfer of activated/cytotoxic CD8+ T cells from CD40L tg mice, indicating that autoreactive T cells are present in these animals.28 To investigate if injection of Foxp3-infected T cells would alter CD8+ T-cell activation or proliferation in CD40L tg mice, CD8+ T-cell numbers and surface marker expression were analyzed. CD8+ T cells from lymph nodes of CD40L tg animals that had been injected with control virus-infected T cells showed high levels of the activation marker CD44 similar to those observed in untreated CD40L tg mice (Figure 6a). In contrast, CD40L tg mice that were treated with Foxp3-infected T cells had strongly reduced overall numbers of CD8+ T cells compared to mice treated with control virus-infected cells or agematched untreated CD40L tg animals (data not shown). Interestingly, these CD8+ T cells were markedly less activated, as demonstrated by the reduced expression of CD44 (Figure 6a). These findings indicate that injection of Foxp3-infected T cells into autoimmune-prone CD40L tg mice was able to control activation and expansion of cytotoxic CD8+ T cells. Furthermore, these data suggest that the development and/or function of autoreactive T cells can be effectively inhibited in vivo by using in vitrogenerated regulatory T cells. Overexpression of CD40L induced also B-cell proliferation and autoantibody production in CD40L tg mice.28 Interestingly, analyses of B-cell numbers from submandibular and cervical lymph nodes of CD40L tg mice that had been injected with Foxp3 virus-, control virus-

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infected T cells or CD4+CD25 T cells revealed that a 6 weeks treatment with Foxp3-infected T cells resulted in reduced B-cell numbers compared to controls as evidenced by CD19 marker staining (Figure 6b).

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Homing pattern of Foxp3-infeced T cells after injection It is currently unknown whether regulatory T cells exert their suppressive function primarily within secondary lymphatic organs or within inflammatory lesions. Migration of regulatory T cells into lymph nodes has been associated with the expression of CD62L by these cells.29 Recent data from our group have suggested that (UVinduced) regulatory T cells can act also locally when injected into the ear skin.30 Since CD40L tg mice with overt autoimmune disease show lymphadenopathy and cutaneous lesions, we were interested in determining the homing pattern of Foxp3 virus- or control virus-infected T cells after injection. Immunofluorescence analyses of lymph nodes and skin lesions of CD40L tg mice that were injected with control virus-infected eGFP+ T cells (CD4 in red) show that yellow cells (overlay green and red) can be detected both in lymph nodes and skin. Similarly, Foxp3-infected T cells were also detectable in dermatitis lesions and lymph nodes of CD40L tg mice at the beginning of treatment (d1; Figure 7a). The number of eGFP+CD4+ cells in the lymph nodes as well as in inflammatory lesions of CD40L tg mice treated with control virus-infected cells remained similar during the 6 weeks of treatment. In contrast, the numbers of eGFP+CD4+ cells decreased continuously in the lymph nodes of CD40L tg mice treated with Foxp3-infected T cells (Figure 7a and b). Moreover, after the healing of dermatitis, injected Foxp3-infected T cells were below detectable levels in the skin of treated mice (d28 and d42; Figure 7a). After termination of injections, low numbers of Foxp3-infected cells were only found in lymph nodes and not in the skin of CD40L tg mice anymore (Figure 7a). At that time, high levels of control virus-infected T cells were still detectable in lymph nodes and the lesional skin of CD40L tg mice since cutaneous inflammation was still present. These results suggest that (a) control virusand Foxp3 virus-infected T cells have comparable homing patterns and (b) that Foxp3-infected T cells did not migrate into the skin after dermatitis had resolved.

Discussion

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Figure 6 (a) Foxp3-transfected T cells inhibit cytotoxic CD8+ T-cell activation in vivo in CD40L tg mice. Flow cytometric analysis of cervical and submandibular lymph nodes of CD40L tg mice after a 2 or 6 weeks treatment period with Foxp3 virus- or control virus-infected CD4+CD25 T cells. Cells were gated for CD8 and percentage of CD44high-expressing cells was evaluated. Data are mean7s.d. and are representative of three different mice at each time point. * indicates statistical significance (Student’s t-test; Po0.05). (b) Reduced numbers of CD19+ B cells in CD40L tg mice treated with Foxp3-infected T cells for 6 weeks compared to control virus-treated T cells. Data are mean7s.d. and are representative of three different mice at each time point. * indicates statistical significance (Student’s t-test; Po0.05).

Regulatory CD4+CD25+ T cells play an increasingly important role in our understanding of immunotolerance and its breakdown. The function of regulatory T cells in mice and humans is quite similar. Hence, complete loss of these cells or impaired function leads to severe autoimmunity, which can be fatal. Therefore, intervention with regulatory T-cell numbers or even function appears to be an intriguing alternative for the control of autoimmune as well as inflammatory disorders. Given the fact that CD4+CD25+ T cells constitute only a small subset of Th cells, transfection of Foxp3 into naı¨ve T cells provides a useful method to generate regulatory T cells in vitro. Foxp3-infected T cells are phenotypically almost indistinguishable from naturally occurring regulatory T cells. These in vitro-generated regulatory T cells can subsequently be utilized to inhibit effectively the progression of an ongoing severe systemic autoimmune Gene Therapy


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0 2 4 6 8 10 12 eGFP+CD4+ cells in 0.1 mm2 LN tissue Figure 7 (a) Detection of eGFP/Foxp3- or eGFP/control virus-infected T cells in skin lesions and lymph nodes of CD40L tg mice at different time points (d1, d14, d28 and d42) after start of treatment. Immunofluorescence staining of cervical lymph nodes and the lesional ear skin of CD40L tg mice injected with GFP/control virus-infected or GFP/Foxp3 virus-infected CD4+ T cells (green: injected virus-infected eGFP+ T cells; red: anti-CD4 staining; overlay: yellow for eGFP+/CD4+ cells; magnification 400); scale bar ¼ 25 mm. (b) Numbers of eGFP/Foxp3- or eGFP/control virus-infected T cells in cervical lymph nodes. Cells were counted in 0.1 mm2 areas and data are representative of 15 different cryosections at each time point. * indicates statistical significance (Student’s t-test; Po0.05).

disease or to suppress allergic CHS in mice. Since in vitro and in vivo analyses revealed that Foxp3- as well as control virus-infected cells die 2–3 weeks after infection, most likely due to activation-induced cell death (AICD), weekly injections of virus-infected cells seemed to be appropriate to suppress ongoing autoimmunity in CD40L tg mice (data not shown). Accordingly, CD40L tg mice continued to develop autoimmune dermatitis 3–4 weeks after discontinuation of treatment. In CD40L tg mice, chronic activation of epidermal dendritic cells (DC), the Langerhans cells, results in the breakdown of immunotolerance and the development of a fatal severe systemic autoimmune disorder.28 This mixed connective tissue disease-like disorder is characterized by the presence of autoreactive CD8+ T cells and antinuclear antibodies produced by activated B cells. The repetitive treatment of mice with overt disease with Foxp3-transfected T cells resulted in a significant reduction of activated T cells and reduced B-cell numbers. Strikingly, no antinuclear antibodies were detectable in CD40L tg mice injected with Foxp3-infected T cells, which was paralleled by strongly reduced immunoglobulin depositions at the glomeruli of the kidneys. As a Gene Therapy

consequence, a marked improvement of renal function could be noted, as evidenced by the reduced proteinuria. Previous studies have indicated that regulatory T cells are not only able to prevent the proliferation of CD4+CD25 T cells but also stimulation of CD8+ T cells.31 We hypothesize that the reduced production of antinuclear antibodies is a consequence of impaired Th function since T cells can provide help to B cells. In vitro, Foxp3-infected T cells were not able to inhibit autoantibody secretion when added to B cells isolated from enlarged lymph nodes of CD40L tg mice (data not shown). Naturally occurring CD4+CD25+ regulatory T cells can migrate to lymph nodes due to the expression of CD62L (L-selectin). From this finding, it was concluded that regulatory T cells exert their suppressor function primarily at sites where T-cell priming is induced. Indeed, studies investigating the homing pattern of regulatory T cells induced by ultraviolet irradiation showed that, upon injection, these cells migrate to lymph nodes, which leads to suppression of the induction but not elicitation of CHS responses since T-cell priming occurs in lymph nodes whereas elicitation occurs in the


periphery, for example, skin.30 When ultraviolet irradiation-induced regulatory T cells were injected into the skin, they were also able to prevent elicitation of CHS responses. In our system, Foxp3-infected T cells were detected upon injection in sites of inflammation and in lymph nodes. Hence, Foxp3-infected T cells abrogated elicitation of CHS responses. Foxp3-infected T cells expressed intermediate to low levels of CD62L (data not shown). From these data, we surmise that Foxp3infected T cells exert their suppressor activity in autoimmune-prone CD40L tg mice locally in the skin, which results in resolution of the dermatitis, as well as in anatomic areas where T-cell activation frequently occurs, that is, the lymph nodes. The latter effect might contribute to the reduced lymph node weight, reduced activation of CD8+ T cells and lower numbers of B cells in CD40L tg mice treated with Foxp3-infected T cells, which was accompanied by significantly suppressed autoantibody secretion. It is currently believed from studies utilizing antigenspecific tg models that regulatory T cells have to be activated in an antigen-specific manner in order to induce suppressor activity. However, once activated, regulatory T cells can regulate in an antigen nonspecific way.32 Upon transfection with Foxp3-containing retrovirus, CD4+ T cells become activated. This activation is further enhanced by the culture conditions that include IL-2 and anti-CD3. Therefore, Foxp3-infected T cells present an activated phenotype, and without additional stimulation, can be directly used for injection to suppress T-cell-mediated immune responses against a variety of different (auto-) antigens. Transcription factors play an important role in the development and lineage commitment of T cells. In Th cells, t-bet has been identified to control Th1 cells and GATA-3 Th2 cells.33 More recently, analyses of the genetic defect in patients and mice suffering from a similar Xlinked recessive autoimmune and inflammatory disorder revealed mutations in a common gene Foxp3.34 The gene Foxp3 encodes a member of the forkhead/winged-helix family of transcription regulators termed scurfin (also known as forkhead box P3).35 In patients suffering from immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX; MIM 304930) mutations in the Foxp3 gene have been identified.36 These mutations affected the forkhead/winged helix domain of the scurfin protein, which is needed for DNA interactions. The genetic equivalent to the IPEX syndrome in humans is the scurfy (sf) mouse. Scurfy is an X-linked recessive mouse mutant resulting in a rapidly fatal lymphoproliferative disorder, which is characterized by overproliferation of CD4+ T cells and extensive multiorgan infiltration.35 A similar phenotype has been reported in mice with a gene-targeted disruption of Foxp3.20 These studies prompted investigations into the role of Foxp3 during regulatory T-cell development. Indeed, CD4+CD25+ T cells could not be detected in Foxp3/ or sf mice.20,22 Moreover, CD4+CD25+ regulatory T cells were found to express high levels of Foxp3, as evidenced by quantitative RT-PCR.21 On the other hand, overexpression of Foxp3 in T cells induced a suppressor phenotype or expansion of CD4+CD25+ T cells.21,22 These findings are in agreement with our data since retroviral transfection of Foxp3 into naı¨ve CD4+CD25 T cells induced functional regulatory T cells. Introduction of an

intact Foxp3 gene appears to be useful in generating regulatory T cells, especially in situations where Foxp3 is mutated and/or no regulatory T cells are present. Parallel to intracellular factors, surface receptors have been identified to participate in regulatory T-cell development. It was shown that Notch3-I-intracellular domain (IC) tg mice, which overexpress the constitutively active Notch3 intracellular domain in thymocytes and T cells, show upregulated expression of CD25 on thymocytes and T cells.37,38 Accordingly, increased numbers of CD4+CD25+ T cells were found in the periphery of these tg mice.37 These CD4+CD25+ T cells displayed suppressor function because streptozotocin-induced autoimmune diabetes failed to develop in Notch3-IC tg animals. The failure to induce diabetes in these tg mice was associated with accumulation of CD4+CD25+ T cells in lymphoid organs as well as pancreas infiltrates and was paralleled by increased production of IL-4 and IL-10. The Notch pathway includes a family of transmembrane receptors and represents a major regulatory network underlying intrathymic T-cell differentiation. Notch3 seems to control the pre-TCR-dependent CD4CD8 double-negative CD25+ to double-positive CD25 transition. It was hypothesized that dysregulated CD25 expression may underlie Notch3-specific events that lead to regulatory Tcell generation.37 In terms of regulatory T-cell accumulation, we have made very similar observations since injected Foxp3-transfected T cells were detectable in the lesional skin and lymph nodes of CD40L tg mice. The results obtained from Notch3-IC tg mice suggest that enhanced Notch3 signaling might be useful to generate or increase the number of regulatory T cells. However, Notch3-IC tg mice have been initially described to develop aggressive T-cell lymphomas.38 Since development of lymphomas after injection of Foxp3-infected T cells has not been observed so far, we favor transfection of Foxp3 to generate regulatory T cells. Besides retroviral transfection of Foxp3 into T cells, several other methods have been previously described to successfully generate regulatory T cells from naı¨ve T cells in vitro. It was shown that engagement of naı¨ve T cells with antigen-presenting cells such as DC induced CD4+CD25+ T cells with regulatory function. Repetitive exposure of CD4+CD25 T cells with immature DC resulted in the development of regulatory T cells, which were able to subsequently suppress the proliferation of CD4+CD25 T cells.39 These T cells were also able to produce IL-10 in significant concentrations. In another study, IL-10 exposure of DC was utilized to induce immature DC, which were than used in coculture with T cells to generate T cells with suppressor function.40 More recently, fully mature DC were shown to be able to expand CD4+CD25+ T cells in vitro as well as in vivo.41 However, these methods all require the culture of DC and usually yield only low numbers of regulatory T cells. Other investigations have centered on the binding of antigen via antibodies directed against the CD205 (DEC 205) surface molecule of DC to induce regulatory T cells.42 These findings demonstrate that differentially treated DC or different DC subsets can also be used to modulate regulatory T-cell numbers and function. In summary, regulatory T cells can be generated in vitro by retroviral transfection of Foxp3. These regulatory T cells can be successfully used to control ongoing severe systemic autoimmunity in mice. These

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findings strengthen the rationale for using regulatory T cells perhaps also in humans suffering from autoimmune diseases with polyclonal T-cell activation, such as lupus erythematosus.

Materials and methods Mice Female C57BL/6 (H-2b) mice at the age of 8 weeks were obtained from the Harlan breeding laboratories (Borchen, Germany). Female CD40L tg mice (DBA/2xC57BL/6 F1) were used at the age of 4 months and the experiments were carried out using heterozygous animals.28 All mice were housed under specific pathogen-free conditions and used according to institutional regulations. Retroviral infection of CD4+CD25 T cells The cDNA encoding murine Foxp3 was amplified by RTPCR from mouse spleen using specific primers (50 -GGAC AAGGACCCGATGCCCAACC-30 and 50 -CCCTGCCCC CACCACCTCTGG-30 ), cloned into pCR2.1 TOPO (Invitrogen, Karlsruhe, Germany), sequenced and inserted into a pMCSV-based retroviral vector encoding eGFP under the control of an internal ribosomal entry site21 (IRES). This construct was used to stably transfect the ecotropic GPE-86+ packaging cell line (kindly provided by Dr W Gerhard, Wistar Institute, Philadelphia, PA, USA). Retrovirus-containing culture supernatants were utilized to infect CD4+CD25 T cells isolated from spleens and lymph nodes of C57/BL6 mice or DBA/ 2xC57BL/6 F1 mice. Control infections were carried out using the empty eGFP vector. At 2 days after preparation and activation of CD4+CD25 T cells using 1 mg/ml plate-bound anti-CD3 (clone 145-2C11, Becton Dickinson, Heidelberg, Germany) and IL-2 (15 U/ml; R&D Systems, Wiesbaden, Germany), cells were infected by resuspension in filtrated retrovirus-containing supernatant supplemented with 20 mM HEPES (PAA, Linz, Austria) and 8 mg/ml polybrene (Sigma-Aldrich, Taufkirchen, Germany) followed by centrifugation at 3000 g for 90 min. Thereafter, cells were transferred to six-well plates and incubated at 371C and 5% CO2. After 7 days expansion with 15 U/ml IL-2, eGFP-expressing cells were used for further experiments. eGFP-expressing cells (eGFP+) were separated from nontransfected cells by cell sorting using a MoFlo cell sorter (DAKOCytomation, Hamburg, Germany). Cell preparation and flow cytometry Single cell suspensions of spleens and lymph nodes were prepared as described and purified with an AutoMACS magnetic cell sorter (Miltenyi, Bergisch Gladbach, Germany).43 Expression of cell surface and intracellular markers was analyzed by multicolor flow cytometry on a FACScalibur cytometer (Becton Dickinson) with the Cell Quest software (Becton Dickinson). Cells were stained in PBS containing 1% FCS (PAA) using the following antibodies from Becton Dickinson: fluorescein isothiocyanate (FITC)-conjugated anti-CD45RB (clone 16A), anti-CD62L (clone Mel-14), anti-CD103 (clone 2E7), anti-CD44 (clone IM7), polyclonal goat anti-rabbit immunoglobulin (Ig); phycoerythrin (PE)-conjugated antiCTLA-4 (clone UC10-4F10-11), anti-CD69 (clone H1.2F3); peridinin chlorophyll protein (PerCP)-conjugated antiGene Therapy

CD4 (clone L3T4 RM4-5), anti-CD3 (clone 145-2C11), anti-CD19 (clone 1D3), allophycocyanin (APC)-conjugated anti-CD25 (clone PC-61), anti-CD8a (clone 53-6.7). Mouse monoclonal anti-Nrp-1 (clone H-286) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Isotype-matched control antibodies were included in each staining.

Cytometric bead array The cytokine concentration in culture supernatants of CD4+CD25 T cells as well as control virus- or Foxp3 virus-infected CD4+CD25 T cells was measured by cytometric bead array (CBA; Becton Dickinson) according to the manufacturer’s instructions. T cells (2 106/ ml) were incubated for 5 days and stimulated with platebound anti-CD3 (clone 145-2C11) and anti-CD28 (clone 37.51, both 1 mg/ml; antibodies were obtained from Becton Dickinson) at 371C and 5% CO2 in 96-well round-bottom plates (BD-Falcon, Heidelberg, Germany) in a volume of 200 ml RPMI supplemented with 10% FCS. Supernatants were collected and subjected to cytokine quantification using CBA kits. Proliferation and suppression assays Freshly isolated CD4+CD25 T cells and CD4+CD25 T cells infected with control virus or Foxp3 virus were cultured in triplicate in 96-well round-bottom plates (2 106/ml alone or mixed at indicated ratios) and stimulated with plate-bound anti-CD3 (clone 145-2C11, 1 mg/ml) and anti-CD28 (clone 37.51, 1 mg/ml) for 5 days. Proliferation and suppression assays were performed in a final volume of 200 ml and 1 mCi/well [3H]thymidine was added for the last 12 h of the experiment. Thymidine incorporation was measured by liquid scintillation counting. In some suppression assays, a Transwell system with 1 mm pore size (BD-Falcon) was used or the assays were supplemented with anti-IL-10 (50 mg/well, kindly provided by Werner Mu¨ller, National Research Center for Biotechnology, Braunschweig, Germany) to analyze the contact as well as IL-10 dependency of suppression. Contact hypersensitivity C57BL/6 mice were sensitized by painting 100 ml of 0.5% DNFB in acetone/olive oil (4/1) on the shaved back on day 0. On day 4, 2 106 CD4+CD25 T cells untreated or infected with control virus and Foxp3 virus were injected i.v. into each recipient mouse. For elicitation of CHS responses, 12 ml of 0.3% DNFB was painted on both sides of the right ear on day 5. CHS was determined by the degree of ear swelling of the challenged right ear compared to the ear thickness on the nonchallenged left ear and measured with a micrometer (Mitutoyo, Tokyo, Japan) 36 h after challenge. Mice that were ear challenged without prior sensitization served as negative controls. Immunofluorescence Immunofluorescence stainings were performed on cryostat sections of ears, lymph nodes and kidneys according to standard methods.44 Renal IgG/IgM depositions were detected on cryostat sections of kidneys stained with FITC-coupled anti-mouse IgG or FITC-coupled antimouse IgM (Becton Dickinson) diluted 1:50 in PBS. Localization of control virus- or Foxp3 virus-infected T


cells was performed by immunofluorescence staining using anti-CD4 (clone L3T4 RM4-5, diluted 1:50 in PBS) and Texas Red-coupled secondary antibody (Molecular Probes, Leiden, The Netherlands). Slides were examined using a Leica TSP-1 confocal microscope (Wetzlar, Germany). eGFP+CD4+ (yellow) cells were counted by two independent investigators in 0.1 mm2 areas of 15 different cryosections for each time point.

Systemic autoimmunity Development of autoimmunity was determined as described.28 Groups of CD40L tg mice were i.v. injected weekly for a period of 6 weeks with 5 106 untreated CD4+CD25 T cells and control virus-infected or Foxp3 virus-infected CD4+CD25 T cells. Mice were evaluated for onset of dermatitis three times per week by two independent investigators. Localization and size of inflammatory skin lesions were documented according to the dermatitis scoring system (one ear red and swollen ¼ 1 point; both ears red and swollen ¼ 2 points; one ear inflamed including slough ¼ 3 points; both ears inflamed including slough ¼ 4 points; both ears and tail inflamed ¼ 5 points; both ears, tail and snout inflamed ¼ 6 points, both ears, tail and snout inflamed, mouse cachexic ¼ 7 points). At 6 weeks after the start of treatment, mice were killed. Subsequently, antinuclear antibodies, renal IgG/IgM depositions, skin lesions and proteinuria were analyzed as previously described.28 Detection of antinuclear antibodies Serum samples of CD40L tg mice injected with untreated CD4+CD25 T cells as well as control virus-infected or Foxp3 virus-infected CD4+CD25 T cells were screened for the presence of antinuclear antibodies by indirect immunofluorescence staining on HEp-2 cells (LD Labor Diagnostika, Germany). Sera were diluted 1:80 in PBS, applied to the slides and incubated for 2 h. Slides were then incubated with FITC-coupled mouse immunoglobulins (Dianova, Hamburg, Germany), washed, mounted and analyzed using an Olympus BX61 microscope and MetaMorphs software (Visitron Systems, Puchheim, Germany). Real-time quantitative RT-PCR Total RNA was isolated from MACS-sorted CD4+CD25 T cells and CD4+CD25 T cells infected with control virus or Foxp3 virus using RNeasy columns (Qiagen, Hilden, Germany). cDNA synthesis was performed with oligodT primers and AMV reverse transcriptase (Promega, Mannheim, Germany) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed in an ABI PRISM 7000 cycler (Applied Biosystems, Darmstadt, Germany) using the murine TGF-b and Foxp3 TaqMan gene expression assays (Applied Biosystems). All reported mRNA levels are normalized to the bactin mRNA level (b-actin primers: 50 -GTGGGGCGC CCCAGGCACCA-30 and 50 -CTCCTTAATGTCACGCAC GATTTC-30 ).

Acknowledgements We thank Maik Voskort and Joachim Windau for excellent technical assistance. This work was funded by

the German Research Association (DFG), Grant SFB293 B8 (to SB).

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