Prolonged skin allograft survival by IL-10 gene-introduced CD4 T cell administration
Takeshi Miyamoto1,2,
Takaaki Kaneko1,
Masakatsu Yamashita1,
Yoshiyuki Tenda1,
Masamichi Inami1,
Akane Suzuki1,
Sohtaro Ishii1,
Motoko Kimura1,
Kahoko Hashimoto3,
Hideaki Shimada2,
Hiroshi Yahata4,
Takenori Ochiai2,
Izumu Saito5,
James DeGregori6 and
Toshinori Nakayama1
1 Department of Immunology and 2 Department of Academic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan
3 Department of Life and Environmental Sciences and High Technology Research Center, Chiba Institute of Technology, Narashino, Tsudanuma, Chiba 275-0016, Japan
4 Second Department of Surgery, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
5 Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
6 Department of Biochemistry and Molecular Genetics, University of Colorado Denver Health Sciences Center, Aurora, CO 80045, USA
Correspondence to: T. Nakayama; E-mail: tnakayama{at}faculty.chiba-u.jp
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Abstract
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Both CD4 and CD8 T cells play crucial roles in immune responses in transplantation. Immunosuppressive drugs, such as FK506 and cyclosporin A, block the priming of alloreactive CD4 Th cells and the subsequent induction of allospecific CD8 cytotoxic effector T cells and inhibit allograft rejection. However, the desire to minimize chronic complications that may arise from the use of immunosuppressive agents drives the search for additional strategies for immunosuppression of allograft rejection. In this study, CD4 or CD8 T cells into which the IL-10 gene is introduced using an adenovirus vector containing human IL-10 (hIL-10) cDNA (Ad-hIL-10) and into mouse T cells transgenic for the Coxsackie virus and adenovirus receptor form a model system to study the effect of administration of IL-10-secreting T cells on the survival of the allogenic skin grafts. Ad-hIL-10-infected CD4 and CD8 T cells secreted a large amount of hIL-10 for 34 days in culture in vitro. Ad-hIL-10-infected CD4 T cells administered in vivo could be detected in the spleen for 7 days post-transfer. Significantly prolonged survival of grafts was observed in animals that received either Ad-hIL-10-infected activated CD4 T cells or Th2-skewed CD4 T cells as compared with controls. Furthermore, substantial enhancement of the effect was observed in B6.C-H2bm1/ByJ transplants. Thus, a direct manipulation of T cells through the introduction of the immunosuppressive cytokine gene IL-10 may be a novel strategy for the control of allograft rejection.
Keywords: Bm1, CAR Tg, gene therapy, skin graft
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Introduction
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CD4 and CD8 T cells play critical roles during allograft rejection in transplantation (1, 2). In various experimental allotransplantation systems, rejection appears to be associated with increased production of Th1 cytokines (IL-2 and IFN
), and the suppressive roles of Th2 cytokines such as IL-4 and IL-10 on rejection have been reported (3, 4). Immunosuppressive drugs, such as FK506 and cyclosporin A (CsA), inhibit the priming of alloreactive CD4 Th cells and the subsequent induction of allospecific CD8 cytotoxic effector T cells very efficiently. Both of these cellular processes are critical in allograft rejection (5, 6). However, the treatment with FK506 and CsA may be accompanied by several side effects, particularly in patients treated for long periods (7, 8). Thus, the establishment of additional strategies for immunosuppression of allograft rejection to minimize complications is desirable.
Several investigators have reported that a strategy for tolerance induction in allograft rejection involves the introduction of immunosuppressive cytokine genes to the host (913). Several viral vector systems have been used for these kinds of gene therapies (14) and among one of the most popular gene-transfer systems is a retrovirus system (15). Retroviruses are integrated in the genome, and the effect of the transgene can be observed for a long time. However, infection by retroviral vectors requires dividing cells, which limits the application for in vivo gene therapy (16, 17). Another system is an adenovirus-mediated gene-transfer system (18). There are several advantages for the adenovirus vector system for the induction of immunosuppression in allograft rejection. (1) Expression is transient and so prospects for side effects due to long-term treatment would be minimal. (2) Preparation of high-titer virus stocks can be easily achieved. (3) Various new adenovirus-related vectors are being investigated, and these may help solve the issue of possible side effects. However, several disadvantages have been reported (19, 20). First, the generation of antibodies against adenoviral antigens may limit the repeated administration of adenovirus vectors or adenovirus-infected cells and may in fact be harmful to the host. Also, CD8 cytotoxic T cells specific for adenovirus may be generated in the host, and infected cells would be eliminated very quickly. Finally, lymphocytes including T cells express a limited amount of Coxsackie virus and adenovirus receptor (CAR) on their cell surface, which may make gene transfer into lymphocytes by adenovirus vectors very difficult from a practical point of view.
IL-10 is produced by T cells, B cells and macrophages and plays potent immunosuppressive roles by inhibiting the production of pro-inflammatory cytokines, the expression of MHC class II antigens and antigen-presenting function (21). In addition, IL-10 can down-regulate IL-12 expression, resulting in a decrease in Th1 cell differentiation (22). Recently, it has been reported that IL-10 drives the generation of a unique CD4 T cell subset, T regulatory cell type-1, which suppresses antigen-specific immune responses and pathologic inflammation in vivo (23, 24).
CAR transgenic (Tg) mice were established in order to overcome the limited expression of CAR on T cells (25). In the present study, we demonstrate the efficient adenovirus vector-mediated IL-10 gene transfer into CAR Tg T cells and we analyzed the distribution of such adenovirus-infected T cells into the lung, liver and spleen following the administration of these cells into mice. Using an allo-skin graft model system, we observed significant immunosuppressive effects following administration of adenovirus vector containing human IL-10 gene (Ad-hIL-10)-infected activated CD4 T cells or Th2 cells. Thus, the introduction of suppressive cytokine genes into CD4 T cells and the subsequent administration of these cells may be a novel cell therapy approach for inducing the long-term survival of allografts.
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Methods
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Animals
C57BL/6 (H-2b) male mice were used as recipients, and BALB/c (H-2d) and B6.C-H2bm1/ByJ (Bm1) male mice were used as skin graft donors. C57BL/6 and BALB/c mice were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Japan) and Bm1 from the Jackson Laboratories. Tg mice expressing CAR under the control of an lck proximal promoter (CAR Tg mice) in the C57BL/6 background have been described previously (25). All mice used in this study were maintained under specific pathogen-free conditions and were used at 712 weeks of age (body weight, 2025 g). Animal care was in accordance with the guidelines of Chiba University.
Flow cytometry analysis
In general, one million cells were stained with antibodies as indicated according to a standard method (26). The reagents used are as follows: anti-CD4FITC (RM4-1FITC), anti-CD8FITC (53.6-72FITC), anti-CD8PE (53.6-72PE) and streptavidinPE purchased from BD-PharMingen (La Jolla, CA, USA). For detecting human Coxsackie virus and adenovirus receptor (hCAR), biotinylated anti-CAR antibody (RmcB) (25) and streptavidinPE were used. Two-color flow cytometric analysis was performed using FACSCalibur (Becton Dickinson, Mountain View, CA, USA).
Adenovirus vectors
AdCMVhIL-10 (Ad-hIL-10) and AdCMVlacZ (adenovirus vector containing lacZ gene, Ad-LacZ) were described previously (11). An empty adenovirus vector AxCAwt (Adex1Cawt) was reported previously (27). The preparation of adenovirus supernatant was performed as described (27). The titer of the viral stocks [infectious units (i.f.u.)] as determined by Adeno-XTM Rapid Titer Kit (BD Bioscience) was 2.8 x 109 i.f.u. ml1.
Cell culture and adenovirus infection into CAR+ T cells
Splenocytes were stained with anti-CD4FITC or anti-CD8FITC, and then the CD4 or CD8 T cells were purified using anti-FITC magnetic beads (Miltenyi Biotec) and an Auto MACS sorter® (Miltenyi Biotec), yielding a purity of >98% as described (28). Naive CD4 and CD8 T cells were stimulated for 6 days under several conditions as described (28, 29): (1) activated CD4 T cellsnaive CD4 T cells (2 x 106) were cultured in a 24-well plate (Corning) with immobilized anti-TCR mAb (1µg ml1) in the presence of IL-2 (25 U ml1) for 2 days. The cultured T cells were transferred to new wells and cultured for another 4 days in the presence of only IL-2 (25 U ml1). (2) Type-2 cell differentiation (Th2 or Tc2)naive CD4 T cells (2 x 106) or CD8 T cells (1.5 x 106) were stimulated with immobilized anti-TCR mAb for 2 days in the presence of IL-2 (25 U ml1), IL-4 (100 U ml1) and anti-IFN
mAb (R4-6A2, 12.5% culture supernatant). The cultured T cells were transferred to new wells and cultured for another 4 days in the presence of only the cytokines present in the initial culture. Ad-hIL-10 virus vector (2.8 x 108 i.f.u.) was added to the T cell culture on day 6. The amounts of secreted human IL-10 (hIL-10) and mouse IL-10 were determined by ELISA (OptEIATM Human IL-10 Set, OptEIATM Set mouse IL-10; BD Bioscience).
Allo-MLR (Mixed lymphocyte reaction)
Responder C57BL/6 CD4 T cells (0.1 x 106 or 0.3 x 106) purified by an Auto MACS sorter® were cultured with irradiated 1 x 106 BALB/c spleen cells (3500 rad). Graded doses of Ad-hIL-10- or Ad-LacZ-infected activated CD4 T cells harvested 12 h after infection were added at the beginning of the culture. [3H]Thymidine (0.5 µCi) was added in the last 16 h of the 5-day culture.
Skin allograft
Full-thickness abdominal skin grafts were transplanted onto the lateral thorax of the recipients and covered with sterile bactericidal gauze. The entire chest was then wrapped with an elastic bandage. The dressings were removed on day 6, and the grafts were inspected daily until the point of graft rejection, which is defined as >90% necrosis of the graft epithelium as described (30, 31).
On post-operation day (POD) 0 and POD 2, we transferred IL-10 gene-transduced T cells (5 x 107) into recipients intravenously. A sub-therapeutic dose of FK506 (0.1 mg kg1 per day, Fujisawa Pharmaceutical Co., Osaka, Japan) was administered by the intra-muscular route in some of the experiments.
Statistical analysis
Statistical analyses were performed using the MannWhitney U test and log-rank test. P < 0.05 was considered as statistically significant.
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Results
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Adenovirus infection of splenic CD4 T cells from CAR Tg mice
In CAR Tg mice, the majority (>90%) of CD4 and CD8 T cells expressed considerable levels of CAR on the cell surface (25). The function of CAR Tg T cells such as anti-TCR mAb-induced proliferation and the production of cytokines (IL-2, IFN
and IL-4) is normal (data not shown). Using an adenovirus vector containing EGFP gene (Ad-GFP), the efficiency of adenovirus infection in CAR Tg naive and activated CD4 T cells with an immobilized anti-TCR mAb for 16 h was assessed, and highly efficient infection (>90%) was observed in both naive and activated CD4 T cells from CAR Tg mice (Fig. 1A).

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Fig. 1. IL-10 production from T cells infected with Ad-hIL-10. (A) A green fluorescence protein (GFP) gene-encoded adenoviral vector (Ad-GFP) was used for evaluating the efficiency of adenovirus infection in CAR Tg CD4 T cells. Naive CD4 T cells from wild-type and CAR Tg mice and activated CD4 T cells with anti-TCR mAb for 16 h were infected with Ad-GFP. Two days later, GFP expression was assessed by flow cytometry analysis. Percentages of GFP-positive cells are depicted in each panel. (B) The amount of hIL-10 produced by Ad-hIL-10-infected CAR Tg T cells in vitro. In vitro-differentiated Th2 and Tc2 cells as well as activated CD4 T cells prepared as described in Methods were infected with 2.8 x 108 i.f.u. of Ad-hIL-10 (arrow), and the amounts of secreted hIL-10 in the culture supernatant were measured by ELISA. (C) The amounts of mouse IL-10 in the culture supernatant of the indicated T cell cultures were measured by ELISA. Each symbol represents the same cell preparations indicated in panel A.
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IL-10 production from T cells infected with an adenovirus vector encoding the hIL-10 gene
In order to establish the most efficient cell-transfer system, three different CAR Tg T cells were prepared for IL-10 gene introduction: activated CD4 T cells with immobilized anti-TCR mAb in the presence of IL-2 for 5 days and in vitro-differentiated CD4 (Th2) and CD8 (Tc2) T cells cultured under type-2-skewed conditions (with immobilized anti-TCR, IL-2 and IL-4) for 5 days. The activated CD4 T cells, Th2 cells and Tc2 cells were infected with 2.8 x 108 i.f.u. of AdCMVhIL-10 virus (Ad-hIL-10) on day 6, and the amount of secreted hIL-10 in the culture supernatants was measured. Very high levels of IL-10 were secreted from all the Ad-hIL-10-infected T cell populations (Fig. 1B). No hIL-10 was produced without Ad-hIL-10 infection. The production of mouse IL-10 from the cultured cells was also measured (Fig. 1C). As we expected, Tc2 cells produced a substantial amount of mouse IL-10, whereas Th2 cells produced less but still significant levels of IL-10. No significant effect on mouse IL-10 production was observed as the consequence of the infection of Ad-hIL-10. Mouse IL-10 production was not detected in the activated CD4 T cells cultured under non-type-2-skewed conditions.
Inhibition of allo-MLR by the addition of Ad-hIL-10-infected CAR Tg CD4 T cells
In order to confirm the immunosuppressive capacity of Ad-hIL-10-infected CD4 T cells, we used the allo-MLR system in which graded doses of Ad-hIL-10-infected CD4 T cells prepared 2 days after infection were added to the C57BL/6 responder and BALB/c stimulator allo-MLR cultures. Efficient inhibition of the MLR was observed (Fig. 2). Only a few percentage of Ad-hIL-10-infected CD4 T cells caused significant inhibition of the response. No inhibition was observed by the presence of control Ad-LacZ-infected CD4 T cells (Fig. 2) or empty vector-infected CD4 T cells (data not shown). These results suggest that Ad-hIL-10-infected CD4 T cells exert a potent immunosuppressive effect on anti-allo responses in vitro.

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Fig. 2. Inhibition of allo-MLR by Ad-hIL-10-infected CAR Tg CD4 T cells. Graded doses of Ad-hIL-10-infected or Ad-LacZ-infected CD4 T cells prepared 12 h after infection were added to the allo-MLR cultures consisting of C57BL/6 CD4 T cell responder (3 x 105) and BALB/c stimulator cells (1 x 106). Relative values of mean [3H]thymidine uptake (% counts per minute) and standard deviations of triplicate cultures are shown.
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Distribution of Ad-hIL-10-infected T cells administered intravenously
It was important to analyze the distribution of adenovirus-infected T cells administered intravenously to ensure that the virus infection would not significantly alter the distribution of the transferred cells. Activated CD4 T cells and in vitro-differentiated CAR Tg Th2 and Tc2 cells were infected with Ad-hIL-10, and 2 days later these cells (5 x 107) were administered to C57BL/6 mice. Cell administration was done twice on days 0 and 2. The percentages of CAR-positive CD4 or CD8 T cells were monitored by flow cytometry 1, 3 and 7 days after the first transfer. Figure 3 shows the percentages of CAR-positive cells among CD4 or CD8 T cells in each of the organs analyzed. Without administration of CAR Tg T cells, essentially no CAR-positive cells were detected in these organs (see top control panel). However, large numbers (
15%) of CAR-positive CD4 T cells were detected in the lung and liver on days 3 and 7 in the mice with administration of Ad-hIL-10-infected activated CD4 T cells (Fig. 3A, middle panel). Substantial numbers of CAR-positive transferred CD4 T cells were detected in the spleen, although the frequency of the cells was significantly lower. The kinetics of CAR-positive population were similar between these three different organs. Furthermore, the introduction of sub-therapeutic dose of FK506 had no obvious effect on the distribution of the administered T cells (compare Fig. 3A, middle and bottom panels). These results suggest that combinational treatment with a sub-therapeutic dose of FK506 does not affect the distribution of Ad-hIL-10-infected activated CD4 T cells in the allograft-transplanted hosts.

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Fig. 3. Distribution of Ad-hIL-10-infected T cells administrated intravenously. Activated CD4 T cells (A), in vitro-differentiated Th2 (B) and Tc2 cells (C) were infected with Ad-hIL-10, and 2 days later the cells (5 x 107) were administered into C57BL/6 mice intravenously on days 0 and 2. The percentages of CAR-positive CD4 or CD8 T cells in the indicated organs were monitored by flow cytometry 1, 3 and 7 days after the initial transfer. Where indicated, host mice were treated with a sub-therapeutic dose of FK506 (0.1 mg kg1 per day). Three mice were used in each group. Mean percentage and standard deviation are shown.
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When we used in vitro-differentiated Ad-hIL-10-infected CAR Tg Th2 cells for transfer, significant numbers (up to 5%) of CAR-positive CD4 T cells were detected in the spleen, liver and lung (Fig. 3B). The levels in the spleen were similar to those of activated CD4 T cells, but those in the lung and liver were significantly lower (compare Fig. 3A and B). The number of CAR-positive cells peaked at the day 3 time point after administration, and the administration of sub-therapeutic doses of FK506 again had no dramatic effects. We also administered non-infected activated CD4 T cells and non-infected Th2 cells and observed essentially the same distribution in these organs (data not shown). In contrast, we failed to detect CAR-positive CD8 T cells when Ad-hIL-10-infected Tc2 cells were used (Fig. 3C). Significant numbers of CAR-positive CD8 T cells were detected in the spleen when we injected Tc2 cells without Ad-hIL-10 or Ad-LacZ infection (data not shown). Thus, these data suggest that adenovirus infection affects the distribution or survival of this T cell sub-population.
Effect of administration of Ad-hIL-10-infected activated CD4 T cells and Th2 cells on BALB/c allo-skin graft survival
The major goal of this study was to determine how the administration of Ad-hIL-10-infected activated CD4 T cells, Th2 cells or Tc2 cells would affect allo-skin graft survival. We first studied a fully allergenic system. BALB/c allo-skin grafts transplanted on C57BL/6 hosts survive for 7 ± 1days in our assay system (see groups with no administration in Table 1 and Fig. 4). No significant differences in allograft survival was observed among the experimental groups of no administration (7 ± 0.8), non-infected activated CD4 T cells (7 ± 0.9), empty vector-infected activated CD4 T cells (7 ± 0.4) and Ad-LacZ-infected activated CD4 T cells (8 ± 1.1) (Table 1, Exp. 1 and Fig. 4A). In comparison, a significant prolongation was detected in the group that received the administration of Ad-hIL-10-infected activated CD4 T cells (9 ± 1.5). In addition, in the group treated with sub-therapeutic doses of FK506 and Ad-hIL-10-infected activated CD4 T cell administration, significant prolongation was detected compared with the groups with a single treatment (11 ± 2.9 versus 8 ± 1.2, P < 0.05 and 11 ± 2.9 versus 9.5 ± 1.5, P < 0.01). These results indicate that hIL-10-secreted CD4 T cell administration induced a significant immunosuppressive effect on allo-skin grafts, and in combination with sub-therapeutic doses of FK506 an enhanced effect was obtained.

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Fig. 4. BALB/c full allo-skin graft and Bm1 allo-skin graft survival after administration of adenovirus-infected activated CD4 T cells or Th2 cells into C57BL/6 hosts. Graft survival curves with Ad-hIL-10-infected activated CD4 T cells (A) with Ad-hIL-10-infected Th2 cells (B) in a BALB/c full allo-skin graft system are presented. Bm1 skin graft survival with Ad-hIL-10-infected activated CD4 T cells is shown in (C). The actual numbers are summarized and presented in Table 1. Where indicated, a sub-therapeutic dose of FK506 (0.1 mg kg1 per day) was administered by an intra-muscular route.
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When Ad-hIL-10-infected Th2 cells were used, significant prolonged graft survival was detected (Table 1, Exp. 2 and Fig. 4B; 10 ± 1.3 versus 7 ± 1.4, P < 0.05). A significant prolongation was also seen in the group with non-infected Th2 cell administration (9.5 ± 1.9 versus 7 ± 1.4, P < 0.01). The addition of the treatment with a sub-therapeutic dose of FK506 appeared to have some effect, but no significant difference was detected. These results suggest that endogenous mouse IL-10 produced by non-infected Th2 cells can have a significant effect on the survival of allo-skin grafts. No significant effect was observed by the administration of Ad-hIL-10-infected CD8 T cells (data not shown). This may be accounted for by the fact that Ad-hIL-10-infected CD8 T cells were not detected in the spleen, lung or liver (Fig. 3C).
Effect of administration of Ad-hIL-10-infected activated CD4 T cells on Bm1 or allo-skin graft survival
We next examined a system in which the allograft only differed in class I. Bm1 skin grafts were transplanted on C57BL/6 hosts and the effect of the administration of Ad-hIL-10-infected activated CD4 T cells was assessed. As can be seen in Fig. 4(C) and Table 1, Exp. 3, the survival was substantially prolonged when Ad-hIL-10-infected activated CD4 T cells were administrated (11 ± 3.1 versus 18 ± 3.7, P < 0.0002). No significant effect was observed with control empty vector-infected activated CD4 T cells (10 ± 2.9) or Ad-LacZ-infected activated CD4 T cells (11 ± 2.8). The addition of the treatment with a sub-therapeutic dose of FK506 appeared to have little effect on survival of Bm1 transplants (11 ± 2.6 versus 11 ± 3.1, non-significant). Even in the presence of a sub-therapeutic dose of FK506, a significant effect of Ad-hIL-10-infected activated CD4 T cells was detected (19.5 ± 1.8 versus 11 ± 2.6, P < 0.001). These results indicate that hIL-10-secreted CD4 T cell administration induced significant immunosuppressive effect on Bm1 allo-skin grafts and may suggest that tolerance induction is more effective in transplantation with class I-mismatched allo-skin grafts compared with those with full allo-skin grafts.
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Discussion
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In this report, we describe efficient IL-10 gene transfer to CAR Tg T cells using an adenovirus vector and the distribution of the modified cells following transfer in vivo. A major goal of this study was to establish an approach to inhibit allograft rejection. To this end we showed that significant immunosuppressive effects could be achieved by the administration of Ad-hIL-10-infected activated CD4 T cells or Th2 cells in allo-skin graft-rejection models. Introduction of the IL-10 gene to CD4 T cells and subsequent administration of such cells may offer a novel cell therapy procedure for the induction of allotransplantation tolerance.
IL-10 is a well-known immunosuppressive cytokine. Recombinant cytokines, including IL-10, are, however, short lived and their effects are not easily observed by simple administration in vivo (3234). The serum half-life of recombinant IL-10 after a single injection was reported to be several hours (32, 33). Because of these issues, gene therapy with cytokines such as IL-10 has been considered as an alternative approach. In fact, IL-10 gene therapy has been performed in several organ transplantation animal models (35), including rat liver (11, 36), mouse heart (9), rat heart (37), sheep cornea (13), rabbit heart (3841) and rat lung (42, 43). Several different vectors, such as retrovirus, adenovirus, plasmid and liposomal vectors, have been used, but in all reports, the IL-10 gene was introduced directly into the graft tissues, and potent immunosuppressive effects were generally observed. Coates et al. used dendritic cells (DCs) for targets of the IL-10 gene transduction (44). They used the bovine IL-10 gene-encoded adenovirus vector, and they observed that DCs transduced with the IL-10 gene inhibited allostimulation and cytolytic activity.
In contrast to the above-mentioned previous experimental systems, we used CD4 T cells as a target of the IL-10 gene introduction. It is well known that in most of the inflammatory legions various levels of lymphocyte infiltration including activated CD4 and CD8 T cells are observed, suggesting their regulatory roles in inflammation. Thus, we wished to determine whether the manipulation of immunoregulatory functions of CD4 and CD8 T cells and their administration is an effective approach for the control of inflammation. The results shown in this report indicate that CD4 T cells secreting the immunosuppressive cytokine IL-10 regulate allo-skin graft responses. In addition, one of the advantages of the use of activated lymphocytes for gene introduction is that the effect is transient. The gene-introduced lymphocytes should undergo apoptotic cell death efficiently, and the incidence of tumor generation as well as possible side effects of inducing an immunosuppressive state in hosts would be minimal.
The infection by adenovirus vectors is mediated by CAR (45) whose tissue distribution varies among organs (46). CAR expression is greatest in the liver, while it is very limited on lymphocytes. We overcame this limitation by using CAR Tg mice in which CAR is highly expressed on T cells. For therapeutic considerations in man, to use this approach it may be necessary to establish a new adenovirus-related vector system that can allow human T cells be infected efficiently or alternatively some more efficient means, including lentivirus vector system, to get the IL-10 gene into human T cells. However, once such an efficient gene introduction system is invented, results obtained from experimental model systems such as using CAR Tg T cells would be helpful. Thus, these studies should provide a proof of concept.
As for mechanisms of immunosuppression, one scenario is that the transferred IL-10-producing T cells migrated in the lymphoid organs and inhibited the priming of allospecific CD4 T cells and the subsequent induction of allospecific CD8 CTLs. This is likely because very efficient inhibition of all MLR by Ad-hIL-10-infected CD4 T cells was observed in vitro (Fig. 2). Another possible mechanism is that Ad-hIL-10-infected CD4 T cells migrated at the site of skin grafts and directly inhibited the rejection at the effector phase, i.e. inhibiting the function of allospecific cytotoxic CD8 T cells. In fact, we detected significant levels of CAR-positive cells (
0.05% of migrated T cells on day 7) in the skin graft tissue by PCR Southern blotting analysis (our unpublished observation). By immunofluorescent staining assay, no significant numbers of CAR+ cells were detected in the Bm1 graft of mice with Ad-hIL-10-infected CD4 T cell administration on day 7 or day 14 (our unpublished observation). This could be due to low frequency of CAR+ administered cells in the skin graft tissue (
0.05%). Moreover, no detectable serum level of IL-10 was observed (1, 4, 8, 24 and 48 h and 4, 7 and 14 days after transfer of Ad-hIL-10-infected CD4 T cells) (our unpublished observation), suggesting a non-systemic action of IL-10. Further investigation is required to clarify the actual mechanism involved. We used allo-skin graft systems for the evaluation of the immunosuppressive effects of the administration of IL-10-secreting CD4 and CD8 T cells; however, it is possible that other transplantation systems may be more appropriate for IL-10 gene-introduced T cell therapy. In addition, it may be necessary to consider more efficient strategies including the combinational use of other inhibitory cytokines such as transforming growth factor ß (47).
We detected a significant enhancing effect by the combined use of administration of low-dose sub-therapeutic FK506 (Fig. 4 and Table 1). This observation would be important because we may be able to reduce the doses of immunosuppressive drugs when transplanted patients are treated in combination with the administration of IL-10-secreting CD4 T cells. Since immune responses during allotransplant rejection consist of various distinct cellular activation processes, the combinational use of inhibitors acting on different processes would be more effective, particularly for minimizing different side effects observed in patients.
In summary, we demonstrate that the administration of adenovirus-delivered IL-10-secreting CD4 T cells inhibits the allograft rejection significantly in a specific graft-rejection model. In addition, the combined use of low-dose sub-therapeutic FK506 led to a substantial enhancement of the therapy. Thus, the direct manipulation of T cells by the introduction of immunosuppressive genes may offer a novel approach for the control of allograft rejection.
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Acknowledgements
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The authors are grateful to Ralph T. Kubo for helpful comments and constructive criticisms in the preparation of the manuscript. The authors also thank Ms Kaoru Sugaya for excellent technical assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (Japan) (Grants-in-Aid for: Scientific Research, Priority Areas Research #13218016 and #16043211; Scientific Research B #14370107; Scientific Research C #16616003 and #15790248 and Special Coordination Funds for Promoting Science and Technology), the Ministry of Health, Labor and Welfare (Japan), the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (Japan) and the Japan Health Science Foundation, Uehara Memorial Foundation, Kanae Foundation and Mochida Foundation.
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Abbreviations
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Ad-GFP | adenovirus vector containing EGFP gene |
Ad-hIL-10 | adenovirus vector containing human IL-10 gene |
Ad-LacZ | adenovirus vector containing lacZ gene |
Bm1 | B6.C-H2bm1/ByJ |
CAR | Coxsackie virus and adenovirus receptor |
CsA | cyclosporin A |
DC | dendritic cell |
hIL-10 | human IL-10 |
i.f.u. | infectious units |
POD | post-operation day |
Tg | transgenic |
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Notes
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Transmitting editor: A. Singer
Received 3 February 2005,
accepted 10 March 2005.
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