Natural Killer T-Cells Participate in Rejection of Islet Allografts in the Liver of Mice
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糖尿病学杂志 2006年第1期
1 Department of Surgery I, Fukuoka University, Fukuoka, Japan
2 Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
3 Department of Laboratory Medicine, Fukuoka University, Fukuoka, Japan
4 Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan
5 Laboratory for Immune Regulation, Riken Research Center for Allergy and Immunology, Yokohama, Japan
-GalCer, -galactosylceramide; IFN-, -interferon; IL, interleukin; mAb, monoclonal antibody; NKT, natural killer T; STZ, streptozotocin
ABSTRACT
A role of natural killer T (NKT) cells in transplant rejection remains unknown. Here, we determined whether NKT cells participate in rejection of islet allografts, using NKT celleCdeficient mice. Survival of islet allografts in streptozotocin-induced diabetic CD1deC/eC mice or V14 NKT celleC/eC mice was significantly prolonged without immunosuppression when grafted into the liver, but not beneath the kidney capsule, compared with wild-type mice. Acceptance of intrahepatic islet allografts was achieved in CD1deC/eC mice by a subtherapeutic dose of rapamycin, which was abrogated in conjunction with the transfer of hepatic mononuclear cells from wild-type, but not from CD1deC/eC, mice at islet transplantation. The second islet grafts from a donor-specific, but not from a third-party, strain in CD1deC/eC mice bearing functional islet allografts were accepted without immunosuppression at 120 days after the initial transplantation. These findings demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice, but that NKT cells are not essential for induction of donor-specific unresponsiveness in this model. The current study indicates that NKT cells might be considered as a target for intervention to prevent islet allograft rejection when the liver is the site of transplantation.
Pancreatic islet transplantation has become a procedure of choice for treatment of insulin-dependent diabetes (type 1 diabetes) since Shapiro et al. (1,2) reported that successful islet transplantation in patients with type 1 diabetes can be achieved by introduction of a novel steroid-free immunosuppressive regime different from that of organ transplantation and designed for islet transplantation. Currently, however, the rate of insulin independence in patients with type 1 diabetes declines with time to <10% by 5 years after transplantation, although it is >70% at 1 year after islet transplantation (3). This indicates that the functional mass of islet grafts in recipients decreases with time, resulting in a requirement for treatment with exogenous insulin after transplantation (4). Among the factors producing the loss of functional islet graft mass, the continuous administration of immunosuppressive agents might be a potential cause because it has been well known that calcineurin inhibitors, such as FK506 and cyclosporin A, which are currently used for prevention of islet allograft rejection in a clinical setting, are toxic to -cells of islets (5eC7), and their long-term use may lead to deterioration of islet grafts without rejection. To overcome this, a novel immunosuppressive regime for islet transplantation that is nontoxic to -cells needs to be developed, and for this purpose, a precise dissection of cellular mechanisms involved in islet allograft rejection is essential to help provide potential targets for intervention.
Natural killer T (NKT) cells have been recently identified as a novel lymphoid subset distinct from conventional T-cells, coexpressing an invariant T-cell receptor and natural killer celleCrelated surface markers, including NK1.1 (8). Unlike conventional T-cells, NKT cells are specific for glycolipid antigen bound with the major histocompatibility complex class I-like molecule CD1d (9,10). NKT cells produce large amounts of -interferon (IFN-) on activation (11,12), mediating protective functions, including prevention of infectious diseases (13,14), experimental tumor metastasis (15), and chemical-induced tumor development (16). NKT cells also mediate regulatory functions in the protection against autoimmune disease development (14), such as experimental allergic encephalitis (17,18) and type 1 diabetes in NOD mice (19eC21).
In the current study, we hypothesized that NKT cells play a significant role in islet allograft rejection because the liver, the site of clinical islet transplantation, contains relatively abundant NKT cells (8) and because NKT cells have been reported to activate CD4 Th1 (T helper 1) cells and CD8 cytotoxic T-cells in the acquired immune system after activation (14,22). To prove this, we used NKT celleCdeficient mice as recipients, and we demonstrated that NKT cells are activated after islet transplantation, leading to rejection of islet allografts in the liver of mice. The current study suggests that NKT cells might be considered as a target for intervention for prevention of islet allograft rejection when the liver is the site of transplantation.
RESEARCH DESIGN AND METHODS
Male C57BL/6 (H-2b, 12eC15 weeks old), BALB/c (H-2d, 9eC12 weeks old), and C3H/HeN (H-2k, 9eC12 weeks old) mice were purchased from Charles River Japan (Kanagawa, Japan) and used as recipients, donors, and third-party donors, respectively. CD1d-deficient (CD1deC/eC) mice and V14NKT celleCdeficient (J281eC/eC) mice (15) with a C57BL/6 background were also used as recipients. V14NKT cells are a major component of NKT cells reactive to CD1d (23). CD1deC/eC mice, originally developed by Dr. Luc Van Kaer (Vanderbilt University), were provided by Dr. Moriya Tsuji (Rockefeller University). All experiments were performed in accordance with the institutional animal care and use committee.
Induction of diabetes in mice.
Diabetes was induced in recipient mice by an intravenous injection of streptozotocin (STZ; 180 mg/kg; Sigma, St. Louis, MO). Plasma glucose levels were measured with a glucose analyzer (Beckman Instruments, Tokyo). After the STZ injection, nonfasting plasma glucose levels exceeded 400 mg/dl by day 2, and mice remained hyperglycemic at the time of islet transplantation.
Islet isolation and transplantation.
Islets were isolated using static collagenase (type S-1; Nitta Gelatin, Osaka, Japan) digestion (24) and Ficoll-Conray gradient separation (25). Isolated islets were incubated overnight at 24°C in a 5% CO2 incubator and then handpicked using a Pasteur pipette with the aid of a dissecting microscope. We transplanted 600 islets beneath the left kidney capsule (26) or into the liver via the portal vein (27) of diabetic mice 3 days after STZ injection. Nonfasting plasma glucose levels and body weight were monitored three times a week, and rejection was defined when two consecutive plasma glucose levels exceeded 200 mg/dl after islet transplantation.
Administration of rapamycin.
Rapamycin was generously provided by Wyeth-Ayerst Research (Princeton, NJ). A solution of 1.0 mg/ml rapamycin in 0.2% carboxymethylcellulose (Sigma) was dispersed after ultrasonic disruption by a Bioruptor UCD-200T (Cosmo Bio, Tokyo) for 5 min and then diluted to the specified concentrations for the experimental groups. Rapamycin was administered intraperitoneally for 7 consecutive days, starting on the day of transplantation.
Morphological study.
For light microscopic examination, the livers were removed and fixed with Bouin’s solution, processed, and embedded in paraffin. The sections were stained with hematoxylin and eosin or aldehyde and fuchsin.
Preparation of hepatic mononuclear cells.
Hepatic mononuclear cells were prepared as described previously (28). In brief, an excised liver was pressed through a stainless steel mesh, and the resulting dissociated liver tissues were suspended in Dulbecco’s modified Eagle medium (DMEM/F-12; Life Technologies, Tokyo) and washed twice. The mixture was resuspended in an isotonic 33% Percoll solution containing heparin (67 units/ml) and centrifuged at 2,000g at 4°C for 15 min. The resulting pellet was suspended in 0.83% ammonium chloride solution to lyse erythrocytes. After counting, these hepatic mononuclear cells were washed twice in PBS and used for further analysis.
Antibodies and flow cytometry analysis.
The antibodies used for flow cytometry analysis were as follows: Fc block (anti-mouse FcRII/III monoclonal antibody [mAb], 2.4G2), fluorescein isothiocyanateeCconjugated anti-CD3 (clone 145-2C11, American hamster IgG1), allophycocyanin-conjugated antieCIFN- mAb (clone XMG1.2, rat IgG1), antieCinterleukin (IL)-4 mAb (clone 11B11, rat IgG1), antieCIL-2 receptor -chain (CD25; clone PC61, rat IgG1), and their isotype control (clone R3-34, rat IgG1) were purchased from PharMingen (San Diego, CA). Phycoerythrin-conjugated -galactosylceramide (-GalCer)-loaded CD1d tetramers were prepared as described previously (29).
The stained cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA), and the data were processed by CELLQuest software (Becton Dickinson). We collected 10,000 viable hepatic mononuclear cells for dot plot to analyze each cell population and 1,000 for histogram plot for each fraction.
Preparation of hepatic mononuclear cells for adoptive transfer experiment.
Hepatic mononuclear cells were prepared from wild-type or CD1deC/eC mice in the same way as flow cytometry analysis under aseptic conditions. We transferred 5 x 106 hepatic mononuclear cells in 200 e蘬 Hanks’ balanced salt solution via the portal vein of CD1deC/eC mice at the time of islet transplantation.
Statistical analysis.
Statistical significance of graft survival was determined by log rank test or Fisher’s exact probability test, and that of flow cytometry data was examined by unpaired Student’s t test. Results were considered statistically different at P < 0.05.
RESULTS
Prolongation of islet allograft survival in the liver but not renal subcapsular space of NKT celleCdeficient mice without immunosuppression.
First, we determined whether NKT cells are involved in acute rejection of islet allografts with the use of CD1deC/eC mice and V14 NKT celleC/eC mice as recipients. Furthermore, the effect of transplant sites, namely the renal subcapsular space versus the liver, on survival of islet allografts was examined because the liver contains relatively abundant NKT cells (8). When BALB/c islets were grafted beneath the kidney capsule of diabetic C57BL/6 mice, all islet allografts were rejected within 3 weeks after transplantation irrespective of the presence or absence of NKT cells (Table 1). Rejection was confirmed morphologically, in which only accumulations of mononuclear cells were seen at the site of islet transplantation, namely beneath the kidney capsule (histology not shown). In contrast, when BALB/c islets were grafted into the liver of diabetic mice, there was a significant prolongation of islet allograft survival in CD1deC/eC and V14 NKT celleC/eC mice compared with wild-type mice (Table 1). The difference in survival between wild-type and CD1deC/eC mice and between wild-type and V14 NKT celleC/eC mice was statistically significant (P < 0.05, log rank test) (Table 1). Morphologically, islet grafts infiltrated with mononuclear cells in the liver of recipient mice were seen at the time of rejection (Fig. 1, right panels).
Upregulation of IFN- production and CD25 expression in NKT cells and T-cells in the liver of wild-type mice rejecting islet allografts.
To evaluate a role of NKT cells in the liver of mice rejecting islet allografts, hepatic mononuclear cells of mice were isolated for examination by flow cytometry. The yield of mononuclear cells from the liver of nave untreated wild-type mice was 25.8 ± 1.2 x105 (n = 3), and that from the liver of wild-type mice that had received isografts or allografts was 40.3 ± 1.3 (n = 3) or 56.7 ± 2.7 x105 (n = 3), respectively, at 8 days after transplantation, when islet allografts are rejected. The difference in the yield of hepatic mononuclear cells between wild-type mice receiving isografts and those receiving allografts was statistically significant (P < 0.05, Student’s t test). The yield of hepatic mononuclear cells from CD1deC/eC mice was 27.3 ± 1.0 x105 (n = 3) and 54.2 ± 4.6 x105 (n = 3) at 8 and 16 days, respectively, after islet transplantation, when islet allografts maintained or ceased to function without or with rejection, respectively.
We then determined whether NKT cells in the liver of wild-type mice are activated in association with rejection. To detect NKT cells, -GalCereCloaded CD1d tetramers (29) were used for fluorescence-activated cell sorting analysis. The percentage of tetramer+ CD3+ NKT cells in the liver of nave wild-type mice was 10.2 ± 1.6% (n = 3), and that in the liver of wild-type mice receiving isografts and allografts was 1.5 ± 0.3% (n = 3) and 5.9 ± 0.6% (n = 3), respectively, at 8 days after transplantation. Tetramer+ CD3+ NKT cells and tetramereC CD3+ T-cells were further gated and examined with respect to the production of IFN- and IL-4 as well as CD25 expression. NKT cells are known to secrete IFN- and IL-4 on activation. The percentage of IFN-eCproducing tetramer+ CD3+ NKT cells in the liver of nave wild-type mice was 2.5 ± 0.2% (n = 3), and that of wild-type mice receiving isografts or allografts was 6.6 ± 2.5% (n = 3) or 20.6 ± 2.9% (n = 3), respectively, at 8 days after islet transplantation. In contrast, there was no upregulation of IL-4 production in NKT cells in association with rejection. The CD25 expression of NKT cells in nave wild-type mice was 15.2 ± 1.1% (n = 3), and that of wild-type mice receiving isografts or allografts was 3.4 ± 1.4% (n = 3) or 42.8 ± 3.7% (n = 3), respectively, at 8 days after transplantation. Representative data of three experiments is shown in Fig. 2A.
Regarding CD3+ T-cells, an analysis similar to that of NKT cells was performed. It was found that IFN- production and CD25 expression of T-cells in the liver of wild-type mice receiving islet allografts became upregulated from 3.6 ± 0.2% (n = 3) to 21.9 ± 2.8% (n = 3) and from 7.4 ± 0.5% (n = 3) to 23.1 ± 1.7% (n = 3), respectively, at 8 days after transplantation, and that those of mice receiving isografts were from 3.6 ± 0.2% (n = 3) to 8.4 ± 3.7% (n = 3) and from 7.4 ± 0.5% (n = 3) to 8.5 ± 1.6% (n = 3), respectively. In marked contrast, the upregulation of IFN- production and CD25 expression of T-cells in the liver of CD1deC/eC mice receiving islet allografts was not seen at 8 days after transplantation, when the islet grafts were rejected in wild-type mice. Representative data of three experiments is shown in Fig. 2A and B.
Acceptance of islet allografts by a subtherapeutic dose of rapamycin in the absence of NKT cells.
We next determined the effect of NKT cells on rejection of islet allografts in the liver of mice treated with rapamycin. The survival rate of islet allografts in wild-type recipient mice treated with 0.2, 1.0, or 3.0 mg/kg rapamycin once a day for 6 days after transplantation was found to be 0% (0 of 9), 12.5% (1 of 8), and 71.7% (5 of 7), respectively, at 90 days after transplantation (Table 2). In marked contrast to wild-type mice, 91.0% (10 of 11) of CD1deC/eC mice and 60% (3 of 5) of V14NKT celleC/eC mice receiving islet allografts and treated with 1.0 mg/kg rapamycin were normoglycemic at 90 days after transplantation (Table 2 and Fig. 3, left panels). There was a statistical significance in the survival rate between islet allografts in CD1deC/eC mice treated with 1.0 mg/kg rapamycin and those in wild-type mice treated similarly (P < 0.01, Fisher’s exact probability test). Morphologically intact islets with well-granulated -cells were seen in the liver of CD1deC/eC mice at 120 days after islet transplantation (Fig. 3, right panels).
Acceptance of islet allografts in CD1deC/eC mice is abrogated by the adoptive transfer of hepatic mononuclear cells from wild-type but not from CD1deC/eC mice.
To further confirm the role of NKT cells in rejection of islet allografts in the liver of mice treated with rapamycin, cell transfer experiments were performed. As described above, rejection of islet allografts is prevented in CD1deC/eC mice treated with 1 mg/kg rapamycin, and therefore CD1deC/eC mice were used for the following study. When 5 x 106 hepatic mononuclear cells from wild-type mice were transferred via the portal vein into the liver of CD1deC/eC mice receiving islets and treated with rapamycin at the time of islet transplantation, all recipient mice (n = 3) became hyperglycemic again after transplantation (Fig. 4A). In marked contrast, when the same number of hepatic mononuclear cells from CD1deC/eC mice was transferred, recipient CD1deC/eC mice (n = 3) remained normoglycemic (Fig. 4B). Morphologically intact or infiltrated islets were seen in the livers of CD1deC/eC mice receiving the transfer of cells from CD1eC/eC or wild-type mice, respectively (histology not shown).
Donor-specific unresponsiveness is induced in CD1deC/eC mice treated with rapamycin and accepting islet allografts.
Finally, we determined whether donor-specific unresponsiveness is induced in CD1deC/eC mice treated with rapamycin and accepting islet allografts. For this purpose, third-party or donor-specific islets were grafted into the liver of CD1deC/eC mice that had been made diabetic again with intravenous injection of STZ at 120 days after the initial islet transplantation. No immunosuppressive agent was administered at the time of the second transplantation. CD1deC/eC mice receiving third-party C3H islets (n = 3) became hyperglycemic at 22, 26, and 36 days (Fig. 5A) because of rejection, whereas CD1deC/eC recipient mice (n = 3) with donor-specific BALB/c islets remained normoglycemic after the second transplantation (Fig. 5B).
DISCUSSION
These findings clearly demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice without immunosuppressive treatment and with immunosuppression by rapamycin, which is currently used in clinical islet transplantation. In the current study, two kinds of NKT celleCdeficient mice, namely CD1deC/eC and V14 NKT celleC/eC mice, were used as recipients to elucidate a role of NKT cells in islet allograft rejection. V14 NKT celleC/eC mice lack only V14NKT cells, which are a major population of CD1d-reactive NKT cells in mice (23). The important finding is that survival of islet allografts is prolonged in CD1deC/eC and V14NKT celleC/eC mice without immunosuppression compared with wild-type mice when the liver is the site of transplantation versus the renal subcapsular space. The finding suggests that NKT cells play a significant role in acute rejection of islet allografts in the liver of mice. Flow cytometry analysis revealed that not only NKT cells but also T-cells in the liver of wild-type mice became activated at the time of rejection. In marked contrast, CD3+ T-cells in the liver of CD1deC/eC mice at 8 days after transplantation, when islet allografts were rejected in wild-type mice but not in CD1deC/eC mice, was not activated. Consequently, CD1deC/eC mice rejected intrahepatic islet allografts with the upregulation of IFN- production and CD25 expression in CD3+ T-cells in the liver. These findings indicate that T-cell activation is delayed in the absence of NKT cells after islet transplantation, which may reflect the prolonged survival of islets in the liver of CD1deC/eC mice, although the precise role of NKT cells in T-cell activation in response to islet allografts remains unclear.
Regarding a role of NKT cells in islet allograft rejection under the immunosuppressive agent rapamycin, rejection was prevented in the absence of NKT cells with a subtherapeutic dose of rapamycin. The frequency of rejection appears to be high in V14 NKT celleC/eC mice compared with CD1deC/eC mice, although the difference has no statistical significance. The potential difference may rely on the disparity between CD1deC/eC and V14 NKT celleC/eC mice (23), in that CD1deC/eC mice lack not only V14NKT cells, a major component of NKT cells, but also the other CD1d-reactive cells, which are less well characterized and whose role in transplant immunity remains unknown. Furthermore, the exact mechanisms involved in the response of NKT cells to donor islets leading to rejection, in relation to cell components other than NKT cells, remain undetermined, which is a matter of interest for future studies.
The current finding that the dosage of rapamycin can be reduced to prevent rejection of islet allografts in the absence of NKT cells raises a possibility that the downregulation of NKT cells may help prevent islet allograft rejection in wild-type mice with reduced dosage of rapamycin. Currently, there is limited number of reports with respect to suppression of NKT cell function. NKT cells secrete large amounts of IFN- on activation (11,12). IFN- production from NKT cells is modulated by the use of their synthetic ligand -GalCer (30), in that single administration of -GalCer upregulates IFN- production, whereas repeated administration downregulates IFN- production (31). Recently, natural ligands of NKT cells have been reported (32eC34). Thus, it is interesting to learn whether procedures targeting NKT cells with the use of their synthetic or natural ligands have any beneficial effects on prevention of islet allograft rejection.
In the current study, it was found that donor-specific unresponsiveness is induced and maintained in CD1deC/eC mice treated with rapamycin and accepting islet allografts. This finding indicates that NKT cells are not required for induction of donor-specific unresponsiveness in this model. Previously, we have shown that CD4+ V14 NKT cells are essential for acceptance of rat islet xenografts in mice treated with anti-CD4 mAb (35). In a cardiac allograft model, tolerance induction after the blockade of CD28/B7 interaction in conjunction with that of LFA-1/ICAM-1 (lymphocyte functioneCassociated antigen 1/intercellular adhesion molecule 1) interaction is dependent on NKT cells (36). NKT cells are reported to be required for induction of allospecific T-regulatory cells and are essential for survival of cornea allografts (37). In contrast to these findings, human CD1d (hCD1d) has been reported to function as a transplantation antigen, and skin grafts are rejected by expression of human CD1d in the major histocompatibility complexeCmatched mouse model (38). Survival of porcine neural xenografts is shown to be enhanced in the brain of CD1deC/eC mice compared with wild-type controls (39). Recently, Oh et al. (40) reported that rejection of H-Y mismatched skin grafts is accelerated in CD1deC/eC mice as recipients compared with wild-type mice. Thus, these findings indicate that NKT cells may have a diverse effect as regulatory and/or effector cells on the outcome of transplants, depending on the type of graft (allograft versus xenograft, organ versus tissue [cell]) and/or the type of immunosuppression.
In summary, the current study affords evidence that NKT cells play a significant role in islet allograft rejection in the liver of mice without immunosuppressive treatment and with immunosuppression by rapamycin, providing a new insight into cellular immune response in association with rejection. Currently, a major obstacle facing clinical islet transplantation is that the rate of insulin independence in patients with type 1 diabetes after islet transplantation declines with time after transplantation (3). Current immunosuppressive regimes include FK506 and rapamycin, which are known to be toxic to -cells of islets and are considered as a major cause of islet graft failure after transplantation. Thus, the current study raises the possibility that introduction of a novel immunosuppressive regime targeting NKT cells may lead to a beneficial outcome of clinical islet transplantation with reduction and/or replacement of FK506 and rapamycin.
ACKNOWLEDGMENTS
This work was supported by funds (0211001) from the Central Research Institute of Fukuoka University, by health science research grants from the Ministry of Health, Labor, and Welfare, Japan, and a grant-in-aid for scientific research (B) from the Japan Society for the Promotion of Science.
We thank Dr. Moriya Tsuji, Rockefeller University, for providing CD1deC/eC mice.
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2 Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
3 Department of Laboratory Medicine, Fukuoka University, Fukuoka, Japan
4 Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan
5 Laboratory for Immune Regulation, Riken Research Center for Allergy and Immunology, Yokohama, Japan
-GalCer, -galactosylceramide; IFN-, -interferon; IL, interleukin; mAb, monoclonal antibody; NKT, natural killer T; STZ, streptozotocin
ABSTRACT
A role of natural killer T (NKT) cells in transplant rejection remains unknown. Here, we determined whether NKT cells participate in rejection of islet allografts, using NKT celleCdeficient mice. Survival of islet allografts in streptozotocin-induced diabetic CD1deC/eC mice or V14 NKT celleC/eC mice was significantly prolonged without immunosuppression when grafted into the liver, but not beneath the kidney capsule, compared with wild-type mice. Acceptance of intrahepatic islet allografts was achieved in CD1deC/eC mice by a subtherapeutic dose of rapamycin, which was abrogated in conjunction with the transfer of hepatic mononuclear cells from wild-type, but not from CD1deC/eC, mice at islet transplantation. The second islet grafts from a donor-specific, but not from a third-party, strain in CD1deC/eC mice bearing functional islet allografts were accepted without immunosuppression at 120 days after the initial transplantation. These findings demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice, but that NKT cells are not essential for induction of donor-specific unresponsiveness in this model. The current study indicates that NKT cells might be considered as a target for intervention to prevent islet allograft rejection when the liver is the site of transplantation.
Pancreatic islet transplantation has become a procedure of choice for treatment of insulin-dependent diabetes (type 1 diabetes) since Shapiro et al. (1,2) reported that successful islet transplantation in patients with type 1 diabetes can be achieved by introduction of a novel steroid-free immunosuppressive regime different from that of organ transplantation and designed for islet transplantation. Currently, however, the rate of insulin independence in patients with type 1 diabetes declines with time to <10% by 5 years after transplantation, although it is >70% at 1 year after islet transplantation (3). This indicates that the functional mass of islet grafts in recipients decreases with time, resulting in a requirement for treatment with exogenous insulin after transplantation (4). Among the factors producing the loss of functional islet graft mass, the continuous administration of immunosuppressive agents might be a potential cause because it has been well known that calcineurin inhibitors, such as FK506 and cyclosporin A, which are currently used for prevention of islet allograft rejection in a clinical setting, are toxic to -cells of islets (5eC7), and their long-term use may lead to deterioration of islet grafts without rejection. To overcome this, a novel immunosuppressive regime for islet transplantation that is nontoxic to -cells needs to be developed, and for this purpose, a precise dissection of cellular mechanisms involved in islet allograft rejection is essential to help provide potential targets for intervention.
Natural killer T (NKT) cells have been recently identified as a novel lymphoid subset distinct from conventional T-cells, coexpressing an invariant T-cell receptor and natural killer celleCrelated surface markers, including NK1.1 (8). Unlike conventional T-cells, NKT cells are specific for glycolipid antigen bound with the major histocompatibility complex class I-like molecule CD1d (9,10). NKT cells produce large amounts of -interferon (IFN-) on activation (11,12), mediating protective functions, including prevention of infectious diseases (13,14), experimental tumor metastasis (15), and chemical-induced tumor development (16). NKT cells also mediate regulatory functions in the protection against autoimmune disease development (14), such as experimental allergic encephalitis (17,18) and type 1 diabetes in NOD mice (19eC21).
In the current study, we hypothesized that NKT cells play a significant role in islet allograft rejection because the liver, the site of clinical islet transplantation, contains relatively abundant NKT cells (8) and because NKT cells have been reported to activate CD4 Th1 (T helper 1) cells and CD8 cytotoxic T-cells in the acquired immune system after activation (14,22). To prove this, we used NKT celleCdeficient mice as recipients, and we demonstrated that NKT cells are activated after islet transplantation, leading to rejection of islet allografts in the liver of mice. The current study suggests that NKT cells might be considered as a target for intervention for prevention of islet allograft rejection when the liver is the site of transplantation.
RESEARCH DESIGN AND METHODS
Male C57BL/6 (H-2b, 12eC15 weeks old), BALB/c (H-2d, 9eC12 weeks old), and C3H/HeN (H-2k, 9eC12 weeks old) mice were purchased from Charles River Japan (Kanagawa, Japan) and used as recipients, donors, and third-party donors, respectively. CD1d-deficient (CD1deC/eC) mice and V14NKT celleCdeficient (J281eC/eC) mice (15) with a C57BL/6 background were also used as recipients. V14NKT cells are a major component of NKT cells reactive to CD1d (23). CD1deC/eC mice, originally developed by Dr. Luc Van Kaer (Vanderbilt University), were provided by Dr. Moriya Tsuji (Rockefeller University). All experiments were performed in accordance with the institutional animal care and use committee.
Induction of diabetes in mice.
Diabetes was induced in recipient mice by an intravenous injection of streptozotocin (STZ; 180 mg/kg; Sigma, St. Louis, MO). Plasma glucose levels were measured with a glucose analyzer (Beckman Instruments, Tokyo). After the STZ injection, nonfasting plasma glucose levels exceeded 400 mg/dl by day 2, and mice remained hyperglycemic at the time of islet transplantation.
Islet isolation and transplantation.
Islets were isolated using static collagenase (type S-1; Nitta Gelatin, Osaka, Japan) digestion (24) and Ficoll-Conray gradient separation (25). Isolated islets were incubated overnight at 24°C in a 5% CO2 incubator and then handpicked using a Pasteur pipette with the aid of a dissecting microscope. We transplanted 600 islets beneath the left kidney capsule (26) or into the liver via the portal vein (27) of diabetic mice 3 days after STZ injection. Nonfasting plasma glucose levels and body weight were monitored three times a week, and rejection was defined when two consecutive plasma glucose levels exceeded 200 mg/dl after islet transplantation.
Administration of rapamycin.
Rapamycin was generously provided by Wyeth-Ayerst Research (Princeton, NJ). A solution of 1.0 mg/ml rapamycin in 0.2% carboxymethylcellulose (Sigma) was dispersed after ultrasonic disruption by a Bioruptor UCD-200T (Cosmo Bio, Tokyo) for 5 min and then diluted to the specified concentrations for the experimental groups. Rapamycin was administered intraperitoneally for 7 consecutive days, starting on the day of transplantation.
Morphological study.
For light microscopic examination, the livers were removed and fixed with Bouin’s solution, processed, and embedded in paraffin. The sections were stained with hematoxylin and eosin or aldehyde and fuchsin.
Preparation of hepatic mononuclear cells.
Hepatic mononuclear cells were prepared as described previously (28). In brief, an excised liver was pressed through a stainless steel mesh, and the resulting dissociated liver tissues were suspended in Dulbecco’s modified Eagle medium (DMEM/F-12; Life Technologies, Tokyo) and washed twice. The mixture was resuspended in an isotonic 33% Percoll solution containing heparin (67 units/ml) and centrifuged at 2,000g at 4°C for 15 min. The resulting pellet was suspended in 0.83% ammonium chloride solution to lyse erythrocytes. After counting, these hepatic mononuclear cells were washed twice in PBS and used for further analysis.
Antibodies and flow cytometry analysis.
The antibodies used for flow cytometry analysis were as follows: Fc block (anti-mouse FcRII/III monoclonal antibody [mAb], 2.4G2), fluorescein isothiocyanateeCconjugated anti-CD3 (clone 145-2C11, American hamster IgG1), allophycocyanin-conjugated antieCIFN- mAb (clone XMG1.2, rat IgG1), antieCinterleukin (IL)-4 mAb (clone 11B11, rat IgG1), antieCIL-2 receptor -chain (CD25; clone PC61, rat IgG1), and their isotype control (clone R3-34, rat IgG1) were purchased from PharMingen (San Diego, CA). Phycoerythrin-conjugated -galactosylceramide (-GalCer)-loaded CD1d tetramers were prepared as described previously (29).
The stained cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA), and the data were processed by CELLQuest software (Becton Dickinson). We collected 10,000 viable hepatic mononuclear cells for dot plot to analyze each cell population and 1,000 for histogram plot for each fraction.
Preparation of hepatic mononuclear cells for adoptive transfer experiment.
Hepatic mononuclear cells were prepared from wild-type or CD1deC/eC mice in the same way as flow cytometry analysis under aseptic conditions. We transferred 5 x 106 hepatic mononuclear cells in 200 e蘬 Hanks’ balanced salt solution via the portal vein of CD1deC/eC mice at the time of islet transplantation.
Statistical analysis.
Statistical significance of graft survival was determined by log rank test or Fisher’s exact probability test, and that of flow cytometry data was examined by unpaired Student’s t test. Results were considered statistically different at P < 0.05.
RESULTS
Prolongation of islet allograft survival in the liver but not renal subcapsular space of NKT celleCdeficient mice without immunosuppression.
First, we determined whether NKT cells are involved in acute rejection of islet allografts with the use of CD1deC/eC mice and V14 NKT celleC/eC mice as recipients. Furthermore, the effect of transplant sites, namely the renal subcapsular space versus the liver, on survival of islet allografts was examined because the liver contains relatively abundant NKT cells (8). When BALB/c islets were grafted beneath the kidney capsule of diabetic C57BL/6 mice, all islet allografts were rejected within 3 weeks after transplantation irrespective of the presence or absence of NKT cells (Table 1). Rejection was confirmed morphologically, in which only accumulations of mononuclear cells were seen at the site of islet transplantation, namely beneath the kidney capsule (histology not shown). In contrast, when BALB/c islets were grafted into the liver of diabetic mice, there was a significant prolongation of islet allograft survival in CD1deC/eC and V14 NKT celleC/eC mice compared with wild-type mice (Table 1). The difference in survival between wild-type and CD1deC/eC mice and between wild-type and V14 NKT celleC/eC mice was statistically significant (P < 0.05, log rank test) (Table 1). Morphologically, islet grafts infiltrated with mononuclear cells in the liver of recipient mice were seen at the time of rejection (Fig. 1, right panels).
Upregulation of IFN- production and CD25 expression in NKT cells and T-cells in the liver of wild-type mice rejecting islet allografts.
To evaluate a role of NKT cells in the liver of mice rejecting islet allografts, hepatic mononuclear cells of mice were isolated for examination by flow cytometry. The yield of mononuclear cells from the liver of nave untreated wild-type mice was 25.8 ± 1.2 x105 (n = 3), and that from the liver of wild-type mice that had received isografts or allografts was 40.3 ± 1.3 (n = 3) or 56.7 ± 2.7 x105 (n = 3), respectively, at 8 days after transplantation, when islet allografts are rejected. The difference in the yield of hepatic mononuclear cells between wild-type mice receiving isografts and those receiving allografts was statistically significant (P < 0.05, Student’s t test). The yield of hepatic mononuclear cells from CD1deC/eC mice was 27.3 ± 1.0 x105 (n = 3) and 54.2 ± 4.6 x105 (n = 3) at 8 and 16 days, respectively, after islet transplantation, when islet allografts maintained or ceased to function without or with rejection, respectively.
We then determined whether NKT cells in the liver of wild-type mice are activated in association with rejection. To detect NKT cells, -GalCereCloaded CD1d tetramers (29) were used for fluorescence-activated cell sorting analysis. The percentage of tetramer+ CD3+ NKT cells in the liver of nave wild-type mice was 10.2 ± 1.6% (n = 3), and that in the liver of wild-type mice receiving isografts and allografts was 1.5 ± 0.3% (n = 3) and 5.9 ± 0.6% (n = 3), respectively, at 8 days after transplantation. Tetramer+ CD3+ NKT cells and tetramereC CD3+ T-cells were further gated and examined with respect to the production of IFN- and IL-4 as well as CD25 expression. NKT cells are known to secrete IFN- and IL-4 on activation. The percentage of IFN-eCproducing tetramer+ CD3+ NKT cells in the liver of nave wild-type mice was 2.5 ± 0.2% (n = 3), and that of wild-type mice receiving isografts or allografts was 6.6 ± 2.5% (n = 3) or 20.6 ± 2.9% (n = 3), respectively, at 8 days after islet transplantation. In contrast, there was no upregulation of IL-4 production in NKT cells in association with rejection. The CD25 expression of NKT cells in nave wild-type mice was 15.2 ± 1.1% (n = 3), and that of wild-type mice receiving isografts or allografts was 3.4 ± 1.4% (n = 3) or 42.8 ± 3.7% (n = 3), respectively, at 8 days after transplantation. Representative data of three experiments is shown in Fig. 2A.
Regarding CD3+ T-cells, an analysis similar to that of NKT cells was performed. It was found that IFN- production and CD25 expression of T-cells in the liver of wild-type mice receiving islet allografts became upregulated from 3.6 ± 0.2% (n = 3) to 21.9 ± 2.8% (n = 3) and from 7.4 ± 0.5% (n = 3) to 23.1 ± 1.7% (n = 3), respectively, at 8 days after transplantation, and that those of mice receiving isografts were from 3.6 ± 0.2% (n = 3) to 8.4 ± 3.7% (n = 3) and from 7.4 ± 0.5% (n = 3) to 8.5 ± 1.6% (n = 3), respectively. In marked contrast, the upregulation of IFN- production and CD25 expression of T-cells in the liver of CD1deC/eC mice receiving islet allografts was not seen at 8 days after transplantation, when the islet grafts were rejected in wild-type mice. Representative data of three experiments is shown in Fig. 2A and B.
Acceptance of islet allografts by a subtherapeutic dose of rapamycin in the absence of NKT cells.
We next determined the effect of NKT cells on rejection of islet allografts in the liver of mice treated with rapamycin. The survival rate of islet allografts in wild-type recipient mice treated with 0.2, 1.0, or 3.0 mg/kg rapamycin once a day for 6 days after transplantation was found to be 0% (0 of 9), 12.5% (1 of 8), and 71.7% (5 of 7), respectively, at 90 days after transplantation (Table 2). In marked contrast to wild-type mice, 91.0% (10 of 11) of CD1deC/eC mice and 60% (3 of 5) of V14NKT celleC/eC mice receiving islet allografts and treated with 1.0 mg/kg rapamycin were normoglycemic at 90 days after transplantation (Table 2 and Fig. 3, left panels). There was a statistical significance in the survival rate between islet allografts in CD1deC/eC mice treated with 1.0 mg/kg rapamycin and those in wild-type mice treated similarly (P < 0.01, Fisher’s exact probability test). Morphologically intact islets with well-granulated -cells were seen in the liver of CD1deC/eC mice at 120 days after islet transplantation (Fig. 3, right panels).
Acceptance of islet allografts in CD1deC/eC mice is abrogated by the adoptive transfer of hepatic mononuclear cells from wild-type but not from CD1deC/eC mice.
To further confirm the role of NKT cells in rejection of islet allografts in the liver of mice treated with rapamycin, cell transfer experiments were performed. As described above, rejection of islet allografts is prevented in CD1deC/eC mice treated with 1 mg/kg rapamycin, and therefore CD1deC/eC mice were used for the following study. When 5 x 106 hepatic mononuclear cells from wild-type mice were transferred via the portal vein into the liver of CD1deC/eC mice receiving islets and treated with rapamycin at the time of islet transplantation, all recipient mice (n = 3) became hyperglycemic again after transplantation (Fig. 4A). In marked contrast, when the same number of hepatic mononuclear cells from CD1deC/eC mice was transferred, recipient CD1deC/eC mice (n = 3) remained normoglycemic (Fig. 4B). Morphologically intact or infiltrated islets were seen in the livers of CD1deC/eC mice receiving the transfer of cells from CD1eC/eC or wild-type mice, respectively (histology not shown).
Donor-specific unresponsiveness is induced in CD1deC/eC mice treated with rapamycin and accepting islet allografts.
Finally, we determined whether donor-specific unresponsiveness is induced in CD1deC/eC mice treated with rapamycin and accepting islet allografts. For this purpose, third-party or donor-specific islets were grafted into the liver of CD1deC/eC mice that had been made diabetic again with intravenous injection of STZ at 120 days after the initial islet transplantation. No immunosuppressive agent was administered at the time of the second transplantation. CD1deC/eC mice receiving third-party C3H islets (n = 3) became hyperglycemic at 22, 26, and 36 days (Fig. 5A) because of rejection, whereas CD1deC/eC recipient mice (n = 3) with donor-specific BALB/c islets remained normoglycemic after the second transplantation (Fig. 5B).
DISCUSSION
These findings clearly demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice without immunosuppressive treatment and with immunosuppression by rapamycin, which is currently used in clinical islet transplantation. In the current study, two kinds of NKT celleCdeficient mice, namely CD1deC/eC and V14 NKT celleC/eC mice, were used as recipients to elucidate a role of NKT cells in islet allograft rejection. V14 NKT celleC/eC mice lack only V14NKT cells, which are a major population of CD1d-reactive NKT cells in mice (23). The important finding is that survival of islet allografts is prolonged in CD1deC/eC and V14NKT celleC/eC mice without immunosuppression compared with wild-type mice when the liver is the site of transplantation versus the renal subcapsular space. The finding suggests that NKT cells play a significant role in acute rejection of islet allografts in the liver of mice. Flow cytometry analysis revealed that not only NKT cells but also T-cells in the liver of wild-type mice became activated at the time of rejection. In marked contrast, CD3+ T-cells in the liver of CD1deC/eC mice at 8 days after transplantation, when islet allografts were rejected in wild-type mice but not in CD1deC/eC mice, was not activated. Consequently, CD1deC/eC mice rejected intrahepatic islet allografts with the upregulation of IFN- production and CD25 expression in CD3+ T-cells in the liver. These findings indicate that T-cell activation is delayed in the absence of NKT cells after islet transplantation, which may reflect the prolonged survival of islets in the liver of CD1deC/eC mice, although the precise role of NKT cells in T-cell activation in response to islet allografts remains unclear.
Regarding a role of NKT cells in islet allograft rejection under the immunosuppressive agent rapamycin, rejection was prevented in the absence of NKT cells with a subtherapeutic dose of rapamycin. The frequency of rejection appears to be high in V14 NKT celleC/eC mice compared with CD1deC/eC mice, although the difference has no statistical significance. The potential difference may rely on the disparity between CD1deC/eC and V14 NKT celleC/eC mice (23), in that CD1deC/eC mice lack not only V14NKT cells, a major component of NKT cells, but also the other CD1d-reactive cells, which are less well characterized and whose role in transplant immunity remains unknown. Furthermore, the exact mechanisms involved in the response of NKT cells to donor islets leading to rejection, in relation to cell components other than NKT cells, remain undetermined, which is a matter of interest for future studies.
The current finding that the dosage of rapamycin can be reduced to prevent rejection of islet allografts in the absence of NKT cells raises a possibility that the downregulation of NKT cells may help prevent islet allograft rejection in wild-type mice with reduced dosage of rapamycin. Currently, there is limited number of reports with respect to suppression of NKT cell function. NKT cells secrete large amounts of IFN- on activation (11,12). IFN- production from NKT cells is modulated by the use of their synthetic ligand -GalCer (30), in that single administration of -GalCer upregulates IFN- production, whereas repeated administration downregulates IFN- production (31). Recently, natural ligands of NKT cells have been reported (32eC34). Thus, it is interesting to learn whether procedures targeting NKT cells with the use of their synthetic or natural ligands have any beneficial effects on prevention of islet allograft rejection.
In the current study, it was found that donor-specific unresponsiveness is induced and maintained in CD1deC/eC mice treated with rapamycin and accepting islet allografts. This finding indicates that NKT cells are not required for induction of donor-specific unresponsiveness in this model. Previously, we have shown that CD4+ V14 NKT cells are essential for acceptance of rat islet xenografts in mice treated with anti-CD4 mAb (35). In a cardiac allograft model, tolerance induction after the blockade of CD28/B7 interaction in conjunction with that of LFA-1/ICAM-1 (lymphocyte functioneCassociated antigen 1/intercellular adhesion molecule 1) interaction is dependent on NKT cells (36). NKT cells are reported to be required for induction of allospecific T-regulatory cells and are essential for survival of cornea allografts (37). In contrast to these findings, human CD1d (hCD1d) has been reported to function as a transplantation antigen, and skin grafts are rejected by expression of human CD1d in the major histocompatibility complexeCmatched mouse model (38). Survival of porcine neural xenografts is shown to be enhanced in the brain of CD1deC/eC mice compared with wild-type controls (39). Recently, Oh et al. (40) reported that rejection of H-Y mismatched skin grafts is accelerated in CD1deC/eC mice as recipients compared with wild-type mice. Thus, these findings indicate that NKT cells may have a diverse effect as regulatory and/or effector cells on the outcome of transplants, depending on the type of graft (allograft versus xenograft, organ versus tissue [cell]) and/or the type of immunosuppression.
In summary, the current study affords evidence that NKT cells play a significant role in islet allograft rejection in the liver of mice without immunosuppressive treatment and with immunosuppression by rapamycin, providing a new insight into cellular immune response in association with rejection. Currently, a major obstacle facing clinical islet transplantation is that the rate of insulin independence in patients with type 1 diabetes after islet transplantation declines with time after transplantation (3). Current immunosuppressive regimes include FK506 and rapamycin, which are known to be toxic to -cells of islets and are considered as a major cause of islet graft failure after transplantation. Thus, the current study raises the possibility that introduction of a novel immunosuppressive regime targeting NKT cells may lead to a beneficial outcome of clinical islet transplantation with reduction and/or replacement of FK506 and rapamycin.
ACKNOWLEDGMENTS
This work was supported by funds (0211001) from the Central Research Institute of Fukuoka University, by health science research grants from the Ministry of Health, Labor, and Welfare, Japan, and a grant-in-aid for scientific research (B) from the Japan Society for the Promotion of Science.
We thank Dr. Moriya Tsuji, Rockefeller University, for providing CD1deC/eC mice.
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