Acceptance of Murine Islet Allografts Without Immunosuppression in Inguinal Subcutaneous White Adipose Tissue Pretreated With bFGF

Prevention of immune rejection without immunosuppression is a major concern and an ultimate goal of transplant immunobiology. In the field of cellular transplantation, such as pancreatic islet transplantation, there are advantages over organ transplantation with respect to the feasibility of pretreatment, not only of donors (1–6) but also of the transplant site (7), prior to transplantation, making the goal of prevention of immune rejection without immunosuppression more likely. Regarding pretreatment of the transplant site, Luan and Iwata showed the beneficial effect on graft survival of pretreatment of the dorsal subcutaneous space of recipient rats as a transplant site with basic fibroblast growth factor (bFGF) (7). However, transplantation efficiency was extremely low in these studies and a similar treatment failed to prevent islet allograft rejection in major allogeneic combinations from BALB/c to C57BL/6 mice (Supplementary Fig. 1). However, the findings suggest that prevention of islet allograft rejection without immunosuppression becomes feasible and realistic when a novel transplantation site is developed, where pretreatment is easily accessible and when a new procedure to create an appropriate local environment for islet allografts to survive without immunosuppression becomes available. In this sense, we have recently reported that the inguinal subcutaneous white adipose tissue (ISWAT) is a novel site of islet transplantation (8) superior to the liver, which is the currently preferred site of clinical islet transplantation (9–12).

In the current study, we demonstrate that C57BL/6 mice with streptozotocin (STZ)-induced diabetes remain normoglycemic for >1 year after transplantation of BALB/c islet allografts without immunosuppression when the ISWAT is the site of islet transplantation and when the site is treated with bFGF prior to transplantation. Mechanistically, the acceptance was mediated through TGF-β–producing mesenchymal stem cells (MSCs) expanded in the ISWAT after the treatment with bFGF, capable of preventing immune rejection without immunosuppression.

Allogeneic islets were grafted in the ISWAT of mice with STZ-induced diabetes, and it was determined whether the pretreatment to the site of transplantation with bFGF produced prevention of islet allograft rejection without immunosuppression.

Male BALB/c and C3H mice were used as donors, and male C57BL/6 mice were used as recipients. All mice were purchased from Charles River Japan (Atsugi, Kanagawa, Japan). IL-10–deficient mice were purchased from The Jackson Laboratory. Mice were kept under specific pathogen-free conditions and used at 6–15 weeks of age.

The procedure for preparation of agarose rods containing bFGF can be found in Supplementary Material. The procedure for implantation of an agarose rod in the ISWAT of mice is identical to that for transplantation of islets in the same site as reported previously (8).

Procedures for islet isolation and transplantation can be found in Supplementary Material. At 14 days after the implantation of a rod, when it was 3 days after the intravenous injection of STZ, BALB/c islets were grafted in the space after removal of the rod in the ISWAT of recipient mice (Fig. 1). Detailed information on blood sampling can be found in Supplementary Material. Rejection was considered to have occurred when the two consecutive blood glucose levels of recipient mice exceeded 200 mg/dL after transplantation. The blood glucose levels were measured with a GLUCOCARD DIA meter (ARKRAY, Kyoto, Japan).

The ISWAT of mice receiving islet allografts were retrieved at an appropriate time after islet transplantation and then fixed in 4% paraformaldehyde (PFA) (Nakalai Tesque, Inc., Kyoto, Japan) and processed for histological analysis. Sections were stained with hematoxylin-eosin (H-E) or for immunofluorescent microscopic analysis. Primary and secondary antibodies as well as fluorescent microscopy used are described in Supplementary Material.

MNCs in the ISWAT were prepared according to a procedure for isolation of the stromal cells in the ISWAT previously described (13). Descriptions for antibodies and equipment used for FACS analysis can be found in Supplementary Material.

MSCs from the left ISWAT of mice were obtained as adherent cells in a tissue culture dish (Corning no. 3295; Corning, NY) after overnight in vitro culture of the MNCs isolated as stromal cells from the ISWAT of mice with the same procedure as described for the FACS analysis.

Isolated MSCs were centrifuged at 190g for 1 min to make a pellet, which was fixed with 4% PFA and processed for immunohistological analysis. For detection of the localization of MSCs in the ISWAT of a mouse before and after treatment with bFGF, the ISWAT with or without a rod containing bFGF was removed at 14 days after implantation, was fixed with 4% PFA, and proceeded to immunohistochemical analysis. Primary and secondary antibodies used for immunehistochemical analysis of MSCs are described in Supplementary Material.

Materials for cotransplantation of islets with MSCs, macrophotography, and histology of cotransplanted islets are described in Supplementary Material.

Normoglycemic C57BL/6 mice bearing functional BALB/c islets with MSCs received a second transplantation of 400 BALB/c or C3H islets beneath the contralateral right kidney capsule at 180–240 days after initial islet transplantation. Blood glucose levels were measured three times per week for 60 days after the second transplantation, when the left kidney with the initial islet grafts was removed. Then, 4–5 days later, the right kidney with the second islet grafts was removed and examined histologically.

Inhibitory MSCs were induced by in vitro culture of MSCs isolated from the ISWAT of naive C57BL/6 mice in the presence of bFGF, described in Supplementary Material.

The statistical significance of FACS data was determined with unpaired Student t test. Values are expressed as means ± SEM from independent experiments. The statistical significance of Kaplan-Meier curves was determined with log-rank test. Statistical analyses were performed with Prism5 software (GraphPad, La Jolla, CA). Any difference with a P value <0.05 was considered significant.

No data sets were generated or analyzed during the current study. No applicable resources were generated or analyzed during the current study.

We first determined whether islet allograft rejection in mice can be prevented without immunosuppression when the site of islet transplantation is ISWAT and is treated with bFGF prior to transplantation. For these purposes, bFGF was administered through an implanted agarose rod containing bFGF in the left ISWAT of C57BL/6 mice (Fig. 1). Since this model includes many variables, such as the concentration of bFGF, the time interval between implantation and transplantation after removal of a rod, and size of the agarose rod, we evaluated individual factors one by one to find an optimal condition in which islet allograft rejection could be prevented without immunosuppression. Ultimately, we found that hyperglycemia of C57BL/6 mice with STZ-induced diabetes was ameliorated after transplantation of 400 BALB/c islets isolated from two donors, in which 11 of 14 recipient mice remained normoglycemic (nonfasting blood glucose levels <200 mg/dL) for >90 days after transplantation when an agarose rod of 2.5 mm (diameter) × 10 mm (length) containing 10 μg bFGF in 50 μL solution was used and the duration of the implantation was 2 weeks (Fig. 2A, left). On removal of the ISWAT bearing functional islet allografts at 90 days after transplantation, recipient mice promptly became hyperglycemic again (* in Fig. 2A, left), indicating that the normoglycemia of recipient mice was dependent on the transplanted islet allografts. On macro- and microscopic examination, islet grafts were found to form large clusters composed of aggregated islets in which intact islet cells without infiltration of mononuclear cells were seen (Fig. 2A, right). In contrast, hyperglycemia of recipient C57BL/6 mice implanted with an agarose rod containing vehicle and receiving 400 BALB/c islets in the ISWAT was ameliorated after transplantation; however, 13 of 17 (76%) recipient mice became hyperglycemic again by 90 days after transplantation (Fig. 2B, left), and the accumulation of mononuclear cells with infiltration into transplanted islets was visible histologically (Fig. 2B, right), indicating that transplanted allogeneic islets had been rejected in the hyperglycemic recipients. A 24% rate of nonrejection of allogeneic islets in recipient mice treated with vehicle appears to be associated with agarose as a delivery device, since all mice without any treatment (n = 6) rejected islet allografts and were hyperglycemic at 14 days after transplantation (Fig. 2C), although the exact mechanisms remain unknown. The difference in the survival of islet allografts between bFGF- and vehicle-treated groups was statistically significant (log-rank test, P < 0.05) (Fig. 2D).

For examination of how long allogeneic islet acceptance is maintained without immunosuppression, another set of transplantation experiments was performed. Among mice accepting islet allografts without immunosuppression, four normoglycemic mice were followed for up to 1 year after transplantation. Remarkably, these mice remained normoglycemic until the end of the observation period, when removal of the ISWAT bearing functional islet allografts promptly resulted in hyperglycemia in the recipient mice (Fig. 2E, * in the left panel). Histologically, large clusters composed of intact islet cells were seen in the tolerant mice (Fig. 2E, right).

For analysis of cellular immune responses to transplanted allogenic islets, mononuclear cells in the ISWAT were isolated and examined with flow cytometry. When mononuclear cells were isolated from the untreated left ISWAT of C57BL/6 mice with STZ-induced diabetes receiving 400 BALB/c islets, CD8⁺ T cells were found to be expanded, with a statistically significant increase in number compared with that in the ISWAT of mice receiving the same number of syngeneic (C57BL/6) islets at 7 days after transplantation (Fig. 3A). Furthermore, the expression of the CD69 activation marker (14,15) on CD8 T cells in the ISWAT of mice rejecting islet allografts was upregulated (Fig. 3B). In marked contrast, CD69⁺CD8⁺ T cells in the ISWAT of mice treated with bFGF did not increase in number (Fig. 3B). These findings indicate that expansion of CD8 T cells in ISWAT in association with islet allograft rejection does not occur after bFGF treatment.

Among cytokines, IL-10 and TGF-β are known to have immunoinhibitory effects (16–19). Therefore, we first determined a role of IL-10, finding that the beneficial effect of the treatment on prevention of islet allograft rejection was not abolished when IL-10–deficient mice were used as recipients (Fig. 4A), which indicates that IL-10 does not play an essential role in the acceptance. Next, we determined whether the administration of anti–TGF-β antibody influenced the beneficial effect of the treatment. In fact, only one of nine recipient mice with the treatment remained normoglycemic at 90 days after transplantation when anti–TGF-β1 antibody was administered to recipient mice after transplantation (Fig. 4B, left), while, in marked contrast, six of seven recipient mice with the treatment remained normoglycemic at 90 days after transplantation when mouse IgG1 as an isotype control was administered after transplantation (Fig. 4B, right). The difference in the survival of islet allografts between the two groups was statistically significant (Fig. 4C) (log-rank test, P < 0.05).

Regarding the mechanisms involved in prevention of islet allograft rejection in ISWAT after treatment, we hypothesized that MSCs in ISWAT may play a role, since it was reported that adipose tissue including ISWAT contains MSCs that have an inhibitory effect on rejection of islet allografts (20–22).

First, we examined the number of MSCs in the ISWAT before and after treatment as follows. The left ISWAT from individual mice was excised (Fig. 5A) and weighed, and then MSCs were isolated. Of importance, it was found that the MSCs in the ISWAT of naive mouse were too few in number for accurate counting (Fig. 5B, upper panels). In marked contrast, there was a striking increase in the number of MSCs in ISWAT after treatment (Fig. 5B, lower panels). Of note, MSCs adherent to the culture dish increased in number to more than double irrespective of the treatment after in vitro culture for 3 days (Fig. 5B). The number of MSCs isolated from ISWAT of mice treated with vehicle and cultured for 3 days was significantly lower than that from ISWAT of mice treated with bFGF (Fig. 5C).

Next, we examined the phenotype and the location where the MSCs expanded in ISWAT after treatment with bFGF. With immunohistochemical staining, these MSCs were found to be positive for CD44, Sca-1, CD29, and CD106 (Fig. 5D), which is consistent with our previous report with respect to the characteristics of MSCs (13). In the naive mice, CD44⁺ MSCs were very few and scarcely localized in the ISWAT (Fig. 5E, upper panels). Of interest, after treatment MSCs positive for CD44, Sca-1, CD29, and CD106 were found to be localized mainly along the outer surface of the agarose rod that had contained bFGF (Fig. 5E, lower panels). Thus, these finding indicate that MSCs may expand directly in response to bFGF.

Next, we determined whether MSCs expanded in ISWAT after treatment had any effect on prevention of islet allograft rejection. To examine this, we used another model of islet transplantation, namely, the renal subcapsular space, as the site of transplantation instead of the ISWAT, since it lacks MSCs and therefore any effect of cotransplanted MSCs on prevention of islet allograft rejection would be clearer.

First, we used 2 × 10⁵ MSCs isolated from treated ISWAT for each transplantation, since the volume of this number of MSCs was almost the same as that of 400 islets in a PE50 tube (Fig. 6A). We observed that seven of eight diabetic recipient mice (88%) cotransplanted with 400 BALB/c islets from two donors and 2 × 10⁵ C57BL/6 MSCs remained normoglycemic >90 day after transplantation without immunosuppression (Fig. 6B, left). On removal of the kidney bearing islet grafts with MSCs, the recipient mice promptly became hyperglycemic again (* in Fig. 6B, left), indicating that normoglycemia of recipient mice depended on the transplanted allogenic islets. Macroscopically, intact islet grafts yellowish in color surrounded by fibrous structures whitish in appearance with neovascularization were observed (Fig. 6C, left). Histologically, intact islet cells surrounded by dense fibrous tissue without mononuclear cell infiltration were detected beneath the kidney capsule (Fig. 6B, middle and right). BALB/c islet allografts with 2 × 10⁵ MSCs isolated from the ISWAT treated with vehicle were used as controls and were rejected in 10 of 11 recipient mice (Fig. 6B, right). The difference in graft survival between the two groups was statistically significant (Fig. 6D) (Kaplan-Meier curve, log-rank test, P < 0.05). The beneficial effect of cotransplantation with MSCs on prevention of islet allograft rejection was found to be dependent on cell dose and culture period, since it was not obtained when MSCs for cotransplantation were reduced in number (Fig. 6E) and cultured for a prolonged period (Fig. 6F and G), respectively. Of note, prevention of islet allograft rejection without immunosuppression was not achieved when allogeneic (BALB/c or C3H) instead of syngeneic MSCs expanded in ISWAT after treatment were used for cotransplantation (Fig. 6H and I).

Regarding a molecule responsible for prevention of islet allograft rejection with cotransplantation of MSCs expanded in ISWAT of mice after treatment with bFGF, TGF-β was found to play an essential role as it did in prevention of islet allograft rejection without immunosuppression in ISWAT of mice after treatment (Fig. 6J–L).

Then, we determined whether MSCs were responsible for production of TGF-β. FACS analysis revealed that TGF-β1 was expressed on expanded MSCs positive for CD44, Sca-1, CD29, and CD106 and negative for lineage markers and class II (Fig. 7A). The finding was confirmed by histological study in which expanded MSCs stained positive for TGF-β1 (Fig. 7B).

Next, we examined whether TGF-β–producing MSCs cotransplanted with allogeneic islets remained present at the site after transplantation beneath the kidney capsule of recipients. For those purposes, TGF-β–producing MSCs were labeled in vitro with fluorescent dye prior to transplantation (Supplementary Fig. 2) and cotransplanted with allogenic islets. Frozen sections revealed that transplanted allogeneic islets were surrounded densely by labeled MSCs at 7 days after transplantation (Fig. 7C). In separate paraffin sections, those MSCs surrounding islets beneath the kidney capsule proved positive for TGF-β1 (Fig. 7D), indicating that cotransplanted MSCs remained present at the site of transplantation. Since fluorescent dye became faded with time, we could not chase labeled MSCs thereafter. Alternatively, we could detect TGF-β–positive cells adjacent to transplanted islets at 30, 90, and 240 days after transplantation (Supplementary Fig. 3).

Since it is important to learn whether the acceptance of islet allografts in this system is mediated through local or systemic unresponsiveness, we next determined whether second donor–specific (BALB/c) or third party (C3H) islet allografts were accepted or rejected when grafted at 180–240 days after the initial transplantation beneath the contralateral right kidney capsule of normoglycemic mice accepting the initial BALB/c islet grafts. When donor-specific BALB/c islets were used as donors for a second transplantation, recipient mice remained normoglycemic at 60 days after the second transplantation (Fig. 7E, left), and when the left kidney bearing the initial BALB/c islet allografts was removed, two-thirds of recipient mice promptly became hyperglycemic again (Fig. 7E, left). Histologically, intact islets were seen beneath the left kidney capsule of all three recipient mice. Foci of mononuclear cells were observed adjacent to islet grafts but without infiltration (Fig. 7E, left in histology panels). Four days after removal of left kidney, right kidney receiving a second islet transplantation was removed and examined histologically. Importantly, no islet cells were identified and only accumulations of mononuclear cells were seen beneath the right kidney capsule of all three recipient mice (Fig. 7E, right in histology panels), indicating that the second islet allografts were rejected. These findings indicate that the second BALB/c islet grafts beneath the right kidney capsule were rejected; however, the initially accepted BALB/c islets beneath the left kidney capsule were still protected against rejection. When C3H islets were used as donors for a second transplantation grafted beneath the right kidney capsule, functional and histological findings similar to those of BALB/c islets used as donors were seen (Fig. 7F). These findings indicate that acceptance of the initial BALB/c islet allografts beneath the left kidney capsule after cotransplantation with MSCs is maintained through local rather than systemic unresponsiveness.

Finally, we determined whether MSCs with a potent inhibitory effect on prevention of rejection without immunosuppression can be generated in vitro. For those purposes, MSCs were isolated from the ISWAT of naive C57BL/6 mice and cultured in vitro in the medium containing bFGF with different concentrations for 1 or 2 weeks and then cotransplanted with BALB/c islets beneath the kidney capsule of C57BL/6 mice with STZ-induced diabetes. Consequently, we found that islet allograft rejection was prevented when naive MSCs were cultured overnight after isolation in the 3 mL medium containing 4 μg bFGF and then for the following 6 days in that containing 0.4 μg bFGF. The dose of 4 μg bFGF was one-half that used for in vivo generation of MSCs in the ISWAT of recipient mice. As a result, 8 of 11 mice were normoglycemic at 30 days after cotransplantation of BALB/c islets with 2 × 10⁵ MSCs (Fig. 8, middle). In marked contrast, only one of eight recipient mice was normoglycemic at 30 days after transplantation when naive MSCs were cultured in the presence of corresponding doses of vehicle for 7 days after isolation and used for the cotransplantation (Fig. 8, left).

The most important finding in the current study is that rejection of murine islet allografts can be prevented without immunosuppression when ISWAT is the site of islet transplantation and when ISWAT is treated with bFGF prior to transplantation. Mechanistically, the acceptance was mediated through TGF-β–producing MSCs expanded in ISWAT after treatment at the site of islet transplantation, enabling islet allografts to survive without immunosuppression.

bFGF is known as an angiogenic factor and is currently used for the local treatment of ischemic diseases such as decubitus and skin ulcer by promoting neovascularity in the affected lesion (23). On the other hand, bFGF has also been reported as an essential factor for the in vitro maintenance of cell proliferation (24,25) such as of induced pluripotent stem/embryonic stem cells. In the current study, we found that bFGF treatment causes in vivo expansion of TGF-β–producing MSCs in ISWAT and that these expanded MSCs possess potent inhibitory activity for preventing rejection without immunosuppression. How MSCs are expanded in response to bFGF and which cellular populations among MSCs expand in ISWAT in response to TGF-β are issues to be investigated in a future study.

It is well-known that MSCs themselves, prepared either from bone marrow or adipose tissue, have inhibitory effects on allograft rejection in a clinical setting in the field of solid organ transplantation (26) and bone marrow transplantation to treat graft versus host disease (27,28). However, these beneficial effects by MSCs all have been achieved under the cover of immunosuppressants. Thus, the current finding is the first to demonstrate that syngeneic MSCs expanded in ISWAT of mice after treatment with bFGF enable islet allografts to survive without immunosuppressive treatment when they are cotransplanted with allogeneic donor islets.

Regarding the unresponsiveness maintained in islet allografts after cotransplantation with MSCs, it was found to be mediated locally rather than systemically. One potential explanation is that the syngeneic inhibitory MSCs cotransplanted with the allogeneic islets remain present at the transplantation site and serve to protect the islet allografts from rejection induced by a second transplant. The findings that TGF-β–producing cells remain present close to transplanted allogeneic islets up to the end of observation period at 240 days after cotransplantation (Supplementary Fig. 2) may support the hypothesis, although exact mechanisms are to be investigated in future studies.

Current approaches for prevention of immune rejection without immunosuppression for transplantation of insulin-producing cells focus on development of micro- and macro-devices equipped with a membrane that mechanically prevents immune cells of recipients from entering inside the device containing the transplanted cells, which at present has been unsuccessful due to an inadequate supply of nutrients and oxygen to transplanted cells caused by interruption of neovascularization by a membrane (29–31). Thus, it would be ideal if a novel membrane, that does not block neovascularization while maintaining immuno-isolation for prevention of rejection in the local environment, were developed. In this sense, our current model fulfils these criteria, offering a new concept, called “biological immuno-isolation,” meaning that prevention of immune rejection without immunosuppression is achieved without synthetic devices but with biological material such as MSCs.

Taken collectively, our current studies provide a new procedure to prevent islet allograft rejection without immunosuppression. Further, we are now investigating to find a way to prevent islet alloimmune and autoimmune rejection by a conditioned local environment with the use of an NOD mouse model, since not only alloimmune but also autoimmune rejection limits the outcome of islet transplantation as a treatment for patients with insulin-dependent diabetes (32–34). In addition to allogeneic islets, insulin-producing cells such as those derived from induced pluripotent stem/embryonic stem cells (35–37) are currently under consideration as potential unlimited sources as donors instead of allogeneic islets from deceased donors for transplantation as a treatment for patients with insulin-dependent diabetes. For these future studies, if “biological immuno-isolation” proposed by the current study becomes a clinical reality, it would afford a tremendous impact on the treatment of patients with diabetes.

Acknowledgments. The authors thank Dr. Peter Burrows (University of Alabama at Birmingham) for helpful comments and constructive criticisms in the preparation of the manuscript. Technical support from Yuriko Hamaguchi and Yuri Otsu, Research Institute for Islet Biology, Fukuoka University Central Research Organization, is greatly appreciated.

Funding. This work was supported by Japan Agency for Medical Research and Development (20bm0404030 to Y.Y., M.G., and H.I.) and by funds from the Central Research Institute of Fukuoka University (no. 932 to Y.Y. and T.I.).

Duality of Interest. The data presented in the study are included in a patent application by Y.Y. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. M.T. and Y.Y. designed the study, collected and analyzed data, and wrote the manuscript. Y.N., N.N., and Y.Y. mainly performed experiments including islet isolation and transplantation. N.N. and T.N. performed the morphological studies. Y.N., K.I., and N.S. participated in FACS analysis. T.I., M.N., M.G., and H.I. participated in research design and data analysis. Y.Y. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the 78th Scientific Sessions of the American Diabetes Association, Orlando, FL, 22–26 June 2018.