Spleen , as an optimal site for islet transplantation 2

Islet transplantation is a cellular replacement therapy to treat severe diabetes mellitus, 11 but its clinical outcome is unsatisfactory at present. One factor in clinical success of this therapy is 12 selection of the most appropriate transplantation site. In this review, we review evidence showing 13 the advantages of the spleen as a transplantation site for islets. The spleen has been studied for a 14 long time as a candidate site for islet transplantation. Its advantages include physiological insulin 15 drainage and regulation of immunity. Recently it has also been shown that the spleen contributes to 16 the regeneration of transplanted islets and that splenic stem cells have the potential to differentiate 17 into islet cells. The spleen also has some disadvantages associated with the transplantation 18 procedure itself (bleeding, thrombosis and splenic infarction). The efficacy of transplantation is not 19 as high as that obtained with intraportal transplantation, which is the current representative 20 method of clinical islet transplantation. Safer and more effective methods of islet transplantation 21 need to be established before the spleen can be effectively used in the clinic to support the 22 engraftment of multiple transplanted islets. 23


Introduction: Islet Transplantation and the Hurdles
Islet transplantation is a cellular replacement therapy to treat severe diabetes mellitus in patients who are unable to control their blood glucose even with intensive insulin treatment.Islet transplantation enables patients to receive an appropriate supply of insulin in response to changes in blood glucose levels.Islet transplantation also can prevent severe hypoglycemia and life-threatening complications including cardiomyopathy, nephropathy, retinopathy and neuropathy [1][2][3].
Although islet transplantation was first established in the clinic in the 1970s [4], the early therapeutic outcome was inadequate, and islet transplantation is still regarded as an "experimental therapy".At the end of the 1990s, fewer than 50% of patients achieved insulin independence at two months after islet transplantation and less than 10 % after one year [5].However, a turning point in islet transplantation was the development of an automated method for islet isolation in the mid-1980s.This method involves the progressive chemical and mechanical digestion of the pancreas in a warm collagenase solution using a digestion chamber known as a "Ricordi chamber" [6].
Purification of islets from the digested pancreatic tissue is performed by density -gradient separation using a blood cell processor IBM 2991 device (sold as COBE 2991®, Terumo BCT, Inc., Lakewood, CO, USA).This advance in digestion and islet purification has enabled the harvesting of large numbers of islets with high purity, and was important to achieving the first clinical success in islet transplantation in 1989 at Washington University in St Louis.This was a thirty six year old woman with type 1 diabetes mellitus, who received transplantation of approximately 800,000 islet equivalents, achieved normoglycemia for 22 days without insulin treatment [7].
The other turning point was the development of an effective immunosuppressive regimen for islet transplantation.In 1990, the Pittsburg group achieved success in prolonging insulin independence for over 3 months in a clinical allogeneic islet transplantation study using tacrolimus (FK506) [8].Tacrolimus is an inhibitor of calcineurin, which is required for T-cell receptor induction of interleukin-2 (IL-2) and for T cell proliferation.Tacrolimus has a superior safety profile compared to cyclosporine, an earlier calcineurin inhibitor [9,10].At the end of 1990s, the Edmonton group developed an islet transplantation protocol using the steroid-free immunosuppressive agents sirolimus, daclizumab and tacrolimus.In a study involving seven patients with severe type 1 diabetes, all were able to function without insulin treatment and no episodes of hypoglycemic coma were reported [11].Sirolimus (rapamycin) inhibits the activation of T and B cells by suppressing the multifunctional serine-threonine kinase mTOR (mammalian target of rapamycin), which is required for efficient production of IL-2 [12,13].Daclizumab is a monoclonal antibody directed against CD25, a component of IL-2 receptor, and thereby blocks the formation of the high-affinity IL-2 receptor.
Daclizumab can prevent acute rejection by inhibiting the expansion of cytotoxic T cells [14].The recommended protocol employed today uses antithymocyte globulin (ATG) plus the recombinant soluble tumor necrosis factor receptor protein etanercept as induction immunosuppressant agents, followed by tacrolimus or cyclosporine along with mycophenolate mofetil (an inhibitor of purine biosynthesis) for immunosuppression maintenance.The Minnesota group tested this protocol on six recipients and four of them became insulin-independent for a mean of 3 years [15].
The outcome for clinical islet transplantation has dramatically improved over the past 50 years due to technological improvements.A report in 2005 by the Edmonton group analyzing the long-term outcomes of their 65 patients showed that approximately 80 percent of them achieved successful islet engraftment at five years after transplantation (i.e.detection of serum C-peptide and reactivity to glucose stimulation), but only 10 % of the patients remained free from insulin treatment [16].A recent report from the Collaborative Islet Transplant Registry (CITR: a registry of clinical islet transplant cases performed in USA, Europe or Australia) indicated that the rates of insulin independence at three years after transplantation have been improving (44 % in 2007 -2010 era vs. 27 % in 1999 -2002 era).The positive fasting C-peptide levels (≥ 0.3 ng/mL) were also significantly higher in the period 2007 -2010 versus 1999 -2002 (90 % vs. 60 % at three years after transplantation) [17].Moreover, it was observed that approximately 80 % of recipients who had received ≥600,000 total islet equivalents achieved insulin independence, compared to 55 % who had received <600,000 islet equivalents [18].Islet transplantation is therefore now considered a practical option for treating severe diabetes mellitus in order to improve endocrinal function and to prevent hypoglycemic attack, but the current clinical outcome is still not satisfactory.The key points in obtaining a positive outcome are the acquisition of large numbers of islets from the donor pancreas, prevention of graft loss in the early stage of transplantation and maintaining engraftment for long period.Another key factor influencing engraftment is the transplant site, and the outcome of clinical islet transplantation could be further improved by utilizing a more optimal transplant site.

Candidate Transplantation Sites for Islets
What would be an optimal site for islet transplantation?We would define it by the following three criteria: 1) sites with an abundant, oxygen-and nutrient-rich blood flow, 2) sites that are privileged immunologically to minimize transplant graft loss, and 3) sites where transplantation can be performed with minimum invasiveness.To date, many organs have been assessed including the liver [19][20][21], renal subcapsular space [19,20], omental pouch [22,23], mesentery [24], gastrointestinal tract [25], skeletal muscle [26], subcutaneous tissue [26], eye [27], brain [28], testis [29,30], bone marrow [31], thymus [32], and spleen [33].However, it has been difficult to find a site that meets all three criteria (Table 1).(percutaneous infusion into intrahepatic portal vein using ultrasonography under local anesthesia) [34].On the other hand, the liver also has some problems as a transplant site.Many islets are destroyed in the early stages of transplantation due, in part, to hypoxia caused by ischemia.The isolated islets are in an avascular state throughout the process of preparation [35] and suffer from hypoxia in the hypo-oxygenized portal venous blood (the mean PO2 of approximately 5 mmHg [36]) until revascularization occurs.Moreover, the islets themselves can be a cause of liver ischemia by embolizing the peripheral portal vein [37,38].Another issue is inflammation and immunity.The transplanted islets are frequently the subject of an innate immune response and are attacked by Kupffer cells, tissue macrophages in the liver [39,40] as well as by natural killer cells [41], and this 4 may in turn induce an adaptive immune response.Furthermore, infusion of islets into the blood stream can trigger the instant blood-mediated inflammatory reaction (IBMIR), which can damage intraportal transplanted islets [42].The IBMIR is triggered by the exposure of islet surface molecules during the process of islet isolation and purification [43,44].One such surface molecule is tissue factor (coagulation factor III), which causes the rapid binding of platelets, leading to coagulation and activation of complement systems.Most of the islets are destroyed by this reaction within 1 hour after transplantation [43].Some immunosuppressants can be more toxic to islets in the liver, as their concentration is higher in the portal vein than in peripheral vessels [45].Other complications of intraportal islet transplantation include portal hypertension and portal vein thrombosis.Portal hypertension can be a risk factor for post-transplant bleeding, portal vein thrombosis and sepsis [46,47].Portal vein thrombosis is a critical complication in islet transplantation and can cause esophageal varices, splenomegaly, mesenteric ischemia, sepsis and death [48].
A common islet transplantation site in experimental studies, especially rodent, is the kidney (i.e. renal subcapsular space).There are some reports of islet transplantation into the kidney that have led to the restoration of normoglycemia.These studies have used relatively small numbers of islets, as it is difficult to transplant large amounts of islets into the human renal subcapsular space because it is rather inelastic and tight [49].This may be why clinical progress in renal subcapsular islet transplantation has lagged [50].Muscle and subcutaneous tissues have also been examined as candidate transplantation sites, as the transplantation procedure and biopsies can be performed easily with minimal invasion and few complications.These sites suffer from hypovascularity and hypoxia, and transplantation efficacy could be improved if these obstacles were overcome, especially in subcutaneous tissue [26].Another problem with these sites is systemic insulin release.
In general, secreted insulin from the pancreas flows into the liver via the portal vein, and therefore smaller amounts of insulin are needed to control blood glucose.This is referred to as physiological insulin secretion, as opposed to systemic insulin release.When islets are transplanted into intramuscular and subcutaneous sites, resulting in systemic insulin release, a much larger amount of insulin needs to be produced, as the insulin does not enter the portal system directly.The large amount of insulin required to be produced by the transplanted islets in order to control blood glucose is similar that required by insulin injection therapy.Another favorable islet transplantation site is the omental pouch.It has advantages in that the insulin drainage is via the portal vein, thus closer to physiological, and this site is highly vascularized [51].There has been much progress in intra-omental pouch islet transplantation in rodent [23], dog [52] and nonhuman primate models [53].In particular, because the omental is highly vascularized, this site has been proposed as an alternative site for encapsulated islet transplantation [54][55][56], but to date no clinical trials have been performed.The mesentery is also considered a candidate islet transplant site due to its rich vascularization and ability to accommodate a large number of islets, however one disadvantage is that if there is any trouble with the graft it would be difficult to remove it without damage to the intestinal tract [57].The submucosal space of the gastrointestinal tract is another candidate site that has a rich vascular supply providing oxygen and nutrients and connects to the same portal system as the liver, spleen and pancreas [51].Hara and colleagues have studied transplantation into this location by endoscopy in a pig model [25,58], but there has been limited demonstration of this concept in large animal models.The brain, testis, the anterior to chamber of the eye, and the thymus are the organs where the immunological response is suppressed and are thus considered "immune privileged" sites.The "immune privilege" of these sites was once assumed to be due to lack of cellular infiltration and lymphatic drainage [59], but more recently it has been shown that this is provided by a complex of immune responses [60].For example, the brain, testis and retina-blood barrier are maintained in an immunosuppressed condition due to a cellular physical shield [60][61][62].
In some cases, regulatory T cells (Tregs) also contribute to immune privilege.Larocque and colleagues showed that the immune response in the brain could be normally activated when CD4+CD25+ Tregs were depleted [63].Hedger further revealed that rodent testes contain significant numbers of immunoregulatory cells, including Tregs [64].And recently, Farooq showed that Tregs contribute to immune tolerance in the rodent anterior chamber when challenged by myelin antigen  [65].Many experimental trials have investigated allo-and xenogeneic islet transplantation into immune privileged sites in using non-human animals.While such studies in animals have demonstrated the effectiveness of transplantation into immune privileged sites [27,28,30,32], little has been done in a human clinical setting.In particular, the brain or eye are problematic sites for transplantation, as it would be difficult to remove a graft without damage in case of graft failure.

Characteristics of the Spleen as an Islet Transplant Site
Among the candidate islet transplant sites, the spleen may come closest to being an ideal site.
The spleen is a highly vascularized organ which receives blood from the splenic artery and drains into the portal venous system.Vascularization is the most important factor determining the success of transplantation, and the spleen provides a rich oxygen and nutrition supply.Another advantage is that islets transplanted into the spleen can achieve physiological levels of insulin secretion, as insulin produced by pancreatic β cells flows into the portal -splenic vein (portal venous circulation) [66].In contrast, insulin provided by a subcutaneous pump or by injection is delivered directly into systemic circulation.Recent advances in these insulin injection systems enable them to achieve close to physiological insulin release profiles (i.e. in the portal system), but there is still a limitation in dayto-day changes in insulin sensitivity [67].As the spleen connects to the portal venous system, as does the liver and pancreas, insulin released from transplanted islets flows into the splenic vein.
The spleen is the site responsible for immune tolerance, and tends to be somewhat immunosuppressed, although this suppression is weaker than that found in immune privileged sites such as the testis or thymus.Previous studies have revealed that the spleen is involved in the suppression of T cell proliferation and antibody production following the induction of immune tolerance [68,69].Other studies have shown that splenic dendritic cells are a good source of suppressor cytokines, including transforming growth factor-β (TGFβ).The splenic T cell population was shown to include suppressor T cells [70], a cell type rebranded today as Tregs [71].Tregs in the spleen prevent antigen presentation by dendritic cells to effector T cells, and suppress proliferation of effector T cells via production of suppressor cytokines including TGFβ, interleukin (IL) 10 and IL-35 [72].Horton and colleagues performed intrasplenic allo-transplantation of islets into lymphoid-irradiated dogs that had received donor bone marrow transplantation before transplantation.In this study, the authors observed that islet graft function was maintained after total pancreatectomy without the use of immunosuppressants [73].Moreover, splenocytes themselves may help regulate autoimmunity.In a previous study, we found that we could rescue non-obese (NOD) mice from a severe diabetic condition by injection of live donor splenocytes with complete Freund's adjuvant (CFA) to eliminate autoimmunity.In contrast, NOD mice that received irradiated splenocytes all became diabetic.Attack against lymphoid cells was minimal when live splenocytes were injected into CFA-infused mice [74,75].Thus it is not too surprising that the spleen can also protect transplanted islets from innate inflammatory responses, which are a major factor contributing to islet graft failure, as are acquired immune responses.Previously, we reported that several kinds of inflammatory cytokines, including monocyte chemotactic protein-1 (MCP-1), granulocyte-colony stimulating factor (G-CSF), and high-mobility group box 1 (HMGB1), were increased in the plasma after intraportal islet transplantation [76][77][78].We also confirmed that these cytokines were significantly lower in intrasplenic transplantation in comparison with intraportal transplantation [79].
Interestingly, the spleen has been shown to be a reservoir of islet stem cells in diabetic mice.We confirmed that CD45-(nonlymphoid) splenocytes could develop into stem cells and further differentiate into islet progenitor cells, thus contributing to islet regeneration [74].Moreover, we found in a subsequent study that adult mice spleens contained putative mesenchymal stem cells expressing Hox11 (known as Tlx1, a marker of splenic stem cell [80]) but not Pdx1, an early pancreatic regeneration marker, and that were CD45-in origin [81].Lee and colleagues have provided additional evidence showing that removal of the spleen in children with severe thalassemias leads to the eventual development of insulin-dependent diabetes [82].Thus, the spleen may facilitate the proliferation of intrasplenic transplanted islets.In 1989, Wohlrab and colleagues first observed proliferation of β cells in intrasplenic transplanted islets at 200 days post-transplantation.They speculated that the proliferative response was the result of a long-term stimulation by slightly enhanced plasma glucose levels at the transplantation site [83].We also observed proliferation of intrasplenic islets transplanted into the renal subcapsule, and these transplanted islets expressed both insulin and ribonucleoside-diphosphate reductase subunit M2 b (Rrm2b) [79].The Rrm2b gene encodes the small subunit of a p53-inducible ribonucleotide reductase.Expression of Rrm2b may therefore contribute to proliferation of the transplanted islets, as this gene has a role in DNA synthesis [84].
In summary, the spleen may be close to an optimal site for islet transplantation due to its rich vascularity, physiological insulin secretion, regulation of immunity including autoimmunity, and potential for islet regeneration (Figure 1).
Du Toit and colleagues performed intrasplenic allogeneic islet transplantation in pancreatomized dogs treated with cyclosporin and showed that survival was extended in comparison with non-immunosuppressed dogs [111].Moreover, this allogeneic transplant dog model helped demonstrate the usefulness of rapamycin in transplantation [113].Andersson reported survival of allogeneic grafts from cultured islets for several weeks without the use of any immunosuppressants [114].In a xenograph model, the Washington group (Paul Lacy) succeeded in prolonging graft survival for more than 100 days using cultured islets in a rat to mouse transplant model where the recipients were treated with anti-rat and/or anti-mouse lymphocyte sera [112].These findings demonstrated the possibility of using the spleen for transplantation of allo-and xenogeneic islets.While the spleen has many advantages over other transplant sites, the efficacy of transplantation has been somewhat unclear.For example, Evans and colleagues showed that transplantation efficacy into spleen was better than that of the liver or kidney in an islet autotransplantation dog model: 90% of animals achieved normoglycemia at one month for spleen compared to 33% for liver and 0% for kidney [92] (Table 3).Using fetal porcine allotransplantation and murine transplantation models, Stokes et al. showed higher transplantation efficacy for spleen compared to liver, although kidney was better [125,126].Many other studies have reported the superiority of spleen compared to liver [96,107] or omental pouch [52,127], although some groups have reported the opposite [93][94][95] (Table 3).
Next, the route of transplantation into the spleen needs to be considered.In the earliest studies, the pulp was used as the transplant site in the spleen [86,88].After various trials, the Warnock  [118] (Table 2).Intravenous transplantation is generally regarded as preferable to intrasplenic transplantation, in part because intrasplenic transplantation carries the risk of IBMIR that can damage the transplanted islets, similar to intraportal transplantation [51].To examine the potential usefulness of the spleen as an islet transplantation site and to try to develop a better procedure for intrasplenic transplantation, we explored the "splenic subcapsular implantation technique" using a rodent syngeneic transplant model and analyzed the transplant efficacy of this method compared to intrahepatic and renal subcapsular transplantation [79].This procedure involved direct puncture from the surface with a 27-gauge needle and implantation of islets under the splenic surface without venous or pulp injury (Figure 2).Amazingly, all of the mice (n = 10) achieved normoglycemia for two months despite having received only 50 islets by intrasplenic transplantation.In contrast, none of the mice achieved normoglycemia when islets were transplanted into the liver or kidney.Thus, not only was transplantation efficacy superior to transplantation into other sites, but three to four diabetic mice could be treated by a single donor mouse using this method (i.e. 150 -200 islets can be harvested from one donor mouse).Normoglycemia could also be achieved using as few as 25 islets transplanted into the spleen when glucose levels were also rigorously managed.By histological assessment, we observed that intrasplenic transplanted islets were enlarged in size.The transplantation efficacy of this model clearly exceeded those previously reported for intrapulp and intravenous transplantation models [95,116].We speculate that this is because intrapulp and intravenous transplantation involves greater tissue damage and consequent exposure of islets to blood, thus inducing IBMIR, compared to intraspenic transplantation.In addition to preventing graft loss, intraspenic transplantation allows the engrafted islets to access a rich oxygen and nutrition supply due to an abundant blood flow.These factors, plus the privileged immune status of this site, may be responsible for a greater success of engraftment and regeneration.

Conclusion: for the Future Clinical Intrasplenic Islet Transplantation
The first intrasplenic islet transplantation was performed in a clinical setting at the University of Leicester 20 years ago.Five chronic pancreatitis patients underwent spleen-preserving total pancreatectomy and intrasplenic islet autotransplantation, and of these, two acquired insulin independence for over a year.However, the patients who underwent this procedure suffered from high morbidity, including splenic infarction and portal thrombosis [33].Du Toit reported that 13 intrasplenic islet transplantation was accompanied by some life-threatening complications including subcapsular hematoma, intrasplenic necrosis and cavitation, capsular perforation, and arteriolar thrombosis [111].However, we believe these complications could be overcome with advances in surgical procedures.In our opinion, implantation into the splenic subcapsular region may minimize the risk of necrosis, thrombosis and hemorrhage by preventing venous and pulp injury.
Laparoscopic surgery could also minimize the surgical stress of the transplantation, as can intraportal transplantation.We would suggest that with the combination of techniques described here, intraspenic transplantation may offer the most optimal approach to islet transplantation among the approaches currently available.
In conclusion, the spleen has historically been an important site for islet transplantation, but its utility could be greatly improved by the application of some recent novel findings and techniques.
We would advocate the development of clinical methods to optimize the safe and effective transplantation of islets into the spleen.

Figure 1 .
Figure 1.Summary of the Characteristics of the Spleen as a Transplantation Site for Islets.The spleen has four advantages as a site of islet transplantation: (A) rich vascularity, (B) physiological insulin secretion, (C) regulation of immunity, and (D) potential for islet regeneration.

Figure 2 .
Figure 2. A. Procedure of splenic subcapsular implantation technique.B. Engrafted islets (indicated by arrows) under the capsule of spleen at 28 days after transplantation.Left: hematoxylin and eosin staining, Right: immunostaining for insulin.

Table 1 .
Candidate Islet Transplantation Sites other than Spleen.