**2.2. Alloimmunity**

**1.2. Post-transplant outcome**

168 Type 1 Diabetes

back the enthusiasm in this field.

transplanted islet mass [6].

**2.1. Early events after islet transplantation**

**2. Molecular mechanism of beta cell dysfunction**

According to a recent report from the Collaborative Islet Transplant Registry, 677 patients have received either an islet transplant alone (ITA) or islets-after-kidney (IAK) transplants [3]. There has been a remarkable improvement in the post-transplant graft function in recent times. Prior to the publication of Edmonton protocol, the achievement of insulin-independ‐ ent status by islet transplant recipients was <10%. Patients treated initially under the Ed‐ monton protocol showed remarkable achievement of 82% insulin-independent status at one year post-transplant. However, this result proved to be unsustainable when the five year in‐ sulin-independence rates fell to 12.5% at the same center [4]. This data resulted in skepticism on the use of allogeneic islet transplantation as a reliable treatment for long-term success. With the introduction of thymoglobulin at induction phase and the combination of prograf, rapamycin and/or mycophenolate mofetil as maintenance immunosuppressive agents, the islet transplant survival rate has significantly improved to 50% at five year post-transplant [5]. Control of inflammatory reaction during peri-transplant period with the use of TNF-α blockers also played a key role in this improvement. These remarkable results necessitated comparison with whole pancreas transplantation which is considered as an established clini‐ cal procedure. Although whole organ treatment achieved high levels of graft survival in the years 1994-1997, the islet survival rate at five years has reached around fifty percent in 2010-2011, comparable to the level of whole pancreas graft success [5]. Moreover, islet cell transplantation seems to confer significantly better glycemic control than maximal medical therapy, and essentially eliminates hypoglycemic unawareness. These results have brought

The liver is the most commonly used site for transplantation of islets. Data supporting the use of this transplant site came from autologous islet transplants in patients with chronic pancreatitis, which showed that islets can function inside the liver for several years. There are several drawbacks associated with the liver as a host site for islets. Major factors affect‐ ing islet function include hypoxia, drug toxicity and instant blood-mediated inflammatory reaction (IBMIR). Together, these events may lead to loss of up to 75% of islet transplant mass. IBMIR is primarily a response of innate immune system to isolated islets. Major char‐ acteristics of IBMIR include activation of coagulation and complement cascades and infiltra‐ tion of inflammatory cells. Several approaches are adopted to minimize the deleterious effects of IBMIR which include infusion of low molecular weight dextran sulfate and also inclusion of anti-inflammatory molecules during the infusion of islets. Besides the innate im‐ mune response, islets transplanted into liver may experience low oxygen tension. Activation of resident Kupffer cells may pose additional risk to islet survival. In addition, high concen‐ trations of immunosuppressive drugs in the portal vein are likely to exert toxic effect on the The exposure of body to allogeneic tissues via organ/cell transplantation, blood transfusions, pregnancy can cause development of anti-human leukocyte antigen (HLA) antibodies [7]. These *de novo* HLA antibodies have been shown to play a significant role in the early graft loss after solid organ transplantation [8]. Currently, HLA matching between the recipients and donors is not performed before islet cell transplantation. Moreover, to achieve and/or maintain insulin independence and good metabolic control in an islet recipient, multiple is‐ let infusions from multiple donors and high doses of immunosuppressants are generally re‐ quired. The requirement of multi-donor infusions and reduction or weaning of immunosuppressants due to significant adverse effects could cause patients eventually to develop HLA antibodies against islet graft.

The issue of sensitization of alloantigens after islet cell transplantation has been raised by the Edmonton group in 2007 [9]. 98 islet transplant recipients were screened for HLA antibodies by flow-based methods. Twenty-nine patients (31%) represented *de novo* donor specific anti‐ bodies following islet transplantation. Among 14 recipients who discontinued immunosup‐ pression, 10 recipients (71%) were largely sensitized with panel reactive antibody ≥50%. On the other hand, only 11 of 69 (16%) recipients who continued immunosuppression became broadly sensitized posttransplant. This study suggested that development of HLA antibod‐ ies after islet transplantation is concerning and withdrawal of immunosuppression complete‐ ly following failed islet transplantation raises the risk for broad sensitization. Along with the report of Edmonton group, there are several studies that have demonstrated that islet alone transplant recipients develop donor-specific and/or nondonor-specific HLA antibodies, espe‐ cially following discontinuation of immunosuppression [10-13].

In contrast, in the report of Geneva group it was shown that multiple islet infusions did not act as a risk factor for appearance of anti-HLA antibodies [14]. The group claimed that trans‐ plantation of islets in liver might cause less immunogenicity. After combined kidney-islet transplantation and continued immunosuppression even with failed islet graft function, pa‐ tients had a low risk for sensitization as long as their kidney remained functional.

It has been known that islets express mainly HLA class I antigens on their surfaces. Previous reports demonstrated that patients develop antibodies posttransplant not only against HLA class I antigens, but also against HLA class II antigens [9]. Jackson et al. showed that there would be an induction of HLA class II expression on human islets under inflammatory con‐ ditions, which in return may be a possible cause of allosensitization [15]. For this aim, the group conducted an experiment in which they had two groups of isolated human islets; group 1 was control group and cultured at 37˚C, whereas group 2 was cultured in the same condition and treated with tumor necrosis factor alpha (TNF-α) and interferon gamma (INFγ). Presence of HLA class II on islet surface was analyzed by real-time polymerase chain re‐ action (PCR), immunofluorescence and flow cytometry. Expression of class II transactivator, HLA-DR-α and HLA-DR-β1 increased maximum 9.38, 18.95 and 46.5 fold respectively in group 2 compared to control group after 24 hours of incubation with TNF-α and INF-γ which is shown by real-time PCR analysis. Fluorescent imaging and flow cytometric analy‐ sis confirmed the significant increase in the expression of HLA class II expression both on islet α and β cells after cytokine treatment. Inflammatory conditions shortly after islet trans‐ plantation up-regulates HLA class II antigens on islet surfaces that trigger alloimmunity. Thus, protocols which provide adequate and efficient control of inflammation after islet transplantation should be considered to improve islet transplant outcome.

Soluble CD30 (sCD30) is a cell membrane protein of tumor necrosis factor receptor family. sCD30 is released into blood with the activation of CD30 + T cells, leading to speculation that it may act as a marker for immune system activation [21]. Although it has been shown to be predictive for acute rejection in lung, kidney, and heart transplantation [22-24], there are not many reports about the role of sCD30 in the prediction of early graft loss following islet transplantation. In the study of Hire et al., 19 allograft islet recipients treated with three different immunosuppression inductions were evaluated retrospectively for the serum sCD30 levels [18]. Pretransplant, early posttransplant day (day 4−7), one month posttrans‐ plant, late posttransplant (day 90−120) sCD30 levels were measured and correlated with islet graft outcomes at 1 year. No correlation between sCD30 levels at any time point and graft function at 1 year was found. However, a greater reduction in SCD30 levels posttransplant was associated with full graft function. Therefore, sCD30 may be of value for immune moni‐

Beta Cell Function After Islet Transplantation http://dx.doi.org/10.5772/ 52952 171

Cytotoxic lymphocyte (CL) genes granzyme, Fas ligand and perforin may play an active role in the course of acute allograft rejection. University of Miami group studied 13 islet trans‐ plant recipients treated with steroid-free immunosuppressive regimen in order to demon‐ strate whether CL gene expression could be a predictor of allogeneic rejection [19]. All patients attained insulin independence; however, 8 of them restarted insulin therapy. Realtime PCR was used to assess CL gene mRNA levels. The group demonstrated that recipients who restarted insulin therapy had a significant elevation of CL gene mRNA levels and the most reliable measure of ongoing graft loss was granzyme B. Hence, increased blood CL

Microparticles (MP) are plasma membrane fragments of apoptotic cells in peripheral blood. The quantity of microparticles is correlated with the degree of cell death, so they are consid‐ ered to be indicators of apoptosis. Kessler et al. demonstrated the elevation of microparticles in peripheral blood at the time of acute rejection following intraportal islet transplantation with a case report [25]. Loss of islet graft function without the presence of GAD65, IA2 or anti-HLA antibodies brought up the diagnosis of acute cellular rejection. With a successful steroid bolus therapy, MPs level declined and the patient regained islet function. In 2011, Toti et al. [20] demonstrated from three islet transplant recipients that in the case of rejec‐ tion, C-peptide and MPs levels exhibited opposite pattern and a decline in C-peptide was related with increased insulin needs. This data suggested an increment in MPs level might indicate allogeneic rejection. Thus, MPs level in peripheral blood might be a useful tool to

Type 1 diabetes is an autoimmune disease in which pancreatic beta cells are destroyed through a T-cell mediated mechanism in genetically susceptible individuals [26]. Autoanti‐ bodies against pancreatic islets comprise anti-glutamate decarboxylase 65 (GAD65), islet cell autoantibody (ICA), anti-insulin autoantibody (IAA), anti-tyrosine phosphatase autoanti‐ body (IA-2) and against zinc transporter ZnT8. Antibodies present in serum against these pancreatic islet antigens are commonly used to predict and or diagnose the disease in clini‐

gene levels might be a potential marker to predict islet allograft loss.

monitor allogeneic rejection after islet transplantation.

**2.3. Autoimmune recurrence**

toring of islet allografts.

Collaborative Islet Transplant Registry reported the sensitization rates against HLA class I antigens pre- and posttransplant in islet alone recipients in 2011 [16]. Data is collected from 303 islet alone recipients between January 1999 and December 2008. Panel reactive antibody (PRA) pretransplant and PRA at 6 months and yearly posttransplant correlated to measures of islet graft failure. Pretransplant PRA showed not to be a predictor of islet graft failure; whereas there was 3.6 fold increased hazard ratio for graft failure when the recipient devel‐ oped PRA ≥20% post-transplant. Each additional islet infusion increased the cumulative number of mismatched HLA alleles from a median of 3 to 9; respectively for one infusion and for 3 infusions. Significantly higher rate of PRA ≥ 20% was observed in recipients who had com‐ plete graft loss with discontinued immunosuppression compared to recipients who had func‐ tioning grafts with continuing immunosuppression. Development of *de novo* HLA class I antibodies is less pronounced in recipients with exposure to repeat HLA class I mismatched than increased class I mismatch. Reducing the number of islet donors used for each patient and repeating HLA I mismatches with consequent islet transplantation without presence of donor specific anti-HLA antibodies are vital factors to decrease the risk of allosensitization.

Currently, there is no clearly defined monitoring tool for alloimmunity in islet cell trans‐ plantation, but researchers have proposed many experimental tools to assess alloreactivity in islet transplanted patients. Alloantibodies, soluble CD30 level, cytotoxic lymphocyte gene expression and microparticles in peripheral blood are the markers which were shown to de‐ tect allogeneic rejection after islet transplantation. Monitoring panel reactive antibody in im‐ munosuppressed recipients had little clinical value to assess islet graft survival [16, 17].


**Table 1.** Immunologic tools to assess alloimmunity after islet cell transplantation

Soluble CD30 (sCD30) is a cell membrane protein of tumor necrosis factor receptor family. sCD30 is released into blood with the activation of CD30 + T cells, leading to speculation that it may act as a marker for immune system activation [21]. Although it has been shown to be predictive for acute rejection in lung, kidney, and heart transplantation [22-24], there are not many reports about the role of sCD30 in the prediction of early graft loss following islet transplantation. In the study of Hire et al., 19 allograft islet recipients treated with three different immunosuppression inductions were evaluated retrospectively for the serum sCD30 levels [18]. Pretransplant, early posttransplant day (day 4−7), one month posttrans‐ plant, late posttransplant (day 90−120) sCD30 levels were measured and correlated with islet graft outcomes at 1 year. No correlation between sCD30 levels at any time point and graft function at 1 year was found. However, a greater reduction in SCD30 levels posttransplant was associated with full graft function. Therefore, sCD30 may be of value for immune moni‐ toring of islet allografts.

Cytotoxic lymphocyte (CL) genes granzyme, Fas ligand and perforin may play an active role in the course of acute allograft rejection. University of Miami group studied 13 islet trans‐ plant recipients treated with steroid-free immunosuppressive regimen in order to demon‐ strate whether CL gene expression could be a predictor of allogeneic rejection [19]. All patients attained insulin independence; however, 8 of them restarted insulin therapy. Realtime PCR was used to assess CL gene mRNA levels. The group demonstrated that recipients who restarted insulin therapy had a significant elevation of CL gene mRNA levels and the most reliable measure of ongoing graft loss was granzyme B. Hence, increased blood CL gene levels might be a potential marker to predict islet allograft loss.

Microparticles (MP) are plasma membrane fragments of apoptotic cells in peripheral blood. The quantity of microparticles is correlated with the degree of cell death, so they are consid‐ ered to be indicators of apoptosis. Kessler et al. demonstrated the elevation of microparticles in peripheral blood at the time of acute rejection following intraportal islet transplantation with a case report [25]. Loss of islet graft function without the presence of GAD65, IA2 or anti-HLA antibodies brought up the diagnosis of acute cellular rejection. With a successful steroid bolus therapy, MPs level declined and the patient regained islet function. In 2011, Toti et al. [20] demonstrated from three islet transplant recipients that in the case of rejec‐ tion, C-peptide and MPs levels exhibited opposite pattern and a decline in C-peptide was related with increased insulin needs. This data suggested an increment in MPs level might indicate allogeneic rejection. Thus, MPs level in peripheral blood might be a useful tool to monitor allogeneic rejection after islet transplantation.

#### **2.3. Autoimmune recurrence**

islet α and β cells after cytokine treatment. Inflammatory conditions shortly after islet trans‐ plantation up-regulates HLA class II antigens on islet surfaces that trigger alloimmunity. Thus, protocols which provide adequate and efficient control of inflammation after islet

Collaborative Islet Transplant Registry reported the sensitization rates against HLA class I antigens pre- and posttransplant in islet alone recipients in 2011 [16]. Data is collected from 303 islet alone recipients between January 1999 and December 2008. Panel reactive antibody (PRA) pretransplant and PRA at 6 months and yearly posttransplant correlated to measures of islet graft failure. Pretransplant PRA showed not to be a predictor of islet graft failure; whereas there was 3.6 fold increased hazard ratio for graft failure when the recipient devel‐ oped PRA ≥20% post-transplant. Each additional islet infusion increased the cumulative number of mismatched HLA alleles from a median of 3 to 9; respectively for one infusion and for 3 infusions. Significantly higher rate of PRA ≥ 20% was observed in recipients who had com‐ plete graft loss with discontinued immunosuppression compared to recipients who had func‐ tioning grafts with continuing immunosuppression. Development of *de novo* HLA class I antibodies is less pronounced in recipients with exposure to repeat HLA class I mismatched than increased class I mismatch. Reducing the number of islet donors used for each patient and repeating HLA I mismatches with consequent islet transplantation without presence of donor specific anti-HLA antibodies are vital factors to decrease the risk of allosensitization.

Currently, there is no clearly defined monitoring tool for alloimmunity in islet cell trans‐ plantation, but researchers have proposed many experimental tools to assess alloreactivity in islet transplanted patients. Alloantibodies, soluble CD30 level, cytotoxic lymphocyte gene expression and microparticles in peripheral blood are the markers which were shown to de‐ tect allogeneic rejection after islet transplantation. Monitoring panel reactive antibody in im‐ munosuppressed recipients had little clinical value to assess islet graft survival [16, 17].

**Team Approach Outcome References**

after islet transplantation.

function at 1 year was found.

rejection of islet allograft.

had little clinical value for islet graft survival.

was associated with full graft function.

Increased CL gene levels could predict islet

A greater reduction in sCD30 levels posttransplant

MPs levels in peripheral blood increase with acute

[9]

[16]

[18]

[19]

[20]

**Edmonton group** Alloantibodies Pretransplant HLA antibodies reduce graft survival

**CITR report** Alloantibodies Monitoring PRA in immunosuppressed patients

**Minnesota group** Soluble CD30 No correlation between sCD30 levels and graft

**GRAGIL group** Microparticles MPs and C-peptide showed opposite pattern.

**Table 1.** Immunologic tools to assess alloimmunity after islet cell transplantation

allograft loss.

**Miami group** Cytotoxic lymphocyte (CL) gene expression

transplantation should be considered to improve islet transplant outcome.

170 Type 1 Diabetes

Type 1 diabetes is an autoimmune disease in which pancreatic beta cells are destroyed through a T-cell mediated mechanism in genetically susceptible individuals [26]. Autoanti‐ bodies against pancreatic islets comprise anti-glutamate decarboxylase 65 (GAD65), islet cell autoantibody (ICA), anti-insulin autoantibody (IAA), anti-tyrosine phosphatase autoanti‐ body (IA-2) and against zinc transporter ZnT8. Antibodies present in serum against these pancreatic islet antigens are commonly used to predict and or diagnose the disease in clini‐ cal practice. For successful islet cell replacement, it is crucial to prevent recurrent destruction of beta cells through existing autoimmune destruction. The graft failure due to recurrent au‐ toimmunity in a pancreas segment transplanted between identical twins was proven with the demonstration of insulitis in the transplanted tissue [27]. Islet specific T cells seem to have a basic role in the process of autoimmune destruction of beta cells [28].

Autoimmunity recurrence might be assessed by monitoring islet specific autoantibodies and T-cell autoreactivity. But the association between autoantibodies and insulin independence and islet graft outcome are variable; increase in autoantibody levels were shown due to au‐ toimmune activity but did not indicate loss of islet graft function [29, 32]. Assays that meas‐ ure anti-islet cellular autoimmunity before and after islet transplantation demonstrated that pre-and posttransplant cellular autoimmunity were related with delayed insulin independ‐ ence and lower levels of circulating C-peptide during the first year posttransplant [30]. Nonetheless, in this study islet allograft outcome did not seem to be affected by autoanti‐

Beta Cell Function After Islet Transplantation http://dx.doi.org/10.5772/ 52952 173

Matsumoto et al. have reported on a global immune assay specific for GAD65 (EpiMax) in order to analyze the property of autoreactive T-cell responses [31]. Five type 1 diabetic pa‐ tients were studied 1 year after allogeneic islet transplantation. All patients achieved insulin independence at 1 year. Three out of five patients maintained long-term insulin independ‐ ence and EpiMax affirmed minimum T-cell responses in these patients. In contrast, the two patients who developed chronic graft failure and lost insulin independence showed broad repertoire of GAD65 specific T-cells secreting various types of cytokines, including IL-5, IL-13, IL-17, TNF- alpha, and IFN-gamma. In addition to those observations, IFN-γ and IL-13 expressing CD4+ T cells and IFN-γ expressing CD8+ T cells were encountered in the other two failed patients. These findings suggested that broad repertoire of islet antigenspecific T cells which secrete variable types of cytokines were related with chronic graft fail‐

ure, preventing islet recipients from maintaining long-term insulin independence.

Following transplantation of islets, administration of immunosuppression is essential to maintain graft function. However, most of the immunosuppressive drugs also have adverse effects on beta cell function. Careful selection of immunosuppressive regimen is critical for

Corticosteroid was a widely used agent as maintenance immunosuppression in the pioneer‐ ing days of islet cell transplantation in 1990's (Table 3). During this decade, majority of islet cell transplants were after or performed simultaneously with kidney transplantation. Corti‐ costeroid has antiinflammatory as well as immunosuppressive effects by direct or indirect actions on various leukocytes, including T lymphocytes, monocytes and macrophages, through glucocorticoid receptor [33, 34]. However, steroid therapy leads to β cell dysfunc‐ tion and insulin resistance. [35, 36] Deterioration of insulin secretion from β cell by steroid treatment has been reported, caused by enhanced α-adrenergic receptor signaling [37], β cell

skeletal muscle by long-term steroid administration are well known clinically and in basic studies [40-42]. Thus, steroid use for the purpose of maintenance immunosuppression has

been averted in the recent decade of islet transplantation (Table 3).

channel [39]. Insulin resistance in liver, adipose tissue and

body levels or cellular alloreactivity.

*Immunosuppression*

prolonged function of transplanted islets.

*2.3.1. Early period of islet cell transplantation*

apoptosis [38] and activated K+

To investigate T-cell allo- and autoreactivities in peripheral blood following islet transplan‐ tation, Roep et al. examined 7 islet allograft recipients [29]. They showed that three patients who got thymoglobulin for induction immunosuppression and retained full islet function for more than 1 year exhibited minor autoreactivites but no alloreactivities. Three patients who did not get thymoglobulin had rapid decline (<3 weeks) in islet function and showed alloreactivities; but one out of these three patients had rapid increase in autoreactivity to several islet autoantigens prior to alloreactivity. One recipient who did not receive thymo‐ globulin exhibited hyperautoreacivity with no detectable alloreaactivity and developed de‐ layed loss of islet graft function consequently (<33 weeks); which indicated that autoimmune recurrence might be the cause of chronic islet graft dysfunction. In this study, because of the excellent outcomes in thymoglobulin group, the authors evaluated allo- and autoimmunity again in a bigger sample sized group in 2008 [30]. 21 islet recipients under thymoglobulin induction and tacrolimus plus mycophenolate mofetil maintenance immuno‐ suppressive regimen were studied. Immunity against allo- and autoantigens were checked at pretransplant and at 1 year posttransplant. The analyses showed that existence of cellular autoimmunity pretransplant and posttransplant was related with delayed insulin independ‐ ence and lower levels of circulating C-peptide during the first year posttransplant. Seven out of eight patients with no previous T-cell autoreactivity achieved insulin independence; whereas none of the four patients with autoantibodies against GAD and IA-2 before trans‐ plantation became insulin independent. Cellular alloreactivity and autoantibody levels did not show significant involvement with the outcome. Based on these findings, the authors commented that thymoglobulin may cope sufficiently with alloimmunity, but insufficient to control islet autoreactivity in an early period. The issue of autoimmunity remains unad‐ dressed and needs further investigation.


**Table 2.** Immunologic tools to assess autoimmunity after islet cell transplantation

Autoimmunity recurrence might be assessed by monitoring islet specific autoantibodies and T-cell autoreactivity. But the association between autoantibodies and insulin independence and islet graft outcome are variable; increase in autoantibody levels were shown due to au‐ toimmune activity but did not indicate loss of islet graft function [29, 32]. Assays that meas‐ ure anti-islet cellular autoimmunity before and after islet transplantation demonstrated that pre-and posttransplant cellular autoimmunity were related with delayed insulin independ‐ ence and lower levels of circulating C-peptide during the first year posttransplant [30]. Nonetheless, in this study islet allograft outcome did not seem to be affected by autoanti‐ body levels or cellular alloreactivity.

Matsumoto et al. have reported on a global immune assay specific for GAD65 (EpiMax) in order to analyze the property of autoreactive T-cell responses [31]. Five type 1 diabetic pa‐ tients were studied 1 year after allogeneic islet transplantation. All patients achieved insulin independence at 1 year. Three out of five patients maintained long-term insulin independ‐ ence and EpiMax affirmed minimum T-cell responses in these patients. In contrast, the two patients who developed chronic graft failure and lost insulin independence showed broad repertoire of GAD65 specific T-cells secreting various types of cytokines, including IL-5, IL-13, IL-17, TNF- alpha, and IFN-gamma. In addition to those observations, IFN-γ and IL-13 expressing CD4+ T cells and IFN-γ expressing CD8+ T cells were encountered in the other two failed patients. These findings suggested that broad repertoire of islet antigenspecific T cells which secrete variable types of cytokines were related with chronic graft fail‐ ure, preventing islet recipients from maintaining long-term insulin independence.

#### *Immunosuppression*

cal practice. For successful islet cell replacement, it is crucial to prevent recurrent destruction of beta cells through existing autoimmune destruction. The graft failure due to recurrent au‐ toimmunity in a pancreas segment transplanted between identical twins was proven with the demonstration of insulitis in the transplanted tissue [27]. Islet specific T cells seem to

To investigate T-cell allo- and autoreactivities in peripheral blood following islet transplan‐ tation, Roep et al. examined 7 islet allograft recipients [29]. They showed that three patients who got thymoglobulin for induction immunosuppression and retained full islet function for more than 1 year exhibited minor autoreactivites but no alloreactivities. Three patients who did not get thymoglobulin had rapid decline (<3 weeks) in islet function and showed alloreactivities; but one out of these three patients had rapid increase in autoreactivity to several islet autoantigens prior to alloreactivity. One recipient who did not receive thymo‐ globulin exhibited hyperautoreacivity with no detectable alloreaactivity and developed de‐ layed loss of islet graft function consequently (<33 weeks); which indicated that autoimmune recurrence might be the cause of chronic islet graft dysfunction. In this study, because of the excellent outcomes in thymoglobulin group, the authors evaluated allo- and autoimmunity again in a bigger sample sized group in 2008 [30]. 21 islet recipients under thymoglobulin induction and tacrolimus plus mycophenolate mofetil maintenance immuno‐ suppressive regimen were studied. Immunity against allo- and autoantigens were checked at pretransplant and at 1 year posttransplant. The analyses showed that existence of cellular autoimmunity pretransplant and posttransplant was related with delayed insulin independ‐ ence and lower levels of circulating C-peptide during the first year posttransplant. Seven out of eight patients with no previous T-cell autoreactivity achieved insulin independence; whereas none of the four patients with autoantibodies against GAD and IA-2 before trans‐ plantation became insulin independent. Cellular alloreactivity and autoantibody levels did not show significant involvement with the outcome. Based on these findings, the authors commented that thymoglobulin may cope sufficiently with alloimmunity, but insufficient to control islet autoreactivity in an early period. The issue of autoimmunity remains unad‐

**Team Approach Outcome References**

independence.

chronic graft failure.

outcome.

activity, but did not indicate loss of graft function.

Pre- and posttransplant cellular autoimmunity

Autoantibody levels did not affect islet allograft

Broad repertoire of islet antigen-specific T cells secreting various cytokines were related with

were associated with delayed insulin

[29]

[30]

[31]

Roep *et al*. Autoantibodies Autoandibodies increased due to autoimmune

have a basic role in the process of autoimmune destruction of beta cells [28].

dressed and needs further investigation.

172 Type 1 Diabetes

Roep *et al*. T-cell autoreactivity

Matsumoto *et al*. GAD65 specific global

immune assay

**Table 2.** Immunologic tools to assess autoimmunity after islet cell transplantation

in peripheral blood

Following transplantation of islets, administration of immunosuppression is essential to maintain graft function. However, most of the immunosuppressive drugs also have adverse effects on beta cell function. Careful selection of immunosuppressive regimen is critical for prolonged function of transplanted islets.

#### *2.3.1. Early period of islet cell transplantation*

Corticosteroid was a widely used agent as maintenance immunosuppression in the pioneer‐ ing days of islet cell transplantation in 1990's (Table 3). During this decade, majority of islet cell transplants were after or performed simultaneously with kidney transplantation. Corti‐ costeroid has antiinflammatory as well as immunosuppressive effects by direct or indirect actions on various leukocytes, including T lymphocytes, monocytes and macrophages, through glucocorticoid receptor [33, 34]. However, steroid therapy leads to β cell dysfunc‐ tion and insulin resistance. [35, 36] Deterioration of insulin secretion from β cell by steroid treatment has been reported, caused by enhanced α-adrenergic receptor signaling [37], β cell apoptosis [38] and activated K+ channel [39]. Insulin resistance in liver, adipose tissue and skeletal muscle by long-term steroid administration are well known clinically and in basic studies [40-42]. Thus, steroid use for the purpose of maintenance immunosuppression has been averted in the recent decade of islet transplantation (Table 3).

The calcineurin inhibitors (CNIs) have been major players in maintenance immunosuppres‐ sion of islet cell transplantation. Cyclosporine A and tacrolimus are currently available CNIs in clinic. They inhibit calcineurin, a serine-threonine phosphatase, which is responsible for dephosphorylation of nuclear factor for activated T cells (NF-AT), which in turn results in inactivation of the transcription of cytokine genes. However, CNIs might have β cell toxicity since calcineurin is expressed in β cell and regulates β cell growth as well as function [43, 44].

**Publication year**

1993 2

**Publication year**

2000 13

2001 2

**Pts no.** **Induction**

✓ATG or ✓Bas

✓ATG or ✓Bas

**Pts no.** **Induction**

✓mALG ✓15-DSG

1997 6 ✓mPred ✓Tac

1997 8 ✓OKT3 ✓mPred ✓CsA ✓Aza

1997 20 ✓ATG ✓Pred ✓CsA ✓Aza

1997 3 ✓ATG ✓Pred ✓CsA ✓MMF

1999 12 ✓ATG ✓Pred ✓CsA ✓Aza

4 ✓Pred ✓Tac

**therapy Maintenance therapy Transplant**

7 ✓Pred ✓Tac SIK M

<sup>1998</sup> <sup>7</sup> ✓ATG (3pts) ✓Pred ✓CsA ✓Aza IAK <sup>M</sup> 2 pts

**therapy Maintenance therapy Transplant**

**Steroid CNIs Other**

✓Pred ✓CsA ✓MMF

✓Pred ✓CsA

**Table 3.** Immunosuppression protocols in clinical islet transplants published in 1990's. \*M: Multiple donor transplants, S: Single donor transplant. \*\* Not achieved II, but positive C-peptide or decreased insulin requirement was confirmed. Abbreviations; 15-DSG: 15-deoxyspergualin, ATG: antithymocyte globulin, Aza: azathioprine, CNIs: Calcineurin inhibitors, CsA: Cyclosporine A, IAK: Islet after kidney transplantation, II: Insulin Independence, ITA: Islet transplantation alone, mALG: Minnesota antilymphoblast globulin, MMF: mycophenolic mofetil, mPred: methylprednisolone, Pred: Prednisone, SIK: Simultaneous islet kidney transplantation, Tac: Tacrolimus.

> ✓Aza or ✓MMF

2000 7 ✓Dac ✓Tac ✓Sir ITA M 100% II [1]

**type**

Simultaneous Islet-Liver transplant

✓Pred ✓CsA ✓Aza SIK <sup>S</sup> 1 pt achieved

Simultaneous Islet-Liver-Bone marrow transplant

IAK (7 pts) or SIK (1 pt)

IAK (7 pts) or SIK (13 pt)

SIK (2 pts) or IAK (1 pt)

IAK (12 pts) or SIK (12pts)

**type**

SIK, IAK or Islet after lung transplant

SIK (5 pts) or IAK (2 pts)

**Donor no.\***

S

**Major outcomes**

Beta Cell Function After Islet Transplantation http://dx.doi.org/10.5772/ 52952

> Partial function\*\*

> Partial function\*\*

II

<sup>S</sup> 3 pts

M/S 2 pts

M/S 7 pts

Partial

Partial

M/S

M/S

**Donor no.\***

M/S

**Refs**

175

[53]

achieved II [54]

achieved II [55]

achieved II [56]

function\*\* [57]

achieved II [58]

function\*\* [59]

**Major outcomes**

M/S 2 pts achieved II

Partial

function\*\* [61]

**Refs**

[60]

Azathioprine is a purine analog, serving as a blocker of de novo pathway in purine synthe‐ sis in actively proliferative cells such as T cells and B cells [45]. Currently this drug is used for immunosuppression in allogeneic transplantation and autoimmune disease like rheumatoid arthritis as well as therapy in hematologic malignancies [46]. Azathioprine may also prevent the onset of diabetes [47, 48] and no major β cell toxicity of azathioprine has been reported.

#### *2.3.2. Edmonton protocol*

Remarkable success in islet transplant survival was achieved by the University of Alberta group using steroid-free immunosuppression regimen that included daclizumab, tacrolimus and sirolimus, resulting in that all 7 recipients achieving insulin independence [1]. The bene‐ fit of Edmonton protocol is to eliminate the risk of steroid-induced β cell toxicity as well as insulin resistance and increasing the dose of transplanted islets. However, the protocol uses tacrolimus that has the effect of β cell deterioration.



The calcineurin inhibitors (CNIs) have been major players in maintenance immunosuppres‐ sion of islet cell transplantation. Cyclosporine A and tacrolimus are currently available CNIs in clinic. They inhibit calcineurin, a serine-threonine phosphatase, which is responsible for dephosphorylation of nuclear factor for activated T cells (NF-AT), which in turn results in inactivation of the transcription of cytokine genes. However, CNIs might have β cell toxicity since calcineurin is expressed in β cell and regulates β cell growth as well as function [43, 44].

Azathioprine is a purine analog, serving as a blocker of de novo pathway in purine synthe‐ sis in actively proliferative cells such as T cells and B cells [45]. Currently this drug is used for immunosuppression in allogeneic transplantation and autoimmune disease like rheumatoid arthritis as well as therapy in hematologic malignancies [46]. Azathioprine may also prevent the onset of diabetes [47, 48] and no major β cell toxicity of azathioprine has been reported.

Remarkable success in islet transplant survival was achieved by the University of Alberta group using steroid-free immunosuppression regimen that included daclizumab, tacrolimus and sirolimus, resulting in that all 7 recipients achieving insulin independence [1]. The bene‐ fit of Edmonton protocol is to eliminate the risk of steroid-induced β cell toxicity as well as insulin resistance and increasing the dose of transplanted islets. However, the protocol uses

**type**

Islet after liver transplant

✓Pred ✓CsA ✓Aza IAK M/S 1 pt achieved

Simultaneous Islet-Liver transplant

**Donor no.\***

M/S 5 pts

**Major outcomes**

achieved II [49]

Rejected 2 weeks after ITA

Partial function\*\*

II

<sup>S</sup> 6 pts

II for 7, 14 and 121 days

achieved II [52]

**Refs**

[50]

[51]

**therapy Maintenance therapy Transplant**

**Steroid CNIs Other**

1991 3 ✓Pred ✓CsA ✓Aza ITA M/S

3 ✓mALG ✓Pred ✓CsA ✓Aza IAK S

3 ✓mALG ✓Pred ✓CsA ✓Aza IAK M

2 ✓ATG ✓Pred ✓CsA ✓Aza SIK M/S

*2.3.2. Edmonton protocol*

174 Type 1 Diabetes

**Publication year**

1991 4

**Pts no.** **Induction**

1990 9 ✓Tac

✓ATG (3 pts)

1992 10 ✓Tac

tacrolimus that has the effect of β cell deterioration.

**Table 3.** Immunosuppression protocols in clinical islet transplants published in 1990's. \*M: Multiple donor transplants, S: Single donor transplant. \*\* Not achieved II, but positive C-peptide or decreased insulin requirement was confirmed. Abbreviations; 15-DSG: 15-deoxyspergualin, ATG: antithymocyte globulin, Aza: azathioprine, CNIs: Calcineurin inhibitors, CsA: Cyclosporine A, IAK: Islet after kidney transplantation, II: Insulin Independence, ITA: Islet transplantation alone, mALG: Minnesota antilymphoblast globulin, MMF: mycophenolic mofetil, mPred: methylprednisolone, Pred: Prednisone, SIK: Simultaneous islet kidney transplantation, Tac: Tacrolimus.



**Publication year**

2008 7

2008 6

2008 6

2009 15

2010 5

2011 3

5

2010 8 ✓ATG

6

**Pts no.** **Induction**

✓Dac ✓Inf ✓Eta

✓ATG ✓Eta

✓Dac ✓Eta

✓Dac ✓Inf

✓Dac or ✓Bas

✓ATG ✓Bela

✓ATG ✓Efa

✓ATG ✓Eta ✓Ana

4 ✓Dac ✓Tac

2008 3 ✓Ale ✓Tac

✓ Pred (2 pts) or mPred (1 pt)

**therapy Maintenance therapy Transplant**

5 ✓ATG ✓Sir ITA M

✓Tac

✓CyA

✓Tac

<sup>2008</sup> <sup>5</sup> ✓ATG ✓Tac ✓Sir ITA <sup>M</sup> 3 pts achieved

<sup>2008</sup> <sup>13</sup> ✓Dac ✓Tac ✓Sir SIK M/S 7 pts achieved

✓Sir ✓MMF (2 pts)

✓Eve →MMF

2008 4 ✓Dac ✓Tac ✓Sir ITA M 100% II [82]

✓Sir ✓Exe

✓Sir ✓MPA

2009 14 ✓Dac ✓Tac ✓Sir ITA M 100% II [85]

✓Sir ✓MMF ✓Efa

2010 8 ✓Dac ✓Tac ✓Sir ITA M/S 100% II [88]

✓Sir or ✓MMF

✓Sir or ✓MMF

3 ✓Dac ✓Tac ✓Sir ITA M 100% II

✓Tac ✓MMF ITA M/S

✓Sir ✓Efa

✓Tac ✓Sir

**type**

**Donor no.\***

II

II

II

II

II

ITA M/S 100% II [87]

<sup>S</sup> 3 pts achieved II

achieved II [86]

100% II after single infusion

100% II after single infusion [89]

100% II after single infusion

100% II after single infusion [90]

IAK M/S 6 pts achieved

ITA M/S 5 pts achieved

ITA M/S 2 pts achieved

ITA M/S 100% II

Islet transplant with Bone marrow

✓Tac ✓Sir IAK M/S 100 pts

ITA S

ITA M/S

ITA M/S

Partial function\*\*

**Major outcomes**

Beta Cell Function After Islet Transplantation http://dx.doi.org/10.5772/ 52952

**Refs**

177

[78]

[79]

[80]

[81]

[83]

[84]


**Publication year**

176 Type 1 Diabetes

2005 8

2005 16

2005 22

2005 10

✓ATG ✓Dac ✓Eta

✓Dac ✓Inf (8pts)

✓Dac/ ✓Bas

✓ATG or ✓Bas

2007 11 ✓Dac ✓Tac

2007 19 ✓Dac ✓Tac

**Pts no.** **Induction**

**therapy Maintenance therapy Transplant**

<sup>2001</sup> <sup>10</sup> ✓Bas ✓Pred ✓CsA ✓MMF IAK M/S 2 pts achieved

<sup>2003</sup> <sup>6</sup> ✓Dac ✓Tac ✓Sir ITA M/S 3 pts achieved

<sup>2004</sup> <sup>6</sup> ✓OKT3γ1 ✓Tac ✓Sir ITA <sup>S</sup> 4 pts achieved

<sup>2004</sup> <sup>10</sup> ✓Dac ✓Tac ✓Sir ITA M/S 5 pts achieved

<sup>2004</sup> <sup>6</sup> ✓Dac ✓Tac ✓Sir SIK <sup>M</sup> 5 pts achieved

✓MMF ✓Sir

✓Sir/ ✓Eve

✓Sir or ✓MMF

2006 8 ✓Dac ✓Tac ✓Sir IAK M/S 100% II [72]

✓Sir or ✓MMF plus ✓Exe

✓Sir or ✓MMF

<sup>2006</sup> <sup>6</sup> ✓Dac ✓Tac ✓Sir ITA <sup>M</sup> 3 pts achieved

<sup>2007</sup> <sup>10</sup> ✓Dac ✓Tac ✓Sir ITA M/S 6 pts achieved

<sup>2006</sup> <sup>36</sup> ✓Dac ✓Tac ✓Sir ITA M/S 16 pts

✓Tac

✓Tac/ ✓CsA

✓Tac

<sup>2005</sup> <sup>65</sup> ✓Dac ✓Tac ✓Sir ITA M/S 44 pts

<sup>2004</sup> <sup>13</sup> ✓Dac ✓Tac ✓Sir ITA (9 pts) or

**type**

IAK (4 pts)

✓Tac ✓Sir ITA M/S 14 pts

ITA S

IAK or ITA M/S 15 pts

ITA M/S 100% II [71]

II

M/S 8 pts achieved II

II

ITA M/S 16 pts

**Donor no.\***

II

II

II

<sup>M</sup> 11 pts

II

II

100% II after single infusion [68]

achieved II [69]

achieved II [70]

achieved II [4]

achieved II [74]

achieved II [77]

**Major outcomes**

**Refs**

[62]

[63]

[64]

[66]

[67]

[73]

[75]

[76]

achieved II [65]


efforts to find effective immunosuppression with less effect on β cell function while enhanc‐ ing β cell function such as exenatide which is a glucagon-like peptide-1 (GLP-1) analog [75].

Beta Cell Function After Islet Transplantation http://dx.doi.org/10.5772/ 52952 179

The islet encapsulation aims to eliminate or reduce the dose of immunosuppression, which is a major obstacle in current islet transplantation, by isolating islets from blood flow and avoiding direct interaction with antibodies and immune cells such as lymphocytes and mac‐ rophages. However, few clinical trials using encapsulation technique have been reported [91, 102]. The University of Peruga group demonstrated the efficacy of microencapsulated human islets with sodium alginate in 4 type 1 diabetic patients, who were able to reduce HbA1c level and the amounts of exogenous insulin injection [91]. Elliot RB et al. showed a case report on xenotransplantation using alginate-encapsulated porcine islets, also allowing reduction of insulin dose [102]. In both reports, islet recipients did not use any immunosup‐ pressants although insulin independence was not achieved, suggesting the advantage and

There are several methods of islet encapsulation; macrocapsular devices, microencapsula‐ tion and surface modification. A macrocapsular device that is composed of polytetrafluoro‐ ethylene membrane enabled delayed onset of diabetes in mice model [103]. Microencapsulation of islets has been prepared using various materials such as alginate, agarose and collagen [104-106]. An issue of microencapsulation is the enlargement of the size of islet mass; micro‐ encapsulation of an islet can increase the size by as much as 3 to 5 folds of the original islet. Alternatively, surface modification of islets is a strategy to reduce the tissue volume. Polyethy‐ lene glycol (PEG) is a hydrogen polymer and can be used for conformal coating to encapsu‐ late islets in the process of polymerization [107]. PEGylation, i.e. PEG conjugation at the islet surface, is the another way of islet encapsulation without significant increase in tissue size [108]. Recently, PEGylation attached with biologically active agents of heparin, activated protein C, urokinase or thrombomodulin has been developed to prevent the local coagulation imme‐ diately after islet infusion [109-112]. These techniques were recently developed and the sus‐

**2.4. Islet encapsulation**

limitation of current encapsulation strategy (Figure 1).

tainability of PEGylation needs to be proven.

**Figure 1.** Benefits and current limitations of islet encapsulation.

**Table 4.** Immunosuppression protocols in clinical islet transplants published after 2000. \*M: Multiple donor transplants, S: Single donor transplant. \*\*Microencapsulated islets transplanted. \*\*\* Not achieved II, but positive Cpeptide or decreased insulin requirement was confirmed. Abbreviations; Ale: Alemtuzumab, ATG: antithymocyte globulin, Aza: azathioprine, Ana: anakinra, Bas: basiliximab, Bela: belatacept, CNIs: Calcineurin inhibitors, CsA: Cyclosporine A, Dac: daclizumab, Efa: efalizumab, Eta: etanercept, Eve: everolimus, Exe: exenatide, IAK: islet after kidney transplantation, II: insulin independence, Inf: infliximab, ITA: islet transplantation alone, mALG: Minnesota antilymphoblast globulin, MMF: mycophenolic mofetil, MPA: mycophenolic acid, mPred: methylprednisolone, Pred: prednisone, SIK: simultaneous islet kidney transplantation, Sir: sirolimus, Tac: tacrolimus

Sirolimus is an inhibitor of mammalian target of rapamycin (mTOR), which plays an impor‐ tant role in cell cycle from late G1 to S phase in T cells [92]. The effect of sirolimus in β cell function is still unclear; impaired β cell proliferation and islet graft function by sirolimus has been reported [93-95] while Melzi et al found no significant adverse effect of sirolimus in is‐ let engraftment [96]. Gao et al reported sirolimus and daclizumab did not show any individ‐ ual or synergistic negative effects on islet proliferation [97]. However, insulin independence in Edmonton protocol was not sustained for a long-term resulting in 12.5% at 5 year after islet transplant [4].

#### *2.3.3. Newer immunosuppression protocols*

Recent clinical trials implementing monoclonal antibodies such as basiliximab (anti-IL-2 re‐ ceptor)[70], efalizumab (anti-LFA-1)[89], alemtuzumab (anti-CD52)[83] have shown high rate of insulin independence after transplant. These monoclonal antibodies are produced as mo‐ lecular targeting agents and considered as less likely to have direct effects on β cell function.

Currently major islet transplant centers are increasingly adopting stronger induction immu‐ nosuppression comprised of T cell depletion using anti-thymocyte globulin, alemtuzumab or OKT3γ1 (anti-CD3) plus anti-TNF-α treatment. This has resulted in significantly im‐ proved long-term maintenance of insulin independence [3, 5].

In maintenance immunosuppression, tacrolimus is still a key medication; although, there is controversy on the use of tacrolimus and its effect to islet graft function as described above (See § 2.5.1). Mycophenolate mofetil (MMF) is also used for maintenance immunosuppres‐ sion, inhibiting proliferation of T and B cells and promoting apoptosis of activated T cells [98, 99]. Gallo et al recently showed that MMF was able to reduce survival of β cells, impair glucose-stimulated insulin secretion and β cell proliferation [100]. Posselt et al reported ex‐ cellent islet transplant outcome using CNI-free immunosuppression that included belata‐ cept [89], which is a fusion protein with Fc fragment of a human IgG linked to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) that allows costimulation blockade of CD80 and CD86 on antigen presenting cells [101]. Overall islet investigators have continued to make efforts to find effective immunosuppression with less effect on β cell function while enhanc‐ ing β cell function such as exenatide which is a glucagon-like peptide-1 (GLP-1) analog [75].

### **2.4. Islet encapsulation**

**Publication year**

178 Type 1 Diabetes

**Pts no.**

islet transplant [4].

*2.3.3. Newer immunosuppression protocols*

**Induction**

**therapy Maintenance therapy Transplant**

**Table 4.** Immunosuppression protocols in clinical islet transplants published after 2000. \*M: Multiple donor transplants, S: Single donor transplant. \*\*Microencapsulated islets transplanted. \*\*\* Not achieved II, but positive Cpeptide or decreased insulin requirement was confirmed. Abbreviations; Ale: Alemtuzumab, ATG: antithymocyte globulin, Aza: azathioprine, Ana: anakinra, Bas: basiliximab, Bela: belatacept, CNIs: Calcineurin inhibitors, CsA: Cyclosporine A, Dac: daclizumab, Efa: efalizumab, Eta: etanercept, Eve: everolimus, Exe: exenatide, IAK: islet after kidney transplantation, II: insulin independence, Inf: infliximab, ITA: islet transplantation alone, mALG: Minnesota antilymphoblast globulin, MMF: mycophenolic mofetil, MPA: mycophenolic acid, mPred: methylprednisolone, Pred:

Sirolimus is an inhibitor of mammalian target of rapamycin (mTOR), which plays an impor‐ tant role in cell cycle from late G1 to S phase in T cells [92]. The effect of sirolimus in β cell function is still unclear; impaired β cell proliferation and islet graft function by sirolimus has been reported [93-95] while Melzi et al found no significant adverse effect of sirolimus in is‐ let engraftment [96]. Gao et al reported sirolimus and daclizumab did not show any individ‐ ual or synergistic negative effects on islet proliferation [97]. However, insulin independence in Edmonton protocol was not sustained for a long-term resulting in 12.5% at 5 year after

Recent clinical trials implementing monoclonal antibodies such as basiliximab (anti-IL-2 re‐ ceptor)[70], efalizumab (anti-LFA-1)[89], alemtuzumab (anti-CD52)[83] have shown high rate of insulin independence after transplant. These monoclonal antibodies are produced as mo‐ lecular targeting agents and considered as less likely to have direct effects on β cell function.

Currently major islet transplant centers are increasingly adopting stronger induction immu‐ nosuppression comprised of T cell depletion using anti-thymocyte globulin, alemtuzumab or OKT3γ1 (anti-CD3) plus anti-TNF-α treatment. This has resulted in significantly im‐

In maintenance immunosuppression, tacrolimus is still a key medication; although, there is controversy on the use of tacrolimus and its effect to islet graft function as described above (See § 2.5.1). Mycophenolate mofetil (MMF) is also used for maintenance immunosuppres‐ sion, inhibiting proliferation of T and B cells and promoting apoptosis of activated T cells [98, 99]. Gallo et al recently showed that MMF was able to reduce survival of β cells, impair glucose-stimulated insulin secretion and β cell proliferation [100]. Posselt et al reported ex‐ cellent islet transplant outcome using CNI-free immunosuppression that included belata‐ cept [89], which is a fusion protein with Fc fragment of a human IgG linked to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) that allows costimulation blockade of CD80 and CD86 on antigen presenting cells [101]. Overall islet investigators have continued to make

2011 4 ITA\*\* M/S

prednisone, SIK: simultaneous islet kidney transplantation, Sir: sirolimus, Tac: tacrolimus

proved long-term maintenance of insulin independence [3, 5].

**type**

**Donor no.\***

**Major outcomes**

function\*\*\* [91]

Partial

**Refs**

The islet encapsulation aims to eliminate or reduce the dose of immunosuppression, which is a major obstacle in current islet transplantation, by isolating islets from blood flow and avoiding direct interaction with antibodies and immune cells such as lymphocytes and mac‐ rophages. However, few clinical trials using encapsulation technique have been reported [91, 102]. The University of Peruga group demonstrated the efficacy of microencapsulated human islets with sodium alginate in 4 type 1 diabetic patients, who were able to reduce HbA1c level and the amounts of exogenous insulin injection [91]. Elliot RB et al. showed a case report on xenotransplantation using alginate-encapsulated porcine islets, also allowing reduction of insulin dose [102]. In both reports, islet recipients did not use any immunosup‐ pressants although insulin independence was not achieved, suggesting the advantage and limitation of current encapsulation strategy (Figure 1).

There are several methods of islet encapsulation; macrocapsular devices, microencapsula‐ tion and surface modification. A macrocapsular device that is composed of polytetrafluoro‐ ethylene membrane enabled delayed onset of diabetes in mice model [103]. Microencapsulation of islets has been prepared using various materials such as alginate, agarose and collagen [104-106]. An issue of microencapsulation is the enlargement of the size of islet mass; micro‐ encapsulation of an islet can increase the size by as much as 3 to 5 folds of the original islet. Alternatively, surface modification of islets is a strategy to reduce the tissue volume. Polyethy‐ lene glycol (PEG) is a hydrogen polymer and can be used for conformal coating to encapsu‐ late islets in the process of polymerization [107]. PEGylation, i.e. PEG conjugation at the islet surface, is the another way of islet encapsulation without significant increase in tissue size [108]. Recently, PEGylation attached with biologically active agents of heparin, activated protein C, urokinase or thrombomodulin has been developed to prevent the local coagulation imme‐ diately after islet infusion [109-112]. These techniques were recently developed and the sus‐ tainability of PEGylation needs to be proven.

**Figure 1.** Benefits and current limitations of islet encapsulation.
