**1.4. Immunotherapy for the prevention and treatment of Type 1 diabetes**

cell differentiation protocol and retroviral expression of the same four transcription factors, it was reported that differentiation into insulin-producing islet-like clusters was possible. Isletlike clusters were obtained from iPSCs using a serum-free, feeder-free protocol [93]. Following initial reports, a number of modifications to the original protocol have been introduced. These included substituting the originally-described transcription factors with oncogenic potential with stable recombinant proteins [94], episomal constructs [95], DNA minicircles [96], modified mRNAs [97], and small molecule compounds with re-programming properties [98]. Despite these efforts, generation of patient-specific cell lines form iPSCs remains inefficient and expensive, hindering progress in this area. Additionally, there seem to be an inconsistency in the methods utilized for the successful differentiation of insulin-producing islet-like cells, leaving the field open for a much wanted universal protocol utilized to generate a wide variety of patient-specific cell lines, much like that developed by ViaCyte for the Pro-Islet™ technol‐ ogy. Then, of course, the risks inherent to the use of iPSC-and ESC-based approaches must be

First, there are the reports of teratoma formation when undifferentiated (cultured *in vitro* for ~12 days) ESCs are utilized in pre-clinical models [89]. Interestingly, when cells cultured under similar conditions for extended period of time were utilized [87], no teratoma formation was observed in recipient animals suggesting that more extensively differentiated ESCs lose their ability for neoplastic transformation. Hence, teratoma formation can probably be controlled through elimination of less differentiated cells via advanced purification techniques, as well

Another critical aspect that deserves serious consideration is related to the full cell complement present at the final ESC differentiation stages. Transplantation of pancreatic progenitor cells results in the development of not only the endocrine cell types, the full complement of which are probably required for fully functional islet structures, but also the exocrine pancreas, i.e. acinar and ductal cells, albeit at much lower frequency [89]. The presence of these cells that have the ability to produce and release various enzymatically active proteins is worrisome. In addition, under conditions of stress caused by inflammation and injury, acinar cells can develop into cells with progenitor-like activity, able to result in neoplastic lesions as a result of oncogenic mutations. While the possibility of such events is small, detailed investigation into these issues needs to continue to assure that the function of ESC-derived endocrine cells are not compromised by the cancer-related risks associated with exocrine cell populations. Another issue that needs to be explored is the immune response of the host following trans‐ plantation of an allogeneic ESC-derived cellular graft. In the last decade or so sophisticated immunosuppressive regiments have been developed to protect allogeneic islet grafts obtained from deceased donors [46-50] long term, following transplantation. In case of ESC-related therapies, not only the graft must be protected from the immune insult by the recipient's immune system, but the cells with tumorigenic capacity need to be isolated and sequestered. This can be, most probably, achieved with the use of sophisticated immunoisolation / encap‐

Finally, what needs to be ascertained is the fact that stem-cell derived β-cells have the same ability to synthesize, store and release insulin in a highly regulated fashion similar to that of

carefully considered, as these seem to be almost identical.

594 Type 1 Diabetes

as more efficient machinery for cell differentiation.

sulation devices that have become available in the last few years [4].

As Type 1 diabetes is an autoimmune disease characterized by the selective and progressive destruction of insulin-producing β-cells via the cumulative attack by autoantigen-specific CD4+ and CD8+ T-cells, autoantibodies, and functionally defective bone marrow derived antigen-specific cells, development of various immunotherapeutic options has been the major focus for prevention and treatment of IDDM. Multiple studies in the NOD mouse model demonstrate that islets are attacked in step-wise manner, with benign insulitis being the starting point of this assault. With time and not fully defined qualitative changes, overt diabetes characterized by the efficient destruction of β-cells ensues. It is generally acknowl‐ edged that diabetes in animal models and men is strongly associated with the changes in more than 20 genetic loci - with genes encoding MHC class II molecules playing the major role, most probably influenced by a number of environmental factors, although it's been quite challeng‐ ing to identify either in detail [11].

Animal studies indicate that a number of pathogenic events contribute to the progressive loss of T-cell tolerance to β-cell proteins, and, therefore, expansion of β-cell specific pathogenic CD4+ and CD8+ T-cells. These events seem to take place during the early stages of the preclinical IDDM. These include defective negative selection in the thymus, inefficient peripheral tolerance characterized by low frequencies of IL-4, IL-10 and TGF-β secreting CD4+ T helper 2 (Th2) cells, as well as diminished numbers of "natural" immunoregulatory FoxP3 expressing CD4+CD25+ Regulatory T (Treg) cells and invariant natural killer T (iNKT) cells. These events, coupled with reduced frequency / function of immunoregulatory effector cells within the islets, reduced sensitivity of T cells to immunoregulation, and increased levels of pro-inflammatory cytokines produced by macrophages and dendritic cells (DCs), result in the severe loss in the balance between pathogenic effector and immunoregulatory T cells, especially during the later stages of the disease [99-101]. Effective prevention / treatment strategies for Type 1 diabetes must focus on the restoration of this balance..

The progression course of diabetes offers obvious time points for interventional immunother‐ apeutic strategies. The first opportunity is presented during the pre-clinical stages of diabetes, when the goal is to suppress the on-going β-cell autoimmune process and prevent the development of overt diabetes. Undiagnosed at risk individuals - family members of patients with the previously diagnosed diabetes - can be monitored by screening for autoantibodies specific for several autoantigens found in the serum. These include insulin, glutamic acid decarboxylase 65 (GAD65), and insulinoma–associated tyrosine phosphate (IA-2) [102]. The second time point for intervention is at clinical onset, in an attempt to preserve 10-15% of the β-cell mass that is usually still present at the time of diagnosis.

There is a definite therapeutic potential through the rescue of the residual β-cells, and there are reports that halting autoimmunity at this stage can potentially lead to β-cell regeneration and/or replication and, in ideal circumstances, remission of diabetes [103,104].

It's been demonstrated that treatment with antigen-based therapy can have a dual effect on au‐ toreactive T-cells: induction of T-cell deletion and the induction of immunoregulatory T- cell population [108,109]. The number of immunoregulatory β-cell specific T-cells induced as result of treatment is critical. As diabetes progresses and the pro-inflammatory milieu is established, a relatively high number of immunoregulatory T-cells would be required to effectively suppress β-cell autoimmunity and to restore the balance between pathogenic effector and immunoregu‐ latory T-cell subsets. The number of inducible immunoregulatory effector cells is, at least in part, dependent on the size of the pool of naive precursors for a given β-cell autoantigen [101,106]. As the pool of β-cell specific T-cell precursors actively involved in the autoimmune process is limited, minimizing the pool of immunoregulatory T-cells that can be induced, it is of critical importance to choose the autoantigen utilized for treatment at late stages of the disease wisely. While in experimental models it's been demonstrated that administration of a combina‐ tion of β-cell autoantigens suppresses β-cell autoimmunity during the late stages of the disease, the same has not been clearly defined in patients [101]. Although some progress has been made towards the development of methods that can be used to detect β-cell specific T-cells, more de‐ velopment is necessary before this approach can become a standard immunotherapeutic ap‐ proach. In addition, as demonstrated by the DPT-1 [107], the efficacy of a given treatment, i.e. βcell autoantigen, may vary significantly between individuals, probably based on the extent of autoimmunity, i.e. β-cell specific T-cell precursors. This means that, similarly to the NOD mod‐ el, immunization with the cocktail of various β-cell specific peptides would be necessary to ach‐ ieve a measurable degree of success in abating the progress of the autoimmune process taking

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The number of immunoregulatory β-cell specific T-cells induced as result of treatment also depends on the efficiency of the process involved in the induction of immunoregulatory T-cell population. What complicates matters is the fact that this induction must take place *in vivo*, under the same conditions that favor the expansion of autoreactive β-cell specific T-cell subsets. Hence, strategies that preferentially promote the expansion of immunoregulatory T-cell populations are necessary. Properties of mucosal tissues [110,111], co-administration of various types of adjuvants and cytokines, as well as manipulation of the way the autoantigen is presented have been investigated in both experimental and clinical settings [107], with some degree of reported success. In addition, variety of inducible immunoregulatory T-cell popu‐ lations has been reported to be of importance as induction of different types of immunoregu‐ latory cells, each with a distinct mode of action, would be expected to increase the overall

Various monoclonal antibodies have been utilized to target a wide range of immune compo‐ nents actively involved in the progressive autoimmune process. Most of these focus on directly or indirectly targeting the T-cell compartment [101], but also include soluble mediators such as cytokines and chemokines, and antigen presenting cells (APC). Several recent reports suggest that B-cells may also represent a useful target to alter the progression of β-cell

place during the advanced stages of Type 1 Diabetes.

efficacy of a given immunotherapy [107].

*1.4.2. Antibody-based immunotherapy*

autoimmune process.

Further down the line, when all β-cells are lost, the likelihood of remission through im‐ munoregulation becomes slim, but recent observation in clinical trials performed in pa‐ tients with undetectable C-peptide suggest that restoration of β-cell function is not impossible in these circumstances. A recent clinical trial conducted in China demonstrated that following treatment with autologous lymphocytes and allogeneic cord blood-derived stem cells, patients with and without residual β-cell function demonstrated improved Cpeptide levels, reduced median Glycated hemoglobin A1C (HbA1C) values, and decreased daily insulin requirements [105].

The development of specific immunotherapeutic strategies that effectively target pathogenic effector cell populations, promote β-cell tolerance, while maintaining a "normal" immune function, i.e. balance between pathogenic effector and immunoregulatory T-cells, is the ultimate goal. This means that different immunotherapeutic strategies, alone or in combina‐ tion, must be considered to effectively suppress β-cell autoimmunity at different stages of the disease progression.

There is sufficient information that deals with various immunotherapeutic strategies to prevent / treat Type 1 diabetes, for which both clinical and experimental findings are available. Two major approaches have received most attention, although others have been discussed, namely, antigen- and antibody-based immunotherapies.

#### *1.4.1. Antigen-based immunotherapy*

Antigen-based immunotherapy has to do with selectively targeting disease-specific T cells to maintain the normal function of the immune system. β-cell antigen-specific vaccination has proved to be an effective strategy for the induction of the immunoregulatory T cells and suppression of autoimmune pre-clinical diabetes in rodent models and NOD mice. Vaccination of 12-week old NOD mice with GAD65 protein resulted in the inhibition of the progression of insulitis and long-term protection mediated by the GAD65-specific CD4+ T cells [106]. Successful application of antigen-based immunotherapies in the clinical setting has yet to be reported, although some evidence does exist of the successful application of this methodology. The Diabetes Prevention Trial-1 (DPT-1), during which participating pre-diabetic subjects received insulin either orally or parentally demonstrated no significant effect on the develop‐ ment of diabetes or β-cell autoimmunity [107]. Although the reason for why the treatment failed to prevent diabetes in the majority of subjects was never clearly identified, it was thought that insufficient dose of insulin administered to trial participants was the main culprit. One interesting observation had to do with the fact that some effect was observed in subjects receiving oral insulin that presented with high titers of insulin autoantibodies. It is, therefore, entirely possible that success or failure, as well as efficacy, of a given antigen-based immuno‐ therapy is related to the severity of the existing autoimmunity.

It's been demonstrated that treatment with antigen-based therapy can have a dual effect on au‐ toreactive T-cells: induction of T-cell deletion and the induction of immunoregulatory T- cell population [108,109]. The number of immunoregulatory β-cell specific T-cells induced as result of treatment is critical. As diabetes progresses and the pro-inflammatory milieu is established, a relatively high number of immunoregulatory T-cells would be required to effectively suppress β-cell autoimmunity and to restore the balance between pathogenic effector and immunoregu‐ latory T-cell subsets. The number of inducible immunoregulatory effector cells is, at least in part, dependent on the size of the pool of naive precursors for a given β-cell autoantigen [101,106]. As the pool of β-cell specific T-cell precursors actively involved in the autoimmune process is limited, minimizing the pool of immunoregulatory T-cells that can be induced, it is of critical importance to choose the autoantigen utilized for treatment at late stages of the disease wisely. While in experimental models it's been demonstrated that administration of a combina‐ tion of β-cell autoantigens suppresses β-cell autoimmunity during the late stages of the disease, the same has not been clearly defined in patients [101]. Although some progress has been made towards the development of methods that can be used to detect β-cell specific T-cells, more de‐ velopment is necessary before this approach can become a standard immunotherapeutic ap‐ proach. In addition, as demonstrated by the DPT-1 [107], the efficacy of a given treatment, i.e. βcell autoantigen, may vary significantly between individuals, probably based on the extent of autoimmunity, i.e. β-cell specific T-cell precursors. This means that, similarly to the NOD mod‐ el, immunization with the cocktail of various β-cell specific peptides would be necessary to ach‐ ieve a measurable degree of success in abating the progress of the autoimmune process taking place during the advanced stages of Type 1 Diabetes.

The number of immunoregulatory β-cell specific T-cells induced as result of treatment also depends on the efficiency of the process involved in the induction of immunoregulatory T-cell population. What complicates matters is the fact that this induction must take place *in vivo*, under the same conditions that favor the expansion of autoreactive β-cell specific T-cell subsets. Hence, strategies that preferentially promote the expansion of immunoregulatory T-cell populations are necessary. Properties of mucosal tissues [110,111], co-administration of various types of adjuvants and cytokines, as well as manipulation of the way the autoantigen is presented have been investigated in both experimental and clinical settings [107], with some degree of reported success. In addition, variety of inducible immunoregulatory T-cell popu‐ lations has been reported to be of importance as induction of different types of immunoregu‐ latory cells, each with a distinct mode of action, would be expected to increase the overall efficacy of a given immunotherapy [107].

#### *1.4.2. Antibody-based immunotherapy*

There is a definite therapeutic potential through the rescue of the residual β-cells, and there are reports that halting autoimmunity at this stage can potentially lead to β-cell regeneration

Further down the line, when all β-cells are lost, the likelihood of remission through im‐ munoregulation becomes slim, but recent observation in clinical trials performed in pa‐ tients with undetectable C-peptide suggest that restoration of β-cell function is not impossible in these circumstances. A recent clinical trial conducted in China demonstrated that following treatment with autologous lymphocytes and allogeneic cord blood-derived stem cells, patients with and without residual β-cell function demonstrated improved Cpeptide levels, reduced median Glycated hemoglobin A1C (HbA1C) values, and decreased

The development of specific immunotherapeutic strategies that effectively target pathogenic effector cell populations, promote β-cell tolerance, while maintaining a "normal" immune function, i.e. balance between pathogenic effector and immunoregulatory T-cells, is the ultimate goal. This means that different immunotherapeutic strategies, alone or in combina‐ tion, must be considered to effectively suppress β-cell autoimmunity at different stages of the

There is sufficient information that deals with various immunotherapeutic strategies to prevent / treat Type 1 diabetes, for which both clinical and experimental findings are available. Two major approaches have received most attention, although others have been discussed,

Antigen-based immunotherapy has to do with selectively targeting disease-specific T cells to maintain the normal function of the immune system. β-cell antigen-specific vaccination has proved to be an effective strategy for the induction of the immunoregulatory T cells and suppression of autoimmune pre-clinical diabetes in rodent models and NOD mice. Vaccination of 12-week old NOD mice with GAD65 protein resulted in the inhibition of the progression of insulitis and long-term protection mediated by the GAD65-specific CD4+ T cells [106]. Successful application of antigen-based immunotherapies in the clinical setting has yet to be reported, although some evidence does exist of the successful application of this methodology. The Diabetes Prevention Trial-1 (DPT-1), during which participating pre-diabetic subjects received insulin either orally or parentally demonstrated no significant effect on the develop‐ ment of diabetes or β-cell autoimmunity [107]. Although the reason for why the treatment failed to prevent diabetes in the majority of subjects was never clearly identified, it was thought that insufficient dose of insulin administered to trial participants was the main culprit. One interesting observation had to do with the fact that some effect was observed in subjects receiving oral insulin that presented with high titers of insulin autoantibodies. It is, therefore, entirely possible that success or failure, as well as efficacy, of a given antigen-based immuno‐

and/or replication and, in ideal circumstances, remission of diabetes [103,104].

daily insulin requirements [105].

*1.4.1. Antigen-based immunotherapy*

namely, antigen- and antibody-based immunotherapies.

therapy is related to the severity of the existing autoimmunity.

disease progression.

596 Type 1 Diabetes

Various monoclonal antibodies have been utilized to target a wide range of immune compo‐ nents actively involved in the progressive autoimmune process. Most of these focus on directly or indirectly targeting the T-cell compartment [101], but also include soluble mediators such as cytokines and chemokines, and antigen presenting cells (APC). Several recent reports suggest that B-cells may also represent a useful target to alter the progression of β-cell autoimmune process.

There is an abundance of literature that discusses the efficacy of monoclonal antibodies targeting T-cells, in a number of experimental models. Following the administration of a short course of depleting CD4 antibody or anti-lymphocyte serum in NOD mice, suppression of βcell autoimmunity and, in some cases, remission of the recent onset of diabetes is achieved [112,113]. There is, however, a drawback to this approach. Depleting antibody immunotherapy resulted in the indiscriminate depletion of not only the pathogenic, but also non-autoimmune T-cell populations, and induced long-term state of immunosuppression. In addition, after the depleting antibody was cleared from the system the number of T-cells that reappeared was significantly reduced, compared to normal levels. At this same time, the use of non-depleting anti-CD4 and CD8 antibodies resulted in tolerance induction in the antigen-specific manner, with the T-cell numbers intact [114], induction of apoptosis in activated T-cells, and activation of the CD4+CD25+FoxP3+ cell population demonstrated to have a suppressive effect on the differentiation of pathogenic effector T-cells [114].

monoclonal anti-CD20 antibody proved beneficial in abrogating diabetes in young NOD mice, and significantly delaying the onset of the disease in older animals [119]. A recent clinical trial conducted by the TrialNet group confirmed these findings by demonstrating that selective and transient depletion of B-lymphocytes with rituximab, an anti-CD20 monoclonal antibody, partially preserved β-cell function in patients with recent onset of Type 1 diabetes, for a period

Cell Replacement Therapy in Type 1 Diabetes

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Although this cell subset with unique immunomodulatory properties has been briefly discussed above, these cells deserve special attention and are discussed in more detail in this section. Ever since the realization that Treg cells have an innate capacity to maintain tolerance to self-antigens in peripheral organs under immune assault, this population has attracted great attention with respect to their potential role in the prevention of autoimmune disorders which include Type 1 diabetes. The interest in these immune traffic regulators peaked when it was demonstrated that they represented an inducible population able to halt the progression of IDDM, while curbing autoimmune responses not only to antigens responsible for the induction of autoimmunity but others involved in this process as well. This represents an attractive therapeutic alternative for IDDM as to date no specific antigen(s) has been identified as a

As discussed elsewhere in this chapter, autoimmune response aimed at the progressive destruction of pancreatic β-cells can be manipulated through antigen-based manipulation and non-antigen-based treatments, possible though the involvement of Treg cell population. Although immunoregulatory capacity has been demonstrated in several T-cell subsets, the main players in the field are "natural" CD4+CD25+, and "adaptive or induced" regulatory Tcells of various phenotypes. "Natural" CD4+CD25+ regulatory T-cells require a variety of costimulatory interactions for their development, and are mainly identified by the FoxP3 transcription factor necessary for the development and function of this cell subset. *In vitro*, natural CD4+CD25+ cells have been demonstrated to have an uncanny ability to inhibit T-cell proliferation and cytokine production, most probably, via cell-cell contact [121]. Despite previously published dissenting reports, there is an agreement that during the development of diabetes, the autoreactive T-cell subsets become unresponsive to CD4+CD25+ mediated suppression mechanism. This could be due to the fact that CD4+CD25+ cell are present in reduced numbers during the development of the IDDM in humans. At the same time, opposing results have been obtained in an NOD model: at the time of diabetes onset, CD4+CD25+ cells exist in equal numbers compared to non-diabetic animals [121]. It's been also demonstrated that while CD4+CD25+ cells are relatively abundant in normal individuals, data obtained from various animal models suggest that antigen-induced Treg cells are present in relatively low numbers. Despite this fact, of most benefit is the data that demonstrated that once induced, Treg cells become activated in the immediate tissue where the given autoantigen is expressed. Of added benefit is the realization that in addition to suppressing the responses of an autoan‐ tigen in question, Treg cells are able to modulate other autoreactive T-cell responses as well, most probably via production of anti-inflammatory soluble cytokines such as IL-4, IL-10 and

of 1 year [120].

*1.4.3. Regulatory R (Treg) cells*

causative agent for the diabetogenic response.

Studies investigating the efficacy of anti-CD3 monoclonal therapy for the treatment of Type 1 diabetes have been generating a lot of interest ever since they've been first reported [104], Chatenoud demonstrated that a short course treatment of NOD mice with low dose anti-CD3 antibody resulted in long-term remission of recent onset diabetes and β-cell specific tolerance [104]. The mode of action of this therapy proved to be multi-faceted. A critical observation was of the anti-CD3 antibody preferentially affecting activated rather than naïve T-cells by downregulating the T-cell receptor and reducing TCR signaling, enhancing apoptosis, and altering T-cell trafficking [115]. This treatment was also demonstrated to promote the expansion of the immunoregulatory T-cells with CD4+CD25+ phenotype. Utilization of a non-mitogenic anti-CD3 antibody in a clinical setting, during the first 6 weeks following diagnosis, resulted in the preservation of C-peptide response over a 2-year period in certain patients relative to untreated controls. The fact that residual β-cell function was reported in some patients at the time of treatment speaks to the importance of therapeutic administration at "earlier" stages in the disease progression [116]. Although efficacy with this treatment was observed, the protection offered by the anti-CD3 antibody treatment was nevertheless transient. This suggests that this type of therapy needs to be refined either in terms of schedule or route of the administration, or the therapeutic dose, before it can be applied to a larger patient population.

Studies with monoclonal-based therapies targeting co-stimulatory pathway of immune activation such as CD40-CD40L, and APCs such as DC's and B-cells have also been reported. Blocking the CD40-CD40L pathway proved highly effective in abrogating T-cell responses in autoimmune and transplantation models [117]. However, before this approach could be investigated further the anti-CD40L antibody was withdrawn from use in various clinical trials due to serious adverse events that came into view as a result of treatment.

Targeting B-cells, whose primary role in Type 1 diabetes is that of APCs to T-cells, was never high on the list of targets for potential immunotherapy. The reason behind this is simple: isletspecific autoantibodies have never been considered the primary culprits of β-cell destruction. However, this pathway may prove to be the indirect approach to targeting β -cell autoreactivity [118]. Despite an initial skepticism, some work has been done in this area. Recent studies performed in an experimental setting reported that depleting B-cells with a short course of monoclonal anti-CD20 antibody proved beneficial in abrogating diabetes in young NOD mice, and significantly delaying the onset of the disease in older animals [119]. A recent clinical trial conducted by the TrialNet group confirmed these findings by demonstrating that selective and transient depletion of B-lymphocytes with rituximab, an anti-CD20 monoclonal antibody, partially preserved β-cell function in patients with recent onset of Type 1 diabetes, for a period of 1 year [120].

### *1.4.3. Regulatory R (Treg) cells*

There is an abundance of literature that discusses the efficacy of monoclonal antibodies targeting T-cells, in a number of experimental models. Following the administration of a short course of depleting CD4 antibody or anti-lymphocyte serum in NOD mice, suppression of βcell autoimmunity and, in some cases, remission of the recent onset of diabetes is achieved [112,113]. There is, however, a drawback to this approach. Depleting antibody immunotherapy resulted in the indiscriminate depletion of not only the pathogenic, but also non-autoimmune T-cell populations, and induced long-term state of immunosuppression. In addition, after the depleting antibody was cleared from the system the number of T-cells that reappeared was significantly reduced, compared to normal levels. At this same time, the use of non-depleting anti-CD4 and CD8 antibodies resulted in tolerance induction in the antigen-specific manner, with the T-cell numbers intact [114], induction of apoptosis in activated T-cells, and activation of the CD4+CD25+FoxP3+ cell population demonstrated to have a suppressive effect on the

Studies investigating the efficacy of anti-CD3 monoclonal therapy for the treatment of Type 1 diabetes have been generating a lot of interest ever since they've been first reported [104], Chatenoud demonstrated that a short course treatment of NOD mice with low dose anti-CD3 antibody resulted in long-term remission of recent onset diabetes and β-cell specific tolerance [104]. The mode of action of this therapy proved to be multi-faceted. A critical observation was of the anti-CD3 antibody preferentially affecting activated rather than naïve T-cells by downregulating the T-cell receptor and reducing TCR signaling, enhancing apoptosis, and altering T-cell trafficking [115]. This treatment was also demonstrated to promote the expansion of the immunoregulatory T-cells with CD4+CD25+ phenotype. Utilization of a non-mitogenic anti-CD3 antibody in a clinical setting, during the first 6 weeks following diagnosis, resulted in the preservation of C-peptide response over a 2-year period in certain patients relative to untreated controls. The fact that residual β-cell function was reported in some patients at the time of treatment speaks to the importance of therapeutic administration at "earlier" stages in the disease progression [116]. Although efficacy with this treatment was observed, the protection offered by the anti-CD3 antibody treatment was nevertheless transient. This suggests that this type of therapy needs to be refined either in terms of schedule or route of the administration,

or the therapeutic dose, before it can be applied to a larger patient population.

due to serious adverse events that came into view as a result of treatment.

Studies with monoclonal-based therapies targeting co-stimulatory pathway of immune activation such as CD40-CD40L, and APCs such as DC's and B-cells have also been reported. Blocking the CD40-CD40L pathway proved highly effective in abrogating T-cell responses in autoimmune and transplantation models [117]. However, before this approach could be investigated further the anti-CD40L antibody was withdrawn from use in various clinical trials

Targeting B-cells, whose primary role in Type 1 diabetes is that of APCs to T-cells, was never high on the list of targets for potential immunotherapy. The reason behind this is simple: isletspecific autoantibodies have never been considered the primary culprits of β-cell destruction. However, this pathway may prove to be the indirect approach to targeting β -cell autoreactivity [118]. Despite an initial skepticism, some work has been done in this area. Recent studies performed in an experimental setting reported that depleting B-cells with a short course of

differentiation of pathogenic effector T-cells [114].

598 Type 1 Diabetes

Although this cell subset with unique immunomodulatory properties has been briefly discussed above, these cells deserve special attention and are discussed in more detail in this section. Ever since the realization that Treg cells have an innate capacity to maintain tolerance to self-antigens in peripheral organs under immune assault, this population has attracted great attention with respect to their potential role in the prevention of autoimmune disorders which include Type 1 diabetes. The interest in these immune traffic regulators peaked when it was demonstrated that they represented an inducible population able to halt the progression of IDDM, while curbing autoimmune responses not only to antigens responsible for the induction of autoimmunity but others involved in this process as well. This represents an attractive therapeutic alternative for IDDM as to date no specific antigen(s) has been identified as a causative agent for the diabetogenic response.

As discussed elsewhere in this chapter, autoimmune response aimed at the progressive destruction of pancreatic β-cells can be manipulated through antigen-based manipulation and non-antigen-based treatments, possible though the involvement of Treg cell population. Although immunoregulatory capacity has been demonstrated in several T-cell subsets, the main players in the field are "natural" CD4+CD25+, and "adaptive or induced" regulatory Tcells of various phenotypes. "Natural" CD4+CD25+ regulatory T-cells require a variety of costimulatory interactions for their development, and are mainly identified by the FoxP3 transcription factor necessary for the development and function of this cell subset. *In vitro*, natural CD4+CD25+ cells have been demonstrated to have an uncanny ability to inhibit T-cell proliferation and cytokine production, most probably, via cell-cell contact [121]. Despite previously published dissenting reports, there is an agreement that during the development of diabetes, the autoreactive T-cell subsets become unresponsive to CD4+CD25+ mediated suppression mechanism. This could be due to the fact that CD4+CD25+ cell are present in reduced numbers during the development of the IDDM in humans. At the same time, opposing results have been obtained in an NOD model: at the time of diabetes onset, CD4+CD25+ cells exist in equal numbers compared to non-diabetic animals [121]. It's been also demonstrated that while CD4+CD25+ cells are relatively abundant in normal individuals, data obtained from various animal models suggest that antigen-induced Treg cells are present in relatively low numbers. Despite this fact, of most benefit is the data that demonstrated that once induced, Treg cells become activated in the immediate tissue where the given autoantigen is expressed. Of added benefit is the realization that in addition to suppressing the responses of an autoan‐ tigen in question, Treg cells are able to modulate other autoreactive T-cell responses as well, most probably via production of anti-inflammatory soluble cytokines such as IL-4, IL-10 and TGF-β. Pre-clinical studies in non-obese diabetic mice have demonstrated that adoptive transfer of Tregs can slow diabetes progression and, in some cases, reverse new onset diabetes. Clinical trials investigating the effect of natural expanded and patient-specific Treg cells on autoreactive T-cell responses, preservation of β-cell function and other outcomes related to diabetes management are in progress at the present time [122].

demonstrated that adult recipient pre-conditioning is necessary to "make space" for the

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It was initially thought that lethal recipient conditioning which leads to complete BM ablation was necessary for engraftment of allogeneic BMC. Over time, however, it has become clear that stable engraftment can be achieved using partial pre-conditioning strategies [125,126]. Conditioning approaches to allow for stable engraftment of donor cells have included total body irradiation, total lymphoid irradiation, cytoreductive approaches, low dose irradiation with polyclonal or monoclonal antibodies, single or multiple infusions of large doses of donor BMC with T-cell co-stimulatory blockade, anti-CD4 and anti-CD8 antibodies with local thymic irradiation, and targeted BM ablation using bone seeking 153Samarium-Lexidronam (153Sm) compound with transient T-cell co-stimulatory blockade [125-127]. The fact that hematopoietic chimerism induces donor-specific tolerance, while preserving third-party reactivity, has been established in experimental animal models, i.e. rodents [127,128], large animals [129], primates [130] and in humans [131]. Using conditioning approaches listed above full or mixed chimer‐ ism leading to stable, long-term donor-specific tolerance has been achieved. Although both full and mixed chimerism can be achieved in animal models, fully chimeric animals demon‐ strate immune-incompetence for antiviral activity and antibody production [125,126]. Mixed allogeneic chimerism is much more preferable in tolerance induction protocols, as both donor

The realization that BM transplantation represents a credible treatment for diabetes came as a result of animal studies that demonstrated the interdependence between BMC transplantation and autoimmune disease: the disease could be transferred from NOD mice to mouse strains resistant to autoimmunity, while BM from disease-resistant mouse strains could prevent the development of autoimmunity in NOD mice [125,126]. BMC-associated tolerance to islet cell grafts has been achieved in a number of animal models and human subjects [125,126,132]. Donor-specific tolerance has been demonstrated in both animals that were first precondi‐ tioned, treated with donor-specific BMC, with the islet graft placed at a later date, and those that received islet grafts 24-48 hours after BMC infusion [125,126]. Over the last several decades a profound contribution has been made to the understanding of underlying processes in the induction of BM-derived tolerance to pancreatic islet grafts in the later stages of diabetes, prevention of recurrence of autoimmunity in the graft, and reversal of overt diabetes once the

Animal Type 1 diabetes models fall into two groups, which deal with etiology of the disease. Diabetes can be induced chemically or surgically, or developed spontaneously as in BB rat or NOD mouse model. In the first case autoimmunity is not an underlying factor of the disease. In the second case, however, the disease progresses spontaneously, similarly to the clinical course of Type 1 diabetes, which is autoimmune in nature. BMC transfer experiments between the NOD mouse and disease-resistant mouse strains discussed earlier have suggested that it is a BMC-derived stem cell that is associated with the development of the autoimmunity observed in Type 1 diabetes. Both unmodified and T-cell depleted NOD-derived BMC can transfer autoimmunity followed by diabetes development [125,126]. Conversely, BMC from diabetes-resistant mouse strains, when transferred to a lethally, or sub-lethally conditioned

successful engraftment of donor BMC and induction of donor-specific chimerism.

and recipient antigen presenting cells can be found in the recipient [125].

pre-diabetic state is identified [125,126].

The effect of antigen-based immunotherapy has been discussed earlier in this chapter. However, to recapitulate, the available data demonstrates that antigen-based immunothera‐ peutics probably favor the induction of immunoregulatory T-cell subsets by reacting with endogenous reactive autoantigens, and halting the progression of diabetes. In animal models of IDDM, amplification of Treg cell responses has been achieved using several self-antigens administered using tolerogenic means such intravenous, intranasal or subcutaneous injection, or oral feeding. It has also been shown that Treg cells are able to exert their modulatory effector function through the action of several cytokines, namely IL-4 and IL-10. The situation with TGF-β is much more complex. When administered as a vaccine, it was shown to confer protection from diabetes in NOD mice, but not in other animal models [123].

"Adaptive or induced" Treg cells comprise a group of heterogeneous T-cell subsets that arise as a function of a specific context in which they are generated [120]. These normally go along with antibody-specific approaches to treating IDDM. For example, treatment with CD3 antibody, a potent treatment option for autoimmune disease, has been associated with a marked increase in Treg cell populations, although the mode of action was never elucidated [101,121]. The results of the administration of non-mitogenic anti-CD3 therapy proved to be encouraging [116]. Recent-onset IDDM patients treated with FcR-nonbinding humanized anti-CD3 monoclonal antibody were found to maintain their insulin production for ~2 years following treatment. Although the mechanism of action is well understood, it was thought that the treatment had a direct effect on pathogenic T-cells and resulted in the induction of Treg cell population, or both [116,121]. Data from several other clinical trials seems to indicate that anti-CD3 monotherapy could neither elicit long-term protection, nor protect from adverse effects. Hence, it is possible that combination of immunotherapeutic options might offer a better sustained protection against the disease over time.

#### **1.5. Bone Marrow Chimerism**

It was Owen, back in 1945, who made an observation that bone marrow cells have the ability to induce transplantation tolerance to donor histocompatibility antigens. Billingham, Brent and Medawar confirmed and expanded on this idea by transplanting Major Histocompatibility Complex (MHC)-disparate bone marrow cells (BMC) into neonatal recipient mice which resulted in the induction of specific, systemic, stable tolerance to the donor, while preserving immunocompetence required to reject genetically disparate third party grafts [124]. Fetuses and neonates, of course, offer an immunoprivileged state, during which pre-conditioning is not required for the successful BMC engraftment that leads to chimerism. The situation changes after that. Over the last several decades, numerous investigators working in the area of bone marrow (BM) conditioning to reduce the immunogenicity of solid and cellular grafts, demonstrated that adult recipient pre-conditioning is necessary to "make space" for the successful engraftment of donor BMC and induction of donor-specific chimerism.

TGF-β. Pre-clinical studies in non-obese diabetic mice have demonstrated that adoptive transfer of Tregs can slow diabetes progression and, in some cases, reverse new onset diabetes. Clinical trials investigating the effect of natural expanded and patient-specific Treg cells on autoreactive T-cell responses, preservation of β-cell function and other outcomes related to

The effect of antigen-based immunotherapy has been discussed earlier in this chapter. However, to recapitulate, the available data demonstrates that antigen-based immunothera‐ peutics probably favor the induction of immunoregulatory T-cell subsets by reacting with endogenous reactive autoantigens, and halting the progression of diabetes. In animal models of IDDM, amplification of Treg cell responses has been achieved using several self-antigens administered using tolerogenic means such intravenous, intranasal or subcutaneous injection, or oral feeding. It has also been shown that Treg cells are able to exert their modulatory effector function through the action of several cytokines, namely IL-4 and IL-10. The situation with TGF-β is much more complex. When administered as a vaccine, it was shown to confer

"Adaptive or induced" Treg cells comprise a group of heterogeneous T-cell subsets that arise as a function of a specific context in which they are generated [120]. These normally go along with antibody-specific approaches to treating IDDM. For example, treatment with CD3 antibody, a potent treatment option for autoimmune disease, has been associated with a marked increase in Treg cell populations, although the mode of action was never elucidated [101,121]. The results of the administration of non-mitogenic anti-CD3 therapy proved to be encouraging [116]. Recent-onset IDDM patients treated with FcR-nonbinding humanized anti-CD3 monoclonal antibody were found to maintain their insulin production for ~2 years following treatment. Although the mechanism of action is well understood, it was thought that the treatment had a direct effect on pathogenic T-cells and resulted in the induction of Treg cell population, or both [116,121]. Data from several other clinical trials seems to indicate that anti-CD3 monotherapy could neither elicit long-term protection, nor protect from adverse effects. Hence, it is possible that combination of immunotherapeutic options might offer a

It was Owen, back in 1945, who made an observation that bone marrow cells have the ability to induce transplantation tolerance to donor histocompatibility antigens. Billingham, Brent and Medawar confirmed and expanded on this idea by transplanting Major Histocompatibility Complex (MHC)-disparate bone marrow cells (BMC) into neonatal recipient mice which resulted in the induction of specific, systemic, stable tolerance to the donor, while preserving immunocompetence required to reject genetically disparate third party grafts [124]. Fetuses and neonates, of course, offer an immunoprivileged state, during which pre-conditioning is not required for the successful BMC engraftment that leads to chimerism. The situation changes after that. Over the last several decades, numerous investigators working in the area of bone marrow (BM) conditioning to reduce the immunogenicity of solid and cellular grafts,

diabetes management are in progress at the present time [122].

600 Type 1 Diabetes

better sustained protection against the disease over time.

**1.5. Bone Marrow Chimerism**

protection from diabetes in NOD mice, but not in other animal models [123].

It was initially thought that lethal recipient conditioning which leads to complete BM ablation was necessary for engraftment of allogeneic BMC. Over time, however, it has become clear that stable engraftment can be achieved using partial pre-conditioning strategies [125,126]. Conditioning approaches to allow for stable engraftment of donor cells have included total body irradiation, total lymphoid irradiation, cytoreductive approaches, low dose irradiation with polyclonal or monoclonal antibodies, single or multiple infusions of large doses of donor BMC with T-cell co-stimulatory blockade, anti-CD4 and anti-CD8 antibodies with local thymic irradiation, and targeted BM ablation using bone seeking 153Samarium-Lexidronam (153Sm) compound with transient T-cell co-stimulatory blockade [125-127]. The fact that hematopoietic chimerism induces donor-specific tolerance, while preserving third-party reactivity, has been established in experimental animal models, i.e. rodents [127,128], large animals [129], primates [130] and in humans [131]. Using conditioning approaches listed above full or mixed chimer‐ ism leading to stable, long-term donor-specific tolerance has been achieved. Although both full and mixed chimerism can be achieved in animal models, fully chimeric animals demon‐ strate immune-incompetence for antiviral activity and antibody production [125,126]. Mixed allogeneic chimerism is much more preferable in tolerance induction protocols, as both donor and recipient antigen presenting cells can be found in the recipient [125].

The realization that BM transplantation represents a credible treatment for diabetes came as a result of animal studies that demonstrated the interdependence between BMC transplantation and autoimmune disease: the disease could be transferred from NOD mice to mouse strains resistant to autoimmunity, while BM from disease-resistant mouse strains could prevent the development of autoimmunity in NOD mice [125,126]. BMC-associated tolerance to islet cell grafts has been achieved in a number of animal models and human subjects [125,126,132]. Donor-specific tolerance has been demonstrated in both animals that were first precondi‐ tioned, treated with donor-specific BMC, with the islet graft placed at a later date, and those that received islet grafts 24-48 hours after BMC infusion [125,126]. Over the last several decades a profound contribution has been made to the understanding of underlying processes in the induction of BM-derived tolerance to pancreatic islet grafts in the later stages of diabetes, prevention of recurrence of autoimmunity in the graft, and reversal of overt diabetes once the pre-diabetic state is identified [125,126].

Animal Type 1 diabetes models fall into two groups, which deal with etiology of the disease. Diabetes can be induced chemically or surgically, or developed spontaneously as in BB rat or NOD mouse model. In the first case autoimmunity is not an underlying factor of the disease. In the second case, however, the disease progresses spontaneously, similarly to the clinical course of Type 1 diabetes, which is autoimmune in nature. BMC transfer experiments between the NOD mouse and disease-resistant mouse strains discussed earlier have suggested that it is a BMC-derived stem cell that is associated with the development of the autoimmunity observed in Type 1 diabetes. Both unmodified and T-cell depleted NOD-derived BMC can transfer autoimmunity followed by diabetes development [125,126]. Conversely, BMC from diabetes-resistant mouse strains, when transferred to a lethally, or sub-lethally conditioned NOD mice and rendering these recipients mixed chimeras, reverses insulitis and the autoim‐ mune process, halting the development of overt diabetes. Ildstad proposed two possible explanations for how allogeneic BMC-derived chimerism can prevent diabetes. First, donor BMC activates a regulatory cell instrumental in suppressing the activation of autoreactive lymphocytes identified as a culprit in the progression of autoimmunity, development of the overt diabetes and fully developed disease. Second, BMC can cause clonal deletion of autor‐ eactive T lymphocytes via donor-specific disease resistant APCs [126].

**1.6. Concluding remarks**

Cell replacement strategies offer an enormous potential for the treatment of patients with Type 1 diabetes, in both clinical and economic terms. The availability of unlimited amounts of functionally competent graft material to treat millions of patients suffering from IDDM and its dreadful, debilitating complications can move this field forward from the experimental stage it has found itself in for the last several decades to the forefront of transplantation medicine. The fact that allogeneic islet transplantation offers the most extensively studied and sensible solution to potential cure for IDDM is clear. However, this therapeutic option is far from a perfect solution, and comes hand-in-hand with several problems in the form of serious shortages of the available organs and resulting tissue to satisfy the ever-growing demand, recurrence of autoimmunity and rejection and life-long immunosuppression. Porcine islets offer a viable substitution or addition to the allogeneic islet therapy, offering both a function‐ ally competent adult cell source with already developed insulin-sensing machinery, and sufficient quantities of tissue immediately available for transplant. However, before persistent problems with immune rejection and destruction of the graft can be overcome, porcine islets do not have a hope of replacing or supplementing allogeneic islet cells as a viable treatment option. Embryonic stem cells have the required proliferative potential, with recent studies clearly demonstrating that ESCs provide a definitive platform for differentiation into insulin producing structures. However, it remains to be seen whether (a) current experimental protocols can be scaled-up to generate sufficient number of cells for transplant; (b) current purification methods offer sufficiently stringent protocols to be able to transplant glucosesensing β-like cells only, all the while unequivocally excluding potentially oncogenic "other types" of cell populations; (c) functional equivalency of the resulting glucose-sensing β-like cells to native β-cells can be clearly confirmed; and (d) the cell graft can be adequately protected to avoid efficient immune surveillance systems of the host. This is where the concept of generating sufficient insulin-producing tissue from an autologous, i.e. patient-specific, source becomes attractive. However, the early promise of this iPSCs has not translated from it early success in the experimental setting to the clinical model, mostly due to the same problems that are associated with ESCs. These, however, are multiplied by the limited proliferative capacity of these cells, as well as issues with inadequate function, i.e. poor insulin expression coupled with very low insulin secretion. The problems that stem from immunogenicity of the graft tissue are intrinsic to cells from various sources, including a tailored patient-specific iPSCsderived approach. With the number of factors impacting the way β-cell autoimmunity can be manipulated, several key issues might be considered when it comes to the development of immunotherapeutic solutions to diabetes. These include the requirement for the suppression of the diabetogenic response early in the course of the development of the disease, as well as clear understanding of the autoreactive antigen(s) that might be defined by particular geno‐ type and/or environmental exposure. The complexity of IDDM means that immunomodula‐ tory therapies, antigen- and antibody-specific, might offer a solution when utilized in combination. The goal here is to preserve the functional capacity of the cellular graft or innate islet cells, while at the same time attempting to restore the unique balance between the pathogenic effector and the immunomodulatory T-cell population eroded by the autoimmune assault brought forth by the onset of the disease. Combination immunotherapy will likely

Cell Replacement Therapy in Type 1 Diabetes

http://dx.doi.org/10.5772/54943

603

Taking into an account that it is a BM stem cell that's involved in the development of autoim‐ mune disease, the timing of BMC administration for the treatment of autoimmune diabetes is critical. Due to the fact that autoimmunity results in the progressive destruction of pancreatic β-cells, the ultimate timing for BMC infusion is during the early stages of the disease, when overt diabetes ensues, exogenous insulin is administered, and return to normoglycemia and even production of endogenous insulin are observed. However, the main drawback for the widespread use of BMC therapy to treat Type 1 diabetes is harsh, often lethal, recipient preconditioning regiments. Although non-lethal conditioning protocols, discussed earlier, have been developed, donor-specific chimerism reported under such circumstances is often transient [126]. However, encouraging results in terms of the induction of stable chimerism in kidney transplant recipients have been recently reported by Leventhal et al [132]. He used mobilized cells enriched for hematopoietic stem cells (HSC) in combination with a graftfacilitating cell (FC) population (CD8dim, CD3+ /CD45R+ /Thy1+ /Class IIdim/intermediate, αβ-TCR- and δλ-TCR- ) and nonmyeloablative conditioning in recipients of MHC mismatched, unrelated kidney grafts. Five out of eight transplant recipients exhibited stable donor-specific chimerism, and were weaned of immunosuppression at 1 year following transplant. None of the transplant recipients were reported to show signs of GVHD. As previously reported by Ildstad [125], the FC is not a stem cell, but this population seems to be necessary to enable successful BMC engraftment in MHC disparate environment. Although the mechanism by which FC aids engraftment is not clear, it was characterized previously and found to be necessary to prevent GVHD and promote engraftment in standard BMC transplant protocols [125,126]. These results are exciting and offer much optimism towards treatment strategies applicable to patients with Type 1 diabetes.

Type 1 diabetes is a multifaceted disease, for which no single arm immunotherapeutic approach is possible. It has been long established that immunotherapies that target early vs. later pre-clinical stages in the disease progression offer a treatment approach with higher likelihood of success. However, even that might not be enough to effectively solve this formidable problem. It is possible that no single immunotherapeutic approach will offer longterm protection from diabetes onset and progressive autoimmune destruction of β-cells, in either prevention or treatment setting. A number of immunological approaches, in combina‐ torial manner, that exploit the strengths and circumvent the adverse events of potential therapies at the same time, might prove to be the answer. At this point such approaches are still in the development stage, albeit many hurdles have been overcome to move this approach forward. Latest developments in this area do offer much optimism.

#### **1.6. Concluding remarks**

NOD mice and rendering these recipients mixed chimeras, reverses insulitis and the autoim‐ mune process, halting the development of overt diabetes. Ildstad proposed two possible explanations for how allogeneic BMC-derived chimerism can prevent diabetes. First, donor BMC activates a regulatory cell instrumental in suppressing the activation of autoreactive lymphocytes identified as a culprit in the progression of autoimmunity, development of the overt diabetes and fully developed disease. Second, BMC can cause clonal deletion of autor‐

Taking into an account that it is a BM stem cell that's involved in the development of autoim‐ mune disease, the timing of BMC administration for the treatment of autoimmune diabetes is critical. Due to the fact that autoimmunity results in the progressive destruction of pancreatic β-cells, the ultimate timing for BMC infusion is during the early stages of the disease, when overt diabetes ensues, exogenous insulin is administered, and return to normoglycemia and even production of endogenous insulin are observed. However, the main drawback for the widespread use of BMC therapy to treat Type 1 diabetes is harsh, often lethal, recipient preconditioning regiments. Although non-lethal conditioning protocols, discussed earlier, have been developed, donor-specific chimerism reported under such circumstances is often transient [126]. However, encouraging results in terms of the induction of stable chimerism in kidney transplant recipients have been recently reported by Leventhal et al [132]. He used mobilized cells enriched for hematopoietic stem cells (HSC) in combination with a graft-

/CD45R+

kidney grafts. Five out of eight transplant recipients exhibited stable donor-specific chimerism, and were weaned of immunosuppression at 1 year following transplant. None of the transplant recipients were reported to show signs of GVHD. As previously reported by Ildstad [125], the FC is not a stem cell, but this population seems to be necessary to enable successful BMC engraftment in MHC disparate environment. Although the mechanism by which FC aids engraftment is not clear, it was characterized previously and found to be necessary to prevent GVHD and promote engraftment in standard BMC transplant protocols [125,126]. These results are exciting and offer much optimism towards treatment strategies applicable to

Type 1 diabetes is a multifaceted disease, for which no single arm immunotherapeutic approach is possible. It has been long established that immunotherapies that target early vs. later pre-clinical stages in the disease progression offer a treatment approach with higher likelihood of success. However, even that might not be enough to effectively solve this formidable problem. It is possible that no single immunotherapeutic approach will offer longterm protection from diabetes onset and progressive autoimmune destruction of β-cells, in either prevention or treatment setting. A number of immunological approaches, in combina‐ torial manner, that exploit the strengths and circumvent the adverse events of potential therapies at the same time, might prove to be the answer. At this point such approaches are still in the development stage, albeit many hurdles have been overcome to move this approach

forward. Latest developments in this area do offer much optimism.

) and nonmyeloablative conditioning in recipients of MHC mismatched, unrelated

/Thy1+

/Class IIdim/intermediate, αβ-TCR- and

eactive T lymphocytes via donor-specific disease resistant APCs [126].

facilitating cell (FC) population (CD8dim, CD3+

patients with Type 1 diabetes.

δλ-TCR-

602 Type 1 Diabetes

Cell replacement strategies offer an enormous potential for the treatment of patients with Type 1 diabetes, in both clinical and economic terms. The availability of unlimited amounts of functionally competent graft material to treat millions of patients suffering from IDDM and its dreadful, debilitating complications can move this field forward from the experimental stage it has found itself in for the last several decades to the forefront of transplantation medicine. The fact that allogeneic islet transplantation offers the most extensively studied and sensible solution to potential cure for IDDM is clear. However, this therapeutic option is far from a perfect solution, and comes hand-in-hand with several problems in the form of serious shortages of the available organs and resulting tissue to satisfy the ever-growing demand, recurrence of autoimmunity and rejection and life-long immunosuppression. Porcine islets offer a viable substitution or addition to the allogeneic islet therapy, offering both a function‐ ally competent adult cell source with already developed insulin-sensing machinery, and sufficient quantities of tissue immediately available for transplant. However, before persistent problems with immune rejection and destruction of the graft can be overcome, porcine islets do not have a hope of replacing or supplementing allogeneic islet cells as a viable treatment option. Embryonic stem cells have the required proliferative potential, with recent studies clearly demonstrating that ESCs provide a definitive platform for differentiation into insulin producing structures. However, it remains to be seen whether (a) current experimental protocols can be scaled-up to generate sufficient number of cells for transplant; (b) current purification methods offer sufficiently stringent protocols to be able to transplant glucosesensing β-like cells only, all the while unequivocally excluding potentially oncogenic "other types" of cell populations; (c) functional equivalency of the resulting glucose-sensing β-like cells to native β-cells can be clearly confirmed; and (d) the cell graft can be adequately protected to avoid efficient immune surveillance systems of the host. This is where the concept of generating sufficient insulin-producing tissue from an autologous, i.e. patient-specific, source becomes attractive. However, the early promise of this iPSCs has not translated from it early success in the experimental setting to the clinical model, mostly due to the same problems that are associated with ESCs. These, however, are multiplied by the limited proliferative capacity of these cells, as well as issues with inadequate function, i.e. poor insulin expression coupled with very low insulin secretion. The problems that stem from immunogenicity of the graft tissue are intrinsic to cells from various sources, including a tailored patient-specific iPSCsderived approach. With the number of factors impacting the way β-cell autoimmunity can be manipulated, several key issues might be considered when it comes to the development of immunotherapeutic solutions to diabetes. These include the requirement for the suppression of the diabetogenic response early in the course of the development of the disease, as well as clear understanding of the autoreactive antigen(s) that might be defined by particular geno‐ type and/or environmental exposure. The complexity of IDDM means that immunomodula‐ tory therapies, antigen- and antibody-specific, might offer a solution when utilized in combination. The goal here is to preserve the functional capacity of the cellular graft or innate islet cells, while at the same time attempting to restore the unique balance between the pathogenic effector and the immunomodulatory T-cell population eroded by the autoimmune assault brought forth by the onset of the disease. Combination immunotherapy will likely prove the most effective by exploring the strength of each approach, while limiting the adverse effects associated with each. Despite significant success attained in this area, most progress so far has been made in experimental models, while clinical applications are still relatively early in their development. Although the challenge of bench-to-bedside technology transfer is significant, success of the last few years give much hope and even optimism for future clinical developments.

[4] Marzarati S, Pileggi A, Ricordi C. Allogeneic islet transplantation. Expert Opin Biol

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[5] Jones PM, Courtney ML, Burns CJ, Persaud SJ. Cell-based treatments of diabetes.

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[9] Centers for Disease Control and Prevention. National diabetes fact sheet: General in‐ formation and national estimates on diabetes in the United States (Rev Ed.). Atlanta:

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Various types of cellular therapies discussed in this chapter might offer multi-faceted and practical approaches to the treatment of diabetes. It is entirely possible that a choice of several different therapeutic options is of great benefit, and might provide a platform to avoid frustrating developmental pains towards a "universal cure". While the prospect of developing patient-specific, i.e. personalized, cellular therapy is appealing, it is complicated, quite expensive and, it's tempting to say, unrealistic to develop. Each of the allogeneic cell replace‐ ment approaches towards a potential therapeutic option discussed here needs to be carefully studied, dissected, and defined regardless of the costs associated with it. Further development in the area of immunotherapeutic approaches and various immunoisolation methodologies, which are beyond the scope of this chapter, will be able to help move cell replacement therapy to the forefront of transplant science. Given the fact that for almost a century administration of exogenous insulin was the only real available therapeutic alternative to the treatment of IDDM, the developments of the past several decades are exiting. It is quite possible that the following decade will see clinical application of a whole gamut of therapeutic options to treat this devastating disease.
