Induction of mixed chimerism with MHC-mismatched but not matched bone marrow transplants results in thymic deletion of host-type autoreactive T-cells in NOD mice

OBJECTIVE

Induction of mixed or complete chimerism via hematopoietic cell transplantation (HCT) from nonautoimmune donors could prevent or reverse type 1 diabetes (T1D). In clinical settings, HLA-matched HCT is preferred to facilitate engraftment and reduce the risk for graft versus host disease (GVHD). Yet autoimmune T1D susceptibility is associated with certain HLA types. Therefore, we tested whether induction of mixed chimerism with major histocompatibility complex (MHC)-matched donors could reverse autoimmunity in the NOD mouse model of T1D.

RESEARCH DESIGN AND METHODS

Prediabetic wild-type or transgenic BDC2.5 NOD mice were conditioned with a radiation-free GVHD preventative anti-CD3/CD8 conditioning regimen and transplanted with bone marrow (BM) from MHC-matched or mismatched donors to induce mixed or complete chimerism. T1D development and thymic deletion of host-type autoreactive T-cells in the chimeric recipients were evaluated.

RESULTS

Induction of mixed chimerism with MHC-matched nonautoimmune donor BM transplants did not prevent T1D in wild-type NOD mice, although induction of complete chimerism did prevent the disease. However, induction of either mixed or complete chimerism with MHC-mismatched BM transplants prevented T1D in such mice. Furthermore, induction of mixed chimerism in transgenic BDC2.5-NOD mice with MHC-matched or -mismatched MHC II^−/− BM transplants failed to induce thymic deletion of de novo developed host-type autoreactive T-cells, whereas induction of mixed chimerism with mismatched BM transplants did.

CONCLUSIONS

Induction of mixed chimerism with MHC-mismatched, but not matched, donor BM transplants re-establishes thymic deletion of host-type autoreactive T-cells and prevents T1D, with donor antigen-presenting cell expression of mismatched MHC II molecules being required.Type 1 diabetes (T1D) is an autoimmune disease in which autoreactive T-cells attack the insulin-secreting islet β-cells and result in insulin deficiency and hyperglycemia (1–3). NOD mouse is still the best animal model for T1D, although the autoimmune abnormality in NOD mice does not totally reflect the abnormality in T1D patients (4–6). The autoimmunity in NOD mice and T1D patients is associated with particular major histocompatibility complex (MHC) or HLA loci such as IAβ^g7 or HLA-DR (7–9). This particular genetic background is associated with central tolerance defects, in which autoreactive thymocytes are resistant to negative selection (10–12), as well as peripheral tolerance defects (13–17).Transgenic expression of protective MHC II molecules in the thymus has been shown to prevent T1D development in mice (18–20). However, this approach cannot readily be translated to humans. Immunomodulation therapies such as administration of anti-CD3 have been shown to reverse new-onset T1D in mouse or ameliorate new-onset T1D in patients via induction of regulatory T-cells (21–25). However, the therapeutic benefit in patients appears to be limited in terms of duration (25). This indicates modulation of peripheral tolerance may not be sufficient for stable re-establishment of immune tolerance in T1D patients, because the defective thymus may constantly export autoreactive T-cells, which can overwhelm peripheral tolerance mechanisms. Therefore, a therapy that can re-establish both central and peripheral tolerance in T1D patients would appear optimal as a means to reverse the autoimmunity associated with T1D.Indeed, autoimmune diseases such as T1D arise from abnormality in the immuno-hematological compartment, and a replacement of the system from a nonautoimmune individual can cure autoimmune T1D or vice versa (26). Therefore, previous studies have proposed that induction of mixed chimerism via hematopoietic cell transplantation (HCT) may provide a curative therapy for autoimmune diseases such as T1D (27). Although it was reported induction of mixed chimerism with bone marrow (BM) transplants from MHC-mismatched or MHC-matched nonautoimmune donors was able to prevent T1D development in NOD recipients conditioned with myelo- or nonmyeloablative total body irradiation (TBI) (28–32), as well as in recipients conditioned with a radiation-free anti-CD3-based regimen (33,34), the mechanisms whereby mixed chimerism reverses such autoimmunity remain largely unknown. So-called mixed chimerism has been defined by the coexistence of donor- and host-type lymphocytes in the periphery such as in the blood or spleen, but it remains unclear whether the host-type cells in the mixed chimeric recipients are de novo developed after HCT or residual mature lymphocytes developed before HCT. In other words, it is not clear whether mixed chimerism can mediate deletion of de novo developed autoreactive T-cells. In addition, although MHC-matched HCT is preferred in clinical settings, it is not yet clear whether induction of mixed chimerism with MHC-matched donor transplants can mediate thymic deletion of de novo developed host-type autoreactive T-cells, because the defect in negative selection is associated with particular MHC II loci (7–9).In the current study, we identified true mixed chimeras by measuring the donor and host-type T-cell precursors in the thymus as well as immature B and myeloid cells in the BM and we evaluated the impact of mixed and complete chimerism with MHC-matched or mismatched donor BM transplants.     

Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell�CAssociated Epitopes Using Combinatorial MHC Multimers

OBJECTIVE

Type 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8⁺ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8⁺ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive.

RESEARCH DESIGN AND METHODS

Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B_10–18, prepro-insulin (PPI)_15–24, islet antigen (IA)-2_797–805, GAD65_114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)_265–273, and prepro islet amyloid polypeptide (ppIAPP)_5–13–specific CD8⁺ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients.

RESULTS

Using this kit, islet autoreactive CD8⁺ T-cells recognizing insulin B_10–18, IA-2_797–805, and IGRP_265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI_15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome.

CONCLUSIONS

A kit was developed that allows simultaneous detection of CD8⁺ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.Type 1 diabetes results from a selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. It is becoming increasingly clear that islet epitope–specific CD8⁺ T-cells play a pivotal role in the destruction process and constitute a significant portion of insulitis (1,2). In accordance, nonobese diabetic mice lacking the expression of major histocompatibility complex (MHC) class I are resistant to autoimmune diabetes (3,4), whereas HLA-A2 transgenic nonobese diabetic mice develop accelerated disease (5). Additionally, transfer of CD8⁺ T-cell clones resulted in transfer of type 1 diabetes (6,7). Thus, detection and monitoring of specific CD8⁺ T-cells may provide a valuable tool to assess the disease activity.Islet cell transplantation has considerable potential as a cure for type 1 diabetes (8). Several groups have reported short-term success, using different islet isolation and immunosuppressive regimens (9–12), but long-term insulin independence is rare (13). The rationale behind transplantation of islet cells is replenishment of destructed cells. Yet, as the insulin-producing cells were destructed by an autoimmune response, islet cell transplantation could also result in reactivation of the autoimmune response. Recently, we have shown that proliferation of CD4⁺ T-cells specific for GAD and IA-2 in patients who underwent islet cell transplantation is associated with clinical outcome (14). Yet, ultimately, the destruction of β-cells is likely to be caused by CD8 T-cells.The epitopes recognized by the diabetes-specific human autoreactive CD8⁺ T-cells are primarily derived from β-cell antigens, most importantly (pre-)(pro-)insulin. Previously, we showed that the presence of CD8⁺ T-cells reactive to the naturally processed insulin–peptide B_10–18 in HLA-A2 correlated with islet cell destruction (15). Recently, another important epitope that was uncovered as the signal peptide of pro-insulin was shown to contain a glucose-regulated CD8⁺ T-cell epitope (prepro-insulin [PPI]_15–24) (16), but many other epitopes derived from insulin and a range of other β-cell–derived antigens, such as GAD65 (17), islet antigen (IA)-2 (18), islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) (19,20), and prepro islet amyloid polypeptide (ppIAPP) (21), have been reported (rev. in 22). Ideally, monitoring for the presence of CD8⁺ T-cells reactive to all of the above-mentioned epitopes simultaneously would be desired, posing considerable constraints on blood volumes accessible for monitoring of islet autoimmunity with conventional immune assays.Currently, monitoring of CD8⁺ T-cells reactive to β-cell–derived antigens requires staining of a large number of, usually fresh, cells with HLA tetramers loaded with a single peptide, or in vitro culture for functional immune assays (proliferation, cytokine production [ELISPOT]). Monitoring multiple epitope-specific CD8⁺ T-cell populations by conventional tetramer technology is generally impossible because of the scarcity of material. Furthermore, detection of islet autoreactive T-cells is hampered by their low precursor frequencies in circulation (23,24), low T-cell receptor (TCR) avidity (15), potentially low binding affinity of peptide epitopes to HLA (25), a wide range of candidate islet epitopes (22), and the existence of regulatory T-cells (26,27).Therefore, we used the recently described combinatorial quantum dot (Qdot) technique (28) to simultaneously detect CD8⁺ T-cells specific for six different β-cell–derived antigens, a naturally occurring HLA-A2 derived peptide, and a mix of viral epitopes in HLA-A2 multimers. Using peripheral blood cells from recent-onset type 1 diabetic patients, their siblings, and control subjects, we validated this technique and established the specificity of these stainings. Subsequently, we monitored the presence of reactive CD8⁺ T-cells before and at several time points after clinical islet cell transplantation. Altogether, we developed a high-throughput and relatively sensitive and specific Diab-Q-kit, allowing simultaneous detection of autoreactive CD8⁺ T-cells to multiple islet epitopes, which is applicable to small volumes of stored blood samples to allow screening in multicenter immune intervention trials.     

Deficiency in B7-H1 (PD-L1)/PD-1 Coinhibition Triggers Pancreatic ��-Cell Destruction by Insulin-Specific, Murine CD8 T-Cells

OBJECTIVE

RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic β-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic β-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells.

RESEARCH DESIGN AND METHODS

DNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1^−/−, PD-1^−/− or bone marrow chimeric mice.

RESULTS

Preproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1^−/− or PD-1^−/− mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic β-cells or of PD-1 in autoreactive CD8 T-cells induced EAD.

CONCLUSIONS

An imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic β-cells can trigger pancreatic β-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the β-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.Insulin-producing β-cells in the pancreatic islets are destroyed by an immune attack in autoimmune type 1 diabetes. Type 1 diabetes is triggered by a poorly defined breakdown in central or peripheral tolerance that allows activation of diabetogenic T-cells (1,2). Preclinical animal models have elucidated some aspects of the priming and effector phase of a diabetogenic immune response (3,4). Mice develop diabetes either spontaneously in the NOD model (5), or in response to transgene-encoded “neo-self” antigens selectively expressed in pancreatic β-cells (6–8). These studies indicated that priming of self-reactive T-cells and β-cell susceptibility to an autoaggressive T-cell attack are distinct steps in the pathogenesis of the disease.Costimulating B7/CD28 family molecules provide critical signals for T-cell activation (9,10). RIP-B7.1 mice express the B7.1 costimulator in pancreatic β-cells under rat insulin promoter (RIP) control (11). We have shown that RIP-B7.1 mice develop CD8 T-cell–dependent experimental autoimmune diabetes (EAD) after immunization with preproinsulin-encoding vectors (12–14). Transgene-driven B7.1 expression in pancreatic β-cells thus makes them susceptible to T-cell–mediated immune attack.Coinhibitory signals generated by “programmed death-1” (PD-1)/“programmed death-ligand-1” (B7-H1 or PD-L1) interaction downmodulated T-cell responses and maintain self-tolerance in autoimmune diabetes (15,16). Inducible or constitutive expression of B7-H1 is found in many peripheral tissues, including the β-cells of the pancreatic islets (17,18). Ligation of PD-1 (expressed by activated T-cells) to B7-H1 (expressed by epitope-presenting cells) downmodulates T-cell proliferation and IFNγ production (19). Furthermore, B7-H1 interacts specifically with the costimulatory B7.1 (CD80) molecule upregulated by activated T-cells and inhibits their responses (20). PD-1/B7-H1 interaction facilitates establishment of self-tolerance, thereby partially controlling diabetes development in NOD mice (15,21–23). Selective, transgene-driven overexpression of B7-H1 by pancreatic β-cells can, however, result in EAD, suggesting operation of a costimulatory B7-H1 pathway (24).We investigated the impact of inhibitory (B7-H1, PD-1) molecules on the pathogenicity of preproinsulin-specific CD8 T-cells. We used RIP-B7.1 mice to characterize the specificity and diabetogenic potential of preproinsulin-specific CD8 T-cell responses. RIP-B7.1 mice were immunized with preproinsulin-encoding vectors, or used as hosts for adoptive T-cell transfers. We further analyzed preproinsulin-specific CD8 T-cell responses and EAD development in C57BL/6 (B6), B7-H1^−/− (25), or PD-1^−/− knockout mice (26), as well as bone marrow chimeras (using different donor T-cell and host β-cell phenotypes of B7-H1/PD-1).     

Activation of Insulin-Reactive CD8 T-Cells for Development of Autoimmune Diabetes

OBJECTIVE

We have previously reported a highly diabetogenic CD8 T-cell clone, G9C8, in the nonobese diabetic (NOD) mouse, specific to low-avidity insulin peptide B15-23, and cells responsive to this antigen are among the earliest islet infiltrates. We aimed to study the selection, activation, and development of the diabetogenic capacity of these insulin-reactive T-cells.

RESEARCH DESIGN AND METHODS

We generated a T-cell receptor (TCR) transgenic mouse expressing the cloned TCR Vα18/Vβ6 receptor of the G9C8 insulin-reactive CD8 T-cell clone. The mice were crossed to TCRCα^−/− mice so that the majority of the T-cells expressed the clonotypic TCR, and the phenotype and function of the cells was investigated.

RESULTS

There was good selection of CD8 T-cells with a predominance of CD8 single-positive thymocytes, in spite of thymic insulin expression. Peripheral lymph node T-cells had a naïve phenotype (CD44^lo, CD62L^hi) and proliferated to insulin B15-23 peptide and to insulin. These cells produced interferon-γ and tumor necrosis factor-α in response to insulin peptide and were cytotoxic to insulin peptide–coated targets. In vivo, the TCR transgenic mice developed insulitis but not spontaneous diabetes. However, the mice developed diabetes on immunization, and the activated transgenic T-cells were able to transfer diabetes to immunodeficient NOD.scid mice.

CONCLUSIONS

Autoimmune CD8 T-cells responding to a low-affinity insulin B-chain peptide escape from thymic negative selection and require activation in vivo to cause diabetes.Type 1 diabetes is a complex, multifactorial disease in which genetic factors interact with environmental modifiers to give rise to immune abnormalities leading to pancreatic β-cell damage and destruction. Both CD4 and CD8 T-cells have a major role in pathogenesis of type 1 diabetes. There are a number of autoantigens that are recognized by CD8 T-cells, including proinsulin (PI) (1), islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP) (2), dystrophia myotonica kinase (3), and glutamic acid decarboxylase (4) in the nonobese diabetic (NOD) animal model of diabetes. In HLA-A2 transgenic mice, both PI and IGRP have also been shown to be important targets (5,6). Furthermore, emerging evidence suggests that CD8 T-cells in humans also recognize these antigens (7–9), although their role in pathogenesis is currently unknown.In humans, CD8 T-cells are present in islet infiltrates at the time diabetes develops (10). CD8 T-cells were predominant in the islet infiltrate when diabetes recurred within 6 weeks after transplantation of a hemipancreas from normal, identical nondiabetic twins into their diabetic cotwins (11). In NOD mouse studies, it is well established that CD8 T-cells play an important role both in early events leading to insulitis and diabetes (12,13) as well as in the final effector stage (14) of diabetes development.PI is an important autoantigen in diabetes, and it has been suggested that it is the “prime” autoantigen (15) recognized by CD8 T-cells, CD4 T-cells, and autoantibodies. There is clear evidence that PI is expressed in the thymus (16) in humans and in mice (17), which influences the expression of autoreactive T-cells to PI/insulin. In humans, although the major histocompatibility complex (MHC) is the most important genetic susceptibility factor, the second most important genetic region (IDDM2) is the insulin 5′VNTR region. This controls expression of PI in the thymus and the pancreas (18). Mice have two types of insulin, produced as proinsulin 1 (PI1), mainly in the pancreas, and proinsulin 2 (PI2) in the thymus and pancreas. Studies using individual proinsulin PI1^−/− and PI2^−/− knockout mice, when backcrossed to the NOD background, showed that PI1^−/− mice have a reduced incidence of diabetes (19), whereas in PI2^−/− mice, diabetes is accelerated with 100% developing diabetes (19,20). In addition, insulin autoantibody production is increased in PI2^−/− mice, and spleen cells from young PI2^−/− animals have an increased ability to transfer diabetes (20). Furthermore, when PI2 was overexpressed in the thymus (and on peripheral antigen-presenting cells [APCs]) on the MHC class II promoter (PI2tg), the incidence of diabetes was decreased (21,22). This suggests that PI2 is important for both central and peripheral tolerance for islet β-cells.We have previously isolated a highly pathogenic CD8 T-cell clone (G9C8) from the islets of young, pre-diabetic NOD mice that is capable of very rapidly causing diabetes (5–10 days) upon adoptive transfer to young or irradiated nondiabetic NOD mice (23). The autoantigen is an insulin B-chain peptide (amino acids 15–23 [B15-23]) (1). The epitope is common to both mouse insulins (and is also conserved in human insulin) and is restricted by MHC-K^d. The insulin peptide is recognized by a small population of cells present in the very early stages of the islet infiltrate, identified using a K^d–B15-23 tetramer (1) and is found in mice aged <5 weeks (1,24). Subsequently, other specificities such as IGRP become increasingly dominant (24–26), and this insulin-specific population becomes a smaller percentage of the infiltrate as the disease progresses (1). To further study the selection and activation of this early population of CD8 T-cells, we have generated a T-cell receptor (TCR) transgenic mouse in which the T-cells express the receptor of the highly pathogenic G9C8 clone. Unlike other CD8 TCR transgenic mice that recognize higher-affinity MHC binding peptides of IGRP (2,27), G9C8 T-cells have low avidity, and G9 transgenic mice do not develop spontaneous diabetes. However, transgenic G9 cells can be highly diabetogenic after activation both in vitro and in vivo.     

Interleukin-21 is critically required in autoimmune and allogeneic responses to islet tissue in murine models

OBJECTIVE

Type 1 diabetes is an incurable chronic autoimmune disease. Although transplantation of pancreatic islets may serve as a surrogate source of insulin, recipients are subjected to a life of immunosuppression. Interleukin (IL)-21 is necessary for type 1 diabetes in NOD mice. We examined the efficacy of an IL-21–targeted therapy on prevention of diabetes in NOD mice, in combination with syngeneic islet transplantation. In addition, we assessed the role of IL-21 responsiveness in islet allograft rejection in mouse animal models.

RESEARCH DESIGN AND METHODS

NOD mice were treated with IL-21R/Fc, an IL-21–neutralizing chimeric protein. This procedure was combined with syngeneic islet transplantation to treat diabetic NOD mice. Survival of allogeneic islet grafts in IL-21R–deficient mice was also assessed.

RESULTS

Evidence is provided that IL-21 is continually required by the autoimmune infiltrate, such that insulitis was reduced and reversed and diabetes inhibited by neutralization of IL-21 at a late preclinical stage. Recovery from autoimmune diabetes was achieved by combining neutralization of IL-21 with islet transplantation. Furthermore, IL-21–responsiveness by CD8+ T-cells was sufficient to mediate islet allograft rejection.

CONCLUSIONS

Neutralization of IL-21 in NOD mice can inhibit diabetes, and when paired with islet transplantation, this therapeutic approach restored normoglycemia. The influence of IL-21 on a graft-mounted immune response was robust, since the absence of IL-21 signaling prevented islet allograft rejection. These findings suggest that therapeutic manipulation of IL-21 may serve as a suitable treatment for patients with type 1 diabetes.In type 1 diabetes, activated immune cells lead to the destruction of the insulin-producing β-cells in the islets of Langerhans of the pancreas (1). Clinical diabetes occurs in the nonobese diabetic (NOD) model after months of chronic pancreatic inflammation, progressing from peri-insulitis to destructive insulitis (2,3). Importantly, therapies designed to modulate lymphocyte activation are compatible with the prevention of the destructive form of insulitis, i.e., the movement of immune cells into the islet, and subsequent loss of insulin production from β-cells (4,5).Insulin replacement is the current standard for treating type 1 diabetes, however, transplantation of pancreatic islets has the potential to serve as a viable alternative (6). Challenges of islet transplantation include finding alternatives to broad-spectrum immunosuppression (7) while preventing graft rejection and recurrence of the underlying autoimmune destruction of pancreatic islets. As T-cells constitute an integral component in both autoimmune responses of diabetes and the rejection of transplanted islet allografts (8,9), therapies addressing modulation of T-cell function may provide an appropriate strategy.IL-21 is a member of the common γ-chain signaling family of cytokines that is necessary for the development of diabetes in the NOD mouse (10–12). The receptor for IL-21, comprising the α unit (IL-21Rα) and the common γ chain, is expressed on immune cells including T-, B-, NK, and dendritic cells, whereas IL-21 expression is largely limited to CD4+ T-cells (13). Several studies demonstrate that IL-21 acts as a lymphocyte costimulator, enhancing the proliferation and effector function of CD8+ T-cells (14,15), and transgenic over-expression of murine IL-21 revealed that IL-21 predominantly expands memory phenotype CD8+ T-cells (16). The prosurvival effect of IL-21 is important for CD8+ T-cells during chronic viral infection (17–19), with IL-21 also potently effecting the activation and differentiation of numerous CD4+ T-helper subsets, including Th17 cells (20,21).Consistent with its actions on lymphocyte populations, IL-21 has been found to contribute to the development of autoimmune diseases in several animal models (22). Likewise, IL-21 has the potential to influence the outcome of islet graft transplantation (23–25). For instance, IL-21 has a well-documented ability to promote the production of granzyme and perforin in differentiating CD8+ cytotoxic T-cells. Direct killing of islet cells by antigen-specific cytotoxic T-cells is an important component of both allograft rejection and the autoimmune destruction of β-cells (26,27). Secondly, IL-21 costimulates the activation and differentiation of antigen specific CD4+ T-cells, and these cells can produce proinflammatory cytokines that are toxic to the islets, such as IL-1β, tumor necrosis factor-α, and γ-interferon (IFNγ) (28–31).In this study, we demonstrate that IL-21 acts on immune cells to elicit autoimmune destruction of endogenous pancreatic islet tissue in autoimmune diabetes and islet graft rejection caused by both autoimmune and allogeneic immune responses. We provide evidence that through the modulation of IL-21, a potential therapeutic intervention for type 1 diabetes may be attainable.     

Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

Enterovirus Infection, CXC Chemokine Ligand 10 (CXCL10), and CXCR3 Circuit: A Mechanism of Accelerated ��-Cell Failure in Fulminant Type 1 Diabetes

OBJECTIVE

Fulminant type 1 diabetes is characterized by the rapid onset of severe hyperglycemia and ketoacidosis, with subsequent poor prognosis of diabetes complications. Causative mechanisms for accelerated β-cell failure are unclear.

RESEARCH DESIGN AND METHODS

Subjects comprised three autopsied patients who died from diabetic ketoacidosis within 2–5 days after onset of fulminant type 1 diabetes. We examined islet cell status, including the presence of enterovirus and chemokine/cytokine/major histocompatibility complex (MHC) expressions in the pancreata using immunohistochemical analyses and RT-PCR.

RESULTS

Immunohistochemical analysis revealed the presence of enterovirus-capsid protein in all three affected pancreata. Extensive infiltration of CXCR3 receptor–bearing T-cells and macrophages into islets was observed. Dendritic cells were stained in and around the islets. Specifically, interferon-γ and CXC chemokine ligand 10 (CXCL10) were strongly coexpressed in all subtypes of islet cells, including β-cells and α-cells. No CXCL10 was expressed in exocrine pancreas. Serum levels of CXCL10 were increased. Expression of MHC class II and hyperexpression of MHC class I was observed in some islet cells.

CONCLUSIONS

These results strongly suggest the presence of a circuit for the destruction of β-cells in fulminant type 1 diabetes. Enterovirus infection of the pancreas initiates coexpression of interferon-γ and CXCL10 in β-cells. CXCL10 secreted from β-cells activates and attracts autoreactive T-cells and macrophages to the islets via CXCR3. These infiltrating autoreactive T-cells and macrophages release inflammatory cytokines including interferon-γ in the islets, not only damaging β-cells but also accelerating CXCL10 generation in residual β-cells and thus further activating cell-mediated autoimmunity until all β-cells have been destroyed.Fulminant type 1 diabetes is characterized by abrupt onset of severe hyperglycemia and ketoacidosis preceded by flu-like symptoms including fever, abdominal pain, and headache (1–3). Due to the rushed clinical course in most cases, patients with fulminant type 1 diabetes are sometimes untreated until becoming comatose and/or entering a critical, life-threatening state (4). Endogenous insulin secretion is completely abolished over time and diabetic microangiopathies develop over a short duration (5,6). The mechanisms underlying the aggressive and rapid destruction of β-cells have remained one of the major questions regarding this subtype of type 1 diabetes. However, in situ human data on affected islets and pancreas and possible mechanisms have been completely lacking for fulminant type 1 diabetes.Viral infection with subsequent immunological mechanisms represents one of the leading candidates for destruction of β-cells in fulminant type 1 diabetes (3,7). Some studies on the mouse model of lymphocytic choriomeningitis virus–induced type 1 diabetes have demonstrated that islet β-cells can be destroyed as follows: within 1 day after virus infection, CXC chemokine ligand 10 (CXCL10) (8), a key chemoattractant for activated T-cells and macrophages, is produced in β-cells and secreted from islets (9). Activated T-cells bearing the receptor for CXCL10, named CXCR3 (8), infiltrate and accumulate in islets secreting CXCL10 (10). Accumulated T-cells at the islets then destroy β-cells through cell-mediated mechanisms (11). With this mechanism, CXCL10 is necessary and sufficient for accelerated T-cell response with complete β-cell destruction and resulting type 1 diabetes (10,12,13). We have recently found that serum CXCL10 levels are increased at the onset of fulminant type 1 diabetes, suggesting a crucial role of the CXCL10-CXCR3 axis in the aggressive β-cell destruction in this syndrome (14). We therefore examined in situ status with regard to enterovirus infection, CXCL10-CXCR3 axis, major histocompatibility complex (MHC) molecule expression, and islet dysfunction in pancreata from patients with fulminant type 1 diabetes who died due to diabetic ketoacidosis within 2–5 days after outset of flu-like symptoms. Our in situ findings for affected pancreata provide new insights into understanding the pathogenesis of and developing interventional strategies against human type 1 diabetes.     

Insulin resistance, defective insulin-mediated fatty acid suppression, and coronary artery calcification in subjects with and without type 1 diabetes: The CACTI study

OBJECTIVE

To assess insulin action on peripheral glucose utilization and nonesterified fatty acid (NEFA) suppression as a predictor of coronary artery calcification (CAC) in patients with type 1 diabetes and nondiabetic controls.

RESEARCH DESIGN AND METHODS

Insulin action was measured by a three-stage hyperinsulinemic-euglycemic clamp (4, 8, and 40 mU/m²/min) in 87 subjects from the Coronary Artery Calcification in Type 1 Diabetes cohort (40 diabetic, 47 nondiabetic; mean age 45 ± 8 years; 55% female).

RESULTS

Peripheral glucose utilization was lower in subjects with type 1 diabetes compared with nondiabetic controls: glucose infusion rate (mg/kg FFM/min) = 6.19 ± 0.72 vs. 12.71 ± 0.66, mean ± SE, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and final clamp glucose and insulin. Insulin-induced NEFA suppression was also lower in type 1 diabetic compared with nondiabetic subjects: NEFA levels (μM) during 8 mU/m²/min insulin infusion = 370 ± 27 vs. 185 ± 25, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and time point insulin. Lower glucose utilization and higher NEFA levels, correlated with CAC volume (r = −0.42, P < 0.0001 and r = 0.41, P < 0.0001, respectively) and predicted the presence of CAC (odds ratio [OR] = 0.45, 95% CI = 0.22–0.93, P = 0.03; OR = 2.4, 95% CI = 1.08–5.32, P = 0.032, respectively). Insulin resistance did not correlate with GHb or continuous glucose monitoring parameters.

CONCLUSIONS

Type 1 diabetic patients are insulin resistant compared with nondiabetic subjects, and the degree of resistance is not related to current glycemic control. Insulin resistance predicts the extent of coronary artery calcification and may contribute to the increased risk of cardiovascular disease in patients with type 1 diabetes as well as subjects without diabetes.Cardiovascular disease (CVD) remains the leading cause of death in individuals with type 1 diabetes (1–4). Although hyperglycemia appears to be the primary mediator of microvascular disease (5,6), its role in macrovascular disease is less clear (4). Tight glycemic control improves, but does not normalize CVD risk, and correlation of GHb to CVD risk remains controversial (7–15). In addition, standard prediction rules for CVD risk do not accurately predict CVD in type 1 diabetic populations (16). Thus, the mechanism of accelerated atherosclerosis in type 1 diabetes is unclear and identification of those patients at highest risk and most in need of aggressive risk factor modification is inaccurate.In the general population, insulin resistance has been implicated as an important contributor to accelerated atherosclerosis (17–25). Although type 1 diabetes is primarily a disease of insulin deficiency, previous studies have demonstrated insulin resistance and suggested that CVD may also be linked to insulin resistance in type 1 diabetes (10,26–32). As early as 1968, Martin et al. (30) demonstrated an “impaired glucose assimilation index” and an inverse association between this index and prevalent macrovascular disease in type 1 diabetic subjects. More recently, the Pittsburgh Epidemiology of Diabetes Complications Study (10) found no correlation between GHb and coronary artery disease outcomes. However, in addition to other known CVD risk factors, estimated glucose disposal rate correlated inversely with these outcomes. Similar correlations of estimated insulin resistance or a surrogate of insulin resistance (waist-to-hip ratio) to coronary artery disease were also found in the Diabetes Control and Complications Trial (DCCT) and the EURODIAB study (33). These data suggest that an estimate of insulin resistance may add to CVD risk prediction in type 1 diabetes. In addition, elevated nonesterified fatty acid (NEFA) levels have been proposed to mediate the increased atherosclerotic risk associated with insulin resistance in the general population (18,34–37). It is not known whether the defects in insulin action in type 1 diabetes extend beyond glucose utilization to NEFA suppression.The Coronary Artery Calcification in Type 1 Diabetes (CACTI) study has followed a cohort of type 1 diabetic subjects and similar nondiabetic controls with electron beam computed tomography for measurement of coronary artery calcification (CAC) and CVD outcomes for 6 years (15,38). We hypothesized that type 1 diabetic subjects would be more insulin resistant than nondiabetic controls in terms of both glucose utilization and NEFA suppression, and that both measures of insulin resistance would correlate with CAC, a marker of the extent of coronary atherosclerosis.     

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVE

Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway.

RESEARCH DESIGN AND METHODS

We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model.

RESULTS

We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR^−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR^−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR^−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators.

CONCLUSIONS

Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.The complement system is an integral part of both the innate and adaptive immune response involved in the defense against invading pathogens (1). Complement activation culminates in a massive amplification of the immune response leading to increased cell lysis, phagocytosis, and inflammation (1). C3 is the most abundant component of the complement cascade and the convergent point of all three major complement activation pathways. C3 is cleaved into C3a and C3b by the classical and lectin pathways, and iC3b is generated by the alternative pathway (2,3). C3a has potent anaphylatoxin activity, directly triggering degranulation of mast cells, inflammation, chemotaxis, activation of leukocytes, as well as increasing vascular permeability and smooth muscle contraction (3). C3a mediates its downstream signaling effects by binding to the C3a receptor (C3aR), a Gi-coupled G protein–coupled receptor. Several studies have demonstrated a role for C3a and C3aR in asthma, sepsis, liver regeneration, and autoimmune encephalomyelitis (1,3). Therefore, targeting C3aR may be an attractive therapeutic option for the treatment of several inflammatory diseases.Increasing literature suggests that metabolic disorders such as diabetes, obesity, and atherosclerosis also possess an important inflammatory component (4–7). Several seminal reports have demonstrated that resident macrophages can constitute as much as 40% of the cell population of adipose tissue (7–9) and can significantly affect insulin resistance (10–18). Several proinflammatory cytokines, growth factors, acute-phase proteins, and hormones are produced by the adipose tissue and implicated in insulin resistance and vascular homeostasis (4–7,19). An integrated genomics approach was performed with several mouse strains to infer causal relationships between gene expression and complex genetic diseases such as obesity/diabetes. This approach identified the C3aR gene as being causal for omental fat pad mass (20). The C3aR^−/− mice were shown to have decreased adiposity as compared with wild-type mice on a regular diet (20). Monocytes and macrophages express the C3aR (21–28). Increased C3a levels also correlate with obesity, diabetes, cholesterol, and lipid levels (29–34). We therefore sought to investigate the specific role of the C3aR in insulin resistance, obesity, and macrophage function utilizing both normal diet and the diet-induced obesity model.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

Enhanced Energy Expenditure, Glucose Utilization, and Insulin Sensitivity in VAMP8 Null Mice

OBJECTIVE

Previous studies have demonstrated that the VAMP8 protein plays a complex role in the control of granule secretion, transport vesicle trafficking, phagocytosis, and endocytosis. The present study was aimed to investigate the role of VAMP8 in mediating GLUT4 trafficking and therefore insulin action in mice.

RESEARCH DESIGN AND METHODS

Physiological parameters were measured using Oxymax indirect calorimetry system in 12-week-old VAMP8 null mice. Dynamic analysis of glucose homeostasis was assessed using euglycemic–hyperinsulinemic clamp coupled with tracer radioactively labeled 2-deoxyglucose. Insulin stimulated GLUT4 protein expressions on muscle cell surface were examined by immunofluorescence microscopy.

RESULTS

VAMP8 null mice display reduced adiposity with increased energy expenditure despite normal food intake and reduced spontaneous locomotor activity. In parallel, the VAMP8 null mice also had fasting hypoglycemia (84 ± 11 vs. 115 ± 4) and enhanced glucose tolerance with increased insulin sensitivity due to increases in both basal and insulin-stimulated glucose uptake in skeletal muscle (0.19 ± 0.04 vs. 0.09 ± 0.01 mmol/kg/min during basal, 0.6 ± 0.04 vs. 0.31 ± 0.06 mmol/kg/min during clamp in red-gastrocnemius muscle, P < 0.05). Consistent with a role for VAMP8 in the endocytosis of the insulin-responsive GLUT4, sarcolemma GLUT4 protein levels were increased in both the basal and insulin-stimulated states without any significant change in the total amount of GLUT4 protein or related facilitative glucose transporters present in skeletal muscle, GLUT1, GLUT3, and GLUT11.

CONCLUSIONS

These data demonstrate that, in the absence of VAMP8, the relative subcellular distribution of GLUT4 is altered, resulting in increased sarcolemma levels that can account for increased glucose clearance and insulin sensitivity.Insulin-stimulated glucose uptake in muscle and adipose tissue is mediated by the insulin-responsive GLUT isoform GLUT4 (1). In the basal state, ∼95% of the GLUT4 protein is sequestered in intracellular membranes (termed the insulin-responsive storage vesicles) and after acute insulin stimulation undergoes a translocation process such that 50% of the GLUT4 protein is cell surface localized (2–5). There is now good evidence that vesicle fusion reactions are initiated by interactions among SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that assemble into a four-helix bundle between the transport and acceptor membranes (6–8). It is generally accepted that the plasma membrane t-SNARE is composed of syntaxin 4 and SNAP23 whereas the v-SNARE present in GLUT4 vesicles is VAMP2 (9–15), although a recent study has suggested that several VAMP proteins can play redundant roles in this process (16). Endobrevin or VAMP8 was originally identified as an endosomal v-SNARE that mediated the homotypic fusion of early and late endosomes (13,17–21). Subsequently, VAMP8 was found to be present on several membrane compartments including early and late endosomes as well as the plasma membrane, trans-Golgi network, clathrin-coated pits, and secretory granules (15,19,22,23). VAMP8 was found to be required for mast cell degranulation, zymogen granule release from pancreatic acinar cells, and dense core granule release from platelets, suggesting that VAMP8 functions in plasma membrane exocytosis (13,19,24–28). On the other hand, it has been recently reported that VAMP8 functions to inhibit phagocytosis in immature dendritic cells, to induce membrane ruffles and bacterial entry into nonphagocytic cells, and for the efficient endocytosis of the plasma membrane GLUT4 protein in adipocytes (16,29). Thus, VAMP8 appears to have several functions that may be dependent upon the particular cell type and/or trafficking cargo material.To further investigate the role of VAMP8, we have taken advantage of the VAMP8 null mouse (19) to examine the integrative physiology of glucose metabolism and GLUT4 trafficking in skeletal muscle in vivo. These data demonstrate that VAMP8 plays multiple roles in the whole-body regulation of metabolism, energy expenditure, and insulin sensitivity. Consistent with a role of VAMP8 in mediating GLUT4 endocytosis, skeletal muscle sarcolemma levels of GLUT4 were elevated in the VAMP8 null mice, directly accounting for the increase in both basal and insulin-stimulated glucose uptakes.     

Unexpected Acceleration of Type 1 Diabetes by Transgenic Expression of B7-H1 in NOD Mouse Peri-Islet Glia

OBJECTIVE

Autoimmune target tissues in type 1 diabetes include pancreatic β-cells and peri-islet Schwann cells (pSC)—the latter active participants or passive bystanders in pre-diabetic autoimmune progression. To distinguish between these alternatives, we sought to suppress pSC autoimmunity by transgenic expression of the negative costimulatory molecule B7-H1 in NOD pSC.

RESEARCH DESIGN AND METHODS

A B7-H1 transgene was placed under control of the glial fibrillary acidic protein (GFAP) promoter. Transgenic and wild-type NOD mice were compared for transgene PD-1 affinities, diabetes development, insulitis, and pSC survival. Mechanistic studies included adoptive type 1 diabetes transfer, B7-H1 blockade, and T-cell autoreactivity and sublineage distribution.

RESULTS

Transgenic and endogenous B7-H1 bound PD-1 with equal affinities. Unexpectedly, the transgene generated islet-selective CD8⁺ bias with accelerated rather than suppressed diabetes progression. T-cells of diabetic transgenics transferred type 1 diabetes faster. There were no earlier pSC losses due to conceivable transgene toxicity, but transgenic pSC loss was enhanced by 8 weeks, preceded by elevated GFAP autoreactivity, with high-affinity T-cells targeting the major NOD K^d-GFAP epitope, p253–261. FoxP3⁺ regulatory T- and CD11c⁺ dendritic cell pools were unaffected.

CONCLUSIONS

In contrast with transgenic B7-H1 in NOD mouse β-cells, transgenic B7-H1 in pSC promotes rather than protects from type 1 diabetes. Here, ectopic B7-H1 enhanced the pathogenicity of effector T-cells, demonstrating that pSC can actively impact diabetes progression—likely through modification of intraislet T-cell selection. Although pSC cells emerge as a new candidate for therapeutic targets, caution is warranted with regard to the B7-H1–PD1 axis, where B7-H1 overexpression can lead to accelerated autoimmune disease.The NOD mouse is a spontaneous model of type 1 diabetes, with genetic and pathophysiological roots comparable with the human disease (1). Pancreatic islets of Langerhans are tightly enveloped by peri-islet Schwann cells (pSC) that express glial fibrillary acidic protein (GFAP), a marker of Schwann cells and astrocytes (2). During pre-diabetes progression, T-cell infiltrates accumulate at the endocrine/exocrine border, constituted by the pSC mantle, where lengthy “peri”-insulitis lasts for weeks to months in NOD mice and likely for years in humans with islet autoimmunity. Eventual breakdown of the pSC mantle initiates pathogenic islet invasion, progressive β-cell loss, insulin deficiency, and overt diabetes development. In NOD mice, CD8⁺ T-cells predominate islet attack until late in this process (3).Islet T-cell infiltrations are heterogeneous in their target autoantigen specificities for not only β-cell–selective autoantigens (e.g., insulin) but also autoantigens shared by β-cells and nervous system tissue, islet-associated autoantigens shared by pSC and β-cells (e.g., S100β) or those that are pSC specific (e.g., GFAP) (4). pSC functions and their importance in type 1 diabetes development have yet to be fully characterized. In NOD mice, pSC-specific T-cell autoreactivities are present by 5 weeks of age. GFAP target epitopes were recently mapped to residues 79–87 and 253–261 for K^d and 96–110, 116–130, and 216–230 for NOD-IA^g7, and fresh ex vivo CD8⁺ cells mediate direct lysis of primary pSC cultures from diabetic NOD mice (5).pSC cells likely have physiological functions similar to conventional Schwann cells of the peripheral nervous system, providing neurotrophic support for islet-innervating neurons as well as the neural crest-derived β-cell (2). For example, nerve growth factor, glial cell—derived neurotrophic factor, and insulin-like growth factor-1 promote β-cell survival and probably regeneration (6–8). Loss of these factors with pSC destruction may amplify β-cell stress, enhancing β-cell susceptibility to inflammatory insults (7). Anatomically, pSC provide a physical barrier to infiltrating T-cells, accumulating at the endo-exocrine islet border and impeding direct β- and T-cell contact.B7-H1, a ligand for programmed death (PD)-1, is expressed by CD4⁺ and CD8⁺ T-cells, B-cells, dendritic cells (DCs), macrophages, mast cells, and nonhemopoietic tissues (9). In nonlymphoid tissue, DC-B7-H1 supports peripheral tolerance, limiting randomly arising autoaggressive lymphocytes and their inflammatory tissue damage (10,11). In tumors, expression of B7-H1 contributes to immune evasion, inducing anergy or apoptosis of tumor-specific T-cells (12–14). Consistently with an inhibitory role, treatment of NOD mice with blocking antibodies to either PD-1 or B7-H1 accelerates diabetes (15), with analogous scenarios in autoimmune (16) and other (12,17,18) models. These systemic manipulations of the PD-1/B7-H1 axis generated the consensus view that B7-H1 ligation keeps potentially damaging autoimmune T-cells in check and serves to downregulate lymphoid effector functions (19).However, conflicting data exist. The B7-H1 pathway can promote T-cell activation and autoimmunity in certain experimental settings, including transgenic expression of B7-H1 in β-cells of C57Bl/six mice (20–22). For these exceptions, an alternative receptor for B7-H1 has been proposed but not identified to date (23,24). We nevertheless felt that the weight of evidence, specifically in NOD mice, suggested that B7-H1 might serve as a tool to selectively suppress NOD pSC autoimmunity, allowing us to learn whether and how pSC cells impact on the β-cell autoimmune progression program: transgenic expression of B7-H1 in NOD β-cells protects from type 1 diabetes (19). We here describe the effects of a pSC B7-H1 transgene. Our finding of type 1 diabetes acceleration emphasizes the complexity of this costimulatory pathway, while the selective, intraislet CD8⁺ bias of high-affinity T-cells demonstrates that pSC cells do impact the β-cell destruction program, culminating in type 1 diabetes.     

Redox Modulation Protects Islets From Transplant-Related Injury

OBJECTIVE

Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant.

RESEARCH DESIGN AND METHODS

The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation.

RESULTS

Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection.

CONCLUSIONS

Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.Hypoxia is the leading cause of β-cell death during islet isolation and transplantation (1), with the highest percentage of islet graft loss and dysfunction occurring just days after transplantation (2,3). Because islets are a cellular transplant, devoid of intrinsic vasculature (1,4), they are exceptionally susceptible to ischemia-reperfusion injury. Islets are also increasingly vulnerable because they have inherently decreased antioxidant capacity (5–10), making them prone to oxidative/nitrosative/free radical damage. The antigen-independent complexities of islet transplantation increase the incidence of primary graft nonfunction and β-cell death, thus requiring protection for islets at early stages of the transplant procedure (11).In addition to antigen-independent innate-mediated inflammatory injury, islet allografts are also plagued by the antigen-dependent T-cell mediated alloimmune response, which necessitates immunosuppressive drugs for allograft survival. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response, although most are harmful to islets and do not protect well from reactive oxygen species and inflammation during islet isolation and ischemia-reperfusion injury (12–14). In their review, Balamurugan et al. (13) concluded that successful islet transplantation in type 1 diabetes necessitates islet-sparing immunosuppressive agents that combat recurrent autoimmunity with low islet toxicity. Predominantly, the field of islet transplantation is devoid of cytoprotective agents that promote islet survival and function by inhibiting nonspecific innate-mediated inflammation during islet isolation and early inflammatory events in islet transplantation (11,13,15–19).The first phase of immunity involves innate immune activation and subsequent proinflammatory signals required for optimal adaptive immune function (20–22), yet the majority of immunosuppressive drugs only target adaptive immune function (17,23), the second phase of immunity. A nontoxic, cell-permeable catalytic antioxidant (CA) redox modulator, FBC-007 [manganese(II) tetrakis (N-ethylpyridium-2-yl)porphyrin], is able to depress free radical and cytokine production by antigen-presenting cells (24) and T cells in transgenic and allospecific mouse models (20,25). Additionally, redox modulation inhibits cytotoxic lymphocyte target cell lysis by reducing the production of intracellular cytolytic molecules (perforin and granzyme B) in a mixed leukocyte reaction without toxicity (25), preserves and promotes human islet function in vitro (15,16), prevents the transfer of diabetes into young NOD.scid mice (26), and inhibits innate-immune nuclear factor (NF)-κB activation (24). Thus, islet-sparing agents, which decrease the production of free radicals and, therefore, inflammatory cytokines, may have a positive impact on islet function post-transplant.Because islet transplantation can benefit from agents that inhibit early inflammatory cascades to preserve islet function (18), we hypothesize that redox modulation holds potential as a therapy in islet transplantation to decrease the incidence of β-cell primary nonfunction. To further test the effects of redox modulation using CA we treated human islets with streptozotocin (STZ) in vitro and treated mice in vivo with STZ, both in the presence or absence of CA, to mimic antigen-independent free radical damage and inflammation of post-transplant ischemia-reperfusion injury. To examine the effects of islet-directed CA treatment on innate-mediated (antigen-independent) primary islet nonfunction in vivo, we performed syngeneic (175 islets/recipient), suboptimal syngeneic (100 islets/recipient), allogeneic (300 islets/recipient), and xenogeneic (400–500 islets/recipient) islet transplants to assess islet function. Additionally, we performed (300 islets/recipient) islet transplants in diabetic recipients to assess islet function in the presence or absence of systemic redox modulation in an allogeneic transplant setting inclusive of both innate (antigen-independent) and adaptive (antigen-dependent) immune responses. Our results demonstrate that islet-directed and systemically delivered redox modulation, administered in the absence of an additional immunosuppressive regimen, preserve islet function post-transplant.     

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

OBJECTIVE

The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets.

RESEARCH DESIGN AND METHODS

Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO–expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice.

RESULTS

IDO-expressing grafts survived significantly longer than controls (41.2 ± 1.64 vs. 12.9 ± 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming.

CONCLUSIONS

Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts.Endocrine replacement therapy by islet transplantation represents a feasible and attractive alternative therapeutic approach for treating type 1 diabetes (1,2). Despite improvement of allogeneic islet engraftment using systemic immunosuppression, islet transplantation is still limited by high rates of rejection. Furthermore, some immunosuppressive agents are prodiabetogenic and associated with adverse side effects (3–6). Finding more efficient and less harmful strategies to protect islet graft is therefore required for improving islet transplantation outcome.Localized expression of immunoregulatory factors using gene transfer to graft is a feasible method to provide an immunoprivileged microenvironment and consequently improves graft survival. Such an on-site delivery system results in more potent local immunosuppression with less systemic side effects (7–9).IDO is a cytosolic enzyme that catalyzes essential amino acid l-tryptophan to kynurenine (10) and has profound effects on T-cell proliferation, differentiation, effector functions, and viability (11). Both the reduction in local tryptophan concentration and the production of immunomodulatory tryptophan metabolites contribute to immunosuppressive effects of IDO (12,13). Broad evidence implicates IDO and the tryptophan catabolic pathway in generation of immune tolerance to antigens in tissue microenvironments. In particular, the role of IDO in fetal tolerance in mammalian pregnancy (14,15), immunologic tolerance to tumors (16,17), and self-tolerance has been documented (18,19). The unique immunoregulatory function of IDO substantiates the application of this enzyme as a strategy to suppress alloimmune responses in transplantation.Our research group has shown that overexpression of IDO in fibroblasts suppresses immune response and improves outcome of skin grafts (20–25) and that bystander IDO-expressing fibroblasts suppress immune response to allogeneic mouse islets in vitro (26). Furthermore, in a recent study we showed that mouse islets and fibroblasts are selectively resistant to IDO-mediated activation of nutrient deficiency stress (27). Here, we engineered a three-dimensional composite islet allograft equipped with IDO-expressing fibroblasts and examined whether local expression of IDO, conferred by adenoviral-mediated gene transfer to bystander syngeniec fibroblasts, prevents the rejection of islet allograft. Our approach here is novel compared with other studies that examined the suppressive effect of IDO in islet transplantation (28,29) because 1) bystander syngeneic fibroblasts were used as the target of gene transfer instead of islets to avoid deleterious effects of adenovirus infection on islets (30–32), 2) islets were embedded within an extracellular matrix that by itself improves islet function and viability (33,34), and 3) cotransplanted fibroblasts are more than just a source of IDO and can enhance islet physiological competence (35,36).     

Mesenchymal Stem Cells Enhance Allogeneic Islet Engraftment in Nonhuman Primates

OBJECTIVE

To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation.

RESEARCH DESIGN AND METHODS

Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection.

RESULTS

MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-β, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood.

CONCLUSIONS

MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.Multipotent mesenchymal stem cells (MSCs) (1,2) can deliver immunomodulatory signals (3–7) that inhibit allogeneic T-cell responses through downregulation of the proinflammatory cytokines TNF-α and IFN-γ and production of the regulatory cytokines/molecules IL-10, hepatocyte growth factor (HGF), TGF-β, vascular endothelial growth factor (VEGF), indoleamine 2,3-dioxygenase, galectin-1, prostaglandin E2, nitric oxide, and matrix metalloproteinase-2 and -9 (3,8–12). Inflammatory signals, such as IFN-γ, can activate and upregulate MSC suppressive activities (9,13). These cells are able to migrate to sites of injury after intravenous injection (14,15). Their use in clinical trials and experimental models is based on their immunomodulatory and regenerative properties (1,7,16). Clinically, MSCs have been observed to enhance donor bone marrow cell (DBMC) engraftment and chimerism (17,18). Therefore, cotransplantation of MSCs that secrete immunomodulatory cytokines and growth factors might enhance islet survival and function. In experimental mouse models, intravenously infused MSCs are capable of migrating to pancreatic islets (19,20). Systemic infusion of MSCs in murine models of diabetes was accompanied by delayed onset of diabetes, improved glycemic levels, reduced pancreatic insulitis, and pancreatic tissue regeneration (19,21–25), as well as prevention of autoimmune destruction of β-cells via induction of regulatory T-cells (Tregs) (26). Cotransplantation of syngeneic MSCs with a marginal mass of allogeneic islets under the kidney capsule of streptozotocin (STZ)-induced diabetic mice resulted in prolonged normoglycemia (11). Cotransplantation of syngeneic MSC with a marginal mass of allogeneic islets has been performed in the omentum (27) and kidney capsule (28) of STZ-induced diabetic rats, with enhanced islet graft survival as compared with animals receiving islets alone. In this study, cynomolgus monkey MSCs were characterized and donor MSCs were examined for the ability to promote intraportal islet engraftment as well as chimerism in recipients of islet/DBMC transplants. In addition, we tested the use of donor or third-party MSCs to reverse episodes of islet allograft rejection.     

Additive Effects of Genetic Variation in GCK and G6PC2 on Insulin Secretion and Fasting Glucose

OBJECTIVE

Glucokinase (GCK) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) regulate the glucose-cycling step in pancreatic β-cells and may regulate insulin secretion. GCK rs1799884 and G6PC2 rs560887 have been independently associated with fasting glucose, but their interaction on glucose-insulin relationships is not well characterized.

RESEARCH DESIGN AND METHODS

We tested whether these variants are associated with diabetes-related quantitative traits in Mexican Americans from the BetaGene Study and attempted to replicate our findings in Finnish men from the METabolic Syndrome in Men (METSIM) Study.

RESULTS

rs1799884 was not associated with any quantitative trait (corrected P > 0.1), whereas rs560887 was significantly associated with the oral glucose tolerance test 30-min incremental insulin response (30′ Δinsulin, corrected P = 0.021). We found no association between quantitative traits and the multiplicative interaction between rs1799884 and rs560887 (P > 0.26). However, the additive effect of these single nucleotide polymorphisms was associated with fasting glucose (corrected P = 0.03) and 30′ Δinsulin (corrected P = 0.027). This additive association was replicated in METSIM (fasting glucose, P = 3.5 × 10⁻¹⁰ 30′ Δinsulin, P = 0.028). When we examined the relationship between fasting glucose and 30′ Δinsulin stratified by GCK and G6PC2, we noted divergent changes in these quantitative traits for GCK but parallel changes for G6PC2. We observed a similar pattern in METSIM.

CONCLUSIONS

Our data suggest that variation in GCK and G6PC2 have additive effects on both fasting glucose and insulin secretion.Genome-wide association (GWA) studies have identified several loci for type 2 diabetes (1–5) and type 2 diabetes–related quantitative traits (6–20). Two of these loci, glucokinase (GCK) (21–23) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) (9,10), regulate the critical glucose-sensing mechanism within pancreatic β-cells. Mutations in GCK confer susceptibility to maturity-onset diabetes of the young (MODY)-2 (24–26), and a −30 GCK promoter variant (rs1799884) has been shown to be associated with β-cell function (21), fasting glucose, and birth weight (23). Chen et al. (9) demonstrated an association between the G6PC2 region and fasting glucose, an observation replicated by Bouatia-Naji et al. (10). Although fasting glucose levels are associated with both GCK and G6PC2, there has been no evidence that genetic variation at these two loci contribute a risk for type 2 diabetes, suggesting contribution to mild elevations in glycemia.GCK phosphorylates glucose to glucose-6-phosphate, whereas G6PC2 dephosphorylates glucose-6-phosphate back to glucose, forming a glucose cycle previously demonstrated to exist within pancreatic β-cells (27,28). The important role of GCK in glucose sensing by pancreatic islets has been demonstrated by numerous studies, and other studies suggest a role for glucose cycling in insulin secretion and diabetes (28–30), implying that the balance between GCK and G6PC2 activity is important for determining glycolytic flux, ATP production, and subsequent insulin secretion. This was validated by a demonstration that direct manipulation of glucose cycling alters insulin secretion (31,32).BetaGene is a study in which we are performing detailed phenotyping of Mexican American probands with recent gestational diabetes mellitus (GDM) and their family members to obtain quantitative estimates of body composition, insulin sensitivity (S_I), acute insulin response (AIR), and β-cell compensation (disposition index) with the goal of identifying genes influencing variations in type 2 diabetes–related quantitative traits (33–35). Based on the evidence that variation in GCK (rs1799884) and G6PC2 (rs560887) are independently associated with fasting glucose concentrations and both are crucial to glucose cycling in β-cells, we hypothesized that interaction between these loci may be associated not just with fasting glucose but also with measures of insulin secretion or β-cell function. We tested this hypothesis in the BetaGene Study and, for replication, in a separate sample of Finnish men participating in the METabolic Syndrome in Men (METSIM) Study (36).     

Differences in Baseline Lymphocyte Counts and Autoreactivity Are Associated With Differences in Outcome of Islet Cell Transplantation in Type 1 Diabetic Patients

OBJECTIVE

The metabolic outcome of islet cell transplants in type 1 diabetic patients is variable. This retrospective analysis examines whether differences in recipient characteristics at the time of transplantation are correlated with inadequate graft function.

RESEARCH DESIGN AND METHODS

Thirty nonuremic C-peptide–negative type 1 diabetic patients had received an intraportal islet cell graft of comparable size under an ATG-tacrolimus–mycophenolate mofetil regimen. Baseline patient characteristics were compared with outcome parameters during the first 6 posttransplant months (i.e., plasma C-peptide, glycemic variability, and gain of insulin independence). Correlations in univariate analysis were further examined in a multivariate model.

RESULTS

Patients that did not become insulin independent exhibited significantly higher counts of B-cells as well as a T-cell autoreactivity against insulinoma-associated protein 2 (IA2) and/or GAD. In one of them, a liver biopsy during posttransplant year 2 showed B-cell accumulations near insulin-positive β-cell aggregates. Higher baseline total lymphocytes and T-cell autoreactivity were also correlated with lower plasma C-peptide levels and higher glycemic variability.

CONCLUSIONS

Higher total and B-cell counts and presence of T-cell autoreactivity at baseline are independently associated with lower graft function in type 1 diabetic patients receiving intraportal islet cells under ATG-tacrolimus–mycophenolate mofetil therapy. Prospective studies are needed to assess whether control of these characteristics can help increase the function of islet cell grafts during the first year posttransplantation.Islet cell tranplantation is a promising therapy for type 1 diabetic patients, but its current state faces several limitations and obstacles (1,2). Insulin independence can be achieved during the first year posttransplantation in up to 80% of selected patients in small, single-center cohorts (3–7), but the success rate is lower in larger studies with less stringent criteria for selection of recipients and donor tissue (8,9). Several factors can account for the observed variability in outcome. Their identification is hindered by the difficulty in standardizing protocols and by the small numbers of patients that have so far been included per protocol. Within these limitations, graft and recipient characteristics have been related with the outcome of clinical islet cell transplantation (10–13). A minimal donor tissue mass was reported to induce insulin independence but is in itself not sufficient (3,10,13); administration of more potent immune suppressants can lower this treshold (14,15), which is lowest in autologous transplantation (16). Using cultured β-cell preparations in an ATG-based protocol, we defined the minimal number of β-cells that reproducibly resulted in circulating signs of a surviving graft 2 months after transplantation (17). In the latter study, achievement of insulin independence also depended on the β-cell mass in the graft but appeared counteracted by the presence of an islet-specific T-cell autoreactivity as measured by in vitro lymphocyte stimulation tests against the islet autoantigens GAD and insulinoma-associated protein 2 (IA2) (18). We have now analyzed a cohort of 30 consecutively transplanted recipients in search for a possible correlation between their baseline characteristics and the clinical outcome of defined islet cell grafts that are intraportally injected under the same ATG-based protocol.