NOD mice have a generalized defect in their response to transplantation tolerance induction.

A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.     

"Indirect" acute islet allograft destruction in nonobese diabetic mice is independent of donor major histocompatibility complex and requires host B lymphocytes

Islet allografts are destroyed rapidly in spontaneously diabetic nonobese diabetic (NOD) mice. However, whether this process is more similar to conventional allograft immunity, islet-specific autoimmune pathogenesis, or both remains controversial. In particular, we sought to determine whether C57BI/6 donor islet major histocompatibility complex (MHC) class I or class II expression was required for islet allograft destruction in autoimmune prone NOD mice versus non-autoimmune-prone BALB/c mice. Results show that islet allografts deficient in both MHC I and II are uniformly accepted in BALB/c mice. In sharp contrast, such MHC-deficient allografts were destroyed acutely in spontaneously diabetic NOD mice. Such donor MHC-independent rejection implicates "indirect" (host MHC-restricted) immunity as a pathway responsible for islet injury. To determine whether host NOD B lymphocytes could contribute to indirect graft recognition, wild-type and MHC I/II-deficient allografts were grafted into B-lymphocyte-deficient (microMT) NOD mice. Whereas wild-type NOD mice could reject MHC-I/II-deficient islet allografts, such grafts were all accepted in B-lymphocyte-deficient NOD mice. Taken together, these results indicate that NOD mice are capable of vigorous donor MHC-independent islet allograft rejection not found in non-autoimmune-prone recipients. Importantly, B lymphocytes may play a key role as antigen-presenting cells in this exuberant host 'indirect' response found in NOD mice.     

Differences in suppressor of cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c and spontaneously-diabetic NOD recipient mice

BACKGROUND: The ability to block interferon signaling represents an important strategy in designing therapies to prevent beta-cell destruction during islet allograft rejection. METHODS: The SOCS proteins regulate cytokine signaling by blocking activation of JAK/STAT proteins. Using islets isolated from SOCS-1 transgenic mice (SOCS-1-Tg; these mice express SOCS-1 under the control of the human insulin promoter and are on the C57BL6/J background), we investigated whether SOCS proteins can prevent the destruction pancreatic islet cells transplanted beneath the kidney capsule of major histocompatibility complex mismatched normal BALB/c and spontaneously-diabetic NOD mouse recipients. RESULTS: Immunohistochemical staining for insulin confirmed the presence of donor SOCS-1-Tg islets in islet allografts harvested at 22 days posttransplant, whereas grafts of control non-Tg islets were destroyed by 14 days. In contrast, SOCS-1-Tg allogeneic islets were not protected from beta-cell destruction in clinically diabetic NOD mice. The islet allografts functioned for 1 week posttransplant; however, hyperglycemia returned after 2 weeks and the grafts were destroyed. Rejection of SOCS-1-Tg and non-Tg islets in autoimmune diabetic NOD mice was associated with an infiltrate of both CD4+ and CD8+ T cells and a T2-type cytokine response (IL-4) rather than the conventional T1-type cytokine response observed during islet allograft rejection. Self-antigen upregulation in response to IFN-gamma stimulation did not appear to be a factor in rejection of the islet allografts. CONCLUSIONS: These results demonstrate that expression of SOCS-1 in islets delays islet allograft rejection but cannot circumvent destruction of the islets by the recurrence of the tissue-specific autoimmune process of spontaneous diabetes.     

A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.     

Allorecognition and effector pathways of islet allograft rejection in normal versus nonobese diabetic mice

Islet transplantation is becoming an accepted therapy to cure type I diabetes mellitus. The exact mechanisms of islet allograft rejection remain unclear, however. In vivo CD4(+) and CD8(+) T cell-depleting strategies and genetically altered mice that did not express MHC class I or class II antigens were used to study the allorecognition and effector pathways of islet allograft rejection in different strains of mice, including autoimmunity-prone nonobese diabetic (NOD) mice. In BALB/c mice, islet rejection depended on both CD4(+) and CD8(+) T cells. In C57BL/6 mice, CD8(+) T cells could eventually mediate islet rejection by themselves, but they produced rejection more efficiently with help from CD4(+) T cells stimulated through either the direct or indirect pathway. In C57BL/6 mice, CD4(+) T cells alone caused islet rejection when only the direct pathway was available but not when only the indirect pathway was available. In contrast, in NOD mice, CD4(+) T cells alone, with only the indirect pathway, could mediate islet and cardiac allograft rejection. These findings indicate that different mouse strains can make use of different pathways for T cell-mediated rejection of islet allografts. In addition, they demonstrate that NOD mice, which develop autoimmunity and are known to be resistant to tolerance induction, have an unusually powerful CD4(+) cell indirect mechanism that can cause rejection of both islet and cardiac allografts. These data shed light on the mechanisms of islet allograft rejection in different responder strains, including those with autoimmunity.     

Intrinsic defects in the T-cell lineage results in natural killer T-cell deficiency and the development of diabetes in the nonobese diabetic mouse.

T-cell-mediated autoimmune diabetes in nonobese diabetic (NOD) mice is closely associated with natural killer T (NKT)-cell deficiency. To determine whether intrinsic defects of the T-cell lineage contribute to the pathogenesis of the disease and NKT cell deficiency, we reconstituted the T-cell compartment in NOD.scid or BALB.scid mice with T-cells from NOD, nonobese diabetes-resistant (NOR), or AKR thymic precursor cells and examined the development of the NKT cell population. NKT cells developed well from AKR thymic precursor cells but not from other precursor cells in both recipient strains. Insulitis and diabetes developed only in the NOD.scid recipients of NOD or NOR precursor cells. When thymic precursor cells of beta2-microglobulin gene-deficient AKR mice, which have a deficient NKT population, were introduced into NOD.scid recipients, both CD4(+) and CD8(+) T-cell populations developed and the recipient mice developed insulitis and diabetes. We conclude that NKT cells originate from a T-cell-committed thymic precursor population and that the deficiency in the NKT cell population in NOD mice results from intrinsic defects within the T-cell lineage and plays a major role in the development of autoimmune diabetes in the presence of both the NOD thymus and antigen-presenting cells.     

Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into na?ve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.     

Role of CD4+ and CD8+ T cells in the rejection of concordant pancreas xenografts

BACKGROUND: We studied the ability of CD4 and CD8 T cells to induce rejection of pancreas xenografts in a concordant combination using rat pancreas xenografts as donors and chemically induced diabetic mice as recipients. METHODS: Lewis rat (2 to 3 weeks old) pancreas xenografts were transplanted into streptozotocin (STZ)-induced diabetic mice. Lymphocyte proliferation and cytokine production were analyzed in vitro. All pancreas xenografts were assessed by functional (blood glucose) and histopathologic examinations. RESULTS: Lewis rat pancreas grafts were rejected within 10 to 13 days, with mononuclear cell infiltrate and tissue necrosis in STZ-induced diabetic mice. A predominant T cell receptor alphabeta -CD4 cell (on day 4) and T cell receptor alphabeta -CD8 cell (on day 8) infiltrate and IgM deposition were found in the pancreas xenografts after transplantation. Anti-CD4 (GK1.5), but not anti-CD8 (YTS169.4), monoclonal antibodies resulted in a prolonged survival of Lewis rat pancreas xenografts. Lewis pancreas xenografts were permanently accepted by CD4 knockout mice but not by CD8 knockout mice. The pancreas xenografts were acutely rejected with a mean survival time of 15.3 days in B cell-deficient mice (microMT/microMT). Transfer of CD4 but not CD8 spleen cells from na?ve C57BL/6 mice into Rag2 mice led to acute rejection of transplanted pancreas xenografts. However, activated CD8 spleen cells elicited rejection of Lewis rat pancreas xenografts in SZT-induced diabetic mice. CONCLUSION: The current results show that CD4 T cells are necessary and sufficient for mediating the rejection of Lewis rat pancreas xenografts in STZ-induced diabetic mice. However, CD8 cells, when activated, can also induce acute rejection of concordant pancreas xenografts.     

Mechanism of allorecognition and skin graft rejection in CD28 and CD40 ligand double-deficient mice

BACKGROUND: It has been shown that simultaneous blockade of CD28- and CD40-mediated costimulatory signals significantly prolongs allograft survival. Although these results led to an expectation of the establishment of specific immunotolerant therapy for organ transplantation, it became evident that these treatments rarely resulted in indefinite allograft survival. To uncover the mechanisms underlying these costimulation blockade-resistant allograft rejections, we studied the process of allogenic skin graft rejection in CD28 and CD40 ligand (L) double-deficient (double-knockout [dKO]) mice. METHODS: Skin grafts from BALB/c or BALB.B mice were transplanted to C57BL/6 background dKO mice. The frequency of CD4+ and CD8+ T cells responding to alloantigens presented by direct or indirect pathways were defined by the use of a cytostaining assay. RESULTS: BALB/c skin grafts were rapidly rejected by dKO mice. This CD28 and CD40L independent allograft rejection was inhibited by the depletion of CD8+ T cells. In vitro studies indicated that CD8+ T cells from BALB/c skin-grafted dKO mice responded to donor antigen presented only by the direct pathway. Unlike major histocompatibility complex (MHC)-mismatched donors, allogenic skin grafts from MHC-matched donors were accepted by dKO mice. CONCLUSION: In the absence of CD28 and CD40 costimulatory signals, CD8+ T cells recognize MHC antigens by the direct pathway, resulting in the rejection of skin grafts from MHC-mismatched donors. In contrast, MHC-matched and non-MHC-mismatched donor skin grafts indefinitely survive in dKO mice. These results indicated that donor-host MHC matching may still be critical to costimulation blockade therapy for organ transplantation.     

Prevention of type 1 diabetes with major histocompatibility complex-compatible and nonmarrow ablative hematopoietic stem cell transplants

Progression to hyperglycemia in young nonobese diabetic (NOD) mice is blocked by the transplantation of hematopoietic cells mismatched at the major histocompatibility complex (MHC). Because the NOD MHC class II allele, I-A(g7), is the primary disease susceptibility gene, it is logical to conclude that MHC-mismatched hematopoietic grafts prevent diabetes by replacement of this susceptibility allele on critical hematolymphoid populations. In this report, transplantation of MHC-matched purified hematopoietic stem cells (HSCs) pre-vented diabetes development in NOD mice, demonstrating that alleles of non-MHC background genes expressed on hematopoietic cells are sufficient to disrupt the autoaggressive process. Nonmarrow ablative conditioning was 100% protective, further showing that elimination of NOD hematopoiesis, including T-cells, was not required for the graft to block diabetes pathogenesis. The current standard clinical practice of hematopoietic cell transplantation uses donor/recipient pairs that are matched at the MHC. In our view, the principles established here using an MHC-matched engineered hematopoietic graft in conjunction with nonmarrow ablative conditioning to successfully block autoimmune diabetes sufficiently reduces the morbidity of the allogeneic transplantation procedure such that a similar approach can be translated to the treatment of human autoimmune disorders.     

Adoptive immunity in immune-deficient scid/scid mice. I. Differential requirements of naive and primed lymphocytes for CD4+ T cells during rejection of minor histocompatibility antigen-disparate skin grafts

Using a model system in which mature lymphocytes were adoptively transferred into immunodeficient C.B17-scid/scid recipients, the requirement for CD4+ T cells in rejection of previously healed-in multiple minor-H-antigen-disparate skin grafts was investigated. Depletion of functional CD4+ cells was accomplished by anti-CD4 antibody + complement treatment prior to adoptive transfer followed by chronic anti-CD4 serotherapy in vivo. Homozygous scid mice harboring nondepleted naive donor cells effectively rejected minor-H-antigen-disparate grafts, whereas scid/scid mice harboring CD4+ T-cell-depleted naive cells did not. Homozygous scid mice harboring minor-H-antigen-primed cells rejected the minor-H-antigen-disparate allografts regardless of whether or not the animals had received anti-CD4 treatment, although rejection by the mice receiving anti-CD4 treatment was retarded compared to mice not receiving anti-CD4 treatment. All the mice that received viable donor cells rejected minor + H-2 disparate allografts, demonstrating effective immunity in this case was not dependent upon the activity of CD4+ T cells. These data suggest that in responses against multiple minor-H-antigen-disparate tissue, primary allograft rejection is absolutely dependent upon CD4+ cells, and secondary allograft rejection is not. The implications of these findings in understanding interactions of T cell subsets in vivo and of the utility of using homozygous scid mice as recipients for exploring the function of transferred lymphoid cell populations are discussed.     

The influence of the major histocompatibility complex on development of autoimmune diabetes in RIP-B7.1 mice

The most important genetic susceptibility factor for type 1 diabetes is encoded in the major histocompatibility complex (MHC). The nonobese diabetic (NOD) mouse, which develops spontaneous diabetes, expresses H-2g7 comprising the MHC class I molecules Kd and Db and the MHC class II molecule I-Ag7. However, neither B6.H-2g7 mice, in which H-2g7 is expressed on the C57BL/6 genetic background, nor the nonobese resistant (NOR) mouse, in which H-2g7 is expressed on a genetic background that is 88% similar to NOD mice, develop diabetes. Immune tolerance can be broken in these diabetes-resistant mice expressing H-2g7 if the costimulatory molecule B7.1 is present on the islet beta cells. This does not occur if only single MHC class I components of the H-2g7 haplotype are present, such as Kd in BALB/c mice or Db in C57BL/6 mice, both of which develop only a low level of diabetes when B7.1 is expressed. The presence of I-Ag7 leads to the development of an autoimmune T-cell repertoire, and local costimulation of CD8 T-cells precipitates aggressive diabetes. This implies that a major role of the MHC class II molecules in diabetes is the development of an autoreactive T-cell repertoire.     

A substantial level of donor hematopoietic chimerism is required to protect donor-specific islet grafts in diabetic NOD mice

BACKGROUND: Mixed chimerism can induce tolerance to alloantigens and restore self-tolerance to autoantigens, thereby permitting islet transplantation. However, the minimal level of donor chimerism that is required to prevent islet allograft rejection and recurrence of autoimmune diabetes has not been established. METHODS: We investigated whether allogeneic Balb/c donor chimerism can be induced in C57BL/6 mice, in prediabetic NOD mice, and in diabetic NOD mice after transplantation of a modest dose of bone marrow by using purine nucleoside analogue, fludarabine phosphate and cyclophosphamide conditioning therapy, followed by short-term anti-CD40 ligand monoclonal antibody and rapamycin posttransplant treatment. We also investigated whether the induced donor chimerism is sufficient to prevent the onset of diabetes in prediabetic NOD mice and protect donor islet grafts in diabetic NOD mice. RESULTS: Allogeneic donor chimerism could be induced under the authors' approach that is nonmyeloablative and radiation-free. Diabetes onset was prevented in chimeric prediabetic NOD mice. The induction of mixed chimerism protected donor-specific islet grafts in diabetic NOD mice. At 60 days after islet transplantation, all donor Balb/c islet grafts survived in diabetic NOD mice whose level of donor-derived lymphocytes was higher than 30% at the time of islet transplantation (n=8). In contrast, Balb/c islet grafts were rejected in five of seven diabetic NOD mice whose level was lower than 30%. CONCLUSIONS: Our data demonstrate that a donor lymphocyte chimerism (>30%) at the time of islet transplantation is required to protect donor-specific islet grafts, and indicate that a strictly non-irradiation-based protocol can be used to achieve this goal.     

Immunotherapy with nondepleting anti-CD4 monoclonal antibodies but not CD28 antagonists protects islet graft in spontaneously diabetic nod mice from autoimmune destruction and allogeneic and xenogeneic graft rejection.

BACKGROUND: T-cell activation and the subsequent induction of effector functions require not only the recognition of antigen peptides bound to MHC molecules by T-cell receptor (TCR) for antigen but also a costimulatory signal provided by antigen presenting cells. CD4 T-cell activation and function require the CD4 molecule as a coreceptor of TCR. The CD28/B7 pathway is a major costimulatory signal for T-cell activation and differentiation. METHODS: The effect of targeting CD4 by nondepleting anti-CD4 monoclonal antibodies (mAbs) versus blocking CD28/B7 by CTLA4Ig, anti-CD80 mAbs, and anti-CD86 mAbs on the prevention of recurrence of autoimmune diabetes after MHC-matched nonobese diabetes-resistant (NOR) islet transplantation in nonobese diabetic (NOD) mice were compared. Whether nondepleting anti-CD4 mAbs prolong allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice were studied. Furthermore, the effect of nondepleting anti-CD4 mAbs combined with CTLA4Ig on allogeneic islet graft survival in NOD mice was investigated. RESULTS: Recurrence of autoimmune diabetes can be prevented by nondepleting anti-CD4 mAbs. Blocking the CD28/B7 costimulatory pathway by CTLA4Ig or by anti-CD80 mAbs and anti-CD86 mAbs cannot prevent recurrence of autoimmune diabetes after islet transplantation. Short-term treatment with nondepleting anti-CD4 mAbs significantly prolongs allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice. But nondepleting anti-CD4 mAbs combined with CTLA4Ig decreased allogeneic islet graft survival. CONCLUSIONS: Nondepleting anti-CD4 mAbs but not CD28 antagonists protect islet grafts in diabetic NOD mice from autoimmune destruction and allogeneic and xenogeneic graft rejection. The efficacy of nondepleting anti-CD4 mAbs is compromised when it combines with CTLA4Ig.     

Transplanted MHC class I-deficient nonobese diabetic mouse islets are protected from autoimmune injury in diabetic nonobese recipients

The injury of transplanted islets may occur by both autoimmune and alloimmune processes directed against MHC targets. To examine the role of MHC class I in islet graft injury, we transplanted syngeneic and allogeneic beta2-microglobulin-deficient islets into diabetic nonobese diabetic (NOD) mice. Loss of graft function was observed within 14 days using allogeneic C57BL/6 and BALB/c MHC class I deficient as well as wild-type MHC class I-bearing NOD donor islets. However, islets isolated from MHC class I-deficient NOD mice (NOD-B2 m-/-) survived indefinitely when transplanted under the kidney capsule of diabetic NOD recipients. Transplanted NOD-B2 m-/- islets were surrounded by a nondestructive periinsular infiltrate that expressed interleukin-4 in addition to interferon-gamma. These studies demonstrate the primary role of MHC class I molecules in causing autoimmune destruction or recurrent diabetes in transplanted islets.     

Systemic transforming growth factor-beta1 gene therapy induces Foxp3+ regulatory cells, restores self-tolerance, and facilitates regeneration of beta cell function in overtly diabetic nonobese diabetic mice

BACKGROUND: Type 1 diabetes results from auto-aggressive T-cell-mediated destruction of beta cells of the pancreas. Recent data suggest that restoration of self-tolerance may facilitate islet-cell regeneration/recovery. In view of the immunoregulatory activity of transforming growth factor (TGF)-beta1, we investigated whether systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity and facilitates regeneration of beta-cell function in overtly diabetic nonobese diabetic (NOD) mice. METHODS: We used site-directed mutagenesis to create cysteine to serine mutation at sites 224 and 226 and constructed a replication deficient adenovirus (Ad) vector encoding active form of human TGF-beta1 (Ad-hTGF-beta1). Overtly diabetic NOD mice received intravenous injection of Ad-hTGF-beta1. Seven to 14 days after the injection, the mice received transplants with 500 syngeneic islets under the kidney capsule. Islet-graft survival and regeneration of endogenous beta-cell function were examined. RESULTS: Syngeneic islet grafts failed by day 17 in all untreated mice, whereas Ad-hTGF-beta1 therapy prolonged survival of islet grafts. Islet grafts from treated mice showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Systemic TGF-beta1 gene therapy was associated with islet regeneration in the native pancreas. Native pancreas of treated mice revealed islets staining strongly for insulin. Similar to what was found in the syngeneic islet graft, there were well-demarcated peri-islet infiltrates that were positive for CD4, TGF-beta1, and Foxp3. CONCLUSIONS: Our data demonstrate that systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity, facilitates islet regeneration, and cures diabetes in diabetic NOD mice.     

Blockade of OX40 signals enhance survival of xenoislet grafts in spontaneously diabetic NOD mice

Role of OX40 blockade was investigated in islet xenograft model in chemically diabetic C57B1/6 and spontaneously diabetic NOD mice. The effect of OX40/OX40L blockade on effector function of diabetogenic T cells was studied in an adoptive transfer model. 2000 IEQ of porcine islets were transplanted under the kidney capsule of diabetic C57B1/6 or NOD mice and were treated i.p. with control Ig, anti-OX40L, or CTLA4Ig. Graft survival was assessed using blood glucose (BG) measurement. Cells from diabetic NOD spleens and pancreatic lymphnodes were injected i.v. into NOD.scid mice. Recipients were treated i.p. with anti-OX40L, CTLA4Ig alone, or in combination; control mice received Ig. Diabetes incidence was assessed using BG measurement. Anti-OX40L treatment delayed xenoislet rejection significantly in chemically diabetic animals, although CTLA4Ig delayed rejection even further. Neither treatment completely prevented rejection. In spontaneously diabetic NOD mice, rejection of xenoislet graft was delayed by anti-OX40L treatment but not by CTLA4Ig. Anti-OX40L alone and in combination with CTLA4Ig significantly delayed diabetes transfer by activated diabetogenic T cells, compared with control and CTLA4Ig treatment. Preliminary mechanistic studies suggest that anti-OX40L treatment preserves Treg numbers, unlike CTLA4Ig that diminishes Tregs. Our studies show that OX40 blockade offers better xenoislet graft survival than CTLA4Ig in spontaneous autoimmune NOD model, suggesting that preactivated T cells use alternate costimulatory pathways, independent of CD28. Results from adoptive transfer studies further support the role of OX40 signaling in effector function of diabetogenic T cells. These data suggest that OX40/OX40L may offer novel therapeutic target for xenoislet graft protection in type 1 diabetic patients.     

Is facilitating pancreatic beta cell regeneration a valid option for clinical therapy?

Type 1 diabetes (T1D) is an autoimmune disease in which the clinical onset most frequently presents in adolescents who are genetically predisposed. There is accumulating evidence that the endocrine pancreas has regenerative properties, that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, and that, in this manner, physiologically sufficient endogenous insulin production can be restored in clinically diabetic NOD mice. Recapitulating what also has been seen sporadically in humans, we set out to test reliable and clinically translatable alternatives able to achieve these same goals. Recently, Tian and colleagues demonstrated that T1D can be prevented in genetically susceptible mice by substituting a "diabetes-susceptible" class II MHC beta chain with a "diabetes-resistant" allelic transgene on their hematopoietic stem cells through gene supplantation. The expression of the newly formed diabetes-resistant molecule in the reinfused hematopoietic cells was sufficient to prevent T1D onset even in the presence of the native, diabetogenic molecule. If this approach to obtain autoimmunity abrogation could facilitate a possible recovery of autologous insulin production in diabetic patients, safe induction of an autoimmunity-free status might become a new promising therapy for T1D.     

The thymus is required for the ability of FTY720 to prolong skin allograft survival across different histocompatibility MHC barriers

The immunosuppressive effect of FTY720 is associated with the reversible sequestration of lymphocytes from the blood and the spleen into secondary lymphoid organs and reduced egress of mature thymocytes from the thymus. This work was designed to dissect the differential effect of FTY720 on CD4 and CD8 T cell-mediated mechanisms of skin graft rejection in the presence (euthymic) or absence (thymectomized) of thymic output. To that end, untreated and FTY720-treated euthymic (Euthy) and thymectomized (ATX) mice received skin allografts across a full, class II or class I major histocompatibility complex (MHC) mismatched (MM) barriers and graft survival was monitored. We demonstrate that a short course of FTY720 treatment significantly augments the survival of full, class I and class II MHC MM skin grafts compared to the nontreated controls. Interestingly, FTY720-treated Euthy recipients showed a significantly prolonged skin allograft survival compared to FTY720-treated ATX mice. These results together show that FTY720 impairs both CD4 and CD8 T cell-mediated mechanisms of rejection and, more importantly, the presence of the thymus is necessary for the ability of FTY720 to modulate skin allograft rejection across different histocompatibility MHC barriers.