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.     

Indefinite survival of neonatal porcine islet xenografts by simultaneous targeting of LFA-1 and CD154 or CD45RB

A variety of transient therapies directed against molecules involved in T-cell activation and function result in long-term islet allograft survival. However, there are relatively few examples of durable islet xenograft survival using similar short-term approaches, especially regarding highly phylogenetically disparate xenograft donors. Previous studies demonstrate that combined anti-lymphocyte function-associated antigen-1 (LFA-1) plus anti-CD154 therapy results in a robust form of islet allograft tolerance not observed with either individual monotherapy. Thus, the aim of this study was to determine whether the perturbation of anti-LFA-1, either alone or in combination with targeting CD154 or CD45RB, would promote neonatal porcine islet (NPI) xenograft survival in mice. NPI xenografts are rapidly rejected in wild-type C57BL/6 mice but reproducibly mature and restore durable euglycemia in diabetic, immune-deficient C57BL/6 rag-1(-/-) recipients. A short course of individual anti-LFA-1, anti-CD154, or anti-CD45RB therapy resulted in long-term (>100 days) survival in a moderate proportion of C57BL/6 recipients. However, simultaneous treatment with anti-LFA-1 plus either anti-CD154 or anti-CD45RB therapy could achieve indefinite xenograft function in the majority of recipient animals. Importantly, prolongation of islet xenograft survival using combined anti-LFA-1/anti-CD154 therapy was associated with little mononuclear cell infiltration and greatly reduced anti-porcine antibody levels. Taken together, results indicate that therapies simultaneously targeting differing pathways impacting T-cell function can show marked efficacy for inducing long-term xenograft survival and produce a prolonged state of host hyporeactivity in vivo.     

Both innate and adaptive major histocompatibility complex class I-dependent immunity impair long-term islet xenograft survival

Natural killer (NK) cells have long been appreciated for their rapid, proinflammatory contribution to host defense. However, more recent studies show an unexpected regulatory role for host major histocompatibility complex (MHC) class I-dependent immunity and NK cells in promoting tolerance induction to islet allografts. It is unclear whether the potential tolerance induction to islet xenografts follows similar requirements to those found in allograft tolerance. In this study, we determined whether induced islet xenograft prolongation also showed a reliance on MHC class I-dependent immune pathways. In particular, we tested whether NK1.1+ cells and/or CD8 T cells were required for the long-term islet xenograft survival in a rat-to-mouse transplant model. Short-term host treatment with combined anti-CD154 plus anti-LFA-1 (CD11a) resulted in prolonged, but not indefinite, survival of WF rat islet xenografts in C57BI/6 mouse recipients. In stark contrast with similar islet allograft studies, adjunct treatment with anti-NK1.1 therapy combined wither anti-CD154/anti-LFA-1 treatment led to long-term (>100 days) survival of the majority of islet xenografts. In parallel studies, we determined whether CD8 T cells also contributed a barrier to xenograft survival. Similar to results found in anti-NK1.1-treated animals, CD8-deficient (knockout) recipients also demonstrated augmented xenograft prolongation after combined anti-CD154/anti-LFA-1 therapy. Taken together, NK1.1+ cells (NK/NKT cells) and CD8 T cells constitute differing MHC class I-dependent immune pathways forming a significant barrier to xenograft prolongation.     

The complement dependent cytotoxicity (CDC) immune effector mechanism contributes to anti-CD154 induced immunosuppression

BACKGROUND: In many situations, anti-CD154 (CD40 ligand) monoclonal antibody (mAb) treatment is very potent in producing allograft tolerance. In accordance to our previously reported results, combined donor specific transfusion (DST)3 plus anti-CD154 mAb (MR1) treatment enables the permanent engraftment of DBA/2 (H-2(d)) islets into B6AF1 (H-2(b/kd)) recipients in all cases. It has been widely assumed that the MR1 anti-154 is a noncytolytic neutralizing mAb, and it exerts immune suppressive effects by blockade of CD40/CD154 signal pathway. In this study, we sought to test the role of complement dependent cytotoxicity (CDC) immune effector mechanism in MR1 anti-CD154 induced immunosuppression. METHODS: We have evaluated the contributions of CDC in the context of the potent tolerizing effects of DST plus anti-CD154 mAb treatment regiment in recipients of islet allografts. We have used CD40 knockout (KO) mice and complement C5 deficient mice DBA/2 as islet allograft recipients as well as cobra venom factor (CVF), a complement blocker, treatment. RESULTS: The absence of direct and indirect CD40/CD154 pathway signals does not prevent islet allograft acute rejection. Interestingly, MR1 anti-CD154 induces islet allograft tolerance in the absence of CD40/CD154 pathway. In a wild-type major histocompatibility complex (MHC) mismatched strain combination, DST results in accelerated islet allograft rejection. Combination of DST and MR1 anti-CD154 treatment prevents presensitization and permits permanent engraftment. However, administration of CVF abolishes the tolerance induction. Moreover, DST plus MR1 anti-CD154 regiment, a potent tolerizing therapy, does not prevent acute islet allograft rejection when complement C5 deficient DBA/2 mice are used as recipients. Thus, the mechanisms of the tolerizing effects by MR1 anti-CD154 are not limited to blockade of CD40/CD154 signals. The CDC immune effector mechanism contributes to MR1 anti-CD154 induced immunosuppression.     

Combination of anti-CD4 with anti-LFA-1 and anti-CD154 monoclonal antibodies promotes long-term survival and function of neonatal porcine islet xenografts in spontaneously diabetic NOD mice

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet beta-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for TIDM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing beta-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.     

The effect of simultaneous CD154 and LFA-1 blockade on the survival of allogeneic islet grafts in nonobese diabetic mice

BACKGROUND: The rate of success in clinical transplantation of islets of Langerhans has dramatically improved with perspectives of wide-scale applicability for patients with type 1 diabetes. One drawback is the need for lifelong immunosuppression, which is associated with significant side effects. Immunomodulatory strategies devoid of side effects and with tolerogenic potential, such as co-stimulatory blockade, would be a great improvement if successful. In this study, the authors have explored the effect of simultaneous blockade of CD40/CD154 and intercellular adhesion molecule (ICAM)/lymphocyte function-associated antigen (LFA)-1 interactions. METHODS: Spontaneously diabetic nonobese diabetic (NOD) mice underwent transplantation with allogeneic (C57BL/6) islets and were treated with anti-CD154 monoclonal antibody (mAb) (500 microg, three doses), anti-LFA-1 mAb (100 microg, three doses), or a combination of both in the early peritransplant period. In another set of experiments, LFA-1 engagement was impaired by transplanting islets isolated from ICAM-1-knockout (KO) mice. RESULTS: Untreated animals rejected their grafts within 10 days. LFA-1 blockade alone did not result in improved islet graft survival, whereas CD154 blockade alone increased graft survival to 18 days. Simultaneous blockade of both pathways led to significantly improved islet graft survival to 30 days (ICAM-1-KO islets plus anti-CD154), 35 days (anti-LFA-1 plus anti-CD154), and 44 days (ICAM-1-KO islets plus anti-LFA-1 plus anti-CD154). CONCLUSIONS: These data suggest that a synergistic effect for prolonged graft survival can be obtained by simultaneously targeting CD154 and LFA-1 in the challenging model of islet allotransplantation in NOD mice. The observation of similar results with anti-LFA-1 mAb and with ICAM-1-KO grafts suggests a key role of direct antigen presentation for the activation of LFA-1-driven signaling.     

Role of CD40-CD154 pathway in the rejection of concordant and discordant xenogeneic islets

BACKGROUND: Costimulatory blockade has been shown to allow long-term survival of xenogeneic islets. The aim of the present study was to evaluate the role of recipient CD40 and CD154 in the rejection process of concordant and discordant islet xenotransplantation (Tx). METHODS: Diabetic C57BL/6 mice, CD40- or CD154 knockout (KO) mice were transplanted with either concordant rat or discordant human islets. Experimental design: group 1, control (ie, C57BL/6 mice received islet Tx without therapy); group 2, C57BL/6 mice received islet Tx with anti-CD154 monoclonal Ab (mAb) therapy; group 3, CD40 KO mice; and group 4, CD154 KO mice were used as recipients without therapy. Mouse anti-rat mixed lymphocyte reactions (MLR) were performed using mouse splenocytes obtained from animals transplanted with rat islets in groups 1 to 4. RESULTS: In group 2, short-term anti-CD154 mAb therapy significantly prolonged rat-to-mouse and human-to-mouse xenograft survival, compared to controls. In CD40-KO and CD154-KO recipients, survival of concordant or discordant islets was not prolonged significantly compared to control groups. Mouse anti-donor rat cellular responses were reduced approximately 50% in group 2 but remained unmodified in groups 3 and 4, when compared to group 1. CONCLUSIONS: Improved graft survival and reduced MLR responses against donor cells in vitro among the anti-CD154 mAb-treated mice could be explained by specific targeting of activated T cells with subsequent inactivation by anergy and/or elimination by apoptosis, or complement- or cellular-mediated mechanisms. Rejection of xenografts and strong MLR responses against donor cells in vitro in CD40 or CD154 KO animals is possible through efficient activation of alternate pathways of costimulation.     

Effect of Triple Costimulation Blockade on Islet Allograft Survival in Sensitized Mice

Background

Islet allograft rejection in sensitized recipients is difficult to control by costimulation blockade using anti-CD154 and cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig). Because leukocyte function antigen (LFA) 1 is highly expressed on memory T cells, adding an LFA-1 blockade may inhibit memory T-cell activities. We examined the effects on islet allograft survival of triple costimulation blockade in presensitized recipient mice.

Methods

C57BL/6 mice were sensitized by transplantation under the kidney capsule or intraperitoneal injection of Balb/c islets. Four weeks after transplantation, sensitization was confirmed by flow-cytometric detection of alloreactive antibodies. Diabetes was induced by a single intravenous injection of streptozotocin. Recipients were transplanted with 200 Balb/c islets under the right kidney capsule. Graft function was assessed by daily blood glucose and body weight records. Transplanted animals were divided into 3 treatment groups: group 1, control antibody; group 2, anti-CD154 and CTLA-4 Ig double therapy; group 3, anti-CD154, CTLA4Ig, and anti-LFA-1 triple therapy. Injections were administered every second day from day −2 to day 8.

Results

Naïve mice rejected islet allografts between days 7 and 29 (mean 16 ± 6 d; n = 5), sensitized mice in group 1 between days 0 and 14 (mean 7 ± 5 d; n = 8), in group 2 between days 4 and 16 (mean 8 ± 4 d; n = 7), and in group 3 between days 4 and 26 (mean 11 ± 7 d; n = 10).

Conclusion

Triple costimulation blockade with anti-CD154, CTLA4Ig, and anti-LFA-1 was not sufficient to improve islet allograft survival in sensitized recipients.     

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.     

Rat xenograft survival in mice treated with donor-specific transfusion and anti-CD154 antibody is enhanced by elimination of host CD4+ cells

BACKGROUND: Treatment with a donor-specific transfusion (DST) and a brief course of anti-mouse CD154 (anti-CD40-ligand) monoclonal antibody (mAb) prolongs the survival of both allografts and rat xenografts in mice. The mechanism by which allograft survival is prolonged is incompletely understood, but depends in part on the presence of CD4+ cells and the deletion of alloreactive CD8+ T cells. Less is known about the mechanism by which this protocol prolongs xenograft survival. METHODS: We measured rat islet and skin xenograft survival in euthymic and thymectomized mice treated with combinations of DST, anti-CD154 mAb, anti-CD4 mAb, and anti-CD8 mAb. Recipients included C57BL/6, C57BL/6-scid, C57BL/6-CD4null, and C57BL/6-CD8null mice. RESULTS: Pretreatment with a depleting anti-CD4 mAb markedly prolonged the survival of both skin and islet xenografts in mice given DST plus anti-CD154 mAb. Comparable prolongation of xenograft survival was obtained in C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb. In contrast, anti-CD8 mAb did not prolong the survival of either islet or skin xenografts in mice treated with DST and anti-CD154 mAb. Thymectomy did not influence xenograft survival in any treatment group. Adoptive transfer of splenocytes from C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb and bearing long-term skin xenografts revealed the presence of residual xenoreactive cells. CONCLUSIONS: These data suggest that treatment with DST and anti-CD154 mAb induces a state of "functional" transplantation tolerance. They also support the hypothesis that both the induction and maintenance of graft survival based on this protocol depend on different cellular mechanisms in allogeneic and xenogeneic model systems.     

Comparison of successful and unsuccessful islet/Sertoli cell cotransplant grafts in streptozotocin-induced diabetic mice

Sertoli cells (SC) protect islet allografts from immune destruction in diabetic rodents. In this study, we examined the difference between successful and rejected islet/SC cografts in order to further improve this procedure for optimal extension of islet allograft survival. We cotransplanted 500 BALB/c islets with 1-8 million BALB/c SC under the kidney capsule of diabetic BALB/c, C3H-HeJ, and C57BL/6 mice. Cotransplantation of islets with up to 8 million SC was not detrimental to long-term islet graft function in syngeneic mice. However, large numbers of SC were detrimental to islet graft survival in allogeneic mice with the optimal dose for cotransplantation of 4 or 1 million SC in C3H-HeJ or C57BL/6 mice, respectively. Examination of successful grafts, from euglycemic recipients, revealed the presence of SC arranged in tubule structures with islets surrounding these tubules. Cellular infiltrate in successful grafts revealed CD4 T cells and macrophages along the periphery and within the grafts, and very few CD8 T cells. Conversely, examination of unsuccessful grafts, harvested from hyperglycemic recipients at the time of rejection, revealed the presence of SC arranged randomly with islets adjacent to the Sertoli cells, when present, and massive CD4 and CD8 T cell as well as macrophage cell infiltration. Prolongation of islet allograft survival appeared to be a function of SC transplant mass and recipient genetic background. A consequence of long-term graft acceptance is the formation of SC tubule structures, which may be an additional requirement for optimal protection of islet allografts.     

Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD154.

Clinical islet cell transplantation has resulted in insulin independence in a limited number of cases. Rejection, recurrence of autoimmunity, and impairment of normal islet function by conventional immunosuppressive drugs, e.g., steroids, tacrolimus, and cyclosporin A, may all contribute to islet allograft loss. Furthermore, intraportal infusion of allogeneic islets results in the activation of intrahepatic macrophages and endothelial cells, followed by production of proinflammatory mediators that can contribute to islet primary nonfunction. We reasoned that the beneficial effects of anti-CD154 treatment on autoimmunity, alloreactivity, and proinflammatory events mediated by macrophages and endothelial cells made it an ideal agent for the prevention of islet allograft failure. In this study, a nonhuman primate model (Papio hamadryas) was used to assess the effect of humanized anti-CD154 (hu5c8) on allogeneic islet engraftment and function. Nonimmunosuppressed and tacrolimus-treated recipients were insulin independent posttransplant, but rejected their islet allografts in 8 days. Engraftment and insulin independence were achieved in seven of seven baboon recipients of anti-CD154 induction therapy administered on days -1, 3, and 10 relative to the islet transplant. Three of three baboons treated with 20 mg/kg anti-CD154 induction therapy experienced delayed rejection episodes, first detected by elevations in postprandial blood glucose levels, on postoperative day (POD) 31 for one and on POD 58 for the other two. Re-treatment with three doses of anti-CD154 resulted in reversal of rejection in all three animals and in a return to normoglycemia and insulin independence in two of three baboons. It was possible to reverse multiple episodes of rejection with this approach. A loss of functional islet mass, as detected by reduced first-phase insulin release in response to intravenous glucose tolerance testing, was observed after each episode of rejection. One of two baboons treated with 10 mg/kg induction therapy became insulin independent post-transplant but rejected the islet graft on POD 10; the other animal experienced a reversible rejection episode on POD 58 and remained insulin independent and normoglycemic until POD 264. Two additional baboon recipients of allogeneic islets and donor bone marrow (infused on PODs 5 and 11) were treated with induction therapy (PODs -1, 3, 10), followed by initiation of monthly maintenance therapy (for a period of 6 months) on POD 28. Rejection-free graft survival and insulin independence was maintained for 114 and 238 days, with preservation of functional islet mass observed in the absence of rejection. Prevention and reversal of rejection, in the absence of the deleterious effects associated with the use of conventional immunosuppressive drugs, make anti-CD154 a unique agent for further study in islet cell transplantation.     

Anti-LFA-1 improves pig islet xenograft function in diabetic mice when long-term acceptance is induced by CTLA4Ig/anti-CD40L

BACKGROUND: It has been previously demonstrated that addition of anti-LFA-1 to a combination of CTLA4Ig and anti-CD40L induces the permanent acceptance of dopaminergic fetal pig xenografts when transplanted into the brain of wild-type mice. The purpose of this study was to test whether this costimulation blockade also can induce acceptance of adult pig islets transplanted to C57BL/6 mice with streptozotocin-induced diabetes. METHODS: Recipients were treated with CTLA4Ig/anti-CD40L+/-anti-LFA-1 or isotype control antibodies during the first week after transplantation. Half of the costimulation blockade-treated recipients had their grafts removed after 8 weeks. The other half was observed up to 5 months. RESULTS: Recipients treated with CTLA4Ig/anti-CD40L/anti-LFA-1 had significantly lower blood glucose and gained more weight than CTLA4Ig/anti-CD40L-treated recipients. CTLA4Ig/anti-CD40L-treated recipients exhibited unstable blood glucose. IPGTT of these recipients revealed a slow recovery to normal blood glucose levels at week 4. In comparison, CTLA4Ig/anti-CD40L/anti-LFA-1 treated recipients exhibited a significantly superior glucose clearance. CTLA4Ig/anti-CD40L+/-anti-LFA-1 treated recipients did not produce anti-pig IgG, whereas control antibody-treated mice did. CD4+ T cells from costimulation blockade-treated recipients proliferated less than CD4+ T cells from control antibody-treated mice when co-cultured with syngeneic antigen presenting cells loaded with pig islet antigens. CONCLUSIONS: CTLA4Ig/anti-CD40L/anti-LFA-1-treated recipients had superior islet function compared with CTLA4Ig/anti-CD40L-treated recipients. However, both costimulation blockade regimens led to islet graft acceptance up to 5 months after a 1-week treatment.     

CTLA4ig induces long-term graft survival of allogeneic skin grafts and totally inhibits T-cell proliferation in LFA-1-deficient mice

BACKGROUND: It was recently shown that some strains of mice are capable of rejecting transplants independently of B7 and CD40L signaling and that this rejection is mediated by CD8(+) T cells. LFA-1 is known to be important for CD8(+) T cell activation and cytotoxicity. Therefore, blockade of LFA-1 could be important in overcoming costimulation blockade, CD8(+) T-cell-mediated, resistant rejection. The purpose of this study was to define the effect of combined blockade of the LFA-1 and B7 costimulation pathways on the alloimmune response in mice. METHODS: Allogeneic skin transplantation was performed using BALB/c mice as donors and C57BL/6J wild-type or LFA-1-deficient (CD11a(-/-)) mice as recipients. CTLA4Ig or anti-LFA-1 was administered either as an induction or a prolonged therapy. Mixed lymphocyte reactions were conducted to study the effect of CTLA4Ig on T-cell proliferation in CD11a(-/-) mice. RESULTS AND CONCLUSIONS: Administration of CTLA4Ig completely inhibits CD11a(-/-) T-cell proliferation in response to alloantigens and significantly improved skin allograft survival in CD11a(-/-) mice. Prolonged treatment of wild-type recipient mice with CTLA4Ig and anti-LFA-1 increased median survival time to 45.5 days compared with 16 days after induction therapy, but it was not sufficient to induce indefinite allograft survival in this model.     

Prolonged survival of microencapsulated neonatal porcine islets in mice treated with a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies

BACKGROUND: The aim of this study was to determine whether short-term administration of a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies can prolong the survival of microencapsulated neonatal porcine islets (NPI) in immunocompetent mice. METHODS: Microencapsulated NPI were transplanted into the peritoneal cavity of streptozotocin-induced diabetic B6 mice that received a short-term treatment of a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies. Blood glucose levels of each recipient were measured for more than 100 days posttransplantation or until graft rejection. Microcapsules were recovered to determine the presence of immune cells using immunoperoxidase staining. In addition, the levels of mouse anti-porcine immunoglobulin (Ig) G antibodies in the serum of each recipient were measured by flow cytometry. RESULTS: Short-term administration of a combination of monoclonal antibodies resulted in significant prolongation of microencapsulated NPI xenograft survival. All treated mice (n = 20) achieved normoglycemia within 10-35 days posttransplantation and 11/20 mice remained normoglycemic for more than 100 days posttransplantation. In contrast, only 1/20 of the untreated mice achieved normoglycemia and this mouse became diabetic at 17 days posttransplantation. Histological examination of the recovered microcapsules from long-term surviving treated mice revealed minimal cellular overgrowth containing intact viable islets, whereas several layers of immune cells surrounding the capsules containing nonviable islets were observed in untreated mice. The levels of mouse anti-porcine IgG was also reduced in treated recipients compared to untreated mice. CONCLUSIONS: These data demonstrate that short-term administration of anti-CD154 and anti-LFA-1 monoclonal antibodies can be effective in promoting long-term survival of microencapsulated NPI in immune-competent mice.     

Regulation of skin and islet allograft survival in mice treated with costimulation blockade is mediated by different CD4+ cell subsets and different mechanisms

BACKGROUND: Donor-specific transfusion (DST) and a brief course of anti-CD154 monoclonal antibody (mAb) induces permanent islet and prolonged skin allograft survival in mice. Induction of skin allograft survival requires the presence of CD4 cells and deletion of alloreactive CD8 cells. The specific roles of CD4 and CD4CD25 cells and the mechanism(s) by which they act are not fully understood. METHODS: We used skin and islet allografts, a CD8 T cell receptor (TCR) transgenic model system, and in vivo depleting antibodies to analyze the role of CD4 cell subsets in regulating allograft survival in mice treated with DST and anti-CD154 mAb. RESULTS: Deletion of CD4 or CD25 cells during costimulation blockade induced rapid rejection of skin but only minimally shortened islet allograft survival. Deletion of CD4 or CD25 cells had no effect upon survival of healed-in islet allografts, and CD25 cell deletion had no effect upon healed-in skin allograft survival. In the TCR transgenic model, DST plus anti-CD154 mAb treatment deleted alloreactive CD8 T cells, and anti-CD4 mAb treatment prevented that deletion. In contrast, injection of anti-CD25 mAb did not prevent alloreactive CD8 T cell deletion. CONCLUSIONS: These data document that (1) both CD4CD25 and CD4CD25 cells are required for induction of skin allograft survival, (2) CD4CD25 T cells are not required for alloreactive CD8 T cell deletion, and (3) CD4CD25 regulatory cells are not critical for islet allograft tolerance. It appears that skin and islet transplantation tolerance are mediated by different CD4 cell subsets and different mechanisms.     

Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD154 antibody

BACKGROUND: Combined treatment with a single donor-specific transfusion (DST) and a brief course of anti-mouse CD154 monoclonal antibody (mAb) to induce co-stimulation blockade leads to long-term murine islet allograft survival. The authors hypothesized that this protocol could also induce long-term survival of neonatal porcine islet cell clusters (NPCC) in chemically diabetic immunocompetent mice and allow their differentiation into functional insulin-producing cells. METHODS: Pancreata from 1- to 3-day-old pigs were collagenase digested and cultured for 8 days. NPCC were recovered and transplanted into the renal subcapsular space. Recipients included chemically diabetic nonobese diabetic (NOD)-scid and C57BL/6 mice that were otherwise untreated, treated with anti-CD154 mAb alone, or treated with DST plus anti-CD154 mAb. Plasma glucose concentration and body weight were measured, and xenografts were examined histologically. RESULTS: NPCC fully differentiated and restored normoglycemia in four of five diabetic NOD-scid recipients but were uniformly rejected by diabetic C57BL/6 recipients. Anti-CD154 mAb monotherapy restored normoglycemia in 4 of 10 (40%) NPCC-engrafted, chemically diabetic C57BL/6 mice, but combined treatment with DST and anti-CD154 mAb restored normoglycemia in 12 of 13 (92%) recipients. Reversal of diabetes required 5 to 12 weeks. Surviving grafts were essentially free of inflammatory infiltrates 15 weeks after transplantation. CONCLUSIONS: Combination therapy with a single DST and a brief course of anti-mouse CD154 mAb without maintenance immunosuppression permits survival and differentiation of NPCC in diabetic C57BL/6 mice. Successful grafts were associated with durable restoration of normoglycemia and the absence of graft inflammation.     

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.     

CD8+ T cells contribute to the development of transplant arteriosclerosis despite CD154 blockade

BACKGROUND: The CD40-CD154 receptor-ligand pair plays a critical role in allograft rejection by mediating the activation of endothelial cells, antigen-presenting cells, and T cells. Blockade of this interaction prevents acute allograft rejection and leads to prolonged allograft survival in numerous experimental models, but in most cases indefinite graft survival is not achieved due to evolving transplant arteriosclerosis. In this study, we have used a model of transplant arteriosclerosis to investigate whether CD4+ and CD8+ T cells are differentially affected by CD154 blockade. METHODS: BALB/c (H2d) aortic grafts were transplanted into C57BL/6 (H2b) recipients treated with anti-CD154 monoclonal antibody in the presence or absence of CD8+ T-cell depletion. Histology and morphometric measurements were performed on day 30 after transplantation. RESULTS: Only combined treatment with anti-CD154 and anti-CD8 monoclonal antibodies resulted in a significant reduction of intimal proliferation (33 +/-10% vs. 67+/-14%; untreated control). Administration of either antibody alone did not produce this effect. Thymectomy did not alter the degree of intimal proliferation observed in any of the treatment groups. CONCLUSIONS: Our data provide direct evidence that CD8+ T cells are not targeted effectively by CD154 blockade and that the transplant arteriosclerosis seen after CD154 blockade is not due to recent thymic emigrant T cells.