Bone marrow-derived hematopoietic stem and progenitor cells infiltrate allogeneic and syngeneic transplants.

Lineage (CD3e, CD11b, GR1, B220 and Ly-76) negative hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) infiltrate islet allografts within 24 h posttransplantation. In fact, lineage(negative) Sca-1(+) cKit(+) ("LSK") cells, a classic signature for HSCs, were also detected among these graft infiltrating cells. Lineage negative graft infiltrating cells are functionally multi-potential as determined by a standard competitive bone marrow transplant (BMT) assay. By 3 months post-BMT, both CD45.1 congenic, lineage negative HSCs/HPCs and classic "LSK" HSCs purified from islet allograft infiltrating cells, differentiate and repopulate multiple mature blood cell phenotypes in peripheral blood, lymph nodes, spleen, bone marrow and thymus of CD45.2 hosts. Interestingly, "LSK" HSCs also rapidly infiltrate syngeneic islet transplants as well as allogeneic cardiac transplants and sham surgery sites. It seems likely that an inflammatory response, not an adaptive immune response to allo-antigen, is responsible for the rapid infiltration of islet and cardiac transplants by biologically active HSCs/HPCs. The pattern of hematopoietic differentiation obtained from graft infiltrating HSCs/HPCs, cells that are recovered from inflammatory sites, as noted in the competitive BMT assay, is not precisely the same as that of intramedullary HSCs. This does not refute the obvious multi-lineage potential of graft infiltrating HSCs/HPCs.

Prevention of nonimmunologic loss of transplanted islets in monkeys.

The nonimmunologic loss of islets in the pre-, peri-, and early post-islet transplant periods is profound. To determine the potential role that transplantation of only a marginal mass of functioning beta cells may play in triggering late nonimmunologic graft loss, we studied the effect of treatment with alpha-1-antitrypsin (AAT) in the autologous cynomolgus islet transplant model. A marginal mass of autologous islets, that is islets prepared from 70% to 80% of the pancreas, was transplanted at 1600-4100 IEQ/kg into subtotal pancreatectomized, streptozotocin-treated and insulin-deficient diabetic hosts. In this marginal mass islet transplant model, islet function is insidiously lost over time and diabetes recurs in all untreated monkeys by 180 days posttransplantation. Short-term treatment with AAT, an acute phase reactant, in the peri-transplant period serves to terminate inflammation through effects upon expression of TGFß, NFκB and AKT and favorably altering expression of cell death and survival pathways, as detected by a system biology approach and histology. These effects enabled functional expansion of the islet mass in transplanted hosts such that graft function improves rather than deteriorating over time.

Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term triple therapy.

Preclinical studies in nonhuman primates (NHP) are particularly useful to evaluate the safety and efficacy of new therapeutic proteins developed for use in clinical transplantation. We hypothesized that a treatment that selectively destroys activated cytopathic donor reactive T cells while sparing resting and immunoregulatory T cells in a mouse model might also produce long-term drug-free engraftment and tolerance without the hazards of lymphopenia in the challenging nonhuman primate islet allograft model. Short-term treatment with a regimen consisting of rapamycin, and IL-2.Ig plus mutant antagonist-type IL-15.Ig cytolytic fusion proteins (triple therapy) posttransplantation results in prolonged, drug-free engraftment of cynomolgus islet allografts. Moreover slow progressive loss of islet function in some recipients was not associated with obvious pathologic evidence of rejection.

Expression of CD39 by human peripheral blood CD4+ CD25+ T cells denotes a regulatory memory phenotype.

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4+ Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood-derived human CD4+ CD25+ CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4+ CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+ CD25+ CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection.

Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: nonimmunologic graft failure?

Conventional drug therapy and several anti-CD154 mAb-based regimens were tested in the nonhuman primate (NHP) islet allograft model and found to be inadequate because islets were lost to rejection. Short-term therapy with an optimized donor-specific transfusion (DST) + rapamycin (RPM) + anti-CD154 mAb regimen enables immunosuppression drug-free islet allograft function for months following cessation of therapy in the NHP islet allograft model. After a substantial period of drug-free graft function, these allografts slowly and progressively lost function. Pathologic studies failed to identify islet allograft rejection as a destructive islet invasive lymphocytic infiltration of the allograft was not detected. To evaluate the mechanism, immunologic versus nonimmunologic, of the late islet allograft loss in hosts receiving the optimized therapeutic regimen, we performed experiments with islet autografts and studied islet function in NHPs with partial pancreatectomy. The results in both experiments utilizing autologous islet allografts and partially pancreatectomized hosts reinforce the view that the presence of a marginal islet mass leads to slowly progressive nonimmunological islet loss. Long-term clinically successful islet cell transplantation cannot be realized in the absence of parallel improvements in tolerizing regimens and in the preparation of adequate numbers of islets.

Effects of streptozotocin on autoimmune diabetes in NOD mice.

Non-obese diabetic (NOD) mice develop autoimmunity that destroys their native beta cells causing diabetes. Their autoimmunity will also destroy syngeneic transplanted islets and transfer both autoimmunity and diabetes via spleen cells to non-diabetic mice. In this report, we studied the effects of streptozotocin (STZ) on the autoimmune diabetes in NOD mice. We transplanted NOD.SCID islets into three groups of NOD mice: (1) spontaneously diabetic NOD mice (NOD-sp.); (2) prediabetic NOD mice made diabetic by streptozotocin (NOD-stz); and (3) diabetic NOD mice also treated with streptozotocin (NOD-sp./stz). In the first group, the transplants were rejected within 3 weeks. In the second and third groups, the transplants survived indefinitely. Alloxan, a drug similar to streptozotocin, did not have the same effect as streptozotocin. The ability of streptozotocin to prevent diabetes in young NOD mice was reversed by anti-CD8 antibody treatment but not by anti-CD4 treatment. Streptozotocin also made spleen cells from diabetic NOD mice less effective transferring diabetes. These results indicate that streptozotocin treatment both prevents and reverses the islet destructive autoimmunity in NOD mice. We postulate that the effects of streptozotocin treatment may be mediated in part by regulatory T cells.

The effect of low versus high dose of streptozotocin in cynomolgus monkeys (Macaca fascilularis).

Streptozotocin (STZ) is often used to induce diabetes in animal models. However, morbidity associated with STZ and its ability to induce diabetes vary with different dosages among different animal species, including nonhuman primates. To find an optimal dose of STZ that would cause diabetes with minimal toxicity, we compared low and high doses of STZ. Male cynomolgus monkeys (3-6 years old) were given a single dose of 100 mg/kg (high dose, 4 animals) or 55 mg/kg (low dose, 20 animals) of STZ. Blood glucose levels, intravenous glucose tolerance test (IVGTT), pancreatic biopsies, liver function tests (LFTs), liver biopsies, kidney function tests, and kidney biopsies were performed periodically. Animals from both groups developed diabetes within 24 h after administration of STZ. Serum C-peptide levels in both groups decreased from 2 to 8 ng/mL before STZ to between 0.01 and 0.6 ng/mL after STZ. Animals with the high dose of STZ developed transient vomiting within minutes after injection. During the first week after STZ injection, high-dose animals developed elevated LFTs, BUN and creatinine. In contrast, low-dose animals had normal liver and kidney function tests. Histological analysis showed that animals given the high dose of STZ developed marked steatosis of the liver and tubular injury in the kidneys, whereas animals given the low dose of STZ had normal-looking liver and kidney histology. The pancreatic islets in both groups were indistinguishable by immunoperoxidase staining for insulin, and showed either no insulin-positive cells or rare insulin-positive cells. Glucagon staining was normal. Over time, low-dose diabetic monkeys remained persistently hyperglycemic with negligible C-peptide stimulation by intravenous glucose. We conclude that low-dose STZ at 55 mg/mL successfully induces diabetes in cynomolgus monkeys with minimal liver and kidney toxicity.

Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade.

The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (I) conventional triple drug immunosuppressive therapy or (2) a non-myeloablative regimen and an anti-CD154 monoclonal antibody (mAb) aimed at tolerance-induction. Group 1 baboons (n = 3) were pancreatectomized prior to intraportal injection of 10,000 porcine islet equivalents (IE)/kg and immunosuppressed with anti-thymocyte globulin (ATG), cyclosporine and azathioprine. In Group 2 (n = 2), non-pancreatectomized baboons underwent induction therapy with whole body and thymic irradiation, and ATG. Extracorporeal immunoadsorption (EIA) of anti-Galalpha1,3Gal (Gal) antibody was carried out. Maintenance therapy was with cobra venom factor, cyclosporine. mycophenolate mofetil, methylprednisolone and anti-CD154 mAb. Porcine islets were injected intraportally (14,000 and 32,000 IE/kg, respectively) and high-dose pig mobilized peripheral blood progenitor cells (3 x 10(10) cells/kg) were infused into a systemic vein. Porcine islets were also implanted in the sternomastoid muscle to facilitate subsequent biopsies. In both groups. porcine C-peptide was measured, and histological examination of liver or sternomastoid muscle biopsies was performed at regular intervals. In Group 1, total pancreatectomy reduccd human C-peptide to < 0.1 ng/ml and induced insulin-requiring diabetes. The transplantation of porcine islets was followed by normalization of glycemia for 15-24 h. Porcine C-peptide was detected only transiently immediately after porcine islet injection (maximum 0.12 ng/ml). Histological examination of liver biopsies taken between days 2 and 19 did not reveal viable islets, but necrotic cell structures with mononuclear cell infiltrates were identified in portal venules. In Group 2, injection of porcine islets into non-pancreatectomized recipients induced a transient hypoglycemia (2-4 h) requiring concentrated intravenous dextrose administration. Porcine C-peptide was detectable for 5 and 3 days (maximum 2.8 and 1.0 ng/ml), respectively. Baboon #4 died on day 12 from small bowel intussusception. Liver and sternomastoid muscle biopsies showed well-preserved porcine islets, staining positive for insulin and glucacon, without signs of rejection. In baboon #5, viable islets were detected in the sternomastoid muscle biopsy on day 14, but not on day 28 or thereafter. A progressive mononuclear cell and macrophage infiltration was seen in the biopsies. In conclusion, conventional immunosuppression allowed survival of porcine islets in baboons for < 24 h. The non-myeloablative regimen prolonged survival of porcine islets for > 14 days. However, despite depletion of T cells, anti-Gal antibody and complement, and CD154-hlockade, porcine islets were rejected by day 28. These results suggest that powerful innate immune responses are involved in rejection of discordant xenogencic islets.

Long-term islet allograft function in the absence of chronic immunosuppression: a case report of a nonhuman primate previously made tolerant to a renal allograft from the same donor.

UNLABELLED: Development of mixed chimerism by donor bone marrow transplantation (DBMT) has led to long-term tolerance of solid organ allografts in nonhuman primates. As an initial attempt to extend this approach to cellular transplant, islet transplant from the same donor was attempted in the recipient previously made tolerant to a kidney allograft. METHODS: After the conditioning with ATG, total body irradiation, thymic irradiation, and splenectomy, DBMT was performed followed by 4 weeks of cyclosporine. Kidney transplantation and native nephrectomies were subsequently performed on day 89. After 2.8 years of DBMT, diabetes was induced by streptozocin (STZ) and islets from bone marrow and kidney donor were transplanted without immunosuppression. RESULTS: After DBMT, the recipient developed chimerism and no evidence of kidney rejection for more than 1000 days. STZ induced diabetes was reversed after the islet transplantation. Islet biopsies demonstrated insulin staining without rejection. Although the recipient became diabetic 300 days after islet transplantation, viable transplanted islets were found in the liver and under the kidney capsule without any evidence of rejection. CONCLUSION: Tolerance with a nonmyeloablative conditioning can allow successful pancreatic islet transplantation without immunosuppression. Because no histological evidence of rejection was identified, recurrent diabetes is presumed to be inadequate islet mass.

Cyclophosphamide, but not CTLA4Ig, prolongs survival of fetal pig islet grafts in anti-T cell monoclonal antibody-treated NOD mice.

Fetal pig islets, xenografted after organ culture into non-immunosuppressed prediabetic NOD mice, are rejected within 10 days. Immunosuppression with anti-T cell (anti-CD4 and anti-CD3) monoclonal antibodies alone is highly effective in delaying graft rejection in this discordant model, but rejection eventually occurs, usually within 80 days, despite marked depletion of T cells. In an attempt to prevent rejection, we used cyclophosphamide (CP), a powerful anti-B cell agent, or CTLA4Ig, an inhibitor of T-cell co-stimulation [via B7-1 (CD80) and B7-2 (CD86)], either given in combination with anti-CD4 (GK1.5) or anti-CD3 (KT3) MAb to the recipient mice. The addition of cyclophosphamide in a dose that significantly depleted B cells in peripheral blood was highly effective in preventing rejection, with xenografts surviving for at least 112 days, when the experiment was terminated. CTLA4Ig, administered alone, did not prevent delayed rejection (rejection occurred in <60 days) and, in contrast to CP, did not prevent delayed rejection when used in combination with GK1.5 and KT3 treatment. Thus, immunosuppressive agents found to be highly effective in other strains, e.g., CTLA4Ig and anti-T cell MAbs, had a lesser effect in NOD mice but the addition of an anti-B cell drug, CP, was useful. This finding may be applicable to patients with IDDM.

Evidence that macrophages are required for T-cell infiltration and rejection of fetal pig pancreas xenografts in nonobese diabetic mice.

BACKGROUND: Host macrophages are abundant within fetal pig pancreas xenografts undergoing rejection, but their role is unknown. Therefore, we examined the effect of host macrophage depletion on xenograft rejection. METHODS: Nonobese diabetic (NOD) mice were given clodronate-loaded liposomes intravenously to deplete macrophages. Controls received phosphate-buffered saline (PBS) or PBS-liposomes. General immune status was assessed after 2, 3, and 7 days by (1) fluorescence-activated cell sorter analysis of peripheral blood, spleen, and lymph node cells, (2) immunohistochemistry on spleens, and (3) mixed lymphocyte reaction. Organ-cultured fetal pig pancreas was transplanted under the kidney capsule of NOD mice 3 days after clodronate or PBS injection. Grafts were assessed histologically at 4, 5, 6, and 8 days after transplantation. RESULTS: Splenic macrophages and peripheral blood monocytes were depleted 2 days after clodronate treatment but had recovered within 11 days. T cell, B cell, and dendritic cell numbers were normal in spleen, peripheral blood, and lymph nodes of clodronate-treated mice, and T cells and antigen-presenting cells from these mice functioned normally in mixed lymphocyte reaction. Clodronate treatment markedly reduced graft infiltration by macrophages, T cells, and eosinophils at 4, 5, and 6 days after transplantation, and was associated with maintenance of endocrine cell viability and insulin expression. However, all grafts were rejected 8 days after transplantation, concordant with reappearance of splenic macrophages. CONCLUSIONS: Short-term, specific depletion of macrophages markedly delayed cellular infiltration and rejection of xenografts. The results provide the first evidence that macrophages promote T-cell infiltration and rejection of fetal pig pancreas xenografts in NOD mice.

Pig islet xenografts are susceptible to "anti-pig" but not Gal alpha(1,3)Gal antibody plus complement in Gal o/o mice.

Hyperacute rejection due to Galalpha(1,3)Gal (Gal) Ab plus complement (C') is a major problem in xenografting vascularized organs from pigs to primates, but the fate of neovascularized xeno islets is unclear. Nonendocrine islet cells are Gal+, and there is a large rise in Gal Abs after transplantation, but graft remnants persist for some days in monkeys and humans. To define the role of alphaGal Ab plus C' in porcine islet graft rejection, cultured porcine fetal islets were grafted to mice lacking the alpha(1,3)galactosyltransferase gene. Anti-Gal Ab plus C' did not cause islet damage or rejection in mice lacking the alpha(1,3)galactosyltransferase gene, even when additional Ab plus C' was given; in addition, hyperimmune mice (titer >1/ 20,000) did not reject pig islets, showing that islets are resistant to Gal Ab plus C'. However, islets can be destroyed by polyclonal mouse anti-pig Abs. Thus, the focus of islet xenografting should not be on Gal Ab plus C'.

Antibodies to ICA512/IA-2 in rodent models of IDDM.

Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior to and soon after the onset of insulin dependency. The non-obese diabetic (NOD) mouse and the diabetes-prone BB rat develop spontaneous diabetes as a consequence of T-cell mediated autoimmune destruction of islet beta-cells, but the occurrence of autoantibodies is controversial. We tested sera from NOD mice and BB-rats for anti-ICA512 by a radioimmunoprecipitation assay (RIP). In sequential serum samples from 20 NOD mice, of which 15 developed diabetes, low levels of anti-ICA512 were demonstrable. Anti-ICA512 appeared close to the onset of hyperglycaemia and was usually transient. Non-diabetic NOD mice also produced anti-ICA512, but at a later age and at lower levels than the diabetic NOD mice. In a cross-sectional analysis of sera from BB rats, low levels of anti-ICA512 were present in 11/20 (55%) of non-diabetic-diabetes prone (DP) BB rats, 0/4 (0%) of diabetic DP BB rats, and 1/6 (17%) of diabetes-resistant BB rats. Anti-ICA512 was not detected in rats of other strains, including three Sprague-Dawley rats with streptozotocin-induced diabetes. In both NOD mice and BB rats the anti-ICA512 reactivity was directed to the cytoplasmic domain of the protein. The transient appearance of anti-ICA512 close to the onset of diabetes in NOD mice and the loss of these antibodies after diabetes onset is consistent with the occurrence of anti-ICA512 in human IDDM. Thus in both human IDDM and rodent models, anti-ICA512 is a marker of the impending onset of diabetes and disappears after diabetes onset.

Expression of both Th1 and Th2 cytokines correlates with the histological rejection of MHC-matched and MHC-mismatched foetal pancreas allografts in mice.

Foetal mouse pancreatic islet grafts were used to investigate differences in the histological appearance and cytokine expression pattern during acute rejection of fully MHC-mismatched, and MHC-matched but minor histocompatibility-mismatched (mH) allografts. Grafts of foetal islet tissue from non-obese diabetic mice under the kidney capsule of non-immunosuppressed MHC and mH-disparate BALB/c mice were rejected by day 9, whereas the rejection of only mH-disparate C3H tissue into CBA mice occurred between 11 and 15 days. In both situations enhanced expression of transcripts for Th1 (IL-2, IFN-gamma, TNF-beta) and Th2 cytokines (IL-4, IL-6, IL-10) was demonstrable at the peak of infiltration of immune cells into the graft site, indicating a close association of these cytokines with graft destruction. Besides these kinetic differences no variation in the expression pattern of the tested cytokines could be demonstrated, indicating that the allograft response in either combination leads to enhanced expression of pro-inflammatory cytokines which could contribute to graft destruction.

Cytokines and autoimmune beta cell destruction in NOD mouse fetal pancreas isografts in cyclophosphamide-induced diabetes.

The role of cytokines in a model of cyclophosphamide (CP)-accelerated beta cell destruction in fetal pancreas isografts transplanted into NOD mice was studied. One group of prediabetic NOD mice was injected with CP at a dose of 300 mg/kg i.p. and 7 days later isografts of organ cultured fetal pancreas (FP) were transplanted under the kidney capsule of these and untreated control mice. The mice were killed at several time points post-transplantation and the histological appearance of the host pancreas used to evaluate the disease progress in the grafts since previous studies had shown good correlation between isograft and native pancreas pathology. Intragraft cytokine gene expression was monitored by reverse-transcriptase polymerase chain reaction (RT-PCR) at the same time points and the expression levels between the experimental groups compared to normal kidney tissue. In comparison to isografts from non-CP injected mice, isografts from CP-treated mice showed increased expression of IFN-gamma, TNF-alpha, TNF-beta, IL-5, and eotaxin but no increase in IL-10 expression. The enhanced transcription of these cytokines correlated with massive infiltration of immune cells and ongoing beta cell destruction in the host pancreas of the CP-treated recipients.

The role of cytokines during rejection of foetal pig and foetal mouse pancreas grafts in nonobese diabetic mice.

The rejection of discordant foetal pig islet xenografts in nonimmunosuppressed nonobese diabetic (NOD) mice is dominated by polymorphonuclear cell infiltration whereas allografts are almost exclusively infiltrated by mononuclear cells. To determine if this variation is due to different proinflammatory factors generated at the graft site, we analysed graft-site mRNA expression of various cytokines, and the eosinophil attractant chemokine, eotaxin, in a renal subcapsular islet transplant model using organ cultured foetal pig (xenograft) and foetal BALB/c (allograft) pancreas in prediabetic NOD mice. Using semiquantitative RT-PCR on samples recovered at multiple time points during the first 15 post-transplantation days from mice transplanted with either allogeneic or xenogeneic tissue, we found increased expression of IL-2, IL-4. TNF-beta and IL-10 mRNAs at the peak of the cellular infiltrate (on day 5) in both xenografts and allografts but, in contrast to the allografts, no enhanced transcription of IFN-gamma mRNA in the rejecting xenografts. When an allograft and a xenograft were placed at the opposite pole of the same kidney the histological appearance of the rejecting allograft site resembled the xenograft site with significant numbers of eosinophils in both, and enhanced expression of eotaxin and iNOS. Additionally, the xenograft response, unlike the allograft response, was marked by an early increased expression of TNF-alpha and IL-S (day 3) and an almost complete absence of IFN-gamma expression. The results suggest a distinct cell-mediated mechanism for rejection of foetal pancrease xenografts compared to the rejection of foetal pancreas allografts.