Nonhuman Primate Transplant Research Articles

Human DAF on pig cells protects against human and non-human primate sera cytotoxicity mediated by exogenous or endogenous complement, as determined by flow cytometry

Expression of human complement regulatory proteins (CRP) in pig cells through transgenesis was proposed to prevent complement activation and the ensuing rejection of pig tissues and organs following pig-to-primate transplantation. Transplantation in non-human primates of organs from transgenic pigs for human decay accelerating factor (hDAF) did not undergo hyperacute rejection, but hDAF could not prevent humoral xenograft rejection (AHXR). A possible explanation for the lack of efficacy of the expression of human complement regulatory proteins in pig cells to prevent AHXR may be interspecies differences between human and non-human complement regulatory system. We assayed the efficacy of transgenic hDAF expressed on porcine cells to inhibit the in vitro complement activity of primate sera. The individual cytotoxicity of sera from seven untreated baboons and of pools of normal human and baboon sera was assayed with endogenous and exogenous complement using a flow-cytometry complement-mediated cytotoxicity assay (FCCA) against peripheral blood lymphocytes (PBL) from hDAF and non-transgenic pigs. We also analyzed the anti-Galα1-3Gal (αGal) antibody titre of the baboon sera by ELISA and the expression of hDAF on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. Cellular expression of hDAF reduced cytotoxicity mediated by endogenous and exogenous complement, although the former was slightly higher. Humoral cytotoxicity was not related to a particular antibody but was inversely related to hDAF expression. The presence of hDAF protected pig cells against lysis by NHS more effectively than against NBS. These results confirm in vitro the protective role of hDAF in pig cells to heterologous complement mediated damage, but they also suggest that the extent of hDAF protection decreases, however, if cells express low levels of hDAF.

Hepatocyte transplantation: Studies in preclinical models

Transplantation of allogeneic or genetically modified autologous hepatocytes may be an alternative to whole-liver transplantation for the treatment of hereditary metabolic liver diseases. Human hepatocytes have already been transplanted in patients, demonstrating the safety and feasibility of both approaches. Although a few cases of allogeneic transplantation have resulted in long-term engraftment and function, only a partial and transient correction of the disease was achieved. This may partly result from a lack of proliferation of transplanted cells. In rodents, transplanted hepatocytes do not proliferate in adult quiescent livers and repopulate recipient livers only when they display a proliferative advantage over resident hepatocytes. Most of these models are not transposable to humans, however. Our aim is to develop preclinical approaches to hepatocyte transplantation in nonhuman primates. We have defined a strategy that increases the engraftment efficiency of transplanted hepatocytes by inducing their proliferation together with that of resident hepatocytes. We have also immortalized simian fetal hepatic progenitor cells and shown that these cells do not proliferate in situ after transplantation into the livers of immunodeficient mice. By contrast early human hepatoblasts repopulate mouse livers more efficiently. However, if we consider the number of cells to be transplanted (one to several billion), the means of expanding and differentiating stem or progenitor cells other than hepatocytes will have to be determined prior to envisaging treating patients.

Keratinocyte Growth Factor Increases Thymic Output of Naïve T-Cells after Total Body Irradiation and Autologous Peripheral Blood Progenitor Cell Transplantation in Rhesus Macaques.

Opportunistic infections have an major impact on morbidity and mortality after peripheral blood progenitor cell transplantation, and are caused by deficient T cell numbers and activities, which correlate with thymic dysfunction. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells from injury induced by chemotherapy and radiation in mice, associated with improved thymopoiesis. We for the first time evaluated the effect of KGF on immune reconstitution after myeloablative total body irradiation (TBI) and autologous transplantation in non-human primates, a clinically relevant large animal model. Four animals received KGF 250mg/kg body weight given once (day −5) before TBI (low dose KGF) and CD34+ purified PBPC transplantation, and two animals received the same dose given 3 times (day −5, −4, −3) before and 3 times (day +1, +2, +3) after TBI (day −2, −1) and transplantation (day 0) (high-dose KGF). These KGF-treated animals were compared to an age and CD34+ cell dose-matched control group of five monkeys transplanted with purified CD34+ cells but without KGF treatment. We evaluated the reconstitution of T and B cells in blood and lymph nodes (LN) for a year post transplantation. All animals engrafted (neutrophils >500/ml) day by day 11 post transplantation. The animals treated with KGF had higher frequencies of naïve T cells in blood and in peripheral LN post transplantation as compared to the control animals, for instance at one month after transplantation the percentage of LN naïve CD4+ T-cells was 35±19% in the high dose KGF group, 14±2% in the low dose KGF group and 15±4% in the control group. At 3 months after transplantation the percentage of naïve CD4+ in LN was 56±5% in the high dose and 59±9% in the low dose KGF groups versus 27±7% in the control group. We observed similar findings in the CD8+ subset. All KGF treated animals had higher peripheral blood TREC levels, broader TCR repertoires as determined by spectratyping of the CDR3 region, and lower frequencies of Ki-67+ proliferating T cells, presumably due to a lower drive for peripheral expansion in animals with better thymic output of naïve T cells. The recovery of circulating B-cells was not altered with KGF treatment compared to the controls. At 12 months after transplantation there was uniform preservation of thymic architecture with proliferating thymocytes in the animals treated with KGF, in comparison to the control group of animals, with thymic atrophy seen in the majority. Thus, peri-TBI treatment with KGF resulted in protection of thymic epithelial cells, improved thymic repopulation, and increased naïve T-cell numbers in secondary lymphatic tissues, and correlated with better immune responses to newly induced (HIV envelope) as well as recall antigens including measles and CMV. KGF-mediated protection and preservation of thymic epithelial cells from TBI-mediated damage, with improved output of naïve T cells post-transplantation may improve immune reconstitution and thus outcomes in patients undergoing autologous or allogeneic transplantation.

Avian Sarcoma Leukosis Virus (ASLV) Derived Retroviral Vectors Efficiently Transduce CD34+ Cells and Have a Distinct Integration Profile in the Rhesus Macaque Transplantation Model.

Retroviral-induced insertional mutagenesis has generated renewed concern due to the occurrence of three cases of T cell lymphoproliferative disease in the otherwise successful French SCID-X1 trial, as well as one instance of myeloid sarcoma in our rhesus macaque transplantation model. Recent genome-wide analysis of retroviral insertion sites for MLV and HIV-1 based vectors has shown that these retroviral vectors prefer to integrate into regions adjacent to (MLV) or within (HIV-1) genes, which likely results in an increased risk of insertional mutagenesis. Thus there is interest in the development of alternative vector systems, particularly for gene transfer to hematopoietic stem cells (HSCs). Intriguingly, the avian sarcoma leukosis virus (ASLV) has been reported to have an unbiased integration pattern in experiments done in a human cell line, but no studies have been done to determine the integration patterns of ASLV in primary cells that would be used in gene therapy, such as HSCs, and this vector has not previously been investigated as a gene transfer modality in primary hematopoietic cells. We tested the gene transfer efficiency and the integration pattern of an ASLV vector using our well-established non-human primate autologous transplantation model. ASLV vectors pseudotyped with an amphotropic MLV envelope and expressing marker genes were produced by transfection of vector DNA into the chicken cell line, DF-1. The resulting RCAS (Replication Competent ALV LTR with A Splice acceptor) vector is able to stably infect human and other mammalian cells but is not replication-competent in these cells. We obtained vector titers (measured on human HEK-293 cells) in the range of 2 to 9 x 106 cfu/ml for vectors carrying either GFP or a neomycin-resistance marker gene. We found that these ASLV vectors can efficiently and stably transduce rhesus macaque as well as human CD34+ hematopoietic progenitor and stem cells at levels up to 33% and 40%, respectively. We analyzed the proviral insertion sites by an optimized linear-amplification mediated PCR (LAM-PCR). 21.43% of the insertions we identified in CD34+ cells prior to transplantation are within gene-coding regions of the genome, which is comparable to random integration data and statistically significant different from MLV and HIV integration patterns we previously reported in primary hematopoietic cells. We are using these ASLV-transduced rhesus macaque CD34+ cells for in vivo autologous transplants to assess long-term stem cell gene transfer efficiency as well as the integration pattern in long-term repopulating cells. Two animals have been transplanted and will be analyzed in detail. We will compare the pattern of integration found in the transplanted cells with that seen prior to transplantation to ask whether the integration pattern is preserved after engraftment. These results, together with our previous findings showing distinctive integration pattern for MLV and HIV based vectors, further reinforce the idea that specific integration patterns of different retroviruses may arise because the different integration complexes bind to distinct cellular components.

The Influence of Baseline Expression of Human Decay Accelerating Factor Transgene on Graft Survival and Acute Humoral Xenograft Rejection

Transgenic pigs expressing human decay accelerating factor (hDAF) have been widely used as donors in various non-human primate transplant models. Despite the use of similar immunosuppressive protocols, there is marked variation in graft survival among centres. The present study was undertaken to determine whether the level of hDAF expression in the pig kidney correlates with the degree of rejection and duration of graft survival. hDAF transgenic pigs were provided from two suppliers: Guelph Imutran Centre (G) and Harlan Sprague Dawley (H). Following a bilateral nephrectomy, a single hDAF pig kidney was implanted in the baboon, which was subsequently treated with conventional immunosuppressive protocols. The pig's contralateral kidney was collected to provide baseline data. The severity of acute humoral xenograft rejection (AHXR) was graded as stage I-III. hDAF expression was measured using morphologic analysis comparing the contralateral and grafted kidneys at the endpoint. Baseline hDAF expression in kidneys from pigs provided by supplier G was significantly higher than that from supplier H (P<0.01). Furthermore, the survival of baboons receiving grafts from G pigs was significantly longer than those receiving grafts from H pigs (P<0.05). In addition, reduction of hDAF expression at the endpoint was associated with a higher degree of AHXR. Severe apoptosis or necrosis was found in grafts with AHXR II-III. Pig kidneys from different suppliers have variable baseline hDAF expression, which may have an influence on graft survival. Reduced expression of hDAF in the terminal graft was associated with the severity of rejection.

The Autoimmune Response to Vimentin After Renal Transplantation in Nonhuman Primates Is Immunosuppression Dependent

Chronic allograft nephropathy (CAN) is a common late complication of kidney transplantation. Antibodies to both human leukocyte antigen and nonhuman leukocyte antigen antigens have been implicated in the development of this condition. Here we investigated the presence of antivimentin antibodies in nonhuman primate recipients of kidney allografts as a possible predictor of CAN and the effects of immunosuppression. Thirty seven rhesus monkeys received a kidney allograft to study the potency of several different immunosuppressive regimens (conventional immunosuppression, n=19, vs. costimulatory blockade, n=18). Monkeys were tested for antivimentin antibody by enzyme-linked immunosorbent assay and for anti-donor antibody by staining donor spleen cells with recipient serum. The appearance of antibodies was correlated with the graft pathology in biopsy and necropsy material. Antivimentin antibodies were found in 31 of 37 animals, whereas only 15 of 32 animals made anti-donor antibodies. Conventional immunosuppression did not prevent antivimentin antibody formation. Costimulation blockade, in particular blocking CD40 and CD86, significantly delayed or prevented antivimentin antibody formation, but did not prevent CAN. Antivimentin antibodies were not significantly associated with development of CAN. We postulate that vimentin acts as an autoantigen after renal transplantation; it elicits an autoimmune response that is not regulated by cyclosporine. This autoimmune response may be part of the complex immunologic events occurring posttransplantation and may contribute to the development of CAN, but cannot be considered as a major cause of CAN because this condition also develops without antivimentin antibodies.

The use of CD34+ mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD 34(+) from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD 34(+) donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.

Tolerability of cyclosphosphamide and methotrexate induction immunosuppression in nonhuman primates

Transplantation in nonhuman primates, in particular using solid organs from porcine donors, requires an efficacious induction immunosuppression. Besides biologicals, the low molecular weight drugs used include cyclophosphamide (CyP) and methotrexate (MTX). As these compounds generally have a narrow therapeutic window, we performed tolerability studies in baboons and cynomolgus monkeys, with/without maintenance immunosuppressants such as cyclosporine A, everolimus, mycophenolate sodium and FTY720. In both species, a four-dose CyP regimen of 40, 20, 30 and 30 mg/kg i.v. on days 1, 2, 4 and 6 is not tolerated, but the regimen is tolerated upon individual adjustment of the third and fourth dose to 18–25 and 8–20 mg/kg, respectively, based on white blood cell count. In cynomolgus monkeys, a 5-day course of MTX i.v. at 0.5 mg/(kg d) is well tolerated, but not MTX at 1.0 mg/(kg d); in combination with maintenance immunosuppression, the 0.5 mg/(kg d) dose can cause adverse effects. Combinations of CyP and MTX are tolerated using the 5-day course of MTX at 0.25 mg/(kg d) and a four-dose regimen of CyP at 10, 2.5, 7.5 and 7.5 mg/kg. These regimens are tolerated in combination with maintenance immunosuppressants. The data provide base values for investigators using nonhuman primates in experimental studies, particularly in xenotransplantation requiring effective induction immunosuppression that is close to maximum tolerated dose levels.

Humoral Immunity to Vimentin Is Associated with Cardiac Allograft Injury in Nonhuman Primates

Immunity to autologous protein has not previously been described following nonhuman primate cardiac transplant. Native hearts and cardiac allografts from cynomolgus monkeys were assessed by immunohistology for vimentin, a highly conserved intermediate filament protein. IgM and IgG to vimentin were measured in serial sera from untreated (n = 4) or cyclosporine (CsA)-treated (n = 8, 2 with ATG) cardiac allograft recipients, and in groups treated with anti-CD154 antibody with (n = 6) or without ATG (n = 28). IgM or IgG reactive with vimentin was elaborated within 30 days with unmodified acute rejection (3/4) or in CsA-treated animals (5/6). CD154 blockade did not prevent anti-vimentin IgM (14/28) but tended to delay the IgG response during therapy (anti-CD154: 8/28, p = 0.10 vs. CsA; anti-CD154+ATG: 2/6). CAV and alloantibody were seen in 25 of 26 animals with grafts surviving over 30 days, including seven animals without increasing anti-vimentin antibody. Anti-vimentin antibodies and vascular complement deposition were found in rejected hearts. Acute and chronic alloimmunity disrupt modulation of autoreactivity to vimentin through pathways, which are resistant to CsA, but may be partially regulated by CD154.

Porcine Hematopoietic Progenitor Cell Transplantation in Nonhuman Primates: A Review of Progress

The critical shortage of human donor organs for transplantation would be overcome if a suitable animal, e.g., the pig, could be used as an organ source. There are, however, several immune barriers that have to date resulted in limited function of pig organs transplanted into nonhuman primates. It would be beneficial, and indeed may be essential, to induce a state of tolerance in the primate recipient to the pig organ. In allotransplantation, the successful transplantation of hematopoietic progenitor cells with the development of mixed chimerism is associated with the induction of tolerance toward a donor-specific organ. For some years, this approach has been explored in the pig-to-nonhuman primate model. This experience is briefly reviewed. The problems of natural and elicited anti-pig antibodies, recipient platelet adhesion to pig hematopietic progenitor cells, and the rapid removal of these cells by the host macrophage-phagocytic system are highlighted. Recent experience with the use of hematopoietic cells from pigs homozygous for alpha1,3-galactosyltransferase gene-knockout is reported.

Gene therapy for the hemoglobin disorders

The hemoglobin disorders of beta-thalassemia and sickle cell disease together constitute the most prevalent group of human monogenic diseases. Although curative allogeneic stem cell transplantation therapy and palliative therapies have been developed for these disorders, the majority of patients still suffer significant morbidity and early mortality. The development of therapeutic approaches based on genetic manipulation of autologous stem cells therefore remains an attractive alternative. In the past 4 years, significant advances have been made toward this goal using lentiviral vectors to obtain high-level expression of complex globin gene cassettes. Therapeutic correction in murine models of both beta-thalassemia and sickle cell anemia has been achieved using this approach. These advances, coupled with progress in the ability to achieve in vivo selection of genetically modified cells, can now be evaluated in the well-developed nonhuman primate autologous transplant model. The goal in these studies is to provide preclinical safety and efficacy data prior to human clinical trials in order to maximize the likelihood of success in the context of an acceptable risk to benefit ratio. Here we review progress in each of these areas.

Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates.

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow-derived, CD34+ -enriched donor cells. Animals treated postnatally received either donor-derived T cell-depleted or CD34+ -enriched marrow cells. Chimerism was determined by traditional and real-time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune-suppressed animals relative to controls (11.3% +/- 2.7% and 5.1% +/- 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence-activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at > or = 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune-suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6-month follow-up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long-term chimerism also was not improved with postnatal donor cell infusion.

The use of mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD34 + from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD34 + donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.

Heterologous immunity: an overlooked barrier to tolerance.

In less than 50 years the field of organ transplantation has transitioned from an experimental concept to clinical commonplace. Notwithstanding the dramatic improvements in patient and allograft outcomes, chronic rejection and the complications from life-long immunosuppressive therapy remain significant problems. The induction of transplantation tolerance, indefinite allograft acceptance independent of chronic immunosuppressive therapy, remains the ultimate objective in transplantation. Many strategies have achieved tolerance to transplanted tissue in rodents; however, few, if any, have shown equal efficacy when tested in non-human primate transplant models or human patients. A critical distinction between specific pathogen-free mice and primates or human patients is the exposure of the latter to environmental pathogens and the resultant-acquired immune history. Recent data has shown that virally induced, alloreactive immune responses can provide a potent barrier to tolerance. In this review, we discuss one of the most robust methods for tolerance, the induction of hematopoietic chimerism as well as the influence of viral infections on the alloimmune response.

Rabbit antithymocyte globulin induction and sirolimus monotherapy supports prolonged islet allograft function in a nonhuman primate islet transplantation model.

We reported that rabbit anti-thymocyte globulin (RATG) induction followed by maintenance immunosuppression with sirolimus supports human kidney allograft survival and asked if this combination would promote islet allograft survival in our primate model. Using intra-arterial streptozotocin infusion, we rendered four cynomolgus primates diabetic with undetectable C-peptide levels. Animals were maintained on insulin therapy for at least 1 month, and then islets from mixed lymphocyte reaction mismatched primates were infused into the portal vein. Immediately before the islet allotransplant and for 6 additional days, primates were infused with RATG (20 mg/kg) and given a sirolimus dose to achieve a 24-hr trough level of 8 to 14 ng/mL. The regimen resulted in profound peripheral and lymph node lymphocyte depletion for up to 1 month. Repopulation was gradual thereafter. One primate remained insulin-independent for 169 days and rejected after a sirolimus-dose reduction. Two primates died on day 23 while insulin independent because of wound dehiscence, and a third died on day 30 with high sirolimus levels. Liver sections revealed well-vascularized islets with no signs of inflammation. Using a nonhuman primate islet transplant model, RATG plus sirolimus supports islet survival as long as proper sirolimus levels are maintained, but the therapy is limited by sirolimus toxicity. Our findings suggest that RATG is not toxic for islets and thus may be considered in future clinical trails while recognizing that sirolimus monotherapy, with its difficult-to-achieve therapeutic dosing, may not be sufficient to maintain long-term islet allograft function in an autoimmune environment.

Heterologous immunity provides a potent barrier to transplantation tolerance.

Many strategies have been proposed to induce tolerance to transplanted tissue in rodents; however, few if any have shown equal efficacy when tested in nonhuman primate transplant models. We hypothesized that a critical distinction between specific pathogen-free mice and nonhuman primates or human patients is their acquired immune history. Here, we show that a heterologous immune response--specifically, virally induced alloreactive memory--is a potent barrier to tolerance induction. A critical threshold of memory T cells is needed to promote rejection, and CD8(+) "central" memory T cells are primarily responsible. Finally, treatment with deoxyspergualin, an inhibitor of NF-kappa B translocation, together with costimulation blockade, synergistically impairs memory T cell activation and promotes antigen-specific tolerance of memory. These data offer a potential explanation for the difficulty encountered when inducing tolerance in nonhuman primates and human patients and provide insight into the signaling pathways essential for memory T cell activation and function.

In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells.

In utero transplantation of hematopoietic stem cells is a promising treatment for immune and hematologic diseases of fetuses and newborns. Unfortunately, there are limited data from nonhuman primates and humans describing optimal transplantation conditions. The purpose of this investigation was to determine the effect of T-cell number on engraftment and the level of chimerism after in utero transplantation in nonhuman primates. CD34(+) allogeneic adult bone marrow cells, obtained from the sire after G-CSF and stem cell factor administration, were transplanted into female fetal recipients. The average CD34(+) cell dose was 3.0 x 10(9)/kg (range, 9.9 x 10(8) to 4.4 x 10(9)) and the T-cell dose ranged from 2.6 x 10(5) to 1.1 x 10(8)/kg. Chimerism was determined in peripheral blood subsets (CD2, CD13, and CD20) and in progenitor cell populations by using polymerase chain reaction. Chimerism was noted in seven of eight live-born animals. The level of chimerism in the progenitor population was related to the fetal T-cell dose (r = 0.64, p < 0.02). At the lowest T-cell dose (2.6 x 10(5)/kg), no chimerism was detected. As the T-cell dose increased to 10(6-7)/kg, the level of chimerism increased. Adjusting the T-cell dose to 1.1 x 10(8)/kg resulted in fatal graft-versus-host disease (GVHD). The results of this study emphasize the importance of T cells in facilitating donor cell engraftment and in producing GVHD in fetal nonhuman primates. Some animals achieved levels of chimerism in the marrow hematopoietic progenitor cell population that would likely have clinical relevance. However, the levels of chimerism in peripheral blood were too low for therapeutic benefit. Further studies are needed to test methods that are likely to enhance donor cell engraftment and peripheral blood levels of donor cells.

Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector

The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice. Gene transfer efficiency as reflected by the number of genetically modified cells in hematopoietic tissues varied among the five monkeys studied from low levels (<1%) in three animals to much higher levels in two (20–60%). An animal that exhibited extremely high levels for several weeks was found by vector genome insertion site analysis to have reconstitution predominantly with a single clone of cells. This variability among animals is in keeping with computer simulations of reconstitution with limiting numbers of stem cells genetically modified at about 10% efficiency. Our studies provide insights into the biology of hematopoietic reconstitution and suggest approaches for increasing stem cell targeted gene transfer efficiency.

Combination induction therapy with monoclonal antibodies specific for CD80, CD86, and CD154 in nonhuman primate renal transplantation.

Antibodies and fusion proteins specific for CD80, CD86, and CD154 have shown promise as agents capable of inducing donor-specific tolerance in rodents. These agents have also been shown to be synergistic with one another in many settings of counter-adaptive immunity. In the nonhuman primate, monoclonal antibodies specific for CD80 and CD86 have prolonged the time to rejection of renal allografts but have not resulted in tolerance. A monoclonal antibody specific for CD154 has resulted in markedly prolonged survival of kidney, islet, cardiac, and skin allografts, but again most animals have eventually developed rejection after prolonged periods of rejection-free survival off therapy. A combination of monoclonal antibodies specific for CD80, CD86, and CD154 were used in a mismatched nonhuman primate renal-allograft model. Doses used were based on optimized treatment protocols for each agent individually. Treatment of four rhesus macaques with this combination yielded a mean rejection-free survival of 565 days (311-911 days), significantly greater than untreated controls (mean survival=7.0 days, P=0.001) and animals treated with only a combination of anti-CD80 and CD86 (mean survival=191 days, P=0.01). The survival of animals treated with this combination of monoclonal antibodies was not significantly greater than those treated with anti-CD154 alone, but the production of alloantibody was delayed compared with monotherapy anti-CD154. These data suggest that a synergy exists between these agents, particularly with regard to T-dependent B-cell responses, but that they fail to induce durable tolerance in nonhuman primates.

Ultrasound score is more predictive than serum creatinine in assessment of cellular rejection in cynomolgus monkey renal allografts.

To investigate whether ultrasound (US), in particular the use of an ultrasound scoring system, can provide more diagnostic information than clinical parameters, such as serum creatinine, for the diagnosis and determination of the degree of cellular rejection in renal allografts in the cynomolgus monkey (Macaca fascicularis). Sixty-eight cynomolgus monkeys with life-supporting renal allografts were examined with a 7.5MHz linear ultrasound transducer. One-hundred fifty two-dimensional, spectral, and power Doppler examinations were performed and four ultrasound parameters, percentage increase in graft volume, cortical thickness, resistive index (RI) of the renal arcuate artery, and power Doppler (PD) scores were recorded from serial examinations. An ultrasound score was assigned to each graft based on the number of those parameters that were abnormal; a score of 1 indicated that all four were normal, and a score of 5 that all four were abnormal. Each parameter and the combined score were compared with serum creatinine values and histology and evaluated statistically using Spearman rank correlation. In animals with dysfunctioning allografts (serum creatinine elevations >200 micromol/L), Spearman rank correlation showed a significant correlation between the US score and the histology score: between 200 and 500 micromol/L, r = 0.309, P = 0.046, n = 31 and if > 500 micromol/L, r = 0.486, P = 0.005, n = 30. In those same animals, no correlation could be shown between serum creatinine values and the US score or between the serum creatinine values and the histologic diagnosis. In contrast to the US score, single ultrasound parameters were not found to correlate to histologic findings. The application of ultrasound imaging in nonhuman primate renal transplant models provides valuable information concerning the presence and severity of cellular rejection in cases of graft dysfunction and the US score has a better predictive value of histology than serum creatinine values alone.