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Transplant Immunology 06/2009; 21(1):56. · 1.46 Impact Factor
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Judith M Thomas,
Juan L Contreras,
Pei X Wang,
Devin E Eckhoff,
Clement Asiedu,
William J Hubbard,
Samuel Cartner,
Francis T Thomas,
Michelle L Robbin,
Steven Nadler,
William J Cook,
Joshua Sharff,
David M Neville,
Joseph Shiloach
Transplantation 04/2008; 85(6):920. · 4.00 Impact Factor
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ABSTRACT: Natural killer T cells (NKT) possess dual functions of innate and adaptive immune systems, controlling viral infections and regulating autoimmune diseases. Non-human primates (NHP) are penultimate models for advancing therapeutic immunoregulatory strategies for translational application in humans, though, little is known about NHP NKT cells. Here we characterized rhesus macaque NKT cells ex vivo.
The frequency, phenotype and intracellular cytokine production of V alpha 24+ 6B11+ invariant NKT (iNKT) cells were analyzed by multi-color flow cytometry. V alpha 24J alpha Q mRNA expression was analyzed by real-time RT-PCR.
The frequencies of peripheral blood (PB) and spleen V alpha 24+ 6B11+ iNKT cells were not significantly different. The iNKT cell subset in spleen was significantly increased for CD4+ CD8+ and CD3+ CD56+ co-expression as well as intracellular interleukin-4 production, which was rarely observed in circulating PB.
Spleen iNKT cells in rhesus macaques are Th2 biased and display phenotypically and functionally distinct profiles from their PB counterpart.
Journal of Medical Primatology 03/2008; 37(1):1-11. · 1.30 Impact Factor
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ABSTRACT: The mechanisms mediating T-cell depletion plus 15-deoxyspergualin (DSG)-induced prolonged allograft survival or tolerance are uncertain. The purpose of this study is to evaluate the role of IL-4 and IL-10 in prolonged allograft survival induced by T-cell depletion plus DSG. MHC mismatched skin allograft transplantation was performed, using wild-type and three separate knockout (i.e., IL-4-/-, Stat6-/-, or IL-1-/ -) mice as recipients. Induction therapy consisted of T-cell depletion and or brief course of DSG. The data demonstrate that monotherapy with T-cell-depleting mAbs or DSG prolonged skin allograft survival, compared to controls, in wild-type Balb/c recipients [median survival time (MST) = 25 and 21 vs. 10 days, p < 0.007]. T-cell depletion plus DSG further augmented skin allograft survival in wild-type animals relative to monotherapy (MST = 35 days vs. 25 and 21 days, p < 0.006 vs. mAbs or DSG only), and was equally effective in IL-4-/- and Stat6-/- recipients. In contrast, combined therapy was no better than monotherapy in IL-10-/- animals (p > 0.05). Furthermore, skin allograft survival after combined therapy was shorter in IL-10-/- versus wild-type recipients (MST 20 and 41 days, respectively, p < 0.001). IL-4-mediated signaling through Stat6 is dispensable for prolonged allograft survival induced by T-cell depletion plus DSG. In contrast, IL-10 appears to be important for prolonged allograft survival induced by combined therapy in this model.
Cell Transplantation 02/2008; 17(6):713-20. · 5.13 Impact Factor
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ABSTRACT: T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.
Protein Engineering Design and Selection 10/2007; 20(9):425-32. · 2.94 Impact Factor
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ABSTRACT: CD4(+)CD25(+) regulatory T cells (Tregs) play an important role in allograft and self-tolerance and thus have potential therapeutic application in transplantation, autoimmunity, and allergy. Although nonhuman primate (NHP) provide the most accepted preclinical models for translational studies in allograft tolerance and infectious diseases, CD4(+)CD25(+) Tregs have been rarely studied in NHP. The low frequencies of Tregs in peripheral blood will likely necessitate ex vivo expansion to enable Tregs adaptive immune therapy in NHP and humans. Tregs were isolated by magnetic and flow sorting and then stimulated weekly with antirhesus CD3 clone FN18 and antihuman CD28-coated Dynal beads plus 100 U/ml rhIL-2. Under these conditions, the Tregs were expanded 300- to 2000-fold in 4 weeks. Expanded CD4(+)CD25(+) Tregs expressed high to moderate levels of FOXP3 as well as CD95, CD62L, CD69, and CCR7 surface antigens. Expanded rhesus Tregs were anergic and suppressed the proliferation of autologous peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion, and the suppression was partially reversed by anti-transforming growth factor (TGF)-beta1 neutralizing antibody (Ab). These results demonstrate that rhesus macaque suppressive regulatory CD4(+)CD25(+)FOXP3(+) Tregs can be efficiently expanded in vitro under rhesus-specific stimulation, which enables preclinical testing of Treg therapy in the NHP model.
Human Immunology 07/2007; 68(6):478-90. · 2.84 Impact Factor
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Annals of the New York Academy of Sciences 12/2006; 685(1):175 - 192. · 3.15 Impact Factor
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The Journal of Immunology 09/2006; 177(3):2023. · 5.79 Impact Factor
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Transplantation 09/2006; 82(4):577. · 4.00 Impact Factor
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ABSTRACT: Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5-10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 +/- 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 +/- 3.0% of CD4 T cells vs 2.8 +/- 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 +/- 0.9% vs 0.4 +/- 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25- T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25- effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.
The Journal of Immunology 01/2006; 175(12):8060-8. · 5.79 Impact Factor
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ABSTRACT: Natural killer T cells are immunoregulatory cells, which have important roles in tolerance and autoimmunity, as demonstrated primarily in mice and humans. In this study, we define the phenotype and function of Valpha24(+) T cells derived from the spleens of rhesus macaques, a species increasingly used in models of immune tolerance. Valpha24(+) cells were isolated and expanded with monocyte-derived immature dendritic cells in the presence of alpha-galactosylceramide, IL-2, and IL-15. Rhesus NKT cells were stained with mAbs against both Valpha24 and the invariant complementarity-determining region 3 epitope of the human Valpha24/JalphaQ TCR. The cells were CD4, CD8 double negative and expressed CD56. Rhesus NKT cells also exhibited moderate to high expression of CD95, CD45RO, CD11a, and beta(7) integrin, but did not express CD45 RA, CD62L, CCR7, CD28, and other activation, costimulatory molecules (CD69 and CD40L). By intracellular staining, >90% of unstimulated rhesus NKT cells expressed IL-10, but not IFN-gamma. However, the latter was strongly expressed after stimulation. Rhesus NKT secreted large amounts of TGF-beta, IL-13, and IL-6, and modest levels of IFN-gamma, whereas IL-10 secretion was negligible and no detectable IL-4 was observed either intracellularly or in culture supernatants. Functionally, the NKT cells and their supernatants suppressed T cell proliferation in allogeneic MLR. We conclude that long-term cultured rhesus macaque spleen-derived Valpha24(+) T cells are semi-invariant double-negative cells with effector memory phenotype. These cells are semianergic, polarized to a uniquely Th3 > T regulatory-1 regulatory cell phenotype, and have regulatory/suppressive function in vitro.
The Journal of Immunology 10/2003; 171(6):2904-11. · 5.79 Impact Factor
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ABSTRACT: We have demonstrated the presence of crystalline particles in University of Wisconsin (UW) solution used to perfuse rhesus monkey kidney transplants. These particles were visible in obstructed blood vessels and associated with immediate graft thrombosis and necrosis. This occurred in 25.7% of kidneys perfused with UW solution and transplanted into young, unsensitized recipients. Two molecular species of crystals were defined by mass spectrometry. The particle size ranged from 3 to greater than 100 microm, with a preponderance of particles less than 25 microm in diameter. Such particles are not removed by 40-microm filtration, but can be removed by centrifugation. With extensive use of UW solution for organ storage, the potential for particle-induced damage in small vessels in both experimental and human transplants needs to be carefully scrutinized.
Transplant International 09/2003; 16(8):491-6. · 2.92 Impact Factor
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ABSTRACT: Donor bone marrow infusion has long been used to enhance graft survival or induce tolerance in T cell depleted solid organ allograft recipients. However, the mechanisms through which bone marrow cells affect tolerance remain obscure. We studied the affect of allogeneic bone marrow cells on the activation of allospecific T cells in vitro.
Carboxyfluorescein-diacetate succinimidyl ester-labeled CBA/Ca strain CD8+ splenocytes, bearing T-cell receptor alpha and beta transgenes from the BM3.3 T-cell clone specific for the major histocompatibility complex class I antigen Kb, were placed in culture with irradiated C57BL/6J stimulator cells in the presence of increasing numbers of C57BL/6J or Balb/cJ bone marrow cells for 1 to 3 days. Responder cells were individually analyzed for proliferative history, expression of activation-associated antigens, and intracellular cytokine production.
Allogeneic bone marrow cells exert a dose-dependent inhibitory effect on proliferation of allospecific CD8+ T cells in mixed lymphocyte culture. However, the inhibited T-cell subpopulations show physiologic changes associate with the early stages of T-cell activation, including expression of CD69 and early decrease of surface T-cell expression. Unlike cells not co-cultured with bone marrow, these cells fail to reexpress the T-cell receptor (TCR) by 72 hr of culture. The observed inhibitory effect is also associated with a decrease in the proportion of CD8+ cells expressing interleukin-2 and interferon-gamma.
Collectively, these results suggest that peripheral allospecific T cells undergo the initial stages of activation on exposure to antigen in the presence of bone marrow cells, but the cell cycle is arrested and TCR reexpression is inhibited. We speculate that bone marrow cells effect this inhibition through a receptor-ligand interaction that modulates the transmembrane signal pathway for the TCR.
Transplantation 08/2003; 76(1):237-43. · 4.00 Impact Factor
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ABSTRACT: Peritransplant treatment with anti-CD3 immunotoxin plus deoxyspergualin induces tolerance to kidney allografts in most rhesus macaque recipients. Tolerant recipients maintain normal function for years without evidence of chronic rejection. Indirect alloantigen presentation is implicated in chronic rejection. Accordingly, we determined if anti-CD3 immunotoxin plus deoxyspergualin induced rejection-free tolerance associates with suppression of anti-donor indirect pathway responses. Tolerant recipients exhibited an early decrease in direct anti-donor responses with recovery to baseline levels by 3 years posttransplantation. In contrast, tolerant monkeys were unresponsive to donor antigens presented by the indirect pathway. Recipients that rejected their allografts retained vigorous direct and indirect anti-donor responses. Therefore, following temporary donor-specific hyporesponsiveness, direct responses recover in tolerant recipients >1.5 years after transplantation. However, tolerant recipients tested at 1.9-4 years posttransplant are specifically unresponsive to donor antigens presented by the indirect pathway. Thus, the rejection-free state of tolerant recipients may depend on mechanisms regulating indirect pathway responsiveness.
Cellular Immunology 06/2003; 223(2):103-12. · 1.97 Impact Factor
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ABSTRACT: The immunosuppressive regimens currently in use have been responsible for major improvements in transplanted organ acceptance, but long-term survival can be compromised by drug toxicity and/or chronic immune deficiency. The ultimate goal for transplantation is tolerance, defined as durable, donor-specific allograft acceptance in the absence of long-term immunosuppression. This review describes current experimental strategies for tolerance induction in primate models that are poised for clinical application.
Current opinion in investigational drugs (London, England: 2000) 06/2003; 4(5):530-5. · 3.31 Impact Factor
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ABSTRACT: Pancreatic islet transplantation (PIT) is an attractive alternative for type 1 diabetic patients. PIT is not yet an effective clinical reality due in part to early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. Precise and durable alpha- and beta-cell function is essential for the success of PIT. Therefore, it is important to establish whether PIT can produce adequate long-term metabolic control, especially in the absence of chronic immunosuppressive therapy (CIT). In the present study, the stability of functional alpha- and beta-cell mass and metabolic function was assessed in streptozotocin (STZ)-induced diabetic primates following PIT in the absence of CIT. Diabetes was induced in rhesus macaques with STZ, 140 mg/kg. Hyperglycemia was reversed rapidly by PIT coupled with a 14-day tolerance induction protocol based on F(Ab)2-IT and DSG (n = 7). Two diabetic animals received the tolerance induction protocol without PIT. Acute rejection was presented in three animals at 70, 353 and 353 days post transplant in the tolerance induction protocol, whereas the controls [F(Ab)2-IT or DSG alone] showed early 10-day function but all lost islet function by days 15-70. One recipient [F(Ab)2-IT or DSG] died euglycemic after a surgical procedure on day 187. At 2 years, three animals studied had a normal FIM evaluated by oral glucose tolerance test, mixed meal test, acute insulin response to glucose, glucose disposal rate, and hyperinsulinemic hypoglycemic clamp. PIT in STZ-induced diabetic primates resulted in restoration of normal alpha- and beta-cell function. Operational tolerance induction was achieved with only peritransplant administration of F(Ab)2-IT and DSG sparing the animals from chronic exposure of diabetogenic immunosuppressive drugs. These results offer an exciting new potential for treatment of type 1 diabetes mellitus.
American Journal of Transplantation 03/2003; 3(2):128-38. · 6.39 Impact Factor
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ABSTRACT: Pancreatic islet transplantation (PIT) is an attractive alternative for type 1 diabetic patients. PIT is not yet an effective clinical reality due in part to early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. Precise and durable - and β-cell function is essential for the success of PIT. Therefore, it is important to establish whether PIT can produce adequate long-term metabolic control, especially in the absence of chronic immunosuppressive therapy (CIT). In the present study, the stability of functional - and β-cell mass and metabolic function was assessed in streptozotocin (STZ)-induced diabetic primates following PIT in the absence of CIT. Diabetes was induced in rhesus macaques with STZ, 140 mg/kg. Hyperglycemia was reversed rapidly by PIT coupled with a 14-day tolerance induction protocol based on F(Ab)2-IT and DSG (n = 7). Two diabetic animals received the tolerance induction protocol without PIT. Acute rejection was presented in three animals at 70, 353 and 353 days post transplant in the tolerance induction protocol, whereas the controls [F(Ab)2-IT or DSG alone] showed early 10-day function but all lost islet function by days 15–70. One recipient [F(Ab)2-IT or DSG] died euglycemic after a surgical procedure on day 187. At 2 years, three animals studied had a normal FIM evaluated by oral glucose tolerance test, mixed meal test, acute insulin response to glucose, glucose disposal rate, and hyperinsulinemic hypoglycemic clamp. PIT in STZ-induced diabetic primates resulted in restoration of normal - and β-cell function. Operational tolerance induction was achieved with only peritransplant administration of F(Ab)2-IT and DSG sparing the animals from chronic exposure of diabetogenic immunosuppressive drugs. These results offer an exciting new potential for treatment of type 1 diabetes mellitus.
American Journal of Transplantation 02/2003; 3(2):128 - 138. · 6.39 Impact Factor
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ABSTRACT: The tolerogenic activity of allogeneic bone marrow cells (BMCs) associates with functional inactivation of alloreactive T cells and has been attributed to a veto effect. Studies in mice and rhesus monkeys indicated that the CD8alpha molecule expressed on a subpopulation of allogeneic BMCs is necessary to induce signal transduction within the BMCs to increase veto effector molecules such as transforming growth factor (TGF)-beta1. In vitro activation of alloreactive cytotoxic T-lymphocyte precursor enhances their susceptibility to veto-mediated functional inactivation by specific alloantigen-bearing BMCs. Accordingly, we examined a hypothesis that mature rhesus monkey (Rh) monocyte-derived dendritic cells (MDDCs) modified by gene transfer to over-express active TGF-beta1 might mediate veto activity without the need to express CD8alpha.
Rh MDDCs were modified by recombinant adenovirus (Ad) transduction and characterized by phenotype and functional studies.
Rh MDDC transduction with Ad vectors using conventional methods was remarkably inefficient. However, a single-chain anti-CD40/soluble Coxsackie and adenovirus receptor-fusion protein (G28/sCAR) permitted high-efficiency transduction of Rh MDDCs by retargeting Ad to Rh MDDC CD40. Mature Rh MDDCs that were transduced to overexpress active TGF-beta1 (AdTGF-beta1 Rh MDDC) significantly suppressed alloimmune responses in [ H]thymidine uptake mixed leukocyte reaction assays. We showed by the carboxyfluorescein succinimidyl ester dilution method that allogeneic mature AdTGF-beta1 Rh MDDCs inhibited proliferation of CD4 and CD8 responder T cells. Notably, AdTGF-beta1 Rh MDDC abrogated alloimmune responses induced by control AdGFP Rh MDDC in an antigen-specific manner.
These results suggest that nonhuman primate mature MDDCs can be genetically engineered to function as alloantigen-specific cellular immunosuppressants, an approach that has potential to facilitate induction of allograft tolerance in vivo.
Transplantation 10/2002; 74(5):629-37. · 4.00 Impact Factor
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ABSTRACT: Background. The tolerogenic activity of allogeneic bone marrow cells (BMCs) associates with functional inactivation of alloreactive T cells and has been attributed to a veto effect. Studies in mice and rhesus monkeys indicated that the CD8α molecule expressed on a subpopulation of allogeneic BMCs is necessary to induce signal transduction within the BMCs to increase veto effector molecules such as transforming growth factor (TGF)-β1. In vitro activation of alloreactive cytotoxic T-lymphocyte precursor enhances their susceptibility to veto-mediated functional inactivation by specific alloantigen-bearing BMCs. Accordingly, we examined a hypothesis that mature rhesus monkey (Rh) monocyte-derived dendritic cells (MDDCs) modified by gene transfer to over-express active TGF-β1 might mediate veto activity without the need to express CD8α.
Methods. Rh MDDCs were modified by recombinant adenovirus (Ad) transduction and characterized by phenotype and functional studies.
Results. Rh MDDC transduction with Ad vectors using conventional methods was remarkably inefficient. However, a single-chain anti-CD40/soluble Coxsackie and adenovirus receptor-fusion protein (G28/sCAR) permitted high-efficiency transduction of Rh MDDCs by retargeting Ad to Rh MDDC CD40. Mature Rh MDDCs that were transduced to overexpress active TGF-β1 (AdTGF-β1 Rh MDDC) significantly suppressed alloimmune responses in [3H]thymidine uptake mixed leukocyte reaction assays. We showed by the carboxyfluorescein succinimidyl ester dilution method that allogeneic mature AdTGF-β1 Rh MDDCs inhibited proliferation of CD4+ and CD8+ responder T cells. Notably, AdTGF-β1 Rh MDDC abrogated alloimmune responses induced by control AdGFP Rh MDDC in an antigen-specific manner.
Conclusions. These results suggest that nonhuman primate mature MDDCs can be genetically engineered to function as alloantigen-specific cellular immunosuppressants, an approach that has potential to facilitate induction of allograft tolerance in vivo.
Transplantation 09/2002; 74(5):629-637. · 4.00 Impact Factor
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Transplantation 04/2002; 73(6):839. · 4.00 Impact Factor
