Makiko Kumagai-Braesch

Human embryonic stem cells are immunogenic in allogeneic and xenogeneic settings

Recent studies have suggested that human embryonic stem cells (HESC) are immune-privileged and may thereby circumvent rejection. The expression of immunologically active molecules was studied by DNA microarray analysis and by flow cytometry. HESC were transplanted into immunologically competent mice and traced by fluorescence in-situ hybridization (FISH) and immunohistochemistry. The ability of HESC to directly and indirectly induce immune responses in CD4+ T-cells from naive and transplanted mice was studied. Their ability to induce immune responses of human CD4+ T-cells, when cultured in the presence of dendritic cells (DC) syngeneic to responder T-cells, was also analysed. HESC demonstrated expression of HLA class I and HLA class II genes, but the cell surface expression of HLA class II molecules was low even after incubation with IFNgamma. In wild-type mice, HESC could be demonstrated by FISH until 3 days after transplantation and were surrounded by heavy infiltrates of T-cells and macrophages. HESC induced a similar immune response as human fibroblast cells (HFib) on naive and immunized T-cells, both directly and in the presence of syngeneic DC. A similar response was observed in the allogeneic setting. It is concluded that HESC are immunologically inert and do not inhibit immune responses during direct or indirect antigen presentation, and they were acutely rejected in a xenogeneic setting.

The ADP/ATP Ratio: A Novel Predictive Assay for Quality Assessment of Isolated Pancreatic Islets

The current standard assays for islet product release criteria are unable to predict the outcome after clinical islet transplantation. Therefore, establishment of reliable and rapid assays enabling pre‐transplantation prediction of islet potency is warranted. In the present study, we have evaluated the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) test, the glucose‐stimulated insulin release, the loss of islets during the first 24 h in culture, and the insulin/deoxyribonucleic acid as predictive assays for the ability of isolated porcine islets to cure athymic mice with streptozotocin‐induced diabetes. From the results presented, it is concluded that the measurement of the ADP/ATP ratio was the only test that correlated with transplantation outcome. In summary, we propose that the ADP/ATP assay is worthwhile as applied to human islet transplantation and seek to validate it as a rapid and potent predictor of transplant outcome.

The TheraCyte™ Device Protects against Islet Allograft Rejection in Immunized Hosts:

Clinically, many candidates for islet transplantation are already immunized, which increases their risk of graft rejection. Encapsulation of pancreatic islets using the TheraCyte™ device has been shown to protect against allograft rejection in nonimmunized recipients. However, the capacity of the TheraCyte™ device to prevent rejection in immunized recipients has not yet been studied. In this study, the protective capacity of the TheraCyte™ device was evaluated in an allogeneic rat model. Lewis rats were used as islet donors, and nonimmunized (control) and alloimmunized, diabetic Wistar–Furth (WF) rats were used as recipients. Graft survival was shorter in immunized recipients than in nonimmunized recipients (mean survival, 5.3 ± 2.7 and 9.3 ± 1.6 days, respectively, p < 0.01) when nonencapsulated islets were transplanted under the kidney capsule. When islets were transplanted into the TheraCyte™ device, graft function was maintained during the 6-month study period in both immunized and nonimmunized rats. In oral glucose tolerance tests performed at 1 month after transplantation, both groups had similar insulin and blood glucose levels indicating similar metabolic functions. Volume densities and absolute volumes of tissue inside the devices 6 months after transplantation were also comparable between the two groups, indicating that both groups maintained similar amounts of endocrine tissue. A higher number of IFN-γ-producing CD8+ T-cells were detected in immunized WF rats compared to control WF rats transplanted with encapsulated islets. This suggests that donor-specific alloreactivity in recipient rats was sustained throughout the study period. This study suggests that the TheraCyte™ device protects islet allografts also in immunized recipients. Our results further highlight the potential for using macroencapsulation to avoid immunosuppressive therapy in clinical islet transplantation.

Adult porcine islets produce MCP-1 and recruit human monocytes in vitro

Abstract:  Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood‐mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet‐infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4+ T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell‐mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post‐isolation, expressed mRNA for monocyte chemoattractant protein‐1 (MCP‐1), IL‐1β and TNF‐α. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti‐human MCP‐1 antibody (Ab). Immunohistochemistry revealed MCP‐1 in the cytoplasm of α‐ and β‐cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL‐1β nor TNF‐α were detected by enzyme‐linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL‐1β and TNF‐α were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP‐1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.

Preimplantation of an Immunoprotective Device Can Lower the Curative Dose of Islets to That of Free Islet Transplantation-Studies in a Rodent Model

Islet graft survival inside macroencapsulation devices is suboptimal. We hypothesized that induction of neovascularization by preimplantation of devices would improve the physiological conditions, thereby lowering the number of islets required for cure. Several rat islets were transplanted to TheraCyte immunoprotective devices implanted subcutaneously in diabetic athymic mice. Cure rates in the groups with preimplanted devices were significantly better than in those with freshly implanted devices (375 islets: 8/8 vs. 1/6, P=0.003; 125 islets: 6/6 vs. 0/7, P=0.001). Morphometric evaluations of the 125 islet groups showed higher fractional and absolute volumes of endocrine tissue in the group with preimplanted devices (P<0.001 and P=0.035, respectively). In the following dose titration study, using preimplanted devices, as low as 50 islets cured diabetic mice (100% cure, n=6). We conclude that preimplantation significantly lowers the curative dose of macroencapsulated islets to levels resembling those of free islets transplanted under the renal capsule.

Costimulation Blockade Induces Tolerance to HESC Transplanted to the Testis and Induces Regulatory T‐Cells to HESC Transplanted into the Heart

In order to study the ability of costimulation blockade to induce tolerance to human embryonic stem cells (HESC), severe combined immunodeficient (SCID), and immunocompetent C57BL/6 mice treated with costimulation blockade received intratesticular and intramyocardial HESC transplants. All SCID mice with intratesticular HESC transplants developed teratoma. When SCID mice were transplanted intramyocardially, only two of five mice developed teratoma‐like tumors. C57BL/6 mice transplanted intratesticularly and treated with costimulation blockade all developed teratoma and were surrounded by CD4+CD25+Foxp3+ T‐cells, while isotype control treated recipients rejected their grafts. Most C57BL/6 mice transplanted intramyocardially and treated with costimulation blockade demonstrated lymphocytic infiltrates 1 month after transplantation, whereas one maintained its graft. Isolation of regulatory T‐cells from intramyocardial transplanted recipients treated with costimulation blockade demonstrated specificity toward undifferentiated HESC and down‐regulated naive T‐cell activation toward HESC. These results demonstrate that costimulation blockade is sufficiently robust to induce tolerance to HESC in the immune‐privileged environment of the testis. HESC specific regulatory T‐cells developed to HESC transplanted to the heart and the success of transplantation was similar to that seen in SCID mice.

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells

ABSTRACT: In vitro studies of human NK cell‐mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3–6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA‐induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo‐ and xeno‐stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL‐2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD 16+ or CD56+ lymphoblasts.

Kinetics and character of xenoantibody formation in diabetic patients transplanted with fetal porcine islet cell clusters

Abstract: Porcine fetal islet‐like cell clusters (ICC) were transplanted to 10 renal transplant patients suffering from long‐standing type I diabetes. Since they had renal grafts, they were given immunosuppression with cyclosporine, prednisolone and azathioprine. Eight patients had the ICC injected intraportally and two had the ICC placed under the renal graft capsule. At the time of the xenotransplantation, ATG or 15‐deoxyspergualin was given as an adjunct. Evidence of engraftment, as reflected by excretion of small amounts of porcine C‐peptide into the urine, was observed in four of the patients. In all patients, irrespective of the type of immunosuppression given and whether graft function was established or not, specific xenoantibodies were formed. Titer increases in lymphocytotoxic antibodies occurred after 10 to 14 days with peak reactivity after 30 to 50 days. High titers of ADCC activity measured against porcine lymphoblasts were found in all patients. Titers were maintained at a high level for at least 100 days. Antibody titers were monitored against pig lymphocytes, erythrocytes and against pig membrane antigens solubilized from peripheral blood mononuclear cells, platelets and erythrocytes. Both IgM and IgG antibodies were formed with identical kinetics. There was no increase in the titers of alloantibodies, as evidenced by panel‐reactive lymphocytotoxic antibodies. In all patients an increase in titers of isohemagglutinins was recorded, especially against blood group B antigens. Absorption studies showed that xenoreactivity present in healthy individuals could not be blocked by absorption with human RBC. However, in all transplanted patients, xenoreactivities against pig antigens were inhibited by absorption with human RBC, in particular with B‐type RBC. These data show that the increase in isohemagglutinins was probably due to cross‐reactivity with xenogeneic antigens. Using an ELISA assay, increased antibody titers were also recorded against purified pig MHC class I antigens. However, as is shown in the accompanying paper (next issue), these antibodies were probably not directed against protein determinants on the MHC molecules but rather against glycosylated side chains of these molecules.

Anticarbohydrate Antibody Repertoires in Patients Transplanted with Fetal Pig Islets Revealed by Glycan Arrays

Ten patients with type I diabetes were transplanted with porcine fetal islet‐like cell clusters (ICC) between 1990 and 1993. A significant rise in the anti‐α‐Gal antibody titers was seen posttransplant, but also non‐α‐Gal‐specific antibodies were detected in some patients. We have reanalyzed the carbohydrate specificity of antibodies in the sera from seven of these patients taken before transplantation, 1, 6 and 12 months posttransplantation using a glycan array with 200 structurally defined glycans. The main findings were: (i) prepig ICC transplantation patients had antibodies reactive with terminal α‐GalNAc (e.g. the Forssman antigen, but not the blood group A determinant in blood group A patients), α‐Gal (except blood group B determinants in B individuals), β3‐linked Gal especially Galβ1,3GlcNAc even if terminally sulfated or sialylated, β‐GlcNAc except if β1,3‐linked and oligomannosyl compounds; (ii) the titers of all carbohydrate‐specific antibodies detected before transplantation rose after transplantation; (iii) the kinetics of the antibody responses differed between patients; (iv) in some patients antibodies reacting with Galα1,3Lex and several structures terminated with Neu5Gc appeared after transplantation. In conclusion, anti‐α‐Gal antibodies are the predominant anticarbohydrate antibodies detected after porcine ICC transplantation, with some patients also developing Neu5Gc‐specific antibodies. Their clinical significance needs to be established.

Aberrant expression of α‐Gal on primary human endothelium does not confer susceptibility to NK cell cytotoxicity or increased NK cell adhesion

The contribution of Galα1,3Gal (α‐Gal) to cell‐mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine α1,3 galactosyltransferase (α1,3GalT) to obtain α‐Gal‐expressing primary human aortic endothelial cells (HAEC) at a frequency of 70–90%. These cells were compared to non‐transduced and mock‐transduced HAEC with regard to their susceptibility to human NK cell‐mediated lysis, ability to stimulate IFN‐γ production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 post‐transduction, and at a multiplicity of infection of 2.5, it reached98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF‐α and IFN‐γ stimulation. No differences were detected between non‐α‐Gal‐ and α‐Gal‐expressing HAEC in terms of their susceptibility to NK cell‐mediated lysis, ability to stimulate IFN‐γ production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the α‐Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.