Human interleukin-10 transduced fetal liver stem cells prolong survival of mouse skin and heart allografts

Interleukin (IL)-10 regulates immune responses, acting as a suppressive cytokine by inhibiting the synthesis of Th1 cytokines, such as IL-2 and interferon (IFN)-gamma. It also strongly down-regulates major histocompatibility complex (MHC) class II determinants on antigen presenting cells (APC). On the other hand, long-term tolerance is well correlated with the persistence of a peripheral microchimerism. In this study, we investigated the synergistic effect of human IL-10 (huIL-10) and hematopoietic microchimerism for the induction of long-term tolerance. Irradiated Balb/c mice (H-2d) were used as recipients (fetal liver stem cells [FLSC], skin and heart) and C57BL/6 (H-2b) mice were used as donors of FLSC, skin and heart. Recipients were simultaneously transplanted with the heart, the skin and with huIL-10 gene-transduced FLSC. Microchimerism was checked using fluorescent flow cytometry, huIL10 production using enzyme-linked immunosorbent assay (ELISA), and graft survival was evaluated by daily observation. Significant level of huIL10 (up to 900 pg/mL) was detected for more than 2 weeks in the serum of mice that underwent transplantation. Four weeks after the FLSC injection, microchimerism was identified in the recipient lymphoid organs (spleen, thymus, and bone marrow) by the presence of donor cells (H-2b). Finally, in the group of mice treated with huIL-10 gene-transduced FLSC, skin allografts survived for 18.9 +/- 1.8 days compared with 9.5 and 9.6 days in the groups of mice treated with nontransduced FLSC or huIL-10 alone, respectively. The same pattern for heart allograft survival was observed. HuIL-10 transduction of donor hematopoietic stem cells resulted in production of huIL-10, cell engraftment, and chimerism. Although full tolerance was not obtained, specific and highly significant (P < .001) prolongation of the survival of donor heart allografts with (more than 2-fold compared with nontreated groups) was observed. The infiltration of the transplanted heart and its late rejection demonstrate that stem cells transduced with huIL-10 gene induce "prope" tolerance in this model.     

Prolonged survival of heart allografts transduced with AAV-CTLA4Ig

Organ grafts transduced with gene-encoding immunosuppressive molecules are a less toxic approach to preventing graft rejection. Adenovirus vectors have been widely tested with unsatisfactory results, while adeno-associated virus (AAV) is smaller and elicits a low host humoral response. We constructed an AAV vector containing the mouse CTLA4Ig gene. B10 (H2(b)) cardiac grafts were transduced with AAV-CTLA4Ig by coronary infusion. AAV-LacZ vectors were used as reporters and controls, and the expression of beta-gal was determined by X-gal staining. Thirty percent to 40% of myocytes displayed strongly positive X-gal staining after infusion with AAV-LacZ. Additional infusion with vascular dilator reagents did not improve the transduction rate. Survival of B10 heart allografts transduced with AAV-CTLA4-Ig was significantly prolonged in C3H (H2(k)) recipients. These data demonstrate that AAV vectors can efficiently be transduced into the mouse myocardium by coronary infusion. Graft transduction with AAV-CTLA4Ig may be a novel approach to preventing allograft rejection.     

Prolonged survival of human skin allografts following thermal injury.

We have studied the immune response of six patients admitted to the San Diego Regional Burn Treatment Center for treatment of major thermal injuries. Three of the patients retained skin allografts from unrelated donors for long periods (37, 47, and 67 days) while the remaining three rejected their grafts at 8, 10, and 12 days, respectively. Allograft survival appeared to be directly related to the immunosuppressive activity of patient sera on phytohemagglutinin-induced blastogenesis of normal lymphocytes in vitro. Survival did not appear to be related to patient lymphocyte number or reduced reactivity, nor was graft prolongation accompanied by reduced immunoglobulin production. Our work thus supports the hypothesis that spontaneous immunosuppression may be of importance in the clinical consequences of thermal injuries.     

Prolonged survival of cultured keratinocyte allografts in the nonimmunosuppressed mouse

The effect of in vitro culture on the survival of allografts of epidermal keratinocytes has been examined using a mouse model. Female BALB/c tail epidermal cells were cultured from single cell suspensions to form confluent sheets that were grafted onto male CBA recipients using a transplantation technique that ensured separation of donor graft from host skin. Animals were killed at defined intervals, and the status of the grafts determined histologically. Full thickness skin allografts rejected at 13-15 days. Allografts of epidermis (obtained by enzymatic cleavage at the dermoepidermal junction) rejected at 19-20 days. Cultured keratinocyte allografts were not rejected at least within 70 days and had a histological appearance identical to syngeneic controls. The expression of MHC class I and class II determinants and the leukocyte common (Ly5) surface marker on the donor cells before and after culture were examined using indirect immunofluorescence and monoclonal antibodies. These and other cytochemical studies showed that freshly dissociated tail epidermal cells contained 0.3% of cells that expressed membrane-bound ATP-ase activity, Ia antigens, and the Ly5 surface antigen. These are the Langerhans' cells of the epidermis. In culture, these cells decrease so that by day 8 of culture, no such cells can be detected. At confluence, there are no Ia positive cells, but all cells express MHC class I antigens and stain with an antikeratin antibody. The loss of Ia expression on culture correlates with a decreased stimulation of the class II H-2d-restricted T cell clone D7.1 by cultured keratinocytes compared with freshly dispersed epidermal cells. Furthermore, cultured keratinocytes fail to prime allogeneic mice as determined by the survival of whole thickness skin grafts, whereas freshly dispersed cells induce an accelerated rejection. The results suggest that the survival of cultured keratinocyte allografts is due to the elimination of cells expressing Ia antigens and supports the passenger leukocyte theory of graft rejection.     

Prolonged disease-free survival after orthotopic liver transplantation plus adjuvant chemoirradiation for cholangiocarcinoma

Orthotopic liver transplantation (OLT) alone for unresectable cholangiocarcinoma is often associated with early disease relapse and limited survival. Because of these discouraging results, most programs have abandoned OLT for cholangiocarcinoma. However, a small percentage of patients have achieved prolonged survival after OLT, suggesting that adjuvant approaches could perhaps improve the survival outcome. Based on these concepts, a protocol was developed at the Mayo Clinic using preoperative irradiation and chemotherapy for patients with cholangiocarcinoma. We report our initial results with this pilot experience. Patients with unresectable cholangiocarcinoma above the cystic duct without extrahepatic or extrahepatic metastases were eligible. Patients initially received external-beam irradiation plus bolus fluorouracil (5-FU), followed by brachytherapy with iridium and concomitant protracted venous infusion of 5-FU. 5-FU was then administered continuously through an ambulatory infusion pump until OLT After irradiation, patients underwent an exploratory laparotomy to exclude metastatic disease. To date, 19 patients have been enrolled onto the study and have been treated with irradiation. Eight patients did not go on to OLT because of the presence of metastasis at the time of exploratory laparotomy (n = 6), subsequent development of malignant ascites (n = 1), or death from intrahepatic biliary sepsis (n = 1). Eleven patients completed the protocol with successful OLT Except for 1 patient, all had early-stage disease (stages I and 11) in the explanted liver. All patients who underwent OLT are alive, 3 patients are at risk at 12 months or less, and the remaining 8 patients have a median follow-up of 44 months (range, 17 to 83 months; 7 of 9 patients > 36 months). Only 1 patient developed tumor relapse. OLT in combination with preoperative irradiation and chemother apy is associated with prolonged disease-free and overall survival in highly selected patients with early-stage cholangiocarcinoma.     

Prolonged survival in rat liver transplantation with mouse monoclonal antibody against an inducible costimulator (ICOS)

BACKGROUND: An inducible costimulator (ICOS), a recently identified costimulatory receptor with a close structural homology to CD28 and CTLA4, is expressed on activated T cells. Interaction with its ligand on antigen-presenting cells stimulates T-cell proliferation to produce a different spectrum of cytokine. The inhibition of ICOS-mediated signal transduction by an anti-ICOS antibody is considered to be capable of protecting against graft rejection in organ transplantation. METHODS: An anti-rat ICOS antibody was intravenously administered into recipients of dark Agouti-to-Lewis liver transplantations. The recipient lymphocytes from mesenteric lymph nodes were harvested on day 7 after transplantation for fluorescence-activated cell sorting analysis, and tissue specimens from the grafts were removed for histologic evaluation. Antigen-specific T-cell proliferation responses were assessed in vitro with anti-ICOS antibody. RESULTS: Monotherapy with the antibody significantly prolonged the graft survival time by inhibiting T-cell activation and its proliferation response. The graft-infiltrating cells, both CD4 and CD8 T cells, were not completely reduced even when rats were administered the antibody, whereas the expression of ICOS almost completely disappeared in these cells. CONCLUSIONS: T-cell activation through the ICOS costimulatory pathway plays an important role in graft rejection, and manipulating its pathway is an effective method for modulating transplantation immunity.     

Prolonged survival of alymphatic skin allografts in the rat. A humoral component.

Immunological mechanisms of increased graft survival in "immunologically privileged" sites were defined by comparing host responses against orthotopic and alymphatic skin allografts in rats. The conventional skin grafts reject by day 8; grafts placed on alymphatic skin pedicles heal in normally, but begin by day 16 to 18 to contract inexorably until only a scar remains by day 35 to 40. Lymphocyte-mediated cytotoxicity rose significantly in spleen and draining lymph nodes 8 to 10 days after orthotopic grafting, but was absent as long as 35 days after skin transplantation to alymphatic pedicles. No significant activity in antibody-dependent lymphocyte-mediated cytotoxicity was noted in either recipient group, while complement-dependent cytotoxicity was slightly elevated 8 to 10 days postoperatively in both groups. Passive transfer of serum from recipients of alymphatic skin grafts, taken 8 and 12 days after grafting, prolonged survival of test cardiac allografts significantly, although neither control serum from recipients of orthotopic skin allografts, or serum taken 28 days after alymphatic skin grafting increased test heart survival. We conclude that prolonged survival of skin grafts on alymphatic sites may be based, at least partially, on the development of host humoral factors.     

Prolonged survival of rat hepatic allografts after total-body irradiation of the donors.

The effect of total-body irradiation of the donor on hepatic allograft survival was studied in the rat, with ACI(RT1a) as the donor and LEW(RT1(1)) as the recipient. LEW recipients of ACI liver transplants experienced severe acute rejection, with a mean survival of only 10.2 +/- 0.3 days. The doses of irradiation were 450, 750, and 1000 rads administered 24 hr prior to harvesting or subsequent transplantation. TBI with a dose of 750 rads significantly prolonged the survival of the hepatic allograft to 30.3 +/- 1.7 days, without concomitant immunosuppression. However, neither 450 rads nor 1000 rads of TBI resulted in successful suppression of graft rejection. TBI appeared to have a beneficial effect on hepatic allograft survival and to have no deleterious effect on isograft survival, suggesting a possible modulation of the immunogenicity of the donor organ. Although the cause of this beneficial effect is not clear, TBI with a dose of 750 rads 24 hr prior to organ harvest seems to be optimal to eliminate-antigen presenting cells in the donor organs.