Peripheral regulation of graft-versus-host alloreactivity in mixed chimeric miniature swine.

BACKGROUND: Despite the presence of circulating donor-derived T cells during the induction of mixed chimerism across MHC barriers in miniature swine, severe graft-versus-host disease was avoided in the majority of animals. In this study, we investigated the possible roles of recipient and donor lymphoid populations in the regulation of donor-anti-recipient alloreactivity. METHODS: Mixed chimerism across a full MHC-mismatch barrier was established in miniature swine using a high-dose allogeneic peripheral blood stem cell protocol. Peripheral blood mononuclear cells from mixed chimeric swine were co-cultured with naïve donor-matched responders and naïve recipient-matched stimulators in mixed lymphocyte reactions. RESULTS: Peripheral blood mononuclear cells from mixed chimeras inhibited donor-anti-recipient proliferation. This suppression was radioresistant to 25 Gy. Suppression of donor-anti-recipient alloreactivity was not observed in mixed lymphocyte co-cultures when donor-derived cells were added in the absence of recipient-derived cells. CONCLUSIONS: These results suggest an association between the presence of an active and relatively radioresistant cell population, demonstrable in vitro, and the regulation of graft-versus-host disease across MHC barriers in mixed chimeric miniature swine.

Assessment of transduction rates of porcine bone marrow.

Although drug resistance is commonly used as an indicator of gene transfer in various cellular contexts, the assessment of drug resistance is often imprecise and over-estimated. To measure accurately transduction efficiencies of the retroviral-mediated transfer of genes encoding the neomycine phosphotransferase (Neo(r)) and porcine major histocompatibility (MHC) class II in pig bone marrow cells (BMC), the fraction of targeted progenitors was evaluated by both colony-forming unit granulocytes/macrophages assays (G418r CFU-GM) and by PCR analysis of the transgenes (Tg). Transduced and untransduced BMC were selected at different concentrations of G418 and revealed high individual variability of drug sensitivity. Comparison of the results obtained by estimating the CFU frequency and the PCR assays on drug-resistant colonies demonstrated a marked overestimation of BM transduction rates when determined by G418 resistance alone, because only approximately one-third of individual colonies were positive for both the Neo(r) and the class II Tg. Because this discrepancy is likely to affect the overall assessment of transduction rates using drug resistance markers, our data attest for the need of a combination of molecular assays to determine transduction efficiencies accurately.

Regulated expression of an MHC class II gene from a promoter-inducible retrovirus.

Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.

Dendritic cells enriched from swine thymus co-express CD1, CD2 and major histocompatibility complex class II and actively stimulate alloreactive T lymphocytes.

Initial characterization and partial purification of thymic dendritic cells (DC) from miniature swine were carried out with the ultimate goal of using these cells to induce transplantation tolerance in this preclinical animal model. Immunohistochemical analysis of swine thymic tissue sections has shown DC to be large cells located in the medullary and the cortico-medullary regions as evidenced by the presence of surrounding Hassal bodies. These cells exhibit membrane processes and express the CD1, granulocyte/macrophage (G/M), and major histocompatibility complex (MHC) class II surface antigens, as well as the S100 cytosolic and nuclear markers found in humans to be specific for DC. Dendritic cells were purified from thymi following collagenase treatment, Percoll gradient centrifugation, and adhesion steps to plastic. Cells similar in morphology and phenotype to those described in tissue sections were detected in the lighter density layers of the gradient and represented 0.02% of the starting cell number. Removal of plastic nonadherent cells showed enrichment levels similar to those reported for murine and human DC. Two-colour flow cytometric analysis of purified pig DC identified these cells as MHC class IIhi, CD1+, CD2+, and G/M+. The dendritic nature of these cells was confirmed by their potent ability to stimulate alloreactive T lymphocytes. Modification of porcine thymic DC by transfer of allogeneic MHC genes and reinjection into the DC donor should permit testing of the role of this DC subset in the induction of transplantation tolerance.

Thymic transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients.

Previous studies in our laboratory have demonstrated that the presence of the thymus is essential for rapid and stable tolerance induction in allotransplant models. We now report an attempt to induce tolerance to kidney allografts by transplanting donor thymic grafts simultaneously with the kidney in thymectomized recipients. Recipients were thymectomized 3 wk before receiving an organ and/or tissues from a class I-mismatched donor. Recipients received 1) a kidney allograft alone, 2) a composite allogeneic thymokidney (kidney with vascularized autologous thymic tissue under its capsule), or 3) separate kidney and thymic grafts from the same donor. All recipients received a 12-day course of cyclosporine. Thymectomized animals receiving a kidney allograft alone or receiving separate thymic and kidney grafts had unstable renal function due to severe rejection with the persistence of anti-donor cytotoxic T cell reactivity. In contrast, recipients of composite thymokidney grafts had stable renal function with no evidence of rejection histologically and donor-specific unresponsiveness. By postoperative day 14, the thymic tissue in the thymokidney contained recipient-type dendritic cells. By postoperative day 60, recipient-type class I positive thymocytes appeared in the thymic medulla, indicating thymopoiesis. T cells were both recipient and donor MHC-restricted. These data demonstrate that the presence of vascularized-donor thymic tissue induces rapid and stable tolerance to class I-disparate kidney allografts in thymectomized recipients. To our knowledge, this is the first evidence of functional vascularized thymic grafts permitting transplantation tolerance to be induced in a large animal model.

Naturally developing memory T cell xenoreactivity to swine antigens in human peripheral blood lymphocytes.

Naturally developing xenospecific Abs are well-documented barriers to xenograft transplantation in humans, but whether analogous xenoreactive T cell immunity develops is not known. We used an enzyme-linked immunospot assay to determine the frequency and cytokine profiles of xenoreactive PBLs from a panel of human volunteers. Because naive T cells produce only IL-2 in short term culture, IFN-gamma production by this approach is a measure of a memory immune response. Stimulation of human PBLs or purified T lymphocytes with stimulator cells from inbred swine revealed a high frequency of IFN-gamma producers with 5-fold fewer IL-2 producers. In contrast, lymphocytes obtained from neonatal umbilical cord blood contained swine-specific IL-2 producers but few IFN-gamma producers, which is what one would expect to find with a naive phenotype. Moreover, PBLs from adults with a history of abstention from pork consumption responded to swine cells with a significantly lower frequency of IFN-gamma producers than PBLs from adults with unrestricted diets did, suggesting that pork consumption may result in priming of swine-specific T cell immunity. Our findings provide the first evidence for naturally occurring xenospecific T cell immunity in humans. The detected strength of this memory response suggests that it will present a formidable barrier to transplantation of swine organs.

Thymic transplantation across an MHC class I barrier in swine.

Thymic tissue transplantation has been performed previously in adult mice to induce donor-specific tolerance across allogeneic and xenogeneic barriers. We have now attempted to extend this technique to a large animal preclinical model and describe here our initial studies of allogeneic thymic transplantation in miniature swine. Two miniature swine were thymectomized before thymic tissue transplantation, and two remained euthymic. Donor thymic tissue was harvested from SLA class I-mismatched juvenile pigs and placed into recipient sternocephalicus muscle, kidney capsule, and omentum. A 12-day course of cyclosporin A was started on the day of transplantation. Allogeneic thymic engraftment could only be achieved in euthymic and not in thymectomized miniature swine using this treatment regimen. Both nonthymectomized animals showed good graft development, with evidence of thymopoiesis, as indicated by positive CD1 and host-type SLA class I immunoperoxidase staining of immature graft-infiltrating cells. Both animals also demonstrated donor-specific T cell hyporesponsiveness, as measured by MLR and cell-mediated lympholysis. The thymic grafts continued to develop despite the appearance of high levels of anti-donor specific cytotoxic IgG Abs. Thus, thymic tissue transplanted across an SLA class I barrier can engraft and support host thymopoiesis in euthymic miniature swine. The presence of the host thymus was required for engraftment. These data support the potential of thymic transplantation as part of a regimen to induce donor-specific tolerance to xenogeneic organ grafts.

Treatment of cyclosporine-related adverse effects by conversion to tacrolimus after liver transplantation.

BACKGROUND: Cyclosporine (CsA)-associated side effects include nephrotoxicity, hypertension, neurological disorders, and hyperlipidemia. A considerable share of early and long-term posttransplant morbidity is likely to be drug related. METHODS: In 31 patients with stable graft function, conversion from CsA to tacrolimus was implemented due to nephrotoxicity (n=19), hypertension (n=9), and neurological disorders (n=8). RESULTS: Three months after conversion, a response was evident in 26 patients (84%), whereas 5 patients (16%) were nonresponsive. In 13 of 19 patients (68%) suffering from nephrotoxicity, serum creatinine levels decreased significantly from 2.0+/-0.5 mg/dl to 1.5+/-0.4 mg/dl (P<0.005), whereas in 6 of 19 patients (32%) no improvement was observed. Antihypertensive therapy was reduced in six of nine patients and neurological disorders improved in six of eight patients. When analyzing all patients, average levels of cholesterol and triglycerides were significantly lower after conversion when compared with at the time of conversion (P<0.05) CONCLUSIONS: Conversion to tacrolimus reduced drug-related side effects in the majority of patients, while graft function remained stable. Conversion to tacrolimus may be considered for CsA-related side effects as a potential beneficial approach.

Famciclovir therapy for recurrent hepatitis B virus infection after liver transplantation.

Between November 1993 and June 1995 18 patients received oral famciclovir (3 x 500 mg) for treatment of hepatitis B virus (HBV) reinfection after liver transplantation. Reinfection was defined as the reoccurrence of HBsAg in the serum. In the first 15 patients, famciclovir therapy was initiated after clinical signs of graft hepatitis, whereas the last 3 patients received treatment immediately after HBV-DNA was detected. Famciclovir was well-tolerated in all patients. HBV-DNA values were decreased to undetectable levels in 8 out of 18 patients. Clinical status improved in 7 patients, whereas 5 patients remained unchanged and 6 patients progressed to deteriorating graft function and death. When famciclovir was initiated early after reinfection, a response rate of approximately 66% was observed. Late onset of therapy in patients with fulminant hepatitis generally failed to provide any clinical benefit.