Judith M. Thomas

A comprehensive definition of the major antibody specificities in polyclonal rabbit antithymocyte globulin

The potent immunosuppressive action of rabbit antithymocyte globulin (RATG) in allotransplant recipients has been recognized for many years. Some of the antibody specificities and immunoregulatory effects of RATG have been described, but a comprehensive definition of RATG components has not been reported previously. In this study, we have identified 23 specificities that are consistent among different clinical RATG batches and represent the major antibody specificities in RATG. These specificities were defined by immunoprecipitation/gel electrophoresis and also antibody blocking/flow cytometry methods. Titration studies performed for semiquantitative analysis of RATG antibodies showed that the antibodies present in highest titer were directed to CD6, CD16, CD18, CD28, CD38, CD40, and CD58 (titer > 1:4000), most of which are not T cell-specific antibodies. In contrast, the RATG antibodies that persisted the longest in vivo in the plasma of rhesus monkeys transplant recipients are antibodies to CD3, CD4, CD8, CD11a, CD40, CD45, CD54, and class I. These antibodies, which are directed at signal transduction and adhesion molecules, were present during the early period of lymphocyte recovery. We suggest that the persistence of these antibodies in vivo is directly related to the prolonged anergy of circulating T cells after RATG treatment and to the unusual potency and complex tapestry of immunological effects in transplantation.

Anoikis, extracellular matrix, and apoptosis factors in isolated cell transplantation

BACKGROUND Isolated cell transplantation (ICT) of pancreatic islets (PI), nerve tissue, hepatocytes, and other cells is an exciting new concept of transplantation. PI transplantation can be successful in reversing diabetes but, unlike whole pancreas, grafts have a unique and unexplained high failure rate, with over 60% loss by 3 to 6 months. We established that PI of rhesus monkeys have a high rate of death within 48 hours of isolation as a result of apoptosis, as measured with the Annexin V assay (Pharmingen, San Diego, Calif). In contrast, PI incompletely separated from the extracellular matrix (ECM) remained viable for prolonged periods in culture and performed superiorly in perifusion assays (insulin secretion of 4.6 +/- 0.8 times basal secretion). METHODS We studied the ability of the anti-AP Bcl-2 molecule, known to block anoikis (a mechanism of AP due to cell-ECM separation), to prevent apoptosis of isolated PI. RESULTS PI transduced with an adenovirus-Bcl-2 gene complex showed a high viability and a low AP rate in culture versus control rhesus PI. CONCLUSION In summary, PI protected from AP by a surrounding ECM mantle or by Adenovirol Vector (AdV) transduction of the Bcl-2 gene showed superior viability without AP in vitro and in vivo evidence of a preserved insulin secretion response to glucose.

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUND Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model. METHODS Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined. RESULTS Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation. CONCLUSIONS The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

Kidney allograft tolerance in primates without chronic immunosuppression--the role of veto cells.

Posttransplant infusion of specific donor bone marrow cells into ATG-treated recipients promotes long survival of allografts in the absence of immunosuppressive drug therapy. DBMC infusion also inhibits antidonor CTL activation in allograft recipients, a trend analogous to the veto phenomenon. The present studies investigated a hypothesis that veto activity in DBMC is involved in the induction of donor-specific unresponsiveness in rhesus monkey kidney transplant recipients given ATG and DBM. Normal monkey BMC were fractionated into subpopulations by depletion with mAbs and immunomagnetic beads. The unfractionated BMC and BMC subsets were tested for veto activity in in vitro MLR-induced CTL and for in vivo tolerance-promoting activity in ATG-treated monkey kidney transplant recipients. In MLR-induced CTL assays, BMC specifically suppressed CTL activity to PBL from the BMC donor. The suppression was mediated by a small population of BMC that expressed a CD2+, CD8+, CD16+, DR-, CD3-, CD38- phenotype. Results of in vivo studies showed a striking correlation with the in vitro results. ATG-treated recipients given either DR- DBMC or DR- CD3- DBMC infusions had significantly prolonged graft survival and a 40-50% incidence of long survivors over 150 days (P less than 0.001 vs. ATG controls). In contrast, in recipients given CD2- DBMC or DR- CD16- DBMC infusions, the tolerance-promoting effect of DBMC was absent, and there were no long-term survivors. The results also showed an association between long survival and suppressed in vivo antidonor CTL activity. Thus acute rejection and in vivo and in vitro antidonor CTL responses were suppressed by a minor subpopulation of DBMC with similar phenotypic markers. We suggest that a veto mechanism may control the induction phase of allograft tolerance in this model, providing a critical period of CTL silence to allow development of host immunoregulatory mechanisms necessary for maintaining graft tolerance.

Further Studies Of Veto Activity In Rhesus Monkey Bone Marrow In Relation To Allograft Tolerance And Chimerism

Infusing the DR-/dim fraction of bone marrow cells (BMC) from an allogeneic kidney donor into rabbit antithymocyte globulin-treated transplant recipients delivers a tolerogenic signal, leading to functional allograft tolerance in rhesus monkeys without additional drug therapy. Our updated results in an expanded series show a median 131-day graft survival of recipients given DR-/dim donor BMC with a 23% 1-year survival (P < 0.00001 vs. rabbit antithymocyte globulin controls). Removing DRbright cells from donor BMC appeared to have a significant effect (P < 0.05). We have further investigated the tolerogenic mechanism within the experimental framework of the veto hypothesis in this preclinical model. In limiting dilution assays, we demonstrated the donor specificity of clonal inactivation of CTL precursors (CTLp) after in vitro or in vivo exposure to DR-/dim donor BMC, confirming specific tolerance. Additionally, in vitro studies confirmed the allogeneic specificity of CTLp inactivation in 3-cell MLR assays; minimal bystander effects were seen on normal CTLp responses to third party stimulator cells, while CTLp responses to the BMC donors cells were abrogated in the same cultures. BMC mediating the veto effect were found to be resistant to L-leucyl-L-leucine methyl ester (Leu-leu-OMe), which excluded BMC-mediated cytotoxicity by NK or lymphokine-activated killer cells, CTL, or activated macrophages. In contrast, veto activity was abolished if the BMC were pretreated with either high dose UV-B light irradiation, mitomycin, or gamma-irradiation, indicating that BMC contained a UV-B-sensitive precursor of the veto effector, and that a proliferative step separated the two. Irradiation of DR-/dim donor BMC or administration of cyclophosphamide after infusion of nonirradiated BMC prevented the tolerogenic effect. Only recipients given nonirradiated DR-/dim donor BMC demonstrated PBL chimerism, which associated with functional deletion of antidonor CTLp and duration of graft survival. The Leu-leu-OMe resistance and the other properties of the allogeneic monkey CD3- CD2+ CD8+ BMC subpopulation that exhibits tolerance-promoting activity in vitro and in vivo lead us to postulate that a donor BMC-derived precursor population, possibly a dendritic cell population, may induce allogeneic unresponsiveness in this model.

Preclinical studies of allograft tolerance in rhesus monkeys: a novel anti-CD3-immunotoxin given peritransplant with donor bone marrow induces operational tolerance to kidney allografts

A major challenge in clinical transplantation today is to design a practical and effective protocol for tolerance induction compatible with cadaver organ transplantation. A preclinical rhesus monkey kidney allograft model using immediate peritransplant anti-CD3 immunotoxin (anti-CD3-IT) and donor bone marrow (DBM) is shown here to induce operational tolerance with prolonged graft survival in the absence of chronic immunosuppressive drugs. Bone marrow harvested from the kidney donor was depleted of mature alloantigen-presenting cells and T cells by removing DR(bright) cells and CD3(bright) cells, respectively. In outbred, major histocompatibility complex-incompatible donor-recipient pairs with high pretransplant mixed lymphocyte response and cytotoxic T lymphocyte precursor activity, four of six allografts survived for periods of 120 days to >1.5 years. Graft acceptance after peritransplant treatment followed robust elimination of both peripheral blood T cells and lymph node T cells. In most recipients given anti-CD3-IT and DBM infusion, anti-donor immunoglobulin G responses were completely inhibited. Microchimerism was observed in all recipients studied, including those not given DBM, but levels of microchimerism did not correlate with graft survival. Anti-CD3-IT induction in combination with modified DBM protocols such as the depletion of mature T cells and DR(bright) antigen-presenting cells may offer new opportunities to improve clinical tolerance protocols beyond those attempted in the clinic to date. Overall, these results with anti-CD3-IT show promise for development of cadaver transplant tolerance induction.

Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic Bcl-2 gene.

Isolated pancreatic islet transplantation is a promising alternative to conventional insulin-dependent diabetes treatment but is not yet a practical clinical therapy. In the first few days after pancreatic islet transplantation, substantial donor pancreatic islet dysfunction and apoptosis commonly occur. Islet apoptosis has been documented after extracellular matrix disruption and exposure to proinflammatory cytokines, and during hypoxia before islet revascularization and rejection. These studies show that targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects these islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, overexpression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation of macaque islets into diabetic severe combined immunodeficency mice. Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass after transplant and reduce the high donor islet requirement currently needed for successful stable reversal of insulin-dependent diabetes.

Reversal of naturally occurring diabetes in primates by unmodified islet xenografts without chronic immunosuppression

BACKGROUND Isolated pancreatic islet transplantation (IPITx) is an attractive alternative for treatment of insulin-dependent diabetes mellitus (IDDM). However, IPITx has been difficult to implement clinically because islets frequently fail to function, have a high incidence of rejection, and are susceptible to autoimmune recurrence and damage by chronic immunosuppressive therapy. Tolerance induction may be a rational approach to resolve several of these limitations. Because anti-CD3 immunotoxin (IT) has been successful in promoting stable primate kidney transplant tolerance in our experience, we considered that tolerance induction with IT might be duplicated in IPITx. MATERIALS AND METHODS Three monkeys with spontaneous IDDM (two Macaca fascicularis and one Ceropithecus aethiops) were treated with xenogeneic pancreatic islets (Macaca mulatta). Intrahepatic islet transplantation was performed at a mean of 13136+/-3860 islet equivalents/kg. Islet xenograft acceptance was accomplished by tolerance induction with two injections of IT given on day 0 at 2 hr before transplantation and on day +1, respectively. IT treatment was supplemented with cyclosporine and steroids administered on days 0 through 4. No additional immunosuppression was given thereafter. Two additional control macaques with spontaneous IDDM received the immunosuppressive protocol without islet infusion. RESULTS All recipients were restored to stable euglycemia, off exogenous insulin, within 1-2 weeks after transplantation. Glucose tolerance, C-peptide, and glycosylated hemoglobin tests confirmed the restoration of normal glucose homeostasis after islet transplantation. All three islet recipients have remained euglycemic at 410, 255, and 100 days of follow-up despite recovery of peripheral T cells to normal levels. In contrast, none of the controls presented changes in the diabetic status 4 and 8 months after treatment. CONCLUSIONS These results represent the first demonstration in nonhuman primates of stable, long-term acceptance of nonencapsulated xenogeneic islets off all immunosuppression, suggesting operational tolerance. The findings have potential implications for islet transplantation as well as improved and more cost-effective therapy for IDDM.

Retraction: Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Long-term incompatible kidney survival in outbred higher primates without chronic immunosuppression.

Transplantation between non-identical humans has been limited by the requirement for chronic immunosuppression (CI). This study demonstrates in a nonhuman primate model that long-term acceptance of incompatible kidney allografts can be achieved without the use of CI. Following incompatible kidney transplantation, rhesus monkey recipients were given a 5-day course of clinical rabbit antithymocyte globulin (RATG). On day 12, unfractionated donor bone marrow (BM) was infused intravenously. Recipients were monitored for T-cell levels and T-cell subset levels with monoclonal antibodies and for responses in one way MLR. Graft survival in untreated control animals was 9.2 +/- 2.8 days. In six animals given RATG only, all died of rejection at a mean 35.8 +/- 5.7 days. Of five animals given RATG and donor BM (mean 2.5 RhLA mismatches, mean MLC 12.7), four are alive at 150 days, 248 days, 342 days, and 401 days (median 248 days). The ATG-BM infused group showed a prolonged imbalance of their OKT4/OKT8 cell ratio and cellular suppression of MLR responsiveness. The long-term survival obtained in these outbred primates is apparently due to a synergistic immunoregulatory effect induced by the RATG and donor BM. The model described is apparently the first report of long-term survival of outbred higher primates without CI and may represent a technique for producing tolerance without CI in the human.