Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dendritic cells activated by lipopolysaccharide after dexamethasone treatment induce donor-specific allograft hyporesponsiveness

BACKGROUND: Immature dendritic cells (imDC) can prolong allograft survival in murine transplantation models. Recent data indicate that semi-mature or alternatively activated DC (aaDC) may be even more tolerogenic. METHODS: We compared the phenotype and regulatory capacity of: a) imDC, cultured in the presence of dexamethasone (DEX), b) mature DC (matDC), activated with LPS, and c) aaDC, activated with LPS after pretreatment with DEX. RESULTS: As compared to imDC, aaDCs displayed a slight upregulation of CD40 while expression levels of MHC-II and CD86 remained low. The production of proinflammatory cytokines, in particular IL-12, by aaDC was much lower than by matDC while both produced similar amounts of the regulatory cytokine IL-10 leading to an increased IL-10/IL-12 ratio for aaDC. After infusion of donor type aaDCs, responder cells isolated from the recipient mice showed donor-specific hyporesponsiveness to restimulation by matDC. Infusion of matDC was immunogenic, while imDC induced partial hyporesponsiveness. Importantly, pretreatment with donor type aaDC (but not imDC) resulted in prolonged survival of a completely MHC-mismatched heart allograft. CONCLUSIONS: Alternatively activated DC are more efficacious than the classical imDC in the regulation of the alloimmune response, which may be related to a distinct cytokine profile characterized by an increased IL-10/IL12 ratio.     

Prolonged survival of donor-specific rat intestinal allograft by administration of bone-marrow-derived immature dendritic cells

It has been reported that intraportal administration of donor antigens induced donor-specific hyporesponsiveness. We studied here the effects of transplantation of BM-derived immature dendritic cells (imDCs) and mature DCs (mDCs) via portal vein on rat small intestinal allograft survival. This study comprised four treatment groups: 1) untreated controls; 2) FK506 alone; 3) intraportal donor-specific BM-derived imDCs transplantation+FK506; 4) mDCs/Tx+FK506. Allograft survival was minimal in control group (5.2+/-0.8 days) and maximal in imDC+FK506 group (28.4+/-3.0 days). The rats in mDC+FK506 group showed systemic inflammatory reaction due to GVHR, and died within 10 days after transplantation. The in vitro MLR reaction using imDCs was also strongly inhibited both in direct and indirect recognition pathways. The impact of imDCs for the specific induction of transplant tolerance may suggest that immunization with donor-specific imDCs has therapeutic potential in organ transplantation.     

Pretransplant portal venous administration of donor antigen and portal venous allograft drainage synergistically prolong rat cardiac allograft survival

The effect of antigen given through the portal vein (PV) before transplantation or continuous drainage of a graft into the PV results in moderate prolongation of allograft survival. This study examines these treatment modalities further. Pretransplant donor antigen as 25 x 10(6) ultraviolet B-irradiated (12,000 joules/m2) donor spleen cells was given 7 days before heart transplantation through either the PV or systemic venous (IV) routes. On day 0, Lewis-to-Buffalo rat cardiac allografts were drained either into the PV or IV. Pretransplant PV donor antigen administration (p less than 0.005), but not by IV administration, significantly prolonged cardiac allograft survival across the strong RT 1 rat histoincompatibility barrier. Similarly PV, but not IV, drainage of the graft prolonged graft survival (p less than 0.005). Pretransplant IV antigen administration had no additive effect on PV drainage graft survival. In contrast, when pretransplant PV donor antigen was combined with PV drainage, 11 of 14 allografts (p less than 0.001) continued to function, free of rejection, after 150 days. Therefore for rat cardiac transplants a clearly synergistic graft-prolonging effect results when pretransplant PV donor antigen is combined with PV drainage of the allograts. These data clarify the potent tolerogenic effects of alloantigen not only administered into the PV but also continuously shed intraportally so that it is first processed by the liver.     

Factors determining prolongation of rat heart allograft survival by perioperative injection of donor spleen cells

Previously, we have shown that a perioperative injection of donor mononuclear cells in combination with cyclosporine treatment on day 2 after transplantation prolongs heart allograft survival in rats. In this study we determined whether the efficacy of this treatment was influenced by the same factors that have been shown to affect the efficacy of preoperative administration of donor cells. The effect of the following factors were investigated: dosage and repetition of the donor cell injection, viability of the donor cells, immunosuppressive drugs other than cyclosporine, and the rat strain combination. We found that there was an optimal dosage of donor cells; dosages of 4 x 10(7) or 1 x 10(8) cells gave the best heart graft survival. Repetition of the donor cell injection was not useful. Reducing viability of the cells by irradiation did not abrogate the prolonged graft survival, whereas killing of the cells did. Methylprednisolone, azathioprine, or cyclophosphamide in combination with the perioperative donor cell injection did not prolong heart graft survival in comparison with treatment with the drug only. The efficacy of this treatment was also influenced by the rat strain combination. In some combinations, this treatment prolonged graft survival, whereas in others an effect was absent or undetectable. Importantly, this treatment never adversely affected graft survival. We conclude that the efficacy of this treatment is influenced by similar factors as found for preoperative treatment with donor cells. A major advantage of this treatment over preoperative blood transfusions is that it avoids sensitization.     

Improved allograft survival in nonidentical living related donor transplants using donor-specific blood transfusions

A total of 78 consecutive HLA nonidentical living related donor transplant recipients with moderate to high stimulating mixed lymphocyte culture indexes underwent a deliberate donor-specific blood transfusion protocol. Of the patients 67 were first and 11 were second allograft recipients. Patients were monitored for immunological responses by cytotoxic B-cold, B-warm and T-warm antibody responses to a random panel of 30 donors, and by serial crossmatches to their donor subset B and T lymphocytes at 5C and 37C before beginning the protocol and after each donor-specific blood transfusion. Patients were followed from 3 months to 2 1/2 years, with allograft survival rates reported by the actuarial method. Survival rates of first allograft recipients were 96.0 plus or minus 2.77, 93.97 plus or minus 3.37, 93.97 plus or minus 3.37 and 90.68 plus or minus 4.50 per cent at 3 and 6 months, and 1 and 2 years, respectively. Of the patients entering the protocol for a primary transplant 20.18 per cent had persistently positive crossmatches. With increasing numbers of previous random blood transfusions a statistically significant sensitization rate was noted. Patient sensitization showed a general pattern of initial development of B-warm lymphocytotoxins resulting in positive B-warm crossmatches, which progressed to T-warm lymphocytotoxins and positive T-warm crossmatches if donor-specific blood transfusions continued. However, on development of B-warm positive crossmatches reversion to a negative crossmatch with successful transplantation was possible upon cessation of transfusions. No patient in the study was rendered nontransplantable due to donor-specific blood transfusions. All 5 patients who were completely disparate suffered amnestic type rejection episodes but following control of the rejection the course mimicked that of mixed lymphocyte culture identical living related donor transplants. Donor-specific blood transfusion is highly successful among first allograft recipients and success in extending the procedure to more disparate relatives is noted.     

Prolongation of rat cardiac allograft survival by donor pretreatment. Impact of dose and timing of drug administration.

We investigated the effect of two variables, the time interval between drug administration and transplant removal and the dosage of the drug, on the prolongation of rat heart allograft survival via donor pretreatment. A maximal or near-maximal survival-prolonging effect was recorded when the drug was given at least 6 hours before removal of the transplant; extension of pretreatment beyond that time had relatively little effect on the outcome of the graft. A maximal survival prolonging effect was obtained with all of the test drugs, cyclophosphamide, methotrexate, hydroxyurea, busulfan and procarbazine, in doses of LD-50. The therapeutical margin was small, however, as doses of 0.5 x LD-50 and 2 X LD-50 were clearly inferior in their effect.     

Extensive prolongation of rat renal allograft survival following donor or nonspecific transfusions and concomitant immunosuppressant

We report here a marked beneficial effect upon rat renal allograft survival transplanted across a strong histocompatibility barrier (BN----LEW) by pretransplant concomitant donor-strain blood transfusion (DST) and CsA treatment. Comparisons between recipient groups treated with pretransplant nonspecific blood (NST) and concomitant cyclosporine (CsA) or azathioprine (Aza) administration were also made. LEW recipients receiving only a BN renal allograft survived for a geometric average time of 8.9 days. Recipients receiving 1 ml of donor blood at weekly intervals, each week for three weeks prior to transplantation, demonstrated a geometric mean survival time (GMST) of 40.5 days. Recipients receiving this same regimen and concurrent CsA cover (5 mg/kg/day) starting 7 days prior to the first transfusion with discontinuation 5 days prior to transplantation showed extensive prolongation (greater than 100 days). Recipients treated with only CsA cover survived for a GMST of 34.4 days. LEW recipients receiving 1 ml of nonspecific blood at weekly intervals (DA, BUF, WKY, respectively) each week for 3 weeks prior to transplantation were prolonged to 27.7 days. Recipients treated with this same regimen while under CsA cover also demonstrated extended prolongation (greater than 100 days). Recipients receiving multiple donor blood transfusions under Aza (2 mg/kg/day) cover demonstrated lesser prolongation (22.8 days). Recipients receiving the multiple nonspecific blood protocol under Aza cover showed similar prolongation (38.6 days). Recipients treated only with Aza did not show prolonged survival (9.3 days). These differences in survival were considered significant among the 9 transplant groups as determined by ANOVA (P less than 0.001). The majority of recipient groups showed relatively poor renal function over their life spans, independent of whether prolongation occurred. Yet, renal function in the NST or particularly the DST groups covered by pretransplant CsA, demonstrated the best renal function in our laboratory over many years of investigations using the BN----LEW combination. In conclusion, there was a dramatic synergistic beneficial effect of prior multiple DST or NST specific to CsA, as opposed to another immunopharmacologic agent, Aza.     

A strategy to achieve donor-specific hyporesponsiveness in cadaver renal allograft recipients by donor haematopoietic stem cell transplantation into the thymus and periphery.

INTRODUCTION: We designed a prospective, randomized clinical trial to evaluate the immune response to thymic and peripheral infusions of donor haematopoietic stem cells (HSCs) to create tolerance in recipients of cadaver renal allografts. METHOD: We divided 24 patients into two equal groups. For group A, 350 ml of unfractionated bone marrow (BM) was aspirated from the anterior iliac crests of donor cadavers. A 2 ml aliquot of concentrated marrow was infused into the thymus of the subject and 100 ml into the BM before surgery; the remaining 250 ml was infused peripherally post-transplantation. The mean nucleated cell count inoculated into the thymus was 3.3 x 10(4) cells/cm(3) and into the periphery 8.6 x 10(7) cells/kg body weight. Group B (controls) underwent renal transplantation directly. Recipients were lymphocytotoxicity cross-match negative in both groups. Group A received low dose prednisolone and cyclosporin; controls also received azathioprine. RESULTS: Over a mean follow-up of 703 days for both groups, group A had significantly better graft function with minimum acute rejection episodes or cytomegalovirus (CMV) infections, a mean serum creatinine (SCr) of 1.23 mg/dl and no graft or patient loss. Group B, with a mean SCr of 2.19 mg/dl had three patients with single acute rejection episodes, two of whom died following uncontrolled rejection-associated infections. The third patient maintained an SCr of 2.5 mg%. Actuarial graft survival was 87.5% in controls at the end of 2 years compared with group A with 100% graft survival at the end of 2 years. CONCLUSION: This novel approach of introducing unfractionated HSCs into the thymus and periphery to create tolerance is safe and efficacious and gives significantly better graft function, minimum acute rejection and no CMV disease with monotherapy.     

The role of the allograft in the induction of donor-specific T cell hyporesponsiveness

BACKGROUND: With adequate immunosuppression the majority of renal allografts are accepted, despite the exceptional vigour of the T cell alloimmune response. Previous work from this laboratory has demonstrated that this is accompanied by significant reductions in the precursor frequencies of anti-donor T cells. We have also shown that parenchymal cells are tolerogenic in vitro. We propose that the reduction in T cell frequencies may be due to the interaction between circulating T cells and potentially tolerogenic graft parenchymal cells. Primed/memory T cells (CD45RO+) are the only subset capable of reaching the allograft and therefore we would predict that T cell hyporesponsiveness would develop predominantly in the CD45RO+ subset due to their trafficking properties. METHODS: Frequencies of IL-2 secreting CD45RA+ and CD45RO+ CD4+ T cells in response to donor and third party stimulator cells were estimated in a series of renal transplant recipients, both before and after transplantation. RESULTS: There were highly significant reductions in the frequencies of donor-specific CD4+CD45RO+ T cells, when adjusted to control for the generalised effects of immunosuppression. There were no significant alterations in the frequencies of donor-specific CD4+CD45RA+ T cells. CONCLUSIONS: In renal transplant recipients, donor-specific CD4+ T cell hyporesponsiveness occurs predominantly in CD4+ CD45RO+ T cells which is the subset capable of trafficking through the graft.     

Clinical significance of in vitro donor-specific hyporesponsiveness in renal allograft recipients as demonstrated by the MLR

A longitudinal study was carried out on 19 recipients of cadaveric renal allografts, monitoring their anti-donor and anti-third party responses in the mixed lymphocyte reaction (MLR) at the time of transplantation and at 3, 6, and 12 months post-transplant. Two patterns of responses were identified: in the first (n=11), patients showed, or later developed, donor-specific hyporesponsivenes, and in the second (n=8), patients had persistent antidonor and anti-third party responses. After 1 year, the serum creatinine, number of episodes of acute rejection and biopsy findings were compared in both groups. In the first group, the mean serum creatinine was 136.4 mmol/l, the total number of acute rejection episodes was three and in nine of the ten available biopsies, there were minimal cellular infiltrates and normal appearance of the glomeruli, tubules and blood vessels. In the second group, the mean serum creatinine was 163 mmol/l, the total number of acute rejection episodes was 12 and in five of the seven biopsies available, evidence of ongoing rejection was obtained. The difference in mean serum creatinine was not statistically significant (P>0.05), but the difference in the numbers of acute rejection episodes was (P>0.05). It is concluded that in some renal allograft recipients, a state of donor-specific hyporesponsiveness develops, and this state may be associated with better graft out-come at 1 year. These data may be useful in selecting patients for reduced immunosuppressive therapy.     

Induction of long-term allograft survival by anti-immunoglobulin, anti-thymocyte serum, and donor antigen.

Heterologous anti-immunoglobulin (AI) is a potent immunosuppressant which prolongs H-2-incompatible skin graft survival and in conjunction with anti-thymocyte serum (ATS) results in greater prolongation of graft life than either antiserum used alone. Combination treatment of recipient mice with AI, ATS, and donor spleen cells produces long-term allograft survival to the lifetime of the host and can result in specific immune unresponsiveness of sufficient strength to permit acceptance of a second set graft.