Donor-specific Hyporeactivity Research Articles

Chimerism and Tolerance Without GVHD in Mismatched Recipients of Combined Hematopoietic Stem Cell/Kidney Transplants: Donor-Specific Hyporeactivity Is Not a Reliable Biomarker for Tolerance.

We recently reported low-intensity conditioning combined with facilitating cell (FC)-enriched hematopoietic stem cell transplantation (FCRx) can safely achieve high levels of durable chimerism in unrelated and related mismatched kidney transplant (KTx) recipients. This is associated with stable renal function, absence of GVHD, and successful withdrawal of immunosuppression (IS). We herein present interim follow-up on 19 subjects and present data to demonstrate that donor specific hypoactivity does not correlate with tolerance to the renal allograft if durable chimerism is not present. HLA-mismatched related (n = 12) and unrelated (n = 7) living donor KTx recipients were conditioned with fludarabine (days -5,-4,-3), cyclophosphamide (50 mg/kg day -3, +3), 200 cGy TBI, (day -1), KTx (day 0), followed by infusion of cryopreserved G-CSF mobilized FCRx (day +1). Multilineage chimerism was assessed by STR analysis. Mixed lymphocyte reaction (MLR) assays were performed to assess donor-specific hyporeactivity. 18 of 19 patients demonstrated peripheral blood macrochimerism at 1 month post-transplant (6% - 100%). The one patient who failed to engraft was highly sensitized (Class I PRA > 40%). No subjects developed GVHD or “engraftment syndrome.” Durable whole blood and T cell chimerism was achieved in 12 subjects. All patients demonstrated in vitro donor-specific hyporesponsiveness (DSH) by MLR +/- CML post-transplant regardless of chimerism status. However, DSH in patients who lost chimerism was not predictive of successful IS weaning, as 3 transiently chimeric patients with DSH had subclinical Banff 1A rejection on protocol biopsy while on IS monotherapy. Recurrence of underlying autoimmune disease has only occurred in subjects who were transiently chimeric. Protocol biopsies in chimeric subjects have exhibited normal histology for up to 3 years. 12 chimeric subjects are completely off IS from 3-42 months. The presence of durable, high level whole blood and T cell chimerism in combined kidney + FCRx recipients is a robust biomarker of tolerance. DSH without donor chimerism does not predict successful IS withdrawal or tolerance. DISCLOSURES:Gallon, L.: Grant/Research Support, NOvartis. Tollerud, D.: Other, regenerex LLC, COO. Elliot, M.: Employee, REGENEREX LLC. Ildstad, S.: Other, regenerex LLC, CEO.

Chimerism and Tolerance Without Gvhd In Mismatched Recipients Of Combined Hematopoietic Stem Cell/Kidney Transplants: Donor-Specific Hyporeactivity Is Not a Reliable Biomarker For Tolerance

We recently reported that low-intensity conditioning combined with facilitating cell (FC)-enriched hematopoietic stem cell transplantation (FCRx) can safely achieve high levels of durable chimerism in unrelated and related mismatched kidney transplant (KTx) recipients. This is associated with stable renal function, complete absence of GHVD, and successful withdrawal of immunosuppression (IS). We herein present interim follow-up of now 19 subjects and present data to demonstrate that donor specific hypoactivity does not correlate with tolerance to the renal allograft if chimerism is not present.HLA-mismatched related (n = 12) and unrelated (n = 8) living donor KTx recipients were conditioned with fludarabine (days -5,-4,-3), cyclophosphamide (50 mg/kg day -3, +3), 200 cGy TBI, (day -1), KTx (day 0), followed by infusion of cryopreserved G-CSF mobilized FCRx (day +1). Multilineage chimerism was assessed prospectively by STR analysis. Mixed lymphocyte reaction (MLR) and cell mediated lympholysis (CML) assays were performed to assess donor-specific hyporeactivity.Subjects ranged in age from 18-65 years. 18 of 19 patients demonstrated peripheral blood macrochimerism at 1 month post-transplant (6% - 100%). The one patient who failed to engraft was highly sensitized (Class I PRA > 40%). No subjects developed acute or chronic GVHD or “engraftment syndrome.” Chimerism was transient in 4 subjects, each of which received either a suboptimal cell dose or had PRA > 20%. Durable whole blood and T cell chimerism was achieved in 14 subjects. All patients demonstrated in vitro donor-specific hyporesponsiveness (DSH) by MLR +/- CML post-transplant regardless of chimerism status. However, DSH in patients who lost chimerism was not predictive of successful IS weaning, as 3 of 4 transiently chimeric patients with DSH had subclinical Banff 1A rejection on protocol biopsy and while on tacrolimus monotherapy. Recurrence of underlying autoimmune disease has only occurred in subjects who were transiently chimeric. Protocol biopsies in chimeric subjects have exhibited normal histology for up to 3 years. Nine chimeric subjects are completely off IS from 1-34 months. The remainder are in the process of weaning.The presence of durable, high level whole blood and T cell chimerism in combined kidney + FCRx recipients is a robust biomarker of tolerance. DSH alone without donor chimerism in the first post-transplant year does not predict successful IS withdrawal or tolerance. Disclosures:Tollerud:Regenerex, LLC: Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.

Tolerance Induction in HLA Disparate Living Donor Kidney Transplantation by Donor Stem Cell Infusion

We recently reported that durable chimerism can be safely established in mismatched kidney recipients through nonmyeloablative conditioning followed by infusion of a facilitating cell (FC)-based hematopoietic stem cell transplantation termed FCRx. Here we provide intermediate-term follow-up on this phase II trial. Fifteen human leukocyte antigen-mismatched living donor renal transplant recipients underwent low-intensity conditioning (fludarabine, cyclophosphamide, 200 cGy TBI), received a living donor kidney transplant on day 0, then infusion of cryopreserved FCRx on day +1. Maintenance immunosuppression, consisting of tacrolimus and mycophenolate, was weaned over 1 year. All but one patient demonstrated peripheral blood macrochimerism after transplantation. Engraftment failure occurred in a highly sensitized (panel reactive antibody [PRA] of 52%) recipient. Chimerism was lost in three patients at 2, 3, and 6 months after transplantation. Two of these subjects had received either a reduced cell dose or incomplete conditioning; the other two had PRA greater than 20%. All demonstrated donor-specific hyporesponsiveness and were weaned from full-dose immunosuppression. Complete immunosuppression withdrawal at 1 year after transplantation was successful in all patients with durable chimerism. There has been no graft-versus-host disease or engraftment syndrome. Renal transplantation loss occurred in one patient who developed sepsis following an atypical viral infection. Two subjects with only transient chimerism demonstrated subclinical rejection on protocol biopsy despite donor-specific hyporesponsiveness. Low-intensity conditioning plus FCRx safely achieved durable chimerism in mismatched allograft recipients. Sensitization represents an obstacle to successful induction of chimerism. Sustained T-cell chimerism is a more robust biomarker of tolerance than donor-specific hyporeactivity.

Role of Donor-Specific Regulatory T Cells in Long-Term Acceptance of Rat Hind Limb Allograft

BackgroundVascularized bone marrow transplantation (VBMT) is widely accepted as an efficient means of establishing chimerism and inducing tolerance. However, the mechanism underlying is poorly understood. Recently, regulatory T cells (Tregs) have been shown to play an important role in regulating immune responses to allogeneic antigens. In this study, we explored the role of Tregs in the induction of tolerance in an allogeneic hind limb transplantation model.Methodology/Principal FindingsForty-eight Lewis rats were divided into 6 groups. They received isografts and allografts from Brown-Norway hind limbs. Recipients in groups 1 and 2 received isografts and those in the other groups received allografts. The bone components of donor limbs were kept intact in groups 1, 3, and 5 but removed before transplantation into groups 2, 4, and 6. Tapered cyclosporin A (CsA) was administered to recipients in groups 5 and 6 after transplantation. During the 100-day observation period, all isografts survived, but the allografts in groups 3 and 4 were rejected within 8 to 12 days. CsA-treated intact allografts survived rejection-free for more than 100 days, and CsA-treated allografts lacking bone elements were rejected within 2 months. Stable peripheral chimerism and myeloid chimerism were observed in group 5. Declining peripheral chimerism and a lack of myeloid chimerism were observed in group 6. Donor-specific Tregs were exclusively detected in both peripheral blood and in the spleens of long-term recipient rats in group 5, with an increased FoxP3 mRNA expression in the allografts. This was further demonstrated to be responsible for donor-specific hyporeactivity by in vitro one-way mixed lymphocyte reaction (MLR).Conclusion/SignificanceBone components in the allogeneic hind limbs can induce myeloid chimerism and donor-specific Tregs may be essential to tolerance induction. The bone-removal hind limb model may be a suitable counterpart to the induction of tolerance in the study of limb transplantation.

Improved long‐term graft function in kidney transplant recipients with donor antigen‐specific hyporeactivity

We investigated the development of donor antigen-specific hyporeactivity by using donor cells as stimulator cells in the MLC and comparing the pre- and post-transplant responses of peripheral blood mononuclear cells. Twenty-two haploidentical pediatric living-relative donor recipients treated with daclizumab, methylprednisone, mofetil mycophenolate and calcineurin inhibitors were tested for study. Of these, 50% of the recipients developed in vitro donor antigen-specific hyporeactivity. The recipients who did so have higher creatinine clearance levels at 12, 24 and 36 months post-transplant (104, 92 and 81 mL/min/1.73 m(2), respectively) than those who remained responsive to donor antigens (77, 74 and 70 mL/min/1.73 m(2)) (p < 0.05). Acute rejection episodes were not observed; however, no recipients with donor-specific hyporeactivity have been diagnosed with CAN, unlike three recipients who remained responsive to donor antigens (0% vs. 27.3%, p = 0.06). Differences in accumulative doses of methylprednisone and mofetil mycophenolate were observed between hyporeactivity- and response-patients to donor antigens at the three years end-point (1.9 +/- 0.8 g/m(2) vs. 4.2 +/- 0.5 g/m(2), and 277 +/- 89 g/m(2) vs. 672 +/- 16.0 g/m(2); p < 0.01 and <0.02, respectively). We conclude that the development of donor antigen-specific hyporeactivity correlate with improved graft function and may permit lower immunosuppression.

Comparative analysis of the fate of donor dendritic cells and B cells and their influence on alloreactive T cell responses under tacrolimus immunosuppression

We have shown that tacrolimus (TAC)-induced liver allograft acceptance is associated with migration and persistence of donor B cells and dendritic cells (DC). To clarify whether these MHC class II + leukocytes have favorable roles in inducing tolerance, we analyzed recipient T cell reactions after allogeneic B or DC infusion. LEW rat B cells localized exclusively in BN host B cell follicles without any direct contact with host T cells. While few donor DC migrated to T cell areas and marginal zones, they were captured by host APC, suggesting that allogeneic MHC class II + cells may induce immune reactions via the indirect pathway. Although DC-infused non-immunosuppressed recipients showed enhanced ex vivo anti-donor responses, persistent in vitro donor-specific hyporeactivity was seen equally with donor DC or B cell infusion under TAC. The results indicate that donor MHC class II + APC are capable of regulating recipient immune reactions under TAC. Possible involvement of the indirect pathway of allorecognition is discussed.

IMMUNOMONITORING IN ALLOGRAFT RECIPIENTS TREATED WITH A TOLERANCE-ENHANCING IMMUNOSUPPRESSIVE REGIMEN

O147* Aims: Recipients of whole organ allografts were treated in accordance with two therapeutic principles of (1) recipient pretreatment with Thymoglobulin (THY, 3-6 mg/kg) or Campath (CAM, 30 mg) before graft reperfusion and (2) minimum posttransplant immunosuppression consistent with graft survival and function using tacrolimus monotherapy (target trough level of 10 ng/ml). Other agents (e.g. steroid, rapamycin) were added only to treat biopsy-proven rejection. This report described changes in immunological and hematological parameters with a minimum followup of >12 months. Methods: Blood samples were obtained at 1, 3, 6, and 12 months after transplantation for four-color flow cytometry of leukocyte lineages, in vitro cell proliferation assays, and ELISPOT using cadaveric donor splenocytes or live donor PBMC as alloantigens. Results: Pretreatment with THY or CAM resulted in significant posttransplant lymphocyte depletion, which slowly recovered over the next 6-12M. CAM had stronger and prolonged impacts on lymphocyte depletion. Recovery of CD4 population was slower than CD8, resulting in remarkably low CD4/CD8 ratios (<0.8) at 6-12M with a higher incidence in the CAM group. Likewise, DC1 and DC2 populations considerably decreased after transplantation. DC1 gradually recovered and became nearly normal by 12M (49% vs. 57% in normal volunteers); however, recovery of DC2 was remarkably slow, particularly in the CAM group, and was 5.5 ± 5.0% at 12M compared to 15.0 ± 9.0% in the normal. CAM, but not THY, also effectively depleted B cells, and significantly low B cell counts persisted for nearly 12M. At 1 year, 48 of 155 (31%) functioning kidney recipients were on daily immunosuppression with single or multiple drugs. The remaining 107 (69%) were on tacrolimus, cyclosporine, or rapamycin monotherapy, with doses spaced from one every other day to once weekly. Of 95 liver recipients, 26 (27%) are on daily therapy with single or multiple agents, while 69 (73%) are on spaced monotherapy. Detailed in vitro studies (MLR, CML, limiting dilution, ELISPOT) were performed in 20 kidney and 16 liver recipients with >1 year follow-up. Kidney recipients on continuous multiple drugs showed global suppression to mitogens and alloantigens. In contrast, two-thirds of recipients on spaced weaning showed donor-specific hyporeactivity with vigorous responses to mitogens and 3rd party alloantigens. The remaining recipients on spaced monotherapy had intact anti-donor reactivity by MLR; but, CML responses were low with CTLp frequencies of less than 1:90,000-300,000. Patients (n=2) with modest killing (10-16%) with CTLp of 1:60,000-150,000 showed a low frequency of γ-IFN positive cells in ELISPOT. On the contrary, although the majority of liver recipients were in more advanced stages of weaning, correlation between immunosuppression and in vitro results was less clear probably due to the complicated immunological status in this population (e.g. hepatitis). Approximately 50% showed donor-specific hyporeactivity or total suppression in MLR and CML. The remaining half showed reactivity to mitogens and alloantigens (donor and 3rd party) in MLR with a significantly higher background. Donor killing in CML was 8-43% with low CTLp frequencies of 1:<200,000. Conclusions: Donor-specific immune modulation was observed in a subset of allograft recipients pretreated with THY or CAM and given minimal posttransplant immunosuppression. In contrast, patients on continuous multiple drugs showed overall suppression.

Analysis of alloreactivity and intragraft cytokine profiles in living donor liver transplant recipients with graft acceptance

Although some previous studies have indicated the possibility of immunosuppression withdrawal in clinical liver transplantation, the mechanism of graft acceptance is not clear. The aim of this study is to elucidate the alloreactivity against the donor and intragraft cytokine profiles in living donor liver transplant (LDLT) recipients with graft acceptance. In October 1999, we had 23 patients who survived without immunosuppression after LDLT with a median drug-free period of 25 months (range: 3–69 months). They consisted of six patients who were electively weaned by an elective weaning protocol and 17 either forcibly or accidentally weaned patients due to various causes but mainly due to infection. We evaluated the alloreactivity against the donor in these patients by a mixed lymphocyte reaction and intragraft cytokine profiles by real-time reverse transcriptase-polymerase chain reaction. The development of donor-specific hyporeactivity was observed in the patients with graft acceptance. The cytokine pattern in the supernatant of the culture medium revealed a down regulation of T helper (Th) 1 cytokine INF γ against the donor while no significant difference was seen in Th2 cytokine IL-10. Regarding the intragraft cytokine profiles, we could find no amplification of Th1 cytokines (IL-2, INF γ) and IL-4 while some of the patients revealed a gene expression of IL-10 with no significant difference from that of the normal, untransplanted liver specimen. In addition, no difference was observed in any other cytokines (IL-1β, IL-8, IL-15, TNFα) compared with those of the normal controls. We propose that the down regulation of Th1 cytokine is one possible mechanism of graft acceptance in LDLT recipients.

Graft hyporeactivity induced by immature donor-derived dendritic cells

Immature dendritic cells (DCs) are deficient in surface co-stimulatory molecules and have been shown to exhibit a ‘tolerogenic’ potential. We investigated the allostimulatory activity of immature DCs in one-way mixed leukocyte reactions and their capacity to inhibit anti-donor cytolytic activity in the sponge matrix allograft model. Immature DCs (CD80 and CD86 deficient) were derived from bone marrow cells propagated in GM-CSF and TGF-β1. Mature DCs (CD80 + and CD86 +) were derived from bone marrow cells propagated in GM-CSF and IL-4. Either 2×10 6 DBA/2J (DBA, H-2 d) immature DCs or 2×10 6 mature DCs were injected intravenously into C57BL/6J (B6, H-2 b) mice 7 days prior to sponge matrix allograft implantation. On day 12, the sponge was harvested and the graft-infiltrating cells were tested in vitro for cytotoxic T lymphocyte (CTL) activity. Immature dendritic cell (DC) infused significantly and markedly inhibited intra-graft CTL activity compared to mature DCs and syngeneic bone marrow control cells. The administration of immature DCs directly into the sponge allograft failed to induce hyporeactivity. Thus, the only systemic infusion of immature donor DCs was able to recapitulate the donor-specific transfusion effect, and the capacity of donor bone marrow cells to induce donor-specific hyporeactivity in the sponge allograft model.

Long-term evaluation of proliferative donor antigen-specific reactivity in cadaveric kidney transplant recipients.

Development of donor-specific proliferative hyporeactivity has been evaluated in many studies for its usefulness in identifying transplant recipients at low risk of immunological complications. These studies often result in controversial conclusions, however. The authors claim that the discrepancy in the predictive value of mixed lymphocyte culture- (MLC) reactivity might partly be due to differences in presentation and interpretation of results. The purpose of this study is to investigate the usefulness of a normalized evaluation of antigen-specific donor-reactivity in a small number of kidney transplant recipients. This could then serve as a basis for an extended clinical study. Ten cadaveric kidney recipients were tested for proliferative reactivity to donor- and third-party antigens up to 20 months posttransplantation. Expressing donor-specific reactivity as a relation between the percentage of pretransplant responses towards donor splenocytes and the percentage of pretransplant responses towards third-party donor cells should minimize influences of e. g. uremia, current immunosuppression or infections on the evaluation of specific reactivity and thus should allow an evaluation of the donor-specificity of T-cell alloresponses independently of fluctuations in global responsiveness. Four of ten recipients acquired a state of donor-specific hyporeactivity ( < 75 % relative specific reactivity) at 20 months post-transplantation (61 +/- 12%, mean +/- SD). Six patients were classified non-hyporeactive (98 +/- 10% mean relative specific reactivity). Relative specific reactivity did not correlate with the levels of general reactivity. Three of the four hyporeactive and four of the six non-hyporeactive patients developed acute rejection. Stable graft function at 20 months posttransplantation (serum creatinine < or = 2 mg/dl) was not closely related to the reactivity status, as five of eight patients with well-functioning grafts did not develop relative specific hyporeactivity. One recipient with chronic rejection was classified hyporeactive. One non-hyporeactive patient lost his graft due to non-immunological causes. Our data suggest that post-transplant relative specific reactivity does not predict acute rejection. Downregulation of donor-specific reactivity might not be a prerequisite for stable graft function but could help identifying recipients who require less immunosuppression. This, however, remains to be established in a prospective immunosuppression-weaning study.

Effects of donor bone marrow infusion in clinical lung transplantation

Background. We have demonstrated that donor cell chimerism is associated with a lower incidence of obliterative bronchiolitis (OB) in lung recipients, and that donor chimerism is augmented by the infusion of donor bone marrow (BM). We herein report the intermediate results of a trial combining the infusion of donor BM and lung transplantation.Methods. Clinical and in vitro data of 26 lung recipients receiving concurrent infusion of donor bone marrow (3.0 to 6.0 × 108 cells/kg) were compared with those of 13 patients receiving lung transplant alone.Results. Patient survival and freedom from acute rejection were similar between groups. Of the patients whose graft survived greater than 4 months, 5% (1 of 22) of BM and 33% (4 of 12) of control patients, developed histologic evidence of OB (p = 0.04). A higher proportion (but not statistically significant) of BM recipients (7 of 10, 70%) exhibited donor-specific hyporeactivity by mixed lymphocyte reaction assays as compared with the controls (2 of 7, 28%).Conclusions. Infusion of donor BM at the time of lung transplantation is safe, and is associated with recipients’ immune modulation and a lower rate of obliterative bronchiolitis.

Infusion of donor leukocytes to induce tolerance in organ allograft recipients.

To further enhance chimerism, 229 primary allograft recipients have received perioperative intravenous infusion of a single dose of 3 to 6 X 10(8) unmodified donor bone marrow (BM) cells/kg body weight. In addition, 42 patients have been accrued in a concurrent protocol involving multiple (up to three) sequential perioperative infusions of 2 x 10(8) BM cells/kg/day from day 0-2 posttransplantation (PTx). Organ recipients (n = 133) for whom BM was not available were monitored as controls. The infusion of BM was safe and except for 50 (18%), all study patients have optimal graft function. Of the control patients, allografts in 30 (23%) have been lost during the course of follow-up. The cumulative risk of acute cellular rejection (ACR) was statistically lower in the study patients compared with that of controls. It is interesting that, 62% of BM-augmented heart recipients were free of ACR (Grade > or = 3A) in the first 6 months PTx compared to controls. The incidence of obliterative bronchiolitis was also statistically lower in study lung recipients (3.8%) compared with the contemporaneously acquired controls (31%). The levels of donor cell chimerism were at least a log higher in the peripheral blood of majority of the study patients compared with that of controls. The incidence of donor-specific hyporeactivity, as determined by one-way mixed leukocyte reaction, was also higher in those BM-augmented liver, kidney, and lung recipients that could be evaluated compared to controls.

CHARACTERIZATION OF DONOR-SPECIFIC HYPOREACTIVITY AFTER DONOR BONE MARROW ADMINISTRATION

238 We previously showed that a single infusion of 107 donor specific bone marrow (DSBM) 7 days before transplantation in the sponge allograft induces graft hyporeactivity (Transplantation 1996;62:1601). The present study was designed to a) to determine whether antigen (Ag) dose or source influenced the hyporeactivity generated after DSBM and b) to determine whether potential regulatory cytokines are generated locally in the graft after DSBM. Methods: a) Dose response and Ag source: On day −7 before grafting, 107, 106, 105 to 104 allogeneic (DBA/2J, H-2d) BM cells, or 107 allogeneic thymocytes or splenocytes were given by tail vein injection to C57BL/6J (B6,H-2b) recipients (n=5 per group). Control animals received 107 syngeneic (B6) BM cells. Seven days after BM administration (day 0), sponges containing 107 allogeneic (DBA) splenocytes were implanted into each group. On day 12 postgrafting, graft-infiltrating cells (GIC) were recovered and assayed for in vitro allospecific cytotoxicity against allogeneic (P815) tumor targets (Table 1). No lytic activity was detected against syngeneic (EL4) targets (not shown). b) Regulatory suppressor cytokines: Nitric oxide (NO) measured as its stable endproduct nitrite (NO2−) and IL-10 production by day 12 GIC from animals receiving syngeneic BM (control) or allogeneic BM (hyporeactive) were measured 72 hrs. after in vitro restimulation with allogeneic splenocytes. Allospecific lytic activity of the same GIC was also determined on the day of GIC recovery (Table 2). Data are mean ± SD of 3 experiments.Table 1Conclusions: a) Donor-specific hyporeactivity correlates with number of transfused donor cells, and is induced by not only BM but also splenocytes and thymocytes b) GIC hyporeactivity induced by DSBM is accompanied by increased production of IL-10 but decreased NO compared with normal GIC, suggesting decreased local immune activation in the graft after DSBM.

Donor-specific hyporeactivity after liver transplantation: prominent decreases in donor-specific cytotoxic T lymphocyte precursor frequencies independent of changes in helper T lymphocyte precursor frequencies or suppressor cell activity.

The development of immunological donor-specific hyporeactivity may account for the low incidence of chronic rejection after clinical liver transplantation. We investigated whether hyporeactivity commonly develops after liver transplantation by analyzing precursor frequencies of donor-reactive cytotoxic (CTLp) and helper (HTLp) T lymphocytes and mixed lymphocyte culture (MLC) reactivity in liver allograft recipients. We further studied whether CTLp hyporeactivity correlated with changes in donor-specific HTLp frequencies or suppressor cell activity. CTLp and HTLp frequencies and MLC reactivity against donor and third-party spleen cells were determined in pre- and posttransplantation peripheral blood samples from 18 recipients with good graft function 2 years after transplantation. By mixing posttransplantation samples (with "putative" suppressor cell activity) with pretransplantation samples (in which normal CTL activity with no suppressor cell activity is expected), the presence of suppressor cell activity in peripheral blood was analyzed. Two years after transplantation, all but one (94%) of the recipients had developed CTLp hyporeactivity as evidenced by reduced donor-specific CTLp frequencies. The development of hyporeactivity was not specific for any particular underlying disease. The occurrence of HTL hyporeactivity, however, was less frequent: 38% and 20% of recipients were HTLp and MLC hyporeactive, respectively. Decreases in CTLp frequencies did not correlate with decreased donor-specific HTL function or suppressor cell activity in peripheral blood samples. Donor-specific CTLp hyporeactivity can develop in the majority of liver allograft recipients, irrespective of underlying disease. Donor-specific HTL hyporeactivity, however, occurs infrequently. A reduction in donor-specific CTLp frequencies was found to be independent of changes in donor-specific HTLp or suppressor cell activity, suggesting that other mechanisms (e.g., clonal deletion) are operative in the reduction of donor-specific CTLp after liver transplantation.

Mutual tolerance after liver and not after heart transplantation? Evaluation of patient-anti-donor and donor-anti-patient responses by mixed lymphocyte culture

The ultimate goal in organ transplantation is the induction of donor-specific transplantation tolerance. The fact that in some patients it is possible to withdraw immunosuppressive therapy completely, suggests that immunological adaptation or donor-specific nonresponsiveness can occur following transplantation. In earlier studies we have shown that after blood transfusion, the mixed lymphocyte reactivity of the donor against patient peripheral blood mononuclear lymphocytes taken after blood transfusion gradually decreased with time. This may reflect the induction of an immunoregulatory mechanism, which protects the recipient against an immune reaction of the donor, enhancing a state of mixed chimerism. A similar phenomenon might also play a role in the immunological mechanism leading to transplantation tolerance. Therefore, we studied responses in patients with a well-functioning liver and heart transplant using a primed lymphocyte test (PLT) and a mixed lymphocyte culture (MLC). Two years after liver transplantation the PLT and MLC responses of patient against donor were decreased significantly compared to the situation before transplantation. The response of donor against patient was also lower two years after transplantation. The decreased responses were donor-specific since responses to third-party cells generally remained unchanged. In heart transplant recipients we could not detect a donor-specific downregulation. The reversed response, of donor against patient, was not different from responses of third-party against patient cells. Therefore, we conclude that donor-specific nonresponsiveness is not induced in patients with well-functioning heart transplant. In contrast, after a successful liver transplantation the response of patient against donor is decreased, as is the reversed response. It may be valuable to test whether in liver transplant patients withdrawing or reducing of maintenance immunosuppression is permitted for patients who appear to have developed two-way donor-specific hyporeactivity.

Donor-specific hyporeactivity in lung transplant recipients

Donor-specific hyporeactivity in lung transplant recipients

Donor-specific hyporeactivity in lung transplant recipients

Donor-specific hyporeactivity in lung transplant recipients

Induction of donor-specific hyporeactivity in augmented microchimerism in rat liver transplantation followed by donor bone marrow

Induction of donor-specific hyporeactivity in augmented microchimerism in rat liver transplantation followed by donor bone marrow

Three years of follow-up of bone marrow-augmented organ transplant recipients: The impact on donor-specific immune modulation

The discovery of the presence of previously unsuspected microchimerism in successful longterm liver and kidney transplant recipients prompted us to postulate that these cells are essential for graft acceptance and the induction of donor-specific hyporeactivity.1–3 This observation provided the basis for the initiation of a new therapeutic strategy which involved infusion of donor bone marrow (BM) cells under conventional immunosuppressive treatment with tacrolimus and prednisolone.4 The initial outcome of sequential in vitro immunologic evaluations performed to determine the development of donor-specific hyporeactivity in the first 15 BM-augmented and 16 contemporaneous controls has been described previously.5,6 We report here the immune profile of 102 BM-augmented and 57 control patients who were at least 6 months posttransplantation.

Sequential mixed lymphocyte culture after kidney transplantation: induction of tolerance or sensitization.

Serial mixed lymphocyte culture (MLC) was used to monitor the evolution of donor-specific responsiveness over the first 2 years after cadaveric renal transplantation. Lymphocytes obtained from 37 patients at 0, 1, 3, 6, 12, 18 and 24 months following transplantation were assayed in a one-way MLC using donor lymphocytes as stimulator cells. Donor-specific hyporesponsiveness developed in 66% of the patients with functioning grafts. Donor-specific sensitization was noted in 2 patients with functioning grafts and the MLC reactivity remained unchanged compared to the pretransplant value in 8 patients. The patients with donor-specific hyporeactivity tended to remain either free of rejection episodes or experienced early rejection episodes only. Thus, using serial MLC we were able to identify patients who developed hyporeactivity or tolerance to donor antigens. These patients may require less immunosuppression.