Infusion Of Donor Bone Marrow Cells Research Articles

Hematopoietic Chimerism Following Non-Myeoloablative Stem Cell Transplantation Is Dependent on NK Cell Tolerance.

NK cells are able to eliminate major mismatched hematopoietic cells and, in addition to T cells, represent a second barrier that prevents engraftment following transplantation over MHC barriers. Here, we studied the role of NK cells in the elimination of major mismatched hematopoietic cells in a Balb/c into B6 transplantation model after anti-CD40L (MR1) treatment. In this model, survival of donor cells was determined using an in-vivo cytotoxicity assay based on the infusion of differentially CFSE-labeled syngeneic and donor splenocytes. Likewise, the efficacy of treatment was determined directly by assessing the level of chimerism three months after transplantation. Four weeks after the infusion of 106 bone marrow cells (BMC), B6 mice rejected donor spleen cells within 3 hours. A combination with anti-CD40L treatment prevented this early elimination and these mice showed the same elimination kinetics as observed in untreated mice (97.0 ± 1.3% vs. 96.6 ± 1.3% in 3 days). These results indicate that anti-CD40L treatment prevents the induction of a memory T cell response after infusion of donor BMC. Nonetheless, elimination of donor cells was still present within 3 days, therefore we hypothesized that the elimination of donor cells was mediated by NK cells, rather than by T cells. A detailed analysis of the elimination kinetics in untreated B6 mice showed that the CFSE-labeled Balb/c splenocytes were gradually eliminated starting from the moment of infusion. Similar elimination kinetics were observed in T cell-deficient B6 nu/nu mice. In addition, in-vivo treatment with a depleting anti-NK cell antibody (PK136) prolonged the survival of donor splenocytes in both B6 and B6 nu/nu mice (54.7 ± 2.8% vs. 8.4 ± 5.9% in 2 days in B6 mice and 72.4 ± 7.2% vs. 19.4 ± 8.6% in 1 day in B6 nu/nu mice). A similarly prolonged survival of donor spleen cells was observed in NK cell-depleted mice that had received 106 BMC 4 weeks earlier in combination with anti-CD40L (77.4 ± 15.6% with NK cell depletion vs. 5.3 ± 0.6% without NK cell depletion in 2 days), while no effect was observed after in-vivo treatment with a depleting anti-CD8 antibody. Infusion of increasing numbers of Balb/c BMC (106, 107, 108) after treatment with anti-CD40L resulted in a dose-dependent prolongation of the survival of donor splenocytes, but up to 108 BMC were needed for complete non-responsiveness. This indicated that transplantation of 108 BMC resulted in tolerization of NK cells, which was also associated with stable chimerism (36.1 ± 14.0% of the GR-1+ fraction). In-vivo depletion of NK cells before transplantation allowed stable chimerism in mice treated with anti-CD40L and only 30 x 106 BMC (5/5 vs. 1/5 without NK cell depletion). These data demonstrate that: 1) The elimination of Balb/c donor splenocytes in untreated B6 recipient mice is mediated by NK cells. 2) In mice treated with donor BMC and anti-CD40L the elimination of donor splenocytes can be delayed by NK cell depletion or by increasing the initial dose of donor BMC. 3) NK cell tolerance over MHC barriers can be induced by transplantation of a high number of BMC (108) and results in sustained engraftment and chimerism. 4) Additional NK cell depletion allows sustained chimerism following transplantation of a lower number (30 x 106) of BMC. We conclude that the induction of NK cell tolerance is dependent on the dose of donor BMC injected. This may explain the high numbers of BMC required for engraftment over MHC barriers.

THYMOGLOBULIN PRECONDITIONING AND TACROLIMUS MONOTHERAPY WITH SUBSEQUENT WEANING IN RENAL TRANSPLANT RECIPIENTS: AN UPDATE

O142* Aims: We have suggested that the heavy conventional immunosuppression used for organ transplantation subverts the seminal tolerogenic mechanism of clonal exhaustion-deletion and thereby contributes to long-term drug dependence. Methods: This self-defeating effect was avoided in 175 adult kidney recipients (mean 49.9 ± 14.3 years) treated between July 2001 and September 2003; 43 (24.6%) had adjunct donor bone marrow cell infusion. Two therapeutic principles were applied: (1.) recipient pretreatment (with 5 mgm/kg Thymoglobulin); (2.) minimal post-transplant immunosuppression (with tacrolimus monotherapy, initial target trough 10 ng/ml). Twenty-five (14.2%) patients were undergoing retransplantation, and 34 (19.4%) had a PRA >20%. Mean HLA mismatches were 3.1 ± 1.7. One hundred kidneys (57%) were cadaveric (mean cold ischemia 25.6 ± .7 hours) and 75 (43%) were live donated. Mean donor age was 39.9 ± 6.3 years (range 1-66). Post-transplant doses of tacrolimus were spaced to every other day or longer at various times after four months. Results: With a mean follow up of 16.9 ± 7.1 months, the actuarial one and two patient survival is 97% and 95% with graft survival of 91% and 84%. Mean current serum creatinine is 1.95 ± 1.14 mg/dl. The incidence of preweaning acute clinical rejection was 34.9% (6.3% steroid resistant). Weaning was attempted in 149 cases (85.1%) and interrupted by rejection in 45 (30.2%). Currently, 111 (63.4%) patients are at various stages of weaning: 25 (14.2%) every other day, 44 (25.1%) three times a week, 28 (16%) two times a week, and 14 (8%) once a week. Ten percent of patients had asymptomatic CMV infection and there was one BK infection, but the incidence of CMV disease, diabetes, and PTLD was 0%. Conclusions: With significant numbers of highly weaned patients beyond the two-year mark, we continue to apply this strategy of immunosuppression, which is neither drug nor organ specific.

PERI-OPERATIVE DONOR BONE MARROW INFUSIONS AND WEANING OF IMMUNOSUPPRESSION IN LIVER ALLOGRAFT RECIPIENTS.

O307 Aims: In this study we attempted to gradually reduce and finally discontinue the immunosuppression in adult liver allograft recipients. Methods: Patients: Eligible patients were at least 30 months post-transplantation with stable liver function, no recent (<12 months) acute rejection episode and no history of autoimmune disease. One hundred and four patients, met these criteria and enrolled to the study. Forty five of them had received donor bone marrow cell (DBMC) infusion at the time of the transplant (study group G1) and 59 patients had not (control group G2). Maintenance immunosuppression was Tacrolimus (n=88), cyclosporine (n=15) or rapamycin (n=1). Weaning of immunosuppression: Upon enrollment, immunosuppression was reduced by 25% (Phase I) and was maintained at that level for 12 months. Subsequently, it was further reduced (Phase II) until it was discontinued (Phase III). Follow up: Liver function tests, peripheral blood and bone marrow chimerism were measured at regular intervals. End points: Patients non compliants with the study protocol and those who had a clinically suspected (persistent increase of the liver function tests) or a biopsy proven acute rejection were taken out of the study. Results: There was no significant difference in the overall incidence or severity of the rejection episodes, between the two groups. However, the freedom of rejection curve shows that there are significantly less rejections in G1 during the initial reduction of immunosuppression until this is reduced at levels below 50% of the enrollment dose. Till the end of March 2004, 10 patients from G1 (26.5%, 9 men/1 woman) are off immunosuppression for 14.2 to 37.1 months (average 25.4 months) and 9 from G2 (19.5%, 8 men/1 woman), for 10.9 to 35 months (average 26.6 months). Graft Loss: A patient in G1 was retransplanted because of chronic rejection. Deaths: There were 4 deaths, 3 in G1 and one in G2. Only one of the patients was still in the study at the time of death (colon necrosis, sepsis). The other deaths, [myocardial infarctus (n=1), complications of dialysis (n=1), complications of surgery for biliary obstruction (stones)(n=1)] occurred long after the patients were taken off the study. Other complications: Two patients in G2 presented renal failure and undergo dialysis. Conclusions: Perioperative donor bone marrow infusion seems to play an immuno-modulatory role in long term liver allograft recipients causing the graft to better tolerate a slight decrease in immunosuppression. However this beneficial effect disappears when the dose is reduced substantially or is discontinued. Successful weaning is possible in about 20 % of liver allograft recipients.

Prevention of obliterative airway disease through tolerance induction following CD40 costimulatory blockade in combination with donor bone marrow cell infusion

Prevention of obliterative airway disease through tolerance induction following CD40 costimulatory blockade in combination with donor bone marrow cell infusion

Nonlethal conditioning for the induction of allogeneic chimerism and tolerance to islet allografts.

We reported that tolerance to skin grafts can be achieved by chimerism induction by way of nonlethal conditioning. In the present study, we evaluated the outcome of islet allografts implanted either simultaneously or after donor bone marrow cell (BMC) infusion when nonlethal conditioning was used. B10 (H-2b) mice were conditioned with antilymphocyte serum (ALS), 100 cGy total body irradiation (TBI), and given 30 x 10(6) allogeneic (B10.BR, H-2k) BMC on day 0. On day 2, cyclophosphamide was given intraperitoneally (IP), followed by a second BMC infusion on day 3. After chimeras were typed for allogeneic BMC engraftment on day 28, animals were rendered diabetic chemically and transplanted under the kidney capsule with islet allografts genetically matched or disparate to the BM. Donor-specific islet grafts were accepted (median survival time [MST] > 180 days, n=6), whereas all major histocompatibility complex (MHC)-disparate third-party BALB/c (H-2d) islet grafts were rejected (MST=13.8 days, n=4). When B10.BR BMC and islets were given simultaneously, graft acceptance (MST >140 days, n=4) was observed. Surprisingly, when MHC-disparate third-party islets (BALB/c) were given together with B10.BR BMC, long-term survival was also observed (MST >100 days, n=3). These findings suggested that conditioning alone at the time of islet implant might induce long-term engraftment without further treatment. However, only chimeric animals accepted a second-set donor-specific graft, whereas all other groups rejected it. Our data indicates that stable allogeneic chimerism and islet indefinite survival can be achieved by the use of a nonmyeloablative protocol. The results of the conditioning-only experiments are consistent with the possibility of graft accommodation.

Baboons undergoing orthotopic concordant cardiac xenotransplantation surviving more than 300 days: Effect of immunosuppressive regimen

Objective: We reviewed long-term survival among hosts in 3 consecutive series of a rhesus monkey-baboon orthotopic cardiac xenotransplantation model with reference to host immune response, including the effectiveness in preventing rejection and limiting toxicity concerning infection, to evaluate specific immunosuppressive regimens for long-term outcomes. Methods: Six juvenile baboons surviving more than 300 days after transplantation were reviewed. Regimen A consisted of splenectomy, FK506, methotrexate, and antilymphocyte globulin. Regimen B consisted of pretransplantation and chronic maintenance with cyclosporine A (INN: ciclosporin), methotrexate, and antithymocyte globulin. Regimen C was the same as regimen B plus pretransplantation total lymphoid irradiation and intraoperative donor bone marrow cell infusion. Rejections were detected by means of echocardiography. Results: Long-term survivors in 3 groups were followed for a range of 332 to 515 days (mean, 436 days). Rejection frequency in regimens A, B, and C was 0.35, 0.58, and 0.18 per month, and rescue therapy days were 23 (4.8%), 123 (9.5%), and 20 (2.4%), respectively (P <.0001). Infection frequency was 0.58, 0.56, and 0.19 per month, and therapy days were 192 (38.2%), 164 (12.6%), and 7 (0.9%), respectively (P <.0001). Concerning the host immune response, interleukin 2-activated T cells of all groups during rejection-free periods showed lower numbers compared with those of control animals (P <.0005), and regimen C was the lowest among 3 groups (P <.01). The production of xenoantibody was sufficiently attenuated in all groups. Conclusion: Regimen C leads to long-term survival with fewer rejection and infection episodes by means of suppression of the interleukin 2 pathway and xenoantibody production.J Thorac Cardiovasc Surg 2003;125:60-70

Real-time sequence-specific primer polymerase chain reaction amplification of HLA class II alleles: a novel approach to analyze microchimerism.

The careful assessment of microchimerism is essential to investigate the effects of donor bone marrow-derived cells in transplantation. We have developed a protocol to assess microchimerism based on the HLA mismatch between the recipient and the donor. Our approach combines real-time polymerase chain reaction (PCR) with sequence-specific primer PCR (SSP-PCR) to selectively amplify and measure the abundance of donor HLA alleles in DNA samples extracted from the recipient after transplant. To optimize and validate the reliability of this method at different levels of microchimerism, we tested serial dilutions of donor DNA into recipient DNA. We demonstrate that donor alleles can be readily detected and reliably measured at concentrations as low as 0.1%. This method is simple and rapid and could find practical application in the assessment of microchimerism in patients receiving organ or cellular transplants in conjunction with donor bone marrow cells infusion.

Converting nonhuman primate dendritic cells into potent antigen-specific cellular immunosuppressants by genetic modification.

T cell depletion plus donor bone marrow cell (BMC) infusion induces long-term kidney allograft survival in a limited number of rhesus macaque recipients. Therefore, there is a need to enhance the tolerogenic activity of donor BMCs. The tolerogenic effect of donor BMCs is ascribed to a veto activity, mediated by a CD8+ subset that upregulates immunoregulatory effector molecules, transforming growth factor-beta1 (TGF-beta), and FasL, after interaction with donor-reactive cytotoxic T lymphocyte precursors (CTLp), leading to clonal inactivation/deletion of donor-reactive CTLp. Of note, the receptors for TGF-beta1- and FasL-induced signal transduction are upregulated in activated T cells. Since mature dendritic cells (DCs) are exceptionally efficient activators of T cells, we postulated that mature DCs modified to overexpress TGF-beta1 and FasL might exert potent veto (i.e., inactivating/deleting) activity independent of CD8 expression. A fusion protein comprising antihuman CD40 single-chain antibody and soluble coxsackie-adenovirus receptor enabled high-efficiency transduction of rhesus monocyte-derived DCs (Rh MDDCs) by recombinant adenovirus (Ad). Mature Rh MDDCs transduced with Ad encoding active TGF-beta1 retained a mature phenotype yet exhibited potent alloantigen-specific cellular immunosuppression. Such modified MDDCs have the potential to promote tolerance induction to allografts in vivo.

Treatment with immunotoxin.

T-cell depletion prior to or beginning at the time of transplantation has been shown to be a valuable adjunct to the induction of immunological unresponsiveness. Both total lymphoid irradiation and anti-lymphocyte globulin have been used for this purpose in experimental models of transplantation as well as in human organ transplant recipients. However, these methods of T-cell depletion are limited in their ability to deplete T cells selectively due to non-specific targeting and limited efficacy. A new anti-CD3 immunotoxin has been developed with a far more potent ability to deplete T cells selectively as measured by flow cytometry analysis of peripheral blood T lymphocytes as well as lymph node lymphocytes. This immunotoxin is well tolerated by rhesus monkeys when administered in vivo. When administered as a single immunosuppressive agent pretransplant, it substantially promotes allograft survival, inducing tolerance in at least one-third of recipients as measured by subsequent acceptance of donor skin grafts and rejection of third-party skin grafts. When administered on the day of transplant in combination with steroid pretreatment and a brief course of deoxyspergualin or mycophenolate mofetil (4 to 14 days), long-term unresponsiveness is also produced and in a more reliable manner than using immunotoxin alone. A new immunotoxin directed at the human CD3epsilon has been developed with excellent potency in T-cell killing and lacking the Fc portion of the CD3 antibody. This construct may be useful for T-cell depletion in humans and has a potential application in tolerance induction in human organ transplantation. Lessons learned from anti-CD3 immunotoxin in the non-human primate model to date include (i) profound (2-3 log) depletion of T-cells can be accomplished safely without inducing lymphoma or infection, (ii) such depletion is a useful adjunct for tolerance induction to allogeneic organ transplants, and (iii) tolerance to both allogeneic renal transplants and xenogeneic islet transplants has been accomplished using such strategies to date in non-human primates and in pigs. Immunotoxin may be useful for the induction of chimerism using strategies that include donor bone marrow infusion. Successful strategies for tolerance induction have also been developed using immunotoxin without the adjunct of donor bone marrow or stem cell infusion. Clinical application of immunotoxin will use a newly engineered construct with the potential for causing cytokine release, less susceptibility to neutralization by anti-diphtheria antibody and not dependent on chemical conjugation of an antibody and toxin. The usefulness of immunotoxin is directly related to its tremendous potency for depleting T cells. Based on results in nonhuman primates, it is anticipated that it will become a useful agent in tolerance induction in humans.

Six-year clinical effect of donor bone marrow infusions in renal transplant patients.

To date, several single- and multicenter clinical trials have attempted to induce specific immunological unresponsiveness using donor bone marrow cell infusions to augment solid organ transplantation, but the outcomes have not been definitive. Between September 1994 and May 1998, 63 cadaver (CAD) renal transplant recipients of either one or two postoperative donor bone marrow cell (DBMC) infusions were prospectively compared with 219 non-infused controls given equivalent immunosuppression. There was at least a 1 HLA DR antigen match present between donors and recipients. The immunosuppressive regimen included a 10-day course of OKT3 induction, and tacrolimus, mycophenolate mofetil, and methylprednisolone maintenance. A total 7.01x10(8)+/-1.9x10(8) (SD) DBMC/kg was infused into the CAD recipients on either days 4 and 11 (n=42) or one half of that dose on day 4 (n=21) postoperatively. Clinical follow-up has ranged from 2.9 to 6.3 years (mean, 4.7 years). Studies were also performed of humoral immunity and quantitative cellular chimerism. There is clear-cut equivalence in immunosuppressive dosaging and in the other major demographic variables in both groups. However, only 2/63 DBMC recipients had biopsy-proven chronic rejection, whereas 41/219 showed chronic rejection in the controls (P = <0.01). In both groups, mortality was not associated with rejection. The actuarial graft survival at 6.3 years in the CAD DBMC group was 84.3% compared with 72.2% in the control group (not statistically significant). However, if death with a functioning graft was excluded, graft survival was 94.1% in the DBMC group and 79.8% in the controls (P=0.039). Forty patients in the control group continue to have deteriorating renal function (increasing serum creatinine concentrations to 2 mg/dl and higher), compared with 2 patients in the DBMC group (P=0.04). In the DBMC group, chimerism in iliac crest marrow aspirates has increased 3-fold in yearly sequential measurements between 1 and 4 years postoperatively averaging 1.3+/-0.36% (SE) most recently. This has not occurred in the controls. There now appears to be more solid long-term evidence, in kidney transplant recipients prospectively receiving DBMC infusions, of an improvement in long-term graft survival, and of the degree of chimerism positively correlating with the absence of graft loss.

Donor bone marrow-derived chimeric cells present in renal transplant recipients infused with donor marrow. I. Potent regulators of recipient antidonor immune responses.

Even though a number of transplant centers have adopted donor-specific bone marrow cell (DBMC) infusions to enhance donor cell chimerism, to date there has been no direct evidence linking chimerism with tolerance induction in human organ transplant recipients. Cells of donor phenotype were isolated 1 year postoperatively from the peripheral blood lymphocytes and iliac crest bone marrow of 11 living-related-donor (LRD) renal transplant recipients, who had received perioperative donor bone marrow cell infusions. These recipient-derived donor (RdD) cells were characterized phenotypically by flow cytometric analysis and functionally as modulators in mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The yield of RdD cells ranged from 0.1 to O.9% of the starting material with the majority being TcRalphabeta, CD3 positive T cells, a substantial percentage of which coexpressed CD28. At 1 year posttransplant almost 50% of the LRD-kidney/DBMC recipients tested so far exhibited donor-specific unresponsiveness in MLR (7/17) and CML (6/13) reactions and this trend was further enhanced at 23 years. In the recipients with residual positive antidonor immune responses, the RdD cells inhibited recipient antidonor MLR and CML responses significantly more strongly than freshly isolated and similarly treated iliac crest bone marrow cells from the donor. RdD cells also inhibited the MLR of the recipient to third party allogeneic stimulator cells; however, this nonspecific effect was significantly weaker than specific inhibition. We also established long-term bone marrow cultures stimulated every 2 weeks with irradiated alogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.

Donor bone marrow cell infusion is effective in inducing tolerance in dogs treated with fractionated lymphoid irradiation and FK506

Donor bone marrow cell infusion is effective in inducing tolerance in dogs treated with fractionated lymphoid irradiation and FK506

Analysis of post-transplant immune status in recipients of liver/bone marrow allografts

The aims of this study were to assess the effect of donor bone marrow infusion on the reactivity of recipient peripheral blood lymphocytes (PBL) to mitogen and to donor and third-party cells after primary liver allotransplantation and to identify any correlation between altered immunoreactivity and HLA mismatches, occurrence of rejection, and immunosuppression. The immunoreactivity of recipient PBL toward frozen donor splenocytes was evaluated in mixed lymphocyte culture (MLC) ( n = 29) and cell-mediated lympholysis (CML) ( n = 27) assays in time intervals ranging from 0.7 to 27 months after transplant. Overall, the mean anti-donor MLC stimulation index (SI) fell from 25.6 ± 5.2 preoperatively to 4.8 ± 1.7 after transplantation ( p < 0.002), with 14 out of 29 (48.3%) patients developing donor-specific MLC hyporeactivity. HLA class II mismatches were significantly associated with recipient post-transplant immune profile ( p < 0.05): MLC donor specific hyporesponsiveness was observed in 70%, versus 37% of patients who shared a class II antigen, versus those that did not. Of the control group, 61.1% developed donor-specific nonreactivity versus 27.2% in the donor bone marrow cells (DBMC) group ( p = 0.02). Donor-specific CML hyporeactivity was observed after transplantation, independent of DBMC infusion, with mean percentage values of pre- and post-transplant donor-specific lysis of 22.4% ± 4.1% versus 3.1% ± 1.6%, p = 0.0004, respectively. Our results suggest that DBMC infusion favors development of nonspecific MLC hyporesponsiveness to donor and third-party alloantigen, with maintenance of reactivity to mitogen and no additional effect on T-cell cytotoxicity.

In vitro immunogenicity of cadaver donor bone marrow cells used for the induction of allograft acceptance in clinical transplantation.

The cascade of immunological effects brought about by donor bone marrow cell (DBMC) infusions to induce allograft acceptance in clinical transplantation is not fully understood. Aside from acting as immune responding and regulatory cells, the infused DBMC also may sensitize the recipient to the donor antigens. To analyze this stimulatory activity of DBMC, in vitro mixed lymphocyte cultures (MLC) and cell-mediated lymphocytotoxicity (CML) culture systems analogous to the transplant model with DBMC infusion were used. When responding peripheral blood lymphocytes (PBL) from normal volunteers were placed in culture with suspensions of Ficoll-purified, T cell-depleted, un-irradiated allogeneic DBMC (NT-DBMC), a reaction was seen in both MLC and CML. However, when compared to allogeneic spleen cells as stimulating cells, the responses to NT-DBMC were of markedly lower magnitude and were not seen when the NT-DBMC was irradiated (3000 R). When responding PBL were stimulated with either NT-DBMC that had been previously cultured with irradiated cells from the responders for 1 week (activated NT-DBMC), NT-DBMC further depleted of CD15+ and glycophorin A-positive cells (NT-LP/DBMC), or purified CD34+ and CD2+ DBMC subsets, stronger lymphoproliferative and cytotoxic responses were observed. Moreover, these responses were not abrogated by irradiation of the stimulating DBMC subpopulations. Depletion of antigen-presenting cells by positive selection of CD3+ cells from the responding PBL abrogated MLC and CML reactivity, even when purified NT-LP/DBMC, the most stimulatory cells, were used. This latter observation was in contrast to the responses seen with cultures containing allogeneic stimulating spleen cell populations. This indicated the requirement for indirect alloantigen presentation, i.e., the failure of these DBMC to stimulate by direct alloantigen presentation. NT-DBMC was able to stimulate responding PBL in secondary MLC and CML responses with an equivalent magnitude, irrespective of whether the stimulators were spleen cells or NT-DBMC. Finally, the MLC and CML responses were inhibited by tacrolimus (FK506), mycophenolic acid (MPA), and cyclosporine (CsA) in a dose dependent manner, in contrast to previously observed refractoriness of DBMC preparations to these agents if DBMC was tested as responder cells or in modulatory assays. These results indicate that DBMC are able to function as effective in vitro stimulators, but only by indirect antigen presentation, and that the immune responses mediated by them can be down-regulated by their own inherent suppressive nature, an effect that can be enhanced by the presence of immunosuppressive drugs.

FOUR YEAR FOLLOW-UP OF HIGH-DOSE DONOR BONE MARROW INFUSIONS TO ENHANCE ALLOGRAFT SURVIVAL

86 Donor bone marrow cell (DBMC) infusions have been proposed to enhance allograft survival. Aim of this clinical trial was to determine the effect of DBMC infusions on patient and graft survival after liver transplantation (LTX). DBMC (from vertebral bodies) were administered in 257 LTX recipients. In 432 LTX recipients donor vertebral bodies were not available (controls). Up to 1 × 109 DBMC/kg were infused in equal aliquots administered at day 5,11 (2 DBMC delayed; n=185), at day 5,14,21,28,90 (5 DBMC; n=36), or at day 0,11 (2 DBMC; n=36). Four year actuarial patient/graft survival (%) was 84.1/81.2 (2 DBMC delayed), 77.4/72.2 (5 DBMC), 74.8/69.2 (2 DBMC) and 79.0/66.1 (Controls). The 4 year results in controls were similar to the 3 year results reported by UNOS (77.4/66.4). Patient survival was not significantly different in all groups. Graft survival in the 2 DBMC delayed group was higher compared to the controls (p<0.001, Wilcoxon). Acute, biopsy proven, rejection was lower in the 2 DBMC delayed group (35.1%) vs. controls (43.3%; p<0.05). The incidence of multiple (3 or more) rejection episodes was 4.9% in the 2 DBMC delayed group, 11.6% in the control group (p=0.01), and 22.2% in the 5 DBMC group (p=0.001). Chronic rejection was 5.8% in the controls and 4.9% in the 2 DBMC delayed group (NS). Two or more biopsies positive for chronic rejection were observed in 0.5% in the 2 DBMC delayed group vs. 2.8% in the controls (NS). In contrast, a significantly higher incidence of chronic rejection was observed in the 5 DBMC group (27.8%). When the DBMC was infused at day 0,11 (2 DBMC group) the incidence of acute and chronic rejection was 50% and 13.9% respectively (NS vs. controls or 2 DBMC delayed). The incidence of EBV infections was higher in the controls and 2 DBMC group compared to the 2 DBMC delayed group (5.7 and 5.7 vs. 1.7 respectively; p<0.05). However PTLD was observed only in 3 cases (2 in the DBMC and 1 in the control groups, NS). The incidence of other infections (bacterial or viral) was not significantly different between groups. Similarly, no significant difference was observed in the distribution of causes of death or graft failure between groups. The incidence of GVHD was higher in the DBMC groups (3.1%) compared to the controls (0.5%; p=0.005), with the highest incidence observed in the 5 DBMC group (8.3%; p=0.001) compared to the 2 DBMC delayed (2.2%; p=0.05). The results indicate that timing of DBMC infusions can result in significantly different outcomes in LTX survival and can also affect the incidence of acute, chronic rejection as well as GVHD.

IMMUNOREGULATION OF BONE MARROW INFUSED LRD RENAL TRANSPLANT RECIPIENTS BY CHIMERIC CELLS OF DONOR PHENOTYPE.

120 A number of transplant centers, including our own, have adopted donor specific bone marrow cell infusions of organ transplant recipients to enhance donor chimerism with the aim of inducing allograft acceptance. However, to date there has been no direct evidence linking chimerism with specific tolerance induction in the transplant recipients. In this study in renal transplant recipients from living related donors (LRD) who had received perioperative donor bone marrow cell infusions, chimeric cells of donor phenotype were isolated from the peripheral blood and the iliac crest bone marrow one year post-operatively, using monoclonal antibodies to donor mismatched HLA class 1 antigens and magnetic microbeads. They were then characterized phenotypically and functionally. In contrast freshly isolated and similarly treated cells from blood and iliac crest marrow of the donors (non-chimeric donor cells) were similarly treated and tested at the same 1 year interval. The yield of such chimeric cells from the recipient marrow by this method of purification ranged as high as 0.5% at this time. Phenotypic characterizations of similarly isolated chimeric cells from the iliac crest marrow of cadaveric kidney recipients 1-3 years postoperatively (the purified chimeric cells from LRD recipients were sufficient only for the functional studies) showed that 51.8±6.3% of the chimeric cells were TcRαβ+ CD2+ CD3-dimly positive. 28.3±7.5% cells were also CD4− CD8−. In addition, 20.4±11.8% were CD4+ CD3+ and 15.0±4.9% were CD8+ CD3+ (n = 4). The regulatory activity of chimeric and non-chimeric donor cells was assessed by testing them as third party cells in MLC and CML responses of the recipients to the allogeneic LRD PBL as stimulating cells. Of the seven cases analyzed so far, two recipients did not respond to the donor in MLC, although this reaction was present preoperatively, so that the role of chimeric cells could not be ascertained. In the five patients with positive anti-donor MLC responses, the purified chimeric cells showed significantly stronger inhibition than non-chimeric donor marrow cells similarly treated (p < 0.01). Modulation experiments on CML responses could be performed only in three cases because of the low yield of the chimeric cells. The chimeric cells were also significantly more potent inhibitors (p < 0.05) of the generation cytotoxic T cells than the non-chimeric donor marrow cells. The inhibitions occurred with chimeric (regulatory) cell concentrations as low as 8% of the responder cells. These results clearly showed that donor bone marrow derived chimeric cells present in transplant recipients have evolved into even more potent immunoregulatory cells by 1 year postoperatively, supporting the notion that chimerism plays a major role in the induction and/or maintenance of transplant tolerance.

CYCLOSPORINE A SUPPRESSES THE TOLEROGENIC EFFECT OF DONOR BONE MARROW CELLS BY INHIBITING TGF-β1-MEDIATED APOPTOSIS

1027 Introduction. Donor bone marrow cell (BMC) infusion can induce prolonged tolerance of allografts in various species, including rhesus monkeys. The tolerogenic effect has been ascribed to a veto mechanism, which associates with functional deletion of donor-specific CTLp. We have postulated that a key CTLp- BMC interaction in this event involves engagement of the BMC CD8α molecule via its natural ligand, MHC Iα3, expressed on the reactive CTLp. Following activation via CD8α, BMC secrete paracrine TGF-β1, which mediates deletion of the reactive CTLp via apoptosis. Earlier murine studies demonstrated that Cyclosporine A (CsA) abolishes veto activity by BMC through an effect on the reactive CTLp rather than on the veto population. To further elucidate the potential mechanism(s) by which CsA inhibits the tolerogenic effect of donor-BMC, we examined the influence of CsA on veto activity of rhesus BMC in vitro and also asked whether CsA impairs TGF-β1 induced apoptosis in activated T cells. Methods. Replicate MLR-induced CTL cultures were set up with and without BMC from the stimulator, in the presence or absence of CsA. Results were calculated as % suppression of CTL by BMC. Additionally, rhesus PHA-activated T cells were treated with and without 2 ug/ml CsA and cocultured with transduced COS cells expressing simian TGF-β1 for 24 h. Apoptosis in the harvested rhesus T cells was assessed by propidium iodide/annexin V FITC staining and flow cytometry. Results. In the absence of CsA, BMC suppressed CTL by 60% while 0.04, 0.5 and 2 ug/ml of CsA decreased the suppression of CTL by BMC to 36%, 20% and 4%, respectively. Although CsA (2 ug/ml) increased TGF-β1 expression, it reduced TGF-β1-induced apoptosis of activated rhesus T cells by 56.6% ± 6.3. CsA had no significant effect on activated rhesus T cell TGF-β1 receptor density, implying that the partial abrogation of TGF-β1-induced apoptosis is not mediated by decreased numbers of TGF-β1 receptors on activated rhesus T cells. Conclusions. We conclude that CsA may inhibit the tolerogenic effect of donor-BMC by suppressing TGF-β1-mediated apoptosis. Suppression of TGF-β1-mediated apoptosis by CsA may involve alteration of post-receptor signaling pathways in donor-reactive CTLp.

Thrombocytopenia after liver transplantation.

Thrombocytopenia after orthotopic liver transplantation (OLT) is a well recognized and prevalent early postoperative complication. The etiology, as well as the effect of this phenomenon on transplant outcome, however, are vague. The aims of this study are to identify factors contributing to thrombocytopenia and to ascertain whether there is any correlation with early rejection and ultimate survival. This study examines 541 OLTs (541 grafts in 494 patients) that were transplanted at the University of Miami during the 3-year period from June 1994 to September 1997. The patients with severe postoperative thrombocytopenia (nadir platelet count [PLT] < 20,000/mm3), as well as the whole group of patients, were analyzed. The preoperative PLT, intra-operative platelet transfusion requirements, cross-match, recipient and donor cytomegalovirus (CMV) status, infusion of donor bone marrow cells (DBMC), occurrence of early rejection episodes (in the first posttransplant month), and re-transplantation were factors examined for any association with thrombocytopenia. Total bilirubin (TB) and direct bilirubin (dB), hematocrit, white blood cell count (WBC), aspartate aminotransferase and alanine aminotransferase, determined on the day that platelets reached a nadir (nadir day), were also analyzed. In 90.9% of the cases, there was a 56.5%+/-23.5% fall in platelets in the immediate posttransplant period (first 2 weeks), but the mean PLT exceeded preoperative levels during the 3rd and 4th postoperative weeks. The nadir of the drop in the PLT most commonly occurred on posttransplant day 4. For preoperative PLT, platelet transfusions during the operation, re-transplantation, early rejection, cross-match, and recipient CMV status, there was significant statistical correlation with any degree of postoperative thrombocytopenia. Four of these factors, preoperative PLT, intra-operative platelet transfusions, re-transplantation, and early rejection, were found to be independently associated with thrombocytopenia in general. None of them was found to be independently correlated with severe thrombocytopenia. A statistically significant correlation between bilirubin and WBC on the nadir day and the degree of thrombocytopenia was observed. No correlation was found between infusion of DBMC or donor CMV serology and thrombocytopenia. Both the nadir PLT and the percentage of the platelet fall were independent predictive factors (p<0.01 and 0.005, respectively) of patient and graft survival. Thrombocytopenia in the immediate posttransplant period is correlated with low preoperative PLT, massive platelet transfusions, and re-transplantation. These factors reflect a poor preoperative condition. There is also a correlation with allograft dysfunction, rejection, and poorer patient and graft survival. A rise in the mean PLT after the 2nd postoperative week reflects proper graft function.

DONOR BONE MARROW, CHIMERISM, HISTOCOMPATIBILITY AND RENAL ALLOGRAFT REJECTION

18 Does donor bone marrow infusion have immunological effects on renal allograft recipients? From September 1994 through December 1997 63 cadaver renal transplant recipients of either 1 (n=21) or 2 (n=42) post-operative donor bone marrow cell (DBMC)-infusions have been followed, compared with 220 non-infused controls given equivalent immunosuppression (Tacrolimus, mycophenolate mofetil, methylprednisolone, and OKT3 induction). Additionally, 16 kidney recipients of DBMC-infusion from living-related donors (LRD) (1-infusion) were followed from November 1996 through December 1997, and given the same immunosuppression. With both CAD and LRD DBMC infusions, the DBMC was similarly processed, frozen under liquid nitrogen, and thawed for administration at the bedside, with similar cell viability in each group. Chimeric analyses were performed by the PCR-Flow technique. There were 8/63 biopsy-proven acute rejection episodes (AR) in the CAD DBMC-infused group, and 33/220 AR in the controls (p=N.S.). However, only 2/63 DBMC recipients had biopsy-proven chronic rejection (CR), while 35/220 showed CR in the controls (p=<.02). Actuarial patient and graft survival (1-40 months) were 93% and 92% in the CAD DBMC-infused group and 96% and 91% in the controls, respectively. If two infusions of CAD DBMC were used, the AR rate was 5% (2/42); while, if one infusion was given, it was 24% (6/21) (p=<0.02). In both groups mortality was not associated with rejection. AR was associated with at least a 5-fold decrease in peripheral blood PCR-Flow chimerism of Total, CD3+, and CD34+ DBMC mononuclear cells (monocyte-lymphocyte gate), i.e., a useful indicator of rejection-not seen with infections of any type. In the LRD DBMC-infused recipients, patient and graft survival were 100% and 100%, respectively (3-15 months), with PCR-Flow DBMC chimerism in peripheral blood being 2× higher than CAD DBMC-infused recipients at serial monthly intervals up to 15 months post-operatively (p=<0.01). However, only 25% of the total average DBMC were initially infused in the LRD group (i.e., 7 × 1010 cells CAD, vs. 1.5 × 1010 cells LRD). These results indicate 1) an effect of histocompatibility on DBMC chimerism, 2) a modulatory effect of DBMC on allograft rejection, 3) the utility of peripheral blood "real time" PCR-Flow as a serial monitoring tool for AR in DBMC-infused patients. We conclude that DBMC infusions have a positive clinical long-term immunoregulatory effect on renal allograft recipients using a heavy induction and early maintenance immunosuppressive regimen.

DONOR BONE MARROW, CHIMERISM, AND KIDNEY TRANSPLANTATION.

422 The role of post-operative specific donor bone marrow cell (DBMC) infusion in solid organ transplantation and the consequent DBMC chimerism established in the recipient has been controversial. We have continued to follow 58 recipients of cadaver donor kidneys transplanted between September 1994 and July 1997 (actuarial followup between 6 and 40 months) who received DBMC, compared with 177 controls given equivalent immunosuppression (consisting of OKT3 induction, tacrolimus, mycophenolate mofetil, and methylprednisolone), but no DBMC. Patient and graft survival in the DBMC-infused group at 40 months was 93% and 93% vs. 96% and 92% in the controls. There were 7/58 biopsy-proven acute rejection episodes (AR) in the DBMC-infused group and 38/177 in the controls (p=.114). There were 1/58 patients with biopsy-proven chronic rejection in the DBMC-infused group vs. 32/177 in the controls (p=.002). Dosaging of tacrolimus and methylprednisolone at 2-years was 0.14±3.9 mg/kg and 0.08±0.04 mg/kg, respectively (n=18), vs. 0.14±0.08 mg/kg and 0.09±0.03 mg/kg, respectively, in the controls (n=29)(p=N.S.). Tacrolimus trough levels were 8.72±3.4 ng/ml in the DBMC-infused group vs. 10.14±3.9 ng/ml in the controls (p=N.S.). PCR-Flow assays of bone marrow aspirates of DBMC-infused recipients were performed sequentially (n=29). In patients who did not have previous AR there was an increase between 1- and 2-years post-operatively in donor total, CD3+, and CD34+ (gated) lymphoid cells from 0.54±0.11, 0.20±0.06, and 0.22±0.05, respectively, to 0.93±0.19, 0.43±0.10, and 0.35±0.07. That is, there was a virtual two-fold increase in PCR-Flow quantitative chimerism. In the DBMC-infused group, early AR was associated with a five-fold decrease in chimerism in bone marrow aspirates at 2-years compared with those patients who did not have AR. In the peripheral blood, donor PCR-Flow chimerism values had decreased between 1-year and 2-years, so that they were an order of magnitude lower in the blood than in the bone marrow. Although other compartments have not been studied as yet, chimeric bone marrow of DBMC-infused recipients appeared to have greater in vitro autoregulatory effects than did marrow of the controls at 2-years post-operatively (i.e., in lymphoproliferative responses to viral protein antigens) (p=<0.05). These clinical and ex vivo observations lend support to the proposal that DBMC infusions and consequent chimerism are a cause of immunoregulation favoring kidney transplant survival.