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Regulatory effects of human donor bone marrow cells on allogeneic cellular immune responses
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4:00 p.m.-5:30 p.m. Abstract Presentation 5 6.4
AUGMENTED DONOR-SPECIFIC CHIMERISM AND IMMUNE MODULATION IN SOLID ORGAN TRANSPLANT RECIPIENTS FOLLOWING INFUSION OF UNMODIFIED DONOR BONE-MARROW. A Zeevi, R Banas, M Pavlick, C Bentlejewski, K Spichty, AS Rao, P Fontes, AJ Demetris, R Shapiro, M Jordan, F Dodson, S Pham, R Keenan, B Griffith, R Corry, F Egidi, JJ Fung and TE Starzl. Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 15261 Background: We have proposed that the donor bone-marrow (BM) cells that migrate from transplanted organs and persist in tissues of recipients may play an important role in graft acceptance and induction of donorspecific immune modulation. This spontaneous chimerism was augmented in 150 whole organ recipients by the preoperative infusion of 3x108 /kg unmodified donor BM. For immunosuppression all patients received Tacrolimus and steroids. A higher incidence (92%) of donor cell chimerism was identified in the BM-augmented group than in the control (52%). From the initiation of the trial, 130 recipients were available for sequential immune monitoring with a minimum of 6 months follow up: 86 were in BM group and 44 were in the control. Methods: Immunological monitoring was performed by 1) serial MLR testing, 2) proliferative responses to mitogens and recall antigen, 3) limiting dilution analysis to determine the frequency for donor-specific T helper (Th) precursor cells and 4) growth assay on protocol endomyocardial biopsies in heart transplant reCipients. Results: Of the BM augmented patients, 41(48%) exhibited donor-specific immune modulation as compared to 14 (32%) in the control. The incidence of donor-specific hyporeactivity was two fold higher in liver (55% vs 34%) and lung (44% vs 22%) transplant recipients than that in the control group. In contrast, there was no difference in the immune modulation of kidney recipients with or without BM. Although the BM augmented heart transplant recipients exhibited a trend towards less donor reactivity, none of the cardiac recipients became donor-specific hyporeactive. Patients who exhibited donor-specific hyporeactivity in MLR had 5 to 10 fold lower Th frequencies than that at the time when they were MLR-reactive. Growth assay on protocol endomyocardial biopsies was negative in the first three months in 6 out of 9 BM augmented heart transplant reCipients who had no evidence of acute cellular rejection. Endomyocardial growth assay was positive in two BM augmented heart transplant recipients who had several episodes of rejection; they also maintained a high frequency of donor-specific Th cells. Conclusions: Augmentation of donor cell chimerism was associated with an increase in the incidence of induction of donor-specific hyporeactivity in liver and lung transplant recipients. It, however, remains to be ascertained if utilization of this strategy would allow us to ultimately reduce or withdraw immunosuppression.
8.6
REGULATORY EFFECTS OF HUMAN DONOR BONE MARROW CELLS ON ALLOGENEIC CELLULAR IMMUNE RESPONSES. JM Mathew, M Carreno, L Fuller, C Ricordi, A Tzakis, V Esquenazi and J Miller. Dept. Surgery, Div.Transplantation, U. Miami and V. A. Hosp., Miami, FL. In order to evaluate the immunoregulatory mechanisms brought about by human cadaveric vertebral-body Donor Bone Marrow Cell (DBMC) infusions accompanying organ transplantation, we have established in vitro culture systems analogous to the transplant model. We have extended our previous observations that DBMC inhibited both MLC and CML responses of allogeneic cells to donor antigens by further characterizing the immunoregulation and analyzing the underlying mechanisms. It was observed that DBMC had to be added within the first two days after the initiation of the cultures for the down-regulation of CML to occur, thus suggesting that the modulation was brought about before the development of effector functions. Physical separation of the DBMC from the responder-stimulator cells using the transwell system abrogated the modulation of MLC and CML, thus indicating the requirement of cell-cell contact for the modulatory effect (n=6). The down-regulation of MLC response might not be antigen specific (n=5). The inhibitory activity by DBMC could not be overcome by the addition of 25% MLR supernatant and/or up to 200U/ml rIL-2 to the MLC and CML cultures (n=5). However, it could be partially abrogated by re-stimulation of the responding cells with donor spleen cells indicating that donor reactive cells were not deleted by DBMC. Both CD34 positive and negative DBMC were able to inhibit MLC and CML of allogenic cells, thus indicating that a cell population that developed from the CD34+ progenitors in culture might be the responsible for the regulatory activity. Addition of DBMC pre-cultured with responder cells even on day 4 after the initiation of the cultures (as opposed to 2 days by uncultured DBMC) inhibited CML and MLC, thus suggesting that "differentiated" DBMC might be the regulatory cells. Furthermore, DBMC and purified CD34+ cells responded to allogeneic stimulation and rapidly gave rise to CD38+ T cell precursors within the 7 days in culture. These results clearly showed a regulatory role for DBMC in the donor specific cellular immune responses, thus indicating a rationale for DBMC to be used for induction of allograft acceptance in clinical transplantation.
Abstracts
4:00 p.m.-5:30 p.m. Abstract Presentation 5 6.4
AUGMENTED DONOR-SPECIFIC CHIMERISM AND IMMUNE MODULATION IN SOLID ORGAN TRANSPLANT RECIPIENTS FOLLOWING INFUSION OF UNMODIFIED DONOR BONE-MARROW. A Zeevi, R Banas, M Pavlick, C Bentlejewski, K Spichty, AS Rao, P Fontes, AJ Demetris, R Shapiro, M Jordan, F Dodson, S Pham, R Keenan, B Griffith, R Corry, F Egidi, JJ Fung and TE Starzl. Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 15261 Background: We have proposed that the donor bone-marrow (BM) cells that migrate from transplanted organs and persist in tissues of recipients may play an important role in graft acceptance and induction of donorspecific immune modulation. This spontaneous chimerism was augmented in 150 whole organ recipients by the preoperative infusion of 3x108 /kg unmodified donor BM. For immunosuppression all patients received Tacrolimus and steroids. A higher incidence (92%) of donor cell chimerism was identified in the BM-augmented group than in the control (52%). From the initiation of the trial, 130 recipients were available for sequential immune monitoring with a minimum of 6 months follow up: 86 were in BM group and 44 were in the control. Methods: Immunological monitoring was performed by 1) serial MLR testing, 2) proliferative responses to mitogens and recall antigen, 3) limiting dilution analysis to determine the frequency for donor-specific T helper (Th) precursor cells and 4) growth assay on protocol endomyocardial biopsies in heart transplant reCipients. Results: Of the BM augmented patients, 41(48%) exhibited donor-specific immune modulation as compared to 14 (32%) in the control. The incidence of donor-specific hyporeactivity was two fold higher in liver (55% vs 34%) and lung (44% vs 22%) transplant recipients than that in the control group. In contrast, there was no difference in the immune modulation of kidney recipients with or without BM. Although the BM augmented heart transplant recipients exhibited a trend towards less donor reactivity, none of the cardiac recipients became donor-specific hyporeactive. Patients who exhibited donor-specific hyporeactivity in MLR had 5 to 10 fold lower Th frequencies than that at the time when they were MLR-reactive. Growth assay on protocol endomyocardial biopsies was negative in the first three months in 6 out of 9 BM augmented heart transplant reCipients who had no evidence of acute cellular rejection. Endomyocardial growth assay was positive in two BM augmented heart transplant recipients who had several episodes of rejection; they also maintained a high frequency of donor-specific Th cells. Conclusions: Augmentation of donor cell chimerism was associated with an increase in the incidence of induction of donor-specific hyporeactivity in liver and lung transplant recipients. It, however, remains to be ascertained if utilization of this strategy would allow us to ultimately reduce or withdraw immunosuppression.
8.6
REGULATORY EFFECTS OF HUMAN DONOR BONE MARROW CELLS ON ALLOGENEIC CELLULAR IMMUNE RESPONSES. JM Mathew, M Carreno, L Fuller, C Ricordi, A Tzakis, V Esquenazi and J Miller. Dept. Surgery, Div.Transplantation, U. Miami and V. A. Hosp., Miami, FL. In order to evaluate the immunoregulatory mechanisms brought about by human cadaveric vertebral-body Donor Bone Marrow Cell (DBMC) infusions accompanying organ transplantation, we have established in vitro culture systems analogous to the transplant model. We have extended our previous observations that DBMC inhibited both MLC and CML responses of allogeneic cells to donor antigens by further characterizing the immunoregulation and analyzing the underlying mechanisms. It was observed that DBMC had to be added within the first two days after the initiation of the cultures for the down-regulation of CML to occur, thus suggesting that the modulation was brought about before the development of effector functions. Physical separation of the DBMC from the responder-stimulator cells using the transwell system abrogated the modulation of MLC and CML, thus indicating the requirement of cell-cell contact for the modulatory effect (n=6). The down-regulation of MLC response might not be antigen specific (n=5). The inhibitory activity by DBMC could not be overcome by the addition of 25% MLR supernatant and/or up to 200U/ml rIL-2 to the MLC and CML cultures (n=5). However, it could be partially abrogated by re-stimulation of the responding cells with donor spleen cells indicating that donor reactive cells were not deleted by DBMC. Both CD34 positive and negative DBMC were able to inhibit MLC and CML of allogenic cells, thus indicating that a cell population that developed from the CD34+ progenitors in culture might be the responsible for the regulatory activity. Addition of DBMC pre-cultured with responder cells even on day 4 after the initiation of the cultures (as opposed to 2 days by uncultured DBMC) inhibited CML and MLC, thus suggesting that "differentiated" DBMC might be the regulatory cells. Furthermore, DBMC and purified CD34+ cells responded to allogeneic stimulation and rapidly gave rise to CD38+ T cell precursors within the 7 days in culture. These results clearly showed a regulatory role for DBMC in the donor specific cellular immune responses, thus indicating a rationale for DBMC to be used for induction of allograft acceptance in clinical transplantation.
Abstracts