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Induction of mixed hematopoietic chimerism in the pig-to-baboon model
Induction of Mixed Hematopoietic Chimerism in the Pig-to-Baboon Model L. Bu¨hler, M. Awwad, S. Treter, M. Basker, T. Ericson, A. Lachance, B. Oldmixon, B. Kurilla-Mahon, S. Gojo, C. Huang, A. Thall, J.D. Down, M.E. White-Scharf, D.H. Sachs, and D.K.C. Cooper
T
O ACHIEVE specific immunologic tolerance to allografts and concordant xenografts, our laboratory has established models in which mixed hematopoietic chimerism has been established in rodents1,2 and nonhuman primates.3,4 In an attempt to extend this approach to a discordant pig-to-baboon combination, we have recently infused high doses of porcine peripheral blood mobilized progenitor cells (PBPC) in baboons treated with a nonmyeloablative conditioning regimen and anti-CD40L mAb. MATERIALS AND METHODS PBPC obtained by leukapheresis from pigs (n ⫽ 9) were transplanted into baboons. Group 1 baboons (n ⫽ 3) underwent whole-body (300 cGy) and thymic (700 cGy) irradiation, T-cell depletion (ATG), complement depletion (CVF), short courses of cyclosporine (CyA), mycophenolate mofetil (MMF), porcine hematopoietic growth factors (pIL-3 and pSCF), and immunoadsorption of anti-Gal antibody before transplantation of high doses (2 to 4 ⫻ 1010/cells/kg) of porcine PBPC. In Group 2 (n ⫽ 4), CyA was replaced by eight doses of anti-CD40L mAb over 14 days. Group 3 (n ⫽ 2) received the Group 1 regimen plus two doses of antiCD40L mAb (on days 0 and 2). Pig cell chimerism was analyzed by FACS and PCR. Colony forming units (CFU) of blood were monitored by PCR for pig DNA.
RESULTS
In Group 1, pig cell chimerism was detected in the blood by FACS for 5 days (with a maximum of 59%) and ⱕ13 days by PCR. In Group 2, pig chimerism was detected for 5 days by FACS (maximum 55%) and ⱕ28 days by PCR. In one of two Group 3 baboons, in which there was no return of Gal IgG for 30 days, engraftement of pig cells was documented by FACS from days 16 to 22. Pig monocytes, coated with IgM only, were detected (maximum at time of transplantation 16% and during engraftment 6%). By PCR, pig DNA was detected for 33 days, and CFUs were positive for pig DNA from days 19 to 33. In the second baboon of Group 3,
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 32, 1101 (2000)
Gal IgG reappeared by day 3 and pig cells were only detectable by FACS until day 4, but by PCR for 30 days. In all groups, pig cells were detectable intermittently by PCR ⬎100 days. DISCUSSION
Macrochimerism detectable by FACS was present for 5 days in all groups. The absence of return of IgG anti-Gal in one baboon allowed engraftment and differentiation of pig monocytes. Microchimerism detectable by PCR was prolonged in Groups 2 and 3. The absence of IgG may be important in allowing chimerism, as these antibodies can destroy cells by ADCC, independent of complement. These results suggest (1) that there is no absolute physiologic barrier to pig hematopoietic engraftment in baboons and (2) that, if the return of anti-Gal IgG can be prevented, pig cell engraftment may be facilitated. REFERENCES 1. Sharabi Y, Abraham VS, Sykes M, et al: Bone Marrow Transplant 9:191, 1992 2. Sharabi Y, Aksentijevich I, Sundt TM, et al: J Exp Med 172:195, 1990 3. Kawai T, Cosimi AB, Colvin RB, et al: Transplantation 59:256, 1995 4. Bartholomew AM, Cosimi AB, Sachs DH, et al: Transplant Proc 29:923, 1997 From Transplantation Biology Research Center (L.B., M.B., S.G., C.H., D.H.S., D.K.C.C.), Massachusetts General Hospital/ Harvard Medical School, Boston, and Biotransplant Inc. (M.A., S.T., T.E., A.L., B.O., A.T., J.D.D., M.E.W.S.), Charlestown, Massachusetts. Address reprint requests to D.K.C. Cooper, MD, PhD, FRCS, Transplantation Biology Research Center, Massachusetts General Hospital, MGH East, Building 149-9019, 13th Street, Boston, MA 02129. E-mail: [email protected].
0041-1345/00/$–see front matter PII S0041-1345(00)01143-X 1101
T
O ACHIEVE specific immunologic tolerance to allografts and concordant xenografts, our laboratory has established models in which mixed hematopoietic chimerism has been established in rodents1,2 and nonhuman primates.3,4 In an attempt to extend this approach to a discordant pig-to-baboon combination, we have recently infused high doses of porcine peripheral blood mobilized progenitor cells (PBPC) in baboons treated with a nonmyeloablative conditioning regimen and anti-CD40L mAb. MATERIALS AND METHODS PBPC obtained by leukapheresis from pigs (n ⫽ 9) were transplanted into baboons. Group 1 baboons (n ⫽ 3) underwent whole-body (300 cGy) and thymic (700 cGy) irradiation, T-cell depletion (ATG), complement depletion (CVF), short courses of cyclosporine (CyA), mycophenolate mofetil (MMF), porcine hematopoietic growth factors (pIL-3 and pSCF), and immunoadsorption of anti-Gal antibody before transplantation of high doses (2 to 4 ⫻ 1010/cells/kg) of porcine PBPC. In Group 2 (n ⫽ 4), CyA was replaced by eight doses of anti-CD40L mAb over 14 days. Group 3 (n ⫽ 2) received the Group 1 regimen plus two doses of antiCD40L mAb (on days 0 and 2). Pig cell chimerism was analyzed by FACS and PCR. Colony forming units (CFU) of blood were monitored by PCR for pig DNA.
RESULTS
In Group 1, pig cell chimerism was detected in the blood by FACS for 5 days (with a maximum of 59%) and ⱕ13 days by PCR. In Group 2, pig chimerism was detected for 5 days by FACS (maximum 55%) and ⱕ28 days by PCR. In one of two Group 3 baboons, in which there was no return of Gal IgG for 30 days, engraftement of pig cells was documented by FACS from days 16 to 22. Pig monocytes, coated with IgM only, were detected (maximum at time of transplantation 16% and during engraftment 6%). By PCR, pig DNA was detected for 33 days, and CFUs were positive for pig DNA from days 19 to 33. In the second baboon of Group 3,
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 32, 1101 (2000)
Gal IgG reappeared by day 3 and pig cells were only detectable by FACS until day 4, but by PCR for 30 days. In all groups, pig cells were detectable intermittently by PCR ⬎100 days. DISCUSSION
Macrochimerism detectable by FACS was present for 5 days in all groups. The absence of return of IgG anti-Gal in one baboon allowed engraftment and differentiation of pig monocytes. Microchimerism detectable by PCR was prolonged in Groups 2 and 3. The absence of IgG may be important in allowing chimerism, as these antibodies can destroy cells by ADCC, independent of complement. These results suggest (1) that there is no absolute physiologic barrier to pig hematopoietic engraftment in baboons and (2) that, if the return of anti-Gal IgG can be prevented, pig cell engraftment may be facilitated. REFERENCES 1. Sharabi Y, Abraham VS, Sykes M, et al: Bone Marrow Transplant 9:191, 1992 2. Sharabi Y, Aksentijevich I, Sundt TM, et al: J Exp Med 172:195, 1990 3. Kawai T, Cosimi AB, Colvin RB, et al: Transplantation 59:256, 1995 4. Bartholomew AM, Cosimi AB, Sachs DH, et al: Transplant Proc 29:923, 1997 From Transplantation Biology Research Center (L.B., M.B., S.G., C.H., D.H.S., D.K.C.C.), Massachusetts General Hospital/ Harvard Medical School, Boston, and Biotransplant Inc. (M.A., S.T., T.E., A.L., B.O., A.T., J.D.D., M.E.W.S.), Charlestown, Massachusetts. Address reprint requests to D.K.C. Cooper, MD, PhD, FRCS, Transplantation Biology Research Center, Massachusetts General Hospital, MGH East, Building 149-9019, 13th Street, Boston, MA 02129. E-mail: [email protected].
0041-1345/00/$–see front matter PII S0041-1345(00)01143-X 1101