Peripheral Blood Microchimerism Does Not Correlate With the State of Graft Acceptance in HLA-DRB1 Mismatched Renal Transplant Recipients K. Sakamoto, K. Matsushita, T. Sakamaki, K. Yamada, Y. Gunji, H. Yamada, H. Kashiwabara, and T. Yokoyama T HE ROLE of peripheral blood microchimerism in organ transplantation has not been clearly under- stood, and there have been controversial discussions con- cerning microchimerism and graft acceptance in renal transplantation. 1 We have examined the microchimeric status in renal transplant recipients by Y-chromosome probes and found its correlation with better HLA-DRB1 (DRB1) matching and lower antidonor response in mixed lymphocyte culture (MLR) as previously reported. 2 In this study, we focused on the microchimeric state in DRB1 mismatched renal transplant recipients for further analysis. MATERIALS AND METHODS Ten kidney allograft recipients (graft survival time, 4.5 to 16 years, average 10.8 years), who underwent DRB1 mismatched living- related or cadaveric renal transplantation at Sakura National Hospital, National Kidney Transplantation Center between Octo- ber 1980 and August 1992 were studied. Five recipients received grafts from living-related donors and five from cadaveric donors. All recipients had good and stable renal function at the time of this study. As maintenance immunosuppression, seven cases were receiving cyclosporine and prednisolone; two cases cyclosporine, azathioprine, and prednisolone, and one case azathioprine and methylprednisolone. Genomic DNA was extracted from the recipients’ peripheral blood cells, and the DRB1 specific sequence of each donor was amplified by nested polymerase chain reaction (PCR) with se- quence specific primers. 3 DNA samples were subjected to 20 amplification cycles (denature, 30 seconds at 96°C; annealing, 50 seconds at 60°C; and extension, 50 seconds at 72°C) in a GeneAmp PE9600 PCR system (Perkin-Elmer) for the first and second PCRs. The detection of microchimerism was carried out without prior knowledge of HLA genotype matching and immunologic assays between donors and recipients. Each PCR amplification was tested at least twice to confirm specificity. Evaluation of DNA PCR intensity was carried out independently by two trained researchers (K.M. and T.S.). Antidonor and antithird party immune responses of each recip- ient were evaluated by MLR as previously reported. MLR was examined in 5 of 10 cases with peripheral blood lymphocytes from HLA-DRB1 genotypically identical healthy volunteers for both antidonor and third party response, but in one we were not able to test antidonor response due to lack of an appropriate donor type volunteer. As a statistical analysis, the two-tailed Student’s t test and chi-square test were used. From the Department of Surgery and Division of Clinical Research, Sakura National Hospital, Sakura, Japan. Address reprint request to Dr Sakamoto, Department of Sur- gery, Sakura National Hospital, 2-36-2 Ebaradai, Sakura-shi, Chiba-ken, 285 Japan. Table 1. Clinical and Immunologic Aspects of Microchimeric and Nonchimeric Patients in DRB1 Mismatched Renal Transplantation Microchimerism Detected (N = 3; CD24, CD37, LD46) Microchimerism Not Detected (N = 7) Donor (cadaver/living related) 2/1 4/3 Age at transplant 25.3  4.2 (24, 22, 30) 29.3  6.3 Percentage of acute rejection 1/3 (33%) 3/7 (43%) Initial immunosuppression Az + Med (1), CyA + Pred (2) CyA + Pred (5), CyA + Az + Pred (2) Graft surviving years 14.3  1.5 (16, 14, 13) 9.3  3.2 Present renal function Serum creatinine (mg/dL) 1.27  0.15 (1.1, 1.3, 1.4) 2.13  1.3 BUN (mg/dL) 23  3.5 (19, 25, 25) 34.3  15.3 Stimulation index in MLR (donor type/third party) 0.74  0.18 (0.61, 0.87) 0.73  0.15 Number of HLA mismatch HLA-A, B 1.33  1.53 (0, 3, 1) 1.86  1.35 HLA-DRB1 1.33  0.58 (2, 1, 1) 1.29  0.49 © 1998 by Elsevier Science Inc. 0041-1345/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(97)01161-5 Transplantation Proceedings, 30, 7–8 (1998) 7