Rejection of Discordant Skin Xenografts by CD4  CD8  TCR  Cells in CD4- and CD8-Deficient Mice M. Umesue, H. Mayumi, Y.-Y. Kong, K. Omoto, H. Muranaka, R. Nakanishi, H. Kohno, K. Yasumoto, K. Kishihara, and K. Nomoto C ELLULAR response to discordant xenogeneic grafts continues after the hyperacute rejection is success- fully suppressed. CD4 + T cells are considered to play a central role in the rejection of discordant xenogeneic skin graft. 1–3 The following study used CD4- and CD8- gene targeting (CD4 -/ - 8 -/ - ) mice and monoclonal antibody (mAb) against TCR  or TCR  to investigate the role of CD4- and CD8- independent pathway in discordant xenograft rejection. MATERIALS AND METHODS CD4 -/- 8 -/- mice were provided by Dr. Tak W. Mak (Toronto, Canada). CD4 -/- 8 -/- mice and C57BL/6 (B6) mice were grafted with rabbit skin. Thymocytes, lymph node cells, and splenocytes of B6 mice and CD4 -/- 8 -/- mice were stained with the following antibodies: CD8-FITC, CD4-PE, CD3-FITC, TCR -biotin, TCR -biotin, and B220-biotin, followed by SA-PE when necessary. Stained cells were analyzed by flow cytometry (FCM). To deplete TCR  + cells or TCR  + cells in recipient CD4 -/- 8 -/- mice, CD4 -/- 8 -/- mice were treated with either depleting anti-TCR  or anti-TCR  mAb. Recipient mice were injected with 400 g of a mAb on day -7 and 200 g on day -2, then grafted with rabbit skin on day 0 and treated weekly with 200 g of the mAb through the experimental period. Completeness of the depletion was confirmed on day 40 by FCM. RESULTS Graft survival in CD4 -/- 8 -/- and B6 recipient mice were 16.7  1.6 (n = 6) and 9.8  1.0 (n = 6) days (mean  SE), respectively (Table 1, experiment 1). CD4 -/- 8 -/- mice showed significant numbers of CD4 - CD8 - TCR  + CD3 + cells and CD4 - CD8 - TCR  + CD3 + cells in the peripheral lymphoid tissue (data not shown), either of which may be responsible for the rejection of the skin grafts. To examine this possibility, we treated CD4 -/- 8 -/- mice with mAb against TCR  or TCR  (Table 1, experiment 2). CD4 -/- 8 -/- mice treated with anti-TCR  mAb accepted the rabbit skin grafts over 100 days (5 of 5). In contrast, three of four CD4 -/- 8 -/- mice treated with anti-TCR  mAb rejected rabbit skin grafts within 1 month. DISCUSSION Most studies on the cellular mechanism of xenograft rejection have been done by depleting the T-cell sub- population of CD4 + or CD8 + T cells with mAbs. These studies involved in vivo depletion of CD4 + T cells or From the Second Department of Surgery (M.U., H.M., R.N., H.K., K.Y.), University of Occupational and Environmental Health, and Department of Immunology (M.U., H.M., Y.-Y.K., K.O., K.K., K.N.), Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. Supported by “Research Grant for Immunology, Allergy and Organ Transplant, Ministry of Health and Welfare, Japan.” Address reprint requests to Dr M. Umesue, University of Occu- pational and Environmental Health, Second Department of Surgery, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu-city, Fukuoka, Japan. Table 1. Survival of Rabbit Skin Grafts in B6 Mice and CD4- and CD8-Gene Targeting Mice Experiment Recipient Mouse mAb Treatment* Graft Survival (day) Mean  SE 1 B6 - 7, 8, 8, 11, 12, 13 9.8  1.0 CD4 -/- 8 -/- - 11, 14, 17, 17, 18, 23 16.6  1.6 2 CD4 -/- 8 -/- - 11, 17, 17, 18, 20 16.6  1.5 CD4 -/- 8 -/- anti-TCR  100  5 100 CD4 -/- 8 -/- anti-TCR  12, 17, 25, 43 24.2  6.8 *In experiment 2, recipient mice were treated with either depleting anti-TCR  or anti-TCR  mAb before and after the skin grafting as described in Materials and Methods. 0041-1345/99/$–see front matter © 1999 by Elsevier Science Inc. PII S0041-1345(98)01821-1 655 Avenue of the Americas, New York, NY 10010 890 Transplantation Proceedings, 31, 890–891 (1999)