Donor AKR bone marrow cells (2107) were inoculated on day 35, 6 hr after whole body irradiation

Donor AKR bone marrow cells (2107) were inoculated on day 35, 6 hr after whole body irradiation. Skin graftingSkin grafting was as described by Kong 005). mAb, anti-CD3 mAb, low-dose irradiation, and AKR BMT exhibited more stable chimerism but had earlier skin graft rejection (mean skin graft survival: 1167176 days) than the mice that did not receive anti-CD3 mAb. These results suggest that anti-TCR mAb, but not anti-CD3 mAb, in combination with low-dose irradiation and BMT, is useful for long-lasting allograft survival after withdrawal from tacrolimus in mice with fully allogeneic skin grafts. INTRODUCTION Organ transplantation has been established as a lifesaving measure for patients with organ failure. It is, however, necessary for transplant recipients to maintain chronic immunosuppression. Recently, tacrolimus and cyclosporin A, which primarily MGC79399 suppress the T-cell immune response by blocking interleukin-2 (IL-2) production, have been shown to be central to immunosuppression after organ transplantation.1,2 The continuous use of these immunosuppressants has been associated with significant morbidity and mortality because Otenabant of opportunistic infections,3 spontaneous neoplasms,4,5 as well as direct drug toxicity and metabolic complications.6,7 It is, therefore, ideal for organ-transplanted recipients to maintain a tolerant state to allografts in a drug-independent manner. Thus, induction of alloantigen-specific immunological tolerance would be a most useful therapeutic strategy, but at present it is not clinically feasible. In this study, we treated grafted mice with various combinations of anti-T-cell receptor (anti-TCR ) and anti-CD3 monoclonal antibodies (mAbs), low dose irradiation and donor bone marrow transfer (BMT) in a fully allogeneic murine skin graft model to induce tolerance after withdrawal from tacrolimus in recipients with allografts. MATERIALS AND METHODS AnimalsC57BL/6 (B6; H-2b) and BALB/c (H-2d) mice were bred and maintained at the Institute for Experimental Animals, Kyushu University (Fukuoka, Japan). AKR (H-2k) mice were obtained from SEAC Yoshitomi, Ltd. (Fukuoka, Japan). Female mice of 7C10 weeks of age were used in this study. Preparation of monoclonal antibodiesAnti-TCR mAb (hybridoma H57-597, hamster immunoglobulin G; IgG)8 and anti-CD3 mAb (hybridoma Otenabant 145-2C11, hamster IgG)9 were prepared as follows. The hybridoma cells were cultured in a serum-free conditioned medium (SFM 101; Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) with sodium bicarbonate (14 g/l), insulin (10 mg/l), transferrin (10 mg/l), monoethanolamine (20 l/l), and gentamicin (10 mg/l). Monoclonal antibodies were precipitated by ammonium sulphate, and dialysed against phosphate-buffered saline. The protein concentration was determined by the Lowry method. Cell preparationTo assay chimerism, peripheral blood cells were collected from the tail vein of recipient mice every week after BMT using heparinized microcapillary tubes. The peripheral blood leucocytes were analysed to determine the percentage of H-2Kk-positive donor-derived cells by flow cytometry described below. Spleen Otenabant cells were prepared as follows. The spleen was surgically removed and then disrupted by pressing their fragments between two glass slides. Erythrocytes were lysed with ammonium chloride buffer. All viable nucleated cells were counted. The viability of the cells was evaluated using trypan blue dye exclusion. Experimental designThe recipient B6 mice were divided into six treatment groups, each consisting of three to five mice (Table 1). To induce the tacrolimus-dependent state, all recipient B6 mice were treated with tacrolimus (Fujisawa Pharmaceutical Co., Osaka, Japan; 5 mg/kg/day i.p.) between day ?3 and day 21, and then every other day for 1 week. Donor AKR skin grafting was performed on day 0 in all of the mice. On day 28 when tacrolimus administration was withdrawn, the recipient B6 mice in groups IICVI were administered 200 g of anti-TCR mAb. Seven days later, the recipients were administered either 200 g (groups III, V) or 400 g (group VI) of anti-CD3 mAb, and irradiated at a dose.