Immunogenetics 37: 139-142, 1993 Brief communications HLA-B51 transgenie mice as recipients for production of polymorphie HLA-A, B-specific antibodies hillllllllO- genetics © Springer-Vertag 1993 Saehiko Karaki 1, Ai Kariyone 1, Noriko Kato ~, Kyoichi Kano 3, Yoichiro Iwakura z, and Masafumi Takiguehi ~ i Departmentsof Tumor Biology and Immunology and 2 Viral Infection, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan 3 Biomedical Research Center, Olympus Optical Co. Ltd. Hachioji-shi, Tokyo192, Japan Received February 3, 1992; revised version received April 2, 1992 Mouse monoclonal antibodies (mAb)s specific for polymorphic determinants of HLA class I antigens are useful for HLA typing and various studies of HLA. Thus far the number of these mAbs has been limited in spite of the fact that many investigators have made efforts to generate them. The most crucial problem in these attempts is that mice produce almost exclusively monomorphic rather than polymorphic antibodies for HLA antigens, because they tend to recognize xenogeneic determinants of HLA antigens. On the other hand, one can expect that HLA transgenic mice (TGM) may produce alloantibodies when they are immunized by HLA antigens since these mice recognize HLA antigens coded by the transgene as "self" and the immunizing antigens as"alloantigens". In fact, re- cent studies have demonstrated that polymorphic mAbs for HLA class I antigens could be generated by using HLA class I TGM (H~mnerling et al. 1990; Tahara et al. 1990). In the present study, we attempted to produce polymor- phic antibodies for HLA-A, B, and C antigens in HLA-B51 TGM. TGM were produced by injecting the 6.4 kilobase (Kb) Eco RI fragment of the HLA-B51 (B*5101) gene into C3H/He one-cell embryos (Hayashi et al. 1989). Surviv- ing embryos were reimplanted into the oviducts of pseudopregnant ICR female mice. The offspring mice were tested for integration of the transgene by dot-blot hybridization of DNA derived from the tail with a 340 base pairs (bp) Beg II-Xba I fragment containing the exons 6 and 7 of the HLA-B51 gene. The surface expression of HLA- B51 antigen was detected on peripheral lymphocyte blood cells (PBL), thymocytes and spleen cells of the HLA-B51 TGM by flow cytometry using W6/32 HLA class I-specific mAb (data not shown). Correspondenceto: M. Taldguchi, Departmentof TumorBiologyand Immunology, Instituteof Medical Science, Universityof Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan. As HLA-B51 antigens were expressed on lymphoid cells on HLA-B51 TGM, it was assumed that these antigens had induced tolerance and had been recognized as self-an- tigen in the TGM. To demonstrate tolerance for HLA-B51, the HLA-B51 TGM along with C3H mice were immunized with L cells expressing HLA-B51. The presence of HLA class I-specific antibodies in the immune sera was examin- ed by using Hmy2C1R cells expressing HLA-All (A*llO1), A24 (A'2401), A31 (A*3101), B51 (B*5101), B35 (B'3501), B38 (B'3801), Cw7 (Cw*0701), Cb-1 (C*1401), or Cb-2 (C*1201) as targets. No antibodies against HLA class I antigens were detected in sera from the immunized TGM, while monomorphic antibodies were seen in sera from the immunized C3H mice (data not shown). These results demonstrated that HLA-B51 TGM are tolerant to HLA-B51 antigen. HLA-B51 TGM were then immunized with L cells ex- pressing HLA-All or HLA-A24. Sera from these immuniz- ed TGM revealed strong reactivity with the HLA-A an- tigens, marginal reactivity with HLA-B38 and no reactivity with HLA-C antigens (Table 1). On the other hand, sera from C3H mice immunized with HLA-All or HLA-A24 reacted strongly with HLA-A and HLA-B antigens and weakly with HLA-C antigens (Table 1). These results in- dicate that alloantibodies might have been produced in HLA-B51 TGM stimulated with HLA-A antigens, although their antisera exhibited a rather strong cross- reactivity among some other HLA-A antigens. Subsequently, HLA-B51 TGM and C3H/He mice were immunized with L cells expressing HLA-B35 or HLA-B38. Sera from HLA-B51 TGM immunized with HLA-B35 an- tigens reacted with the HLA-B antigens, but not with HLA- A and HLA-C antigens, while sera from C3H/He mice im- munized with the same HLA antigen broadly reacted with all the HLA class I antigens (Table 1). Similarly, sera from HLA-B51 TGM immunized with HLA-B38 reacted strong-