An antibody specific for Tcrb-V10a and Tcrb-V10c

Abstract

Although two antibodies specific for mouse Tcrb-V10 have been developed, they only recognize Tcrb-V10 from TcrbVb haplotype mice (Necker et al. 1991; Tomonari et al. 1992). To establish an antibody specific for Tcrb-V10a, a Sprague-Dawley rat was hyperimmunized with a T-cell clone, S2, bearing Tcrb-V10a (confirmed by DNA sequencing, data not shown), derived from a Tcrb-Va haplotype SJL mouse. Spleen cells from this rat were fused with NSO myeloma cells, and the resultant hybridoma cells were screened for their ability to activate Tcrb-V10a+ S2 cells. A hybridoma secreting a mitogenic antibody, KT10a (IgG2b), was selected because of its ability to stain a population of T cells from SJL mice. Specificity of the KT10a antibody was examined with a panel of 29 T-cell clones bearing various Tcrb-V elements (Table 1). These cells were incubated with antibodies, KT10a or KT3 (antiCD3; Tomonari 1988), for 30 min. After two washes, cells were treated with fluorescein-isothiocyanate (FITC)coupled rabbit F(ab)92 fragments specific for mouse immunoglobulin (DAKO, Glostrup, Denmark) for 30 min. The cells were then washed three times, and fixed with phosphate-buffered saline containing 1% formaldehyde, and 1 104 cells were analyzed with an Epics XL flow cytometer (Hialeah, FL). KT10a recognized only the TcrbV10a+ cells, S2 and F11TH17. Various strains of mice were examined for their levels of peripheral T cells expressing the KT10a epitope (Fig. 1, Table 2). Mesenteric lymph node cells (1 106) were stained with KT10a antibody, fixed with formaldehyde, and examined with an EPICS XL cell analyzer as described in Fig. 1. Mice were obtained from Clea Japan (Tokyo), SLC Japan (Hamamatsu), Charles River Japan (Yokohama), National Institute for Genetics (Mishima, Japan), Jackson Laboratory (Bar Harbor, ME.), and our own facility. None of the Tcrb-Vb haplotype or Czech II mice with an unclassified Tcrb-V haplotype had KT10a+ T cells. However, Tcrb-Va haplotype mice (Czech-Vba, C57BR/cdJ, C57L/J, NOD-Vba, SJL/J, and SWR/J) and the Tcrb-Vc haplotype RIIIS/J mice expressed varying levels of KT10a+ T cells. To confirm that the KT10a antibody recognizes a product of a gene located in the Tcrb-V region, [C57BL/6 (SWR C57BL/6)F1] backcross mice were examined for their levels of KT10a+ and Tcrb-V17a+ T cells (Fig. 2). KT10a+ and Tcrb-V17a+ T cells are present in SWR (TcrbVa), but absent in C57BL/6 (Tcrb-Vb) mice. Fourteen backcross animals had both KT10a+ and Tcrb-V17a+ T cells, whereas 21 mice had neither KT10a+ nor Tcrb-V17a+ T cells. As no recombination was observed between KT10a and Tcrb-Va, it is likely that KT10a is closely linked to the Tcrb-Va region. Further evidence for the Tcrb-V10a specificity comes from the observation that unlike NOD mice, NOD-Vba mice can be induced to proliferate in in vitro culture in the presence of KT10a antibody. In addition, these cells were exclusively stained with this antibody, but not with any other Tcrb-V-specific antibodies. Furthermore, no skewing of the Tcra-V repertoire was observed in these proliferated T cells, indicating that the antibody is specific for a Tcrb-V. Thus, KT10a recognizes Tcrb-V10 from TcrbVa and Tcrb-Vc haplotype mice. To date no superantigen(s) specific for Tcrb-V10a or Tcrb-V10c has been found, as no deletion of KT10a+ T cells was observed in various inbred or F1 mice (Table 2). In contrast, higher levels of KT10a+CD4+ T cells in H2E+ C57BR/cdJ and RIIIS/J mice may suggest that these T cells are being positively selected by H2E molecules. This phenomenon has already been observed with Tcrb-V10b+ T cells (Tomonari et al. 1992). Of the six amino acid differences between Tcrb-V10a and Tcrb-V10b, three are located within the complementarity determining region 1 (CDR1) at positions 24, 28, and 31 (Smith et al. 1990). This could suggest that the level of positive selection of Tcrb-V10a+ and TcrbV10b+ T cells in the heterozygous Tcrb-Va/Tcrb-Vb haploK. Tomonari ( ) ? O. A. Rosenwasser Department of Immunology and Parasitology, Fukui Medical School, Matsuoka, Fukui, 910-11, Japan