Expression and Characterization of HHD Monochain

Expression of the HHD monochain and lack of H-2D^b and H-2K^b were documented by indirect immunofluorescence analyses using B9.12.1 (anti–HLA class I), B22.249.R.19 (anti–H-2D^b), and 20.8.4S unlabeled mAb, detected with F(ab)′2 FITC-conjugated goat anti–mouse IgG. Percentages of single CD4⁺ and CD8⁺ T lymphocytes were determined by double staining using phycoerythrin-labeled anti–mouse CD4 (CALTAG Labs., South San Francisco, CA) and biotinylated anti–mouse CD8 (CALTAG Labs.) detected with streptavidin– Perc-P (CALTAG Labs.). Expression of the different Vβ TCR were similarly analyzed using phycoerythrin-labeled anti-CD8 mAb (PharMingen, San Diego, CA) and purified, FITC-labeled Vβ2 (B.20.6), Vβ3 (KJ.25), Vβ4 (KT.10.4), Vβ5.1,.2 (MR.9.4), Vβ6 (44.22), Vβ7 (TR 130), Vβ8.1,.2,.3 (F.23.1), Vβ9 (MR. 10.2), Vβ10 (B.21.5), Vβ11 (RR.3.15), Vβ13 (MR.12.4), and Vβ17 (KJ.23.1)- specific mAb. Splenocytes from three individual D^b−/−, β2m^−/−, HHD⁺, or HHD⁻ mice were red blood cell depleted and enriched in T lymphocytes by wheat germ agglutinin (Sigma Chemical Co., St Louis, MO) precipitation of B lymphocytes and NK cells as described (18 (link)). Staining of 10⁶ cells was performed in 100 μl of PBS with 0.02% sodium azide for 30 min on ice. Purified mAb or F(ab)′2 were used at 10 μg/ml and F(ab)′2 FITCconjugated goat anti–mouse IgG was used 1:100 diluted. A total of 25,000 1% paraformaldehyde-fixed cells per sample was subjected to one- or two-color analysis on FACScan^®.