Fig. 7

Escape from thymic deletion of polyclonal CD8⁺ T cells specific for hematopoietic H60. a A schematic illustration depicting the experimental design. TCD-BM cells (5 × 10⁶) from CD45.1⁺B6 mice were transplanted into B6, Con-H60, and Act-H60 mice that were lethally irradiated. Six weeks after BMT, CD4 cells MACS-purified from the thymocytes and splenocytes of the recipients were subjected to flow cytometric analysis or MLC. b Detection of H60-cognate cells ex vivo from BMT recipients. CD4-negative and H60-tetramer-binding cells were enriched via MACS from the thymocytes and splenocytes of the recipients and were stained with H60-tetramer-PE, anti-CD45.1-FITC, and anti-CD8-allophycocyanin antibodies. Representative FACS profiles are shown after CD45.1⁺ cell gating. The values in the FACS profiles indicate the percentages of H60-tetramer⁺ cells among the CD45.1⁺ CD8⁺ cells. The numbers of H60-cognate CD8 T cells (estimated based on the FACS data) and MFI values from H60-tetramer staining are presented as bar graphs. c Detection of H60-cognate cells ex vivo from normal mice. The CD4⁻H60-tetramer⁺ cells were enriched from the B6, Con-H60, and Act-H60 mice at 8 weeks old, as described above, and stained with H60-tetramer-PE, anti-CD3ε-FITC, and anti-CD8-allophycocyanin antibodies. Representative FACS profiles are shown after CD3ε⁺ cell gating; the percentages of H60-tetramer⁺ cells are indicated. Estimated numbers of H60-cognate CD8 T cells and MFI values of H60-tetramer staining are plotted. The data from b, c represent three independent experiments (n = 2/group/experiment) and are presented as means ± s.e.m. P-values were generated by Student’s t-test