Fig. 4

Presentation by H60⁺ cells in BMT recipients is responsible for negative selection of J15 thymocytes. a–e Flow cytometric analysis of thymocytes in recipients of β2m^−/−J15 BMT. β2m^−/−J15 BM cells were transplanted into B6, Con-H60, Act-H60, or β2m^−/− B6 recipients (left); or (β2m^−/−→B6), (β2m^−/−→Con-H60), or (β2m^−/−→Act-H60) chimera recipients (right). Thymocytes from the recipients were analyzed by flow cytometry 6 weeks after β2m^−/−J15 BMT. a CD4-PE.Cy5/CD8-APC.Cy7 FACS profiles are shown after gating on H-2K^b (Pacific Blue)⁻Vβ8.3 (FITC)⁺ cells; the percentage values of different stages are indicated. b The numbers of thymocytes, c percentages of DP and CD8⁺ SP cells, d percentages of DN4 cells among the DN population, and e MFI values from CD5-PE staining are plotted. f–j CD45.1⁺J15 BM cells were transplanted into B6, Con-H60, and Act-H60 recipients on a β2m^−/− background (β2m^−/− B6, β2m^−/− Con-H60, and β2m^−/− Act-H60). f Immunostaining of thymus tissue. Thymus tissue sections from recipients were stained with anti-β2m-PE and anti-keratin 5 (K5)-Alexa Fluor 647 antibodies. Images were overlaid and examined for co-localization of β2m and K5. White dotted lines are drawn around K5⁺ cells to identify the medulla (M). C indicates the cortex. The bar indicates 100 μm. In the immunostaining procedure, β2m^−/− J15→ β2m^−/− B6 BMT was included as a negative control for β2m-staining. g Representative FACS profiles of CD4-PE.Cy5/CD8-APC.Cy7 staining and H60-tetramer-PE staining in CD8⁺ SP cells are shown. h The numbers of thymocytes and percentages of DP and CD8⁺ SP cells, i percentages of DN4 cells among the DN population, and j MFI values from CD5-PE staining were plotted. The data from a–j represent three independent experiments (n = 3/group/experiment) and are presented as means ± s.e.m. P-values were generated by Student’s t-test