Fig. 5

Escape from thymic deletion of lower avidity T cells and their effector differentiation. a Analysis of splenic CD8⁺ T cells from CD45.1⁺J15 BMT recipients. The numbers of splenic CD8⁺ T cells and the frequencies of H60-tetramer-binding cells among the splenic CD8 T cells are plotted. Representative FACS data show H60-tetramer-PE staining in splenic CD8 T cells after gating on CD45.1⁺ cells; the percentages of H60-tetramer-binding cells are indicated. b TCR avidity assay with intracytoplasmic IFN-γ-staining. Splenic CD8 T cells from three different recipients were stimulated in vitro in the presence of different concentrations of H60 or VSV peptides and then subjected to intracytoplasmic anti-IFN-γ staining. c–e Flow cytometric analysis of the leukocytes infiltrating the spleen, intestines, and liver in the recipients of CD45.1⁺J15 BMT at 6 weeks post-BMT. c CD8β-PE.Cy7 by CD8α-APC.Cy7 and d H60-tetramer-PE by CD44-APC profiles of the CD45.1⁺H60-tetramer⁺CD8α⁺ cells are shown; the percentage values of each quadrat are indicated. e Representative single histograms indicating the origin of leukocytes based on CD45.1 expression. Percentage values of CD45.1⁻ cells (recipient origin) and CD45.1⁺ cells (donor origin) are indicated. f In vivo cytotoxicity assay. A 1:1 mixture of CFSE-labeled and H60 (CFSE^hi) and VSV (CFSE^low) peptide-loaded target cells was injected into CD45.1⁺J15 BMT recipients. A representative single histogram of PBLs from the recipients is shown for the presence or absence of CFSE-labeled target cells. In vivo cytotoxicity was expressed as the percentage of specific lysis, calculated by the equation (1–(% H60-loaded targets/% VSV-loaded targets)) × 100. Percentage cytotoxicity values are plotted. All data (a–f) represent more than three independent experiments (n = 3/group/experiment) and are presented as means ± s.e.m. P-values were generated by Student’s t-test