NKT cells in Elf-1^−/−mice display normal proliferation but increased cell death during early development. (A) WT and Elf-1^−/− mice were injected with BrdU and analyzed 3 days later. iNKT cell–enriched thymocytes were stained with CD1d/αGalCer tetramer and mAb against TCRβ, CD44, and NK1.1, and then analyzed by flow cytometry. Tetramer⁺TCRβ⁺ cells were further gated to distinguish stage I (CD44^lowNK1.1⁻, ST1), stage II (CD44^highNK1.1⁻, ST2), and stage III (CD44^highNK1.1⁺, ST3) NKT cells (left). BrdU incorporation by iNKT cells at each of these stages is shown in the right panel. Data shown are representative of 2 independent experiments. (B-E) Thymocytes were isolated from WT and Elf-1^−/− mice, stained with CD1d-αGalCer tetramer and mAb against various cell surface markers including annexin V, and then analyzed by flow cytometry. (B) Percentages of annexin V⁺ cells within the tetramer⁺TCRβ⁺ (left) and tetramer⁺DP^dull (right) gates. Data shown are representative of 3 independent experiments. (C) Bar graphs depict means ± SD for the proportion of annexin V⁺ cells within the tetramer⁺DP^dull population (n = 4 for each group; *P < .05). (D) The proportions of annexin V⁺ cells within each iNKT-cell stage. Data shown are representative of 3 independent experiments. (E) Bar graphs depict the means ± SD for the proportion of annexin V⁺ cells within the stage I gate (n = 4 for each group; ***P < .001).