KLF13 is critical for the maintenance of CD4⁻CD8⁺CD24⁻ T cells in BALB/c thymus. (A) CD4 and CD8 expression in thymocytes from wild-type and Klf13^−/− BALB/c mice. (B) Absolute numbers of CD4⁺CD8⁻ single positive (CD4⁺ SP) and CD4⁻CD8⁺ (CD8⁺ SP) cells in the thymi of wild-type and Klf13^−/− BALB/c mice. P < 0.05 is considered significant. Error bars are SDs. (C) Purified CD4⁺CD8⁺ double-positive (DP) cells were differentiated with PMA, ionomycin, and IL-7 to CD8⁺ SP cells in vitro. (D) Expression of CD24 and TCRβ in the indicated thymic populations from BALB/c and C57BL/6 mice. Gates were based on an isotype control antibody. (E) Expression of CD44 and CD62L in CD4⁺ SP and CD8⁺ splenocytes from BALB/c mice. Data are representative of three (B and C) and four (A, C, and E) independent experiments with three mice in each genotype group.

IL-4 is both essential and sufficient to sustain memory-like CD8⁺ T cells in BALB/c thymus. (A) Expression of Eomes after culture of thymocytes from wild-type and Klf13^−/− BALB/c mice with IL-4, IL-7, IL-15, or medium. (B) Eomes expression in CD8⁺ SP cells from thymi of Klf13^−/− BALB/c mice after injection with 1 µg IL-4 immune complex or vehicle every other day for 10 d. (C) Relative abundance of IL-4, IL-7, or IL-15 mRNA relative to HPRT1 in thymi from the indicated stains of mice as measured by real-time RT-PCR. Error bars are SDs from triplicate PCR. (D) IL-4 and TCRβ expression in thymi from the indicated strains of mice after treatment with ionomycin and PMA in the presence of brefeldin A for 4 h. (E) FACS analysis of the expression of interferon-γ and IL-4 in wild-type and Il4^−/− thymus from BALB/c mice. Thymocytes were treated with ionomycin and PMA in the presence of brefeldin A for 4 h before analysis. (F) Expression of Eomes, IFN-γ, and CD24 in CD8⁺ SP thymocytes from wild-type and Il4^−/− BALB/c mice. Data are representative of two (A and C) or three (B–F) independent experiments with two (B) or three (D, E, and F) mice in each genotype group.

KLF13 regulates iNKT cells in BALB/c mice. (A) iNKT cells (Tet-PBS57⁺) in thymi of wild-type and Klf13^−/− on BALB/c or C57BL/6 backgrounds. (B) Absolute number of thymic iNKT cells in indicated mice. (C) Minority iNKT cells (Tet-PBS57⁺) from lethally irradiated mice adoptively transferred with unequally mixed bone marrow cells, as indicated. (D) Expression of indicated genes in DP thymocytes from indicated mice measured by Western blot. (E) Abundance of indicated genes in FACS-sorted iNKT cells from indicated mice measured by quantitative real-time PCR. Error bars are generated from real-time PCR triplicates from the same RNA sample. (F) PLZF expression in thymic iNKT cells from the indicated mice (left). The mean fluorescence intensity (MFI) of PLZF in iNKT cells relative to CD8⁺ SP cells was determined using three mice for each point (right). (G) BrdU uptake and Annexin V binding to thymic iNKT cells from wild-type and Klf13^−/− BALB/c mice. (H) Expression of IL-4, CD69, and CD44 by thymic iNKT cells from wild-type and KLF13^−/− BALB/c mice. IL-4 was analyzed 4 h after stimulation with Ionomycin and PMA in the presence of brefeldin A. Data are representative of three independent experiments (A–H) with three mice in each genotype group (A–C and F–H). Error bars are SDs (B and F) or SDs from triplicate (E).

CD8⁺ memory-like T cells exist in the thymi of BALB/c, but not C57BL/6 mice. (A) Abundance of Eomes and T-bet mRNA relative to HPRT1 in splenic CD8⁺ T cells and total thymocytes from wild-type and Klf13^−/− mice on BALB/c or C57BL/6 backgrounds measured by quantitative real time PCR. Error bars are generated from real-time PCR triplicates from the same RNA sample. Error bars are SDs from triplicate PCR. (B) Expression of CD24 and Eomes in CD8⁺ SP thymocytes from wild-type BALB/c mice. (C) Expression of Eomes and IFN-γ in CD8⁺ SP thymocytes from BALB/c and C57BL/6 mice. CD4⁺ SP thymocytes from BALB/c mice are used as negative control. (D) Expression of cell surface markers on CD4⁺ SP and CD8⁺ SP thymocytes from indicated strains of mice. (E) Expression of congenic surface markers Thy1.1 and Thy1.2 in thymocytes and Eomes in CD8⁺ SP cells from lethally irradiated mice adoptively transferred with unequally mixed bone marrow cells, as indicated. Data are representative of two (A and B) or three (C, D, and E) independent experiments with three mice in each genotype group (B–E).

iNKT cells are the source of IL-4 in the thymus. (A) Representation of iNKT cells and γδ T cells in gates of total thymocytes, IL-4⁺ thymocytes, and IL-4⁻ thymocytes from wild-type BALB/c mice after stimulation with ionomycin and PMA in the presence of brefeldin A for 4 h. Tet, CD1d tetramer. (B) iNKT cells from the thymi of wild-type or IL4^−/− BALB/c mice as measured by Tetramer-PB557 binding. (C) Absolute number of iNKT cells from the thymi of wild-type or IL4^−/− BALB/c mice. Error bars are SDs. (D) Thymocytes from wild-type and Cd1d^−/− BALB/c mice stained with Tetramer-PB557, anti–IFN-γ, anti–IL-4, and anti-TCRβ after stimulation as in A. (E) Expression of Eomes, IFN-γ, and CD24 on CD8⁺ SP cells from wild-type and Cd1d^−/− BALB/c mice. Data are representative of three independent experiments with three mice in each genotype group (A–E).