Fig 1.
Rbpj in T cells is required for TCRαβ+CD8αα+ IELs.
The (A) frequency and (B) total cell number of IELs or (A) CD90 and granzyme B expression on TCRαβ+CD8αα+ IELs in Rbpj+/+ and Rbpj−/− mice at the age of 8 wk were evaluated by flow cytometry. The data of (A) and (B) are representative of four independent experiments. The data are shown as mean ± S.D., and ** or *** indicates p < 0.01 or p < 0.001, respectively (n = 5). (C) Sections of small intestine from Rbpj+/+ or Rbpj−/− mice were stained with anti-TCRβ, CD8α, CD8β.2, and laminin, and TCRαβ+CD8αα+ IELs were detected as TCRβ+CD8β-CD8α+ (×400). The data are shown as mean ± S.D., and *** indicates p < 0.001 (n = 9).The data are representative of three independent experiments. (D) The number of TCRαβ+CD8αα+ cells in Rbpj−/− and Rbpj+/+ mice in lamina propria was evaluated by flow cytometry. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; Rbpj, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
Fig 2.
Intrinsic Rbpj in T cells is required for TCRαβ+CD8αα+ IELs.
Bone marrow cells (1.5 × 107) and a 1:1 ratio from Rbpj−/− (CD45.2) and Rbpj+/+ (CD45.1) mice were transplanted into irradiated (9.5 Gy) C57BL/6 mice (CD45.1+CD45.2+). (A) The ratio of intraepithelial CD45+ cells between Rbpj−/− and Rbpj+/+ mice. (B) After 6 wk posttransplantation, the number and frequency of TCRαβ+CD8αα+ IELs from Rbpj−/− (CD45.2) and Rbpj+/+ (CD45.1) mice were evaluated. The data are shown as mean ± S.D., and *** indicates p < 0.001. The data in (A) and (B) are representative of three independent experiments with n = 5 mice in each experiment. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; Rbpj, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
Fig 3.
Notch1 and Notch2 are major receptors that regulate TCRαβ+CD8αα+ IELs.
(A) Expression of Notch1, Notch2, Notch3, or Notch 4 on TCRαβ+CD8αα+ IELs evaluated by flow cytometry. Shadow: isotype control; solid line: Notch antibody. (B) The frequency of TCRαβ+CD8αα+ IELs or CD90/granzyme B expression on TCRαβ+CD8αα+ IELs in Notch1−/−, Notch2−/−, and Notch1/2−/− mice. The data are representative of three independent experiments and are shown as mean ± S.D., and *, **, or *** indicates p < 0.05, p < 0.01, or p < 0.001, respectively. (C) The frequency of TCRαβ+CD8αα+ IELs in Dll1−/−-Villin, Jagged1−/−-Villin, and Dll1/Jagged1−/−-Villin mice. (D) The frequency of TCRαβ+CD8αα+ IELs in Dll1−/−-CD11c, Jagged1−/−-CD11c, and Dll1/Jagged1−/−-CD11c mice. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). Dll1, Delta-like 1; IEL, intraepithelial lymphocyte; N.S.; not significant; Rbpj, recombination signal binding protein for immunoglobulin kappa J region.
Fig 4.
Notch deficiency does not affect the development of precursors of TCRαβ+CD8αα+ IELs.
(A) The frequency and total number of PD-1+ and PD-1− cells in CD25−CD1d-tetramer−CD4−CD8α−TCRβ+CD5+ thymocytes in Rbpj+/+ and Rbpj−/− mice were evaluated by flow cytometry. The data are representative of three independent experiments and are shown as mean ± S.D. (B) The B220−NK1.1−CD4−CD8α−TCRγδ−TCRβ+CD5+ thymocytes from Rbpj+/+ and Rbpj−/− mice were cultured in the presence of IL-15 (50 ng/ml), and the differentiation of CD8α cells was tested by flow cytometry after 8 d of culture. (C) The expression of α4β7, CD103, and CD69 on TCRαβ+CD8αα+ IELs in Rbpj−/− and Rbpj+/+ mice. Shadow: isotype control; solid: Rbpj+/+; dotted: Rbpj−/−. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; PD-1, programmed death-1; Rbpj, recombination signal binding protein for immunoglobulin kappa J region.
Fig 5.
Atp8a2 is a Notch target gene in TCRαβ+CD8αα+ IELs.
(A) Enriched gene ontology of genes in TCRαβ+CD8αα+ IELs. Genes for which expression is more than four times less in Rbpj−/− compared with Rbpj+/+ mice were analyzed. A heat map of genes (>5 times reduction for Rbpj deficiency) in TCRαβ+CD8αα+ IELs in Rbpj−/− mice compared with Rbpj+/+ mice is shown. (B) Expression of Heyl, Dtx1, and Atp8a2 in TCRαβ+CD8αα+ IELs of Rbpj+/+ or Rbpj−/− mice evaluated by real-time PCR. (C) Expression of Dtx1 and Atp8a2 in DO.11.10 T-cell hybridoma cells infected with control retrovirus (“EV”) or retrovirus carrying the intracellular domain of Notch1 (“N1IC”). (D) Expression of Atp11a and Atp11c in TCRαβ+CD8αα+ IELs of Rbpj+/+ or Rbpj−/− mice evaluated by real-time PCR. (E) Bone marrow cells of Rbpj−/− mice infected with control retrovirus or retrovirus carrying Atp8a2 were transplanted in irradiated wild-type mice (n = 4). Six weeks after transplantation, the development of TCRαβ+CD8αα+ IELs was evaluated by flow cytometry. The data in (A–E) are representative of three independent experiments and are shown as mean ± S.D., and ** indicates p < 0.01 (n = 4). (F) Atp8a2, Atp11a, and Atp11c expression in TCRαβ+CD8αα+ IELs, TCRγδ+CD8αα+ IELs, and splenic CD4 or CD8 T cells from Rbpj+/+ or Rbpj−/− mice evaluated by PrimeFlow analysis. Control (“Ctrl.”); shadow; control staining, Rbpj+/+ mice; solid line, Rbpj−/− mice; dotted line. The data in this figure are representative of three independent experiments. Data associated with this figure can be found in the supplemental data file (S1 Data). IEL, intraepithelial lymphocyte; N.S.; not significant; Rbpj, recombination signal binding protein for immunoglobulin kappa J region; TCR, T-cell receptor.
Fig 6.
Rbpj-deficient TCRαβ+CD8αα+ IELs have low flippase activity.
(A) TCRαβ+CD8αα+ IELs or splenic CD4+ or CD8α+ T cells from Rbpj+/+ or Rbpj−/− mice were incubated with fluorescence-conjugated phospholipid (NBD-PS, NBD-PE, or NBD-PC), and the fluorescence was evaluated 10 min later. Shadow: unincubated control; solid line: NBD-PS, NBD-PE, or NBD-PC. (B) Expression of cleaved caspase3/7 in TCRαβ+CD8αα+ IELs from Rbpj+/+ or Rbpj−/− mice. (C) Sorted TCRαβ+CD8αα+ IELs (CD45.2) from Rbpj+/+ or Rbpj−/− mice were incubated with peritoneal macrophages (CD45.1/45.2) for 2 h. The number of CD45.2-positive TCRαβ+CD8αα+ IELs was counted in the presence or absence of macrophages by flow cytometry. Data are shown as the ratio of the number of TCRαβ+CD8αα+ IELs with macrophages to the number of TCRαβ+CD8αα+ IELs without macrophages. The data are representative of three independent experiments and are shown as mean ± S.D., and ** indicates p < 0.01. (D) Flow cytometry of CD11c+MHCclassII+CD11b+F4/80+ macrophages from the small intestine in wild-type mice treated with anti-CSF1R Ab every other day for a total of six times. Cells were analyzed 1 d after final treatment. The data are representative of three independent experiments and are shown as mean ± S.D., and ** indicates p < 0.01. (E) Histological section of the small intestine in CX3CR1gfp/+ mice treated with anti-CSF1R Ab every other day, for a total of two times, was stained with anti-GFP Ab (green) and DAPI (white) (×200). Mice were analyzed 1 d after final treatment. (F) Rbpj−/− mice were treated with anti-CSF1R Ab every other day, for a total of six times. The number of TCRαβ+CD8αα+ IELs was evaluated 1 d after final anti-CSF1R Ab treatment. The data are representative of three independent experiments. Data are shown as mean ± S.D., and * or ** indicates p < 0.05 or p < 0.01, respectively. Data associated with this figure can be found in the supplemental data file (S1 Data). Ab, antibody; CSF1R, colony stimulating factor 1 receptor; GFP, green fluorescent protein; IEL, intraepithelial lymphocyte; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; Rbpj, recombination signal binding protein for immunoglobulin kappa J region.