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. 2014 Nov 17;211(12):2425-38.
doi: 10.1084/jem.20141207. Epub 2014 Nov 10.

Limitation of Immune Tolerance-Inducing Thymic Epithelial Cell Development by Spi-B-mediated Negative Feedback Regulation

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Limitation of Immune Tolerance-Inducing Thymic Epithelial Cell Development by Spi-B-mediated Negative Feedback Regulation

Nobuko Akiyama et al. J Exp Med. .
Free PMC article

Abstract

Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B-mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage-specific negative feedback regulation. OPG-mediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL-Spi-B-OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.

Figures

Figure 1.
Figure 1.
RANK signaling up-regulates Spi-B expression through an NIK-dependent pathway in mTECs. (A) Fetal thymic stromal organ cultures (2DG-FTOCs) were prepared and stimulated with recombinant RANKL protein. Thymic lobes isolated from an E15 mouse fetus were cultured in the presence of 2DG to eliminate hematopoietic cells, yielding 2DG-FTOCs. Addition of recombinant RANKL induced mTECs expressing Aire and TSAs in 2DG-FTOCs. Bottom panels show immunohistochemical staining of 2DG-FTOC cryosections with UEA-1, a mature mTEC marker, and keratin-5 (K5) before and at days 1, 2, 3, and 4 after RANKL stimulation. Bars, 200 µm. (B) qPCR analysis of Spib mRNA expression in 2DG-FTOCs at 4 d after incubation without (control) or with 1 µg/ml recombinant RANKL. For blocking experiments (RANKL + RANK-Fc), 5 µg/ml RANK-Fc was added to the culture medium. The values are arbitrary units (A.U.) normalized to Gapdh expression. (C) Expression of Spib, Aire, and Spt1 in 2DG-FTOC at 1–4 d after incubation with 1 µg/ml recombinant RANKL or before RANKL stimulation (day 0) were analyzed by qPCR. The values are normalized to Gapdh expression. (D) RANKL stimulation induces Spib expression in an established mTEC line. Expression of Spib in the RANK-mTEC line at 2 h and at 2 d after RANKL stimulation was determined by qPCR. Values are normalized to Gapdh expression and further normalized to the control values. (E) The UEA-1+EpCAM+ CD45TER119 (mTEC fraction), UEA-1EpCAM+ CD45TER119 (cTEC-rich fraction), and EpCAMCD45TER119 (non-TEC fraction) fractions were sorted from 6-wk-old C57BL/6 mice. The expression levels of Spib and Rank in each fraction were determined by qPCR. Values are arbitrary units normalized to Gapdh expression. (F) qPCR analysis of expression of Spib1 and Spib2, transcripts from two different promoters of Spib, in 2DG-FTOCs after 1–4 d of incubation with 1 µg/ml recombinant RANKL or before stimulation (day 0). Values are normalized to Gapdh expression and further normalized to the control values. (G) Expression of Spib1 and Spib2 in thymic stromal cells of adult mice. mTEC, cTEC-rich, and non-TEC fractions were sorted from 6-wk-old C57BL/6 mice. Expression levels of Spib1 and Spib2 in each fraction were determined by qPCR. Values are arbitrary units normalized to Gapdh expression. (H) Human and mouse Spib genes were compared using the VISTA algorithm. The arrowhead indicates the transcriptional start site for Spib1 (transcribed from Spib-P1). The CNS shows a conserved noncoding sequence upstream of the Spib1 initiation site. The black bars (Spib-P1 and Spib-P2) indicate the promoter regions isolated for the reporter assay. After RANK-mTECs were transfected with Spib-P1 luciferase reporter plasmid and a β-galactosidase plasmid and stimulated with 1 µg/ml recombinant RANKL, luciferase activities were determined. The fold induction was calculated on the basis of the luciferase activity of unstimulated cells. (I) Overexpression of NIK or RelB complex, but not TRAF6, activates Spib-P1. HEK293T cells were transfected with expression vectors encoding NIK, TRAF6, or a combination of RelB and p50 together with a Spib-P1 luciferase reporter plasmid or artificial NF-κB promoter (3κB) and a β-galactosidase plasmid as an internal control for transfection efficiency. The 3κB promoter contains three typical NF-κB–binding sites. The fold induction was calculated on the basis of the vector control sample. Representative data from three independent triplicate experiments are shown in the graphs. (J) RANKL-mediated Spib expression in 2DG-FTOCs is dependent on functional NIK. qPCR analysis of Spib expression in aly/+ and aly/aly 2DG-FTOCs at 4 d after incubation with 1 µg/ml recombinant RANKL was performed. Error bars represent mean ± one SD for three independent experiments (B–H and J) or triplicate experiments (I). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Student’s t test).
Figure 2.
Figure 2.
Spi-B limits development of mature mTECs and up-regulates CD80 and CD86 expression in mature mTECs. (A) Thymic stromal cells (CD45TER119) among total thymic cells of 3-wk-old WT and Spib−/− mice were analyzed by staining with MHC II (I-A/I-E) antibody and UEA-1 lectin, an mTEC marker (left). Flow cytometric data are summarized in the graphs. The percentages of MHC IIhiUEA-1+ (mTEChi), MHC IIloUEA-1+ (mTEClo), and UEA-1MHC II+ (mainly cTEC) cells among thymic stroma cells and their numbers in the thymus are shown. (B) 2DG-FTOC prepared from a Spib−/− or WT fetus (six lobes each) were transplanted into the kidneys of nude mice. The grafted thymuses were analyzed by flow cytometry at 6 wk after transplantation. The numbers of MHC II+UEA-1+, MHC IIhiUEA-1+ (mTEChi), MHC IIloUEA-1+ (mTEClo), and UEA-1MHC II+ (mainly cTEC) cells in the transplanted thymuses are shown. (C) Expression levels of Aire in WT and Spib−/− thymuses of 3-wk-old mice were determined by qPCR. Values are arbitrary units (A.U.) normalized to Gapdh expression. (D) Flow cytometric profiles of Aire-expressing mTECs in WT and Spib−/− TECs (left) in 3-wk-old mice. TECs (EpCAM+CD45TER119) were analyzed by staining with a combination of UEA-1 lectin and Aire antibody. The percentage of Aire+ mTECs among TECs and cell number of Aire+ mTECs in the thymus are shown in the graphs on the right. (E) Flow cytometric profiles of Aire expression in Aire-expressing mTECs of 3-wk-old WT and Spib−/− mice. Expression of Aire in Aire+UEA-1+EpCAM+CD45TER119 of WT and Spib−/− mice was analyzed. Expression levels of Aire as means of fluorescence intensity (MFI) are summarized in the graph below. (F) Flow cytometric profiles of Aire-expressing mTECs in WT and Spib−/− TECs (left). TECs (EpCAM+CD45TER119) of 3-wk-old mice were analyzed by staining with a combination of CD80 and Aire antibodies. The percentage of Aire+CD80hi mTECs among TECs is shown in the graph on the right. (G) Flow cytometric profiles of CD80 expression in mTECs of 3-wk-old WT and Spib−/− mice. Expression of CD80 in mTECs expressing high levels of MHC II (MHC IIhi) and mTECs expressing low levels of MHC II (MHC IIlo) of WT and Spib−/− mice were analyzed. Expression levels of CD80 as means of fluorescence intensity are summarized in the graph on the right. (H) Flow cytometric profiles of MHC II+CD86+ cells in mTECs (UEA-1+EpCAM+CD45TER119) of WT and Spib−/− mice (left). The percentage of MHC IIhiCD86hi cells (left, rectangles) among mTECs is shown in the graph on the right. (I) Flow cytometric profiles of PD-L1 and CD40 expression in mTECs expressing high levels of MHC II (MHC IIhi) of WT and Spib−/− mice. Error bars represent mean ± one SD for four (A and D–H) or three (B and C) independent experiments. *, P < 0.05, **, P < 0.01; and ***, P < 0.001 (Student’s t test).
Figure 3.
Figure 3.
Spi-B promotes expressions of decoy receptor OPG and thereby limits mTEC development. (A) Expression of Opg, Rank, Ccl19, Spt1, Col2, and Csnb in mTECs sorted from the thymuses of Spib−/− and WT mice was analyzed by qPCR. Salivary protein 1, collagen 2, and casein-β are TSAs promiscuously expressed in mTECs. Values are arbitrary units (A.U.) normalized to Gapdh expression. Per experiment, the mTECs were isolated and pooled from three WT or three Spib−/− mice (3–4 wk old). (B) Thymic cryosections of 3-wk-old WT and Spib−/− mice were immunostained with anti-OPG and UEA-1 antibodies. White arrows indicate OPG expression. Figures are representatives of three independent experiments. Bars, 200 µm. (C, left) qPCR analysis of Opg expression in 2DG-FTOCs at 4 d after incubation with 1 µg/ml recombinant RANKL (+RANKL) or without RANKL (control). For blocking experiments (+RANKL + RANKL-Fc), 5 µg/ml RANK-Fc was added to the culture medium. Values are arbitrary units normalized to Gapdh expression. (right) Expression of Opg in 2DG-FTOC at 1–4 d after incubation with 1 µg/ml recombinant RANKL (+RANKL) or before RANKL stimulation (day 0) was analyzed by qPCR. Values are normalized to Gapdh expression and further normalized to the control values. (D) qPCR analysis was performed to determine Opg expression levels in WT and Spib−/− 2DG-FTOCs at 4 d after incubation with 1 µg/ml recombinant RANKL. Values are arbitrary units normalized to Gapdh expression. (E) DNA methylation analysis of Opg in the mTEC and EpCAM stromal cell fractions sorted from the thymuses of 3-wk-old Spib−/− and WT mice. Each T-DMR contains four CpG sites. The DNA methylation status of CpGs in T-DMRs is indicated as open (unmethylated) and closed (methylated) circles. Percentages of unmethylated CpGs in T-DMR are indicated under each panel. T-DMRs and the transcription start site of Opg are schematically shown at the bottom of the panels. Data of methylated CpGs were obtained from three independent pairs of Spib−/− and WT mice and are summarized in the graphs on the right. (F) Typical flow cytometric profiles of TECs prepared from 3-wk-old Opg−/− and Spib−/−Opg−/− mice (C57BL/6 background). Flow cytometric analyses of Opg−/− and Spib−/−Opg−/− thymic stromal cells are summarized in the graphs. The percentage of MHC II+UEA-1+, MHC IIhiUEA-1+ (mTEChi), MHC IIloUEA-1+ (mTEClo), and UEA-1MHC II+ (mainly cTEC) cells among thymic cells and their cell numbers are shown. Red and green dotted lines indicate data from 3-wk-old Spib−/− mice and WT mice, respectively. Error bars represent mean ± one SD for four (A and F) or three (C–E) independent experiments. *, P < 0.05, **, P < 0.01; and ***, P < 0.001 (Student’s t test).
Figure 4.
Figure 4.
Spi-B–OPG system fine-tunes mTEC development from neonates via negative feedback regulation. (A) Flow cytometric analysis of mTECs prepared from WT and Spib−/− mice at E15.5 (WT, n = 9; and Spib−/−, n = 6; P > 0.05), at E17.5 (WT, n = 8; and Spib−/−, n = 8; P > 0.05), PN1 (WT, n = 7; and Spib−/− mice, n = 9; P = 1.52 × 10−6), and PN8 (WT, n = 4; and Spib−/−, n = 5; P = 3.03 × 10−5). Typical flow cytometric profiles of MHC II (I-A/I-E) and UEA-1 lectin–positive cells, which are also EpCAM+, among thymic stroma cells (CD45+TER119) are shown. Percentages of mTECs among thymic stroma cells are summarized in the graph on the right. (B) Immunostaining of thymic sections from WT and Spib−/− mice at E17.5, PN1, and PN8. Thymic cryosections were immunostained with keratin-5 and UEA-1 lectin antibodies. Figures are representative of three independent experiments. Bars, 200 µm. (C) Expression levels of Aire in whole thymus from WT and Spib−/− mice at E17.5 (WT, n = 4; and Spib−/−, n = 3; P > 0.1), PN1 (WT, n = 7; and Spib−/−, n = 9; P = 7.4 × 10−4), and PN8 (WT, n = 4; and Spib−/−, n = 4; P = 0.045) were determined by qPCR. The values are arbitrary units (A.U.) normalized to 36B4 expression. (D) Flow cytometric profiles of thymic cells prepared at E15.5 (WT, n = 5; and Opg−/−, n = 4; P > 0.5), E17.5 (WT, n = 8; and Opg−/−, n = 4; P = 0.0022), PN1 (WT, n = 8; and Opg−/−, n = 6; P = 4.91 × 10−8), and PN8 (WT, n = 8; and Opg−/−, n = 4; P = 1.94 × 10−8). Typical flow cytometric profiles of MHC II (I-A/I-E)– and UEA-1 lectin–positive cells, which are also EpCAM+ (not depicted), among thymic stroma cells (CD45+TER119) are shown on the left. Percentages of mTECs among thymic stroma cells are summarized in the graph on the right. (E) Flow cytometric profiles of thymic cells prepared at E15.5 (WT, n = 5; and Opg−/−, n = 4; P > 0.2), E17.5 (WT, n = 8; and Opg−/−, n = 4; P > 0.8), PN1 (WT, n = 8; and Opg−/−, n = 6; P = 1.19 × 10−5), and PN8 (WT, n = 8; and Opg−/−, n = 4; P = 1.21 × 10−7). Typical flow cytometric profiles of CD80- and UEA-1 lectin-positive cells, which are also EpCAM+ (not depicted), among thymic stroma cells (CD45+TER119) are shown on the left. Percentages of CD80hi mature mTECs in thymic stroma cells are summarized in the graph on the right. (A and C–E) Black bars indicate mean values. (F) qPCR analysis of Opg in mature mTECs (CD80hiUEA-1+EpCAM+CD45TER119), immature mTECs (CD80loUEA-1+EpCAM+CD45TER119), and cTECs (CD80loUEA-1EpCAM+CD45TER119) sorted from thymuses of WT mice at PN1 (cTECs, n = 6; immature mTECs, n = 7; and mature mTECs, n = 5). Values are arbitrary units normalized to 36B4 expression. Per experiment, the mTECs were isolated from three neonates and pooled for RNA preparation. (G) qPCR analysis of Opg in mature mTECs (CD80hiUEA-1+EpCAM+CD45TER119) sorted from thymuses of WT mice at E17.5 (n = 3), PN1 (n = 5), PN4 (n = 4), and PN14 (n = 4). Values are arbitrary units normalized to 36B4 expression. Per experiment, the mTECs were isolated from several embryos and neonates and pooled for RNA preparation. Error bars represent mean ± one SD. (H) qPCR analysis of Spib in immature mTECs (CD80loUEA-1+EpCAM+CD45TER119) and mature mTECs (CD80hiUEA-1+EpCAM+CD45TER119) sorted from thymuses of Opg−/− and WT mice at PN1 (n = 3). Per experiment, mTECs were isolated from several neonates and pooled for RNA preparation. Values are arbitrary units normalized to 36B4 expression. (F and H) Error bars represent mean ± one SD for four independent experiments. *, P < 0.05, **, P < 0.01; and ***, P < 0.001 (Student’s t test).
Figure 5.
Figure 5.
OPG attenuates TSA expressions and frequency of T reg cells in the thymus. (A) Expression of Aire, Spt1, Col2, and Csnb in the whole thymus of Opg−/− and WT mice was analyzed by qPCR. *, P < 0.05; and **, P < 0.01 (Student’s t test). Values are arbitrary units (A.U.) normalized to Gapdh expression. Error bars represent mean ± one SD for three independent experiments. (B) Flow cytometric analysis of thymocytes of WT and Opg−/− mice at PN1 (WT, n = 3; and Opg−/−, n = 3), PN3 (WT, n = 8; and Opg−/−, n = 5), PN8 (WT, n = 6; and Opg−/−, n = 6), and PN18 (WT, n = 5; and Opg−/−, n = 5). Typical flow cytometric profiles at PN18 are shown on the left. Percentages of Foxp3+CD25+ in CD4SP are summarized in the graph on the right. P-values were determined by Student’s t test between Opg−/− and WT mice: **, P < 0.01; ***, P < 0.001 (P = 3.6 × 10−4 at PN8 and P = 4.6 × 10−3 at PN18). Error bars represent mean ± one SD. T reg cell numbers are shown in the graphs below. P-values were determined by Student’s t test between Opg−/− and WT mice: **, P < 0.01 (P = 0.052 at PN8 and P = 4.2 × 10−3 at PN18). (C) Flow cytometric analysis of thymocytes of WT and Opg−/− mice at PN20 (WT, n = 8; and Opg−/−, n = 8). The percentages of GITR+CD25+ in Foxp3CD4SP and numbers of GITR+CD25+Foxp3CD4SP are summarized in the graphs. P-values were determined by Student’s t test between Opg−/− and WT mice: *, P = 0.036; ***, P = 8.1 × 10−4. (D) mTECs and T reg cells of in vitro FTOC from WT and Opg−/− mice were analyzed by flow cytometry. Percentages of mTECs in stroma cells (CD45TER119) and T reg cells in CD4SP cells and numbers of mTECs and T reg cells per thymic lobe in WT and Opg−/− FTOC are summarized in the graphs (WT FTOC, n = 4; and Opg−/− FTOC, n = 3). The fetal thymuses of WT or Opg−/− mice were cultured for 7 d. Per experiment, FTOCs from three to four embryos were pooled. Cell numbers per thymic lobe were exhibited in graphs. P-values were determined by Student’s t test between Opg−/− and WT mice: *, P = 0.036 for Foxp3+ Treg; *, P = 0.013 for mTEC; ***, P = 1.3 × 10−4 for Foxp3+ Treg; and ***, P = 4.1 × 10−4 for mTEC. (E) 2DG-FTOCs prepared from Opg−/− or WT fetuses were transplanted into the kidney of nude mice (Opg−/−/nude or WT/nude). The grafted thymuses and spleen were analyzed by flow cytometry 8 wk after transplantation. Typical flow cytometric profiles are shown at the top. The ratios of CD4SP cells to total thymocytes, Foxp3+CD25+ cells to CD4SP cells, CD4+ and CD8+ T cells to splenocytes, and Foxp3+CD4+ T cells to splenocytes are summarized in the graphs (WT/nude, n = 4; and Opg−/−/nude, n = 3). P-values were determined by Student’s t test between Opg−/− and WT mice: *, P < 0.05. (B–E) Black bars indicate mean values.
Figure 6.
Figure 6.
OPG expressed in thymic stromal cells attenuates tumor development and growth. (A) Chimeric nude mice receiving Opg−/− or WT 2DG-FTOCs were examined once a week for tumor development after MCA injection. The red line indicates the tumor-free rate of the chimeric nude mice receiving Opg−/− 2DG-FTOCs (n = 14), and the black line indicates that of chimeric mice receiving WT 2DG-FTOCs (n = 13). P = 0.033: log-rank test between Opg−/− and WT chimeric mice sets regarding the tumor-free rate. (B) Tumor cells (Meth A; female, BALB/c background) were subcutaneously transferred to nude mice receiving Opg−/− (tumor → Opg−/−/nude; n = 7) or WT 2DG-FTOCs (tumor → WT/nude; n = 11). The left panel shows a typical image of tumors that developed in these mice. Tumor volumes were measured on days 10, 12, and 15 (middle graph) after tumor transfer. Asterisks indicate statistical significance: *, P < 0.05 (Student’s t test). Error bars represent one SEM. Tumor weight was measured after sacrificing the mice (right graph; asterisk indicates significant difference by Mann–Whitney U test: *, P < 0.05). (C) Tumors developed from Meth A cells were analyzed. Tumor-infiltrating lymphocytes in nude mice receiving Opg−/− (tumor → Opg−/−/nude) or WT 2DG-FTOC (tumor → WT/nude) were analyzed by flow cytometry. The number of CD4+ T and CD8+ T cells in 1 g of tumor from nude mice receiving Opg−/− 2DG-FTOCs (tumor → Opg−/−/nude) and WT 2DG-FTOCs (tumor → WT/nude) was determined on day 15 after tumor cell (Meth A) transfer. Asterisks indicate statistical significance: *, P < 0.05 (Student’s t test). Error bars represent mean ± one SD for the indicated number of independent experiments (WT, n = 5; and KO, n = 4). (D) The ratio of Foxp3+CD25+ cells to CD4+ cells in the lymph nodes of nude mice receiving Opg−/− (tumor → Opg−/−/nude) or WT 2DG-FTOCs (tumor → WT/nude) was determined on day 15 after tumor cell (Meth A) transfer. Asterisks indicate statistical significance: **, P < 0.01 (Student’s t test). Error bars represent mean ± one SD for the indicated number of independent experiments (WT, n = 11; and KO, n = 7). (E) Serum antibodies obtained from the nude mice receiving Opg−/− (tumor → Opg−/−/nude) or WT 2DG-FTOCs (tumor → WT/nude) were analyzed for tumor reactivity after tumor cell (Meth A) transfer. Meth A cells were immunostained with mouse sera (left: green, sera; red, propidium iodide for nuclear staining). Immunostaining data of Meth A with sera from tumor-transplanted mice are shown in the two top panels. Immunostaining data with WT/nude sera (without tumor transplantation) and nude sera (without transplantation of 2DG-FTOCs and tumor) are shown in bottom panels. Data are representative of three independent experiments. Western blot analysis of Meth A cell lysate using the serum from tumor → Opg−/−/nude or WT tumor → WT/nude mice (middle). Cell numbers of Meth A loaded as cell lysate are indicated at the top of the panels. Data are representative of three independent experiments. Arrows indicate protein bands that were reduced or disappeared in Western blots using sera of Opg−/−/nude mice receiving tumors. Molecular masses (kilodaltons) are indicated between the two panels. The intensity of each band indicated by a green arrow was quantified and normalized to tubulin levels (right graph). Error bars represent mean ± one SD for three independent experiments. *, P < 0.05 (Student’s t test). Bars: (B) 1 cm; (E) 50 µm.

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