Memet Fuat, Piraye ve Nazım

Taha Toros Arşivi, Dosya Adı: Nazım Hikmetİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

HTLV-1 bZIP Factorは共抑制分子TIGITを誘導し、抗ウイルス免疫を抑制する

京都大学0048新制・課程博士博士(医学)甲第19594号医博第4101号新制||医||1014(附属図書館)32630京都大学大学院医学研究科医学専攻(主査)教授 竹内 理, 教授 河本 宏, 教授 朝長 啓造学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

HBZ suppresses Fgl2 and CD226 expression.

<p>(A) <i>Fgl2</i> mRNA was quantified in HBZ-expressing murine primary CD4⁺ T cells and control cells by realtime PCR. This experiment was carried out in triplicate, and the mean values ± SD are shown. Three independent experiments were performed and the representative results are shown. (B) The levels of <i>Fgl2</i> mRNA were measured in CD4⁺ T cells from non-Tg (n = 4) and TIGIT⁺CD4⁺ and TIGIT^-CD4⁺ T cells from HBZ-Tg (n = 3) mice. (C) Jurkat cells were cotransfected with 500 ng of FGL2-promoter-Luc, pME18Sneo-sHBZ, 500 ng of pcDNA-C/EBPα and pGL4-TK. Luciferase activity was measured after 24 hours. Luciferase assay was carried out in triplicate, and the mean values ± SD are shown. Three independent experiments were performed and the representative results were shown. (D) The <i>CD226</i> gene transcript was quantified in HBZ-expressing murine primary CD4⁺ T cells and control cells. This assay was carried out in triplicate and the mean values ± SD are shown. Three independent experiments were performed and the representative results were shown. (E) The <i>CD226</i> gene expression was measured in CD4⁺ T cells from HBZ-Tg (n = 7) and non-Tg (n = 7) mice by realtime PCR. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

HBZ upregulates TIGIT expression.

<p>(A) <i>TIGIT</i> mRNA was increased in CD4⁺ T cells of HBZ-Tg mice (n = 7) compared with those of non-Tg mice (n = 7). (B) TIGIT expression was enhanced on resting and activated CD4⁺ T cells from HBZ-Tg mice by flow cytometry (FCM). The representative histogram of two independent experiments is shown. (C) A retrovirus expressing HBZ or control retrovirus was transduced into mouse primary CD4⁺ T cells. <i>TIGIT</i> transcripts were measured by realtime PCR. Results are the mean ± SD in triplicate. Three independent experiments were performed and the representative results are shown. (D) Expression of TIGIT on HBZ transduced mouse primary CD4⁺ T cells was measured by FCM. The representative result was shown for two independent experiments. (E) Expression of TIGIT on CD3^lowCD4⁺ T cells from ATL patients (n = 5) and CD4⁺CADM1⁺ T cells from HAM/TSP patients (n = 6) and healthy donors (HD, n = 6) were measured by FCM. (F) Expression of TIGIT on HBZ-transduced murine primary CD4⁺ cells and control cells in the presence or absence of TGF-β was measured by FCM. Two independent experiments were performed and the representative results are shown. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

TIGIT suppresses T-cell response to Tax.

<p>(A) The production of IL-10 and IL-12 by DCs was modulated in HBZ-Tg mice. Splenocytes from HBZ-Tg mice and non-Tg mice were stimulated by LPS for 12 hours, and then DCs were sorted by FACS Aria II. The levels of <i>IL-10</i> and <i>IL-12</i> mRNA in DCs were measured by realtime PCR. Transcripts were quantified in triplicate and the mean values ± SD are shown. (B) Splenocytes from HBZ-Tg and non-Tg mice were stimulated by LPS overnight followed by sorting for DCs. Sorted cells were then cultured in the presence of LPS for 24 hours. The protein levels of IL-10 in cultured supernatants were measured by Cytometric Bead Array (CBA). Three independent experiments were performed and the representative result was shown. (C) Mice were immunized with Tax protein and CpG at day 0 and 14, and then sacrificed at day 21 for ELISPOT analysis. (D) Splenocytes from a Tax-immunized mouse were subjected to ELISPOT assay using Tax peptide stimulation along with TIGIT-Fc or control Fc conjugated beads. This assay was performed in triplicate and the results shown are the mean ± SD. The representative result was shown from two independent experiments. (E) HBZ-Tg mice (n = 4) and non-Tg mice (n = 4) were immunized by Tax protein and CpG, and then T-cell responses were measured by ELISPOT assay. (F) PBMCs from seven HAM/TSP patients (n = 7) were subjected to ELISPOT assay with Tax peptide stimulation in the presence or absence of anti-TIGIT and/or anti-PD-1 antibodies. The number of IFN-γ secreting cells normalized to the negative control is shown. Each symbol represented each patient. Results shown are the mean ± SD. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Effect of HBZ on the <i>TIGIT</i> gene promoter.

<p>(A) A retrovirus expressing wild-type HBZ, HBZ-RNA (TTG), HBZ-protein (SM) or control retrovirus was transduced into mouse primary CD4⁺ T cells. TIGIT expression on transduced T cells was analyzed by FCM. Two independent experiments were performed, and the representative results are shown. (B) <i>TIGIT</i>-promoter-Luc vector was transfected to Jurkat cells with wt or mutant HBZ. Luciferase activity was measured 24 hours after transfection. (C) <i>TIGIT</i>-promoter-Luc vector was transfected to Jurkat cells with 600 ng of wt or mutant HBZ. Luciferase activity was measured four hours after PMA/ionomycin (P/I) stimulation. (D) Jurkat cells were cotransfected with TIGIT-promoter-Luc, 300 ng of Tax expression vector, 100 ng of vectors expressing wt or mutant HBZ, and pGL4-TK. Two independent experiments were performed, and the representative results are shown. Results are the mean ± SD. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

TIGIT transcription is regulated by HBZ.

<p>(A) Volcano plots of p-value (y-axis) against log₂ fold change (x-axis: HBZ expressing murine primary T cells vs. control cells) for RNA-seq data. (B) RNA-seq and ChIP-seq results of three Treg related genes. RNA-seq was performed twice and ChIP-seq was performed once for each. (C) The enrichment of the <i>TIGIT</i> promoter region by the indicated histone marks or isotype control was measured by ChIP-qPCR. The % input of HBZ expressing murine primary CD4⁺ T cells or control cells is shown. (D) The enrichment of the TIGIT promoter regions by anti-FLAG was analyzed by ChIP-seq in HBZ-FLAG expressing cells. The detected peak in the TIGIT promoter region was shown as the dotted box. The enrichment of the TIGIT promoter regions by anti-FLAG or isotype control was measured by ChIP-qPCR using the primers indicated. Results shown are the mean ± SD in triplicate (C, D).</p

HBZ enhances IL-10 production.

<p>(A) The levels of <i>Blimp1</i> and <i>IL-10</i> mRNA were measured in HBZ-transduced murine primary CD4⁺ T cells and control cells by realtime PCR. This experiment was carried out in triplicate, and the mean values ± SD are shown. Three independent experiments were performed and the representative data are shown. (B) The levels of <i>Blimp1</i> were measured in CD4⁺ T cells from HBZ-Tg (n = 10) and non-Tg (n = 10) mice. (C) The levels of <i>IL-10</i> mRNA were measured in CD4⁺ T cells from HBZ-Tg (n = 7) and non-Tg (n = 7) mice using realtime PCR. (D) The levels of <i>Blimp1</i> and <i>IL-10</i> mRNA were measured in CD4⁺ T cells from non-Tg (n = 4) and TIGIT⁺CD4⁺ and TIGIT^-CD4⁺ T cells from HBZ-Tg (n = 3) mice. (E) Expression of IL-10 was measured by FCM in CD4⁺ T cells from HD (n = 3) and CD4⁺CADM1⁺ and CD4⁺CADM1^- cells from HAM/TSP patients (n = 5). IL-10 MFI ratio to isotype control was analyzed. (F) CD4⁺ T cells from HBZ-Tg mice and from non-Tg mice were cultured with or without plate-coated anti-CD3 mAb (1 μg/ml) and soluble anti-CD28 mAb (1 μg/ml) for 24 hours. IL-10 production was measured by ELISA. N.D., not detected. (G) CD4⁺ T cells from HBZ-Tg mice and from non-Tg mice were cultured in the presence of plate-coated anti-CD3 mAb (1 μg/ml) with or without plate-coated CD155 (1 μg/ml) for 12 hours. Results shown are the mean ± SD. The representative result was shown from two independent experiments.*<i>P</i> < 0.05, **<i>P</i> < 0.01.</p