HTLV-1 induces T cell malignancy and inflammation by viral antisense factor-mediated modulation of the cytokine signaling

Abstract

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a T cell neoplasm and several inflammatory diseases. A viral gene, HTLV-1 bZIP factor (HBZ), induces pathogenic Foxp3-expressing T cells and triggers systemic inflammation and T cell lymphoma in transgenic mice, indicating its significance in HTLV-1-associated diseases. Here we show that, unexpectedly, a proinflammatory cytokine, IL-6, counteracts HBZ-mediated pathogenesis. Loss of IL-6 accelerates inflammation and lymphomagenesis in HBZ transgenic mice. IL-6 innately inhibits regulatory T cell differentiation, suggesting that IL-6 functions as a suppressor against HBZ-associated complications. HBZ up-regulates expression of the immunosuppressive cytokine IL-10. IL-10 promotes T cell proliferation only in the presence of HBZ. As a mechanism of growth promotion by IL-10, HBZ interacts with STAT1 and STAT3 and modulates the IL-10/JAK/STAT signaling pathway. These findings suggest that HTLV-1 promotes the proliferation of infected T cells by hijacking the machinery of regulatory T cell differentiation. IL-10 induced by HBZ likely suppresses the host immune response and concurrently promotes the proliferation of HTLV-1 infected T cells.

Keywords: HBZ; HTLV-1; IL-10; IL-6; JAK/STAT signaling pathway.

Fig. 1.

Loss of IL-6 accelerates inflammation and lymphomagenesis in HBZ-Tg mice. (A) Incidence of dermatitis in WT (blue; n = 65), HBZ-Tg (yellow; n = 39), IL-6 KO (green; n = 72), and HBZ-Tg/IL-6 KO (red; n = 42) mice. These mice were observed for 1 y (log-rank test). (B) Overall survival of each strain: WT, n = 13; HBZ-Tg, n = 13; IL-6 KO, n = 14; HBZ-Tg/IL-6 KO, n = 24. These mice were observed for 2 y (log-rank test). (C) Incidence of lymphoma in each strain at age 24 wk. (D) Histopathological analysis of primary lymphoma in lymph node and spleen of an HBZ-Tg/IL-6 KO mouse. (E) Incidence of dermatitis in IL-6R^fl/fl (blue; n = 15), HBZ-Tg/IL-6R^fl/fl (yellow; n = 10), CD4-Cre/IL-6R^fl/fl (green; n = 7), and HBZ-Tg/CD4-Cre/IL-6R^fl/fl (red; n = 8) mice (log-rank test). (F) Incidence of dermatitis in IL-6R^fl/fl (blue; n = 20), HBZ-Tg/IL-6R^fl/fl (yellow; n = 14), LysM-Cre/IL-6R^fl/fl (green; n = 23), and HBZ-Tg/LysM-Cre/IL-6R^fl/fl (red; n = 17) mice (log-rank test).

Fig. 2.

IL-10–producing CD4⁺ T cells are increased in HBZ-Tg/IL-6 KO mice. (A) Flow cytometry analysis of T cell subsets. Mouse splenocytes were collected from WT, HBZ-Tg, IL-6 KO, and HBZ-Tg/IL-6 KO mice at age 4 wk. Cells were stained with anti-CD4, anti-CD44, and anti-CD62L antibodies for naïve and effector memory T cells, and with anti-Foxp3. (B) Cytokine production by CD4⁺ T cells from 4-wk-old mice. Splenocytes were stimulated with PMA/ionomycin in the presence of protein transport inhibitor for 5 h and then stained with specific antibodies. (C) Expression of Treg-related molecules in CD4⁺ cells collected from 16-wk-old mice. Splenocytes were stimulated with PMA/ionomycin in the presence of protein transport inhibitor for 5 h and then stained with specific antibodies. (D) Immunohistochemical analysis for Foxp3 in lymph node and spleen of HBZ-Tg and HBZ-Tg/IL-6 KO mice (original magnification 40×). (E) Percentage of cells that are Foxp3⁺ in primary lymphomas of HBZ-Tg (n = 3) and HBZ-Tg/IL-6 KO (n = 10) mice. The data were obtained from immunohistochemical analysis. (F) Foxp3 expression in CD4⁺CADM1⁺ T cells from HTLV-1–infected subjects. AC, asymptomatic carrier (n = 16); HAM/TSP, HTLV-1–associated myelopathy/tropical spastic paraparesis (n = 28); ATL, adult T cell leukemia/lymphoma (n = 20).

Fig. 3.

Expression profiles of splenic CD4⁺ T cells in each strain. (A) Volcano plots obtained from RNA-seq analysis of splenic CD4⁺ T cells. Red spots indicate genes that are significantly up-regulated in both a comparison of WT mice (n = 2) and HBZ-Tg mice (n = 2) and in a comparison of HBZ-Tg and HBZ-Tg/IL-6 KO mice (n = 2), while blue spots represent genes that are significantly down-regulated in both experiments (FDR >0.1). (B) Venn diagrams of the overlap of significantly up-regulated and down-regulated genes in WT vs. HBZ-Tg and HBZ-Tg vs. HBZ-Tg/IL-6 KO mice. (C) Heat map of expression profiles for the 26 significant genes (23 up-regulated and 3 down-regulated). (D) Expression tiling of Il10 in each sample. Genomic views of transcription at the Il10 gene locus in WT, HBZ-Tg, and HBZ-Tg/IL-6 KO mice are shown. The y-axis represents the number of reads at the locus, and the maximum read count is set to 500 for all samples. (E) Venn diagrams of significantly up-regulated KEGG pathways in WT vs. HBZ-Tg mice and in HBZ-Tg vs. HBZ-Tg/IL-6 KO mice. (F) Bar plots of significantly (P < 0.05) up-regulated KEGG pathways.

Fig. 4.

HBZ modifies the IL-10/JAK/STAT signaling pathway. (A) Fluorescence dilution assay of CD4⁺ T cells from WT or HBZ-Tg mice. Splenic CD4⁺ T cells were labeled with 5 μM CellTrace Violet and stimulated by anti-CD3 antibodies with or without IL-10. At 48 h after stimulation, CellTrace Violet was measured by flow cytometry (two-way ANOVA with Turkey’s multiple comparisons). (B) Coimmunoprecipitation of HBZ and human STAT1 or STAT3. The indicated expression vectors were cotransfected into HEK293T cells, and protein interactions were analyzed by immunoprecipitation. (C) Interaction of HBZ mutants with STAT1 or STAT3 was analyzed by immunoprecipitation. (D and E) Interaction between HBZ and STAT proteins in ATL-43T(−). A protein extract of ATL-43T(−) cells was subjected to immunoprecipitation with anti-HBZ antibody or control IgG, and STAT proteins were detected by anti-STAT1, anti-STAT3, anti-STAT5, or anti-STAT6 antibody. (F) Colocalization of HBZ and STAT proteins. HBZ-myc and STAT1 or STAT3-3xFLAG were transfected into Jurkat cells by electroporation. Staining was performed using antibodies against myc (green) and FLAG (red). Nuclei were stained with DAPI (blue).

Fig. 5.

HBZ modulates IL-10/JAK/STAT signaling. (A) Flow cytometry analysis of IL10RA in HEK293-IL10R. Cell were stained by PE-conjugated anti-IL10RA antibody (black line) or isotype control (gray filled); two-tailed unpaired Student’s t test. (B–D, Top) Luciferase assay of ISRE, GAS, and SIE. HEK293-IL10R cells were transfected with HBZ, each reporter, and a reporter plasmid driven by cytomegalovirus immediate-early promoter as an internal control, and then stimulated with IL-10 (100 ng/mL). At 24 h after stimulation, luciferase activities were measured (two-way ANOVA with Turkey’s multiple comparisons). (Bottom) Immunoblotting of HBZ, STAT1, pSTAT1, STAT3, pSTAT3, and tubulin. (E) Volcano plots obtained from RNA-seq analysis of splenic CD4⁺ T cells of WT or HBZ-Tg (n = 3) cultured with or without IL-10 for 48 h. Green spots indicate differentially expressed genes (P < 0.05) between IL-10–treated and untreated cells. The results from WT and HBZ-Tg mice are shown in the left and right diagrams, respectively. (F) Enriched gene sets modulated by HBZ and IL-10 using GSEA. The top 2,000 differentially expressed genes in IL-10–treated cells compared with untreated cells from WT or HBZ-Tg mice were analyzed. (G and H) GSEA enrichment plots and heat maps of gene expression in CD4⁺ T cells from HBZ-Tg mice, with or without IL-10 stimulation.

Fig. 6.

Schema of HBZ-mediated T cell proliferation. HBZ and loss of IL-6 enhances Treg differentiation and IL-10 production. Increased IL-10 activates STAT proteins, and HBZ modulates the IL-10/JAK/STAT signaling toward proinflammatory and proliferative properties.