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Cited by 319T-cell and Retrovirus StudiesT-cell and B-cell ImmunologyViral-associated cancers and disorders

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HTLV-1 bZIP Factor Induces T-Cell Lymphoma and Systemic Inflammation In Vivo

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Satou, Yorifumi, Yasunaga, Jun-ichirou, Zhao, Tiejun, Yoshida, Mika, Miyazato, Paola, Takai, Ken, Shimizu, Kei, Ohshima, Koichi, Green, Patrick L., Ohkura, Naganari, Yamaguchi, Tomoyuki, Ono, Masahiro, Sakaguchi, Shimon, Matsuoka, Masao·2011·PLoS Pathogens
DOI 10.1371/journal.ppat.1001274PMC PMC3037353
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Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of a neoplastic disease of CD4+ T cells, adult T-cell leukemia (ATL), and inflammatory diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis, dermatitis, and inflammatory lung diseases. ATL cells, which constitutively express CD25, resemble CD25+CD4+ regulatory T cells (T(reg)). Approximately 60% of ATL cases indeed harbor leukemic cells that express FoxP3, a key transcription factor for T(reg) cells. HTLV-1 encodes an antisense transcript, HTLV-1 bZIP factor (HBZ), which is expressed in all ATL cases. In this study, we show that transgenic expression of HBZ in CD4+ T cells induced T-cell lymphomas and systemic inflammation in mice, resembling diseases observed in HTLV-1 infected individuals. In HBZ-transgenic mice, CD4+Foxp3+ T(reg) cells and effector/memory CD4+ T cells increased in vivo. As a mechanism of increased T(reg) cells, HBZ expression directly induced Foxp3 gene transcription in T cells. The increased CD4+Foxp3+ T(reg) cells in HBZ transgenic mice were functionally impaired while their proliferation was enhanced. HBZ could physically interact with Foxp3 and NFAT, thereby impairing the suppressive function of T(reg) cells. Thus, the expression of HBZ in CD4+ T cells is a key mechanism of HTLV-1-induced neoplastic and inflammatory diseases.

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Figure 1 HBZ-Tg mice spontaneously develop inflammatory diseases in skin and lung. (A) Cell-type specific transcription of the transgene in line 12 was confirmed by RT-PCR in each sorted cell population. (B) The expression of HBZ protein in CD4+ splenocytes was confirmed by Western blotting. (C) Transcripts of the HBZ gene in CD4+ splenocyte of HBZ-Tg mice or ATL cell lines were quantified by real time PCR. ATL-43T and MT-1 are derived from ATL cells. (D) An HBZ-Tg mouse with typical skin symptom (Arrow indicates skin lesion). (E) The incidence of dermatitis in HBZ-Tg (line 12) and non-Tg mice. (F and G) Histological findings of the skin and the lung in HBZ-Tg mice. Lymphocytes massively infiltrated the dermis and epidermis (F) and the alveolar septum (G) (Arrows present infiltration of lymphocytes). Infiltration of CD3+, CD4+ T cells into these tissues was shown by immunohistochemistry compared with non-Tg mice as control.
Figure 1 HBZ-Tg mice spontaneously develop inflammatory diseases in skin and lung. (A) Cell-type specific transcription of the transgene in line 12 was confirmed by RT-PCR in each sorted cell population. (B) The expression of HBZ protein in CD4+ splenocytes was confirmed by Western blotting. (C) Transcripts of the HBZ gene in CD4+ splenocyte of HBZ-Tg mice or ATL cell lines were quantified by real time PCR. ATL-43T and MT-1 are derived from ATL cells. (D) An HBZ-Tg mouse with typical skin symptom (Arrow indicates skin lesion). (E) The incidence of dermatitis in HBZ-Tg (line 12) and non-Tg mice. (F and G) Histological findings of the skin and the lung in HBZ-Tg mice. Lymphocytes massively infiltrated the dermis and epidermis (F) and the alveolar septum (G) (Arrows present infiltration of lymphocytes). Infiltration of CD3+, CD4+ T cells into these tissues was shown by immunohistochemistry compared with non-Tg mice as control.CC BY · PubMed Central
Figure 2 HBZ-Tg mice develop T-cell lymphoma after a long latent period. (A) BrdU was injected into mice twice a day for three days, and splenocytes were stained with antibodies to BrdU, CD4, CD8, and B220. (B) Incidence of T-cell lymphoma in HBZ-Tg mice was statistically significant compared with that in non-Tg mice (P<0.001 by the logrank test). (C) Pleomorphic lymphoma in the cervical lymph node in a representative HBZ-Tg mouse. Infiltrations of lymphoma cells into lung, bone marrow, spleen and liver are also shown. (D) Expression of CD3 and CD4 in lymphoma cells was shown by immunohistochemical staining. (E) Immunohistochemical staining for Foxp3 in primary lymphomas of HBZ-Tg mice.
Figure 2 HBZ-Tg mice develop T-cell lymphoma after a long latent period. (A) BrdU was injected into mice twice a day for three days, and splenocytes were stained with antibodies to BrdU, CD4, CD8, and B220. (B) Incidence of T-cell lymphoma in HBZ-Tg mice was statistically significant compared with that in non-Tg mice (P<0.001 by the logrank test). (C) Pleomorphic lymphoma in the cervical lymph node in a representative HBZ-Tg mouse. Infiltrations of lymphoma cells into lung, bone marrow, spleen and liver are also shown. (D) Expression of CD3 and CD4 in lymphoma cells was shown by immunohistochemical staining. (E) Immunohistochemical staining for Foxp3 in primary lymphomas of HBZ-Tg mice.CC BY · PubMed Central
Figure 3 Transgenic expression of HBZ in CD4+ T cells increases Foxp3+ Treg cells with impaired suppressive function. (A and B) Mouse splenocytes were stained with the indicated antibodies, and analyzed by flow cytometry. Representative dot plots gated on the CD4+ population are shown. For these experiments, HBZ-Tg mice without any symptoms were used. Tables show the mean ± SD (n = 5 for A, n = 3 for B). (C) The absolute number of Foxp3+ or Foxp3− CD4+ T cells in HBZ-Tg and non-Tg mice. The results shown are the mean ± SD (n = 3). (D) Flow cytometric analysis for the Foxp3 expression in CD4 single positive thymocytes. Representative dot plots gated on the CD4 single positive population are shown from three independent analyses. (E) Flow cytometric analyses of CD4+ T cells for Treg related molecules. Numbers in dot plots indicate mean fluorescence intensity (MFI) of each molecule in the rectangular gates. (F) Suppressive activity of Treg cells from HBZ- or non-Tg mice on T-cell proliferation. Sorted Foxp3+ T cells were cultured with CD4+CD25− cells of non-Tg mice as responder cells for 72 h with ConA or soluble anti-CD3 antibody and x-irradiated antigen presenting cells (APCs), and [3H] thymidine incorporation during the last 6 hours was measured. Results are means ± SD for triplicate cultures. (G) In vivo BrdU incorporation in total CD4+, Foxp3+CD4+, or Foxp3−CD4+ T cells. The results shown are the mean ± SD (n = 3). (H) Sorted Foxp3+ cells were labeled with CFSE and cultured with anti-CD3 antibody and x-irradiated APCs. After 96 hours, the cells were stained with anti-Foxp3, and CFSE dilution was analyzed for Foxp3+ cells. *, P<0.01; **, P<0.001 by two-tailed Student t-test.
Figure 3 Transgenic expression of HBZ in CD4+ T cells increases Foxp3+ Treg cells with impaired suppressive function. (A and B) Mouse splenocytes were stained with the indicated antibodies, and analyzed by flow cytometry. Representative dot plots gated on the CD4+ population are shown. For these experiments, HBZ-Tg mice without any symptoms were used. Tables show the mean ± SD (n = 5 for A, n = 3 for B). (C) The absolute number of Foxp3+ or Foxp3− CD4+ T cells in HBZ-Tg and non-Tg mice. The results shown are the mean ± SD (n = 3). (D) Flow cytometric analysis for the Foxp3 expression in CD4 single positive thymocytes. Representative dot plots gated on the CD4 single positive population are shown from three independent analyses. (E) Flow cytometric analyses of CD4+ T cells for Treg related molecules. Numbers in dot plots indicate mean fluorescence intensity (MFI) of each molecule in the rectangular gates. (F) Suppressive activity of Treg cells from HBZ- or non-Tg mice on T-cell proliferation. Sorted Foxp3+ T cells were cultured with CD4+CD25− cells of non-Tg mice as responder cells for 72 h with ConA or soluble anti-CD3 antibody and x-irradiated antigen presenting cells (APCs), and [3H] thymidine incorporation during the last 6 hours was measured. Results are means ± SD for triplicate cultures. (G) In vivo BrdU incorporation in total CD4+, Foxp3+CD4+, or Foxp3−CD4+ T cells. The results shown are the mean ± SD (n = 3). (H) Sorted Foxp3+ cells were labeled with CFSE and cultured with anti-CD3 antibody and x-irradiated APCs. After 96 hours, the cells were stained with anti-Foxp3, and CFSE dilution was analyzed for Foxp3+ cells. *, P<0.01; **, P<0.001 by two-tailed Student t-test.CC BY · PubMed Central
Figure 4 HBZ directly induces Foxp3 expression in CD4+ T cells. (A) Schematic diagrams of retrovirus vectors used in this study. (B) Mouse CD4+CD25− T cells transduced with retrovirus vector encoding HBZ or empty vector with or without TGF-β were stained with anti-Foxp3 antibody and analyzed by flow cytometry. (C) To study the effect of HBZ on promoter activity of the Foxp3 gene, EL4 cells were transfected with Foxp3 reporter plasmid and/or HBZ expressing plasmid. Representative data shown are firefly luciferase activities normalized to those of renilla luciferase (mean ± SD).
Figure 4 HBZ directly induces Foxp3 expression in CD4+ T cells. (A) Schematic diagrams of retrovirus vectors used in this study. (B) Mouse CD4+CD25− T cells transduced with retrovirus vector encoding HBZ or empty vector with or without TGF-β were stained with anti-Foxp3 antibody and analyzed by flow cytometry. (C) To study the effect of HBZ on promoter activity of the Foxp3 gene, EL4 cells were transfected with Foxp3 reporter plasmid and/or HBZ expressing plasmid. Representative data shown are firefly luciferase activities normalized to those of renilla luciferase (mean ± SD).CC BY · PubMed Central
Figure 5 HBZ physically interacts with Foxp3 and NFAT. (A) The expression vectors of the indicated proteins were co-transfected into 293FT cells, and their interactions were analyzed by immunoprecipitation (IP). (B) Nuclear extract of ATL-43T cells was subjected to IP with anti-HBZ antibody or control IgG, and detected by anti-FoxP3 antibody. (C and D) The interactions of HBZ and Foxp3 were analyzed by IP using HBZ mutants (C) or Foxp3 mutants (D). A schematic diagram of Foxp3 mutants is shown. ZnF, zinc finger; LZ, leucine zipper; FH, forkhead domain. Asterisks (* or **) show responsible region for each molecular interaction. (E) The interactions among HBZ, Foxp3 and NFATc2 were analyzed with or without EtBr.
Figure 5 HBZ physically interacts with Foxp3 and NFAT. (A) The expression vectors of the indicated proteins were co-transfected into 293FT cells, and their interactions were analyzed by immunoprecipitation (IP). (B) Nuclear extract of ATL-43T cells was subjected to IP with anti-HBZ antibody or control IgG, and detected by anti-FoxP3 antibody. (C and D) The interactions of HBZ and Foxp3 were analyzed by IP using HBZ mutants (C) or Foxp3 mutants (D). A schematic diagram of Foxp3 mutants is shown. ZnF, zinc finger; LZ, leucine zipper; FH, forkhead domain. Asterisks (* or **) show responsible region for each molecular interaction. (E) The interactions among HBZ, Foxp3 and NFATc2 were analyzed with or without EtBr.CC BY · PubMed Central
Figure 6 HBZ inhibites Foxp3-mediated CTLA-4 and GITR expression in vitro. (A) Mouse CD4+CD25− T cells co-transduced with the retroviral vectors were stained with the indicated antibodies. Mean fluorescence intensity (MFI) of CD25, GITR, and CTLA-4 in GFP/NGFR double-positive cells are shown as mean ± SD. for triplicate culture. *, P<0.01; **, P<0.001 by two-tailed Student t-test. (B and C) CD4+CD25− T cells transduced with the pMXs-Ig vector encoding wild-type or mutant HBZ, and pGCSamIN-Foxp3 vector were stained with anti-GITR (B) or anti-CTLA-4 (C) antibody in addition to anti-NGFR antibody, and then analyzed by flow cytometry. Left, numbers in density plots indicate MFI of GITR (B) or CTLA-4 (C) in GFP/NGFR double-positive cells. Representative data from three independent experiments are shown. Right, relative MFI of wild type or mutated HBZ compared to mock transduced cells was shown as mean ± SD (n = 3). (D) HBZ transduction in Foxp3+ Treg cells inhibited the endogenous expression of Treg associated molecules. Mean fluorescence intensity (MFI) of CD25, GITR, and CTLA-4 in CD4+Foxp3+NGFR+ cells are shown as mean ± SD. for triplicate culture. *, P<0.05; **, P<0.01 by two-tailed Student t-test.
Figure 6 HBZ inhibites Foxp3-mediated CTLA-4 and GITR expression in vitro. (A) Mouse CD4+CD25− T cells co-transduced with the retroviral vectors were stained with the indicated antibodies. Mean fluorescence intensity (MFI) of CD25, GITR, and CTLA-4 in GFP/NGFR double-positive cells are shown as mean ± SD. for triplicate culture. *, P<0.01; **, P<0.001 by two-tailed Student t-test. (B and C) CD4+CD25− T cells transduced with the pMXs-Ig vector encoding wild-type or mutant HBZ, and pGCSamIN-Foxp3 vector were stained with anti-GITR (B) or anti-CTLA-4 (C) antibody in addition to anti-NGFR antibody, and then analyzed by flow cytometry. Left, numbers in density plots indicate MFI of GITR (B) or CTLA-4 (C) in GFP/NGFR double-positive cells. Representative data from three independent experiments are shown. Right, relative MFI of wild type or mutated HBZ compared to mock transduced cells was shown as mean ± SD (n = 3). (D) HBZ transduction in Foxp3+ Treg cells inhibited the endogenous expression of Treg associated molecules. Mean fluorescence intensity (MFI) of CD25, GITR, and CTLA-4 in CD4+Foxp3+NGFR+ cells are shown as mean ± SD. for triplicate culture. *, P<0.05; **, P<0.01 by two-tailed Student t-test.CC BY · PubMed Central

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Authors
Satou, Y., Yasunaga, J.-I., Zhao, T., Yoshida, M., Miyazato, P., Takai, K., Shimizu, K., Ohshima, K., Green, P. L., Ohkura, N., Yamaguchi, T., Ono, M., Sakaguchi, S., & Matsuoka, M.
Affiliations
  • Kyoto University (JP)
  • Kurume University (JP)
  • The Ohio State University (US)
Corresponding author
Matsuoka, Masao
ORCID
Venue
PLoS Pathogens
Publisher
Public Library of Science (PLoS)
Published
2011-02-10
Volume
7
Issue
2
Pages
e1001274
ISSN
  • 1553-7366 (ISSN-L)
  • 1553-7374
DOI
10.1371/journal.ppat.1001274
PMC
PMC3037353
OpenAlex
W1966278500
Cited by
319
References
58
Citations by year
2026: 10 · 2025: 18 · 2024: 11 · 2023: 15 · 2022: 27 · 2021: 31 · 2020: 21 · 2019: 19 · 2018: 14 · 2017: 32 · 2016: 21 · 2015: 19 · 2014: 19 · 2013: 21 · 2012: 22
FWCI
11.50
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Pathogens (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Institutional Repositories DataBase (IRDB) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • UCL Discovery (University College London) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
  • Kyoto University Research Information Repository (Kyoto University) (submittedVersion, cc-by)
Funders
  • Naito Foundation
  • Sumitomo Foundation
  • Uehara Memorial Foundation
  • National Institutes of Health
  • Japan Society for the Promotion of Science (22390193)
  • National Cancer Institute (CA077556, R01CA077556)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and Retrovirus Studies
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Health Sciences › Medicine › Oncology › Viral-associated cancers and disorders
Keywords
HTLV-1, bZIP transcription factors, HBZ, adult T-cell leukemia/lymphoma, T-cell lymphoma, regulatory T cells, Foxp3, NFAT, systemic inflammation, CD4+ T cells, transgenic mice, HTLV-1-associated myelopathy/tropical spastic paraparesis, transcriptional regulation
MeSH terms
Adult, Animals, Cell Transformation, Viral, Cells, Cultured, Gene Expression Regulation, Humans, Inflammation, Mice, Inbred C57BL, Mice, Transgenic, Human T-lymphotropic virus 1, HTLV-I Infections, CD4-Positive T-Lymphocytes, Lymphoma, T-Cell, Basic-Leucine Zipper Transcription Factors, Mice

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