Research ArticleTHYMUS

Tumor suppressor BAP1 is essential for thymic development and proliferative responses of T lymphocytes

See allHide authors and affiliations

Science Immunology  20 Apr 2018:
Vol. 3, Issue 22, eaal1953
DOI: 10.1126/sciimmunol.aal1953
  • Fig. 1 BAP1 deficiency results in thymic atrophy and loss of thymocyte populations.

    (A and B) Thymi (A) and hematoxylin and eosin staining of thymi (B) from Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 mice 2.5 weeks after tamoxifen treatment. (C) Thymocyte numbers in Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 mice 1 week after tamoxifen treatment (n = 6 to 7). (D) Cell numbers of the indicated thymocyte populations in Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 mice 1 week after tamoxifen treatment, as determined by flow cytometry analysis. All data are representative of at least three independent experiments. For (C) and (D), each symbol represents an individual mouse and the line shows the median. *P < 0.05, **P < 0.01, ****P < 0.0001 based on unpaired Student’s t test.

  • Fig. 2 Loss of BAP1 leads to alterations in early hematopoiesis and B cell development.

    (A to D) Cell numbers of progenitor populations (A), neutrophils and monocytes (B), lymphocyte progenitor populations (C), and B cell populations (D) in the bone marrow of Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 mice 1 week after tamoxifen treatment (n = 5 to 7). Each symbol represents an individual mouse, and the line shows the median. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on unpaired Student’s t test.

  • Fig. 3 Loss of BAP1 blocks T cell differentiation at the DN3 stage in vitro.

    Bone marrow progenitor cells from Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 mice were cultured on OP9-DL1 stromal cells for the indicated number of days, with deletion of BAP1 induced at day 2 by 4-OHT addition (n = 3). (A) Staining profiles of GFP CD45+ lineage CD3 TCRβ CD4 CD8 cells (left) and GFP CD45+ lineage cells (right) at the indicated time points. Data are representative of three independent experiments. (B and C) Proportions within GFP CD45+ cells (B) and absolute numbers (C) of T cell populations at the indicated time points. (D) RNA-seq was performed on BAP1 WT and KO DN3 cells (CD45+ lineage CD3 TCRβ CD4 CD8 CD44 c-Kit CD25+) isolated from OP9-DL1 cocultures at day 14. A volcano plot of logFC (KO-WT) versus nominal P values (−log10) is shown. Differentially expressed genes (1.5-fold, FDR < 0.1) are highlighted in dark blue, and genes in the hallmark E2F target gene set are highlighted in orange. (E) GSEA of BAP1 WT versus KO DN3 cell data sets. Gene set–level heat maps (left) and FDR (right) of gene sets that are differentially enriched in BAP1 WT versus KO DN3 data sets with an FDR less than 20% are shown. (F) Densities of logFC values of each gene in the indicated gene data set (orange) compared with the logFC values of all genes (blue). Each green dot represents one gene in the indicated data set.

  • Fig. 4 Deubiquitinating activity, but not HCF-1 binding, is required for BAP1 function in thymocytes.

    (A) Coimmunoprecipitation (IP) of BAP1 with various members of the BAP1 core complex and with members of the PRCs in thymocytes of C57BL/6 mice. (B) Western blot showing protein levels of EZH2, HCF-1, and OGT in 4-OHT–treated Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 DN3 cells isolated from OP9-DL1 cocultures at day 14. (C) Bap1fl/flRosa26CreERT2 and Bap1wt/wtRosa26CreERT2 bone marrow progenitor cells were transduced with empty vector, full-length BAP1 (WT), HCF-1 interaction mutant BAP1 (4A), or catalytically dead BAP1 (C91A). Transduced cells were then cultured with OP9-DL1 stromal cells, and BAP1 deletion was induced at day 2 as described in Fig. 3. Staining profiles of GFP+ CD45+ lineage cells at day 18 are shown. Data are representative of at least three biological replicates in two independent experiments. (D) Proportion of DP cells within the GFP+ CD45+ lineage gate at day 20 of coculture (n = 3). (E) Histogram profiles of global H2AK119ub and H3K27me3 levels in WT and KO DN3 cells (CD45+ lineage CD3 TCRβ CD4 CD8 CD44 CD25+) from OP9-DL1 cocultures at day 13. (F) Histone mass spectrometry of BAP1 WT and KO DN3 cells sorted from OP9-DL1 cultures at day 14. Heat maps depicting the relative abundance of each posttranslational modification normalized to WT abundance levels are shown.

  • Fig. 5 Increase of global H2AK119ub levels in bone marrow and thymocyte progenitors after loss of BAP1.

    (A) Flow cytometry–based detection of BAP1 expression [measured by mean fluorescence intensity (MFI)] in various hematopoietic progenitors in the bone marrow (top) and in T cell populations in the thymus (bottom) of Bap1wt/wtRosa26CreERT2 mice (n = 6). (B and C) MFI of global H2AK119ub (B) and H3K27me3 (C) levels in the same populations shown in (A) of Bap1fl/flRosa26CreERT2 (in red) and Bap1wt/wtRosa26CreERT2 (in black) mice 1 week after tamoxifen treatment (n = 6). Each symbol represents the median, and the error bars show the range. The gray shaded area on each graph shows the range of MFI levels in thymocytes. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on unpaired Student’s t test.

  • Fig. 6 BAP1 is required to maintain T cells in the periphery.

    (A and B) Numbers of total CD4+ and CD8+ T cells (A) and naïve (N), central memory (CM), and effector memory (EM) CD4+ and CD8+ T cells (B) in spleens of 8-week-old Bap1fl/flCd4-Cre+ and Bap1wt/wtCd4-Cre+ mice (n = 5). (C) Flow cytometry–based detection of BAP1 levels in T cells from the spleen of Bap1wt/wtRosa26CreERT2 mice (n = 5). (D and E) Global H2AK119ub and H3K27me3 expression levels in T cells from the spleens of Bap1fl/flRosa26CreERT2 (in red) and Bap1wt/wtRosa26CreERT2 (in black) mice 1 week after tamoxifen treatment (D; n = 6) and 8-week-old Bap1fl/flCd4-Cre+ (in red) and Bap1wt/wtCd4-Cre+ (in black) mice (E; n = 5). (F) Western blot analysis of total EZH2 protein levels (left) and RNA expression levels (right) in naïve CD4 T cells from Bap1fl/flCd4-Cre+ and Bap1wt/wtCd4-Cre+ mice (n = 5). Data are representative of at least three independent experiments. (A and B) Each symbol represents an individual mouse, and the line shows the median. (C to E) Each symbol represents the median, and the error bars show the range. The gray shaded area on each graph shows the range of MFI levels in thymocytes (Fig. 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on unpaired Student’s t test.

  • Fig. 7 BAP1 is required for homeostatic and antigen-driven expansion of peripheral T cells.

    (A) Sorted CD4+ T cells from Bap1fl/flRosa26CreERT2 or Bap1wt/wtRosa26CreERT2 mice 1 week after tamoxifen injection were transplanted into Rag2−/− recipients either separately or at a 1:1 ratio. Numbers of total splenic CD4+ T cells in recipient mice 3 weeks after transfer (n = 3 to 4) are shown. Data are representative of two independent experiments. Error bars represent SD. *P < 0.05, **P < 0.01 based on unpaired Student’s t test. (B) GSEA of WT versus KO nonpolarized, TH2, and TH17 data sets. Gene set–level heat maps (left) and FDR (right) of gene sets that are differentially enriched in WT versus KO with an FDR less than 20% are shown. (C) Number of cells recovered after 6 days of culture of naïve CD4 T cells from Bap1fl/flCd4-Cre+ (red) and Bap1wt/wtCd4-Cre+ (black) mice stimulated with anti-CD3 and anti-CD28 (n = 2). (D) Cell cycle analysis of naïve CD4 T cells from Bap1fl/flCd4-Cre+ or Bap1wt/wtCd4-Cre+ mice stimulated with anti-CD3 and anti-CD28 for 1 or 2 days (left) and proportion of cells in each phase of the cell cycle (right) (n = 3) are shown. ****P < 0.000001 based on Fisher’s exact test. (E) CellTrace-labeled naïve CD4 T cells from Bap1fl/flCd4-Cre+ or Bap1wt/wtCd4-Cre+ mice were stimulated with anti-CD3 and anti-CD28 for the indicated number of days (n = 8). CellTrace profiles (left) and number of live cells recovered (right) are shown. ****P < 0.0001 based on unpaired Student’s t test. 7-AAD, 7-aminoactinomycin D.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/22/eaal1953/DC1

    Materials and Methods

    Fig. S1. Loss of γδ T cells in the thymus of BAP1-deficient mice.

    Fig. S2. Thymocyte defect in BAP1-deficient mice is intrinsic to the hematopoietic compartment.

    Fig. S3. RNA expression levels of members of the PRCs in BAP1-deficient DN3 T cells.

    Fig. S4. Loss of BAP1 in myeloid and B cell progenitors results in increase of global H2AK119ub levels.

    Fig. S5. Tamoxifen-induced deletion of BAP1 results in reduced numbers of peripheral T cell populations.

    Fig. S6. Thymocyte development is grossly normal in Bap1fl/flCd4-Cre+ mice.

    Fig. S7. BAP1 deficiency impairs maintenance of peripheral T cells.

    Fig. S8. BAP1 is not required for early TH cell differentiation.

    Fig. S9. BAP1 deficiency impairs peripheral T cell proliferation and effector function.

    Fig. S10. BAP1-deficient CD4 T cells have cell-intrinsic proliferative defect.

    Fig. S11. Deubiquitinating activity, but not HCF-1 binding, is required for BAP1 function in peripheral T cells.

    Table S1. Raw data sets.

    Table S2. Definition of cell populations and gating strategy.

  • Supplementary Materials

    Supplementary Material for:

    Tumor suppressor BAP1 is essential for thymic development and proliferative responses of T lymphocytes

    Teresita L. Arenzana, Steve Lianoglou, Akiko Seki, Celine Eidenschenk, Tommy Cheung, Dhaya Seshasayee, Thijs Hagenbeek, Arivazhagan Sambandam, Rajkumar Noubade, Ivan Peng, Justin Lesch, Jason DeVoss, Xiumin Wu, Wyne P. Lee, Patrick Caplazi, Joshua Webster, Jinfeng Liu, Victoria C. Pham, David Arnott, Jennie R. Lill, Zora Modrusan, Anwesha Dey,* Sascha Rutz*

    *Corresponding author. Email: dey.anwesha{at}gene.com (A.D.); saschar{at}gene.com (S.R.)

    Published 20 April 2018, Sci. Immunol. 3, eaal1953 (2018)
    DOI: 10.1126/sciimmunol.aal1953

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Loss of γδ T cells in the thymus of BAP1-deficient mice.
    • Fig. S2. Thymocyte defect in BAP1-deficient mice is intrinsic to the hematopoietic compartment.
    • Fig. S3. RNA expression levels of members of the PRCs in BAP1-deficient DN3 T cells.
    • Fig. S4. Loss of BAP1 in myeloid and B cell progenitors results in increase of global H2AK119ub levels.
    • Fig. S5. Tamoxifen-induced deletion of BAP1 results in reduced numbers of peripheral T cell populations.
    • Fig. S6. Thymocyte development is grossly normal in Bap1fl/flCd4-Cre+ mice.
    • Fig. S7. BAP1 deficiency impairs maintenance of peripheral T cells.
    • Fig. S8. BAP1 is not required for early TH cell differentiation.
    • Fig. S9. BAP1 deficiency impairs peripheral T cell proliferation and effector function.
    • Fig. S10. BAP1-deficient CD4 T cells have cell-intrinsic proliferative defect.
    • Fig. S11. Deubiquitinating activity, but not HCF-1 binding, is required for BAP1 function in peripheral T cells.
    • Table S2. Definition of cell populations and gating strategy.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data sets.

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article