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Post-translational control of T cell development by the ESCRT protein CHMP5

Nature Immunology volume 18, pages 780–790 (2017)Cite this article

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Abstract

The acquisition of a protective vertebrate immune system hinges on the efficient generation of a diverse but self-tolerant repertoire of T cells by the thymus through mechanisms that remain incompletely resolved. Here we identified the endosomal-sorting-complex-required-for-transport (ESCRT) protein CHMP5, known to be required for the formation of multivesicular bodies, as a key sensor of thresholds for signaling via the T cell antigen receptor (TCR) that was essential for T cell development. CHMP5 enabled positive selection by promoting post-selection thymocyte survival in part through stabilization of the pro-survival protein Bcl-2. Accordingly, loss of CHMP5 in thymocyte precursor cells abolished T cell development, a phenotype that was 'rescued' by genetic deletion of the pro-apoptotic protein Bim or transgenic expression of Bcl-2. Mechanistically, positive selection resulted in the stabilization of CHMP5 by inducing its interaction with the deubiquitinase USP8. Our results thus identify CHMP5 as an essential component of the post-translational machinery required for T cell development.

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Figure 1: CHMP5 expression in thymocytes.
Figure 2: CHMP5 is required for T cell development.
Figure 3: Cell-intrinsic requirement for CHMP5 in positive selection.
Figure 4: Normal TCR signaling in CHMP5-deficient thymocytes.
Figure 5: Assessment of ESCRT-machinery activity in thymocytes.
Figure 6: Post-transcriptional stabilization of Bcl-2 by CHMP5 ensures the survival of post-selection in thymocytes.
Figure 7: Post-translational regulation of CHMP5 expression by TCR signaling.
Figure 8: Stabilization of CHMP5 is dependent on the deubiquitinase USP8.

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Acknowledgements

We thank A. Singer (US National Institutes of Health) for Bcl2-transgenic mice; S. Dimmeler (J.W. Goethe University, Frankfurt, Germany) for Bcl-2-encoding plasmids; J. Kaplan (University of Utah) for anti-LIP5; M. Greenblatt for critical reading of the manuscript; J. McCormick (Weill Cornell Medicine) for sorting by flow cytometry; L. Cohen-Gould, J. Cohen and J. Jimenez for histology and electron microscopy; and the NIH Tetramer Core Facility (Emory University) for tetramers. Supported by the US National Institutes of Health (R01AR068983 to J.H.S., and R01CA112663 to L.H.G.).

Author information

Author notes

  1. Kwang Hwan Park

    Present address: Department of Orthopedic Surgery, Yonsei University College of Medicine, Seoul, South Korea

Authors and Affiliations

  1. Department of Cancer Immunology and Virology, Dana Farber Cancer Institute, Boston, Massachusetts, USA

    Stanley Adoro & Laurie H Glimcher

  2. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Stanley Adoro & Laurie H Glimcher

  3. Department of Pathology, Weill Cornell Medicine, New York, New York, USA

    Kwang Hwan Park

  4. Quentis Therapeutics, New York, New York, USA

    Sarah E Bettigole & Hee Rae Shin

  5. Department of Genetic Medicine, Ansary Stem Cell Institute, Weill Cornell Medicine, New York, New York, USA

    Raphael Lis

  6. Division of Biomedical Informatics, Seoul National University College of Medicine, Seoul, South Korea

    Heewon Seo & Ju Han Kim

  7. Institute for Neuropathology, University of Freiburg, Freiburg, Germany

    Klaus-Peter Knobeloch

  8. Division of Rheumatology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Jae-Hyuck Shim

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Contributions

S.A. designed study, performed experiments, analyzed data and wrote the manuscript; K.H.P., S.E.B., R.L. and H.R.S. assisted with experiments; H.S. and J.H.K. performed bioinformatics; K.-P.K. provided mice with loxP-flanked Usp8 alleles; J.-H.S. designed the study, performed experiments, analyzed data and wrote the manuscript; and L.H.G. designed the study, analyzed data, provided supervision and wrote the manuscript.

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Correspondence to Jae-Hyuck Shim or Laurie H Glimcher.

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Competing interests

L.H.G. is on the board of directors of and holds equity in Bristol Myers Squibb. L.H.G. is founder of and S.E.B. is a co-founder of Quentis Therapeutics, and S.E.B. and H.R.S. are employed by Quentis Therapeutics.

Integrated supplementary information

Supplementary Figure 1 CHMP5 expression in thymocytes.

(a) Validation of rabbit anti-CHMP5 antibody in Jurkat T-cells depleted of CHMP5 with two independent shRNA and LacZ control shRNA. (b) Immunoblot of MG132-treated wild-type thymocytes. Protein band intensities relative to untreated samples are indicated. (c) Immunoblot for ESCRT proteins with relative band intensities. (d) Chmp5 mRNA in preselection DP and intermediate (Int) thymocytes from WT, MHCII-selection (B2m–/–) and MHCI-selection (H2Ab1–/–) mice. Error bar, s.d. (e) Chmp5 mRNA levels in the thymocyte populations sorted from WT and CKO mice revealing Chmp5 deletion. Error bar, s.d. (f) mRNA expression of selected ESCRT genes in WT and CKO mice relative to Actin. Each circle represents one mouse. Error bar, s.d. DN, double negative; DP, double positive; Int, intermediate thymocytes; SP, single positive. Data are representative of at least three independent experiments.

Supplementary Figure 2 CHMP5 is essential for thymocyte positive selection.

(a) Representative flow cytometry plot of CD24 versus TCRb expression on CD4 and CD8 single positive (SP) thymocytes. Numbers indicate the proportion of cells within each gate. (b) Immunoblot of CHMP5 protein in residual peripheral CD4 and CD8 T cells from WT and CKO mice. (c) CD44 expression on peripheral CD4 and CD8 T cells. (d,e) Characterization of WT and CKO mice expressing the OT-II TCR transgene. Representative flow cytometry plot of CD4 versus CD8 (top) and CD24 versus Vβ5 thymocytes (bottom) on total thymocytes (d) and Vβ5 expression on splenocytes (e) are shown. Numbers indicate the proportion of cells in each gate. Data are representative of two (b) and five independent experiments (a,c-e).

Supplementary Figure 3 Cell-intrinsic role for CHMP5 in T cell generation.

(a) Mixed bone marrow chimera strategy. (b) Lentiviral vector constructs and strategy for CHMP5 transgenesis rescue experiment. (c, d) Representative flow cytometer plot of thymocyte CD24 versus TCRβ expression (c) and splenic TCRβ versus CD19 expression (d) on GFP– and GFP+ cells within CD45.2+ donor-derived cells from mice reconstituted with control (LV-Ctrl) and CHMP5-transduced (LV-C5) bone marrow cells. Dotted line, untransduced WT thymocytes. Numbers indicate cell proportion within each gate. Data are representative of three independent experiments.

Supplementary Figure 4 Characterization of TCR signaling in CHMP5-deficient thymocytes.

(a) Flow cytometer phenotypic definition and sort gating of intermediate thymocytes. (b) Surface expression of positive selection markers IL-7Rα and CCR7 on preselection DP and intermediate (Int) thymocytes from WT and CKO mice. (c) Proportion of CD69+ thymocytes after overnight (18 hours) stimulation with anti-CD3ɛ alone (top) or with anti-CD2 antibodies (bottom). Error bar, s.e.m., n = 4 mice each. Data are representative of three independent experiments.

Supplementary Figure 5 Expression of thymocyte positive selection genes in wild-type and CKO thymocytes.

(a) Gata3 mRNA (top) and Gata3 protein (bottom) expression. Note immunoblot for Grb2 protein as loading control. Western blot is representative of two independent experiments. (b) mRNA expression of TCR-regulated genes: Nfatc1, Egr1 and Tox. (c) mRNA levels of CD4 and CD8 lineage-specification genes Zbtb7b (Th-POK) and Runx3. (d) Relative NF-κB activity assessed by p65 binding to consensus NF-κB oligonucleotides determined by ELISA. Error bar, s.e.m; four mice each. P values are shown on graph, Students t-Test. Representative of two experiments. (e) NF-κB target gene expression. DP, preselection CD4+CD8+ thymocytes; Int, intermediate thymocytes; CD4SP, CD4 single-positive thymocytes; CD8SP, single-positive thymocytes. All mRNA levels were normalized to Actin. For qPCR data, each circle represents an average of duplicate of individual mice.

Supplementary Figure 6 Regulation of Bcl-2 protein by CHMP5.

(a) Relative mRNA levels of Bcl-xl, Bim, Nur77 and Bid. (b) Immunoblot of Bcl-2 and Bim proteins in sorted thymocyte subsets. (c, d) Representative histogram (c) and bar graph (d) of mean fluorescence intensity (MFI) of intracellular Bcl-2 protein in gated thymocyte subsets. Error bar, s.e.m; four mice each. Representative of three independent experiments. (e) Intracellular Bcl-2 MFI in intermediate OT-II TCR transgenic thymocytes. Each circle represents one mouse. (f,g) Intracellular Bcl-2 (f) and phosphorylated STAT5 (g) in IL-7-stimulated intermediate thymocytes. Error bar, s.e.m.; four mice each. (h) Intracellular Bcl-2 expression in WT and CKO intermediate thymocytes after overnight (18 hours) culture in cycloheximide (CHX) containing medium. Error bar, s.e.m.; three mice each. (i,j) Co-immunoprecipitation of epitope-tagged CHMP5 and BCL-2 in HEK293 cells (j) and in cell-free assays with GST-tagged BCL-2 and His-tagged CHMP5 (j). Numbers indicate band intensity relative to “GST” lane. Representative of two independent experiments. (k) Total thymocyte ROS levels determined by CellRox Deep Red staining. Error bar, s.e.m.; four mice each. *, P < 0.05; **, P < 0.001; ***, P < 0.0001, Student’s t-tests.

Supplementary Figure 7 Genetic augmentation of prosurvival pathway restores T cell development in CKO mice.

Representative flow cytometry plot of CCR7 versus TCRβ expression and corresponding thymocyte numbers in WT and CKO mice deficient for Bim (a,b) or expressing transgenic Bcl-2 (c,d). Numbers in plots indicate proportion of cells within gates. Each data point circle represents one mouse. Fold-times difference between CKO groups are indicated. **, P < 0.001; ***, P < 0.0001, Student’s t-tests.

Supplementary Figure 8 Differential regulation of CHMP5 by TCR signaling.

(a) Immunoblot (top) and qPCR (bottom) analyses of CHMP5 expression in stimulated pre-selection (CD69–) DP thymocytes. Relative band intensities (normalized to unstimulated (medium) control) are shown. (b,c) Chmp5 mRNA levels (normalized to Actin) in PMA/ionomycin stimulated pre-selection (CD69–) DP thymocytes (b) or kb-peptide tetramer-stimulated OT-I.B2m–/– thymocytes (c). Error bar, s.d. (d) Anti-HA (CHMP5) immunoblot of Nickel-immunoprecipitates of PMA/ionomycin-treated HEK293 cells co-transfected with HA-tagged CHMP5 and His-tagged ubiquitin. (e) Ubiquitin immunoblot of anti-HA immunoprecipitates showing inhibition of Bcl-2 ubiquitination by the antioxidant NAC in PMA+ionomycin-treated HEK293 cells expressing HA-tagged Bcl-2. (f) Ubiquitin blot of anti-CHMP5 immunoprecipitates from unstimulated (none) or Jurkat T-cells activated by CD3e plus CD28 antibody crosslinking or PMA plus ionomycin. Data are representative of two (d–f) and three (a–c) independent experiments.

Supplementary Figure 9 Interaction between CHMP5 and USP8 in thymocytes that have undergone TCR selection.

(a) USP8 expression in thymocyte subsets from WT and CKO mice. (b) Immunoblot of CHMP5 and USP8 in isotype and anti-CHMP5 antibody immunoprecipitates from pre-selection CD4+CD8+ (DP) and intermediate (Int) thymocytes. (c) Anti-USP8 immunoprecipitation to determine the effect of PMA plus ionomycin dose on the interaction between CHMP5 and USP8 in DP thymocytes. *, non-specific band. (d) Usp8 and Bcl2 mRNA levels (normalized to Actin) in USP8-deficient (Usp8CD4cre) or littermate control (Usp8f/f) intermediate thymocytes. All data in a–d are representative of two independent experiments each. (e) Summarized hypothetical model of CHMP5-USP8 axis during thymocyte development.

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Adoro, S., Park, K., Bettigole, S. et al. Post-translational control of T cell development by the ESCRT protein CHMP5. Nat Immunol 18, 780–790 (2017). https://doi.org/10.1038/ni.3764

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