Activation of the Hippo pathway by CTLA-4 regulates the expression of Blimp-1 in the CD8+ T cell - PubMed (original) (raw)

Activation of the Hippo pathway by CTLA-4 regulates the expression of Blimp-1 in the CD8+ T cell

James E D Thaventhiran et al. Proc Natl Acad Sci U S A. 2012.

Abstract

During the primary response, the commitment of the CD8(+) T cell to Blimp-1 expression and the terminal differentiation that Blimp-1 induces must be timed so as not to impair the process of clonal expansion. We determined whether the Hippo pathway, which links cell-cell contact to differentiation in other cell lineages, controls Blimp-1 expression. Activating the CD8(+) T cell with antigen and IL-2 causes expression of the core Hippo pathway components, including the pivotal transcriptional cofactor Yap. Contact between activated CD8(+) T cells induces Hippo pathway-mediated Yap degradation and Blimp-1 expression; a Hippo-resistant, stable form of Yap suppresses Blimp-1 expression. Cytotoxic T lymphocyte antigen 4 (CTLA-4) and CD80 comprise the receptor-ligand pair that mediates contact-dependent Hippo pathway activation. In vivo, CD8(+) T cells expressing Hippo resistant-Yap or lacking CTLA-4 have diminished expression of the senescence marker, KLRG1, during a viral infection. The CTLA-4/Hippo pathway/Blimp-1 system may couple terminal differentiation of CD8(+) T cell with the magnitude of clonal expansion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Expression of core components of the Hippo pathway by OT-I cells. (A) Immunoblot analysis of components of the Hippo pathway in whole-cell lysates prepared from naïve OT-I cells and OT-I cells stimulated for 24 h with 0.1 nM SIINFEKL peptide alone or with IL-2 (TCR + IL-2). (B) Bar graph showing the mRNA levels of these components in identically treated samples of OT-I cells relative to those levels for CD3ε (given an arbitrary level of one). *P < 0.05; ***P < 0.001. Data presented are the mean ± SEM (n = 3).

Fig. 2.

Fig. 2.

The regulation of Yap expression in OT-I cells. (A) Bar graph showing the mRNA levels, relative to the levels for CD3ε, for Yap in naive OT-I cells and OT-I cells stimulated for timed intervals with 0.1 nM SIINFEKL peptide in the absence or presence of actinomycin D. Data presented are the mean ± SEM. (B) Immunoblot analysis of Yap in whole-cell lysates prepared from naïve OT-I cells and OT-I cells stimulated for 24 h with 0.1 nM SIINFEKL peptide alone (TCR) or together with IL-2 (TCR + IL-2). (C) Levels of Yap protein (Lower) and Yap mRNA levels (Upper) in whole-cell lysates prepared from naïve Thy1.2+ OT-I cells and Thy1.2+ OT-I cells that had been stimulated with SIINFEKL peptide and IL-2 for 24 h alone or in the presence of 10-fold excess of nonactivated Thy1.1+ C57BL/6 CD8+ T cells. The Thy1.2+ OT-I cells were recovered by MACS purification, and Yap protein and mRNA were assessed. Data presented are the mean ± SEM. (D) Immunoblot analysis using antihemagglutin (anti-HA) of whole-cell lysates prepared from retrovirally transduced OT-I cells that expressed ectopic WT Yap tagged with HA (Yap-HA) and Yap in which serine 112 had been mutated to alanine (Yap S112A-HA) or serine-382 had been mutated to alanine (Yap S382A-HA). Lysates were cultured for 48 h with IL-2. ***P < 0.001.

Fig. 3.

Fig. 3.

The role of CD80/86 in triggering the Hippo pathway during contact between activated CD8+ T cells. (A) Bar graph showing the mRNA levels of CD80 and CD86 relative to the levels of CD3ε in naïve OT-I cells and OT-I cells stimulated for 24 h with 0.1 nM SIINFEKL peptide alone (TCR) or together with IL-2 (TCR + IL-2). Data presented are the mean ± SEM. (B) Naïve and TCR + IL-2–stimulated OT-I cells were also assessed by FACS for membrane expression of CD80 and CD86 (shaded histogram, isotype control; black line, specific antibody). (C) Immunoblot analysis of Yap in cell lysates prepared from naïve OT-I cells or OT-I cells that had been stimulated for 24 h with SIINFEKL peptide in the absence or presence IL-2 and the presence of isotype control antibodies or blocking antibodies to CD80 and CD86. (D) Analysis by confocal microscopy of Yap expression by naïve OT-I cells or OT-I cells that had been stimulated for 24 h with SIINFEKL peptide and IL-2 in the presence of isotype control antibodies or blocking antibodies to CD80 and CD86 (green, anti-Yap; blue, DAPI). (Scale bars: 50 μm.) ***P < 0.001.

Fig. 4.

Fig. 4.

Plasma membrane expression of CTLA-4 requires contact between activated CD8+ T cells. (A) Bar graph showing the mRNA levels of CTLA-4 relative to the levels for CD3ε in naïve OT-I cells and OT-I cells stimulated for 24 h with 0.1 nM SIINFEKL peptide alone (TCR) or together with IL-2 (TCR + IL-2). Data presented are the mean ± SEM. (B) Immunoblots showing CTLA-4 and actin protein in lysates of naïve OT-I cells and OT-I cells stimulated for 24 h with 0.1 nM SIINFEKL peptide alone (TCR) or together with IL-2 (TCR + IL-2). (C) FACS analysis of CTLA-4 expression by TCR- and IL-2–stimulated Thy1.2+ OT-I cells that had been cocultured for 30 h alone or with increasing numbers of resting polyclonal Thy1.1+ CD8+ T cells. The numbers represent the proportion of intact and detergent-permeabilized Thy1.2+ cells, respectively, showing specific staining with anti–CTLA-4. (D) FACS analysis of CTLA-4 expression by TCR and IL-2–stimulated Thy1.2+ OT-I cells that had been cultured in the top compartment of a transwell chamber in which the bottom compartment contained activated Thy1.2+ OT-I cells cocultured with 100-fold excess polyclonal CD8+ T cells. The numbers represent the proportion of intact, nonpermeabilized Thy1.2+ cells staining with anti–CTLA-4. *P < 0.05; ***P < 0.001.

Fig. 5.

Fig. 5.

Activation of the Hippo pathway by CTLA-4 in vitro and in vivo. (A) Immunoblot analysis of lysates from activated OT-I cells cultured in the presence of antibodies to CD80 and CD86 and incubated for 1 h with beads bearing isotype control antibody or antibody to CTLA-4. Blots were developed with antibodies specific for phosphothreonine-183 of Mst1 and phosphoserine-909 of Lats1. Staining of actin is shown as a loading control. (B) Immunoblot analysis with antiphosphoserine-112 of Yap of lysates from activated OT-I cells expressing Yap S382A-HA that had been cultured in the presence of blocking antibodies to CD80 and CD86 and incubated for 1 h with beads bearing isotype control antibody or antibody to CTLA-4. HA staining is shown as a loading control. (C) Immunoblot analysis of WT Yap in cell lysates from OT-I cells activated in the presence of antibodies to CD80 and CD86 and incubated for 3 h with beads bearing various ratios of isotype control antibody and antibody to CTLA-4. Staining of actin is shown as a loading control. (D) Analysis by confocal microscopy of anti-Yap–stained OT-I cells that had been activated in the presence of blocking antibodies to CD80 and CD86 and incubated for 3 h with beads bearing isotype control antibody or antibody to CTLA-4 (green, anti-Yap; blue, DAPI). (Scale bars: 50 μm.) (E) Immunoblot analysis of Yap protein in lysates from CTLA-4−/− OT-I cells and WT OT-I cells that had been activated with SIINFEKL peptide and IL-2 for 24 h. (F) Analysis by confocal microscopy of Yap protein in CD8+ and CD4+ cells from the lymph nodes of CTLA-4−/− and CTLA-4−/+ 16- to 20-d-old mice. (Scale bars: 10 μm.)

Fig. 6.

Fig. 6.

Preventing Hippo pathway activation in CD8+ T cells affects Blimp-1 expression and differentiation. (A) OT-I CD8+ T cells were transduced with the Yap 5SA-expressing retrovirus or the pMig vector control and analyzed for Eomes protein by staining of permeabilized cells with anti-Eomes relative to naïve, unstimulated OT-I cells 16 h after restimulation with IL-2 and SIINFEKL peptide. Numbers represent the Eomes-specific mean fluorescence intensity ± SEM. (B) Prdm1gfp/+ CD8+ T cells that had been transduced with control pMig (black line) or the pMig vector-expressing Yap 5SA (red line) and WT CD8+ T cells transduced with pMig (shaded) were restimulated for 48 h with anti-CD3ε and IL-2 and assessed for GFP fluorescence. Numbers are the GFP-specific mean fluorescence intensities ± SEM. (C and D) OT-I cells transduced with a retrovirus expressing the Lats-resistant Yap 5SA or pMig control vector (C) and CTLA-4+/+ or CTLA-4−/− OT-I cells (D) were analyzed for Blimp-1 mRNA 48 h after restimulation with IL-2 and SIINFEKL peptide. The levels of Blimp-1 mRNA relative to the levels of naïve unstimulated OT-I cells are shown. Data presented are the mean ± SEM. (E and F) Thy1.2+ OT-I cells that had been transduced with the Yap 5SA-expressing retrovirus or control pMig (E), and Thy1.2+ CTLA-4−/− OT-I cells or CTLA-4+/+ OT-I cells (F) were adoptively transferred to Thy1.1+ C57BL/6 mice, and 2 d later, the mice were infected with γ-MHV-68/OVA. On days 8 and 11 postinfection, peripheral blood Thy1.2+ OT-I cells were assessed for expression of KLRG1. Numbers represent the percentage KLRG1+ cells (means ± SEM). *P < 0.05.

Fig. P1.

Fig. P1.

Quorum sensing model of CD8+ T-cell differentiation. Clonal expansion increases the frequency of activated antigen-specific cells and the likelihood of cell–cell contact leading to the surface expression and ligation of CTLA-4 to CD80, which triggers differentiation.

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