SIKs Regulate HDAC7 Stabilization and Cytokine Recall in Late-Stage T Cell Effector Differentiation

Abstract

Understanding the mechanisms underlying the acquisition and maintenance of effector function during T cell differentiation is important to unraveling how these processes can be dysregulated in the context of disease and manipulated for therapeutic intervention. In this study, we report the identification of a previously unappreciated regulator of murine T cell differentiation through the evaluation of a previously unreported activity of the kinase inhibitor, BioE-1197. Specifically, we demonstrate that liver kinase B1 (LKB1)-mediated activation of salt-inducible kinases epigenetically regulates cytokine recall potential in effector CD8+ and Th1 cells. Evaluation of this phenotype revealed that salt-inducible kinase-mediated phosphorylation-dependent stabilization of histone deacetylase 7 (HDAC7) occurred during late-stage effector differentiation. HDAC7 stabilization increased nuclear HDAC7 levels, which correlated with total and cytokine loci-specific reductions in the activating transcription mark histone 3 lysine 27 acetylation (H3K27Ac). Accordingly, HDAC7 stabilization diminished transcriptional induction of cytokine genes upon restimulation. Inhibition of this pathway during differentiation produced effector T cells epigenetically poised for enhanced cytokine recall. This work identifies a previously unrecognized target for enhancing effector T cell functionality.

Figure 1.. Administration of BioE-1197 during T cell differentiation durably enhances effector cytokine production upon re-stimulation in CD8⁺ T cells, independent of PASK.

(A) Representative intracellular cytokine staining dot plots of IFNγ (top) and TNFα (bottom) production upon re-stimulation of CD8⁺ T cells on day six after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. (B) Quantification of the percent of IFNγ (top) and TNFα (bottom) cytokine positive cells represented in A across independent experiments. (C) Quantification of the fold change in geometric mean fluorescence intensity (gMFI) of IFNγ (top) and TNFα (bottom) production by CD8⁺ T cells represented in A across independent experiments. (D) Quantification of IFNγ (top) and TNFα (bottom) production in the supernatants of re-stimulated CD8⁺ T cells across independent experiments by ELISA. (E) Representative dot plots of IFNγ (left) and TNFα (right) production upon re-stimulation of WT (top) or PASK knockout (KO) (bottom) CD8⁺ T cells differentiated in control (DMSO) or BioE-1197 (50μM) conditions. (F) Quantification of the fold change in gMFI of IFNγ (top) and TNFα (bottom) production by WT and PASK KO CD8⁺ T cells represented in E across independent experiments. IFNγ: F_genotype p = 0.3640, F_BioE-1197 p = 0.0012. TNFα: F_genotype p = 0.9442, F_BioE-1197 p = 0.0133. (G) Schematic representing the experimental design for park and recall co-adoptive transfer of control (DMSO) and BioE-1197 (50μM) differentiated P14 CD8⁺ T cells. (H) Quantification of the percent of cytokine positive cells for IFNγ and TNFα upon ex vivo peptide re-stimulation and intracellular cytokine staining across ten mice. (I) Quantification of the fold change in gMFI for IFNγ (left) and TNFα (right) upon ex vivo peptide re-stimulation and intracellular cytokine staining across ten mice. Each dot represents values from an independent experiment, summary data are presented as the mean (black line) with SEM error bars (B-D, F). Each dot represents an individual mouse within one independent experiment and presented results are representative of two independent experiments (H, I). Paired t test (B, C, D). Two-way ANOVA, Sidak’s multiple comparisons (F). Paired t test (H, I – IFNγ). Wilcoxon test (I-TNFα). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 2.. BioE-1197 activity is dependent on ARKs.

(A) Representative dot plots of Live/Dead staining of control or LKB1 sgRNA edited CD8⁺ T cells on day six after initial activation and differentiation in control (DMSO) or BioE-1197 (50 μM) conditions. (B) Quantification of percent live cells on day six after activation represented in A across independent experiments. (C) Representative dot plots of IFNγ (left) or TNFα (right) production in control (top) or LKB1 sgRNA (bottom) edited CD8⁺ T cells on day six after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. (D) Quantification of fold change in IFNγ (top) and TNFα (bottom) gMFI represented in C across independent experiments. (E) Representative western blot of control or LKB1 sgRNA edited CD8⁺ T cells on day six after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. (F) Quantification of the fold change in pHDAC7/Actin (left) and HDAC7/Actin (right) represented in E across independent experiments. Each dot represents values from an independent experiment, summary data are presented as the mean (black line) with SEM error bars (B, D, F). Two-way ANOVA, Sidak’s multiple comparison test (B, D). Two-way ANOVA, Tukey’s multiple comparisons test (F). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant.

Figure 3.. SIKs regulate phosphorylation dependent stabilization of HDAC7 and lineage specific cytokine production in CD8⁺ T cells.

(A) Representative western blot of phosphorylated and total HDAC7 by CD8⁺ T cells on day four after activation and differentiation in either control (DMSO), BioE-1197 (50 μM), HG-9-91-01 (HG, 25 nM) or YKL-05–099 (YKL, 270 nM) differentiation conditions. (B) Quantification of the fold change in phosphorylated (left) and total HDAC7 (right) production in CD8⁺ T cells represented in A across independent experiments. (C) Representative intracellular cytokine staining dot plots of IFNγ (top) and TNFα (bottom) production by CD8⁺ T cells on day six after activation and differentiation in either control (DMSO), BioE-1197 (50 μM), HG-9-91-01 (HG, 25 nM) or YKL-05–099 (YKL, 270 nM) conditions. (D) Quantification of the fold change in gMFI of IFNγ (left) and TNFα (right) production in CD8⁺ T cells represented in C across independent experiments. Each dot represents values from an independent experiment, summary data are presented as the mean (black line) with SEM error bars (B, D). Repeated measures one-way ANOVA, Dunnet’s multiple comparisons test (B). Friedman test, Dunn’s multiple comparisons test (D). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant.

Figure 4.. Phosphorylation dependent stabilization enhances nuclear availability of HDAC7 and epigenetically restricts effector cytokine loci.

(A) Fold change in HDAC7 mRNA levels of CD8⁺ T cells on day six after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. (B) Representative western blot of HDAC7 from subcellular fractionation of CD8⁺ T cells on day three after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. Lamin B and IκBα serve as nuclear and cytosolic compartment loading controls, respectively. (C) Quantification of the fold change in HDAC7 localization represented in B across independent experiments in CD8⁺ T cells on day three after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. (D) Quantification of the fold change in global H3K27Ac levels for control (DMSO) and BioE-1197 (50μM) differentiated CD8⁺ T cells prior to re-stimulation and two hours after re-stimulation on day six after activation and differentiation across independent experiments. (E) Fold change of Ifng, Tnf, Gzmb, and Prf1 mRNA transcript abundance prior to re-stimulation and two hours after re-stimulation in CD8⁺ T cells on day six after activation and differentiation in control (DMSO) or BioE-1197 (50μM) across independent experiments. (F) H3K27Ac mark deposition within the Ifng loci in control and BioE-1197 (50μM) differentiated CD8⁺ T cells on day six after activation and differentiation. (G) H3K27Ac mark deposition within the Tnf loci in control and BioE-1197 (50μM) differentiated CD8⁺ T cells on day six after activation and differentiation. Each dot represents values from an independent experiment, summary data are presented as the mean (black line) with SEM error bars (A, C, D, E). Results are representative of three, independent biological replicates, additional replicates presented in Supplemental Figure 3 (F,G). Paired t-test (A,C). Two-way ANOVA, Sidak’s multiple comparisons test (D, E). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant.

Figure 5.. BioE-1197 enhances effector cytokine production and regulates HDAC7 stability and localization in CD4⁺ Th1 cells.

(A) Representative intracellular cytokine staining dot plots of IFNγ (top) and TNFα (bottom) production upon re-stimulation of Th1 cells on day six after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. (B) Quantification of the percent of IFNγ (top) and TNFα (bottom) cytokine positive Th1 cells represented in A across independent experiments. (C) Quantification of the fold change in gMFI of IFNγ (top) and TNFα (bottom) production by Th1 cells represented in A across independent experiments. (D) Representative intracellular staining dot plots of T-bet in Th1 cells on day six after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. (E) Quantification of the percent of T-bet positive cells represented in D across independent experiments. (F) Quantification of the fold change in gMFI of T-bet by CD8⁺ T cells represented in D across independent experiments. (G) Representative western blot of HDAC7 from subcellular fractionation of Th1 cells on day three after activation and differentiation in control (DMSO) or BioE-1197 (50μM) conditions. Lamin B and IκBα serve as nuclear and cytosolic compartment loading controls, respectively. (H) Quantification of the fold change in HDAC7 localization represented in G across independent experiments in Th1 cells on day three after activation and differentiation in control (DMSO) or BioE-1197 (50μM) condition. Each dot represents values from an independent experiment, summary data are presented as the mean (black line) with SEM error bars (B-C, E-F, H). Wilcoxon test (B-IFNγ, C-IFNγ, E). Paired t-test (B-TNFα, C-TNFα, F, H). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant.

Figure 6.. SIKs regulate phosphorylation dependent stabilization of HDAC7 and lineage specific cytokine production in Th1 cells.

(A) Representative western blot of phosphorylated and total HDAC7 by Th1 cells on day four after activation and differentiation in either control (DMSO), BioE-1197 (50 μM), HG-9-91-01 (HG, 25 nM) or YKL-05-099 (YKL, 270 nM) differentiation conditions. (B) Quantification of the fold change in phosphorylated (left) and total HDAC7 (right) production in Th1 cells represented in A across independent experiments. (C) Representative intracellular cytokine staining dot plots of IFNγ (top) and TNFα (bottom) production by Th1 cells on day six after activation and differentiation in either control (DMSO), BioE-1197 (50 μM), HG-9-91-01 (HG, 25 nM) or YKL-05–099 (YKL, 270 nM) conditions. (D) Quantification of the fold change in gMFI of IFNγ (top) and TNFα (bottom) production in Th1 cells represented in C across independent experiments. Each dot represents values from an independent experiment, summary data are presented as the mean (black line) with SEM error bars (B, D). Repeated measures one-way ANOVA, Dunnet’s multiple comparisons test (B). Mixed effects analysis, Dunnet’s multiple comparisons test (D). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant.

Figure 7.. Phosphorylation dependent stabilization of HDAC7 occurs during late-stage differentiation of CD8⁺ and Th1 effector.

(A) Representative western blots for control (DMSO) and BioE-1197 (50μM) differentiated CD8⁺ (left) and Th1 (right) cells for phosphorylated and total HDAC7. (B) Quantification of the fold change in phosphorylated (top) and total (bottom) HDAC7 in CD8⁺ (left) and Th1 (right) cells represented in A across independent experiments. (C) Quantification of global H3K27Ac marks over time in CD8⁺ (left) and Th1 (right) cells differentiated in control (DMSO) and BioE-1197 (50μM) conditions. Each dot represents values form an independent experiment, summary data are presented as the mean (black line) with SEM error bars (B). The mean and standard error of measurement (SEM) are presented for 12 independent experiments (C). Two-way ANOVA, Sidak’s multiple comparisons test (B). Mixed effects analysis, Sidak’s multiple comparisons test (C). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant.

Figure 8.. Late-stage class IIa HDAC inhibition elevates effector cytokine production.

(A) Representative dot plots of IFNγ (top) and TNFα (bottom) production by CD8⁺ T cells on day six after activation and differentiation in either control (DMSO), BioE-1197 (50μM), or TMP269 (TMP, 12.5 μM) conditions. (B) Quantification of the fold change in gMFI of IFNγ (top) and TNFα (bottom) production in CD8⁺ T cells represented in A across independent experiments. (C) Representative dot plots of IFNγ (top) and TNFα (bottom) production by Th1 cells on day six after activation and differentiation in either control (DMSO), BioE-1197 (50μM), or TMP269 (TMP, 12.5 μM)) conditions. (D) Quantification of the fold change in gMFI of IFNγ (top) and TNFα (bottom) production in Th1 cells represented in C across independent experiments. Each dot represents values from an independent experiment, summary data are presented as the mean (black line) with SEM error bars (B,D). Friedman test, Dunn’s multiple comparisons test (B,D). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 9.. LKB1 activated SIK function mediates phosphorylation dependent stabilization of HDAC7 and lineage specific cytokine recall in CD8⁺ and Th1 cells.

(A) Model depicting LKB1-SIK mediated phosphorylation dependent stabilization of HDAC7 leading to enhanced HDAC7 nuclear import due to increased HDAC7 protein levels, removal of H3K27Ac marks and restriction of effector cytokine production, epigenetically. (B) Model depicting BioE-1197 mediated inhibition of SIK dependent HDAC7 phosphorylation dependent stabilization which maintains low nuclear HDAC7 levels compared to the control condition, persistence of H3K27Ac levels, and the generation of cells epigenetically poised for enhanced effector cytokine production.