CD4(+) T cell lineage integrity is controlled by the histone deacetylases HDAC1 and HDAC2

Abstract

Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II-selected CD4(+) helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2-deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8(+) effector T cell program in a manner dependent on Runx-CBFβ complexes, whereas TH2 cells repressed features of the CD8(+) lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFβ complexes that otherwise induce a CD8(+) effector T cell-like program in CD4(+) T cells.

Figure 1

Altered T cell subsets in mice with a T cell-specific loss of HDAC1 and HDAC2. (a) Flow cytometry analysis of B220, TCRβ, CD4 and CD8α expression on splenocytes isolated from WT and HDAC1-2 cKO mice. (b) Absolute numbers of all cells, B220⁺ B cells, TCRβ⁺ T cells, CD4⁺ T cells, CD8⁺ T cells and CD4⁺CD8⁺ T cells in the spleens of WT and HDAC1-2 cKO mice. (c) Flow cytometry analysis of CD8β expression in CD4⁺CD8⁺ T cells isolated from the spleen of HDAC1-2 cKO mice. (d-e) Representative flow cytometry analysis (d) and summary (e) of Annexin V and 7-AAD staining on peripheral splenic CD4⁺ T cells, CD8⁺ T cells and CD4⁺CD8⁺ T cells isolated from WT and HDAC1-2 cKO mice. (a, d) Numbers indicate the percentage of cells in the respective quadrants. (b,e) Thick horizontal bars indicate the mean. (b) *P < 0.05 and ***P < 0.001 (unpaired two-tailed Student’s t-test, except for B220⁺ cells (unpaired Mann-Whitney test)). (e) **P < 0.01 (unpaired two-tailed Mann-Whitney test). Data are representative (a,c,d) or show the summary (b,e) of five mice (a,c), of eight mice (except for B220⁺ cells with four mice) and of ten mice (e) that were analyzed in two (a,b) and three (c-e) independent experiments.

Figure 2

Mature TCRβ^hiCD24^lo SP cells are reduced in HDAC1-2 cKO mice. (a) Flow cytometry analysis of CD4 and CD8α expression on WT and HDAC1-2 cKO thymocytes, and of CD5, CD69 and CD24 expression on DP, TCRβ^hiCD4⁺ and TCRβ^hiCD8⁺ thymocyte subsets. (b) Absolute thymocyte numbers in WT and HDAC1-2 cKO mice. (c,d) Percentages (c) and absolute cell numbers (d) of TCRβ^hi, TCRβ^hiCD24^hi and TCRβ^hiCD24^lo WT and HDAC1-2 cKO thymocyte subsets. (e) Flow cytometry analysis of CD4 and CD8α expression on WT and HDAC1-2 cKO TCRβ^hiCD24^lo thymocytes. (f) Summary of BrdU incorporation in TCRβ^loCD24^hi, TCRβ^hiCD24^hi and TCRβ^hiCD24^lo thymocyte subsets in WT and HDAC1-2 cKO mice. (g) Percentage of Annexin V⁺ cells in TCRβ^loCD24^hi, TCRβ^hiCD24^hi and TCRβ^hiCD24^lo WT and HDAC1-2 cKO thymocyte subsets. (a,e) Numbers in the contour plot quadrants indicate the percentage of cells. (b-d,f,g) Horizontal bars indicate the mean. Data are representative (a,e) or show the summary (b-d,f,g) of thirteen (WT) and nineteen (HDAC1-2 cKO) mice except for CD5 (four mice) and CD69, CD24 (five mice) (a-d), of ten mice (e), of four mice (f) and of six (WT) and eleven (HDAC1-2 cKO) mice (g) that were analyzed in two to five (a), four (b-d), two (f) and three (g) independent experiments. (c) *P < 0.05 (unpaired two-tailed Student’s t-test). (d) **P < 0.001 (unpaired two-tailed Mann-Whitney test). (g) NS, not significant (unpaired two-tailed Mann-Whitney test).

Figure 3

HDAC1-2 cKO CD4⁺CD8⁺ T cells are MHC class II-selected and up-regulate CD154 upon activation. (a) Flow cytometry analysis of CD4 and CD8α expression on TCRβ⁺ splenocytes isolated from WT and HDAC1-2 cKO BM chimeric B2m^−/− mice. (b) Flow cytometry analysis of CD4 and CD8α expression on H2-Ab1^−/−WT or H2-Ab1^−/−HDAC1-2 cKO TCRβ⁺ splenocytes. (c) Flow cytometry analysis of CD154 and CD69 expression on WT and HDAC1-2 cKO CD4⁺ T cells and on HDAC1-2 cKO CD4⁺CD8⁺ T cells and CD8⁺ T cells isolated from WT and HDAC1-2 cKO BM chimeric B2m^−/− mice, or on CD8⁺ T cells isolated from C57BL/6 mice. Cells were activated with anti-CD3 and anti-CD28 for 24 hours. (d) Flow cytometry analysis of intracellular HDAC1 and HDAC2 expression in WT and HDAC1-2 cKO CD4⁺ T cells, and in HDAC1-2 cKO CD4⁺CD8⁺ T cells. The CD4 and CD8α contour plots of WT and HDAC1-2 cKO TCRβ⁺ splenocytes indicate gating areas. (e) Percentage of HDAC1 and HDAC2 expressing WT CD4⁺ T cells, HDAC1-2 cKO CD4⁺ T cells and HDAC1-2 cKO CD4⁺CD8⁺ T cells. Cells were gated on the upper-right quadrant of the HDAC1-HDAC2 contour plots shown in (d). (f) Mean fluorescence intensity (MFI) of HDAC1 and HDAC2 expression in WT CD4⁺ T cells, and in HDAC1-2 cKO CD4⁺ T cells and CD4⁺CD8⁺ T cells. (a,b,d) Numbers in quadrants indicate the percentage of cells. Data are representative (a-d) or show the summary (e,f) of six BM chimeric B2m^−/− mice (a), of three mice (b), of two independent samples (c) and of nine (WT) and ten (HDAC1-2 cKO) mice (d-f) analyzed in two (a-c) or three (d-f) independent experiments. (e) **P < 0.01 (unpaired two-tailed Mann-Whitney test). (f) *P < 0.05 and **P < 0.001 (unpaired two-tailed Mann-Whitney test). Mean ± SD is shown.

Figure 4

CD4⁺ T cells from HDAC1-2 cKO mice up-regulate CD8 upon activation. (a) Flow cytometry analysis showing cell divisions of CFSE-labeled WT and HDAC1-2 cKO CD4⁺ T cells activated with anti-CD3 and anti-CD28 for 2.5 days. (b) Flow cytometry analysis of CD69 and CD25 expression on WT and HDAC1-2 cKO CD4⁺ T cells activated with anti-CD3 and anti-CD28 for 16 hours. Grey areas indicate non-activated CD4⁺ T cells. (c) Flow cytometric analysis of CD4 and CD8α expression on WT and HDAC1-2 cKO CD4⁺ and CD8⁺ T cells activated with anti-CD3 and anti-CD28 for 24 and 48 hours. (d,e) Representative flow cytometry analysis (d) and summary (e) of CD4 and CD8α expression on TCRβ⁺ splenocytes from E8_I^+/+HDAC1-2 cKO (E8_I^+/+) and E8_I^−/−HDAC1-2 cKO (E8_I^−/−) mice. (f) Mean fluorescence intensity (MFI) of CD8α expression on E8_I^+/+HDAC1-2 cKO and E8_I^−/−HDAC1-2 cKO CD4⁺CD8⁺ T cells. CD4⁺CD8⁺ T cells were gated on the upper right quadrants of the CD4-CD8α contour plots shown in (d). (g) Flow cytometric analysis showing Annexin V and 7-AAD stainings on WT and HDAC1-2 cKO CD4⁺ T cells activated with anti-CD3 and anti-CD28 for 48 and 72 hours. (b,c,d,g) Numbers indicate the percentage of cells in the respective quadrant (c,d,g) or histogram regions (b). Data are representative of two (a,b,g) and three (c) independent samples and of five mice (d-f) that were analyzed in two (a,b,d-g) and three (c) experiments. (e) Horizontal bars indicate the mean. (f) Mean ± SD is shown. (e,f) *P < 0.01 (unpaired two-tailed Mann-Whitney test).

Figure 5

A CD8 effector program is induced in activated HDAC1-2 cKO CD4⁺ T cells. (a) IFN-γ in supernatants of WT and HDAC1-2 cKO CD4⁺ and CD8⁺ T cells activated with anti-CD3 and anti-CD28 for 60 hours. *P < 0.05 (unpaired two-tailed Mann-Whitney test). Mean ± SD is shown. (b) qRTPCR analysis showing the expression of several genes in WT and HDAC1-2 cKO CD4⁺ and CD8⁺ T cells activated as described in (a). Expression relative to Hprt is shown. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student’s t-test). Mean ± SD is shown. (c,d) Flow cytometric analysis showing intracellular expression of IFN-γ and Eomesodermin and of GATA-3 and T-bet (c) and of Granzyme B (Gz B) and surface expression of CD8α (d) in WT and HDAC1-2 cKO CD4⁺ and CD8⁺ T cells activated as described in (a). (e) Flow cytometry analysis showing cell divisions of cell division dye Efluor-V450-labeled naive HDAC1-2 cKO CD4⁺ T cells activated as described in (a). Cells were gated on CD4⁺CD8α⁻ and CD4⁺CD8α⁺ subsets. (f) qRTPCR analysis with primers specific for Runx3, Eomes and Cd8a promoter regions and for Cd8 enhancer E8_I from chromatin of non-activated (N) and activated (A; anti-CD3 plus anti-CD28 for 60 hours) WT and HDAC1-2 cKO CD4⁺ T cells immunoprecipitated with anti-H3K9Ac or anti-H3K27me3 antibodies (or IgG as control). Mean ± SD is shown. Values are corrected to H3 levels and shown as % input. (c, e) Numbers in contour plots indicate the percentage of cells in the respective quadrant. Data are representative (c-f) or show the summary (a,b) of four (a,e), of three (CD4⁺ T cells) and two (CD8⁺ T cells) (b) and of two (c,d,f) independent samples that were analyzed in three (a), in two (CD4⁺ T cells) and one (CD8⁺ T cells) (b) and in two (c-f) independent experiments.

Figure 6

A CD8 effector program is induced in HDAC1-2 cKO T_H1 but not in T_H2 cells. (a) Flow cytometry analysis showing intracellular expression of IFN-γ and Eomesodermin and of GATA-3 and T-bet in naive WT and HDAC1-2 cKO CD4⁺ T cells activated with anti-CD3 and anti-CD28 for 60 hours in the presence of T_H1- and T_H2-polarizing conditions. (b) Flow cytometry analysis showing intracellular Granzyme B (Gz B) and surface CD8α expression in WT and HDAC1-2 cKO CD4⁺ T cells activated as described in (a). (c) Flow cytomety analysis showing CD8α expression on naive HDAC1-2 cKO CD4⁺ T cells activated with anti-CD3 and anti-CD28 for 60 hours in the presence of IFN-γ, IL-12, IL-4 or without the addition of cytokines (control; Ctrl). (a-c) Numbers indicate the percentage of cells in the (a) respective quadrant or in (b,d) histograms. (a-c) Data are representative of two independent samples analyzed in two (a,b) or one (c) experiments.

Figure 7

Th-POK expression in HDAC1-2 cKO CD4⁺ T cells. (a) Immunoblot showing Th-POK expression in WT CD4⁺ and CD8⁺ T cells and in HDAC1-2 cKO CD4⁺, CD8⁺ and CD4⁺CD8⁺ T cells. Whole cell lysates from approx. 3 × 10⁵ cells were loaded and β-Actin was used as loading control. (b) Quantification of the immunoblot shown in (a). Th-POK expression was normalized to β-Actin. Th-POK expression in WT CD4⁺ was set as 1 and relative intensities are shown. (c) Flow cytometry analysis showing GFP expression in WT CD4⁺ and CD8⁺ T cells and in HDAC1-2 cKO CD4⁺, CD8⁺ and CD4⁺CD8⁺ T cells. The CD4 and CD8α contour plots of Zbtb7b^+/GFPWT and Zbtb7b^+/GFPHDAC1-2 cKO TCRβ⁺ splenocytes indicate the gating regions. Numbers indicate the percentage of cells. (a-c) Data are representative of three independent samples (a,b) and of five (Zbtb7b^+/GFPWT) and ten (Zbtb7b^+/GFPHDAC1-2 cKO) mice (c) performed in three (a,b) and two (c) independent experiments.

Figure 8

The induction of CD8 lineage genes in HDAC1-2 cKO CD4⁺ T cells is dependent on Runx-CBFβ complexes. (a) Immunoblot showing Runx3 expression in non-activated (N) and anti-CD3 with anti-CD28 activated for 48h (A) WT and HDAC1-2 cKO CD4⁺ T cells. Whole cell lysates were loaded. Non-activated Runx3^f/fCd4-Cre CD4⁺ T cells (Runx3-null) or wild-type CD8⁺ T cells (CD8⁺) were used as controls. Runx3 expression in activated wild-type CD4⁺ T cells treated with MS-275 (MS) or DMSO as carrier control (C) is shown at the right. Diagram shows Runx3 expression (normalized to β-Actin) in activated HDAC1-2 cKO CD4⁺ T cells relative to activated WT CD4⁺ T cells (mean ± SD) (b,c) Flow cytometry analysis showing intracellular expression of IFN-γ and Eomesodermin (Eomes) (b) and intracellular expression of Granzyme B (Gz B) and T-bet expression and surface expression of CD8α (c) in naïve WT, HDAC1-2 cKO and CBFβ-HDAC1-2 cKO CD4⁺ T cells activated with anti-CD3, anti-CD28 and hrIL-2 for 60 hours. (d) qRTPCR analysis with primers specific for Prf1, GzmB, Infg and Tcrb promoter regions and for Cd8 enhancer E8_I from chromatin of activated (anti-CD3 with anti-CD28 for 48 hours) WT CD4⁺ and HDAC1-2 cKO CD4⁺ T cells and from activated (anti-CD3 and anti-CD28 for 2 days, followed by 4 days rest) wild-type CD8⁺ T cells immunoprecipitated with anti-CBFβ antibody or with IgG as control. Values are shown as % input. (e) Flow cytometry analysis showing surface CD8α and intracellular Granzyme B (Gz B), Eomesodermin (Eomes), IFN-γ and T-bet expression on GFP⁺ wild-type CD4⁺ T cells 48 hours after transduction with either “empty” MIGR-control or with a Runx3d-containing retroviral vector. MS-275 (or DMSO as carrier control) or anacardic acid were added one day after transduction. (f) Flow cytometry analysis showing CD4 and CD8α expression on WT and HDAC1-2 cKO CD4⁺ T cells activated with anti-CD3 and anti-CD28 for 48 hours. Anacardic acid (or EtOH as carrier control; Ctrl.) was added 24 hours after activation. (g) Flow cytometry analysis of CD4 and CD8α expression on wild-type (C57BL/6), E8_I^−/− and Cbfb^f/fCd4-Cre CD4⁺ T cells activated with anti-CD3 and anti-CD28 activated for 48 hours. MS-275 was added 24h after activation. Data are representative (a-c,e-g) or show the summary (d) of two (a-c,f), of two (CD4⁺ T cells) and one (CD8⁺ T cells, two independent ChIP assays) (d), of at least three (except two for IFN-γ) (e) and of six (WT), three (E8_I^−/−) and four (Cbfb^f/fCd4-Cre CD4⁺) independent samples (g) that were analyzed in (a-d,f) two and two to three (e,g) independent experiments.