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Epigenetic plasticity of Cd8a locus during CD8(+) T-cell development and effector differentiation and reprogramming.

Harland KL, Day EB, Apte SH, Russ BE, Doherty PC, Turner SJ, Kelso A - Nat Commun (2014)

Bottom Line: Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen.Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8(+) effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus.This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8(+) T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Victoria 3010, Australia [2].

ABSTRACT
Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen. Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8(+) effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus. These epigenetic modifications are maintained long-term in vivo following adoptive transfer. Even after extended type 2 polarization, however, some CD8(low) effector cells respond to interferon-γ by re-expressing CD8 and a type 1 cytokine profile in association with partial Cd8a demethylation. Cd8a methylation signatures in naive, polarized and repolarized cells are distinct from those observed during the initiation, maintenance and silencing of CD8 expression by developing T cells in the thymus. This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8(+) T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.

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IFN-γ induces CD8 re-expression on some CD8low cells.(a) Surface CD8 expression by 7-day type 2 CD8low cells and neutral CD8high cells before and after purification and restimulation with anti-receptor Ab and IL-2 for 8 days in the indicated conditions. (b) CD8 expression by clones grown from single twice-purified 7-day type 2 CD8low cells and neutral CD8high cells cultured with anti-receptor Ab and IL-2 in reversal (IFN-γ and anti-IL-4 Ab), type 2 or neutral conditions for 13 days. Cells expanded 911-fold during primary activation (≥9–10 cell divisions). Cloning efficiencies were CD8low→reversal, 13.1%; CD8low→type 2, 6.6%; CD8low→neutral, 14.3%; CD8high→neutral, 15.8%. Upper panels show clones estimated to contain ≥100 cells from two independent experiments (filled and empty symbols) ranked by % CD8low cells (dashed line: median % CD8low). Lower panels show representative CD8low clones cultured in reversal conditions (isotype control staining of pooled clones in grey). (c) Percent CD8low cells among twice-purified CD8low cells from primary type 2 cultures after re-culture in reversal conditions with or without anti-receptor Ab (n=3, except day 4 primary cells re-cultured for 2 days with (n=2) or without (n=1) restimulation) (representative of three independent experiments). (d) Percent CD8low cells among twice-purified CD8low cells from 7-day type 2 cultures after re-culture for 8 days in reversal conditions with the indicated immobilized Ab (***P<0.001, one-way ANOVA with Bonferroni’s post test for multiple comparisons) (representative of three independent experiments).
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f5: IFN-γ induces CD8 re-expression on some CD8low cells.(a) Surface CD8 expression by 7-day type 2 CD8low cells and neutral CD8high cells before and after purification and restimulation with anti-receptor Ab and IL-2 for 8 days in the indicated conditions. (b) CD8 expression by clones grown from single twice-purified 7-day type 2 CD8low cells and neutral CD8high cells cultured with anti-receptor Ab and IL-2 in reversal (IFN-γ and anti-IL-4 Ab), type 2 or neutral conditions for 13 days. Cells expanded 911-fold during primary activation (≥9–10 cell divisions). Cloning efficiencies were CD8low→reversal, 13.1%; CD8low→type 2, 6.6%; CD8low→neutral, 14.3%; CD8high→neutral, 15.8%. Upper panels show clones estimated to contain ≥100 cells from two independent experiments (filled and empty symbols) ranked by % CD8low cells (dashed line: median % CD8low). Lower panels show representative CD8low clones cultured in reversal conditions (isotype control staining of pooled clones in grey). (c) Percent CD8low cells among twice-purified CD8low cells from primary type 2 cultures after re-culture in reversal conditions with or without anti-receptor Ab (n=3, except day 4 primary cells re-cultured for 2 days with (n=2) or without (n=1) restimulation) (representative of three independent experiments). (d) Percent CD8low cells among twice-purified CD8low cells from 7-day type 2 cultures after re-culture for 8 days in reversal conditions with the indicated immobilized Ab (***P<0.001, one-way ANOVA with Bonferroni’s post test for multiple comparisons) (representative of three independent experiments).

Mentions: IFN-γ counteracts IL-4-induced CD8 downregulation during primary CD8+ T-cell effector differentiation in vivo and in vitro18. To test whether IFN-γ could also act later to reverse CD8 downregulation in type 2-polarized CD8low cells, in vitro generated CD8low cells were restimulated with anti-receptor Ab and IL-2 with or without IFN-γ, IL-4 and/or anti-cytokine Ab for 8 days (Fig. 5a). CD8low cells restimulated in type 2 (IL-4 and anti-IFN-γ Ab) or neutral conditions (none) remained CD8low as previously reported17. In neutralizing concentrations of anti-IL-4 Ab, a small proportion of cells (6.3%) re-expressed CD8, suggesting that endogenous IL-4 maintained the CD8low phenotype of some cells under neutral conditions. IFN-γ induced CD8 re-expression on 34% of cells, but CD8 re-expression was highest (41%) when both IFN-γ and anti-IL-4 Ab were present. CD8 was re-expressed as CD8αβ heterodimers (Supplementary Fig. 5a). Thus, IFN-γ and IL-4 reciprocally regulate CD8 expression not only during primary CD8+ T-cell activation1819 but also during restimulation of type 2-polarized CD8low cells.


Epigenetic plasticity of Cd8a locus during CD8(+) T-cell development and effector differentiation and reprogramming.

Harland KL, Day EB, Apte SH, Russ BE, Doherty PC, Turner SJ, Kelso A - Nat Commun (2014)

IFN-γ induces CD8 re-expression on some CD8low cells.(a) Surface CD8 expression by 7-day type 2 CD8low cells and neutral CD8high cells before and after purification and restimulation with anti-receptor Ab and IL-2 for 8 days in the indicated conditions. (b) CD8 expression by clones grown from single twice-purified 7-day type 2 CD8low cells and neutral CD8high cells cultured with anti-receptor Ab and IL-2 in reversal (IFN-γ and anti-IL-4 Ab), type 2 or neutral conditions for 13 days. Cells expanded 911-fold during primary activation (≥9–10 cell divisions). Cloning efficiencies were CD8low→reversal, 13.1%; CD8low→type 2, 6.6%; CD8low→neutral, 14.3%; CD8high→neutral, 15.8%. Upper panels show clones estimated to contain ≥100 cells from two independent experiments (filled and empty symbols) ranked by % CD8low cells (dashed line: median % CD8low). Lower panels show representative CD8low clones cultured in reversal conditions (isotype control staining of pooled clones in grey). (c) Percent CD8low cells among twice-purified CD8low cells from primary type 2 cultures after re-culture in reversal conditions with or without anti-receptor Ab (n=3, except day 4 primary cells re-cultured for 2 days with (n=2) or without (n=1) restimulation) (representative of three independent experiments). (d) Percent CD8low cells among twice-purified CD8low cells from 7-day type 2 cultures after re-culture for 8 days in reversal conditions with the indicated immobilized Ab (***P<0.001, one-way ANOVA with Bonferroni’s post test for multiple comparisons) (representative of three independent experiments).
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Related In: Results  -  Collection

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f5: IFN-γ induces CD8 re-expression on some CD8low cells.(a) Surface CD8 expression by 7-day type 2 CD8low cells and neutral CD8high cells before and after purification and restimulation with anti-receptor Ab and IL-2 for 8 days in the indicated conditions. (b) CD8 expression by clones grown from single twice-purified 7-day type 2 CD8low cells and neutral CD8high cells cultured with anti-receptor Ab and IL-2 in reversal (IFN-γ and anti-IL-4 Ab), type 2 or neutral conditions for 13 days. Cells expanded 911-fold during primary activation (≥9–10 cell divisions). Cloning efficiencies were CD8low→reversal, 13.1%; CD8low→type 2, 6.6%; CD8low→neutral, 14.3%; CD8high→neutral, 15.8%. Upper panels show clones estimated to contain ≥100 cells from two independent experiments (filled and empty symbols) ranked by % CD8low cells (dashed line: median % CD8low). Lower panels show representative CD8low clones cultured in reversal conditions (isotype control staining of pooled clones in grey). (c) Percent CD8low cells among twice-purified CD8low cells from primary type 2 cultures after re-culture in reversal conditions with or without anti-receptor Ab (n=3, except day 4 primary cells re-cultured for 2 days with (n=2) or without (n=1) restimulation) (representative of three independent experiments). (d) Percent CD8low cells among twice-purified CD8low cells from 7-day type 2 cultures after re-culture for 8 days in reversal conditions with the indicated immobilized Ab (***P<0.001, one-way ANOVA with Bonferroni’s post test for multiple comparisons) (representative of three independent experiments).
Mentions: IFN-γ counteracts IL-4-induced CD8 downregulation during primary CD8+ T-cell effector differentiation in vivo and in vitro18. To test whether IFN-γ could also act later to reverse CD8 downregulation in type 2-polarized CD8low cells, in vitro generated CD8low cells were restimulated with anti-receptor Ab and IL-2 with or without IFN-γ, IL-4 and/or anti-cytokine Ab for 8 days (Fig. 5a). CD8low cells restimulated in type 2 (IL-4 and anti-IFN-γ Ab) or neutral conditions (none) remained CD8low as previously reported17. In neutralizing concentrations of anti-IL-4 Ab, a small proportion of cells (6.3%) re-expressed CD8, suggesting that endogenous IL-4 maintained the CD8low phenotype of some cells under neutral conditions. IFN-γ induced CD8 re-expression on 34% of cells, but CD8 re-expression was highest (41%) when both IFN-γ and anti-IL-4 Ab were present. CD8 was re-expressed as CD8αβ heterodimers (Supplementary Fig. 5a). Thus, IFN-γ and IL-4 reciprocally regulate CD8 expression not only during primary CD8+ T-cell activation1819 but also during restimulation of type 2-polarized CD8low cells.

Bottom Line: Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen.Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8(+) effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus.This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8(+) T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Victoria 3010, Australia [2].

ABSTRACT
Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen. Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8(+) effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus. These epigenetic modifications are maintained long-term in vivo following adoptive transfer. Even after extended type 2 polarization, however, some CD8(low) effector cells respond to interferon-γ by re-expressing CD8 and a type 1 cytokine profile in association with partial Cd8a demethylation. Cd8a methylation signatures in naive, polarized and repolarized cells are distinct from those observed during the initiation, maintenance and silencing of CD8 expression by developing T cells in the thymus. This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8(+) T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.

Show MeSH