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A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance.

Feng Y, van der Veeken J, Shugay M, Putintseva EV, Osmanbeyoglu HU, Dikiy S, Hoyos BE, Moltedo B, Hemmers S, Treuting P, Leslie CS, Chudakov DM, Rudensky AY - Nature (2015)

Bottom Line: In addition to Treg cells, TCR-agonist-driven selection results in the generation of several other specialized T-cell lineages such as natural killer T cells and innate mucosal-associated invariant T cells.Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs.We show that the intronic Foxp3 enhancer conserved noncoding sequence 3 (CNS3) acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Immunology Program, Ludwig Center at Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
T-cell receptor (TCR) signalling has a key role in determining T-cell fate. Precursor cells expressing TCRs within a certain low-affinity range for complexes of self-peptide and major histocompatibility complex (MHC) undergo positive selection and differentiate into naive T cells expressing a highly diverse self-MHC-restricted TCR repertoire. In contrast, precursors displaying TCRs with a high affinity for 'self' are either eliminated through TCR-agonist-induced apoptosis (negative selection) or restrained by regulatory T (Treg) cells, whose differentiation and function are controlled by the X-chromosome-encoded transcription factor Foxp3 (reviewed in ref. 2). Foxp3 is expressed in a fraction of self-reactive T cells that escape negative selection in response to agonist-driven TCR signals combined with interleukin 2 (IL-2) receptor signalling. In addition to Treg cells, TCR-agonist-driven selection results in the generation of several other specialized T-cell lineages such as natural killer T cells and innate mucosal-associated invariant T cells. Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs. Here we explore in mice whether a specialized mechanism enables agonist-driven selection of Treg cells with a diverse TCR repertoire, and the importance this holds for self-tolerance. We show that the intronic Foxp3 enhancer conserved noncoding sequence 3 (CNS3) acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to control self-reactive T cells effectively, especially when thymic negative selection is genetically impaired. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.

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CNS3 acts as an epigenetic switch for the Foxp3 promoter poisinga, ChIP-qPCR of H3K4me1 at the Foxp3 locus and control loci (Hspa2, Rpl30 and Gm5069) in B cells, DP thymocytes, naïve CD4+ T (Tn) and Treg cells.b, c, H3K4me1 at CNS3 in DN and DP thymocytes (b), HSC, ESC, macrophages (Mφ) and dendritic cells (DC) (c).d, H3K4me1 at the Foxp3 promoter in DP, immature CD4 SP (imCD4SP, Foxp3−CD69hiCD62Llo), mature CD4 SP (mCD4SP, Foxp3−CD69loCD62Lhi) thymocytes, and naïve CD4+ T cells.e, f,CNS3 dependent H3K4me1 at the Foxp3 promoter in mature CD4 SP thymocytes (e) and naïve CD4+ T cells (f).g, h, HDAC inhibitor butyrate enhances H3K27Ac at the Foxp3 promoter (g) and rescues impaired Treg differentiation of CNS3-deficient T cells in vitro (h).Two-tailed unpaired t-test. Mean ± SEMs, represent triplicate cultures in one of ≥ 2 experiments.
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Figure 1: CNS3 acts as an epigenetic switch for the Foxp3 promoter poisinga, ChIP-qPCR of H3K4me1 at the Foxp3 locus and control loci (Hspa2, Rpl30 and Gm5069) in B cells, DP thymocytes, naïve CD4+ T (Tn) and Treg cells.b, c, H3K4me1 at CNS3 in DN and DP thymocytes (b), HSC, ESC, macrophages (Mφ) and dendritic cells (DC) (c).d, H3K4me1 at the Foxp3 promoter in DP, immature CD4 SP (imCD4SP, Foxp3−CD69hiCD62Llo), mature CD4 SP (mCD4SP, Foxp3−CD69loCD62Lhi) thymocytes, and naïve CD4+ T cells.e, f,CNS3 dependent H3K4me1 at the Foxp3 promoter in mature CD4 SP thymocytes (e) and naïve CD4+ T cells (f).g, h, HDAC inhibitor butyrate enhances H3K27Ac at the Foxp3 promoter (g) and rescues impaired Treg differentiation of CNS3-deficient T cells in vitro (h).Two-tailed unpaired t-test. Mean ± SEMs, represent triplicate cultures in one of ≥ 2 experiments.

Mentions: These findings raised the question of how, mechanistically, CNS3 could selectively facilitate the initiation, but not the maintenance of Foxp3 expression. To begin addressing this problem, we sought to identify the stage of thymocyte differentiation at which the CNS3 region first acquires features characteristic of a poised enhancer. We previously found that CNS3 is marked by lysine 4 monomethylation of histone H3 (H4K4me1) in DP thymocytes11. Unexpectedly, we found increased H3K4me1 at CNS3 at DN1 stage and in hematopoietic stem cells (HSC), comparable to the levels observed in DP, CD4 SP thymocytes, and naïve CD4+ and CD8+ T cells (Fig. 1a-c and data not shown). In contrast, CNS3 chromatin was not enriched for H3K4me1 in embryonic stem cells (ESC), macrophages (Mϕ), or dendritic cells (DC) (Fig. 1b, c). These results indicate that the poised state of CNS3 is established at a very early stage of hematopoiesis, but is lost in “non-T” cell lineages. Because CNS3 appeared to be the earliest epigenetically modified region in the Foxp3 locus, it might exert its function by facilitating chromatin remodeling at the Foxp3 promoter.


A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance.

Feng Y, van der Veeken J, Shugay M, Putintseva EV, Osmanbeyoglu HU, Dikiy S, Hoyos BE, Moltedo B, Hemmers S, Treuting P, Leslie CS, Chudakov DM, Rudensky AY - Nature (2015)

CNS3 acts as an epigenetic switch for the Foxp3 promoter poisinga, ChIP-qPCR of H3K4me1 at the Foxp3 locus and control loci (Hspa2, Rpl30 and Gm5069) in B cells, DP thymocytes, naïve CD4+ T (Tn) and Treg cells.b, c, H3K4me1 at CNS3 in DN and DP thymocytes (b), HSC, ESC, macrophages (Mφ) and dendritic cells (DC) (c).d, H3K4me1 at the Foxp3 promoter in DP, immature CD4 SP (imCD4SP, Foxp3−CD69hiCD62Llo), mature CD4 SP (mCD4SP, Foxp3−CD69loCD62Lhi) thymocytes, and naïve CD4+ T cells.e, f,CNS3 dependent H3K4me1 at the Foxp3 promoter in mature CD4 SP thymocytes (e) and naïve CD4+ T cells (f).g, h, HDAC inhibitor butyrate enhances H3K27Ac at the Foxp3 promoter (g) and rescues impaired Treg differentiation of CNS3-deficient T cells in vitro (h).Two-tailed unpaired t-test. Mean ± SEMs, represent triplicate cultures in one of ≥ 2 experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4862833&req=5

Figure 1: CNS3 acts as an epigenetic switch for the Foxp3 promoter poisinga, ChIP-qPCR of H3K4me1 at the Foxp3 locus and control loci (Hspa2, Rpl30 and Gm5069) in B cells, DP thymocytes, naïve CD4+ T (Tn) and Treg cells.b, c, H3K4me1 at CNS3 in DN and DP thymocytes (b), HSC, ESC, macrophages (Mφ) and dendritic cells (DC) (c).d, H3K4me1 at the Foxp3 promoter in DP, immature CD4 SP (imCD4SP, Foxp3−CD69hiCD62Llo), mature CD4 SP (mCD4SP, Foxp3−CD69loCD62Lhi) thymocytes, and naïve CD4+ T cells.e, f,CNS3 dependent H3K4me1 at the Foxp3 promoter in mature CD4 SP thymocytes (e) and naïve CD4+ T cells (f).g, h, HDAC inhibitor butyrate enhances H3K27Ac at the Foxp3 promoter (g) and rescues impaired Treg differentiation of CNS3-deficient T cells in vitro (h).Two-tailed unpaired t-test. Mean ± SEMs, represent triplicate cultures in one of ≥ 2 experiments.
Mentions: These findings raised the question of how, mechanistically, CNS3 could selectively facilitate the initiation, but not the maintenance of Foxp3 expression. To begin addressing this problem, we sought to identify the stage of thymocyte differentiation at which the CNS3 region first acquires features characteristic of a poised enhancer. We previously found that CNS3 is marked by lysine 4 monomethylation of histone H3 (H4K4me1) in DP thymocytes11. Unexpectedly, we found increased H3K4me1 at CNS3 at DN1 stage and in hematopoietic stem cells (HSC), comparable to the levels observed in DP, CD4 SP thymocytes, and naïve CD4+ and CD8+ T cells (Fig. 1a-c and data not shown). In contrast, CNS3 chromatin was not enriched for H3K4me1 in embryonic stem cells (ESC), macrophages (Mϕ), or dendritic cells (DC) (Fig. 1b, c). These results indicate that the poised state of CNS3 is established at a very early stage of hematopoiesis, but is lost in “non-T” cell lineages. Because CNS3 appeared to be the earliest epigenetically modified region in the Foxp3 locus, it might exert its function by facilitating chromatin remodeling at the Foxp3 promoter.

Bottom Line: In addition to Treg cells, TCR-agonist-driven selection results in the generation of several other specialized T-cell lineages such as natural killer T cells and innate mucosal-associated invariant T cells.Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs.We show that the intronic Foxp3 enhancer conserved noncoding sequence 3 (CNS3) acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Immunology Program, Ludwig Center at Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

ABSTRACT
T-cell receptor (TCR) signalling has a key role in determining T-cell fate. Precursor cells expressing TCRs within a certain low-affinity range for complexes of self-peptide and major histocompatibility complex (MHC) undergo positive selection and differentiate into naive T cells expressing a highly diverse self-MHC-restricted TCR repertoire. In contrast, precursors displaying TCRs with a high affinity for 'self' are either eliminated through TCR-agonist-induced apoptosis (negative selection) or restrained by regulatory T (Treg) cells, whose differentiation and function are controlled by the X-chromosome-encoded transcription factor Foxp3 (reviewed in ref. 2). Foxp3 is expressed in a fraction of self-reactive T cells that escape negative selection in response to agonist-driven TCR signals combined with interleukin 2 (IL-2) receptor signalling. In addition to Treg cells, TCR-agonist-driven selection results in the generation of several other specialized T-cell lineages such as natural killer T cells and innate mucosal-associated invariant T cells. Although the latter exhibit a restricted TCR repertoire, Treg cells display a highly diverse collection of TCRs. Here we explore in mice whether a specialized mechanism enables agonist-driven selection of Treg cells with a diverse TCR repertoire, and the importance this holds for self-tolerance. We show that the intronic Foxp3 enhancer conserved noncoding sequence 3 (CNS3) acts as an epigenetic switch that confers a poised state to the Foxp3 promoter in precursor cells to make Treg cell lineage commitment responsive to a broad range of TCR stimuli, particularly to suboptimal ones. CNS3-dependent expansion of the TCR repertoire enables Treg cells to control self-reactive T cells effectively, especially when thymic negative selection is genetically impaired. Our findings highlight the complementary roles of these two main mechanisms of self-tolerance.

Show MeSH
Related in: MedlinePlus