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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 facilitates Foxp3 induction and shapes Treg cell repertoirea, Differential impact of CNS3 on Treg cell in vitro development of mature non-Treg CD4 single positive T cells. CD4 single positive thymocytes (CD4+CD8−TCRβhiGFP−CD25−CD69loCD62Lhi) were pooled and sorted from male Foxp3gfp and Foxp3ΔCNS3-gfp littermates (n=7 each group) for in vitro Treg induction performed with titrated CD3 antibody and lethally irradiated antigen presenting cells isolated from wild type B6 spleens in the presence of TGFβ and recombinant IL-2. Foxp3 expression was analyzed four days later and the relative changes of the ratios of Foxp3 expressing cells in the absence of CNS3 was calculated by comparing to CNS3-sufficient groups. Data depict means ± SEMs of five replicate cultures and represent one of three independent experiments.b, Flow cytometric analysis of Nur77 protein expression in CNS3-deficient and -sufficient Treg cells. (n=5 each group). Two-tailed unpaired Mann Whitney test. The data represent one of >2 independent experiments.c, Elevated Nur77 protein levels in CNS3-deficient Treg cells developed after conditional ablation of CNS3 upon tamoxifen induced activation of UBCCre-ERT2. Bone marrow of CD45.1 Foxp3gfp and CD45.2 UBCCre-ERT2 Foxp3CNS3fl-gfp R26Y mice were collected from donor mice treated with tamoxifen, mixed at a 1:1 ratio and transferred into lethally irradiated Tcrb−/−Tcrd−/− recipients. CD45.1+CD4+GFP+, CD45.2+YFP−GFP+ and CD45.2+YFP+GFP+ cells were sorted for flow cytometric analysis of Nur77 protein levels 10 weeks after BM transfer (n=5). Unpaired Mann Whitney tests were used to compare CD45.2+YFP+GFP+ and CD45.2+YFP−GFP+ or CD45.2+YFP+GFP+ and control (CD45.1+CD4+GFP+) groups. The data show medians of individual mice and represent more than three independent experiments.d, Nur77 expression levels in thymic Treg precursors (CD25+Foxp3−), immature (CD69hiCD62Llo) and mature (CD69loCD62Lhi) CD4 SP thymocytes, and peripheral Foxp3−CD4+ and CD8+ T cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test was used for statistical analysis. The data show medians of individual mice and represent more than three independent experiments.e, Differential Nur77 expression in peripheral resting (CD62LhiCD44lo) and activated (CD62LloCD44hi) Treg cells (wild type Foxp3gfp). The data represent one of more than three independent experiments.f, g, Upregulation of Nur77 expression in resting (CD62LhiCD44lo) (f) and activated (CD44hiCD62Llo) (g) CNS3-deficient Treg cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test; the data represent at least three experiments.h, i, CTLA4 (h) and Ki67 (i) expression by CNS3-deficient and -sufficient Treg cells in Foxp3gfp (n=9) and Foxp3ΔCNS3-gfp (n=11) mice (h). n=5 Foxp3gfp, n=4 Foxp3ΔCNS3-gfp (i). Two-tailed unpaired Mann Whitney test. The data represent one of >3 independent experiments.
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Figure 8: CNS3 facilitates Foxp3 induction and shapes Treg cell repertoirea, Differential impact of CNS3 on Treg cell in vitro development of mature non-Treg CD4 single positive T cells. CD4 single positive thymocytes (CD4+CD8−TCRβhiGFP−CD25−CD69loCD62Lhi) were pooled and sorted from male Foxp3gfp and Foxp3ΔCNS3-gfp littermates (n=7 each group) for in vitro Treg induction performed with titrated CD3 antibody and lethally irradiated antigen presenting cells isolated from wild type B6 spleens in the presence of TGFβ and recombinant IL-2. Foxp3 expression was analyzed four days later and the relative changes of the ratios of Foxp3 expressing cells in the absence of CNS3 was calculated by comparing to CNS3-sufficient groups. Data depict means ± SEMs of five replicate cultures and represent one of three independent experiments.b, Flow cytometric analysis of Nur77 protein expression in CNS3-deficient and -sufficient Treg cells. (n=5 each group). Two-tailed unpaired Mann Whitney test. The data represent one of >2 independent experiments.c, Elevated Nur77 protein levels in CNS3-deficient Treg cells developed after conditional ablation of CNS3 upon tamoxifen induced activation of UBCCre-ERT2. Bone marrow of CD45.1 Foxp3gfp and CD45.2 UBCCre-ERT2 Foxp3CNS3fl-gfp R26Y mice were collected from donor mice treated with tamoxifen, mixed at a 1:1 ratio and transferred into lethally irradiated Tcrb−/−Tcrd−/− recipients. CD45.1+CD4+GFP+, CD45.2+YFP−GFP+ and CD45.2+YFP+GFP+ cells were sorted for flow cytometric analysis of Nur77 protein levels 10 weeks after BM transfer (n=5). Unpaired Mann Whitney tests were used to compare CD45.2+YFP+GFP+ and CD45.2+YFP−GFP+ or CD45.2+YFP+GFP+ and control (CD45.1+CD4+GFP+) groups. The data show medians of individual mice and represent more than three independent experiments.d, Nur77 expression levels in thymic Treg precursors (CD25+Foxp3−), immature (CD69hiCD62Llo) and mature (CD69loCD62Lhi) CD4 SP thymocytes, and peripheral Foxp3−CD4+ and CD8+ T cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test was used for statistical analysis. The data show medians of individual mice and represent more than three independent experiments.e, Differential Nur77 expression in peripheral resting (CD62LhiCD44lo) and activated (CD62LloCD44hi) Treg cells (wild type Foxp3gfp). The data represent one of more than three independent experiments.f, g, Upregulation of Nur77 expression in resting (CD62LhiCD44lo) (f) and activated (CD44hiCD62Llo) (g) CNS3-deficient Treg cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test; the data represent at least three experiments.h, i, CTLA4 (h) and Ki67 (i) expression by CNS3-deficient and -sufficient Treg cells in Foxp3gfp (n=9) and Foxp3ΔCNS3-gfp (n=11) mice (h). n=5 Foxp3gfp, n=4 Foxp3ΔCNS3-gfp (i). Two-tailed unpaired Mann Whitney test. The data represent one of >3 independent experiments.

Mentions: While CNS3-dependent poising of the Foxp3 promoter could facilitate Foxp3 induction in a probabilistic manner, it might also enable lower strength TCR signals to promote Treg cell differentiation. To address this possibility we tested whether impaired Foxp3 induction in CNS3-deficient naïve CD4+ T cells could be rescued by increasing amounts of CD3 antibody under in vitro Treg cell differentiation conditions. We found that the relative difference in the efficiency of Foxp3 induction between CNS3-sufficient and - deficient CD4+ T cells was markedly decreased in the presence of higher amounts of CD3 antibody (Fig. 2a and Extended Data Fig. 4a), suggesting that increased TCR signal strength can partially compensate for the lack of Foxp3 promoter poising in the absence of CNS3, and imply that differentiation of Treg precursors receiving lower TCR stimulation might be disproportionally impeded by CNS3 deficiency. A corollary of this notion is that the mature Treg cells differentiated from CNS3-deficient precursors are enriched for TCRs at the higher end of the self-reactivity spectrum, and depleted of those with lower self-reactivity.


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 facilitates Foxp3 induction and shapes Treg cell repertoirea, Differential impact of CNS3 on Treg cell in vitro development of mature non-Treg CD4 single positive T cells. CD4 single positive thymocytes (CD4+CD8−TCRβhiGFP−CD25−CD69loCD62Lhi) were pooled and sorted from male Foxp3gfp and Foxp3ΔCNS3-gfp littermates (n=7 each group) for in vitro Treg induction performed with titrated CD3 antibody and lethally irradiated antigen presenting cells isolated from wild type B6 spleens in the presence of TGFβ and recombinant IL-2. Foxp3 expression was analyzed four days later and the relative changes of the ratios of Foxp3 expressing cells in the absence of CNS3 was calculated by comparing to CNS3-sufficient groups. Data depict means ± SEMs of five replicate cultures and represent one of three independent experiments.b, Flow cytometric analysis of Nur77 protein expression in CNS3-deficient and -sufficient Treg cells. (n=5 each group). Two-tailed unpaired Mann Whitney test. The data represent one of >2 independent experiments.c, Elevated Nur77 protein levels in CNS3-deficient Treg cells developed after conditional ablation of CNS3 upon tamoxifen induced activation of UBCCre-ERT2. Bone marrow of CD45.1 Foxp3gfp and CD45.2 UBCCre-ERT2 Foxp3CNS3fl-gfp R26Y mice were collected from donor mice treated with tamoxifen, mixed at a 1:1 ratio and transferred into lethally irradiated Tcrb−/−Tcrd−/− recipients. CD45.1+CD4+GFP+, CD45.2+YFP−GFP+ and CD45.2+YFP+GFP+ cells were sorted for flow cytometric analysis of Nur77 protein levels 10 weeks after BM transfer (n=5). Unpaired Mann Whitney tests were used to compare CD45.2+YFP+GFP+ and CD45.2+YFP−GFP+ or CD45.2+YFP+GFP+ and control (CD45.1+CD4+GFP+) groups. The data show medians of individual mice and represent more than three independent experiments.d, Nur77 expression levels in thymic Treg precursors (CD25+Foxp3−), immature (CD69hiCD62Llo) and mature (CD69loCD62Lhi) CD4 SP thymocytes, and peripheral Foxp3−CD4+ and CD8+ T cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test was used for statistical analysis. The data show medians of individual mice and represent more than three independent experiments.e, Differential Nur77 expression in peripheral resting (CD62LhiCD44lo) and activated (CD62LloCD44hi) Treg cells (wild type Foxp3gfp). The data represent one of more than three independent experiments.f, g, Upregulation of Nur77 expression in resting (CD62LhiCD44lo) (f) and activated (CD44hiCD62Llo) (g) CNS3-deficient Treg cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test; the data represent at least three experiments.h, i, CTLA4 (h) and Ki67 (i) expression by CNS3-deficient and -sufficient Treg cells in Foxp3gfp (n=9) and Foxp3ΔCNS3-gfp (n=11) mice (h). n=5 Foxp3gfp, n=4 Foxp3ΔCNS3-gfp (i). Two-tailed unpaired Mann Whitney test. The data represent one of >3 independent experiments.
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Figure 8: CNS3 facilitates Foxp3 induction and shapes Treg cell repertoirea, Differential impact of CNS3 on Treg cell in vitro development of mature non-Treg CD4 single positive T cells. CD4 single positive thymocytes (CD4+CD8−TCRβhiGFP−CD25−CD69loCD62Lhi) were pooled and sorted from male Foxp3gfp and Foxp3ΔCNS3-gfp littermates (n=7 each group) for in vitro Treg induction performed with titrated CD3 antibody and lethally irradiated antigen presenting cells isolated from wild type B6 spleens in the presence of TGFβ and recombinant IL-2. Foxp3 expression was analyzed four days later and the relative changes of the ratios of Foxp3 expressing cells in the absence of CNS3 was calculated by comparing to CNS3-sufficient groups. Data depict means ± SEMs of five replicate cultures and represent one of three independent experiments.b, Flow cytometric analysis of Nur77 protein expression in CNS3-deficient and -sufficient Treg cells. (n=5 each group). Two-tailed unpaired Mann Whitney test. The data represent one of >2 independent experiments.c, Elevated Nur77 protein levels in CNS3-deficient Treg cells developed after conditional ablation of CNS3 upon tamoxifen induced activation of UBCCre-ERT2. Bone marrow of CD45.1 Foxp3gfp and CD45.2 UBCCre-ERT2 Foxp3CNS3fl-gfp R26Y mice were collected from donor mice treated with tamoxifen, mixed at a 1:1 ratio and transferred into lethally irradiated Tcrb−/−Tcrd−/− recipients. CD45.1+CD4+GFP+, CD45.2+YFP−GFP+ and CD45.2+YFP+GFP+ cells were sorted for flow cytometric analysis of Nur77 protein levels 10 weeks after BM transfer (n=5). Unpaired Mann Whitney tests were used to compare CD45.2+YFP+GFP+ and CD45.2+YFP−GFP+ or CD45.2+YFP+GFP+ and control (CD45.1+CD4+GFP+) groups. The data show medians of individual mice and represent more than three independent experiments.d, Nur77 expression levels in thymic Treg precursors (CD25+Foxp3−), immature (CD69hiCD62Llo) and mature (CD69loCD62Lhi) CD4 SP thymocytes, and peripheral Foxp3−CD4+ and CD8+ T cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test was used for statistical analysis. The data show medians of individual mice and represent more than three independent experiments.e, Differential Nur77 expression in peripheral resting (CD62LhiCD44lo) and activated (CD62LloCD44hi) Treg cells (wild type Foxp3gfp). The data represent one of more than three independent experiments.f, g, Upregulation of Nur77 expression in resting (CD62LhiCD44lo) (f) and activated (CD44hiCD62Llo) (g) CNS3-deficient Treg cells in 6-7 week old Foxp3gfp (n=5) and Foxp3ΔCNS3-gfp (n=4) littermates. Unpaired Mann Whitney test; the data represent at least three experiments.h, i, CTLA4 (h) and Ki67 (i) expression by CNS3-deficient and -sufficient Treg cells in Foxp3gfp (n=9) and Foxp3ΔCNS3-gfp (n=11) mice (h). n=5 Foxp3gfp, n=4 Foxp3ΔCNS3-gfp (i). Two-tailed unpaired Mann Whitney test. The data represent one of >3 independent experiments.
Mentions: While CNS3-dependent poising of the Foxp3 promoter could facilitate Foxp3 induction in a probabilistic manner, it might also enable lower strength TCR signals to promote Treg cell differentiation. To address this possibility we tested whether impaired Foxp3 induction in CNS3-deficient naïve CD4+ T cells could be rescued by increasing amounts of CD3 antibody under in vitro Treg cell differentiation conditions. We found that the relative difference in the efficiency of Foxp3 induction between CNS3-sufficient and - deficient CD4+ T cells was markedly decreased in the presence of higher amounts of CD3 antibody (Fig. 2a and Extended Data Fig. 4a), suggesting that increased TCR signal strength can partially compensate for the lack of Foxp3 promoter poising in the absence of CNS3, and imply that differentiation of Treg precursors receiving lower TCR stimulation might be disproportionally impeded by CNS3 deficiency. A corollary of this notion is that the mature Treg cells differentiated from CNS3-deficient precursors are enriched for TCRs at the higher end of the self-reactivity spectrum, and depleted of those with lower self-reactivity.

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