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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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Compromised suppressive function of CNS3-deficient Treg cellsa, Autoimmune diseases in Foxp3ΔCNS3-gfp AireKO/KO (DKO) mice. Arrow indicates the inflammatory lesions in the tail of a three week-old mouse with an early onset of autoimmunity (n>11) (i). Depigmentation in a six week-old mouse with delayed onset of autoimmunity (n>16) (ii).b, Analysis of serum Ig isotypes in Foxp3ΔCNS3-gfp AireKO/KO and littermate control mice using ELISA (n=8 each group). Mean ± SEMs. The statistical analysis was performed using a two-way ANOVA. c, Flow cytometric analysis of Foxp3 expression by Treg cells. The data show one of at least three mice per group and represent more than three independent experiments.d-i, Analysis of the ability of CNS3-deficient and -sufficient Treg cells to control CNS3 Aire DKO effector T cells upon adoptive transfer into T cell deficient recipients. Flow cytometric analysis of non-Treg CD4+ T cell numbers (d), Treg cell numbers (e), Foxp3 expression level (f), and IFNγ production (g), IL-17 production (h), and serum IgG1 and IgG2b levels (i) in recipient mice transferred with CNS3 Aire DKO effector T cells (Foxp3−CD4+ and CD8+) at a 10:1 ratio with Treg cells from Aire-sufficient Foxp3gfp or Foxp3ΔCNS3-gfp mice. P values show the comparisons between the two groups performed using two-tailed unpaired Mann Whitney tests (d-h) or unpaired t-test (i). Mean ± SEMs (i). The recipient mice were analyzed 7 weeks after adoptive T cell transfer (n=5 per group).
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Figure 11: Compromised suppressive function of CNS3-deficient Treg cellsa, Autoimmune diseases in Foxp3ΔCNS3-gfp AireKO/KO (DKO) mice. Arrow indicates the inflammatory lesions in the tail of a three week-old mouse with an early onset of autoimmunity (n>11) (i). Depigmentation in a six week-old mouse with delayed onset of autoimmunity (n>16) (ii).b, Analysis of serum Ig isotypes in Foxp3ΔCNS3-gfp AireKO/KO and littermate control mice using ELISA (n=8 each group). Mean ± SEMs. The statistical analysis was performed using a two-way ANOVA. c, Flow cytometric analysis of Foxp3 expression by Treg cells. The data show one of at least three mice per group and represent more than three independent experiments.d-i, Analysis of the ability of CNS3-deficient and -sufficient Treg cells to control CNS3 Aire DKO effector T cells upon adoptive transfer into T cell deficient recipients. Flow cytometric analysis of non-Treg CD4+ T cell numbers (d), Treg cell numbers (e), Foxp3 expression level (f), and IFNγ production (g), IL-17 production (h), and serum IgG1 and IgG2b levels (i) in recipient mice transferred with CNS3 Aire DKO effector T cells (Foxp3−CD4+ and CD8+) at a 10:1 ratio with Treg cells from Aire-sufficient Foxp3gfp or Foxp3ΔCNS3-gfp mice. P values show the comparisons between the two groups performed using two-tailed unpaired Mann Whitney tests (d-h) or unpaired t-test (i). Mean ± SEMs (i). The recipient mice were analyzed 7 weeks after adoptive T cell transfer (n=5 per group).

Mentions: Both negative selection and Treg cell generation are driven by self-antigen recognition in the thymus and likely play complementary roles in self-tolerance1,2,25,26. We reasoned that the relatively mild impairment in suppressive capacity of Treg cells from CNS3-deficient mice on a B6 genetic background, resistant to autoimmunity, may not fully reveal the biological significance of CNS3-dependent broadening of the Treg cell repertoire because of efficient negative selection. Therefore, we assessed the consequences of combined deficiency in CNS3 and Aire (Autoimmune Regulator), a nuclear factor required for thymic negative selection and optimal Treg cell generation. Aire deficiency leads to diminished expression of a subset of tissue-restricted antigens in the thymus and, consequently, an enlarged self-reactive effector T cell pool and diminished Treg repertoire25,27-29. In contrast to a late-onset and rather mild autoimmunity observed in Aire deficient mice on a B6 genetic background, double deficiency in CNS3 and Aire resulted in fatal early-onset aggressive autoimmune lesions in multiple tissues as early as 3-4 weeks of age, when detectable autoimmune inflammation was lacking in littermates with a single deficiency in Aire or CNS3 (Fig. 4a and Extended Data Fig. 7a). We noticed a 100% (n>35) penetrance with a stochastic gender independent variation in manifestations expected from perturbations in randomly generated repertoires of self-reactive T cells as well as the probabilistic nature of negative selection30 (Extended Data Fig. 7a and data not shown). This was accompanied by significant increases in CD4+ T cell activation, IFNγ production (Fig. 4b, c), serum Ig levels (Extended Data Fig. 7b), and autoantibody production (Fig. 4d). Combined Aire and CNS3 deficiency resulted in a further reduction in thymic Treg cell frequency in comparison to the single deficient mice (1.66 ± 0.28% and 0.91 ± 0.35% in Foxp3ΔCNS3-gfp AireKO/WT and Foxp3ΔCNS3-gfp AireKO/KO mice, respectively)25 (Fig. 4e). However, peripheral Treg cells reached normal levels in young Foxp3ΔCNS3-gfpAireKO/KO mice prior to development of clinical signs of disease likely due to homeostatic proliferation (Fig. 4f). Despite their normal quantities and Foxp3 expression, these Treg cells were unable to suppress pathogenic self-reactive T cells resulting from impaired negative selection in the absence of Aire (Fig.4b-d, Extended Data Fig. 7c). Since diminished thymic Treg cell numbers and their skewed TCR repertoire likely contributed to disease severity in Foxp3ΔCNS3-gfpAireKO/KO mice, we directly assessed the ability of CNS3-sufficient and -deficient Treg cells developed in the presence of Aire to control Foxp3ΔCNS3-gfpAireKO/KO effector T cells when adoptively transferred into T cell deficient hosts. Although the negative impact of CNS3 deficiency on the TCR repertoire was likely mitigated by Treg cell expansion in lymphopenic settings, CNS3-deficient Treg cells still exhibited compromised ability to suppress the responses of transferred Foxp3ΔCNS3-gfpAireKO/KO effector T cells and resident B cells in comparison to the controls (Extended Data Fig. 7d-i). These results suggest that control of broad self-reactive T cells requires a diverse CNS3-dependent repertoire of Treg cells.


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)

Compromised suppressive function of CNS3-deficient Treg cellsa, Autoimmune diseases in Foxp3ΔCNS3-gfp AireKO/KO (DKO) mice. Arrow indicates the inflammatory lesions in the tail of a three week-old mouse with an early onset of autoimmunity (n>11) (i). Depigmentation in a six week-old mouse with delayed onset of autoimmunity (n>16) (ii).b, Analysis of serum Ig isotypes in Foxp3ΔCNS3-gfp AireKO/KO and littermate control mice using ELISA (n=8 each group). Mean ± SEMs. The statistical analysis was performed using a two-way ANOVA. c, Flow cytometric analysis of Foxp3 expression by Treg cells. The data show one of at least three mice per group and represent more than three independent experiments.d-i, Analysis of the ability of CNS3-deficient and -sufficient Treg cells to control CNS3 Aire DKO effector T cells upon adoptive transfer into T cell deficient recipients. Flow cytometric analysis of non-Treg CD4+ T cell numbers (d), Treg cell numbers (e), Foxp3 expression level (f), and IFNγ production (g), IL-17 production (h), and serum IgG1 and IgG2b levels (i) in recipient mice transferred with CNS3 Aire DKO effector T cells (Foxp3−CD4+ and CD8+) at a 10:1 ratio with Treg cells from Aire-sufficient Foxp3gfp or Foxp3ΔCNS3-gfp mice. P values show the comparisons between the two groups performed using two-tailed unpaired Mann Whitney tests (d-h) or unpaired t-test (i). Mean ± SEMs (i). The recipient mice were analyzed 7 weeks after adoptive T cell transfer (n=5 per group).
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Figure 11: Compromised suppressive function of CNS3-deficient Treg cellsa, Autoimmune diseases in Foxp3ΔCNS3-gfp AireKO/KO (DKO) mice. Arrow indicates the inflammatory lesions in the tail of a three week-old mouse with an early onset of autoimmunity (n>11) (i). Depigmentation in a six week-old mouse with delayed onset of autoimmunity (n>16) (ii).b, Analysis of serum Ig isotypes in Foxp3ΔCNS3-gfp AireKO/KO and littermate control mice using ELISA (n=8 each group). Mean ± SEMs. The statistical analysis was performed using a two-way ANOVA. c, Flow cytometric analysis of Foxp3 expression by Treg cells. The data show one of at least three mice per group and represent more than three independent experiments.d-i, Analysis of the ability of CNS3-deficient and -sufficient Treg cells to control CNS3 Aire DKO effector T cells upon adoptive transfer into T cell deficient recipients. Flow cytometric analysis of non-Treg CD4+ T cell numbers (d), Treg cell numbers (e), Foxp3 expression level (f), and IFNγ production (g), IL-17 production (h), and serum IgG1 and IgG2b levels (i) in recipient mice transferred with CNS3 Aire DKO effector T cells (Foxp3−CD4+ and CD8+) at a 10:1 ratio with Treg cells from Aire-sufficient Foxp3gfp or Foxp3ΔCNS3-gfp mice. P values show the comparisons between the two groups performed using two-tailed unpaired Mann Whitney tests (d-h) or unpaired t-test (i). Mean ± SEMs (i). The recipient mice were analyzed 7 weeks after adoptive T cell transfer (n=5 per group).
Mentions: Both negative selection and Treg cell generation are driven by self-antigen recognition in the thymus and likely play complementary roles in self-tolerance1,2,25,26. We reasoned that the relatively mild impairment in suppressive capacity of Treg cells from CNS3-deficient mice on a B6 genetic background, resistant to autoimmunity, may not fully reveal the biological significance of CNS3-dependent broadening of the Treg cell repertoire because of efficient negative selection. Therefore, we assessed the consequences of combined deficiency in CNS3 and Aire (Autoimmune Regulator), a nuclear factor required for thymic negative selection and optimal Treg cell generation. Aire deficiency leads to diminished expression of a subset of tissue-restricted antigens in the thymus and, consequently, an enlarged self-reactive effector T cell pool and diminished Treg repertoire25,27-29. In contrast to a late-onset and rather mild autoimmunity observed in Aire deficient mice on a B6 genetic background, double deficiency in CNS3 and Aire resulted in fatal early-onset aggressive autoimmune lesions in multiple tissues as early as 3-4 weeks of age, when detectable autoimmune inflammation was lacking in littermates with a single deficiency in Aire or CNS3 (Fig. 4a and Extended Data Fig. 7a). We noticed a 100% (n>35) penetrance with a stochastic gender independent variation in manifestations expected from perturbations in randomly generated repertoires of self-reactive T cells as well as the probabilistic nature of negative selection30 (Extended Data Fig. 7a and data not shown). This was accompanied by significant increases in CD4+ T cell activation, IFNγ production (Fig. 4b, c), serum Ig levels (Extended Data Fig. 7b), and autoantibody production (Fig. 4d). Combined Aire and CNS3 deficiency resulted in a further reduction in thymic Treg cell frequency in comparison to the single deficient mice (1.66 ± 0.28% and 0.91 ± 0.35% in Foxp3ΔCNS3-gfp AireKO/WT and Foxp3ΔCNS3-gfp AireKO/KO mice, respectively)25 (Fig. 4e). However, peripheral Treg cells reached normal levels in young Foxp3ΔCNS3-gfpAireKO/KO mice prior to development of clinical signs of disease likely due to homeostatic proliferation (Fig. 4f). Despite their normal quantities and Foxp3 expression, these Treg cells were unable to suppress pathogenic self-reactive T cells resulting from impaired negative selection in the absence of Aire (Fig.4b-d, Extended Data Fig. 7c). Since diminished thymic Treg cell numbers and their skewed TCR repertoire likely contributed to disease severity in Foxp3ΔCNS3-gfpAireKO/KO mice, we directly assessed the ability of CNS3-sufficient and -deficient Treg cells developed in the presence of Aire to control Foxp3ΔCNS3-gfpAireKO/KO effector T cells when adoptively transferred into T cell deficient hosts. Although the negative impact of CNS3 deficiency on the TCR repertoire was likely mitigated by Treg cell expansion in lymphopenic settings, CNS3-deficient Treg cells still exhibited compromised ability to suppress the responses of transferred Foxp3ΔCNS3-gfpAireKO/KO effector T cells and resident B cells in comparison to the controls (Extended Data Fig. 7d-i). These results suggest that control of broad self-reactive T cells requires a diverse CNS3-dependent repertoire of Treg cells.

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