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The requirement for pre-TCR during thymic differentiation enforces a developmental pause that is essential for V-DJβ rearrangement.

Hathcock KS, Farrington L, Ivanova I, Livak F, Selimyan R, Sen R, Williams J, Tai X, Hodes RJ - PLoS ONE (2011)

Bottom Line: We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement.These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression.However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. hathcock@exchange.nih.gov

ABSTRACT
T cell development occurs in the thymus and is critically dependent on productive TCRβ rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (β checkpoint), which is uniquely permissive for V-DJβ recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRβ rearrangement and pre-TCR expression enforces suppression of TCRβ rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRβ product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the β checkpoint, would suppress all V-DJβ rearrangement. We previously reported that premature CD28 signaling in murine CD4(-)CD8(-) (DN) thymocytes supports differentiation of CD4(+)CD8(+) (DP) cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRβ rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

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Accessibility of DJβ and Vβ regions are differentially regulated during thymic development.(A) Fold-enrichment H3K4Me3 analyzed by ChIP for Vβ (2, 5, and 11) Dβ1, and Jβ (1.1 and 2.7) regions. Each data point represents the mean ± SEM of six independent lysates prepared from RAG2KO (black bar) or CD28/B7/RAG2KO (grey bar) DN cells. H3K4Me3 of β-actin was used as a normalization control. (B) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells from mice previously injected with either PBS (dark brick bar) or CD3ε mAb (open brick bar). These results represent the mean ± SEM of three to five mice per group analyzed in two separate experiments. (C) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells (black bar), CD28/B7/RAG2KO DN cells (grey bar) or CD28/B7/RAG2KO DP cells (black striped bar). These results represent the mean ± SEM of five to six mice per genotype analyzed in two separate experiments. For both (B) and (C) data shown are normalized to transcription measured in RAG2KO DN cells using β-actin transcription as a loading control for each group. For (A), (B), and (C) the order of the data points reflects the 5′–3′ genomic organization of the TCRβ region and brackets identify comparisons that are significantly different (p<0.05): * indicates p≤0.03, ** indicates p≤0.02, and *** indicates p≤0.001.
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pone-0020639-g006: Accessibility of DJβ and Vβ regions are differentially regulated during thymic development.(A) Fold-enrichment H3K4Me3 analyzed by ChIP for Vβ (2, 5, and 11) Dβ1, and Jβ (1.1 and 2.7) regions. Each data point represents the mean ± SEM of six independent lysates prepared from RAG2KO (black bar) or CD28/B7/RAG2KO (grey bar) DN cells. H3K4Me3 of β-actin was used as a normalization control. (B) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells from mice previously injected with either PBS (dark brick bar) or CD3ε mAb (open brick bar). These results represent the mean ± SEM of three to five mice per group analyzed in two separate experiments. (C) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells (black bar), CD28/B7/RAG2KO DN cells (grey bar) or CD28/B7/RAG2KO DP cells (black striped bar). These results represent the mean ± SEM of five to six mice per genotype analyzed in two separate experiments. For both (B) and (C) data shown are normalized to transcription measured in RAG2KO DN cells using β-actin transcription as a loading control for each group. For (A), (B), and (C) the order of the data points reflects the 5′–3′ genomic organization of the TCRβ region and brackets identify comparisons that are significantly different (p<0.05): * indicates p≤0.03, ** indicates p≤0.02, and *** indicates p≤0.001.

Mentions: We next asked if, in addition to suppressing RAG2 expression, development in the absence of pre-TCR expression also induces epigenetic modifications in the TCRβ locus that may inhibit accessibility of the locus to recombination or transcription machinery [27]. Tri-methylation of histone 3 at lysine 4 (H3K4Me3) is a physical modification that has been associated with RAG recruitment to accessible antigen receptor loci [28]–[30]. We used ChIP coupled to real-time PCR to compare H3K4Me3 across the entire unrearranged TCRβ locus in DN cells isolated from either RAG2KO mice or from CD28/B7/RAG2KO mice (Figure 6A) [7]. In DN cells from both RAG2KO and CD28/B7/RAG2KO mice, levels of H3K4Me3 were equivalently high in Dβ and Jβ regions. In contrast, analysis of Vβ loci indicated that H3K4Me3 modification of Vβ5 and Vβ11, but not Vβ2 regions was suppressed in CD28/B7/RAG2KO relative to RAG2KO DN cells. By this criterion the Dβ and Jβ but not Vβ regions of the TCRβ locus are available for recombination in CD28/B7 DN cells. These observations suggest that in addition to inhibition of RAG2 expression, impaired accessibility of Vβ gene segments also contributes to the inhibition of V-DJβ recombination in these developing DN cells.


The requirement for pre-TCR during thymic differentiation enforces a developmental pause that is essential for V-DJβ rearrangement.

Hathcock KS, Farrington L, Ivanova I, Livak F, Selimyan R, Sen R, Williams J, Tai X, Hodes RJ - PLoS ONE (2011)

Accessibility of DJβ and Vβ regions are differentially regulated during thymic development.(A) Fold-enrichment H3K4Me3 analyzed by ChIP for Vβ (2, 5, and 11) Dβ1, and Jβ (1.1 and 2.7) regions. Each data point represents the mean ± SEM of six independent lysates prepared from RAG2KO (black bar) or CD28/B7/RAG2KO (grey bar) DN cells. H3K4Me3 of β-actin was used as a normalization control. (B) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells from mice previously injected with either PBS (dark brick bar) or CD3ε mAb (open brick bar). These results represent the mean ± SEM of three to five mice per group analyzed in two separate experiments. (C) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells (black bar), CD28/B7/RAG2KO DN cells (grey bar) or CD28/B7/RAG2KO DP cells (black striped bar). These results represent the mean ± SEM of five to six mice per genotype analyzed in two separate experiments. For both (B) and (C) data shown are normalized to transcription measured in RAG2KO DN cells using β-actin transcription as a loading control for each group. For (A), (B), and (C) the order of the data points reflects the 5′–3′ genomic organization of the TCRβ region and brackets identify comparisons that are significantly different (p<0.05): * indicates p≤0.03, ** indicates p≤0.02, and *** indicates p≤0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108609&req=5

pone-0020639-g006: Accessibility of DJβ and Vβ regions are differentially regulated during thymic development.(A) Fold-enrichment H3K4Me3 analyzed by ChIP for Vβ (2, 5, and 11) Dβ1, and Jβ (1.1 and 2.7) regions. Each data point represents the mean ± SEM of six independent lysates prepared from RAG2KO (black bar) or CD28/B7/RAG2KO (grey bar) DN cells. H3K4Me3 of β-actin was used as a normalization control. (B) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells from mice previously injected with either PBS (dark brick bar) or CD3ε mAb (open brick bar). These results represent the mean ± SEM of three to five mice per group analyzed in two separate experiments. (C) Relative germline transcription of the unrearranged TCRβ region in RAG2KO DN cells (black bar), CD28/B7/RAG2KO DN cells (grey bar) or CD28/B7/RAG2KO DP cells (black striped bar). These results represent the mean ± SEM of five to six mice per genotype analyzed in two separate experiments. For both (B) and (C) data shown are normalized to transcription measured in RAG2KO DN cells using β-actin transcription as a loading control for each group. For (A), (B), and (C) the order of the data points reflects the 5′–3′ genomic organization of the TCRβ region and brackets identify comparisons that are significantly different (p<0.05): * indicates p≤0.03, ** indicates p≤0.02, and *** indicates p≤0.001.
Mentions: We next asked if, in addition to suppressing RAG2 expression, development in the absence of pre-TCR expression also induces epigenetic modifications in the TCRβ locus that may inhibit accessibility of the locus to recombination or transcription machinery [27]. Tri-methylation of histone 3 at lysine 4 (H3K4Me3) is a physical modification that has been associated with RAG recruitment to accessible antigen receptor loci [28]–[30]. We used ChIP coupled to real-time PCR to compare H3K4Me3 across the entire unrearranged TCRβ locus in DN cells isolated from either RAG2KO mice or from CD28/B7/RAG2KO mice (Figure 6A) [7]. In DN cells from both RAG2KO and CD28/B7/RAG2KO mice, levels of H3K4Me3 were equivalently high in Dβ and Jβ regions. In contrast, analysis of Vβ loci indicated that H3K4Me3 modification of Vβ5 and Vβ11, but not Vβ2 regions was suppressed in CD28/B7/RAG2KO relative to RAG2KO DN cells. By this criterion the Dβ and Jβ but not Vβ regions of the TCRβ locus are available for recombination in CD28/B7 DN cells. These observations suggest that in addition to inhibition of RAG2 expression, impaired accessibility of Vβ gene segments also contributes to the inhibition of V-DJβ recombination in these developing DN cells.

Bottom Line: We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement.These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression.However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. hathcock@exchange.nih.gov

ABSTRACT
T cell development occurs in the thymus and is critically dependent on productive TCRβ rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (β checkpoint), which is uniquely permissive for V-DJβ recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRβ rearrangement and pre-TCR expression enforces suppression of TCRβ rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRβ product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the β checkpoint, would suppress all V-DJβ rearrangement. We previously reported that premature CD28 signaling in murine CD4(-)CD8(-) (DN) thymocytes supports differentiation of CD4(+)CD8(+) (DP) cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRβ rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

Show MeSH
Related in: MedlinePlus