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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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CD28/B7-dependent development suppresses RAG2 expression in DN but not DP cells.(A) Histograms show GFP+ (RAG2) staining on the FCM gated cell populations from WT GFP+ (solid line) or CD28/B7 GFP+ (dashed line) mice. Gates used to identify early and late DN3 subsets are as described in Figure 1B. The grey filled histograms show FL1 staining of the indicated DP and DN subsets analyzed from GFP− mice. (B) This graph depicts the median channel number of GFP+ cells shown in panel (A) and is representative of three experiments performed. (C) This graph summarizes real-time PCR quantification of RAG2 expression in FCM-sorted DN2/3, DN4 and DP cells from B6 and CD28/B7/CD3εKO thymi. RNA was prepared and cDNA was synthesized as described in Materials and Methods. RAG2 cDNA was amplified using SABiosciences RT2qPCR primers. RAG2 expression was normalized against β-actin expression. Real-time analysis was performed on three independent cDNA samples for each genotype. Data are expressed as mean fold increase ± SE relative to B6 DN2/3 cells.
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pone-0020639-g005: CD28/B7-dependent development suppresses RAG2 expression in DN but not DP cells.(A) Histograms show GFP+ (RAG2) staining on the FCM gated cell populations from WT GFP+ (solid line) or CD28/B7 GFP+ (dashed line) mice. Gates used to identify early and late DN3 subsets are as described in Figure 1B. The grey filled histograms show FL1 staining of the indicated DP and DN subsets analyzed from GFP− mice. (B) This graph depicts the median channel number of GFP+ cells shown in panel (A) and is representative of three experiments performed. (C) This graph summarizes real-time PCR quantification of RAG2 expression in FCM-sorted DN2/3, DN4 and DP cells from B6 and CD28/B7/CD3εKO thymi. RNA was prepared and cDNA was synthesized as described in Materials and Methods. RAG2 cDNA was amplified using SABiosciences RT2qPCR primers. RAG2 expression was normalized against β-actin expression. Real-time analysis was performed on three independent cDNA samples for each genotype. Data are expressed as mean fold increase ± SE relative to B6 DN2/3 cells.

Mentions: To study RAG2 expression we bred mice expressing both CD28 and B7-2 transgenes to a RAG2 transcriptional reporter mouse (RAG2-GFP) [26] and analyzed GFP expression in littermates that were RAG-GFP+ and either negative (WT) or positive for CD28/B7 transgenes (CD28/B7) (Figure 5A and B). In WT thymocytes undergoing pre-TCR dependent development, peak GFP expression is detected in DN3 cells, the stage in which TCRβ is actively recombining, and decreases as cells express a pre-TCR and differentiate to DN4 cells. GFP expression increases again as thymocytes differentiate to DP cells where TCRα undergoes recombination. Notably, GFP expression in DN3 and DN4 cells from CD28/B7 mice is substantially lower than that observed in comparable DN cells from WT littermates. Since TCRβ rearrangement normally begins in early DN3 cells, we evaluated GFP expression in the early and late DN3 subsets. As compared to GFP expression in early DN3 cells from WT littermates, GFP expression is significantly reduced beginning in early DN3 cells from CD28/B7 mice. GFP expression is comparably high in DP cells from both WT and CD28/B7 mice. These results demonstrate that the differentiation program induced by premature CD28/B7 expression inhibits RAG2 transcription beginning in early DN3 cells and that this inhibition continues throughout DN development. In contrast, CD28/B7-driven development does not prevent RAG2 re-expression in DP 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)

CD28/B7-dependent development suppresses RAG2 expression in DN but not DP cells.(A) Histograms show GFP+ (RAG2) staining on the FCM gated cell populations from WT GFP+ (solid line) or CD28/B7 GFP+ (dashed line) mice. Gates used to identify early and late DN3 subsets are as described in Figure 1B. The grey filled histograms show FL1 staining of the indicated DP and DN subsets analyzed from GFP− mice. (B) This graph depicts the median channel number of GFP+ cells shown in panel (A) and is representative of three experiments performed. (C) This graph summarizes real-time PCR quantification of RAG2 expression in FCM-sorted DN2/3, DN4 and DP cells from B6 and CD28/B7/CD3εKO thymi. RNA was prepared and cDNA was synthesized as described in Materials and Methods. RAG2 cDNA was amplified using SABiosciences RT2qPCR primers. RAG2 expression was normalized against β-actin expression. Real-time analysis was performed on three independent cDNA samples for each genotype. Data are expressed as mean fold increase ± SE relative to B6 DN2/3 cells.
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Related In: Results  -  Collection

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pone-0020639-g005: CD28/B7-dependent development suppresses RAG2 expression in DN but not DP cells.(A) Histograms show GFP+ (RAG2) staining on the FCM gated cell populations from WT GFP+ (solid line) or CD28/B7 GFP+ (dashed line) mice. Gates used to identify early and late DN3 subsets are as described in Figure 1B. The grey filled histograms show FL1 staining of the indicated DP and DN subsets analyzed from GFP− mice. (B) This graph depicts the median channel number of GFP+ cells shown in panel (A) and is representative of three experiments performed. (C) This graph summarizes real-time PCR quantification of RAG2 expression in FCM-sorted DN2/3, DN4 and DP cells from B6 and CD28/B7/CD3εKO thymi. RNA was prepared and cDNA was synthesized as described in Materials and Methods. RAG2 cDNA was amplified using SABiosciences RT2qPCR primers. RAG2 expression was normalized against β-actin expression. Real-time analysis was performed on three independent cDNA samples for each genotype. Data are expressed as mean fold increase ± SE relative to B6 DN2/3 cells.
Mentions: To study RAG2 expression we bred mice expressing both CD28 and B7-2 transgenes to a RAG2 transcriptional reporter mouse (RAG2-GFP) [26] and analyzed GFP expression in littermates that were RAG-GFP+ and either negative (WT) or positive for CD28/B7 transgenes (CD28/B7) (Figure 5A and B). In WT thymocytes undergoing pre-TCR dependent development, peak GFP expression is detected in DN3 cells, the stage in which TCRβ is actively recombining, and decreases as cells express a pre-TCR and differentiate to DN4 cells. GFP expression increases again as thymocytes differentiate to DP cells where TCRα undergoes recombination. Notably, GFP expression in DN3 and DN4 cells from CD28/B7 mice is substantially lower than that observed in comparable DN cells from WT littermates. Since TCRβ rearrangement normally begins in early DN3 cells, we evaluated GFP expression in the early and late DN3 subsets. As compared to GFP expression in early DN3 cells from WT littermates, GFP expression is significantly reduced beginning in early DN3 cells from CD28/B7 mice. GFP expression is comparably high in DP cells from both WT and CD28/B7 mice. These results demonstrate that the differentiation program induced by premature CD28/B7 expression inhibits RAG2 transcription beginning in early DN3 cells and that this inhibition continues throughout DN development. In contrast, CD28/B7-driven development does not prevent RAG2 re-expression in DP 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