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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-driven development inhibits TCRβ rearrangement in DN thymocytes.100, 30, and 10 ng of DNA was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangement, (B) Vβ5-DJβ2 rearrangement, (C) Vβ8.1-DJβ2 rearrangement, or (D) Vβb14.1-DJβ2 rearrangement. (E) 10, 3, and 1 ng of DNA was analyzed by PCR to detect unrearranged IgM as a loading control. The arrow in panel A denotes the amplification of germline or non-rearranged Dβ2-Jβ2 PCR products. These results presented here comparing VDJβ rearrangements in CD3ε and CD28/B7/CD3εKO DN cells were analyzed in the same experiment and are representative of three experiments analyzing DNA prepared from a total of six CD3εKO and CD28/B7/CD3εKO mice.
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pone-0020639-g002: CD28/B7-driven development inhibits TCRβ rearrangement in DN thymocytes.100, 30, and 10 ng of DNA was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangement, (B) Vβ5-DJβ2 rearrangement, (C) Vβ8.1-DJβ2 rearrangement, or (D) Vβb14.1-DJβ2 rearrangement. (E) 10, 3, and 1 ng of DNA was analyzed by PCR to detect unrearranged IgM as a loading control. The arrow in panel A denotes the amplification of germline or non-rearranged Dβ2-Jβ2 PCR products. These results presented here comparing VDJβ rearrangements in CD3ε and CD28/B7/CD3εKO DN cells were analyzed in the same experiment and are representative of three experiments analyzing DNA prepared from a total of six CD3εKO and CD28/B7/CD3εKO mice.

Mentions: Expression of TCRβ protein occurs only after successful D-Jβ and V-DJβ rearrangement are completed [19]. Following TCRβ protein expression and pre-TCR formation, a series of differentiation events is initiated, and a critical component of this differentiation program is the suppression of additional V-DJβ rearrangement [6]. Since CD28/B7 interactions in early DN cells can induce differentiation in the absence of pre-TCR expression, we examined the effects of this differentiation program on TCRβ rearrangement and allelic exclusion. We began by analyzing TCRβ rearrangement in purified DN cells isolated from either pre-TCR deficient CD3εKO mice or from CD28/B7/CD3εKO mice. As previously reported [20], the absence of CD3ε precludes pre-TCR expression, and CD3εKO thymocytes, although arrested at the early DN3 stage of development, undergo extensive D-Jβ and V-DJβ rearrangement (Figure 2A). As compared to CD3εKO mice, Dβ2–Jβ2 rearrangement is substantially inhibited in all DN cells or in DN2/3 and DN4 subsets from CD28/B7/CD3εKO mice (Figure 2A). Further, analysis of Vβ5-, Vβ8-, and Vβ14.1-DJβ2 rearrangements in these same DN thymocytes reveal that V-DJβ rearrangement is undetectable in CD28/B7/CD3εKO DN cells (Figure 2 B–D). Thus, in the absence of pre-TCR expression the differentiation program induced by CD28/B7 expression in immature DN thymocytes results in at least a 10-fold inhibition of both Dβ2–Jβ2 and V–DJβ2 rearrangement in 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)

CD28/B7-driven development inhibits TCRβ rearrangement in DN thymocytes.100, 30, and 10 ng of DNA was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangement, (B) Vβ5-DJβ2 rearrangement, (C) Vβ8.1-DJβ2 rearrangement, or (D) Vβb14.1-DJβ2 rearrangement. (E) 10, 3, and 1 ng of DNA was analyzed by PCR to detect unrearranged IgM as a loading control. The arrow in panel A denotes the amplification of germline or non-rearranged Dβ2-Jβ2 PCR products. These results presented here comparing VDJβ rearrangements in CD3ε and CD28/B7/CD3εKO DN cells were analyzed in the same experiment and are representative of three experiments analyzing DNA prepared from a total of six CD3εKO and CD28/B7/CD3εKO mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108609&req=5

pone-0020639-g002: CD28/B7-driven development inhibits TCRβ rearrangement in DN thymocytes.100, 30, and 10 ng of DNA was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangement, (B) Vβ5-DJβ2 rearrangement, (C) Vβ8.1-DJβ2 rearrangement, or (D) Vβb14.1-DJβ2 rearrangement. (E) 10, 3, and 1 ng of DNA was analyzed by PCR to detect unrearranged IgM as a loading control. The arrow in panel A denotes the amplification of germline or non-rearranged Dβ2-Jβ2 PCR products. These results presented here comparing VDJβ rearrangements in CD3ε and CD28/B7/CD3εKO DN cells were analyzed in the same experiment and are representative of three experiments analyzing DNA prepared from a total of six CD3εKO and CD28/B7/CD3εKO mice.
Mentions: Expression of TCRβ protein occurs only after successful D-Jβ and V-DJβ rearrangement are completed [19]. Following TCRβ protein expression and pre-TCR formation, a series of differentiation events is initiated, and a critical component of this differentiation program is the suppression of additional V-DJβ rearrangement [6]. Since CD28/B7 interactions in early DN cells can induce differentiation in the absence of pre-TCR expression, we examined the effects of this differentiation program on TCRβ rearrangement and allelic exclusion. We began by analyzing TCRβ rearrangement in purified DN cells isolated from either pre-TCR deficient CD3εKO mice or from CD28/B7/CD3εKO mice. As previously reported [20], the absence of CD3ε precludes pre-TCR expression, and CD3εKO thymocytes, although arrested at the early DN3 stage of development, undergo extensive D-Jβ and V-DJβ rearrangement (Figure 2A). As compared to CD3εKO mice, Dβ2–Jβ2 rearrangement is substantially inhibited in all DN cells or in DN2/3 and DN4 subsets from CD28/B7/CD3εKO mice (Figure 2A). Further, analysis of Vβ5-, Vβ8-, and Vβ14.1-DJβ2 rearrangements in these same DN thymocytes reveal that V-DJβ rearrangement is undetectable in CD28/B7/CD3εKO DN cells (Figure 2 B–D). Thus, in the absence of pre-TCR expression the differentiation program induced by CD28/B7 expression in immature DN thymocytes results in at least a 10-fold inhibition of both Dβ2–Jβ2 and V–DJβ2 rearrangement in 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