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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 permits Dβ2-Jβ2 rearrangement but substantially inhibits V-DJβ2 rearrangement in DP thymocytes.100, 30, and 10 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangements, (B) Vβ5-DJβ2, or (C) Vβ8.1-DJβ2 rearrangements. The PCR for unrearranged IgM, the loading control, was performed using 10, 3, and 1 ng of DNA (D). The results shown are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3eKO mice.
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pone-0020639-g004: CD28/B7-dependent development permits Dβ2-Jβ2 rearrangement but substantially inhibits V-DJβ2 rearrangement in DP thymocytes.100, 30, and 10 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangements, (B) Vβ5-DJβ2, or (C) Vβ8.1-DJβ2 rearrangements. The PCR for unrearranged IgM, the loading control, was performed using 10, 3, and 1 ng of DNA (D). The results shown are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3eKO mice.

Mentions: Since the recombination machinery necessary for TCRα rearrangement was intact in DP cells from CD28/B7/CD3εKO mice, we reexamined TCRβ rearrangement in these DP cells. In fact, Dβ2-Jβ2 rearrangement in DP cells from CD28/B7/CD3εKO mice was equivalent to that measured in pre-TCR-dependent DP cells from WT B6 mice (Figure 4A). However, in marked contrast, both Vβ5- and Vβ8-DJβ2 rearrangements were substantially inhibited in these same DP cells (Figure 4B–C). Since Dβ2 -Jβ2 rearrangement is deficient in DN thymocytes from CD28/B7/CD3εKO mice (Figure 2), this result demonstrates that during CD28/B7-induced development, D-Jβ2 rearrangement is delayed but occurs efficiently in DP cells. Importantly, however, even when D-Jβ rearrangement is successful, the developmental window in which V-DJβ rearrangement can occur is highly restricted. Suppression of V-DJβ rearrangement does not require pre-TCR expression but rather is developmentally enforced when DN cells exit DN3 and mature to DN4 and 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 permits Dβ2-Jβ2 rearrangement but substantially inhibits V-DJβ2 rearrangement in DP thymocytes.100, 30, and 10 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangements, (B) Vβ5-DJβ2, or (C) Vβ8.1-DJβ2 rearrangements. The PCR for unrearranged IgM, the loading control, was performed using 10, 3, and 1 ng of DNA (D). The results shown are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3eKO mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108609&req=5

pone-0020639-g004: CD28/B7-dependent development permits Dβ2-Jβ2 rearrangement but substantially inhibits V-DJβ2 rearrangement in DP thymocytes.100, 30, and 10 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect (A) Dβ2-Jβ2 rearrangements, (B) Vβ5-DJβ2, or (C) Vβ8.1-DJβ2 rearrangements. The PCR for unrearranged IgM, the loading control, was performed using 10, 3, and 1 ng of DNA (D). The results shown are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3eKO mice.
Mentions: Since the recombination machinery necessary for TCRα rearrangement was intact in DP cells from CD28/B7/CD3εKO mice, we reexamined TCRβ rearrangement in these DP cells. In fact, Dβ2-Jβ2 rearrangement in DP cells from CD28/B7/CD3εKO mice was equivalent to that measured in pre-TCR-dependent DP cells from WT B6 mice (Figure 4A). However, in marked contrast, both Vβ5- and Vβ8-DJβ2 rearrangements were substantially inhibited in these same DP cells (Figure 4B–C). Since Dβ2 -Jβ2 rearrangement is deficient in DN thymocytes from CD28/B7/CD3εKO mice (Figure 2), this result demonstrates that during CD28/B7-induced development, D-Jβ2 rearrangement is delayed but occurs efficiently in DP cells. Importantly, however, even when D-Jβ rearrangement is successful, the developmental window in which V-DJβ rearrangement can occur is highly restricted. Suppression of V-DJβ rearrangement does not require pre-TCR expression but rather is developmentally enforced when DN cells exit DN3 and mature to DN4 and 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