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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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TCRα rearrangement is equivalent in DP cells generated via pre-TCR- or CD28/B7-dependent development.300, 100, and 30 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect the indicated Vα-Jα rearrangements. The PCR reaction for unrearranged IgM was performed using 10 ng DNA. The results presented here comparing TCRα rearrangements in B6 and CD28/B7/CD3εKO DP cells were analyzed in the same experiment and are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3εKO mice.
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pone-0020639-g003: TCRα rearrangement is equivalent in DP cells generated via pre-TCR- or CD28/B7-dependent development.300, 100, and 30 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect the indicated Vα-Jα rearrangements. The PCR reaction for unrearranged IgM was performed using 10 ng DNA. The results presented here comparing TCRα rearrangements in B6 and CD28/B7/CD3εKO DP cells were analyzed in the same experiment and are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3εKO mice.

Mentions: During conventional thymic development, only DN cells that successfully rearrange TCRβ and express a pre-TCR can differentiate to DP cells and rearrange TCRα [3], [21]. We asked if CD28/B7-driven development would support TCRα rearrangement in DP cells that were generated from DN cells which failed to rearrange TCRβ and lack pre-TCR signaling. Despite the substantial inhibition of TCRβ rearrangement observed in DN cells from CD28/B7/CD3εKO mice (Figure 2), Vα-Jα rearrangement in DP cells from these same mice was not inhibited and, in fact, appeared comparable to that detected in DP cells isolated from WT B6 mice (Figure 3). This result demonstrates that CD28/B7-driven development is not universally inhibitory to TCR rearrangement and that efficient TCRα rearrangement in DP cells can occur in the absence of expressed TCRβ or pre-TCR signaling.


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)

TCRα rearrangement is equivalent in DP cells generated via pre-TCR- or CD28/B7-dependent development.300, 100, and 30 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect the indicated Vα-Jα rearrangements. The PCR reaction for unrearranged IgM was performed using 10 ng DNA. The results presented here comparing TCRα rearrangements in B6 and CD28/B7/CD3εKO DP cells were analyzed in the same experiment and are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3εKO mice.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020639-g003: TCRα rearrangement is equivalent in DP cells generated via pre-TCR- or CD28/B7-dependent development.300, 100, and 30 ng of DNA prepared from FCM sorted DP cells was analyzed by PCR to detect the indicated Vα-Jα rearrangements. The PCR reaction for unrearranged IgM was performed using 10 ng DNA. The results presented here comparing TCRα rearrangements in B6 and CD28/B7/CD3εKO DP cells were analyzed in the same experiment and are representative of three experiments performed using DNA prepared from five to six B6 or CD28/B7/CD3εKO mice.
Mentions: During conventional thymic development, only DN cells that successfully rearrange TCRβ and express a pre-TCR can differentiate to DP cells and rearrange TCRα [3], [21]. We asked if CD28/B7-driven development would support TCRα rearrangement in DP cells that were generated from DN cells which failed to rearrange TCRβ and lack pre-TCR signaling. Despite the substantial inhibition of TCRβ rearrangement observed in DN cells from CD28/B7/CD3εKO mice (Figure 2), Vα-Jα rearrangement in DP cells from these same mice was not inhibited and, in fact, appeared comparable to that detected in DP cells isolated from WT B6 mice (Figure 3). This result demonstrates that CD28/B7-driven development is not universally inhibitory to TCR rearrangement and that efficient TCRα rearrangement in DP cells can occur in the absence of expressed TCRβ or pre-TCR signaling.

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