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Lineage commitment in the thymus: only the most differentiated (TCRhibcl-2hi) subset of CD4+CD8+ thymocytes has selectively terminated CD4 or CD8 synthesis.

Punt JA, Suzuki H, Granger LG, Sharrow SO, Singer A - J. Exp. Med. (1996)

Bottom Line: However, the molecular event(s) that defines lineage commitment is controversial.The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5hiCD69hiTCRhibcl-2hi).In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Lineage commitment is a developmental process by which individual CD4+CD8+ (double positive, DP) thymocytes make a decision to differentiate into either CD4+ or CD8+ T cells. However, the molecular event(s) that defines lineage commitment is controversial. We have previously proposed that lineage commitment in DP thymocytes can be molecularly defined as the selective termination of CD4 or CD8 coreceptor synthesis. The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5hiCD69hiTCRhibcl-2hi). In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule. In addition, the present results identify three distinct sub-populations of DP thymocytes that define the developmental progression of the lineage commitment process and demonstrate that lineage commitment is coincident with upregulation of TCR and bcl-2. Thus, this study supports a molecular definition of lineage commitment and uniquely identifies TCRhibcl-2hi DP thymocytes as cells that are already committed to either the CD4 or CD8 T cell lineage.

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CD4+CD8+ thymocytes are heterogeneous for expression of  CD5 and CD69. Freshly isolated thymocytes from B6 mice were analyzed by three color flow cytometry for surface expression of CD4, CD8  and CD5 (a) or CD69 (b). Gates subdividing cells into low, intermediate  and high expression of CD5 (a) or CD69 (b) are indicated. Contour plots  displaying CD4 and CD8 expression of cells in each gate are shown below each gate and the frequency of cells falling into CD4+CD8+,  CD4−CD8+ and CD4+CD8− subpopulations are indicated. Solid line indicates CD5 or CD69 staining and dashed line indicates negative control  (Leu 4) staining.
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Figure 2: CD4+CD8+ thymocytes are heterogeneous for expression of CD5 and CD69. Freshly isolated thymocytes from B6 mice were analyzed by three color flow cytometry for surface expression of CD4, CD8 and CD5 (a) or CD69 (b). Gates subdividing cells into low, intermediate and high expression of CD5 (a) or CD69 (b) are indicated. Contour plots displaying CD4 and CD8 expression of cells in each gate are shown below each gate and the frequency of cells falling into CD4+CD8+, CD4−CD8+ and CD4+CD8− subpopulations are indicated. Solid line indicates CD5 or CD69 staining and dashed line indicates negative control (Leu 4) staining.

Mentions: Surface expression of CD5 and CD69 is markedly heterogeneous among DP thymocytes, with DP thymocytes contained within cell populations expressing low, intermediate, and high levels of each marker (Fig. 2, a and b). More importantly, surface CD5 and CD69 expression is directly correlated with developmental maturity such that the least differentiated DP thymocytes express low amounts of CD5 and CD69 and the most differentiated DP thymocytes express high levels of CD5 and CD69 (17, 19, 20). Consequently, to determine if termination of coreceptor synthesis occurred in a developmentally restricted subpopulation of DP thymocytes, we performed the coreceptor reexpression assay on thymocytes that were electronically purified by 3-color cell sorting into DP thymocytes expressing low/intermediate versus high levels of surface CD5 and CD69 (Fig. 3, a and b). All DP thymocytes expressing low/intermediate levels of CD5 or CD69 either synthesized both coreceptor molecules or synthesized no coreceptor molecule (Fig. 3, a and b, upper panels); essentially none of these cells selectively synthesized only one coreceptor molecule (Fig. 3, a and b). In contrast, DP thymocytes expressing high levels of surface CD5 or CD69 did contain cells that were selectively synthesizing only one coreceptor molecule (Fig. 3, a and b, lower panels). DP thymocytes selectively synthesizing CD8 usually outnumbered thymocytes selectively synthesizing CD4. We suspect this is because thymocytes that have selectively terminated coreceptor synthesis lose surface CD8 expression more quickly than surface CD4 expression, a possibility consistent with observations by other investigators (22, 23). We also observed a significant number of cells that have terminated or diminished synthesis of both CD4 and CD8. The fate of these cells is not yet known but they may represent precursors of mature DN T cells. Most importantly, these data indicate that cells selectively synthesizing only one coreceptor molecule (either CD4 or CD8) are not present among the vast majority of DP thymocytes, but are only present among DP thymocytes expressing high surface levels of CD5 and CD69.


Lineage commitment in the thymus: only the most differentiated (TCRhibcl-2hi) subset of CD4+CD8+ thymocytes has selectively terminated CD4 or CD8 synthesis.

Punt JA, Suzuki H, Granger LG, Sharrow SO, Singer A - J. Exp. Med. (1996)

CD4+CD8+ thymocytes are heterogeneous for expression of  CD5 and CD69. Freshly isolated thymocytes from B6 mice were analyzed by three color flow cytometry for surface expression of CD4, CD8  and CD5 (a) or CD69 (b). Gates subdividing cells into low, intermediate  and high expression of CD5 (a) or CD69 (b) are indicated. Contour plots  displaying CD4 and CD8 expression of cells in each gate are shown below each gate and the frequency of cells falling into CD4+CD8+,  CD4−CD8+ and CD4+CD8− subpopulations are indicated. Solid line indicates CD5 or CD69 staining and dashed line indicates negative control  (Leu 4) staining.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196385&req=5

Figure 2: CD4+CD8+ thymocytes are heterogeneous for expression of CD5 and CD69. Freshly isolated thymocytes from B6 mice were analyzed by three color flow cytometry for surface expression of CD4, CD8 and CD5 (a) or CD69 (b). Gates subdividing cells into low, intermediate and high expression of CD5 (a) or CD69 (b) are indicated. Contour plots displaying CD4 and CD8 expression of cells in each gate are shown below each gate and the frequency of cells falling into CD4+CD8+, CD4−CD8+ and CD4+CD8− subpopulations are indicated. Solid line indicates CD5 or CD69 staining and dashed line indicates negative control (Leu 4) staining.
Mentions: Surface expression of CD5 and CD69 is markedly heterogeneous among DP thymocytes, with DP thymocytes contained within cell populations expressing low, intermediate, and high levels of each marker (Fig. 2, a and b). More importantly, surface CD5 and CD69 expression is directly correlated with developmental maturity such that the least differentiated DP thymocytes express low amounts of CD5 and CD69 and the most differentiated DP thymocytes express high levels of CD5 and CD69 (17, 19, 20). Consequently, to determine if termination of coreceptor synthesis occurred in a developmentally restricted subpopulation of DP thymocytes, we performed the coreceptor reexpression assay on thymocytes that were electronically purified by 3-color cell sorting into DP thymocytes expressing low/intermediate versus high levels of surface CD5 and CD69 (Fig. 3, a and b). All DP thymocytes expressing low/intermediate levels of CD5 or CD69 either synthesized both coreceptor molecules or synthesized no coreceptor molecule (Fig. 3, a and b, upper panels); essentially none of these cells selectively synthesized only one coreceptor molecule (Fig. 3, a and b). In contrast, DP thymocytes expressing high levels of surface CD5 or CD69 did contain cells that were selectively synthesizing only one coreceptor molecule (Fig. 3, a and b, lower panels). DP thymocytes selectively synthesizing CD8 usually outnumbered thymocytes selectively synthesizing CD4. We suspect this is because thymocytes that have selectively terminated coreceptor synthesis lose surface CD8 expression more quickly than surface CD4 expression, a possibility consistent with observations by other investigators (22, 23). We also observed a significant number of cells that have terminated or diminished synthesis of both CD4 and CD8. The fate of these cells is not yet known but they may represent precursors of mature DN T cells. Most importantly, these data indicate that cells selectively synthesizing only one coreceptor molecule (either CD4 or CD8) are not present among the vast majority of DP thymocytes, but are only present among DP thymocytes expressing high surface levels of CD5 and CD69.

Bottom Line: However, the molecular event(s) that defines lineage commitment is controversial.The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5hiCD69hiTCRhibcl-2hi).In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Lineage commitment is a developmental process by which individual CD4+CD8+ (double positive, DP) thymocytes make a decision to differentiate into either CD4+ or CD8+ T cells. However, the molecular event(s) that defines lineage commitment is controversial. We have previously proposed that lineage commitment in DP thymocytes can be molecularly defined as the selective termination of CD4 or CD8 coreceptor synthesis. The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5hiCD69hiTCRhibcl-2hi). In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule. In addition, the present results identify three distinct sub-populations of DP thymocytes that define the developmental progression of the lineage commitment process and demonstrate that lineage commitment is coincident with upregulation of TCR and bcl-2. Thus, this study supports a molecular definition of lineage commitment and uniquely identifies TCRhibcl-2hi DP thymocytes as cells that are already committed to either the CD4 or CD8 T cell lineage.

Show MeSH
Related in: MedlinePlus