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Identification of a committed T cell precursor population in adult human peripheral blood.

Bruno L, Res P, Dessing M, Cella M, Spits H - J. Exp. Med. (1997)

Bottom Line: At a molecular level, they express the pre-T cell receptor alpha (pT alpha) gene, CD3-gamma, CD-delta and CD-epsilon, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-beta locus (D-J).Moreover, low levels of CD3 epsilon protein, but not of TCR-beta chain, can be detected in their cytoplasm.Our results suggest that CD4+CD3- cells identified in peripheral blood are different from CD3-CD4+CD8- thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, Switzerland.

ABSTRACT
Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4+CD3+ alpha beta + mature T cells. These cells, which represent 0.1-0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor alpha (pT alpha) gene, CD3-gamma, CD-delta and CD-epsilon, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-beta locus (D-J). Moreover, low levels of CD3 epsilon protein, but not of TCR-beta chain, can be detected in their cytoplasm. Our results suggest that CD4+CD3- cells identified in peripheral blood are different from CD3-CD4+CD8- thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.

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pTα expression in  the CD2− and CD2+ of  CD4+CD3− CD14− cells. Limited amounts (50, 20, 10, 5) of  CD4+CD3−CD14− cells were  directly sorted in 96-well plates  and analyzed for the expression  of pTα gene by two rounds of  PCR using the same primers (top  right and left). The analysis was  first performed on different cell  numbers in duplicate (top left)  and then on the same cell number 10 times (top right). In the  bottom panel, the same analysis  on different cell numbers (20,  10, 5, 1) in duplicate of CD2−  and CD2+ fractions of CD4+  CD3−CD14− cells is shown.  CD4+CD3−CD14− total sorted  cell population and water were  the positive and negative controls, respectively.
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Figure 6: pTα expression in the CD2− and CD2+ of CD4+CD3− CD14− cells. Limited amounts (50, 20, 10, 5) of CD4+CD3−CD14− cells were directly sorted in 96-well plates and analyzed for the expression of pTα gene by two rounds of PCR using the same primers (top right and left). The analysis was first performed on different cell numbers in duplicate (top left) and then on the same cell number 10 times (top right). In the bottom panel, the same analysis on different cell numbers (20, 10, 5, 1) in duplicate of CD2− and CD2+ fractions of CD4+ CD3−CD14− cells is shown. CD4+CD3−CD14− total sorted cell population and water were the positive and negative controls, respectively.

Mentions: When we initially restricted our analysis to duplicates of each sorted cell aliquots (50, 20, 10, 5, cells; Fig. 6, top left), a positive reaction could be detected in all the wells, down to the ones that contained 5 cells/well. To try and get a more accurate and reliable estimate of the frequency, ten separate PCR reactions were run, each on a well containing 5 cells (Fig. 6, top right). The analysis of the results showed a positive signal in 50% of the samples. Assuming that our PCR was 100% efficient on these 5-cell samples, a frequency of pTα+ cells of 1/7 can be calculated using Poisson's equation.


Identification of a committed T cell precursor population in adult human peripheral blood.

Bruno L, Res P, Dessing M, Cella M, Spits H - J. Exp. Med. (1997)

pTα expression in  the CD2− and CD2+ of  CD4+CD3− CD14− cells. Limited amounts (50, 20, 10, 5) of  CD4+CD3−CD14− cells were  directly sorted in 96-well plates  and analyzed for the expression  of pTα gene by two rounds of  PCR using the same primers (top  right and left). The analysis was  first performed on different cell  numbers in duplicate (top left)  and then on the same cell number 10 times (top right). In the  bottom panel, the same analysis  on different cell numbers (20,  10, 5, 1) in duplicate of CD2−  and CD2+ fractions of CD4+  CD3−CD14− cells is shown.  CD4+CD3−CD14− total sorted  cell population and water were  the positive and negative controls, respectively.
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Related In: Results  -  Collection

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Figure 6: pTα expression in the CD2− and CD2+ of CD4+CD3− CD14− cells. Limited amounts (50, 20, 10, 5) of CD4+CD3−CD14− cells were directly sorted in 96-well plates and analyzed for the expression of pTα gene by two rounds of PCR using the same primers (top right and left). The analysis was first performed on different cell numbers in duplicate (top left) and then on the same cell number 10 times (top right). In the bottom panel, the same analysis on different cell numbers (20, 10, 5, 1) in duplicate of CD2− and CD2+ fractions of CD4+ CD3−CD14− cells is shown. CD4+CD3−CD14− total sorted cell population and water were the positive and negative controls, respectively.
Mentions: When we initially restricted our analysis to duplicates of each sorted cell aliquots (50, 20, 10, 5, cells; Fig. 6, top left), a positive reaction could be detected in all the wells, down to the ones that contained 5 cells/well. To try and get a more accurate and reliable estimate of the frequency, ten separate PCR reactions were run, each on a well containing 5 cells (Fig. 6, top right). The analysis of the results showed a positive signal in 50% of the samples. Assuming that our PCR was 100% efficient on these 5-cell samples, a frequency of pTα+ cells of 1/7 can be calculated using Poisson's equation.

Bottom Line: At a molecular level, they express the pre-T cell receptor alpha (pT alpha) gene, CD3-gamma, CD-delta and CD-epsilon, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-beta locus (D-J).Moreover, low levels of CD3 epsilon protein, but not of TCR-beta chain, can be detected in their cytoplasm.Our results suggest that CD4+CD3- cells identified in peripheral blood are different from CD3-CD4+CD8- thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, Switzerland.

ABSTRACT
Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4+CD3+ alpha beta + mature T cells. These cells, which represent 0.1-0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor alpha (pT alpha) gene, CD3-gamma, CD-delta and CD-epsilon, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-beta locus (D-J). Moreover, low levels of CD3 epsilon protein, but not of TCR-beta chain, can be detected in their cytoplasm. Our results suggest that CD4+CD3- cells identified in peripheral blood are different from CD3-CD4+CD8- thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.

Show MeSH