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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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(A) FACScan® analysis of presorted and sorted cells from PBMC. Mononuclear cells were separated from total PB by standard Ficoll gradient  centrifugation, CD3 depleted using FITC–TR66 mAb and stained with CD4–PE- and CD14–FITC-specific mAbs. Top left, CD3/CD14 versus CD4  staining pattern of cells gated through R1 (FSC versus SSC, bottom left). R1 determined by backgating R2 into FSC versus SSC (see also size pattern, bottom right). Top and bottom right, cells after sorting (purity close to 100%). (B) CD4+CD3−CD14− sorted cells express pTα. RT–PCR analysis for the expression of pTα (top) and β-actin (bottom) was performed on total PBMC, CD4+CD3−CD14− sorted cells according to gates shown in A and total sorted  PBMC gating out CD4+ CD3−CD14− (PBMC, CD4+CD3−CD14−). To ascertain that PCR products shown were derived from pTα cDNA, gels were  blotted and probed with a pTα-specific oligonucleotide (data not shown).
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Figure 1: (A) FACScan® analysis of presorted and sorted cells from PBMC. Mononuclear cells were separated from total PB by standard Ficoll gradient centrifugation, CD3 depleted using FITC–TR66 mAb and stained with CD4–PE- and CD14–FITC-specific mAbs. Top left, CD3/CD14 versus CD4 staining pattern of cells gated through R1 (FSC versus SSC, bottom left). R1 determined by backgating R2 into FSC versus SSC (see also size pattern, bottom right). Top and bottom right, cells after sorting (purity close to 100%). (B) CD4+CD3−CD14− sorted cells express pTα. RT–PCR analysis for the expression of pTα (top) and β-actin (bottom) was performed on total PBMC, CD4+CD3−CD14− sorted cells according to gates shown in A and total sorted PBMC gating out CD4+ CD3−CD14− (PBMC, CD4+CD3−CD14−). To ascertain that PCR products shown were derived from pTα cDNA, gels were blotted and probed with a pTα-specific oligonucleotide (data not shown).

Mentions: By PCR analysis of retrotranscribed mRNA of PBMC of adult donors we were able to detect low levels of pTα message in all of the samples tested (data not shown; Fig. 1 B, lane 1). Depletion of CD3+ T cells from PBMC did not abrogate the detection of pTα expression by PCR. This result was expected, because mature T cells have completely lost expression of pTα (6–8). In view of our previously demonstration (6) that the thymic subset expressing the highest levels of pTα is CD3−CD4+CD8− (immature single positive, ISP), we investigated whether expression of CD4 was shared by pTα+ cells in PBMC. Fig. 1 demonstrates that this is indeed the case, because sorted CD4+, CD3− cells (Fig. 1 A, shown in the box, R2) expressed pTα (Fig. 1 B, lane 2). No pTα message could be detected in total PBMC depleted of CD4+CD3−CD14− cells (Fig. 1 B, lane 3). Monocytes, which also express low levels of CD4 but are CD3−, were excluded from the selection by staining with a specific mAb (CD14) and on the basis of their size (Fig. 1 A). Interestingly, the physical parameters (FSC versus SSC) of the pTα+ subset differ substantially from those of mature lymphocytes and monocytes, in that pTα+ cells have a size and a cell complexity intermediate between lymphocytes and monocytes (Fig. 1 A, bottom). This cell population represents, in different healthy adult individuals analyzed (25–35 yr old), 3–9% of gated cells (R1) and 0.1–0.5% of the total PBMC, obtained by Ficoll preparation.


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)

(A) FACScan® analysis of presorted and sorted cells from PBMC. Mononuclear cells were separated from total PB by standard Ficoll gradient  centrifugation, CD3 depleted using FITC–TR66 mAb and stained with CD4–PE- and CD14–FITC-specific mAbs. Top left, CD3/CD14 versus CD4  staining pattern of cells gated through R1 (FSC versus SSC, bottom left). R1 determined by backgating R2 into FSC versus SSC (see also size pattern, bottom right). Top and bottom right, cells after sorting (purity close to 100%). (B) CD4+CD3−CD14− sorted cells express pTα. RT–PCR analysis for the expression of pTα (top) and β-actin (bottom) was performed on total PBMC, CD4+CD3−CD14− sorted cells according to gates shown in A and total sorted  PBMC gating out CD4+ CD3−CD14− (PBMC, CD4+CD3−CD14−). To ascertain that PCR products shown were derived from pTα cDNA, gels were  blotted and probed with a pTα-specific oligonucleotide (data not shown).
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Related In: Results  -  Collection

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Figure 1: (A) FACScan® analysis of presorted and sorted cells from PBMC. Mononuclear cells were separated from total PB by standard Ficoll gradient centrifugation, CD3 depleted using FITC–TR66 mAb and stained with CD4–PE- and CD14–FITC-specific mAbs. Top left, CD3/CD14 versus CD4 staining pattern of cells gated through R1 (FSC versus SSC, bottom left). R1 determined by backgating R2 into FSC versus SSC (see also size pattern, bottom right). Top and bottom right, cells after sorting (purity close to 100%). (B) CD4+CD3−CD14− sorted cells express pTα. RT–PCR analysis for the expression of pTα (top) and β-actin (bottom) was performed on total PBMC, CD4+CD3−CD14− sorted cells according to gates shown in A and total sorted PBMC gating out CD4+ CD3−CD14− (PBMC, CD4+CD3−CD14−). To ascertain that PCR products shown were derived from pTα cDNA, gels were blotted and probed with a pTα-specific oligonucleotide (data not shown).
Mentions: By PCR analysis of retrotranscribed mRNA of PBMC of adult donors we were able to detect low levels of pTα message in all of the samples tested (data not shown; Fig. 1 B, lane 1). Depletion of CD3+ T cells from PBMC did not abrogate the detection of pTα expression by PCR. This result was expected, because mature T cells have completely lost expression of pTα (6–8). In view of our previously demonstration (6) that the thymic subset expressing the highest levels of pTα is CD3−CD4+CD8− (immature single positive, ISP), we investigated whether expression of CD4 was shared by pTα+ cells in PBMC. Fig. 1 demonstrates that this is indeed the case, because sorted CD4+, CD3− cells (Fig. 1 A, shown in the box, R2) expressed pTα (Fig. 1 B, lane 2). No pTα message could be detected in total PBMC depleted of CD4+CD3−CD14− cells (Fig. 1 B, lane 3). Monocytes, which also express low levels of CD4 but are CD3−, were excluded from the selection by staining with a specific mAb (CD14) and on the basis of their size (Fig. 1 A). Interestingly, the physical parameters (FSC versus SSC) of the pTα+ subset differ substantially from those of mature lymphocytes and monocytes, in that pTα+ cells have a size and a cell complexity intermediate between lymphocytes and monocytes (Fig. 1 A, bottom). This cell population represents, in different healthy adult individuals analyzed (25–35 yr old), 3–9% of gated cells (R1) and 0.1–0.5% of the total PBMC, obtained by Ficoll preparation.

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