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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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Cell surface analysis of pTα+ cells. Mononuclear cells were  separated with standard Ficoll gradient and depleted of CD3+ cells with  FITC–TR66 mAb. Three-color analysis was then performed using CD4-  and CD14-specific mAbs in combination with a third antibody of interest  (histograms of which are shown in the figure): in the case of biotinylated  antibodies (CD2, CD10, and HLA-DR), CD4–R-PE and CD14–FITC  and, subsequently, APC were used; in the case of directly PE-labeled antibodies (CD5, CD7, CD33, and CD34), CD4–tricolor and CD14–FITC  were used. When staining with CD33 or B7-2 mAbs we had to sort  CD4+CD3−CD14− cells, as shown in Fig. 1, and restain them with one  of the two antibodies that were then detected with mouse anti–human  IgG2.
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Figure 4: Cell surface analysis of pTα+ cells. Mononuclear cells were separated with standard Ficoll gradient and depleted of CD3+ cells with FITC–TR66 mAb. Three-color analysis was then performed using CD4- and CD14-specific mAbs in combination with a third antibody of interest (histograms of which are shown in the figure): in the case of biotinylated antibodies (CD2, CD10, and HLA-DR), CD4–R-PE and CD14–FITC and, subsequently, APC were used; in the case of directly PE-labeled antibodies (CD5, CD7, CD33, and CD34), CD4–tricolor and CD14–FITC were used. When staining with CD33 or B7-2 mAbs we had to sort CD4+CD3−CD14− cells, as shown in Fig. 1, and restain them with one of the two antibodies that were then detected with mouse anti–human IgG2.

Mentions: CD4+CD3−CD14− cells were isolated by flow cytometry as described above (see Fig. 1) and subsequently stained with a set of monoclonal antibodies specific for cell surface markers. Fig. 4 shows that sorted cells homogeneously expressed high levels of CD45RA, intermediate levels of MHC class II HLA-DR, low levels of the costimulatory molecule B7-2, but did not express the hematopoietic progenitor cell surface antigen CD34 or the neutral endopeptidase CD10, both present on a T, B, NK, and DC cell precursor population recently described in human bone marrow (17). Only a very small fraction, around 2–5% of sorted cells, expressed CD5 and CD7, found on mature and immature T cells. A higher fraction, 15–40% depending on the different individuals analyzed, expressed the T- and NK-specific molecule CD2. CD33, expressed on myeloid progenitors and monocytes, also presented a bimodal distribution with most of the cells expressing low levels of the marker (CD33dim). Both CD33+ and CD33− cells expressed pTα (data not shown). CD4+CD3−CD14− cells did not exhibit detectable levels of the lineage-specific cell markers CD1a, CD8, CD16, and CD56, CD19 and CD20, γδ TCR and CD83 (data not shown).


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)

Cell surface analysis of pTα+ cells. Mononuclear cells were  separated with standard Ficoll gradient and depleted of CD3+ cells with  FITC–TR66 mAb. Three-color analysis was then performed using CD4-  and CD14-specific mAbs in combination with a third antibody of interest  (histograms of which are shown in the figure): in the case of biotinylated  antibodies (CD2, CD10, and HLA-DR), CD4–R-PE and CD14–FITC  and, subsequently, APC were used; in the case of directly PE-labeled antibodies (CD5, CD7, CD33, and CD34), CD4–tricolor and CD14–FITC  were used. When staining with CD33 or B7-2 mAbs we had to sort  CD4+CD3−CD14− cells, as shown in Fig. 1, and restain them with one  of the two antibodies that were then detected with mouse anti–human  IgG2.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: Cell surface analysis of pTα+ cells. Mononuclear cells were separated with standard Ficoll gradient and depleted of CD3+ cells with FITC–TR66 mAb. Three-color analysis was then performed using CD4- and CD14-specific mAbs in combination with a third antibody of interest (histograms of which are shown in the figure): in the case of biotinylated antibodies (CD2, CD10, and HLA-DR), CD4–R-PE and CD14–FITC and, subsequently, APC were used; in the case of directly PE-labeled antibodies (CD5, CD7, CD33, and CD34), CD4–tricolor and CD14–FITC were used. When staining with CD33 or B7-2 mAbs we had to sort CD4+CD3−CD14− cells, as shown in Fig. 1, and restain them with one of the two antibodies that were then detected with mouse anti–human IgG2.
Mentions: CD4+CD3−CD14− cells were isolated by flow cytometry as described above (see Fig. 1) and subsequently stained with a set of monoclonal antibodies specific for cell surface markers. Fig. 4 shows that sorted cells homogeneously expressed high levels of CD45RA, intermediate levels of MHC class II HLA-DR, low levels of the costimulatory molecule B7-2, but did not express the hematopoietic progenitor cell surface antigen CD34 or the neutral endopeptidase CD10, both present on a T, B, NK, and DC cell precursor population recently described in human bone marrow (17). Only a very small fraction, around 2–5% of sorted cells, expressed CD5 and CD7, found on mature and immature T cells. A higher fraction, 15–40% depending on the different individuals analyzed, expressed the T- and NK-specific molecule CD2. CD33, expressed on myeloid progenitors and monocytes, also presented a bimodal distribution with most of the cells expressing low levels of the marker (CD33dim). Both CD33+ and CD33− cells expressed pTα (data not shown). CD4+CD3−CD14− cells did not exhibit detectable levels of the lineage-specific cell markers CD1a, CD8, CD16, and CD56, CD19 and CD20, γδ TCR and CD83 (data not shown).

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