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Downregulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development.

Res P, Blom B, Hori T, Weijer K, Spits H - J. Exp. Med. (1997)

Bottom Line: Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a.CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells.Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.

View Article: PubMed Central - PubMed

Affiliation: Netherlands Cancer Institute, Amsterdam, Netherlands.

ABSTRACT
We have investigated whether in the human thymus transition of CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a- cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II-positive, but not MHC class II-negative, mouse thymic microenvironments support differentiation of human progenitors into TCR alpha beta+CD4+ SP cells, indicating that mouse MHC class II can positively select TCR alpha beta +CD4+ SP human cells. Strikingly, these SP are arrested in the CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.

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Three parameter analysis of neonatal cord blood cells. Cord  blood lymphocytes were obtained by centrifugation over Lymphoprep  (Nycomed Pharma, Oslo, Norway). Monocytes/macrophages and contaminating erythrocytes were depleted with goat anti–mouse IgG coated  magnetic beads (Dynal Inc.) using monoclonal antibodies against CD14  and glycophorin. The remaining cells were stained with CD4 PE and  CD8 TRC against the indicated FITC conjugated antibodies.
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Figure 7: Three parameter analysis of neonatal cord blood cells. Cord blood lymphocytes were obtained by centrifugation over Lymphoprep (Nycomed Pharma, Oslo, Norway). Monocytes/macrophages and contaminating erythrocytes were depleted with goat anti–mouse IgG coated magnetic beads (Dynal Inc.) using monoclonal antibodies against CD14 and glycophorin. The remaining cells were stained with CD4 PE and CD8 TRC against the indicated FITC conjugated antibodies.

Mentions: As indicated in Fig. 1, the thymus contains expandable CD1a DP cells. Although not shown here, we were able to clone DP cells and these clones maintained a persistent DP phenotype upon long-term culture in accord with data published (13, 14). The presence of DP cells, expressing several characteristics of maturity, raises the question whether these cells are able to migrate from the thymus. DP cells could be observed in T cell samples from neonatal cord blood (Fig. 7) in percentages ranging from 0.5 to 3% of the total number of CD3+ T cells (n = 4). All DP cord blood cells express CD3 and CD27, and they lack CD1a or the activation antigen CD69 (Fig. 7). Further analysis of these cells indicate that the majority of these cells express CD45RA, and are negative for CD45RO and Fas (Fig. 7) suggesting that these DP cells represent naive, not memory, cells. Moreover, like in the thymus (42), both CD8α+β−CD4+ and CD4+CD8α+β+ cells could be observed. These observations are compatible with the notion that DP cells can migrate out of the thymus.


Downregulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development.

Res P, Blom B, Hori T, Weijer K, Spits H - J. Exp. Med. (1997)

Three parameter analysis of neonatal cord blood cells. Cord  blood lymphocytes were obtained by centrifugation over Lymphoprep  (Nycomed Pharma, Oslo, Norway). Monocytes/macrophages and contaminating erythrocytes were depleted with goat anti–mouse IgG coated  magnetic beads (Dynal Inc.) using monoclonal antibodies against CD14  and glycophorin. The remaining cells were stained with CD4 PE and  CD8 TRC against the indicated FITC conjugated antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196108&req=5

Figure 7: Three parameter analysis of neonatal cord blood cells. Cord blood lymphocytes were obtained by centrifugation over Lymphoprep (Nycomed Pharma, Oslo, Norway). Monocytes/macrophages and contaminating erythrocytes were depleted with goat anti–mouse IgG coated magnetic beads (Dynal Inc.) using monoclonal antibodies against CD14 and glycophorin. The remaining cells were stained with CD4 PE and CD8 TRC against the indicated FITC conjugated antibodies.
Mentions: As indicated in Fig. 1, the thymus contains expandable CD1a DP cells. Although not shown here, we were able to clone DP cells and these clones maintained a persistent DP phenotype upon long-term culture in accord with data published (13, 14). The presence of DP cells, expressing several characteristics of maturity, raises the question whether these cells are able to migrate from the thymus. DP cells could be observed in T cell samples from neonatal cord blood (Fig. 7) in percentages ranging from 0.5 to 3% of the total number of CD3+ T cells (n = 4). All DP cord blood cells express CD3 and CD27, and they lack CD1a or the activation antigen CD69 (Fig. 7). Further analysis of these cells indicate that the majority of these cells express CD45RA, and are negative for CD45RO and Fas (Fig. 7) suggesting that these DP cells represent naive, not memory, cells. Moreover, like in the thymus (42), both CD8α+β−CD4+ and CD4+CD8α+β+ cells could be observed. These observations are compatible with the notion that DP cells can migrate out of the thymus.

Bottom Line: Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a.CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells.Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.

View Article: PubMed Central - PubMed

Affiliation: Netherlands Cancer Institute, Amsterdam, Netherlands.

ABSTRACT
We have investigated whether in the human thymus transition of CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a- cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II-positive, but not MHC class II-negative, mouse thymic microenvironments support differentiation of human progenitors into TCR alpha beta+CD4+ SP cells, indicating that mouse MHC class II can positively select TCR alpha beta +CD4+ SP human cells. Strikingly, these SP are arrested in the CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.

Show MeSH
Related in: MedlinePlus