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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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CD1a+CD4+ SP thymocytes differentiate into CD1a− cells upon coculture with thymic stromal cells (A), and part of these cells upregulate  CD8 expression (B). Thymocytes were labeled with CD1a FITC, CD4 PE, and CD8 TRC. CD1a+ and CD1a− CD4high SP cells were sorted and part of the  cells were used to check CD3 expression by staining with CD3 TRC (all CD1a− and >99% of CD1+ CD4 SP thymocytes were CD3+). In experiment  A, 2 × 105 CD1a+CD4 SP (>98% purified) and 2 × 105 CD1a−CD4 SP cells were cultured on a monolayer of human thymic stromal cells. After 4 and  12 d, cells were tested for CD1a expression. The cell numbers of wells started with CD1a+ and CD1a−CD4 SP cells were both reduced to 8 × 104 after  4 d, whereas at day 12, 2.5 × 104, and 7.0 × 104 (CD1a−) cells were recovered from the cultures started with CD1a+ and CD1a−CD4 SP thymocytes,  respectively. In experiment B, sorted CD1a+ and CD1a− CD4 SP thymocytes were cultured for 7 d on a monolayer of thymic stromal cells, assayed for  CD4 and CD8 expression, and expanded in vitro with feeder cells, PHA, and IL-2. Expanded cells were also analysed for CD4 against CD8 expression.
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Figure 6: CD1a+CD4+ SP thymocytes differentiate into CD1a− cells upon coculture with thymic stromal cells (A), and part of these cells upregulate CD8 expression (B). Thymocytes were labeled with CD1a FITC, CD4 PE, and CD8 TRC. CD1a+ and CD1a− CD4high SP cells were sorted and part of the cells were used to check CD3 expression by staining with CD3 TRC (all CD1a− and >99% of CD1+ CD4 SP thymocytes were CD3+). In experiment A, 2 × 105 CD1a+CD4 SP (>98% purified) and 2 × 105 CD1a−CD4 SP cells were cultured on a monolayer of human thymic stromal cells. After 4 and 12 d, cells were tested for CD1a expression. The cell numbers of wells started with CD1a+ and CD1a−CD4 SP cells were both reduced to 8 × 104 after 4 d, whereas at day 12, 2.5 × 104, and 7.0 × 104 (CD1a−) cells were recovered from the cultures started with CD1a+ and CD1a−CD4 SP thymocytes, respectively. In experiment B, sorted CD1a+ and CD1a− CD4 SP thymocytes were cultured for 7 d on a monolayer of thymic stromal cells, assayed for CD4 and CD8 expression, and expanded in vitro with feeder cells, PHA, and IL-2. Expanded cells were also analysed for CD4 against CD8 expression.

Mentions: The presence of CD1a+ and CD1a− SP thymocytes raises the question whether CD1a+ SP cells are the direct precursors of CD1a− SP cells. An alternative possibility would be that the CD1a− SP cells are derived from the CD1a− DP cells and that CD1a+ SP cells represent a dead-end lineage. To investigate this, we cocultured purified CD1a+CD4+ SP cells with short term cultured human thymic stromal cells. This coculture resulted in a gradual downregulation of CD1a which was completed on day 12 (Fig. 6 A). Phenotypic analysis of these cells reveals that they express high levels of TCR αβ and CD4. Unexpectedly, many of these cells also express CD8α (Fig. 6 B). These differentiated CD1a− cells could be expanded in vitro and the phenotype did not alter upon expansion (Fig. 6 B). These data indicate that CD1a+ CD4+ SP cells can differentiate to expandable CD1a−CD4+ SP cells and also to expandable CD1a− CD4+CD8α+ cells.


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)

CD1a+CD4+ SP thymocytes differentiate into CD1a− cells upon coculture with thymic stromal cells (A), and part of these cells upregulate  CD8 expression (B). Thymocytes were labeled with CD1a FITC, CD4 PE, and CD8 TRC. CD1a+ and CD1a− CD4high SP cells were sorted and part of the  cells were used to check CD3 expression by staining with CD3 TRC (all CD1a− and >99% of CD1+ CD4 SP thymocytes were CD3+). In experiment  A, 2 × 105 CD1a+CD4 SP (>98% purified) and 2 × 105 CD1a−CD4 SP cells were cultured on a monolayer of human thymic stromal cells. After 4 and  12 d, cells were tested for CD1a expression. The cell numbers of wells started with CD1a+ and CD1a−CD4 SP cells were both reduced to 8 × 104 after  4 d, whereas at day 12, 2.5 × 104, and 7.0 × 104 (CD1a−) cells were recovered from the cultures started with CD1a+ and CD1a−CD4 SP thymocytes,  respectively. In experiment B, sorted CD1a+ and CD1a− CD4 SP thymocytes were cultured for 7 d on a monolayer of thymic stromal cells, assayed for  CD4 and CD8 expression, and expanded in vitro with feeder cells, PHA, and IL-2. Expanded cells were also analysed for CD4 against CD8 expression.
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Figure 6: CD1a+CD4+ SP thymocytes differentiate into CD1a− cells upon coculture with thymic stromal cells (A), and part of these cells upregulate CD8 expression (B). Thymocytes were labeled with CD1a FITC, CD4 PE, and CD8 TRC. CD1a+ and CD1a− CD4high SP cells were sorted and part of the cells were used to check CD3 expression by staining with CD3 TRC (all CD1a− and >99% of CD1+ CD4 SP thymocytes were CD3+). In experiment A, 2 × 105 CD1a+CD4 SP (>98% purified) and 2 × 105 CD1a−CD4 SP cells were cultured on a monolayer of human thymic stromal cells. After 4 and 12 d, cells were tested for CD1a expression. The cell numbers of wells started with CD1a+ and CD1a−CD4 SP cells were both reduced to 8 × 104 after 4 d, whereas at day 12, 2.5 × 104, and 7.0 × 104 (CD1a−) cells were recovered from the cultures started with CD1a+ and CD1a−CD4 SP thymocytes, respectively. In experiment B, sorted CD1a+ and CD1a− CD4 SP thymocytes were cultured for 7 d on a monolayer of thymic stromal cells, assayed for CD4 and CD8 expression, and expanded in vitro with feeder cells, PHA, and IL-2. Expanded cells were also analysed for CD4 against CD8 expression.
Mentions: The presence of CD1a+ and CD1a− SP thymocytes raises the question whether CD1a+ SP cells are the direct precursors of CD1a− SP cells. An alternative possibility would be that the CD1a− SP cells are derived from the CD1a− DP cells and that CD1a+ SP cells represent a dead-end lineage. To investigate this, we cocultured purified CD1a+CD4+ SP cells with short term cultured human thymic stromal cells. This coculture resulted in a gradual downregulation of CD1a which was completed on day 12 (Fig. 6 A). Phenotypic analysis of these cells reveals that they express high levels of TCR αβ and CD4. Unexpectedly, many of these cells also express CD8α (Fig. 6 B). These differentiated CD1a− cells could be expanded in vitro and the phenotype did not alter upon expansion (Fig. 6 B). These data indicate that CD1a+ CD4+ SP cells can differentiate to expandable CD1a−CD4+ SP cells and also to expandable CD1a− CD4+CD8α+ cells.

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