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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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RAG-1 expression of total  unseparated thymocytes and CD1a−  and CD1a+ CD8 (A) and CD4 SP  (B) postnatal thymocytes. Thymocytes  were depleted with magnetic beads for  >97% of CD4 or CD8 positive cells.  CD4− cells were labeled with CD8  FITC, CD1a PE, and CD3 TRC and  sorted from CD1a+ and CD1a−  CD3+CD8+ SP cells (A). Likewise,  CD1a+ and CD1a− CD4+ SP cells  were sorted from CD8− cells stained  with CD4 FITC, CD1a PE, and CD3  TRC (B).
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Figure 2: RAG-1 expression of total unseparated thymocytes and CD1a− and CD1a+ CD8 (A) and CD4 SP (B) postnatal thymocytes. Thymocytes were depleted with magnetic beads for >97% of CD4 or CD8 positive cells. CD4− cells were labeled with CD8 FITC, CD1a PE, and CD3 TRC and sorted from CD1a+ and CD1a− CD3+CD8+ SP cells (A). Likewise, CD1a+ and CD1a− CD4+ SP cells were sorted from CD8− cells stained with CD4 FITC, CD1a PE, and CD3 TRC (B).

Mentions: The results of the limiting dilution experiments indicate that CD1a+CD3high SP thymocytes are functionally immature. The observation that the great majority of the CD3high cells in the thymus express the activation marker CD69, which is induced after positive selection (26–28) suggests, however, that positive selection signals have been delivered to a significant proportion of the CD1a+ SP cells. To further substantiate whether the CD1a+ SP cells have been submitted to selection signals, we investigated not only expression of CD69, but also Bcl-2 which is associated with positive selection as well (29, 30). In addition, expression of CD27 was analyzed. CD27 is expressed on most CD3high human thymocytes, and may also be associated with positive selection (31). Three parameter analysis of CD1a+ SP cells confirms that the majority of these cells express CD69, Bcl-2, and CD27 (Table 2). These data are consistent with expression profiles of CD69 (25), Bcl-2 (32), and CD27 (31) on total human CD3high thymocytes published previously. Besides upregulation of CD69, positive selection also results in downregulation of RAG-1 (28, 33). A semi-quantitative reverse transcriptase-PCR of the CD1a+ and CD1a− SP populations revealed that CD8+CD1a+ cells still express levels of RAG-1 which are comparable to that of total thymocytes (Fig. 2 A). The levels of RAG mRNA in CD1a+ CD4+ SP cells, however, are much lower than that of total cells (Fig. 2 B). Taken together, these data can be interpreted to indicate that CD1a+ SP cells express some, but not all, features of cells that have received a TCR-mediated positive selection signal.


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)

RAG-1 expression of total  unseparated thymocytes and CD1a−  and CD1a+ CD8 (A) and CD4 SP  (B) postnatal thymocytes. Thymocytes  were depleted with magnetic beads for  >97% of CD4 or CD8 positive cells.  CD4− cells were labeled with CD8  FITC, CD1a PE, and CD3 TRC and  sorted from CD1a+ and CD1a−  CD3+CD8+ SP cells (A). Likewise,  CD1a+ and CD1a− CD4+ SP cells  were sorted from CD8− cells stained  with CD4 FITC, CD1a PE, and CD3  TRC (B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: RAG-1 expression of total unseparated thymocytes and CD1a− and CD1a+ CD8 (A) and CD4 SP (B) postnatal thymocytes. Thymocytes were depleted with magnetic beads for >97% of CD4 or CD8 positive cells. CD4− cells were labeled with CD8 FITC, CD1a PE, and CD3 TRC and sorted from CD1a+ and CD1a− CD3+CD8+ SP cells (A). Likewise, CD1a+ and CD1a− CD4+ SP cells were sorted from CD8− cells stained with CD4 FITC, CD1a PE, and CD3 TRC (B).
Mentions: The results of the limiting dilution experiments indicate that CD1a+CD3high SP thymocytes are functionally immature. The observation that the great majority of the CD3high cells in the thymus express the activation marker CD69, which is induced after positive selection (26–28) suggests, however, that positive selection signals have been delivered to a significant proportion of the CD1a+ SP cells. To further substantiate whether the CD1a+ SP cells have been submitted to selection signals, we investigated not only expression of CD69, but also Bcl-2 which is associated with positive selection as well (29, 30). In addition, expression of CD27 was analyzed. CD27 is expressed on most CD3high human thymocytes, and may also be associated with positive selection (31). Three parameter analysis of CD1a+ SP cells confirms that the majority of these cells express CD69, Bcl-2, and CD27 (Table 2). These data are consistent with expression profiles of CD69 (25), Bcl-2 (32), and CD27 (31) on total human CD3high thymocytes published previously. Besides upregulation of CD69, positive selection also results in downregulation of RAG-1 (28, 33). A semi-quantitative reverse transcriptase-PCR of the CD1a+ and CD1a− SP populations revealed that CD8+CD1a+ cells still express levels of RAG-1 which are comparable to that of total thymocytes (Fig. 2 A). The levels of RAG mRNA in CD1a+ CD4+ SP cells, however, are much lower than that of total cells (Fig. 2 B). Taken together, these data can be interpreted to indicate that CD1a+ SP cells express some, but not all, features of cells that have received a TCR-mediated positive selection signal.

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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Related in: MedlinePlus