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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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Expression of CD1a on CD4+CD8+DP, CD4+SP, and  CD8+SP postnatal thymocyte populations. Total postnatal thymocytes  were stained with CD1a FITC, CD8 PE, and CD4 TRC. The dot plots  show the pattern of CD4 against CD8 staining of total thymocytes gated  on CD1a− (R1) and CD1a+ (R2) thymocytes.
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Figure 1: Expression of CD1a on CD4+CD8+DP, CD4+SP, and CD8+SP postnatal thymocyte populations. Total postnatal thymocytes were stained with CD1a FITC, CD8 PE, and CD4 TRC. The dot plots show the pattern of CD4 against CD8 staining of total thymocytes gated on CD1a− (R1) and CD1a+ (R2) thymocytes.

Mentions: CD1a is a marker that is expressed on the great majority of DP thymocytes and part of the SP cells (25). Since this marker is not present on mature peripheral T cells, it is generally assumed that thymic emigrants are CD1a−, and that therefore, CD1a+ thymocytes are immature. Since a proportion of the SP cells is CD1a+, a linear model of differentiation predicts that virtually all DP cells would be CD1a+. A nonlinear differentiation model, however, would predict existence of CD1a− cells among both DP and SP thymic populations. To examine this issue, we performed three parameter flow cytometric analysis of CD1a, CD4, and CD8, which confirmed earlier data that the vast majority of DP cells, and around 40% of the SP cells, express CD1a (Fig. 1). A very small percentage of the DP cells, however, is clearly negative for CD1a. Interestingly, the levels of CD4 and CD8 on the CD1a− DP cells are lower than on total DP thymocytes (Fig. 1), suggesting that downregulation of either one of these coreceptors had already been initiated before completion of CD1a downregulation. To address whether the CD1a− cells are functionally mature, we performed a limiting dilution of CD1a− and CD1a+ cells. Table 1 shows that the cloning efficiencies of CD1a− DP, CD4+, and CD8+ SP thymocytes in one representative experiment were 24, 41, and 34%, respectively. In sharp contrast, virtually none of the CD1a+ DP or CD1a+ SP cells could be cloned. The lack of clonogenic potential of CD1a+ subsets was not due to an inhibiting effect of the anti-CD1a antibody, since cloning efficiencies of unseparated SP cells plated in presence or absence of anti-CD1a mAb were virtually identical (results not shown). The in vitro expandable DP thymocytes could be cloned, and the majority of the DP clones maintained their DP phenotype for a prolonged period of time (results not shown) which is consistent with data published previously (13, 14). These data conclusively demonstrate that the clonogenic potential of SP and DP thymocytes resides exclusively in the CD1a− subset, and that functional maturation, as defined by the ability to clonally expand, can already be manifested at the DP stage of thymocyte maturation.


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)

Expression of CD1a on CD4+CD8+DP, CD4+SP, and  CD8+SP postnatal thymocyte populations. Total postnatal thymocytes  were stained with CD1a FITC, CD8 PE, and CD4 TRC. The dot plots  show the pattern of CD4 against CD8 staining of total thymocytes gated  on CD1a− (R1) and CD1a+ (R2) thymocytes.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Expression of CD1a on CD4+CD8+DP, CD4+SP, and CD8+SP postnatal thymocyte populations. Total postnatal thymocytes were stained with CD1a FITC, CD8 PE, and CD4 TRC. The dot plots show the pattern of CD4 against CD8 staining of total thymocytes gated on CD1a− (R1) and CD1a+ (R2) thymocytes.
Mentions: CD1a is a marker that is expressed on the great majority of DP thymocytes and part of the SP cells (25). Since this marker is not present on mature peripheral T cells, it is generally assumed that thymic emigrants are CD1a−, and that therefore, CD1a+ thymocytes are immature. Since a proportion of the SP cells is CD1a+, a linear model of differentiation predicts that virtually all DP cells would be CD1a+. A nonlinear differentiation model, however, would predict existence of CD1a− cells among both DP and SP thymic populations. To examine this issue, we performed three parameter flow cytometric analysis of CD1a, CD4, and CD8, which confirmed earlier data that the vast majority of DP cells, and around 40% of the SP cells, express CD1a (Fig. 1). A very small percentage of the DP cells, however, is clearly negative for CD1a. Interestingly, the levels of CD4 and CD8 on the CD1a− DP cells are lower than on total DP thymocytes (Fig. 1), suggesting that downregulation of either one of these coreceptors had already been initiated before completion of CD1a downregulation. To address whether the CD1a− cells are functionally mature, we performed a limiting dilution of CD1a− and CD1a+ cells. Table 1 shows that the cloning efficiencies of CD1a− DP, CD4+, and CD8+ SP thymocytes in one representative experiment were 24, 41, and 34%, respectively. In sharp contrast, virtually none of the CD1a+ DP or CD1a+ SP cells could be cloned. The lack of clonogenic potential of CD1a+ subsets was not due to an inhibiting effect of the anti-CD1a antibody, since cloning efficiencies of unseparated SP cells plated in presence or absence of anti-CD1a mAb were virtually identical (results not shown). The in vitro expandable DP thymocytes could be cloned, and the majority of the DP clones maintained their DP phenotype for a prolonged period of time (results not shown) which is consistent with data published previously (13, 14). These data conclusively demonstrate that the clonogenic potential of SP and DP thymocytes resides exclusively in the CD1a− subset, and that functional maturation, as defined by the ability to clonally expand, can already be manifested at the DP stage of thymocyte maturation.

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