Limits...
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.

Show MeSH

Related in: MedlinePlus

Multiparameter analysis of cells harvested from one FTOC  with thymi of MHC class II–positive mice (A and D) and two FTOC  with MHC class II–deficient mice (B and C). FTOC were set up as indicated in Materials and Methods with 25,000 CD34+CD1a− postnatal thymocytes per lobe and incubated for 4 wk. Cell numbers harvested from  the FTOC cultures were 150,000 cells per lobe in the culture with MHC  class II–positive thymic lobes (A and D), and 175,000 (B) and 100,000  cells (C) per lobe in the cultures with MHC class II–negative thymic  lobes. Cells harvested from the FTOC cultures were stained for threecolor analysis with CD4 FITC, TCR αβ PE, and CD8 TRC. Human  cells were gated on the basis of forward and side scatter profile; all cells  within this gate were positive for human CD45. CD4 against CD8 staining is indicated as dot plots. The histograms show the expression of TCR  αβ on the CD4+CD8− and CD8+CD4− thymocytes. Numbers in histograms represent the percentages of CD4+ or CD8+ TCR αβ+ SP cells in  the total population of human cells derived from the FTOC.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196108&req=5

Figure 3: Multiparameter analysis of cells harvested from one FTOC with thymi of MHC class II–positive mice (A and D) and two FTOC with MHC class II–deficient mice (B and C). FTOC were set up as indicated in Materials and Methods with 25,000 CD34+CD1a− postnatal thymocytes per lobe and incubated for 4 wk. Cell numbers harvested from the FTOC cultures were 150,000 cells per lobe in the culture with MHC class II–positive thymic lobes (A and D), and 175,000 (B) and 100,000 cells (C) per lobe in the cultures with MHC class II–negative thymic lobes. Cells harvested from the FTOC cultures were stained for threecolor analysis with CD4 FITC, TCR αβ PE, and CD8 TRC. Human cells were gated on the basis of forward and side scatter profile; all cells within this gate were positive for human CD45. CD4 against CD8 staining is indicated as dot plots. The histograms show the expression of TCR αβ on the CD4+CD8− and CD8+CD4− thymocytes. Numbers in histograms represent the percentages of CD4+ or CD8+ TCR αβ+ SP cells in the total population of human cells derived from the FTOC.

Mentions: Recently it was demonstrated that human progenitor cells can develop in mouse thymic organs in a FTOC (18, 34–37). Human progenitor cells developed into SP cells, but human stromal cells were not detectable in such cultures (36). Human CD4 can replace mouse CD4 in development of mouse MHC class II–restricted T cells (38). To address the question of whether interaction of human CD4 with mouse MHC class II can select human CD4+ T cells, FTOC were performed with thymi from MHC class II–positive and MHC class II–deficient mice. The mouse thymi were reconstituted with CD34+CD1a− postnatal thymocytes that include the most primitive thymic T cell precursors (39, 40). After incubation in a MHC class II–positive murine thymic microenvironment, 6.5% of the harvested cells were TCR αβ+ CD4+ SP (Fig. 3 A). By contrast, the number of TCRαβ+CD4+ SP T cells recovered from thymi of MHC class II–deficient mice was reduced considerably to 0.46% in experiment 1 (Fig. 3 B) and 0.05% in experiment 2 (Fig. 3 C), compared to that recovered from thymi of MHC class II–positive mice (6–10% in four independent experiments). A significant portion of the TCRαβ+ cells that developed in a class II MHC–positive thymic environment expressed CD69 (Fig. 3 D), indicating that some cells were activated, presumably as a consequence of selection via the TCR. These findings indicate that mouse MHC class II antigens can positively select human CD4+ cells. It is noteworthy that very few human CD8+TCRαβ+ SP cells could be recovered from the FTOC with human CD34+CD1a− cells and the mouse thymi. There were more CD3+CD8+ SP cells present and >90% of those cells express TCR γδ (results not shown). One possible reason for this is that mouse MHC class I does not efficiently select human CD8+ T cells, despite the fact that human CD8 is able to functionally interact with the α3 domain of mouse H2Kb (41). Another explanation is that in addition to class I MHC, other signals are required for selection of CD8+ T 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)

Multiparameter analysis of cells harvested from one FTOC  with thymi of MHC class II–positive mice (A and D) and two FTOC  with MHC class II–deficient mice (B and C). FTOC were set up as indicated in Materials and Methods with 25,000 CD34+CD1a− postnatal thymocytes per lobe and incubated for 4 wk. Cell numbers harvested from  the FTOC cultures were 150,000 cells per lobe in the culture with MHC  class II–positive thymic lobes (A and D), and 175,000 (B) and 100,000  cells (C) per lobe in the cultures with MHC class II–negative thymic  lobes. Cells harvested from the FTOC cultures were stained for threecolor analysis with CD4 FITC, TCR αβ PE, and CD8 TRC. Human  cells were gated on the basis of forward and side scatter profile; all cells  within this gate were positive for human CD45. CD4 against CD8 staining is indicated as dot plots. The histograms show the expression of TCR  αβ on the CD4+CD8− and CD8+CD4− thymocytes. Numbers in histograms represent the percentages of CD4+ or CD8+ TCR αβ+ SP cells in  the total population of human cells derived from the FTOC.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Multiparameter analysis of cells harvested from one FTOC with thymi of MHC class II–positive mice (A and D) and two FTOC with MHC class II–deficient mice (B and C). FTOC were set up as indicated in Materials and Methods with 25,000 CD34+CD1a− postnatal thymocytes per lobe and incubated for 4 wk. Cell numbers harvested from the FTOC cultures were 150,000 cells per lobe in the culture with MHC class II–positive thymic lobes (A and D), and 175,000 (B) and 100,000 cells (C) per lobe in the cultures with MHC class II–negative thymic lobes. Cells harvested from the FTOC cultures were stained for threecolor analysis with CD4 FITC, TCR αβ PE, and CD8 TRC. Human cells were gated on the basis of forward and side scatter profile; all cells within this gate were positive for human CD45. CD4 against CD8 staining is indicated as dot plots. The histograms show the expression of TCR αβ on the CD4+CD8− and CD8+CD4− thymocytes. Numbers in histograms represent the percentages of CD4+ or CD8+ TCR αβ+ SP cells in the total population of human cells derived from the FTOC.
Mentions: Recently it was demonstrated that human progenitor cells can develop in mouse thymic organs in a FTOC (18, 34–37). Human progenitor cells developed into SP cells, but human stromal cells were not detectable in such cultures (36). Human CD4 can replace mouse CD4 in development of mouse MHC class II–restricted T cells (38). To address the question of whether interaction of human CD4 with mouse MHC class II can select human CD4+ T cells, FTOC were performed with thymi from MHC class II–positive and MHC class II–deficient mice. The mouse thymi were reconstituted with CD34+CD1a− postnatal thymocytes that include the most primitive thymic T cell precursors (39, 40). After incubation in a MHC class II–positive murine thymic microenvironment, 6.5% of the harvested cells were TCR αβ+ CD4+ SP (Fig. 3 A). By contrast, the number of TCRαβ+CD4+ SP T cells recovered from thymi of MHC class II–deficient mice was reduced considerably to 0.46% in experiment 1 (Fig. 3 B) and 0.05% in experiment 2 (Fig. 3 C), compared to that recovered from thymi of MHC class II–positive mice (6–10% in four independent experiments). A significant portion of the TCRαβ+ cells that developed in a class II MHC–positive thymic environment expressed CD69 (Fig. 3 D), indicating that some cells were activated, presumably as a consequence of selection via the TCR. These findings indicate that mouse MHC class II antigens can positively select human CD4+ cells. It is noteworthy that very few human CD8+TCRαβ+ SP cells could be recovered from the FTOC with human CD34+CD1a− cells and the mouse thymi. There were more CD3+CD8+ SP cells present and >90% of those cells express TCR γδ (results not shown). One possible reason for this is that mouse MHC class I does not efficiently select human CD8+ T cells, despite the fact that human CD8 is able to functionally interact with the α3 domain of mouse H2Kb (41). Another explanation is that in addition to class I MHC, other signals are required for selection of CD8+ T 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