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Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.

Heemskerk MH, Blom B, Nolan G, Stegmann AP, Bakker AQ, Weijer K, Res PC, Spits H - J. Exp. Med. (1997)

Bottom Line: The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer.In contrast, development into NK cells in an FTOC is enhanced.Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

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CD56+ cells generated in the FTOC from Id3 transduced CD34+ fetal liver cells are  NK cells. (a) Id3- and control-transduced cells, incubated in an  FTOC, were harvested 4 wk  later, and expanded with a feeder  cell mixture. Percentages of GFP+  cells were 30% for the Id3- and  40% for the control-transduced  cells. (b) GFP+ cells of the Id3-transduced culture were isolated  by sorting with a FACStar plus®  and tested for cytotoxic activity  against NK-sensitive K562 cells  and NK-insensitive EBV 225  cells in a 51Cr–release assay.
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Figure 2: CD56+ cells generated in the FTOC from Id3 transduced CD34+ fetal liver cells are NK cells. (a) Id3- and control-transduced cells, incubated in an FTOC, were harvested 4 wk later, and expanded with a feeder cell mixture. Percentages of GFP+ cells were 30% for the Id3- and 40% for the control-transduced cells. (b) GFP+ cells of the Id3-transduced culture were isolated by sorting with a FACStar plus® and tested for cytotoxic activity against NK-sensitive K562 cells and NK-insensitive EBV 225 cells in a 51Cr–release assay.

Mentions: To study the role of bHLH factors in T cell development, we overexpressed the dominant negative HLH protein Id3 in T cell progenitors and monitored the fate of the transduced cells. Id3 has a very broad tissue distribution and CD34+ fetal liver and thymic progenitor cells express Id3 messenger RNA as determined by reverse transcription PCR (results not shown). Purified CD34+ fetal liver cells were cultured with a combination of SCF and IL-7 for 24 h. The cells were then transduced by overnight coculture with supernatants of packaging cells producing recombinant viruses harboring either Id3-IRES-GFP or IRES-GFP, and cultured in an FTOC for 4 wk (22). The flow cytometric analysis shown in Fig. 1 demonstrates that the GFP marker was transferred to 23% of the progeny of the IRES-GFP and 13% of the Id3-IRES-GFP–transduced progenitor cells. The patterns of CD3, CD4, and CD5 stainings of the GFP+ cells from the IRES-GFP–transduced cells are identical to that of the nontransduced GFP− cells (Fig. 1). The great majority of the GFP+ cells express CD1a, and very few CD56+ cells were present in these samples. By contrast, cells harvested from the FTOC with Id3-IRES-GFP–transduced fetal liver cells did not express CD1a, and 32% of these cells were positive for the NK cell marker CD56. Almost no CD3+ cells were observed (Fig. 1). Not only the proportions of CD56+ cells were different, but also the absolute numbers since >30-fold more GFP+CD56+ cells were found in the FTOC, populated with Id3-transduced CD34+ cells. These data indicate that CD34+ fetal liver cells, overexpressing Id3, develop preferentially into CD3−CD56+ cells in the FTOC. It was important to ensure that these GFP+CD3−CD56+ cells represent functional NK cells. To obtain enough cells for testing their cytolytic activity, we cultured the cells harvested from the FTOC with a feeder mixture consisting of irradiated PBMCs, the EBV cell line JY, PHA and IL-2. Under these conditions we can expand NK cells and TCR-γ/δ, but not TCR-α/β cells from an FTOC (24); the failure to obtain TCR-α/β cells is because the mouse thymic environment is unable to induce functional maturation in these cells (24). Fig. 2 demonstrates that the GFP+ cells expanded from the control FTOC contained GPF+ TCR γ/δ+ cells and a few CD56+CD3− cells. By contrast, GFP+ cells expanded from the FTOC with the Id3+ progenitor cells were CD56+ and did not express CD3, underscoring the strong inhibitory effect of Id3 on generation of TCR-γ/δ+ cells. The GFP+CD3−CD56+ cells were sorted from the Id3-IRES-GFP cultures and were shown to be highly cytotoxic for the NK sensitive target cell K562, but not for an NK-resistant cell line (Fig. 2 b). These results confirm that the CD56+CD3−GFP+ cells observed in these FTOC are functional NK cells.


Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.

Heemskerk MH, Blom B, Nolan G, Stegmann AP, Bakker AQ, Weijer K, Res PC, Spits H - J. Exp. Med. (1997)

CD56+ cells generated in the FTOC from Id3 transduced CD34+ fetal liver cells are  NK cells. (a) Id3- and control-transduced cells, incubated in an  FTOC, were harvested 4 wk  later, and expanded with a feeder  cell mixture. Percentages of GFP+  cells were 30% for the Id3- and  40% for the control-transduced  cells. (b) GFP+ cells of the Id3-transduced culture were isolated  by sorting with a FACStar plus®  and tested for cytotoxic activity  against NK-sensitive K562 cells  and NK-insensitive EBV 225  cells in a 51Cr–release assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199115&req=5

Figure 2: CD56+ cells generated in the FTOC from Id3 transduced CD34+ fetal liver cells are NK cells. (a) Id3- and control-transduced cells, incubated in an FTOC, were harvested 4 wk later, and expanded with a feeder cell mixture. Percentages of GFP+ cells were 30% for the Id3- and 40% for the control-transduced cells. (b) GFP+ cells of the Id3-transduced culture were isolated by sorting with a FACStar plus® and tested for cytotoxic activity against NK-sensitive K562 cells and NK-insensitive EBV 225 cells in a 51Cr–release assay.
Mentions: To study the role of bHLH factors in T cell development, we overexpressed the dominant negative HLH protein Id3 in T cell progenitors and monitored the fate of the transduced cells. Id3 has a very broad tissue distribution and CD34+ fetal liver and thymic progenitor cells express Id3 messenger RNA as determined by reverse transcription PCR (results not shown). Purified CD34+ fetal liver cells were cultured with a combination of SCF and IL-7 for 24 h. The cells were then transduced by overnight coculture with supernatants of packaging cells producing recombinant viruses harboring either Id3-IRES-GFP or IRES-GFP, and cultured in an FTOC for 4 wk (22). The flow cytometric analysis shown in Fig. 1 demonstrates that the GFP marker was transferred to 23% of the progeny of the IRES-GFP and 13% of the Id3-IRES-GFP–transduced progenitor cells. The patterns of CD3, CD4, and CD5 stainings of the GFP+ cells from the IRES-GFP–transduced cells are identical to that of the nontransduced GFP− cells (Fig. 1). The great majority of the GFP+ cells express CD1a, and very few CD56+ cells were present in these samples. By contrast, cells harvested from the FTOC with Id3-IRES-GFP–transduced fetal liver cells did not express CD1a, and 32% of these cells were positive for the NK cell marker CD56. Almost no CD3+ cells were observed (Fig. 1). Not only the proportions of CD56+ cells were different, but also the absolute numbers since >30-fold more GFP+CD56+ cells were found in the FTOC, populated with Id3-transduced CD34+ cells. These data indicate that CD34+ fetal liver cells, overexpressing Id3, develop preferentially into CD3−CD56+ cells in the FTOC. It was important to ensure that these GFP+CD3−CD56+ cells represent functional NK cells. To obtain enough cells for testing their cytolytic activity, we cultured the cells harvested from the FTOC with a feeder mixture consisting of irradiated PBMCs, the EBV cell line JY, PHA and IL-2. Under these conditions we can expand NK cells and TCR-γ/δ, but not TCR-α/β cells from an FTOC (24); the failure to obtain TCR-α/β cells is because the mouse thymic environment is unable to induce functional maturation in these cells (24). Fig. 2 demonstrates that the GFP+ cells expanded from the control FTOC contained GPF+ TCR γ/δ+ cells and a few CD56+CD3− cells. By contrast, GFP+ cells expanded from the FTOC with the Id3+ progenitor cells were CD56+ and did not express CD3, underscoring the strong inhibitory effect of Id3 on generation of TCR-γ/δ+ cells. The GFP+CD3−CD56+ cells were sorted from the Id3-IRES-GFP cultures and were shown to be highly cytotoxic for the NK sensitive target cell K562, but not for an NK-resistant cell line (Fig. 2 b). These results confirm that the CD56+CD3−GFP+ cells observed in these FTOC are functional NK cells.

Bottom Line: The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer.In contrast, development into NK cells in an FTOC is enhanced.Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

Show MeSH
Related in: MedlinePlus