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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: 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.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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Enforced expression of Id3 in CD34+CD1a−  thymocytes inhibits induction of  D-Jβ rearrangements after incubation in IL-7 and SCF. CD1a−  and CD1a+, CD34+ cells were  isolated, transduced, and incubated for 5 d in IL-7 plus SCF. After the incubation, GFP+ and GFP− cells were isolated from both samples using a  FACStar®. Based on GFP expression, the purity of the sorted populations  was >99%. DNA was prepared after the sorting and analyzed for the  presence of D-Jβ rearrangements by PCR. The intensities of the hybridized band were determined with a phosphorimager (Fujix Bas 2000;  Raytest Benelux BV, Straubenherdt, FRG). The ratios of D-J/genomic  control were: lane 1, 0; lane 2, 0.0025; lane 3, 0; lane 4, 0.035; lane 5,  0.062; and lane 6, 0.028.
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Figure 5: Enforced expression of Id3 in CD34+CD1a− thymocytes inhibits induction of D-Jβ rearrangements after incubation in IL-7 and SCF. CD1a− and CD1a+, CD34+ cells were isolated, transduced, and incubated for 5 d in IL-7 plus SCF. After the incubation, GFP+ and GFP− cells were isolated from both samples using a FACStar®. Based on GFP expression, the purity of the sorted populations was >99%. DNA was prepared after the sorting and analyzed for the presence of D-Jβ rearrangements by PCR. The intensities of the hybridized band were determined with a phosphorimager (Fujix Bas 2000; Raytest Benelux BV, Straubenherdt, FRG). The ratios of D-J/genomic control were: lane 1, 0; lane 2, 0.0025; lane 3, 0; lane 4, 0.035; lane 5, 0.062; and lane 6, 0.028.

Mentions: T cells are defined by TCR gene rearrangements; in NK cells, these genes are in the germ line configuration (1). The fact that Id3 inhibits generation of CD3+ cells and stimulates that of NK cells, raises the possibility that bHLH factors are required for processes that result in TCR rearrangements. To test this, we cultured transduced CD1a−CD34+ and CD1a+CD34+ cells for 7 d in IL-7 and SCF, sorted GFP+ and GFP− cells, and analyzed for the presence of D-Jβ rearrangements with a sensitive PCR technique (25). Fig. 5 shows that Id3 completely blocked IL-7 and SCF-mediated induction of D-Jβ rearrangement in CD1a−CD34+ cells. One trivial explanation for this observation is that SCF and IL-7 induce growth of a very small population of contaminating CD1a+CD34+ cells that have already undergone D-Jβ rearrangements; overexpression of Id3 could inhibit the growth of these contaminating cells. This is unlikely because D-Jβ rearrangements were still detectable in Id3-transduced CD1a+CD34+ cells recovered after 7 d of culture (Fig. 5). The ratios of the intensities of the bands hybridized with the D-Jβ and genomic DNA control (RAG-2) probes in the starting CD1a+ CD34+ (lane 4) and in the Id3-CD1a+CD34+ (lane 6) populations were the same. This ratio is increased in the untransduced CD1a+CD34+ cells. These data indicate that Id3 inhibits the IL-7 and SCF-mediated increase DJβ rearrangements in CD1a+CD34+ cells. It appears, therefore, that Id3 overexpression inhibits induction of D-Jβ rearrangement. It remains to be determined whether the rearrangement process itself is blocked or whether an earlier step is inhibited. Our results raise the possibility that the SCF receptor (ckit) and/or the IL-7R communicate with bHLH factors as suggested previously (14).


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)

Enforced expression of Id3 in CD34+CD1a−  thymocytes inhibits induction of  D-Jβ rearrangements after incubation in IL-7 and SCF. CD1a−  and CD1a+, CD34+ cells were  isolated, transduced, and incubated for 5 d in IL-7 plus SCF. After the incubation, GFP+ and GFP− cells were isolated from both samples using a  FACStar®. Based on GFP expression, the purity of the sorted populations  was >99%. DNA was prepared after the sorting and analyzed for the  presence of D-Jβ rearrangements by PCR. The intensities of the hybridized band were determined with a phosphorimager (Fujix Bas 2000;  Raytest Benelux BV, Straubenherdt, FRG). The ratios of D-J/genomic  control were: lane 1, 0; lane 2, 0.0025; lane 3, 0; lane 4, 0.035; lane 5,  0.062; and lane 6, 0.028.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199115&req=5

Figure 5: Enforced expression of Id3 in CD34+CD1a− thymocytes inhibits induction of D-Jβ rearrangements after incubation in IL-7 and SCF. CD1a− and CD1a+, CD34+ cells were isolated, transduced, and incubated for 5 d in IL-7 plus SCF. After the incubation, GFP+ and GFP− cells were isolated from both samples using a FACStar®. Based on GFP expression, the purity of the sorted populations was >99%. DNA was prepared after the sorting and analyzed for the presence of D-Jβ rearrangements by PCR. The intensities of the hybridized band were determined with a phosphorimager (Fujix Bas 2000; Raytest Benelux BV, Straubenherdt, FRG). The ratios of D-J/genomic control were: lane 1, 0; lane 2, 0.0025; lane 3, 0; lane 4, 0.035; lane 5, 0.062; and lane 6, 0.028.
Mentions: T cells are defined by TCR gene rearrangements; in NK cells, these genes are in the germ line configuration (1). The fact that Id3 inhibits generation of CD3+ cells and stimulates that of NK cells, raises the possibility that bHLH factors are required for processes that result in TCR rearrangements. To test this, we cultured transduced CD1a−CD34+ and CD1a+CD34+ cells for 7 d in IL-7 and SCF, sorted GFP+ and GFP− cells, and analyzed for the presence of D-Jβ rearrangements with a sensitive PCR technique (25). Fig. 5 shows that Id3 completely blocked IL-7 and SCF-mediated induction of D-Jβ rearrangement in CD1a−CD34+ cells. One trivial explanation for this observation is that SCF and IL-7 induce growth of a very small population of contaminating CD1a+CD34+ cells that have already undergone D-Jβ rearrangements; overexpression of Id3 could inhibit the growth of these contaminating cells. This is unlikely because D-Jβ rearrangements were still detectable in Id3-transduced CD1a+CD34+ cells recovered after 7 d of culture (Fig. 5). The ratios of the intensities of the bands hybridized with the D-Jβ and genomic DNA control (RAG-2) probes in the starting CD1a+ CD34+ (lane 4) and in the Id3-CD1a+CD34+ (lane 6) populations were the same. This ratio is increased in the untransduced CD1a+CD34+ cells. These data indicate that Id3 inhibits the IL-7 and SCF-mediated increase DJβ rearrangements in CD1a+CD34+ cells. It appears, therefore, that Id3 overexpression inhibits induction of D-Jβ rearrangement. It remains to be determined whether the rearrangement process itself is blocked or whether an earlier step is inhibited. Our results raise the possibility that the SCF receptor (ckit) and/or the IL-7R communicate with bHLH factors as suggested previously (14).

Bottom Line: 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.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