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Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo.

Brocker T, Riedinger M, Karjalainen K - J. Exp. Med. (1997)

Bottom Line: Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types.In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed.Thus negative, but not positive, selection events can be induced by DC in vivo.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, Switzerland.

ABSTRACT
It is well established that lymphoid dendritic cells (DC) play an important role in the immune system. Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II+ "interdigitating cells" in the thymus during thymocyte development. Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments. The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors. Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling. Here we present a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4+ T cells, and to a less complete extent, CD8+ T cells. In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed. Thus negative, but not positive, selection events can be induced by DC in vivo.

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Restriction map of the transgenic construct CD11c-Eαd.  MHC class II I-Eαd cDNA (striped box) was placed under the control of a  mouse CD11c promoter–containing DNA segment (gray box). The rabbit  β-globin gene fragment providing the cDNA with an intron and a polyadenylation signal (A(n)) is displayed as a white box. B, BamHI; Bs, BspHI;  E, EcoRI; N, NotI; S, SstI; X, XhoI; restriction sites in parentheses have  been destroyed by blunt-end cloning.
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Figure 1: Restriction map of the transgenic construct CD11c-Eαd. MHC class II I-Eαd cDNA (striped box) was placed under the control of a mouse CD11c promoter–containing DNA segment (gray box). The rabbit β-globin gene fragment providing the cDNA with an intron and a polyadenylation signal (A(n)) is displayed as a white box. B, BamHI; Bs, BspHI; E, EcoRI; N, NotI; S, SstI; X, XhoI; restriction sites in parentheses have been destroyed by blunt-end cloning.

Mentions: To isolate the regulatory elements controlling the expression of CD11c in mice, we screened a mouse genomic library with a probe corresponding to human CD11c exon 7 to 11 (see Materials and Methods). The clone we isolated was partially analyzed by DNA sequencing and showed a high homology to the published (24, 30) human CD11c genomic sequences (data not shown). We isolated a 5-kb fragment of the 5′ region that we expected to contain the promoter region (see Materials and Methods) and used it as the controlling element to express the Eαd cDNA in C57Bl/6 mice (Fig. 1). Two founder lines were obtained and the transgene expression was monitored with 14.4.4S, a mAb specific for the α chain of the I-E MHC class II antigen (31). Thereafter, these mice are referred to as “B6CD11c-Eαd.”


Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo.

Brocker T, Riedinger M, Karjalainen K - J. Exp. Med. (1997)

Restriction map of the transgenic construct CD11c-Eαd.  MHC class II I-Eαd cDNA (striped box) was placed under the control of a  mouse CD11c promoter–containing DNA segment (gray box). The rabbit  β-globin gene fragment providing the cDNA with an intron and a polyadenylation signal (A(n)) is displayed as a white box. B, BamHI; Bs, BspHI;  E, EcoRI; N, NotI; S, SstI; X, XhoI; restriction sites in parentheses have  been destroyed by blunt-end cloning.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196043&req=5

Figure 1: Restriction map of the transgenic construct CD11c-Eαd. MHC class II I-Eαd cDNA (striped box) was placed under the control of a mouse CD11c promoter–containing DNA segment (gray box). The rabbit β-globin gene fragment providing the cDNA with an intron and a polyadenylation signal (A(n)) is displayed as a white box. B, BamHI; Bs, BspHI; E, EcoRI; N, NotI; S, SstI; X, XhoI; restriction sites in parentheses have been destroyed by blunt-end cloning.
Mentions: To isolate the regulatory elements controlling the expression of CD11c in mice, we screened a mouse genomic library with a probe corresponding to human CD11c exon 7 to 11 (see Materials and Methods). The clone we isolated was partially analyzed by DNA sequencing and showed a high homology to the published (24, 30) human CD11c genomic sequences (data not shown). We isolated a 5-kb fragment of the 5′ region that we expected to contain the promoter region (see Materials and Methods) and used it as the controlling element to express the Eαd cDNA in C57Bl/6 mice (Fig. 1). Two founder lines were obtained and the transgene expression was monitored with 14.4.4S, a mAb specific for the α chain of the I-E MHC class II antigen (31). Thereafter, these mice are referred to as “B6CD11c-Eαd.”

Bottom Line: Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types.In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed.Thus negative, but not positive, selection events can be induced by DC in vivo.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, Switzerland.

ABSTRACT
It is well established that lymphoid dendritic cells (DC) play an important role in the immune system. Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II+ "interdigitating cells" in the thymus during thymocyte development. Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments. The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors. Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling. Here we present a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4+ T cells, and to a less complete extent, CD8+ T cells. In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed. Thus negative, but not positive, selection events can be induced by DC in vivo.

Show MeSH