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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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Immunofluorescence staining of B6 (a and b), B6-Eαd (c and d), and B6CD11c-Eαd (e and f ) thymus with the medullary epithelial marker UEA-1  labeled with FITC (a, c, and e) or double immunofluorescence analysis with UEA-1–FITC plus 14.4.4S specific for I-E (biotinylated and detected with  Avidin-Texas red) (b, d, and f ). The B6 thymus shows only green labeling (a and b) indicating the natural absence of I-E expression in this mouse strain.  The B6-Eαd thymus (c and d) expresses both antigens in the wild-type pattern and the green UEA-1 positive cells (c) become yellow-orange in the double  fluorescence filter (d). In addition, red single positive cells become evident and are most likely nonepithelial cells expressing the I-E transgene as well as  UEA-1 weak positive cell processes of epithelial cells (36). In the B6CD11c-Eαd thymus (e and f ), both mAbs clearly stain two different cell types and no  I-E staining is visible on the UEA-1–labeled (green single positives) medullary epithelial cells ( f ), while other nonepithelial I-E positive cells (red single positives) of dendritic shape become evident in the medulla (    f   ).
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Figure 4: Immunofluorescence staining of B6 (a and b), B6-Eαd (c and d), and B6CD11c-Eαd (e and f ) thymus with the medullary epithelial marker UEA-1 labeled with FITC (a, c, and e) or double immunofluorescence analysis with UEA-1–FITC plus 14.4.4S specific for I-E (biotinylated and detected with Avidin-Texas red) (b, d, and f ). The B6 thymus shows only green labeling (a and b) indicating the natural absence of I-E expression in this mouse strain. The B6-Eαd thymus (c and d) expresses both antigens in the wild-type pattern and the green UEA-1 positive cells (c) become yellow-orange in the double fluorescence filter (d). In addition, red single positive cells become evident and are most likely nonepithelial cells expressing the I-E transgene as well as UEA-1 weak positive cell processes of epithelial cells (36). In the B6CD11c-Eαd thymus (e and f ), both mAbs clearly stain two different cell types and no I-E staining is visible on the UEA-1–labeled (green single positives) medullary epithelial cells ( f ), while other nonepithelial I-E positive cells (red single positives) of dendritic shape become evident in the medulla (    f   ).

Mentions: To discriminate I-E expression between medullary epithelial cells and thymic DC we carried out double immunofluorescence analysis looking at the expression of the medullary epithelial marker UEA-1 alone (Fig. 4, a, c, and e) or its colocalization with the mAb specific for the transgenic I-E, 14.4.4S (Fig. 4, b, d, and f  ). UEA-1 positive medullary epithelial cells were stained green, while the cells expressing the I-E transgene stained red after detection with biotinylated mAb 14.4.4S plus avidin-TxR. While in the B6 thymus, the medulla does not show I-E expression (Fig. 4, a and b), and the B6-Eαd medullary areas do express I-E (Fig. 4 d). In this case, the UEA-1–positive medullary epithelial cells (Fig. 4 c, green staining) coexpress both markers and consequently appear orange in the double fluorescence analysis (Fig. 4 d). Since UEA-1 produces very dense punctate stainings of the cell center but very weak stainings of the cell processes (36), only the central areas of epithelial cells appear orange and the cell processes of these cells appear red single positive (Fig. 4 d). In contrast, the UEA-1–positive medullary epithelial cells of the B6CD11c-Eαd thymus do not express I-E and only stain green (Fig. 4, e and f  ), while red single positive cells of typical dendritic shape (extremely large elongated cells with long cell processes) express transgenic I-E (Fig. 4 f  ). An identical pattern has been revealed with MTS-10 (27), another mAb specific for medullary epithelial cells (data not shown) and confirmed the absence of transgenic I-E on medullary epithelial cells.


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)

Immunofluorescence staining of B6 (a and b), B6-Eαd (c and d), and B6CD11c-Eαd (e and f ) thymus with the medullary epithelial marker UEA-1  labeled with FITC (a, c, and e) or double immunofluorescence analysis with UEA-1–FITC plus 14.4.4S specific for I-E (biotinylated and detected with  Avidin-Texas red) (b, d, and f ). The B6 thymus shows only green labeling (a and b) indicating the natural absence of I-E expression in this mouse strain.  The B6-Eαd thymus (c and d) expresses both antigens in the wild-type pattern and the green UEA-1 positive cells (c) become yellow-orange in the double  fluorescence filter (d). In addition, red single positive cells become evident and are most likely nonepithelial cells expressing the I-E transgene as well as  UEA-1 weak positive cell processes of epithelial cells (36). In the B6CD11c-Eαd thymus (e and f ), both mAbs clearly stain two different cell types and no  I-E staining is visible on the UEA-1–labeled (green single positives) medullary epithelial cells ( f ), while other nonepithelial I-E positive cells (red single positives) of dendritic shape become evident in the medulla (    f   ).
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Figure 4: Immunofluorescence staining of B6 (a and b), B6-Eαd (c and d), and B6CD11c-Eαd (e and f ) thymus with the medullary epithelial marker UEA-1 labeled with FITC (a, c, and e) or double immunofluorescence analysis with UEA-1–FITC plus 14.4.4S specific for I-E (biotinylated and detected with Avidin-Texas red) (b, d, and f ). The B6 thymus shows only green labeling (a and b) indicating the natural absence of I-E expression in this mouse strain. The B6-Eαd thymus (c and d) expresses both antigens in the wild-type pattern and the green UEA-1 positive cells (c) become yellow-orange in the double fluorescence filter (d). In addition, red single positive cells become evident and are most likely nonepithelial cells expressing the I-E transgene as well as UEA-1 weak positive cell processes of epithelial cells (36). In the B6CD11c-Eαd thymus (e and f ), both mAbs clearly stain two different cell types and no I-E staining is visible on the UEA-1–labeled (green single positives) medullary epithelial cells ( f ), while other nonepithelial I-E positive cells (red single positives) of dendritic shape become evident in the medulla (    f   ).
Mentions: To discriminate I-E expression between medullary epithelial cells and thymic DC we carried out double immunofluorescence analysis looking at the expression of the medullary epithelial marker UEA-1 alone (Fig. 4, a, c, and e) or its colocalization with the mAb specific for the transgenic I-E, 14.4.4S (Fig. 4, b, d, and f  ). UEA-1 positive medullary epithelial cells were stained green, while the cells expressing the I-E transgene stained red after detection with biotinylated mAb 14.4.4S plus avidin-TxR. While in the B6 thymus, the medulla does not show I-E expression (Fig. 4, a and b), and the B6-Eαd medullary areas do express I-E (Fig. 4 d). In this case, the UEA-1–positive medullary epithelial cells (Fig. 4 c, green staining) coexpress both markers and consequently appear orange in the double fluorescence analysis (Fig. 4 d). Since UEA-1 produces very dense punctate stainings of the cell center but very weak stainings of the cell processes (36), only the central areas of epithelial cells appear orange and the cell processes of these cells appear red single positive (Fig. 4 d). In contrast, the UEA-1–positive medullary epithelial cells of the B6CD11c-Eαd thymus do not express I-E and only stain green (Fig. 4, e and f  ), while red single positive cells of typical dendritic shape (extremely large elongated cells with long cell processes) express transgenic I-E (Fig. 4 f  ). An identical pattern has been revealed with MTS-10 (27), another mAb specific for medullary epithelial cells (data not shown) and confirmed the absence of transgenic I-E on medullary epithelial cells.

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
Related in: MedlinePlus