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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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Expression of the  CD11c-Eαd transgene leads to I-E  expression on thymic DC, but  not on thymic B cells. DC:  Thymi of B6, B6-Eαd, and  B6CD11c-Eαd mice were collagenase digested and DC were  isolated after a low density gradient as described in Materials and  Methods. Cells were stained with  mAbs specific for CD11c (PE)  and I-E or I-A (FITC), respectively. B cells: thymi of the three  mouse types were complement  depleted for CD4 and CD8 to  enrich non–T cells (see Materials  and Methods) and then stained  with mAbs specific for the pan B  cell antigen CD19 (PE) and I-E  or I-A (FITC), respectively.
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Figure 5: Expression of the CD11c-Eαd transgene leads to I-E expression on thymic DC, but not on thymic B cells. DC: Thymi of B6, B6-Eαd, and B6CD11c-Eαd mice were collagenase digested and DC were isolated after a low density gradient as described in Materials and Methods. Cells were stained with mAbs specific for CD11c (PE) and I-E or I-A (FITC), respectively. B cells: thymi of the three mouse types were complement depleted for CD4 and CD8 to enrich non–T cells (see Materials and Methods) and then stained with mAbs specific for the pan B cell antigen CD19 (PE) and I-E or I-A (FITC), respectively.

Mentions: To further characterize the cells expressing the I-E transgene in the thymus, we isolated the light density cell fraction from collagenase-digested thymi as described earlier (29), and performed flow cytometric analysis. The major cell type obtained in the low buoyant density fraction from thymus were cells with relatively high forward scatter (FS) and intermediate side scatter (SS) signals (data not shown); in contrast, thymocytes (from the high density fraction) had both low FS and SS (data not shown). When the low density fraction was stained for CD11c expression, 80–90% of the cells fulfilling the above described FS/SS criteria were CD11cpositive (see Fig. 5, dendritic cells). While more than 60% of the CD11c-positive cells from C57Bl/6 mice stained brightly for MHC class II I-A molecules, they were all negative for I-E (Fig. 5, B6). In contrast, the CD11c-positive cells from B6-Eαd mice were positive for I-A, but as expected, also showed a remarkably high expression of transgenic I-E molecules (Fig. 5, B6-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)

Expression of the  CD11c-Eαd transgene leads to I-E  expression on thymic DC, but  not on thymic B cells. DC:  Thymi of B6, B6-Eαd, and  B6CD11c-Eαd mice were collagenase digested and DC were  isolated after a low density gradient as described in Materials and  Methods. Cells were stained with  mAbs specific for CD11c (PE)  and I-E or I-A (FITC), respectively. B cells: thymi of the three  mouse types were complement  depleted for CD4 and CD8 to  enrich non–T cells (see Materials  and Methods) and then stained  with mAbs specific for the pan B  cell antigen CD19 (PE) and I-E  or I-A (FITC), respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Expression of the CD11c-Eαd transgene leads to I-E expression on thymic DC, but not on thymic B cells. DC: Thymi of B6, B6-Eαd, and B6CD11c-Eαd mice were collagenase digested and DC were isolated after a low density gradient as described in Materials and Methods. Cells were stained with mAbs specific for CD11c (PE) and I-E or I-A (FITC), respectively. B cells: thymi of the three mouse types were complement depleted for CD4 and CD8 to enrich non–T cells (see Materials and Methods) and then stained with mAbs specific for the pan B cell antigen CD19 (PE) and I-E or I-A (FITC), respectively.
Mentions: To further characterize the cells expressing the I-E transgene in the thymus, we isolated the light density cell fraction from collagenase-digested thymi as described earlier (29), and performed flow cytometric analysis. The major cell type obtained in the low buoyant density fraction from thymus were cells with relatively high forward scatter (FS) and intermediate side scatter (SS) signals (data not shown); in contrast, thymocytes (from the high density fraction) had both low FS and SS (data not shown). When the low density fraction was stained for CD11c expression, 80–90% of the cells fulfilling the above described FS/SS criteria were CD11cpositive (see Fig. 5, dendritic cells). While more than 60% of the CD11c-positive cells from C57Bl/6 mice stained brightly for MHC class II I-A molecules, they were all negative for I-E (Fig. 5, B6). In contrast, the CD11c-positive cells from B6-Eαd mice were positive for I-A, but as expected, also showed a remarkably high expression of transgenic I-E molecules (Fig. 5, B6-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