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Targeted expression of MHC class II genes to dendritic cells in vivo
Immunology Letters 57 (1997) 155 – 158
Targeted expression of MHC class II genes to dendritic cells in vivo Mireille Riedinger, Klaus Karjalainen, Thomas Brocker * Basel Institute for Immunology, Grenzacherstrasse 487, CH-4058 Basel, Switzerland
Keywords: MHC class II gene; Dendritic cells; Bone marrow
1. Introduction
2. Methods
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 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 MHC restriction of thymocytes. Most of our knowledge about DC was obtained using highly invasive and manipulatory experimental protocols such as cell transfer experiments, thymus reaggregation cultures, suspension cultures, thymus grafting and bone marrow (BM) 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 not to be identical to those of DC in vivo [1], we intended to establish a system that would allow us to investigate DC-function avoiding artificial interferences due to handling. We developed a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. This system allowed us to create transgenic mice, where different genes of interest are expressed exclusively in DC. These mice will be helpful to elucidate maturation, migration patterns and functions of DC during ontogeny, immune response and after organ transplantation.
A cDNA-pool from four human T-cell clones, all expressing human CD11c, was used as template for PCR-amplification of a human CD11c gene-fragment that served as a probe to screen a mouse genomic library. We isolated two overlapping phages that were characterized by restriction mapping and partial sequencing of their inserts. DNA-sequence analysis of approximately 1000 bp around the initiation codon showed 68.8% identity to the corresponding sequence of the human CD11c gene and were therefore accepted to represent mouse CD11c. We used a 5.5 kb fragment that contained the 5% region of mouse CD11c gene to drive the expression of the I-Eda-cDNA. This transgenic construct was injected into fertilized eggs of C57BL/6 mice, naturally lacking Ea -expression. Expression of cell surface proteins was assayed by immunofluorescence analysis. Organs were teased through a mesh and cell suspensions of 1 × 105 viable cells were stained with 20 mg/ml mAb that was directly labelled. After washing, cells were analyzed using a FACScan. For generation of dendritic cells from bone marrow, total bone marrow was cultured at 5 × 105 cells/ml in culture medium containing 25 ng/ml GM-CSF. Cells were cultured in a total volume of 10 ml in 90 mm tissue culture treated petri dishes. Maximal yield of dendritic cells was obtained between day 6 and 8 of culture.
3. Results
* Corresponding author. 0165-2478/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 5 - 2 4 7 8 ( 9 7 ) 0 0 0 7 4 - 6
Two different mouse strains were used as controls. An I-E transgenic line created by Widera et al. [2] was
Fig. 1. Cell suspensions of different origin were analyzed with mAbs specific for B220, Mac-1, CD11c, I-E, I-A. B-cells from spleen were stained with anti-B220, -I-A and -I-E. Macrophages from peritoneum were stained with anti-Mac-1, -I-A and -I-E, while splenic DC from low density gradients were analyzed with anti-CD11c, -I-A and -I-E. Triple immunofluorescence was performed and the gates set on either B220+ , Mac-1+ or CD11c + cells respectively. Shown are I-E stainings (grey histograms) and I-A stainings (white histograms) only.
156 M. Riedinger et al. / Immunology Letters 57 (1997) 155–158
M. Riedinger et al. / Immunology Letters 57 (1997) 155–158
157
Fig. 2. Three colour FACS analysis of bone marrow GM-CSF cultures. Gates were set on the different subpopulations of the I-A vs. I-E stainings as indicated (1,2 or 3). The CD11c-expression of the gated populations is shown as histograms on the right side with the corresponding gate numbers indicated.
used as a ‘positive’ control. This transgenic line 107.1 (here called B6-Eda) expresses an I-Eda transgene under the control of a segment of the I-E MHC class II promoter. The expression pattern previously described for the B6-Eda line corresponded to wildtype I-E expression in I-E + strains including cortical and medullary
thymic epithelial cells, as well as the BM-derived thymic fraction. Negative control mice were C57Bl/6-mice (‘B6’) that do not express the I-Ea genes, and therefore, no complementation with the bchain can occur, resulting in absence of I-E MHC class II surface expression [3].
M. Riedinger et al. / Immunology Letters 57 (1997) 155–158
158
To analyze transgene expression in B-lymphocytes we prepared cell suspensions from spleen and performed double immunofluorescence analysis with anti-B220 as a B-cell marker and either anti-I-E or anti-I-A. As shown in Fig. 1 (B220 + ), B-cells from B6CD11c-Eda mice do not express I-E (gray histograms), while B-cells from B6-Eda transgenic mice do express I-E. As expected, B-cells from all three mice did express endogenous I-A (white histograms). Furthermore we analyzed peritoneal macrophages from the three mouse types 5 days after intraperitoneal thiolglycollate injection. The macrophages were cultured for additional 48h in the presence of IFN-g to upregulate MHC class II expression. The FACS analysis in Fig. 1 shows that the Mac-1 positive cells from the three different mice all express similar levels of I-A (white histograms). However while macrophages from B6-Eda transgenic mice do express transgenic I-E, the majority of macrophages from B6CD11c-Eda transgenic mice does not express transgenic I-E (grey histograms). Only a few cells (usually 5 – 8%) from these preparations do express low levels of I-E (Fig. 1, Mac-1 + ). When we isolated DC from spleen using a low buoyant density gradient, we find that usually 60 – 80% of the CD11c + cells do express the I-E transgene in B6CD11c-Eda transgenic mice (Fig. 1, CD11c + ). When the thymus of these animals was analyzed for I-E expression, we could not detect any I-E expression on cortical or medullary epithelial cells in the B6CD11c-Eda transgenic mice (data not shown); there the I-E expression was restricted to the medulla and medullary-cortical junctions only (data not shown), the areas where thymic DC had been localized previously [4,5]. To test if bone marrow precursors of transgenic animals would give rise to I-E positive DC, we performed bone marrow cultures in the presence of GMCSF. The FACS-analysis of these cultures is shown in Fig. 2. As expected the bone marrow from non-transgenic littermates gives rise to DC that are I-A + I-E − with all DC expressing the DC-marker CD11c (Fig. 2, B6, gate 2). In bone marrow cultures from mice expressing I-E under the MHC class II promoter, all DC express I-A and the transgenic I-E and are all CD11c positive (Fig. 2, B6-Eda, gate 2). When the cultures from B6 CD11c-Eda mice were analyzed, the DCs could be subdivided into two phenotypes: most of the DC ( \ 85%) did express I-A and transgenic I-E and were CD11c positive (Fig. 2, B6 CD11c-Eda, gate 2). A smaller subpopulation was I-A positive, I-E negative
.
.
but CD11c positive (Fig. 2, B6 CD11c-Eda, gate 3). We are currently investigating, if this subpopulation represents a more immature form of DC that will express I-E when maturing further, or if our transgene is in fact not expressed in 100% of DCs, despite their expression of CD11c. When the DCs were used as stimulators in mixed lymphocyte reactions, they were strong inducers of anti I-E alloresponses and no difference could be observed between DC from B6 CD11c-Eda and B6-Eda mice (data not shown). Apparently the 5.5 kb fragment of the mouse CD11c 5% untranslated region is a valuable tool to explore function of DC in vivo, since it allows to express genes of interest exclusively in DC and not in other lymphoid or epithelial cells. We now do have a system to study more precisely the role of DC’s in negative and positive selection of thymocytes, during immunoresponses and will investigate DC-function during immunization, transplantation and memory in a mouse where B-cells are devoid of I-E expression.
Acknowledgements The author would like to thank M. Dessing for advice with FACS analysis, H. Stahlberger for graphic art work, U. Mueller for microinjections and Dr C. Ruedl for help with DC isolations. The Basel Institute for Immunology was founded and is supported by Hoffmann-La Roche, Basel, Switzerland.
References [1] G. Girolomoni, J.C. Simon, P.R. Bergstresser, P. Cruz Jr., Freshly isolated spleen dendritic cells and epidermal Langerhans cells undergo similar phenotypic and functional changes during short-term culture, J. Immunol. 145 (1990) 2820 – 2826. [2] G. Widera, L.C. Burkly, C.A. Pinkert, E.C. Bottger, C. Cowing, R.D. Palmiter, R.L. Brinster, R.A. Flavell, Transgenic mice selectively lacking MHC class II (I-E) antigen expression on B-cells: an in vivo approach to investigate Ia gene function, Cell 51 (1987) 175 – 187. [3] D.B. Mathis, C. Williams, V. Kanter, H. McDevitt, Several mechanisms can account for defective Ea gene expression in different mouse haplotypes, Proc. Natl. Acad. Sci. 80 (1983) 273 – 277. [4] P.J. Fairchild, J.M. Austyn, Thymic dendritic cells: phenotype and function, Int. Rev. Immunol. 6 (1990) 187 – 196. [5] A.N. Barclay, G. Mayrhofer, Bone marrow origin of Ia-positive Cells in the medulla of rat J. Exp. Med 153 (1981) 1666 – 1671.
Targeted expression of MHC class II genes to dendritic cells in vivo Mireille Riedinger, Klaus Karjalainen, Thomas Brocker * Basel Institute for Immunology, Grenzacherstrasse 487, CH-4058 Basel, Switzerland
Keywords: MHC class II gene; Dendritic cells; Bone marrow
1. Introduction
2. Methods
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 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 MHC restriction of thymocytes. Most of our knowledge about DC was obtained using highly invasive and manipulatory experimental protocols such as cell transfer experiments, thymus reaggregation cultures, suspension cultures, thymus grafting and bone marrow (BM) 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 not to be identical to those of DC in vivo [1], we intended to establish a system that would allow us to investigate DC-function avoiding artificial interferences due to handling. We developed a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. This system allowed us to create transgenic mice, where different genes of interest are expressed exclusively in DC. These mice will be helpful to elucidate maturation, migration patterns and functions of DC during ontogeny, immune response and after organ transplantation.
A cDNA-pool from four human T-cell clones, all expressing human CD11c, was used as template for PCR-amplification of a human CD11c gene-fragment that served as a probe to screen a mouse genomic library. We isolated two overlapping phages that were characterized by restriction mapping and partial sequencing of their inserts. DNA-sequence analysis of approximately 1000 bp around the initiation codon showed 68.8% identity to the corresponding sequence of the human CD11c gene and were therefore accepted to represent mouse CD11c. We used a 5.5 kb fragment that contained the 5% region of mouse CD11c gene to drive the expression of the I-Eda-cDNA. This transgenic construct was injected into fertilized eggs of C57BL/6 mice, naturally lacking Ea -expression. Expression of cell surface proteins was assayed by immunofluorescence analysis. Organs were teased through a mesh and cell suspensions of 1 × 105 viable cells were stained with 20 mg/ml mAb that was directly labelled. After washing, cells were analyzed using a FACScan. For generation of dendritic cells from bone marrow, total bone marrow was cultured at 5 × 105 cells/ml in culture medium containing 25 ng/ml GM-CSF. Cells were cultured in a total volume of 10 ml in 90 mm tissue culture treated petri dishes. Maximal yield of dendritic cells was obtained between day 6 and 8 of culture.
3. Results
* Corresponding author. 0165-2478/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 5 - 2 4 7 8 ( 9 7 ) 0 0 0 7 4 - 6
Two different mouse strains were used as controls. An I-E transgenic line created by Widera et al. [2] was
Fig. 1. Cell suspensions of different origin were analyzed with mAbs specific for B220, Mac-1, CD11c, I-E, I-A. B-cells from spleen were stained with anti-B220, -I-A and -I-E. Macrophages from peritoneum were stained with anti-Mac-1, -I-A and -I-E, while splenic DC from low density gradients were analyzed with anti-CD11c, -I-A and -I-E. Triple immunofluorescence was performed and the gates set on either B220+ , Mac-1+ or CD11c + cells respectively. Shown are I-E stainings (grey histograms) and I-A stainings (white histograms) only.
156 M. Riedinger et al. / Immunology Letters 57 (1997) 155–158
M. Riedinger et al. / Immunology Letters 57 (1997) 155–158
157
Fig. 2. Three colour FACS analysis of bone marrow GM-CSF cultures. Gates were set on the different subpopulations of the I-A vs. I-E stainings as indicated (1,2 or 3). The CD11c-expression of the gated populations is shown as histograms on the right side with the corresponding gate numbers indicated.
used as a ‘positive’ control. This transgenic line 107.1 (here called B6-Eda) expresses an I-Eda transgene under the control of a segment of the I-E MHC class II promoter. The expression pattern previously described for the B6-Eda line corresponded to wildtype I-E expression in I-E + strains including cortical and medullary
thymic epithelial cells, as well as the BM-derived thymic fraction. Negative control mice were C57Bl/6-mice (‘B6’) that do not express the I-Ea genes, and therefore, no complementation with the bchain can occur, resulting in absence of I-E MHC class II surface expression [3].
M. Riedinger et al. / Immunology Letters 57 (1997) 155–158
158
To analyze transgene expression in B-lymphocytes we prepared cell suspensions from spleen and performed double immunofluorescence analysis with anti-B220 as a B-cell marker and either anti-I-E or anti-I-A. As shown in Fig. 1 (B220 + ), B-cells from B6CD11c-Eda mice do not express I-E (gray histograms), while B-cells from B6-Eda transgenic mice do express I-E. As expected, B-cells from all three mice did express endogenous I-A (white histograms). Furthermore we analyzed peritoneal macrophages from the three mouse types 5 days after intraperitoneal thiolglycollate injection. The macrophages were cultured for additional 48h in the presence of IFN-g to upregulate MHC class II expression. The FACS analysis in Fig. 1 shows that the Mac-1 positive cells from the three different mice all express similar levels of I-A (white histograms). However while macrophages from B6-Eda transgenic mice do express transgenic I-E, the majority of macrophages from B6CD11c-Eda transgenic mice does not express transgenic I-E (grey histograms). Only a few cells (usually 5 – 8%) from these preparations do express low levels of I-E (Fig. 1, Mac-1 + ). When we isolated DC from spleen using a low buoyant density gradient, we find that usually 60 – 80% of the CD11c + cells do express the I-E transgene in B6CD11c-Eda transgenic mice (Fig. 1, CD11c + ). When the thymus of these animals was analyzed for I-E expression, we could not detect any I-E expression on cortical or medullary epithelial cells in the B6CD11c-Eda transgenic mice (data not shown); there the I-E expression was restricted to the medulla and medullary-cortical junctions only (data not shown), the areas where thymic DC had been localized previously [4,5]. To test if bone marrow precursors of transgenic animals would give rise to I-E positive DC, we performed bone marrow cultures in the presence of GMCSF. The FACS-analysis of these cultures is shown in Fig. 2. As expected the bone marrow from non-transgenic littermates gives rise to DC that are I-A + I-E − with all DC expressing the DC-marker CD11c (Fig. 2, B6, gate 2). In bone marrow cultures from mice expressing I-E under the MHC class II promoter, all DC express I-A and the transgenic I-E and are all CD11c positive (Fig. 2, B6-Eda, gate 2). When the cultures from B6 CD11c-Eda mice were analyzed, the DCs could be subdivided into two phenotypes: most of the DC ( \ 85%) did express I-A and transgenic I-E and were CD11c positive (Fig. 2, B6 CD11c-Eda, gate 2). A smaller subpopulation was I-A positive, I-E negative
.
.
but CD11c positive (Fig. 2, B6 CD11c-Eda, gate 3). We are currently investigating, if this subpopulation represents a more immature form of DC that will express I-E when maturing further, or if our transgene is in fact not expressed in 100% of DCs, despite their expression of CD11c. When the DCs were used as stimulators in mixed lymphocyte reactions, they were strong inducers of anti I-E alloresponses and no difference could be observed between DC from B6 CD11c-Eda and B6-Eda mice (data not shown). Apparently the 5.5 kb fragment of the mouse CD11c 5% untranslated region is a valuable tool to explore function of DC in vivo, since it allows to express genes of interest exclusively in DC and not in other lymphoid or epithelial cells. We now do have a system to study more precisely the role of DC’s in negative and positive selection of thymocytes, during immunoresponses and will investigate DC-function during immunization, transplantation and memory in a mouse where B-cells are devoid of I-E expression.
Acknowledgements The author would like to thank M. Dessing for advice with FACS analysis, H. Stahlberger for graphic art work, U. Mueller for microinjections and Dr C. Ruedl for help with DC isolations. The Basel Institute for Immunology was founded and is supported by Hoffmann-La Roche, Basel, Switzerland.
References [1] G. Girolomoni, J.C. Simon, P.R. Bergstresser, P. Cruz Jr., Freshly isolated spleen dendritic cells and epidermal Langerhans cells undergo similar phenotypic and functional changes during short-term culture, J. Immunol. 145 (1990) 2820 – 2826. [2] G. Widera, L.C. Burkly, C.A. Pinkert, E.C. Bottger, C. Cowing, R.D. Palmiter, R.L. Brinster, R.A. Flavell, Transgenic mice selectively lacking MHC class II (I-E) antigen expression on B-cells: an in vivo approach to investigate Ia gene function, Cell 51 (1987) 175 – 187. [3] D.B. Mathis, C. Williams, V. Kanter, H. McDevitt, Several mechanisms can account for defective Ea gene expression in different mouse haplotypes, Proc. Natl. Acad. Sci. 80 (1983) 273 – 277. [4] P.J. Fairchild, J.M. Austyn, Thymic dendritic cells: phenotype and function, Int. Rev. Immunol. 6 (1990) 187 – 196. [5] A.N. Barclay, G. Mayrhofer, Bone marrow origin of Ia-positive Cells in the medulla of rat J. Exp. Med 153 (1981) 1666 – 1671.