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Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice
Veterinary Immunology and Immunopathology 87 (2002) 417±421
Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice George C. Russella,*, Robert A. Olivera, Susan Craigmilea, Vish Neneb, Elizabeth J. Glassa b
a Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK International Livestock Research Institute, P.O. Box 30709, Nairobi, Kenya
Abstract Major histocompatibility complex (MHC) class I restricted cellular immune responses play an important role in immunity to intracellular pathogens. By binding antigenic peptides and presenting them to T cells, class I molecules impose signi®cant selection on the targets of immune responses. Candidate vaccine antigens for cellular immune responses should therefore be analysed in the context of MHC class I antigen presentation. Transgenic mice expressing human MHC (HLA) genes provide a useful model for the identi®cation of potential cytotoxic T lymphocyte (CTL) antigens. To facilitate the analysis of candidate CTL vaccines in cattle, we have produced transgenic mice expressing a common bovine MHC (BoLA) class I allele. The functional BoLA-A11 gene, carried on a 7 kb genomic DNA fragment, was used to make transgenic mice by pronuclear microinjection. Three transgenic mouse lines carrying the BoLA-A11 gene were established. Expression of the BoLA-A11 gene was found in RNA and the A11 product could be detected on the surface of spleen and blood cells. Functional analysis of the A11 transgene product, and its ability to act as an antigen presenting molecules in the mouse host will be discussed. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Major histocompatibility complex; Cattle; BoLA; Transgene
1. Introduction Theileria parva and T. annulata account for substantial losses of cattle productivity in much of the tropics and in sub-Saharan Africa. Theileriosis also presents a serious barrier to the improvement of cattle because the more productive taurine cattle breeds are generally more susceptible to disease. Treatment with anti-theilerial drugs is effective, but is too expensive for use in developing countries. Both parasites can also be controlled by live vaccines, but these are costly *
Corresponding author. Tel.: 44-131-527-4354; fax: 44-131-440-0434. E-mail address: [email protected] (G.C. Russell).
to produce, require a cold chain for delivery and give variable degrees of protection. The production of effective synthetic or recombinant vaccines for T. parva and T. annulata has been the subject of a considerable international research effort for some years. Major histocompatibility complex (MHC) class I restricted CD8 cytotoxic T lymphocytes (CTL) play an important role in immunity to theilerial infection (Preston et al., 1999; McKeever et al., 1999). MHC class I molecules bind antigenic peptides and present them to T cells, so MHC±peptide speci®city imposes the ®rst level of selection for potential vaccine candidates. Antigens containing peptides that are bound by MHC molecules and which elicit a productive CTL response are good vaccine
0165-2427/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 0 9 5 - 8
418
G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
candidates. However, while MHC±peptide interactions can be predicted from motif data (Rammensee et al., 1999) or assayed directly (Jameson and Bevan, 1992), the induction of protective CTL responses by candidate antigens can be measured only in vivo. The expense and dif®culty involved in performing such experiments and vaccine trials in cattle mean that only a few candidate antigens can be rigorously analysed. The availability of a tractable small animal model would allow a rapid initial screening of vaccine candidate molecules, and would provide the justi®cation required for large-scale vaccine trials of those molecules that give encouraging results. HLA class I-transgenic mice are an important resource for the understanding of human cellular immune responses (reviewed by Faulkner et al., 1998). Such mice have also been used to assess the immunogenicity of candidate CTL epitopes, identi®ed either by their similarity to a known MHC class I peptide motif sequence or by in vitro analysis. HLA-A transgenic mice immunised with speci®c peptides or recombinant vaccinia viruses (Ressing et al., 1995; Shirai et al., 1995) produced antigen-speci®c HLArestricted CTL responses, and those epitopes which were identi®ed as inducing good CTL responses in the mouse model were also found to induce CTL responses in MHC matched human PBMC. Despite the differences in T cell receptor genes and responses, the epitope speci®city determined by the HLA molecule appeared to be a critical factor in determining the CTL repertoire (Shirai et al., 1995; Engelhard et al., 1991). These ®ndings indicate that the determination of in vivo epitope immunogenicity in MHC-transgenic mice could provide a good model for the pre-screening of potential CTL epitopes. In cattle, over 20 full-length MHC class I genes have been characterised by cDNA sequencing. Of these, fewer than half have been characterised by transfection, and peptide-binding motifs have currently been determined for ®ve class I bovine lymphocyte antigens (BoLA), representing the BoLA-A11, A18, A20 and A31 haplotypes (reviewed by Ellis and Ballingall, 1999). The genomic clone pBoLA-19 was selected for transgenesis because cDNA clones do not express reliably in transgenic mice, and because this cloned genomic fragment represented a convenient cassette for manipulation. The cloned 7 kb genomic DNA fragment carries the complete A11 gene with
approximately 1 kb of upstream sequence, including the class I promoter, and about 3 kb of downstream ¯anking DNA (Fig. 1A). This genomic clone is expressed in transfected mouse cells and functions as a restriction element for BoLA-A11 speci®c CTL (Sawhney et al., 1995). Studies in HLA-transgenic mice showed that as little as 260 bp (base pairs) of upstream sequence were suf®cient for ef®cient expression (Kushida et al., 1997), indicating that the 7 kb pair BoLA-A11 gene fragment was a good candidate for transgenesis. Comparison between the pBoLA-19 promoter region and the HLA-A upstream sequence also showed considerable sequence similarity, with over 70% identity within the 250 bp `minimal HLA promoter' region (Fig. 1B), lending further support to the view that pBoLA-19 would be expressed reliably. 2. Materials and methods The BoLA-A11 transgene was prepared by digestion of the pBoLA-19 plasmid with restriction enzymes SalI and BamHI, followed by gel puri®cation of the 7 kb insert band. The DNA was microinjected into the pronuclei of CBA/C57BL F1 fertilised mouse eggs, and the surviving eggs were implanted into surrogate mothers. Viable offspring were tested for transgene insertion by PCR analysis of crude tail biopsy DNA preparations, using primers in exon 7 (50 -GGC TCT GAT GTG TCT CTC ACG-30 ) and exon 8 (50 -GAT GMA GCA TCA CTC AGT CCC-30 ) which did not co-amplify mouse class I genes. Founder mice identi®ed as carrying the bovine transgene were mated with CBA/C57BL F1 mice to produce transgenic lines carrying each independent transgene insertion. Positive mice in each litter were identi®ed using the BoLA-speci®c PCR test. Transcription and processing of the transgene were assayed by speci®c reverse transcription RT-PCR of total spleen RNA, using primers in exon 1 (50 -ATG CGR GTY ATG RGG CCG SGA RSC CT-30 ) and the 30 -untranslated region (50 -TGG GCA GGG GTG ACC AGT C-30 ) of the BoLA-A11 sequence. Control RNA samples from non-transgenic mice and from an A11 steer were included. Protein expression on the surface of spleen cells from transgenic mice and non-transgenic littermates
G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
Fig. 1. (A) Structure of the 7 kb pBoLA-19 construct, containing a 4 kb BoLA-class I gene with its own promoter and about 3 kb of downstream ¯anking DNA. (B) Alignment of the promoter sequences of pBoLA-19 and HLA-A. Identities (71% total) are shown as dashes (- - -) in the HLA-A sequence. Gaps inserted to maintain alignment are shown by dots (. . .). Numbering is from the HLA-A transcription initiation site and regulatory sequences, as de®ned by Van den Elsen et al. (1998), are in bold and underlined as follows: Enh A, Enhancer A with NF-kB sites; ISRE, interferon stimulated response element; Site a, constitutive regulatory element; Enh B, Enhancer B.
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G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
was analysed by ¯ow cytometry using the bovine class I-speci®c mAb IL-A88. To avoid cross-reaction of secondary anti-mouse Ig with cell surface Ig, puri®ed IL-A88 was labelled with biotin and detected using steptavidin-FITC. 3. Results and discussion The microinjection experiment yielded about 100 viable offspring, which were tested for the presence of the transgene by PCR. A BoLA class I exon 7±exon 8 fragment could be ampli®ed from mouse tail lysates in only three of the offspring (Fig. 2), and this was con®rmed by southern blotting of genomic DNA. These mice were bred with CBA/C57BL F1 mice to establish lines carrying the BoLA-A11 transgene. One line was used for expression studies on the basis of the number of animals available for analysis. Expression of the BoLA-A11 gene was initially analysed by RT-PCR of total RNA extracted from mouse spleen cells. Primers in exon 1 and the 30 untranslated region were used to amplify the fulllength coding region of the pBoLA-19 clone. PCR products were only ampli®ed from the transgenic mice and the cattle control sample (Fig. 3) and had the size expected for the correctly spliced mRNA (about 1.2 kb). The product bands were gel puri®ed and directly sequenced, con®rming that the BoLA-A11 gene was correctly expressed (data not shown). Cell surface expression of the transgene product was analysed by ¯ow cytometry. Spleen cells from transgenic mice and non-transgenic littermates were stained with biotinylated IL-A88. This mAb ef®ciently detects the BoLA-A11 product and has been
Fig. 2. Agarose gel electrophoresis of PCR products from transgenic () or non-transgenic ( ) mouse tail DNA samples. The exon 7±exon 8 product is 200 bp.
Fig. 3. Agarose gel electrophoresis of RT-PCR products from transgenic () or non-transgenic ( ) mouse spleen RNA, or control cattle blood (C). The expected full-length BoLA-A11 spliced product (S) was about 1.2 kb. The size of the unspliced product (U) was about 4 kb. M, size markers with relevant band sizes indicated at the side of the gel; kb, kilobase pairs.
shown not to cross-react with mouse class I molecules (Sawhney et al., 1995). The transgenic mice showed a clear increase in ¯uorescence relative to the control (Fig. 4), indicating that the bovine class I molecule was correctly assembled, loaded with peptide and transported to the cell surface. The small number of transgenic mice currently available have precluded functional testing, but previous results suggest that the BoLA-A11 molecule is capable of interacting with the class I assembly and transport machinery (Sawhney et al., 1995). Mouse cells transfected with pBoLA-19 can act as targets for A11-speci®c bovine CTLs, and it will be an important feature of future research to determine whether the bovine A11 molecule can interact with the mouse T cell receptor and co-receptors. The ability of the 900 bp promoter region from pBoLA-19 to direct expression of the adjacent class I gene in transgenic mice suggests that this construct could be a useful transgene vector. Cloning genomic DNA fragments encoding other class I genes into pBoLA-19 may provide a rapid means to produce transgenic mice expressing a range of BoLA speci®cities for analysis of the immunology underlying cattle
G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
421
Fig. 4. Flow cytometric analysis of BoLA-A11 expression on mouse spleen cells. Panels (a) and (b) are transgenic mice; panels (c) and (d) are non-transgenic littermates. IL-A88 ¯uorescence is the ®lled curves, while non-speci®c ¯uorescence is the open curves.
MHC class I restriction, with potential for rapid screening of cattle CTL vaccine candidates. Acknowledgements This work was supported by the UK Department for International Development (Animal Health Programme grant R7163) and the BBSRC. The authors are grateful to Zoe Webster and Dr. Maria Alexiou at the Embryonic Stem Cell Facility, MRC Clinical Sciences Centre, Hammersmith Hospital, London, for initial microinjection experiments. References Ellis, S.A., Ballingall, K.T., 1999. Cattle MHC: evolution in action? Immunol. Rev. 167, 159±168. Engelhard, V.H., Lacy, E., Ridge, J.P., 1991. In¯uenza A-speci®c, HLA-A2.1-restricted cytotoxic T lymphocytes from HLA-A2.1 transgenic mice recognise fragments of the M1 protein. J. Immunol. 146, 1226±1232. Faulkner, L., Borysiewicz, L.K., Man, S., 1998. The use of human leukocyte antigen class I transgenic mice to investigate human gene function. J. Immunol. Meth. 221, 1±16. Jameson, S.C., Bevan, M.J., 1992. Dissection of major histocompatibility complex (MHC) and T cell receptor contact residues in a Kb-restricted ovalbumin peptide and an assessment of the predictive power of MHC-binding motifs. Eur. J. Immunol. 22, 2663±2667. Kushida, M.M., Dey, A., Zhang, X.-L., Campbell, J., Heeney, M., Carlyle, J., Ganguly, S., Ozato, K., Vasavada, H., Chamberlain,
J.W., 1997. A 150-base pair 50 region of the MHC class I HLAB7 gene is suf®cient to direct tissue-speci®c expression and locus control region activity. J. Immunol. 159, 4913±4929. McKeever, D.J., Taracha, E.L.N., Morrison, W.I., Musoke, A.J., Morzaria, S.P., 1999. Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies. Parasitol. Today 15, 263±267. Preston, P.M., Hall, F.R., Glass, E.J., Campbell, J.D.M., Dargouth, M.A., Ahmed, J.S., Sheils, B.R., Spooner, R.L., Jongejan, F., Brown, C.G.D., 1999. Innate and adaptive immune responses co-operate to protect cattle against Theileria annulata. Parasitol. Today 15, 268±274. Rammensee, H.-G., Bachmann, J., Emmerich, N.P.N., Bachor, O.A., Stevanovi, S., 1999. SYFPEITHI: databse for MHC ligands and peptide motifs. Immunogenetics 50, 213±219. Ressing, M.E., Sette, A., Brandt, R.M.P., Ruppert, J., Wentworth, P.A., Hartman, M., Oseroff, C., Grey, H.M., Melief, C.J.M., Kast, W.M., 1995. Human CTL epitopes encoded by human papillomavirus type-16 E6 and E7 identi®ed through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides. J. Immunol. 154, 5934±5943. Sawhney, S.M.S., Hasima, N.N., Glass, E.J., Al-Murrani, S.W.K., Nichani, A.K., Spooner, R.L., Williams, J.L., Russell, G.C., 1995. Transfection, expression and DNA sequence of a gene encoding a BoLA-A11 antigen. Immunogenetics 41, 246±250. Shirai, M., Arichi, T., Nishioka, M., Nomura, T., Ikeda, K., Kawanashi, K., Engelhard, V.H., Feinstone, S.M., Berzofsky, J.A., 1995. CTL responses of HLA-A2.1-transgenic mice speci®c for hepatitis C viral peptides predict epitopes for CTL of humans carrying HLA-A2.1. J. Immunol. 154, 2733± 2742. Van den Elsen, P.J., Gobin, S.J.P., van Eggermond, M.C.J.A., Peijnenburg, A., 1998. Regulation of MHC class I and II gene transcription: differences and similarities. Immunogenetics 48, 208±221.
Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice George C. Russella,*, Robert A. Olivera, Susan Craigmilea, Vish Neneb, Elizabeth J. Glassa b
a Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK International Livestock Research Institute, P.O. Box 30709, Nairobi, Kenya
Abstract Major histocompatibility complex (MHC) class I restricted cellular immune responses play an important role in immunity to intracellular pathogens. By binding antigenic peptides and presenting them to T cells, class I molecules impose signi®cant selection on the targets of immune responses. Candidate vaccine antigens for cellular immune responses should therefore be analysed in the context of MHC class I antigen presentation. Transgenic mice expressing human MHC (HLA) genes provide a useful model for the identi®cation of potential cytotoxic T lymphocyte (CTL) antigens. To facilitate the analysis of candidate CTL vaccines in cattle, we have produced transgenic mice expressing a common bovine MHC (BoLA) class I allele. The functional BoLA-A11 gene, carried on a 7 kb genomic DNA fragment, was used to make transgenic mice by pronuclear microinjection. Three transgenic mouse lines carrying the BoLA-A11 gene were established. Expression of the BoLA-A11 gene was found in RNA and the A11 product could be detected on the surface of spleen and blood cells. Functional analysis of the A11 transgene product, and its ability to act as an antigen presenting molecules in the mouse host will be discussed. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Major histocompatibility complex; Cattle; BoLA; Transgene
1. Introduction Theileria parva and T. annulata account for substantial losses of cattle productivity in much of the tropics and in sub-Saharan Africa. Theileriosis also presents a serious barrier to the improvement of cattle because the more productive taurine cattle breeds are generally more susceptible to disease. Treatment with anti-theilerial drugs is effective, but is too expensive for use in developing countries. Both parasites can also be controlled by live vaccines, but these are costly *
Corresponding author. Tel.: 44-131-527-4354; fax: 44-131-440-0434. E-mail address: [email protected] (G.C. Russell).
to produce, require a cold chain for delivery and give variable degrees of protection. The production of effective synthetic or recombinant vaccines for T. parva and T. annulata has been the subject of a considerable international research effort for some years. Major histocompatibility complex (MHC) class I restricted CD8 cytotoxic T lymphocytes (CTL) play an important role in immunity to theilerial infection (Preston et al., 1999; McKeever et al., 1999). MHC class I molecules bind antigenic peptides and present them to T cells, so MHC±peptide speci®city imposes the ®rst level of selection for potential vaccine candidates. Antigens containing peptides that are bound by MHC molecules and which elicit a productive CTL response are good vaccine
0165-2427/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 0 9 5 - 8
418
G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
candidates. However, while MHC±peptide interactions can be predicted from motif data (Rammensee et al., 1999) or assayed directly (Jameson and Bevan, 1992), the induction of protective CTL responses by candidate antigens can be measured only in vivo. The expense and dif®culty involved in performing such experiments and vaccine trials in cattle mean that only a few candidate antigens can be rigorously analysed. The availability of a tractable small animal model would allow a rapid initial screening of vaccine candidate molecules, and would provide the justi®cation required for large-scale vaccine trials of those molecules that give encouraging results. HLA class I-transgenic mice are an important resource for the understanding of human cellular immune responses (reviewed by Faulkner et al., 1998). Such mice have also been used to assess the immunogenicity of candidate CTL epitopes, identi®ed either by their similarity to a known MHC class I peptide motif sequence or by in vitro analysis. HLA-A transgenic mice immunised with speci®c peptides or recombinant vaccinia viruses (Ressing et al., 1995; Shirai et al., 1995) produced antigen-speci®c HLArestricted CTL responses, and those epitopes which were identi®ed as inducing good CTL responses in the mouse model were also found to induce CTL responses in MHC matched human PBMC. Despite the differences in T cell receptor genes and responses, the epitope speci®city determined by the HLA molecule appeared to be a critical factor in determining the CTL repertoire (Shirai et al., 1995; Engelhard et al., 1991). These ®ndings indicate that the determination of in vivo epitope immunogenicity in MHC-transgenic mice could provide a good model for the pre-screening of potential CTL epitopes. In cattle, over 20 full-length MHC class I genes have been characterised by cDNA sequencing. Of these, fewer than half have been characterised by transfection, and peptide-binding motifs have currently been determined for ®ve class I bovine lymphocyte antigens (BoLA), representing the BoLA-A11, A18, A20 and A31 haplotypes (reviewed by Ellis and Ballingall, 1999). The genomic clone pBoLA-19 was selected for transgenesis because cDNA clones do not express reliably in transgenic mice, and because this cloned genomic fragment represented a convenient cassette for manipulation. The cloned 7 kb genomic DNA fragment carries the complete A11 gene with
approximately 1 kb of upstream sequence, including the class I promoter, and about 3 kb of downstream ¯anking DNA (Fig. 1A). This genomic clone is expressed in transfected mouse cells and functions as a restriction element for BoLA-A11 speci®c CTL (Sawhney et al., 1995). Studies in HLA-transgenic mice showed that as little as 260 bp (base pairs) of upstream sequence were suf®cient for ef®cient expression (Kushida et al., 1997), indicating that the 7 kb pair BoLA-A11 gene fragment was a good candidate for transgenesis. Comparison between the pBoLA-19 promoter region and the HLA-A upstream sequence also showed considerable sequence similarity, with over 70% identity within the 250 bp `minimal HLA promoter' region (Fig. 1B), lending further support to the view that pBoLA-19 would be expressed reliably. 2. Materials and methods The BoLA-A11 transgene was prepared by digestion of the pBoLA-19 plasmid with restriction enzymes SalI and BamHI, followed by gel puri®cation of the 7 kb insert band. The DNA was microinjected into the pronuclei of CBA/C57BL F1 fertilised mouse eggs, and the surviving eggs were implanted into surrogate mothers. Viable offspring were tested for transgene insertion by PCR analysis of crude tail biopsy DNA preparations, using primers in exon 7 (50 -GGC TCT GAT GTG TCT CTC ACG-30 ) and exon 8 (50 -GAT GMA GCA TCA CTC AGT CCC-30 ) which did not co-amplify mouse class I genes. Founder mice identi®ed as carrying the bovine transgene were mated with CBA/C57BL F1 mice to produce transgenic lines carrying each independent transgene insertion. Positive mice in each litter were identi®ed using the BoLA-speci®c PCR test. Transcription and processing of the transgene were assayed by speci®c reverse transcription RT-PCR of total spleen RNA, using primers in exon 1 (50 -ATG CGR GTY ATG RGG CCG SGA RSC CT-30 ) and the 30 -untranslated region (50 -TGG GCA GGG GTG ACC AGT C-30 ) of the BoLA-A11 sequence. Control RNA samples from non-transgenic mice and from an A11 steer were included. Protein expression on the surface of spleen cells from transgenic mice and non-transgenic littermates
G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
Fig. 1. (A) Structure of the 7 kb pBoLA-19 construct, containing a 4 kb BoLA-class I gene with its own promoter and about 3 kb of downstream ¯anking DNA. (B) Alignment of the promoter sequences of pBoLA-19 and HLA-A. Identities (71% total) are shown as dashes (- - -) in the HLA-A sequence. Gaps inserted to maintain alignment are shown by dots (. . .). Numbering is from the HLA-A transcription initiation site and regulatory sequences, as de®ned by Van den Elsen et al. (1998), are in bold and underlined as follows: Enh A, Enhancer A with NF-kB sites; ISRE, interferon stimulated response element; Site a, constitutive regulatory element; Enh B, Enhancer B.
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G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
was analysed by ¯ow cytometry using the bovine class I-speci®c mAb IL-A88. To avoid cross-reaction of secondary anti-mouse Ig with cell surface Ig, puri®ed IL-A88 was labelled with biotin and detected using steptavidin-FITC. 3. Results and discussion The microinjection experiment yielded about 100 viable offspring, which were tested for the presence of the transgene by PCR. A BoLA class I exon 7±exon 8 fragment could be ampli®ed from mouse tail lysates in only three of the offspring (Fig. 2), and this was con®rmed by southern blotting of genomic DNA. These mice were bred with CBA/C57BL F1 mice to establish lines carrying the BoLA-A11 transgene. One line was used for expression studies on the basis of the number of animals available for analysis. Expression of the BoLA-A11 gene was initially analysed by RT-PCR of total RNA extracted from mouse spleen cells. Primers in exon 1 and the 30 untranslated region were used to amplify the fulllength coding region of the pBoLA-19 clone. PCR products were only ampli®ed from the transgenic mice and the cattle control sample (Fig. 3) and had the size expected for the correctly spliced mRNA (about 1.2 kb). The product bands were gel puri®ed and directly sequenced, con®rming that the BoLA-A11 gene was correctly expressed (data not shown). Cell surface expression of the transgene product was analysed by ¯ow cytometry. Spleen cells from transgenic mice and non-transgenic littermates were stained with biotinylated IL-A88. This mAb ef®ciently detects the BoLA-A11 product and has been
Fig. 2. Agarose gel electrophoresis of PCR products from transgenic () or non-transgenic ( ) mouse tail DNA samples. The exon 7±exon 8 product is 200 bp.
Fig. 3. Agarose gel electrophoresis of RT-PCR products from transgenic () or non-transgenic ( ) mouse spleen RNA, or control cattle blood (C). The expected full-length BoLA-A11 spliced product (S) was about 1.2 kb. The size of the unspliced product (U) was about 4 kb. M, size markers with relevant band sizes indicated at the side of the gel; kb, kilobase pairs.
shown not to cross-react with mouse class I molecules (Sawhney et al., 1995). The transgenic mice showed a clear increase in ¯uorescence relative to the control (Fig. 4), indicating that the bovine class I molecule was correctly assembled, loaded with peptide and transported to the cell surface. The small number of transgenic mice currently available have precluded functional testing, but previous results suggest that the BoLA-A11 molecule is capable of interacting with the class I assembly and transport machinery (Sawhney et al., 1995). Mouse cells transfected with pBoLA-19 can act as targets for A11-speci®c bovine CTLs, and it will be an important feature of future research to determine whether the bovine A11 molecule can interact with the mouse T cell receptor and co-receptors. The ability of the 900 bp promoter region from pBoLA-19 to direct expression of the adjacent class I gene in transgenic mice suggests that this construct could be a useful transgene vector. Cloning genomic DNA fragments encoding other class I genes into pBoLA-19 may provide a rapid means to produce transgenic mice expressing a range of BoLA speci®cities for analysis of the immunology underlying cattle
G.C. Russell et al. / Veterinary Immunology and Immunopathology 87 (2002) 417±421
421
Fig. 4. Flow cytometric analysis of BoLA-A11 expression on mouse spleen cells. Panels (a) and (b) are transgenic mice; panels (c) and (d) are non-transgenic littermates. IL-A88 ¯uorescence is the ®lled curves, while non-speci®c ¯uorescence is the open curves.
MHC class I restriction, with potential for rapid screening of cattle CTL vaccine candidates. Acknowledgements This work was supported by the UK Department for International Development (Animal Health Programme grant R7163) and the BBSRC. The authors are grateful to Zoe Webster and Dr. Maria Alexiou at the Embryonic Stem Cell Facility, MRC Clinical Sciences Centre, Hammersmith Hospital, London, for initial microinjection experiments. References Ellis, S.A., Ballingall, K.T., 1999. Cattle MHC: evolution in action? Immunol. Rev. 167, 159±168. Engelhard, V.H., Lacy, E., Ridge, J.P., 1991. In¯uenza A-speci®c, HLA-A2.1-restricted cytotoxic T lymphocytes from HLA-A2.1 transgenic mice recognise fragments of the M1 protein. J. Immunol. 146, 1226±1232. Faulkner, L., Borysiewicz, L.K., Man, S., 1998. The use of human leukocyte antigen class I transgenic mice to investigate human gene function. J. Immunol. Meth. 221, 1±16. Jameson, S.C., Bevan, M.J., 1992. Dissection of major histocompatibility complex (MHC) and T cell receptor contact residues in a Kb-restricted ovalbumin peptide and an assessment of the predictive power of MHC-binding motifs. Eur. J. Immunol. 22, 2663±2667. Kushida, M.M., Dey, A., Zhang, X.-L., Campbell, J., Heeney, M., Carlyle, J., Ganguly, S., Ozato, K., Vasavada, H., Chamberlain,
J.W., 1997. A 150-base pair 50 region of the MHC class I HLAB7 gene is suf®cient to direct tissue-speci®c expression and locus control region activity. J. Immunol. 159, 4913±4929. McKeever, D.J., Taracha, E.L.N., Morrison, W.I., Musoke, A.J., Morzaria, S.P., 1999. Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies. Parasitol. Today 15, 263±267. Preston, P.M., Hall, F.R., Glass, E.J., Campbell, J.D.M., Dargouth, M.A., Ahmed, J.S., Sheils, B.R., Spooner, R.L., Jongejan, F., Brown, C.G.D., 1999. Innate and adaptive immune responses co-operate to protect cattle against Theileria annulata. Parasitol. Today 15, 268±274. Rammensee, H.-G., Bachmann, J., Emmerich, N.P.N., Bachor, O.A., Stevanovi, S., 1999. SYFPEITHI: databse for MHC ligands and peptide motifs. Immunogenetics 50, 213±219. Ressing, M.E., Sette, A., Brandt, R.M.P., Ruppert, J., Wentworth, P.A., Hartman, M., Oseroff, C., Grey, H.M., Melief, C.J.M., Kast, W.M., 1995. Human CTL epitopes encoded by human papillomavirus type-16 E6 and E7 identi®ed through in vivo and in vitro immunogenicity studies of HLA-A*0201-binding peptides. J. Immunol. 154, 5934±5943. Sawhney, S.M.S., Hasima, N.N., Glass, E.J., Al-Murrani, S.W.K., Nichani, A.K., Spooner, R.L., Williams, J.L., Russell, G.C., 1995. Transfection, expression and DNA sequence of a gene encoding a BoLA-A11 antigen. Immunogenetics 41, 246±250. Shirai, M., Arichi, T., Nishioka, M., Nomura, T., Ikeda, K., Kawanashi, K., Engelhard, V.H., Feinstone, S.M., Berzofsky, J.A., 1995. CTL responses of HLA-A2.1-transgenic mice speci®c for hepatitis C viral peptides predict epitopes for CTL of humans carrying HLA-A2.1. J. Immunol. 154, 2733± 2742. Van den Elsen, P.J., Gobin, S.J.P., van Eggermond, M.C.J.A., Peijnenburg, A., 1998. Regulation of MHC class I and II gene transcription: differences and similarities. Immunogenetics 48, 208±221.