The recombinant human interferon-γ receptor is fully functional in a human × murine hybrid containing human chromosome 21

0 INSTITUTPASTEUR/ELSEVIER Paris 1991

Re.S. Irmrmunol. 1991, 142, 765-772

M.R. Bono(‘)(*), C. Alcdide-Loridan(‘), B. Letouze(‘), H. Jouin(2), S.J.P. Gobin

and M. Fellou#

(‘jlnstitut National de la Sante’ et de la Recherche Mkdicale U.276 and UniversitP Paris 7, Laboratoire d’Immunog&&ique humaine, Institut Pasteur, 75724 Paris Cedex 15, and (‘)Cen tre National de la Recherche Scientijique URA .P46, Laboratoire de Parasitologie experimentale, Institut Pasteur, 75724 Paris Cedex 15

SUMMARY

It has previously been shown that stimulation of class I MHC antigen expression by hums?? interferon gamma (Hu-IFNy) depends on the presence of both human chromosome* 6 and 21 a Evsn although the presence of human chromosome 6 results in normal expression of the !?TNyreceptor on the cell surface, chromosome 21 is required for this biological response. Here, we transfected the cDNA encoding the Hu-IFNy receptor into a mouse cell Sine, L(tk- ), and into a mouse x human hybrid, GM08, containing the human chromosome 21 as the only human genetic material. Two clones, L-yR.8 and GM68-yR.9, were tested for different biological responses to Hu-IFNy. Only the hybrid containing the human chromosome 21 (GM08-yR.9) exhibited a biological response when HC antigen expression by Hu-IFNy. assayed for the stimulation of murine class I Moreover, transcriptional induction of 2’-5’-oligoadenylate synthetase and l-8 genes, as well as antiviral protection, were observed only in the GM08-yR.9 transfectants. These results show that human chromosome 21 encodes a factor which mediates different biological activities of Hu-IFNy.

Key-words: IFNy, Chromosome 2 1, Transfection, Transduction tection, Somatic cell hybrids, Induction.

INTRODUCTION

Interferon-y (IFNy) has numerous properties including immunomodulation (Trinchieri and Perussia, 1985) and antiviral protection (Petska et al., 1987). At the cellular level, IFNy has the ability to induce transcription of a number of genes (Revel and Chebath, 1986) among

;

Signal, Antiviral pro-

which are the MHC antigens (Wallach et al., 1982)) and the 2’-5’-oligoadenylate synthetaseencoding genes (Benech et al., 1987). It is well established that these biological activities follow the binding of IFNy to a membrane receptor (see Langer and Petska, 1988, for a review). Recently, the gene encoding the Hu-

Submitted October 2, 1991, accepted October 29, 1991. *Correspondence to M.R. Bono, Laboratoire d’lmmunog~nktique

humaine, Institut Pasteur, 75724 Paris Cedex 15 (France).

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M.R. BONO E T AL.

IFN'r receptor has been cloned (Aguet et al., 1988). Somatic cell hybrids have been used to localize the gene on human chromosome 6 (Rashidbaigi et al., 1986). In situ hybridization experiments have more precisely located the gene at 6q23-q24 (Le Coniat et al., 1989). Both the gene and cDNA coding for the HuIFN-f receptor have been transfected into mufine cell lines (Aguet et al., 1988; Hemmi et al., 1989). These transfectants expressed the h u m a n receptor at the cell surface and were able to bind radiolabelled Hu-IFN~,. However, binding of Hu-IFN~, did not result in the induction of murine MHC class I antigens or 2'-5'-oligoadenylate synthetase and did not ensure antiviral protection in the transfected cells (Aguet et al., 1988 ; Hemmi et al., 1989). The use of rodent somatic cell hybrids and human cell lines of non-haematopoietic origin has demonstrated that at least two speciesspecific factors are necessary to obtain a biological response to Hu-IFN~. in these heterologous systems. It was shown that these factors are encoded by human chromosomes 6 and 21 (Jung et al., 1987; Bono et al., 1989). Some hybrids of haematopoietic origin show different chromosomal requirements and do not need chromosome 2! (Bono et al., 1989). We report here on the transfection of cDNA encoding the Hu-IFN~, receptor into a murine hybrid contaimng human chromosome 21 as the only human genomic material. We demonstrate that this stable transfectant expresses the human receptor at the cell surface. In addition, we show that Hu-IFNy stimulates murine class I M H C antigen expression, induces transcription of IFNresponsive genes and permits the development of antiviral protection under certain conditions. We conclude that the Hu-IFN~, receptor is fully functional when expressed in a murine hybrid containing human chromosome 21.

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foetal calf serum. fluorescence isothiocyanate. interferon. human IFN gamma. monoclonal antibody.

MATERIALS

AND METHODS

Interferons

Recombinant Hu-IFN-r and Mu-IFN~- were kindly given by Roussel-Uclaf (Paris, France). Their specific activities were estimated to be 10 6 units/ml for HuIFN-f and 10 7 units/ml for Mu-IFN-f as determined by inhibition of the cytopathic effect of vesicular stomatitis virus (VSV) on WISH and L(tk-) cells, respectively (Roussel-Uclaf). Human IFN~ (HuIFN~3) was kindly given by Interpharm Laboratories (Rehovot, Israel). Its specific activity was estimated as 5 × 10 7 units/ml when assayed by antiviral protection on WISH cells. Mouse IFNa/13 (Mu-IFN0c/[3) was a kind gift from Dr I. Gresser (Vi!!ejuif, France). Its specific activity was assessed to be 1.6 x 10 6 units/ml when assayed for antiviral protection using the L(tk-) mouse cell line.

Cell lines

L(tk-) is a murine cell line of fibroblastic origin, and HeLa 7A is a human cell line of epithelial origin. The human × mouse somatic cell hybrid GM08 (GM08854 repository number,, called WAV-17) was obtained from the National Institute of General Medical Sciences (NIGMS). GM08 cells are hybrids obtained after fusion of A9 cells with the human fibroblast WI-38. This hybrid contains the human chromosome 21 as the sole human genetic material. The presence of human chromosome 21 which en-. codes the Hu-iFN~ receptor (Slate el ai., i978) was repeatedly checked using the antiviral properties of Hu-IFN[3 against VSV. All cells were cultured in DMEM supplemented with 10% f~tal calf serum (FCS).

Monoclonal antibodies (mAb)

The Hu-IFN 7 receptor was detected by mAb A6C5 (Aguet and Merlin, 1987) used diluted to 1/4 from culture supernatant. The mAb l l.4.1 and W6/32 were used for the detection of mouse MHC class I (H-2 k) antigens and human MHC class I antigens, respectively, as described previously (Bono et al., 1991).

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murine IFNT. murine IFN alpha and beta. human IFN beta. major histocompatibility complex. vesicular stomatitis virus.

T H E R E C O M B I N A N T H U M A N INTERFERON-'~ R E C E P T O R

Expression vector Hu-IFNT receptor cDNA was subcloned into the expression plasmid pHMG to give rise to pHMG-16' (Hemmi et al., 1989) (kindly given by M. Aguet, Ztirich, Switzerland), which was used in the transfection experiments.

767

nique (Feinberg and Vogelstein, 1982) using the "Multiprime Labelling Kit" from Amersham. The specific activities of the probes ranged from 5 to 10 × 108 cpm/lzg. A [3-actin probe (Gunning et al., 1983) was used to control the amount of RNA per lane. Probes

Induction of surface MHC class I antigens by IFNT and detection by indirect immunofluorescence Subconfluent cell cultures were treated with HuIFN'r (500 units/ml) or Mu-IFN'f (250 units/ml) for 48 h. Cells were then trypsinized and washed with PBS-5% FCS (PBS-FCS), and 10 6 ceils were incuLated with 11.4.1 or W6/32 mAb for 45 min at 4°C. After washing with PBS-FCS, cells were incubated with FITC-labelled goat anti-mouse IgG (1/200) for 30 min at 4°C. The cells were analysed by "FACScan" analyser (Becton-Dickinson) using the "Consort 30" program. Transfections and selection of cells expressing the Hu-IFN~, receptor GM08 and L(tk-) cell lines were cotransfected with pSV2Neo (encoding a neomycine resistance marker (Southern and Berg, 1982) and pHMG16' plasmids at a 1/10 molar ratio. Cells were transfected by CaPO 4 coprecipitation (Chen and Okayama, 1987). Transfected cells were selected in medium containing 1 mg/ml G418 (Geneticin, Gibco). About 30°/0 of the neomycine-resistant cells expressed the HuIFNT receptor (A6C5+), as determined by immunofluorescence. This cell population was enriched for the expression of the Hu-IFNT receptor (A6C5 ÷) by cell sorting (FCStar, Becton Dickenson). This step was followed by subcloning at limiting dilution, to obtain a homogeneous cell population. Two clones, named L-TR.8 (a subclone of transfected L(tk-) cells) and GM08-~,R.9 (a sub,.'lone of transfected GM08 cells) were further studied. Northern blot analysis Total RNA was isolated from subconfluent cultures with the guanidium thiocyanate method (Sambrook et al., 1989). RNA samples (10 l~g per lane) were loaded on an agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane (Nylon N, Amersham). Hybridization was performed at 65°C, in a 0.5 M sodium phosphate buffer (pH 7.2) containing 7°7o SDS, 100 ~tg/ml sonicated salmen sperm DNA and 1 ng probe/ml (about 106 cpm/ml). Probes were labelled by the random-priming tech-

The murine synthetase probe was a kind gift from Dr J. Chebath (Weizmann Institute, Israel). This probe is a 1.4-kb cDNA encoding the 40-kDa murine 2'-5'-oligoadenylate synthetase (Cohen et al., 1988) which hybridizes to a 1.7-kb mRNA. The 1-8 probe is a 0.6-kb cDNA isolated by Friedman et al. (1984) which hybridizes to a family of related mRNA of about 0.8 kb. Antivirai assays This assay was performed with the VSV kindly provided by Dr. G. Uz6. Cells were seeded on a 96-well plate at a concentration of 10,000 cells per well; 24 h later, cells were preincubated for 4 h with 3 U/ml Mu-IFNa/[3. Hu- or Mu-IFN'r were then serially diluted from 1,000 U/ml to 5 U/ml and added to the cells. After a 16-h incubation at 37°C, the virus was added to the cells. After 48 h, cells were studied for lysis by light-inverted microscopy. HeLa cells were used as positive control for Hu-IFNy, and L(tk-) as negative control for Hu-IFN'r and positive control for Mu-IFN~,. RESULTS Transfectants cDNA encoding the Hu-IFN~, receptor was previously cloned under the control of a housekeeping gene promoter giving rise to pHMG-16' (Hemmi et al., 1989). This vector was cotransfected with a plasmid encoding a selection marker (pSV2neo) into GM08, a mouse × human hybrid containing the human chromosome 21, and L ( t k - ) , a murine cell line. Transfectants (L--fR and GM08-'rR) were analysed by indirect immunofluorescence using mAb (A6C5) directed against the human receptor. This antibody is highly species-specific and does not cross-react with the Mu-IFNT receptor (fig. 1B). Immunofluorescence analysis showed that, among the neomycine-resistant transfectants, about 30°70 of the cells expressed the human receptor.

768

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A step in cell sorting, using the same antibodies, enabled us to obtain cell populations for which about 70°70 of the cells expressed the human receptor. These enriched cell populations were subcloned by limiting dilutions, and two suSclones named L-yR.8 (originating from the transfected L(tk-) cells) and GM08-yR.9 (from the transfected GM08 cells) were used for further study. Both transfectants, L-yR.8 (fig. 1C) and GM08-yR.9 (fig. 1D), were homogenous for Hu-IFNy-receptor expression and expressed the receptor at levels similar to that of the human cell line HeLa (fig. 1A).

MHC-antigen stimulation Cell surface expression of MHC class I antigens was detected by indirect immunofluores-

cence techniques. Mu-IFNy induced murine MHC class I (H-2) antigen expression on L-yR.8 and GM08-yR.9 transfectants, demonstrating that the H-2-induction pathway was still functional in both cell lines (data not shown). HuIFNy is highly species-specific, and therefore the murine cell line L(tk-) (data not shown) and the hybrid GM08 (fig. 2B) were not responsive to Hu-IFN-f as concerns the stimulation of H-2-antigen expression. However, figure 2D shows that the transfected Hu-IFNy receptor was fully functional in GM08-yR.9 cells (containing human chromosome 2I) since class I antigen expression was stimulated in all cells by Hu-IFNy. In contrast, L-yR.8 cells could not be stimulated to express class I antigens after Hu-IFNy treatment (fig. 2C) despite normal expression of the HuIFNy receptor.

THE R E C O M B I N A N T H U M A N ! N T E R F E R O N - T R E C E P T O R

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Transcriptional induction

Antiviral protection

By Northern blot analysis we then studied the transcriptional induction of two IFN-responsive genes, the 1-8 gene family, and the 2'-5'-oligoadenylate synthetase gene (fig. 3). Total RNA was extracted from the L-TR.8 and GM08-TR.9 transfectants after treatment with either Hu- or Mu-IFNT for 8 or 16 h or with Mu.IFNoJ[3 for 16 h. Both genes were induced either by Mu-IFN~/[3 or Mu-IFNy in each cell line. However, neither the 1-8 nor the 2'-5'-oligoadenylate synthetase mRNA were induced in LTR.8 cells after Hu-IFNT treatment, whereas both mRNA levels increased in GM08-TR.9 cells.

Due to the high stability of the vector encoding the Hu-IFNT receptor, the transfectants were highly homogeneous ( > 95070, see fig. 2D). This enabled us to analyse the antiviral activity of HuIFNT in both cell lines. L(tk-) or GM08 parental cell lines were protected against VSV by Mu-IFNct/[~ doses as low as 5 U/ml. In contrast, these cells were not protected by Mu-IFNT against viral infection at doses up to 2,500 U/ml (data not shown). It was previously established by Lewis et al. (1989) that IFN[3 and IFNT can act synergistically to produce an antiviral state in cells resistant to each individual interferon. We therefore tested whether

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Mu-IFNa/[3 pre-treatment (at a non-protective dose) could provoke antiviral protection by MuIFNy. Full protection was indeed obtained when cells were incubated with Mu-IFN=/[B (2 U/ml) for 4 h, followed by treatment with MuLIFN~, (I> 10 U/ml; data not shown).

DISCUSSION

matic cell hybrids (Rashidbaigi et aL, 1986). Despite normal binding of Hu-IFN~, to its receptor, a biological response, measured as the stimulation of MHC class I expression, was not observed on these hybrids (Rashidbaigi et al., 1986). Similar data were obtained when cDNA coding for the human receptor was transfected into a murine cell line (Aguet et al., 1988). Furthermore, somatic cell hybrids were used to demonstrate that, in addition to chromosome 6, chromosome 21 was req~fired for the stimulation of class I in response to Hu-IFN~ (Jung et al., 1987; Bono et al., 1989). Recently, these results were corroborated by different studies transfecting cDNA coding for the Hu-IFNT receptor into hybrids containing human chromosome 21 (Jung et al., 1990; Fisher et aL, 1990).

The gene encoding the Hu-IFN-r receptor was previously localized on chromosome 6 using so-

Here, we transfected cDNA encoding the HuIFNT receptor into a murine cell line (L(tk-)) and into a mouse x human hybrid (GM08)

This protocol was then used to test antiviral protection by Hu-IFNT of the L-~-R.8 and GM08-~,R.9 transfectants. GM08--rR.9 cells were protected against VSV infection at concentrations of Hu-IFNT i> 50 U/ml. In contrast, concentrations of Hu-IFNT as high as 1,000 U/ml did not ensure antiviral protection of L-~-R.8 cells.

THE RECOII/IBINANT HUMAN INTERFERON-.f RECEPTOR which contained human chromosome 21 as the sole human genetic material. Two transfectants obtained from each cell line, L-3,R.8 and GM08--rR.9, respectively, were analysed. Both transfectants were examined by indirect immunofluorescence techniques for the stimulation of the expression of murine class I MHC cell surface antigens after Hu-IFN~- t r e a t m e n t . GM08-~'R.9 transfectants expressed an increased level of class I antigens, whereas no stimulation was observed in L-~,R.8 cells. Thus, we confirmed that chromosome 21 is required to ensure the stimulation of MHC class I expression by Hu-IFNy. We then investigated whether the product encoded by chromosome 21 was also required for the other biological activities of IFN~-. Thus, we examined transcriptional induction of IFN-inducible genes and antiviral protection. Transcription of 2'-5'-oligoadenylate synthetase and 1-8 genes was induced by Hu-IFN~- in the GM08-TR.9 cells and not in the L-3,R.8 transfectants. When treated with Hu-IFN~', GM08-~,R.9 cells were protected against viral infection, whereas L-'rR.8 cells remained sensitive. Concordant with the results of class I stimular,~-, measured as t r a n ~.A~JJL, r e s p o n s i v e n e s s tO ,wrr~L_ *'-'-'~ scriptional induction and viral protection, was only detected for the GM08-~,R.9 transfectant containing h u m a n chromosome 21. Another important biological property of IFN~' is the induction of MHC class II antigens. However, class II induction could not be analysed in these transfectants as the parental GM08 and L ( t k - ) cells were not inducible for I-A or for I-E antigen expression by Mu-IFN7 (Bono et al., unpublished results). Nevertheless, using other somatic cell hybrids, we had previously demonstrated that the presence of chromosome 21 is required for M H C class II antigen induction (Bono et aL, 1991). In conclusion, we have shown that a product encoded by h u m a n chromosome 21 complements the signal transduction of the Hu-IFN~, receptor for all biological activities tested. Recently Hemmi & Aguet (1991) have shown that a hybrid IFN~- receptor (human extracellu-

77 !

lar domain, murine transmembrane and intracellular domains), was functional only in a human × murine hybrid containing human chromosome 21. In this context, it can be hypothesized that the product of chromosome 21 forms a complex or a temporary association with the extracellular domain of the ligand-binding protein. This hypothesis is conceivable as a similar structure (ligand-binding subunit/ signal-transducing subunit) has previously been suggested for the IL6 receptor (Hibi et aL, 1990).

Acknowledgements

This work was supported by the Institut National de la Sant6 et de la Recherche M6dicale and by grant 90.C.0398 from the Minist~rede la Rechercheet de la Technologie. Dr. S. Gobin is in receipt of a grant from the D616gation R6gionale de l'Education Nationaie.

Le r~cepteur de I'interf~ron-~. humain recombinant est totalement fonctionnel dans ~es cellules hybrides h o m m e x souris cc, ntenant ie chromosome 2i humain

Des hybrides homme x souris contenant le chromosome 6 humain expriment le r6cepteur de l'interf6ron-~ humain (Hu-IFN~.). Bien que de tels hybrides soient capables de lier I'Hu-IFN~', ils ne pr6sentent pas de r6ponse cellulaire/l cet IFN, mesur6e par la stimulation des antig6nes de surface du CMH murin de classe I. Par contre, des hybrides cellulaires contenant les chromosomes 6 et 21 humains sont capables de pr6senter une telle r6ponse. De m~me, lorsque I'ADNc codant pour le r6cepteur de I'HuIFN~ est transfect6 dans des cellules murines, aucune stimulation des antig6nes du CMH murin n'est d6tect6e. Dans le pr6sent travail, nous avons transfect6 I'ADNc codant pour le r~cepteur de I'Hu-IFN3, dans des cellules murines ainsi que dans un hybride somatique contenant le chromosome 21 comme seul mat6riel g6n6tique humain. L'analyse compar6e de ces deux types de transfectants a permis de montrer que la stimulation des antig/mes du CMH murin de classe I n'est observ6e que dans le transfectant contenant le chromosome 21 humain. De plus, nous avons analys6 d'autres propri6t6s de rIFN~., telles que la protection antivirale et l'induction transcriptionnelle de g6nes r6gul6s par les IFN. A nouveau, ces

772

M.R. BONO ET AL.

activit6s n'ont pu ~tre d6cel~es que pour le transfectant contenant le chromosome 21 humain. Mots-cl~s: IFN'r, Chromosome 21, Transfection, Transduction; Signal, Protection antivirale, Hybrides cellulai~'es somatiques, Induction.

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