GB2280186A

GB2280186A - Human MHC Class II transgenes

Info

Publication number: GB2280186A
Application number: GB9414833A
Authority: GB
Inventors: Joanne Lesley Whittaker; John Edward Norris Morten
Original assignee: Zeneca Ltd
Current assignee: Syngenta Ltd
Priority date: 1993-07-23
Filing date: 1994-07-22
Publication date: 1995-01-25
Also published as: GB9414833D0; WO1995003331A1; EP0710250A1; JPH09501315A; AU7231494A; GB9315303D0

Abstract

There is disclosed a human MHC Class II HLA alpha, beta-DR double transgene, preferably selected from the DR1, DR4 and DR10 sub-types, together with a human beta HLA-DR4Dw4 transgene, which may be used in the transfection of non-human animals. The polypeptide, expressed by the double transgene, may be used in the identification of agents which modulate the production or release of an immunologically-active mediator, involving formation of a complex of polypeptide, putative agent and a factor, which complex interacts with T cells, thereby releasing the mediator. The chromasomal integration site of a transgene, especially alpha HLA-DR4Dw4, may be determined, in a non-human mammal, by in situ fluorescence hybridisation.

Description

POLYPEPTIDE Hajor histocompatibility complex (HHC) class II gene products are cell surface heterodimeric glycoproteins composed of 34 kilodalton a- and 29 kilodalton 6-chains. These proteins play a crucial role in the immunological processes of self-tolerance, HHC restriction and antigen presentation. Each of these processes involves the interaction of NHC molecules with antigen, T cell a-ss receptors (TCRs) and accessory molecules such as CD4. The relationship between these interactions and the biology of tolerance induction and NHC-restricted antigen presentation is, as yet, not fully understood.

Both HHC and TCR gene products are characterised by extensive structural complexity (Natural History of the Hajor Histocompatibility Complex, 1986, J. Klein - Wiley & Sons). An important functional consequence of this complexity is diversity in antigen recognition. In the evolution of independent mammalian species, HHC and TCR complexes have diverged significantly in gene structure, number and regulation.

Lawrance et al (Cell, 1989, 58, 583-594) report that despite considerable divergence between the amino acid sequences of the human DR-a and murine IEa-chains of HHC class II gene complex it is possible to construct transgenic DRa-Ess mice. Such mice express the human DRa gene with no apparent modulation of immune function.

Nishimura et al (J. Immunology, 1990, 145, 353-360) describe a line of transgenic HLA-DQw6 mice. A mixture of DQw6A and DQw6B DNA was injected into 40 fertilised eggs from C57BL/6 mice. Four neonate pups were obtained out of which one mouse carried both the DQw6A and DQw6B genes. However despite the desirability of further HHC class II double transgenes there have been no further reports of these.

In a first aspect of the present invention we now provide a transgenic non-human mammal which is a,ss-double transgenic for an mIC class II HLA-DR gene complex.

Examples of convenient HHC class II HLA-DR gene complexes include those disclosed by Kappes and Strominger in Ann. Rev.

Biochem., 1988, 57, 991-1028; Narsh and Bodmer-in "Gene Registry, HLA class II nucleotide sequences, 1991"; Lanchbury in Clinical and Experimental Perspectives in Cyclosporin Therapy, 1992, 2 (1), 15-17; Uordsworth et al in Am. J. Hum. Genet., 1992, 51, 585-591 and P.N.A.S., 1989, 86, 10049-10053. Such complexes include DR1, DRwlO and DR4 subtypes including DR4Dw4, DR4Dw13, DR4Dw14.1, DR4Dw 14.2 and DR4Dw15. A particular complex is DR4Dw14. A further particular complex is the human HLA-DR4Dw4-a,ss complex.

The mammal is any convenient non-human mammal, for example as used in laboratory test procedures, such as rodents for example mice or rats, more conveniently mice.

The transgenic non-human mammal of the invention may be either heterozygous or homozygous for the desired a,ss-transgene, but is conveniently homozygous.

A preferred non-human mammal of the invention is a mouse transgenic for the human HLA-DR4Dw4-a,ss complex, and preferably homozygous for the a and ss transgenes.

The transgenic non-human mammal of the invention is conveniently obtained by mating appropriate individual non-human mammals who are a- transgenic and ss-transgenic respectively for the desired HHC class II DR gene complex. Conventional mating and breeding procedures may be employed. The desired double transgenic progeny are preferably back-crossed into a background strain which does not have any significant deletion in either the a- or ss- gene.

This is to minimise any unwanted human/non-human gene pairings and to promote reliable expression of the desired human a,ss-transgene.

The level of a,B-transgene expression is conveniently at least l/lOth that of mouse HHC class II and more conveniently the same.

In a further aspect of the present invention we now provide a human ss-DR4Dw4 transgene.

The transgenic non-human mammals of the invention have a number of uses including the study of any aspect of the immune system relating to MMC class II as well as drug and vaccine assays for example to determine the interaction of compounds with these cell surface proteins. By way of example tissue can be removed and (i) cultured (eg. "Culture of Animal Cells - A Manual of Basic Technique" - R. Ian Freshney, A.R. Liss Inc. New York), (ii) used in assays, (iii) immortalised in cell lines using procedures such as those outlined by Caroline MacDonald in Critical Reviews in Biotechnology, 1990, 10 (2), 155-178 or (iv) used to prepare stem cells such as those outlined in "Teratocarcinomas and Embryonic Stem Cells - a practical approachs Ed. E.J. Robertson, IRL Press.

In a further aspect of the invention we provide a mammalian cell line which is heterozygous, homozygous or chimeric for expression of an MHC class II cx,g-double transgene. Examples of convenient MMC class II a,ss-double transgenes include a,ss-double transgenes of the DR class of sequences, especially of DR1; the Dw class of sequences, especially of Dw4, Dw14.1 and Dw 14.2. A particular a,ss-double transgene is of DR4Dw14. A more particular transgene is the a,ss-double trans gene of human HLA-DR4Dw4.

Any convenient cell line may be used. These include those disclosed as above such as immortalised cell lines. The cell line may be pluripotent and/or totipotent.

According to a further aspect of the present invention we provide a polypeptide expressed by the a,-double transgene of the invention according to any preceeding aspect of the invention.

It is known that DR MHC class II gene products expressed by the transgenic non-human mammals of the present invention may interact with other factors to induce proliferation in human T cells.

Therefore in a further aspect of the present invertion we provide a method for the identification of compounds which modulate the production or release of an immunologically active mediator which method comprises contacting a test compound with a polypeptide expressed by the a,ss-double transgene of the invention and a factor which binds to the polypeptide to form a complex, which complex interacts with T cells and leads to the production or release of the immunologically active mediator, and detecting any modulation of production or release.

Whilst we do not wish to be bound by theoretical considerations the test compound may modulate the production or release of the immunologically active mediator by inhibiting binding of the polypeptide to the factor or any subsequent event.

Examples of convenient immunological events which may be used to detect any modulation of production or release of an immunologically active mediator include T cell proliferation, antibody production, and an inflammatory response.

Any convenient test compound may be used. For control purposes or otherwise, antibodies such as monoclonal antibodies may be used to block the polypeptide expressed by the ,double transgene.

The factor is conveniently a peptide, conveniently a synthetic peptide. A particular peptide for use in the above method in conjunction with the HLA-DR4Dw4 trans gene is FHA307-320 (Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr-Gly).

The invention will now be illustrated but not limited by reference to the following Figures and Examples wherein: Figure 1 shows the construction of the DRa clone T9C. The boxes represent exons of the DRa gene and the arrow indicates the direction of transcription. 5'SS is the signal sequence. al and a2 are the extracellular domains. TC are the transmembrane and cytoplasmic domains. 3'UT is the 3' untranslated region. ssl is an exon of the DRss gene. Restriction maps for the EcoRI, BamHI and SalI restriction enzymes are also shown. The 22kb BamHI-SalI fragment, including the 5 DRa exons, was microinjected into mouse oocytes to generate a DRa transgenic.

Figure 2 shows the generation of the DRss construct. The boxes represent exons of the mouse lEa construct and the arrow indicates the direction of transcription. L is the leader peptide.

and 2 are the extracellular domains. TC are the transmembrane and cytoplasmic domains. 3'UT is the 3' untranslated region. Restriction sites for the BamH1 and XbaI restriction enzymes are also shown. The human DRss cDNA was cloned into the first exon of the mouse IEa gene, between the transcription initiation site and the translation start codon.

Figure 3(a) shows an autoradiograph of a Southern Blot including T9C DNA, liver DNA from non-transgenic mice, BSH DNA and DNA from the tails of transgenic and non-transgenic progeny hybridised to DRa cDNA.

Figure 3(b) shows an autoradiograph of a Southern Blot including UE32ss DNA, liver DNA from non-transgenic mice and DNA from transgenic and non-transgenic progeny hybridised to DRss cDNA.

Figure 4(a) shows an autoradiograph of a Northern Blot including RNA from liver, spleen, lung and kidney of DRa transgenic 1530 and RNA from BSM cell line hybridised to DR-a cDNA.

Figure 4(b) shows an autoradiograph of a Northern Blot including liver, lung, spleen and brain RNA from DRss transgenic 1499; liver, lung and heart RNA from DRss transgenic 1502, plus RNA from BSM cell line hybridised to DRss oligonucleotide.

Figure 5 shows the FACS profiles of blood cells from a mouse containing HLA-DR4Dw4 and trans genes stained with antibodies directed towards HLA-DR (MAb L243) and mouse MMC Class II protein (MAb 10-3.6-2L Figure 6 shows two FACS histograms of spleen cells stained with MAb L234 plus phycoerythrin-conjugated goat anti-maouse IgG (Fc)FITC followed by FITC-conjugated rat-anti-IA (Serotec clone) or FITC-conjugated rat-anti-Thy- (Serotec clone) MAbs. The expressing I-A molecules also express HLA-DR4Dw4 molecules (population B of Figure 6a), while the HLA-DR4Dw4-negative cells also do not express I-A molecules. Furthermore, mouse T cells (CD3-positive spleen cells) which do not express mouse MHC Class II Molecules, also do not express HLA-DR4Dw4 molecules (Population B of Figure 6b).

Figure 7 shows the responses of LN T cells obtained from 10 individual mice (5 HLA-DR4Dw4-transgenic mice; 5 non-transgenic littermates) immunised with FHA307-320. It can be seen that addition of FHA307-320 to primed LN T cells from HLA-DR4Dw4-transgenic mice produces a dose-dependent proliferative response, whereas primed LN T cells from non-transgenic littermates do not proliferate when FHA307-320 is added.

Figure 8 shows the effect of anti-HLA-DR monoclonal antibodies on recognition of FHA307-320 by primed T cells from HLA-DR4Dw4-transgenic mice. Spleen cells and primed LN T cells were prepared as described for Example 4, and a stimulatory concentration of FHA307-320 (10 yM) was added. Monoclonal antibody L243 which binds HLA-DR4Dw4 molecules, but not mouse MHC Class II molecules were also added, and the cultures incubated, harvested and counted as described.

It can be seen that addition of MAb L243 completely inhibits prolifereation of primed LN T cells from transgenic mice. In contrast, a control monoclonal antibody (MKD6) which would not react with HLA-DR4Dw4 molecules has no inhibitory activity, showing that the effect is specific to HLA-DR4Dw4.

Figure 9 shows that HLA-DR4Dw4 transgenic mice produce recall delayed type hypersensitity responses to the HLA-DR4Dw4-binding peptide FHA307-320. It can be seen that administration of an HLA-DR4Dw4-binding antagonist peptide to HLA-DR4Dw4 transgenic mice results in measurable inhibition of the delayed type hypersensitivity response. In contrast, an antagonist of mouse MHC Class II molecules (SNase61-80) demonstrated no inhibitory activity. These results confirm that the rsponse is HLA-DR4Dw4 restricted, and that this method can be used to detect HflA-DR4 antagonist peptides.

GENERATION OF TRANSGENIC MICE EXPRESSING HUMAN HLA DR4Dw4 Generation of a transgenic mouse which expreses human HLA DR4Dw4 requires the addition of two human genes, a nonpolymorphic a gene and a polymorphic ss gene.

Source of the human DRa gene A cosmid clone, T9C, containing the whole of the human DR4 a gene was provided by T. Spies (Spies et al., Proc. Natl. Acad. Sci.

USA 82:5165 1985). A 22Kb BamH1 to SalI fragment containing the 7Kb gene and 5' and 3' flanking sequence was generated from T9C (Figure 1) and isolated from agarose gels for microinjection (cf. Hogan et al., Manipulating the mouse embryo - A laboratory manual, Cold Spring Harbour Laboratory Press, 1986) at a concentration of 2nag/1.

Construction of a human DR4Dw4 ss cDNA construct Human DR4Dw4 ss chain cDNA was amplified from a human ss-lymphoblastoid cell line (BSH - a transformed Epstein-Barr virus obtained from R. Bolhuis, ETNO, Rijswijk, NL). Total RNA was prepared from BSN cells and Poly A+ RNA selected by passing 50pug of total RNA down an oligonucleotide dT cellulose column. Sug of polyA+ RNA was used as a template to generate double stranded cDNA. The cDNA was then used in a PCR reaction with oligonucleotides 238 (GTCGAATTCTCCAGCATG & TGTGTCTGAAG) and 239(CATGTCGACTTCAGCTCAGGAATCCTGTTG) to generate an 820 bp product.

This was subsequently cloned between the EcoRI and SalI sites of the bacteriophage cloning vectors n13mp18 and n13mp19 (Boehringer Mannheim) and sequenced. Double standed (RF) DNA was prepared from the mp18 clone, and the insert subcloned into eukaryotic expression vector pNSGCNVa [pNSG - Pharmacia, modified by (i) removal of the MNTV-LTR and replaced by a cytomegalovirus (CHV) immediate early promoter/enhancer (as disclosed by W. Schaffner, Cell, 41, 521-530 and isolated on a 619 bp ThaI fragment from pcm5029 - Gene, 45, 101-105) and (ii) replacement of gpt by the pSV2 neo marker provided by P. Berg (J. of Hol. and App. Genetics, 1982, 1, 327-341)]. The following linker was then ligated at the EcoRI site 5' to the cDNA AATTAGATCT TCTAGATTAA thus generating a BglII site. The construct comprising the DRss cDNA, SV40 splice site and poly A recognition sequence was purified on a BglII to BamHI fragment and susequently subcloned into the BamHI site of plasmid pWE32 provided by D. Hathis, Stanford University (Dorn et al, Proc. Natl. Acad. Sci.USA, 1987, 84, 6249-6253). pWE32 contains the mouse IE gene modified, for cloning purposes, by removal of a BamHI site from the first intron and introduction of a BamHI site in the untranslated region of the first exon. The IEa regulatory sequences and gene now including the DRIPS cDNA in the first exon (Figure 2) were isolated by BglII digestion.

The 9.4 Kb fragment was then gel purified and resuspended at 2ng/yl in TE (0.5 mM Tris - pH 7.5, 0.1 mM EDTA) for microinjection.

Generation of Transenic mice All manipulations were performed as described by Hogan et al., Manipulating the mouse embryo - a laboratory manual, Cold Spring Harbour Laboratory Press, 1986.

CBA-Ca x C57B1/6 (F1) female mice (ZENECA - Alderley Park) were superovulated and mated with F1 males. Fertilzed oocytes were microinjected with either the 22Kb BamHI - SalI fragment containing the DRa gene, or the 9.4 Kb BglI fragment containing the DRB gene and incubated overnight. Viable embryos were transferred to the oviducts of pseudopregnant F1 females (mated with vasectomised males) and allowed to develop to term.

Analysis of Transgenic mice The resulting progeny were analysed to determine whether the transgene was present. Approximately 0.5cm of tail was obtained from each animal, sliced with a scalpel blade, and digested overnight at 550C in 0.5 ml lysis buffer (100 mm Tris.HCl pH 8.5, 5mM EDTA, 0.26 SDS, 200 mM NaCl, 100 g Proteinase K/ml. The solution was extracted with phenol/chloroform and DNA was precipatated. DNA was precipitated with an equal volume of isopropanol, washed in 70% ethanol and resuspended in 500 yl TE (0.lM Tris pH8, 0.01M EDTA) at 550C. PCR analysis using DRa or DRB specific primers was used to identify transgenic animals. Approximately 100 ng of tail DNA were used in PCR reactions with Taq polymerase (Cetus - Amplitaq) using conditions described by the manufacturer (Perkin-Elmer Cetus) on a Techne thermal cycling machine. The amplimers used were: DRa 5'AAGAACATGTGATCATCCAGGCC (EXON), 5' CCCCAGTGCTTGAGAAGAGGCTCA (EXON 2) at 920C for 2 min, 650C for 2 min and 720C for 2 min for 35 cycles giving a 1018 bp product.

DRss 5'GAGATGCATCCAGCAATAAGGAG ( -500 bp in 5' flanking sequence of mouse IEa), 5' TGCACTGTGAAGCTCTCACCAAC (374 bp from translation start site of human DR4Dw4 ss cDNA) at 920C for 2 min, 640C for 2 mins, 720C for 2 min for 35 cycles giving' an 886 bp product.

Transgenic animals were confirmed by Southern analysis of EcoRI digested tail DNA. DNA (10 yG) was transferred to Hybond membrane (Amersham) using 20 x SSC (3M NaCl, 0.3 M tri-sodium citrate - Na C H 0 .2H20), W fixed, prehybridised and hybridised according to the manufacturers recommendations with a 32P labelled probe of either the DRa or DRIPS cDNA using standard protocols. The results are shown in Figures 3 and 4.

Founder animals Six 'founder' DRa animals were identified, two transmitted the transgene with high frequency (DRaO8 and DRalO) a third animal (DRal2) produced transgenic offspring although the litter size was small. Of the other three 'founders' two did not transmit (DRal9 and DRa93) and one gave rise to progeny which appeared to contain rearranged sequences (DRα21). DRα08 was chosen to establish a transgenic line and further reference to DRa relates to DRα0.

Microinjection of the DRss construct resulted in 3 'founders', B325, ss380 and g356. ss325 and ss380 did not transmit the transgene to their progeny. A line was therefore established from mouse/human hydrid ss356 and further reference to DRIPS relates to this line.

Northern Analysis Total RNA was extracted from mouse tissues using the method of Chirgwin et. al., Biochemistry 1979, 18 No. 24: 5294. 10 g of RNA were loaded onto formaldehyde gels and electrophoresis carried out at 100 volts for 4 hours. RNA was blotted onto Hybond membrane (Amersham) using 20 x SSC and W fixed. Prehydridisation and hybridisation were carried out in either 0.5M sodium phosphate pH 7.2, 7%SDS, lmM EDTA (for DRa cDNA probe) or 0.144 M Na HPO 0.056M 2 PO 4 NaH2P04, lmM EDTA, 1% BSA, 7.2t SDS, 15 deionised formamide (for DRB oligonucleotide probe probe 5'ACTGTGAAGCTCTCACCAACCCCG). Highest levels of DRa and DRss expression were found in spleen and mg.

Fluorescent in situ hybridisation (FISH) Integration of the transgenes in the mouse genome was observed by fluorescent in situ hybridisation (FISH). Either cosmid T9C DNA of pWE32 was biotinolated (BRL- Bionick Kit, according to the manufacturers instructions). Metaphse chromosome spreads from fibroblasts of transgenic animals were treated with RNase (100 yg/ml in 2xSSC) for 60 minites at 370C, washed and dehydrated through an alcohol gradient, denatured in 70% formamide, 2xSSC at 750C for 3-5 minutes and dehydrated (denaturation may also be performed at 900C for 5 minutes). 100 ng of labelled DNA was dissolved in 25 yl of hydridisation mix (10% dextran sulphate, 50% formamide, 2xSSC, 0.1 mM EDTA, 0.05mM Tris pH7.5, 100 yg/ml denatured salmon sperm DNA) and denatured at 800C for 10 minutes. Denatured probe was hybridised to the slides under a coverslip at 370C for 48 hours. Slides were washed 3x5 minutes in 50% formamide/2xSSC pH7.0, at 420C and 3x5 minutes in 2xSSC at 420C and blocked for 60 minutes with 5% non-fat dried milk (Marvel - Premier Brands UK Ltd) in 4xSSC, 0.05k Triton X 100.

Detection was by sequential incubation in fluoresicein isothiocyanate (FITC - Vector Laboratories) - avidin (DCS - D cell sorter grade) followed biotinylated anit-avidin (Vector Laboratories), both at 5;ig/ml.in 4xSSC pH7.0, 0.05% Triton-x 100, 3%BSA, at 370C for 20 minutes with 3x3 minute washed in 4XSSC/0.056 TRigton between each layer. After 304 layers of avidin, slides were washed in phosphate buffered saline (lxPBS -0.137 mM NaCL, 2.7mM KC1, 8.1 mM Na2HPO4.2H20, 1.5 mM KH2P04 - pH 7.4) counter stained with propidium iodide and mounted in 90% glycerol 20 mM Tris pH 8.0, 2.3 1,4-diazobicyclo-[2.2.2)octane (DABCO]. The DRn transgene was seen to have integrated at a single site in one of the mouse chromosomes. DRa however, was detected in two different mouse chromosomes, one proximal to the centromere and one distal. DRa transgenics were crossed with CBA's to segregate the two integration events into separate lines.

Each was tested by fluorescence antibody cell sorter (FACS) analysis.

The distral integration site expressed at a higher level than the proximal integration site.

αss crosses DRa08 transgenic animals were mated with DRss356 animals to generate progeny carrying both transgenes.

Example 2 DETECTION OF HLA-DR4Dw4 PROTEINS EXPRESSED BY BLOOD CELLS FROM HLA-DR4Dw4 TRANSGENIC MICE.

Blood from an incision in the tail was drawn into a 100p1 heparinised tube (BDH), and immediately blown out into lml of ice cold PBS. The sample was then centrifuged (2000rpm for 5 minutes), the pellet resuspended in 200p1 of ice cold PBS containing 20X normal rabbit serum plus 0.2X sodium azide, and 50ul portions pipetted into LP4 tubes (Luckham). After 15 minutes incubation in a refrigerator, 50p1 of pre-titrated monoclonal antibody-containing supernatant diluted (typically 1:5) in PBS + 20Z normal rabbit serum + 0.2X sodium azide was added to the cell suspension. After a further 20-45 minute incubation in the cold, the cells were washed by addition of 450us of cold PBS + 2Z foetal calf serum (FCS) + 0.2Z sodium azide followed by centrifugation (2000rpm for 5 minutes) at 40C. The supernatant was then decanted by suction, and the pellet resuspended by vortexing for 2-3 seconds. SOul of FITC-conjugated Goat anti-mouse IgG F(c) antibody (Serotec) (preabsorbed for 30 minutes with 50Z mouse -spleen cell suspension) diluted 1:75 in PBS + 20Z rabbit serum + 0.2Z sodium azide was added, and the pellet resuspended by vortexing.

After 20 minutes incubation in the cold, the cells are washed twice in PBS + 2Z FCS + 0.2Z sodium azide. Erythrocytes are removed by addition of FACS Lysis Buffer (Becton Dickinson - according to the manufacturers instructions), and the fluorescing cells enumerated on a FACS-SCAN flow cytometer (Becton Dickinson).

Figure 5 shows the FACS profiles of blood cells from a mouse containing HLA-DR4Dw4 and transgenes stained with antibodies directed towards HLA-DR (MAb L243 - ATCC HB55) and mouse MHC Class II protein (MAb 10-3.6.2 - ATCC TIB92). It can be seen that similar numbers of cells from HLA-DR4Dw4 transgenic mice are stained with antibodies to mouse and human MHC Class II proteins, respectively.

Example 3 DETECTION OF HLA-DR4Dw4 PROTEINS EXPRESSED BY SPLEEN CELLS FROM TRANSGENIC MICE.

Spleens were excised from transgenic mice, placed in a 50mm diameter sterile plastic petri dish containing 3 ml of cold sterile PBS, and cut into small (approx 3mm) pieces. The pieces were squashed using the plunger of a 2ml plastic syringe (Asik) to release the spleen cells, and a further 7ml of ice cold PBS were added using a lOml pipette. The suspension was pipetted up and down vigorously ten times, and the mixture filtered through a Cell Strainer (Becton Dickinson) into a 25ml sterile glass beaker to remove debris. The cell suspension was poured into a 15ml sterile plastic conical tube (Falcon), and centrifuged at room temperature (1400rpm for 5 mins).

The cell pellet was then resuspended in 3ml PBS, and 2ml of Lympholyte H (Jackson Labs) layered underneath. The cells were centrifuged at 2000rpm for 15 mins at room temperature, and the cells at the interface representing the mononuclear population were then collected and diluted into lOml ice cold PBS in a 15ml conical tube. The cells were washed twice in PBS before being counted.

For staining, cells were resuspended (10-20x106/ml) in PBS + 20X rabbit serum + 0.2Z sodium azide, and processed as disclosed in Example 2, except that the treatment with FACS Lysis Buffer was omitted.

From the profiles shown in Figure 6, it can be seen that similar numbers of spleen mononuclear cells express HLA-DR molecules and mouse I-A molecules. To further characterise the cells expressing HLA-DR molecules, spleen cells were stained with MAb L243 plus phycoerythrin-conjugated goat anti-mouse IgG (Fc) followed by FITC-conjugated rat-anti-I-A (Serotec clone MCA 46F) or FITC-conjugated rat-anti-CD3 (Serotec MCA 500F) NAbs. The two colour FACS histograms show that the spleen cells expressing I-A molecules also express HLA-DR4Dw4 molecules (population B of Figure 6a), while the HL -DR4Dw4-negative cells also do not express I-A molecules.

Furthermore, mouse T cells (CD3-positive spleen cells) which do not express mouse MHC Class II molecules, also do not express HLA-DR4Dw4 molecules (Population A of Figure 6b).

Thus, the pattern of expression of the HLA-DR4Dw4 transgenes in lymphoid cells of the spleen is similar to that of endogenous MHC Class II products.

Example 4 IN VITRO RESPONSES OF T CELLS FROM PEPTIDE-INNUNISED TRANSGENIC NICE.

In order to examine the function of the HLA-DR4Dw4 transgene products, mice were immunised with 10nmol of peptide FHA307-320 (Sequence : Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr-Gly) mixed 1:1 in Complete Freund's Adjuvant (Difco Labs). O.lml of the mixture was administered sub-cutaneously in a single site in the flank using a lml plastic syringe and 23 gauge needle. Mice were sacrificed 7-10 days following immunisation with peptide, and the enlarged draining ingunal lymph node, and spleen were dissected from each mouse.

Lymph node (LN) cell suspensions were prepared by passing through a cell s warm culture medium, counted, and further adjusted to a density of 4x106/ml.

Spleen mononuclear cell suspensions were prepared as described in Example 3. In some experiments, spleen cells were treated with anti-Thy-1 plus complement to deplete T cells.

Specifically, spleen mononuclear cells were suspended (5xl06/ml) in culture medium containing a 1:10,000 dilution of anti-Thy-l HAb (Serotec) for 30 minutes, washed once in cold PBS, and resuspended (5xl06/ml) in Low-tox rabbit complement (Serotec) for 45 minutes at 37"C. The cells were washed twice with warm culture medium, resuspended in 2ml warm culture medium, counted, and adjusted to a density of 4x106/ml.

To assess the degree of T cell responses, LN T cells (50p1/well) and spleen cells (50p1/well) are added to 1001 of medium containing peptides (eg. FHA307-320) at the indicated concentrations in 96-well microtitre plates (Nunc), and incubated for 4 days at 370C in a humidified atmosphere of 5X CO2 in air. Responding T cells are 521 quantified by adding lCi/ml 3H-thymidine (Amersham) for 6 hours, and the plates harvested and counted on a Beta-plate counter (LKB).

-Results are expressed as the mean (+SEM) of triplicate cultures.

Peptide FHA307-320 is known to bind to HLA-DR4Dw4 and induce proliferation in primed human T cells of appropriate specificity.

Example 5 EFFECT OF ANTI-HLA-DR HONOCLONAL ANTIBODIES ON RECOGNITION OF FHA307-320 BY PRIMED T CELLS FROM HLA-DR4DW4-TRANSGENIC MICE.

To confirm that the proliferation of primed LN T cells from transgenic mice observed on addition of FHA307-320 is stimulated by HLA-DR4Dw4, monoclonal antibodies able to block the interaction of T cells with HLA-DR4Dw4 were added. Spleen cells and primed LN T cells were prepared as described for Example 4, and a stimulatory concentration of FHA307-320(1opM)was added. Honoclonal antibody L243 which binds HLA-DR4Dw4 molecules, but not mouse MMC Class II molecules was also added, and the cultures incubated, harvested and counted as described above. The results are shown in Figure 8; from which it can be seen that addition of MAb L243 completely inhibits proliferation of primed LN T cells from transgenic mice. In contrast, a control monoclonal antibody (MUD6 - ATCC HB3) which would not react with HLA-DR4Dw4 molecules has no inhibitory activity, showing that the effect is specific to HLA-DR4Dw4.

Example 6 EFFECT OF HLA-DR4DV4-SPECIFIC BLOCKING PEPTIDES ON RECOGNITION OF FHA307-320 BY PRIMED T CELLS FROM HLA-DR4DV4-TRANSGENIC MICE.

A further proof that the proliferative response of primed In T cells to FHA307-320 is due to the presence of HLA-DR4Dw4 in transgenic mice is the demonstration that the response is blocked by peptides which bind to HLA-DR4Dw4 molecules and are able to displace the stimulating FHA307-320 peptide. Primed LN T cells were prepared as described for Example 4.

In these experiments, spleen cells were prepared as for Example 4, and "fixed" with gluteraldehyde prior to addition of peptides. Specifically, anti-Thy-l plus complement-treated spleen mononuclear cells (as prepared in Example 4 above) were washed in PBS, and resuspended at 5x10 6cells/ml for 30 seconds in 0.5Z gluteraldehyde. An equal volume of 0.4 mM lysine in PBS is added for 3 minutes, and the cells pelleted and washed twice in culture medium.

The fixed cells were resuspended to 4x106/ml in serum-free tissue culture medium (RPNI-1640 medium, Gibco), and 100p1 added to 100p1 of serum-free RPMI1640 medium containing 1iiM FHA307-320 and blocking peptides at concentrations between 0.1-100H in 96-well microtitre culture plates. After 2 hours incubation, the-cells were pelleted, the medium removed by suction, and 200p1 of culture medium added. The cells were again pelleted, the' medium removed, and a further 200p1 of medium added. After pelleting the cells and removing the medium once more, 2x105 primed LN T cells were added in 200p1 of warm culture medium. Cultures are then treated as described in Example 4.

A number of HLA-DR4Dw4-binding peptides are able to inhibit the presentation of FHA307-320 to primed In T cells from transgenic mice. In contrast, peptides able to compete for binding to mouse MMC Class II molecules (I-Ak ; 1-Ek) do not prevent activation of primed LN T cells by FHA307-320. This will confirm that presentation of FHA307-320 is mediated solely by binding to HLA-DR4Dw4 in transgenic mice.

Example 6 INHIBITION OF IN VIVO RESPONSES IN HLA-DR4DU4-TRANSGENIC MICE BY HLA-DR-ANTAGONIST PEPTIDES The ability of antagonist peptides to inhibit binding of stimulatory peptides to HLA-DR4Dw4, and subsequent activation of T cells in vivo can be tested in HLA-DR4Dw4-transgenic mice essentially according to the method of Adorini et al (1988; Nature 334:623). Transgenic mice are immunised with a mixture of 10 nmoles of FHA307-320 mixed with an effective amount (eg 1-500 nmoles) of the antagonist peptide in 1001 of a 1:1 mixture of Freund's Complete Adjuvant and PBS. After 7-14 days, mice are killed, and spleen and LN T cell suspensions made and assayed for reactivity to 1M FHA307-320 as described in Example 4.

HLA-DR4Dw4 antagonist peptides are able to inhibit the priming of LN T cells in transgenic mice, demonstrating that they are able to antagonise the function of HLA-DR4Dw4 in vivo.

INHIBITION OF IN VIVO DELAYED TYPE HYPERSENSITITY RESPONSE IN HLA-DR4Dw4 TRANGENIC MICE BY HAL-DR-ANTAGONISTS PEPTIDES The ability of antagonist peptides and non-peptides to inhibit binding of stimulatory peptides to HLA-DR4Dw4, and subsequent activation of T cells in HLA-DR4Dw4 transgenic mice can also be tested by the following method. HLA-DR transgenic mice are immunised with a mixture of an effective amout of FHA307-320 (100 ng in this example) in 109 mictrolitres of a 1:1 mixture of Freund's Complete adjuvant and PBS. After 7-14 days, approximately 30 microlitrues of a 100 micromolar solution of FHA307-320 is injected sub-plantar into one hind foofpad, and the resultant immune response determined by measuring foot swelling wit hmicronic calipers. HLA-DR4Dw4-binding peptides administered either by admixing with the sub-plantar challenge injection, or given by other standard route, or by slow-release depots such as osmotic mini-pumps can be shown to inhibit the swelling induced by FHA307-320, showing that they are able to antagonise the function of HLA-DR4Dw4 in vivo (Figure 8).

SEOBENCS LISTING (1) GENERAL INFORMATION (i) APPLICANT: ZENECA Limited (ii) TITLE OF INVENTION: POLYPEPTIDES (iii) NUMBER OF SEQUENCES: 10 (iv) CORRHSPONDENCE ADDRESS: (A) ADDRESSEE: Group Patents Services Department (B) STREET: Bessemer Road (C) CITY: Welwyn Garden City (D) STATE: Hertfordshire (E) COUNTRY: United Kingdom (F) ZIP: GB-AL7 1HD (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 mB storage (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS 3.20 (D) SOFTWARE: ASCII from WPS-PLUS (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NO. GB 9315303.9 (B) FILING DATE: 23-Ju1-1993 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GTCGAATTCT CCAGCATGGT GTGTCTGAAG 30 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CATGTCGACT TCAGCTCAGG AATCCTGTTG 30 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AATTAGATCT 10 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TCTAGATTAA 10 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AAGAACATGT GATCATCCAG GCC 23 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CCCCAGTGCT TGAGAAGAGG CTCA 24 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GAGATGCATC CAGCAATAAG GAG 23 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGCACTGTGA AGCTCTCACC AAC 23 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ACTGTGAAGC TCTCACCAAC CCCG (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly

Claims

Claims: 1. A human MHC Class II HLA alpha,beta-DR double transgene.
2. A human alpha,beta double transgene as claimed in claim 1 and selected from the DR1, DR10 and DR14 subtypes.
3. A human alpha,beta double transgene as claimed in claim 2.which is DR4Dw4.
4. A non-human mammal which comprises a human alpha,beta double transgene as claimed in any one of claims 1-3.
5. A non-human mammal as claimed in claim 4 which is a mouse.
6. A non-human mammal as claimed in claim 4 or claim 5 wherein the level of expression of the gene product of the human alpha,beta double transgene is at least 10t of that of the corresponding mammalian gene.
7. A mammalian cell line capable of expressing a human alpha,beta double transgene as claimed in any one of claims 1-3.
8. A polypeptide which is the gene product of a human alpha,beta double transgene as claimed in any one of claims 1-3.
9. A method for the identification of compounds which modulate the production or release of an immunologically active mediator which method comprises contacting a test compound with a polypeptide as claimed in claim 8 and a factor which binds to form a complex, which complex interacts with T cells and leads to the production or release of the immunologically active mediator, and detecting any modulation of production or release of the mediator.
10. The use of a human transgene as claimed in any one of claims 1-3 in the identification of compounds which modulate immune activity by interacting with the transgene and are useful in the treatment of disease in which MHC Class II molecules play a pathophysiological role.
11. The use of a human transgene as claimed in any one of claims 1-3 to identify a peptide antagonist of that human HLA-DR gene.
12. The use of a human transgene as claimed in any one of claims 1-3 to identify a non-peptide antagonist of that human HLA-DR gene.
13. A human beta HLA-DR4Dw4 trazisgene.
14. A non-human mammal which comprises a human transgene as claimed in claim 13.
15. A construct for expression of the human transgene as claimed in claim 13 which comprises DNA for expression of human beta DR4Dw4 under the control of a tissue specific promoter.
A A construct as claimed in claim 15 wherein the DNA for expression of human beta DR4Dw4 is inserted in plasmid pWE32.
17. The use of fluorescence in situ hybridisation to determine the chromosomal integration site(s) of a transgene in a non-human mammal.
18. A non-human mammal which comprises an HLA-DR transgene whose chromosomal integration site(s) has been selected for increased expression of the transgene using the method of claim 17.
19. A non-human mammal as claimed in claim 18 wherein the transgene is alpha HLA-DR4Dw4.
20. A method for the preparation of a human HLA-DR alpha,beta double transgene which method comprises mating appropriate alpha and beta non-human mammals at least one of which is as claimed in claim 17 or 18 and isolating said double transgene from the resulting progeny.
21. A method as claimed in claim 20 wherein a non-human mammal as claimed in claim 19 is mated with a non-human mammal as claimed in claim 14 to yield a transgene as claimed in claim 3.
22. A human double transgene substantially as hereinbefore described with reference to the description and Figures.
23. A non-human mammal substantially as hereinbefore described with reference to the description and Figures.
24. A mammalian cell line substantially as hereinbefore described with reference to the description and Figures.
25. A polypeptide substantially as hereinbefore described with reference to the description and Figures.
26. A method for the identification of compounds substantially as hereinbefore described with reference to the description and Figures.
27. The use of a human double transgene substantially as hereinbefore described with reference to the description and Figures.
28. A construct for expression of a human double transgene substantially as hereinbefore described with reference to the description and Figures.
29. The use of fluorescence in situ hybridisation substantially as hereinbefore described with reference to the description and Figures.