Expression of a hybrid immunoglobulin-T cell receptor protein in transgenic mice

Cell, Vol. 58, 91 l-921,

September

8, 1989, Copyright

0 1989 by Cell Press

Expression of a Hybrid ImmunoglobulinT Cell Receptor Protein in Transgenic Mice Michael L. 6. Becker,’ Richard Near,7 Meredith Mudgett-Hunter,7 Michael N. Margolies,* Ralph T. Kubo,li Jonathan Kaye: and Stephen M. Hedrick’ *Department of Biology and Cancer Center University of California at San Diego La Jolla, California 92093 TMassachusetts General Hospital Cellular Molecular Research Laboratory Boston, Massachusetts 02114 *Department of Surgery Massachusetts General Hospital Boston, Massachusetts 02114 IIDepartment of Medicine National Jewish Center for Immunology and Respiratory Medicine Denver, Colorado 80206 and Departments of Microbiology and Immunology and Medicine University of Colorado Health Sciences Center Denver, Colorado 80262

Summary We have constructed a hybrid immunoglobulin (Vl&)T cell receptor (C,) gene using the VDJH exon from a digoxin-specific antibody. This gene was used to make a line of transgenic mice. The hybrid V&-C, protein is expressed on a subset of T cells in these mice, and we have shown that it forms part of a functional TCR complex by the criteria of coprecipitation and comodulation of CD3 and TCR p chain components and 1 cell activation with anti-idiotypic antibodies or digoxin. Furthermore, in cells expressing the hybrid protein, there is allelic exclusion of endogenous TCR a genes. We discuss the implications for the comparative structure of T cell receptors and immunoglobulins. Introduction The immune specificity of T lymphocytes is mediated by a multi-chain receptor complex termed the T cell receptor (TCR; Allison et al., 1982; Haskins et al., 1983; Meuer et al., 1983; Samelson et al., 1985; Oettegen et al., 1986). This receptor is important in determining the selection of maturing T cell precursors within the thymus as well as mediating the effector functions of cytotoxic and helper T cells (Dembic et al., 1986; Saito and Germain, 1986). The combining specificity of the TCR is manifest in a disulfidebonded a/f3 chain heterodimer that is evolutionarily related ‘to immunoglobulin (lg) molecules. In fact, analyses of the primary sequences of the TCR a and 6 chains indicate that the three-dimensional structure of the TCR may be very similar to an immunoglobulin Fab fragment with each of the chains anchored in the cell membrane

(Beale and Coadwell, 1986; Novotny et al., 1986; Kronenberg et al., 1986). Despite the structural similarities predicted for TCR and lg molecules, the two receptors have different functional specificities: T cells are specific for polypeptide antigen fragments bound to major histocompatibility complex (MHC) molecules, whereas lg molecules are specific for determinants present on a wide variety of molecules. The basis for this difference in specificity is poorly understood. The development and function of the immune system is dependent on cellular interactions that are sbictly dependent on the recognition of MHC molecules by ithe TCR. Experiments using radiation bone marrow chimeras (Bevan, 1977; Zinkernagel et al., 1978a, 1978b) and more recently using transgenic mice (Kisielow et al., 1988a and 1988b; Sha et al., 1988), have shown that the mature T cell population is selected during residence in the thymus for selfMHC recognition, and the specificity of this recognition almost always involves allelic determinants. However, once outside the thymus, these same T cells react: to self-MHC molecules only when they have bound a foreign antigenic peptide. In order to approach this enigmatic phenomenon of MHC-dependent selection and activation of T cells, we wished to create a population of T cells that expressed an aberrant receptor, namely one without MHC specificity. To produce, a population of T cells expressitng an MHCindependent specificity, we have taken advantage of the structural similarities between lg and TCR molecules to construct chimeric Ig-TCR genes. These genes are composed~of heavy and light chain lg variable region exons from a digoxin-specific antibody (Mudgett-Hunter et, al., 1985) recombined with’s or 6 chain constant r;egion exons. Our g&l was to produce transgenic mice with a large percentage of T cells bearing a receptor iwith specificity for digoxin. The lg VDJ regions were shuffled onto ‘the TCR C, and C, genes lin both combinat[ons (~VDdn-C, + VJ,-C, or VD&-Cb + VJ,-‘C,) and were !shown to be trans’cribed’and translated when transfected ikito the Bcell myeloma’P3X63. The VDJ&, construct lwas used to create a line ‘of transgenic mice, ,an,d;in these mice the VDJ&,#gene product cont$b;utes to :the repertoire of a chains associated $ith endogen’ous :@chains an’d forms a functiona! T,T,ceil!rdceptor. Fi!rrt~her’more,T cells ‘expressing, the’hybrid chain can bei ectivai’e’d /‘by digoxin. These ex~periments have~impjications~conceriiing characteristics of’the~~struct&of i’CR and’ fg’domains and concernin,g th:e ;processes’~of T icell maturation ,a& development. Results D,erivation of Transgenic Mouse Line 9a34 The hybridoma 40-140 secretes an tgG& with specificity for digoxin and a K, = 5 x 10lO/M (Mudgett-Hunter et al., 1985). A hybrid immunoglobulin-T cell mceptor gene was constructed (Figure 1A) ‘by ligating a 5 kb EcoRl genomic fragment carrying the rearranged VDJH(40-140)

Cell 912

3? ; neo

pBR322

“H

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Cccl-4 I

lkb

Figure 1. The Hybrid T Cell Receptor

Gene and Translation

Product

(A) A schematic of the plasmid pVDJu-C,, which contains the hybrid gene, showing the location of the immtmoglobulin and TCR gene segments. The Sall and Pvul restriction sites that were used to excise vector sequences, and the location of the various probes employed are shown. (B)Transcription of the transgene in various tissues of the mouse 9a34. Radiolabeled antisense RNA was synthesized from the L-V probe shown above and hybridized to 5 ug of total RNA isolated from the following tissues: bone marrow (lane I), thymus (lane 2) spleen (lane 3), lymph node (lane 4) liver (lane 5) heart (lane 6), nontransgenic ConAstimulated thymocyte blasts (lane 7), and nontransgenic Thy-lsplenocytes (lane 8). One microgram of total RNA from the hybridoma 40-140 (lane 9) was used as a positive control. (C) Thymocytes were isolated from a 14 week nontransgenic mouse (left panel) and a transgenic littermate (right panel) and stained with either FITC-anti-mouse lg (solid line), or anti-(40-140) idiotype followed by FITC-anti-rabbit lg (fiiled histogram). The cells were fixed by washing in 1% formaldehyde and analyzed by fluorescence-activated flow cytometry.

gene (Panka and Margolies, 1987; Near and Haber, 1989) to a 12 kb Sstl-BamHI genomic fragment carrying the TCR C, gene (F Ivars, unpublished data), in a vector consisting of pBR322 modified by addition of the neomycin resistance gene from pSV2’-neo (Southern and Berg, 1982). The chimeric gene thus includes the immunoglobulin heavy chain promoter and enhancer and 2 kb of sequence upstream of the lg promoter. The gene construct also includes sequences between the most proximal J, region and 4 kb downstream of the C, polyadenylation site. Although an enhancer sequence has been mapped in the J,-C, intron of the human TCR a gene (Luria et al., 1987), this has not been confirmed for the murine gene. An enhancer has been recently mapped to a region 3 kb 3’of the murineTCR a gene (Winoto and Baltimore, 1989), and this sequence is included in pVDJu-C,. The splicing between the VDJu exon and the first C, exon occurs within a codon; the first nucleotide is contributed by Ju and the second and third by C,. This pattern of, splicing is conserved in TCR genes, so that a simple exon shuffle maintains the appropriate reading frame. To determine the size of the protein encoded by pVDJn-C,, the B cell myeloma P3X63-Ag8.653, which does’ not express intracellular immunoglobulin: chains (Kearney et al., 1979), was transfected with pVDJ~-C,. G418-resistant P3X63 clones were screened for the expression of VDJu(40-140) messenger RNA, and the most strongly positive clone, P3D5, was examined for the presence of the chimeric protein. A metabolically labeled cell

lysate was immunoprecipitated with protein A-bound MAb H28-710 (R. T. Kubo, M. Pigeon, S. M. Hedrick, and M. L. B. Becker, unpublished data) directed against the constant region of the TCR a chain. In data not shown, a single 40 kd band was specifically immunoprecipitated. No such band was detected in P3X63 clone P3D4 vexpressing a similar amount of message from an analogous construct pVDJu-CB. A fragment 17.5’kb in length was excised from the pVDJu-C, construct (as ,indicated in Figure 1A) and injetted into fertilized eggs from a mating of (CBA/CaJ x BlO.BR)F, parental mice. DNA from each live birth was digested with Hindlll, Southern‘blotted, and analyzed with either a pBR322-derived probe or a 390 bp EcoRl probe from the J,-C, intron. The former probe indicates the presence of a transgene. The latter shows the enddgenous homologous fragments at 8.7 kb and 7.9 kb (there is evidently allelic polymorphism of Hindlll sites in this region) and the expected transgenic fragment at 5.1 kb. ‘Cf 53 living offspring, 2 mice, 9a25 and 9a34, were’healthy transgenic males. Quantitative densitometry of the blot hybridized with the J, probe indicated that mouse .9c$4 had about ten copies of the transgene integrated intolits genome, while 9a25 had a single copy (data not shown). The 9a34 founder mouse was backcrossed to BlO.,BR mice, and transgenic mice at each generation we’re’similarly backcrossed. The founder mouse and all of the prog.eny were homozygous for the H-.2k haplotype. Expression of the transgene in different tissues was as-

Expression 913

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Figure 2. lmmunoprecipitation genie Protein 7

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of the Trans-

Surface-labeled Id+ (lanes 4-6 and 10-12) and nontransgenic (lanes l-3 and 7-9) splenocyte blasts were lysed with 1% digitonin (lanes 1, 3, 4, 6, 7, 9, 10, and 12) or 0.5% MP-40 (lanes 2, 5, 8, and 11) and IO7 cell-equivalents per sample were immunoprecipitated with: anti-CD3 (lanes 1, 4, 7, and lo), anti-C, (lanes 2, 5, 8, and ll), or anti-ld.+rk,, (lanes 3, 6, 9, and 12). The samples were separated by nonreducing (lanes l-6) or reducing (lanles 7-12) 8% SDS-PAGE. The positions alnd molecular weights of 14C-labeled standards are indicated.

30

sayed by RNAase protection using a probe derived from VH(40-140) which spans the transcription initiation site and extends past the leader-v region intron (Figure 1B). There was no transcription detected in the 9a25 line, and these mice were not analyzed further. The sizes of the protected fragments from 9a34 transgenic tissues (126 bp and 98 bp) are identical to those found in message from the hybridoma 40-140 (Figure 18, lane 9) and correspond to the Suntranslated leader exon and the first 98 bp of the VDJH exon. This indicates that transcription was correctly initiated and present in all lymphoid organs tested (bone marrow, thymus, spleen, and lymph nodes) at a level ~20% of that in the parent hybridoma 40-140. Since this hybridoma is typical in that it secretes immunoglobulin at a level of about 10 ug/ml in culture, lg40-140mRNA probably represents over 1% of total mRNA in th,e plasmacytoma. Thus, the VDJH(40-140) message accounts for at least 0.2% of total mRNA in transgenic lymphoid cells, assuming that all cells have a similar level of transcription. A low level of expression was seen in heart muscle, but #not in liver. To determine whether this chain can be expressed on the surface of T cells, thymic lymphocytes were analyzed by flow cytometry for VDJH(40-140) expression (Figure 1C). The staining reagent .used was a purified rabbit a&i-(40-140) idiotypic antibody (anti-ld4,,-140) that had beenaextensively absorbed on murine Ig-Sepharose and nontransgenic mouse splenocytes. A fluorescein isothiocyanateconjugated goat anti-rabbit IgG antibody was used for asecond reagent. After staining with anti-ld40-140, O.i%of the nontransgenic thymocytes were brighter than cells stained with FITC-anti-rabbit lg alone, whereas the figurefor the transgenic thymocytes was 3.50/o, representing a discrete population of ld40-140positive cells. ‘ld40+, positive cells were cytofluorometrically sorted from transgenic thymocytes and splenocytes under sterile conditions and grown in culture by stimulation with either

phorbol ester plus calcium ionophore or solid-phase antild40140, followed after 2 days by expansion of activated T cells in IL-2. After a week in culture, the cells were analyzed for surface ld40-140 and were between 60% and 90% positive for the ld40-140determinant. The presence of the ld4014D determinant was stable indefinitely in cells stimulated with solid phase anti-(40-140) idiotypic antibody. In addition, these cells expressed the 7’cell markers CD3 and Thy-l, and responded to concanavalin A (ConA) and IL-2 (data not shown). Cells grown in this manner were used for biochemical analyses shown below. The specificity of the staining activity in this rabbit antiidiotypic antibody was tested on a cultured line of Id+ cells, and all of the staining activity could be removed by absorption of the anti-idiotypic reagent with purified lg40-140directly coupled to agarose beads, but not by absorption with the beads alone (data not shown). Characterization of ld40140 Bearing Pro’tein as Part of a TCR-like Structure The TCR exists at the cell surface as an a6 disulfidebonded heterodimer noncovalently associated with five polypeptides of the CD3 complex (Samelson et al., 1985; Oettegen et al., 1986). Evidence that the transgenederived protein is part of such a TCR complex was obtained by resolving the peptides immunoprecipitated with anti-CD3 antibody before and after reductioh with dithiothreitot (Figure 2). Lysates of surface-labeled ld40-140+ and nontransgenic splenic T cell lines were immunoprecipitated with either anti-CD9 anti-TCR C, 011‘anti-fd40-140. The autoradiogram in Figure 2 shows that both anti-CD3 and anti-C, (lanes 1, 2, 4, and 5) precipitated polypeptides with an apparent unreduced molecular ieight of 85 kd from nontransgenic and transgenic: ld+ T cells, whereas anti’ld40-140 (lanes 3 and 6) precipitated a band of the same relative mobility only from ‘the transgenic T cells. Upon reduction, there was a broad band at approx-

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Figure 3. The Transgenic Chains

15.5

Protein Is Associated

with CD3 and TCR 6

Metabolically labeled Id’ splenocyte blasts were lysed in 1% digitonin (lanes 2-3) or 0.5% NP-40 (lanes 6 and 7) and lo7 cell-equivalents per sample were immunoprecipitated with: F23.1 (lane 2) anti-ldao_,do (lanes 3 and 7), anti-CD3 (lane 4) anti-KLH (lane 5) and anti-C, (lane 6). The samples were separated by 10% reducing SDS-PAGE. The enhanced gel was exposed for 18 hr. The molecular weights of W-labeled standards (lane 1) are shown on the left, and the measured molecular weights of the precipitated proteins are shown on the right.

imately 40-47 kd from nontransgenic cells (Figure 2, lanes 7 and 8) or 37-47 kd from transgenic cells (lanes 10-12). The difference between the nontransgenic and transgenie samples presumably reflects the lower molecular weight of the hybrid V&Z, chain (40 kd) as compared with endogenous a chains (42-45 kd), and it is important to note that this is true of the proteins precipitated by antiCD3 and anti-C, as well as by anti-ld40-140. If the transgenic VDJu-C, chain assembles as a TCR, then anti-CD3 and antiTRC 6 chain antibodies should immunoprecipitate a multi-chain complex from Id+ cells that includes the transgenic protein. The autoradiogram pictured ,in Figure 3 shows an electrophoretic separation of immunoprecipitations from a metabolically labeled splenit Id+ T cell line using anti-(40-140) anti-CD8 anti-VP8 (F23.1), and anti-C, antibodies. The transgenic VDJ&, polypeptide as expressed in a transfected plasmacytoma separates on SDS-PAGE as a discrete band with an apparent molecular weight of 40 kd (data not shown). The same anti-C, antibody immunoprecipitates a strong band with a relative mobility of 40 kd from an ld40-140positive T cell line (Figure 6, lane 6). The rabbit anti-ld40-140 antibody also precipitated a 40 kd band (Figure 6, lanes 3 and 71, ,although no specific precipitation was seen with rabbit antirKLH antibody (lane 5) apart from background bands at 49 i!kd and 51 kd. Importantly, the anti-CD3 antibody (Figure 6, lane 4) similarly precipitated a 40 kd band, a diffuse band between 42-45 kd, which probably represents TCR f3 chains, and in addition, at least four other bands that correspond in size to the known CD3 polypeptides:

6, E, y, and <, which have molecular weights of 28, 25, 21, and 16 kd, respectively (Clevers et al., 1988). Anti-Vg8 antibody also precipitated a 40 kd band and an equally dense band at 45 kd (Figure 6, lane 2), that probably represents the VP&bearing TCR 6 chain. The CD3 polypeptides were visible in lanes 2 and 3 (Figure 6) after a long exposure of the gel. These data indicate that the VDJH-C, transgenic polypeptide can be immunoprecipitated by antibodies specific for CD3 and TCR b chain polypeptides, and thus that the chimeric VDJH-C, chain is physically associated with CD3 polypeptides and TCR 6 chains on the surface of transgenic Id+ T cells. A second way to show that the transgenic a chain is expressed as T cell receptor is to examine the comodulation of the ld40140 determinant and CD3. In this experiment, Id+ splenocytes grown by stimulation with anti-ld40-140 were incubated overnight in medium, or medium in the presence of saturating concentrations of antibodies, either anti-ld40-140 or anti-CD8 The next day, the cells were washed and second antibodies, either anti-rabbit lg or anti-hamster lg, was added for 3 hr to allow cross-linking and internalization of the antibody-antigen complexes. The cells were then stained with anti-ld4,,-140, anti-CD3, anti-Vp8, or anti-CD8 (Figure 4). As a control, nontransgenie ConA-stimulated splenocytes were stained with anti-CD3 before and after modulation with either antild40-140or anti-CD3 (Figure 4A). Treatment of these cells with anti-CD9 but not anti-ld40-140, significantly decreased the subsequent staining with anti-CD3 (compare Figure 4A, panels 2 and,3). It is important to note that not all of the CD3 was modulated from the surface, although the entire population was shifted about 8-fold (three divisions on the X axis) in brightness. The background shown in Figure 4A, panels l-3 (solid line), is the profile of bnmodulated nontransgenic cells stained with FITC-antihamster lg alone. The background in Figure 4A, panel 4, is the profile of anti-CD3 modulated nontransgenic cells stained with FITC-anti-hamster lg. The staining of these cells was not increased’ by a second incubation of antiCD3 before addition of FITC-anti-hamster lg. This shows that surface CD3 on these cells was saturated by anti-CD3 during the overnight incubation. In the unmodulated tfansgenic Id+ cells (Figure 46), the population was mainly ld40-140and CD3 positive. The positive and negative histograms overlap for both anti: ld40-{40 and anti-C,D3 staining, and thus the mean level of TCR expressed in the ld40-140+population grown on antild40+0 antibody appears to be lower than that of the non transgenic ConA-stimulated line. The Id+ cells wereapproximately 20% 1Vs8+ (Figure 48, panel 3), and’ this is consistent with a [random distribution of Vs8 expression in the Id+ cells. when these cells were incubated overnight with anti-CD3 (Fi$ure 4C), the mean levels’ of ld40140, CD3, and VP8 ;were significantly reduced,1 ;although there wa.s a tail :d brightly staining Id+ cells ‘remaining. The data’in Figure 4D,,indicate that anti-ld40_14q was at least as effective ‘at modulating ld4,-+1m,CD3, and VB8 expression as was anti-CD3 antibody (compare Figures 4C and 4D). CD6 expression was not affected by either anti-ld40-140 or anti-CD3 (Figures 4C and 4D, panel

Expression 915

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A Con A stimulated non-transgenic T cells

Id+ transgenic T cells

e Id+ transgenic T cells

Id+ transgenic T ceils

Figure 4. Comodulation

of Id 40- 140 and Other Components

of the TCR Complex

Actively growing ConA-stimulated normal splenic T cell blasts and transgenic Id+ splenic T cell blasts were incubated overnight #at37% with either anti-CD3 or anti-ld40140, washed once, and then incubated for 3 hr at 37% with anti-hamster lg (A, third and fourth panels; C) o#ranti-rabbit lg (A, second panel; D), respectively. These cells and unmodulated cells were then stained with the indicated antibodies and analyzed by flow cytometry. Histograms of cell number versus cell brightness are shown. Each division on the X axis represents a 2-fold increase in brightness, The backgrounds (solid lines) shown are: (A) first three panels unmodulated nontransgenic splenic blasts stained with FITC-anti-hamster lg; (A, fourth panel) anti-CD3 modulated nontransgenic splenid blasts stained with FITC-anti-hamster lg; (B) unmodulated transgenic Id+ splenic blasts stained with FITC-antirabbit lg; (C) anti-CD3 modulated transgenic Id+ splenic blasts stained with FITC-anti-hamster lg; (D) anti-ld40_,4a modulated transgenic Id+ splenic blasts stained with FITC-anti-rabbit lg.

4). The most important point in this experiment is that antild40-140 modulation appears to reduce the level of CD3 and VP8 expression’in the entire population as effectively as anti-CD3 modu/ation. From -this experiment we conclude that most of the CD3 molecules on each Id+ T cell are associated with the VDJ&, chain. Furthermore, since modulation by anti-Id.+140 comodulated all of the detectable TCR V,$ from the cell surface, this conclusion holds for VDJH+, and TCR V,8 chains, and by extension, for all TCR fi chains: These data cannot rule out the possibiljty that the cells express a minor percentage of the CD3 molecules associated with a different (endogenously encoded) TCR a chain. Alielic Exclusion The effects of an abnormal TCR on the selection and function of T cells would be most pronounced if the transgenic

VDJu-C, protein were expressed to the exclusion of endogenous TCR a chains. To approach this question, we took advantage of the fact that rearrangement of the endogenous TCR a genes results in the deletion of the TCR 6 gene elements (Chien et al., 1987; de Villartay et al., ‘1988). In fact, most T cells that express an up receptor have deleted both TCR 6 alleles (Lindsten et al., 1987). To determine whether the endogenous TCR a genes had rearranged in T cells expressing the transgenic VDJ&, chain, DNA samples from Id+ lines and clones were analyzed by Southern blotting using a 3’ Js2 genomic fragment as a probe (Figure 5A). This detected a 4.5 kb fragment in EcoRI-digested DNA from AKR liver (Figure 5A, lane 12) and the TCR y5 expressing clone G8 (Bluestone et al., 1988; lane 7), both of which have two copies of the TCR 6, locus. No hybridization was detected to DNA from the TCR ab expressing clone DlO.G4.1 (Kaye et al., 1983;

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Figure 5. Id+ T Cells Have Allelically rangement

Excluded

TCR a Gene Rear-

(A) DNA was isolated from lines of Id+ thymocytes or splenocytes, and from Id+ clones, digested with EcoRI, and analyzed by Southern blotting (Southern 1975). The probe consisted of 2 kb of sequence 3’to the TCR J&2 locus. Included as positive controls were DNA from AKR liver (lane 12) and the TCR y&expressing clone GE (lane 7; see test). A negative control was DNA from the AKR clone DiO.G4.1 (lane 11; Kaye et al., 1983), which expresses a and 8 chains of the TCR. The other DNA samples were: lanes 1 and 2, transgenic splenocytes stimulated with anti-CD3 and anti-Id, respectively; lanes 3 and 4, transgenic Id+ thymocytes and splenocytes, respectively; lanes 5 and 6, nontransgenie ConA-stimulated thymocytes and splenocytes, respectively; lanes 8, 9, and 10, three Id+ clones derived from a line of Id+ splenocytes. Lanes 1-6 were loaded with 13 ug of DNA, while lanes 7-12 contained 3 ug. Radiolabeled Hindlll-digested h DNA provided size markers. (6) The same blot as in (A) after stripping and reprobing with a TCR C8 probe.

Figure 5A, lane 11). Barely detectable signals were seen in samples from nontransgenic, ConA-stimulated T cell lines from thymus and spleen (Figure 5A, lanes 5 and 6). However, DNA from three clones derived from a line of Id+ splenocytes showed the Jg2 band (Figure 5A, lanes 8-10) as did DNA from lines of Id+ thymocytes and splenocytes (lanes 3 and 4). DNA from transgenic splenocytes stimulated with anti-ld40-140 (Figure 5A, lane 2) showed the Js2 band, indicative of an unrearranged TCR a locus, as did DNA from T cells stimulated with anti-CD3 (lane l), even though only 20% of the anti-CD3 stimulated T cells were Id+. The signal from the CDB-stimulated T cells was somewhat reduced compared with T cells stimulated with anti-ld40-j40, but there did not appear to be a 5-fold reduction that would correspond to the number of ld40-140positive cells in the population. This may indicate that TCR a gene rearrangements are inhibited, even in the Id- population. By comparing the signals produced with the 3’ JS2 probe (Figure 5A) and a TCR C, probe (Figure 58) which hybridizes with a 9.3 kb EcoRl fragment, we deduced that at least one and most likely two copies ,per genome of the Js2 region are present in the Id+ cells of both the transgenic lines and the clones. These experiments indicate that the transgenic VDJn-C, chain can, to a large extent, exclude the expression of endogenous TCR a chain expression. None of the experiments can rule out double TCR a chain expression in a small percentage of cells, but the data indicate that there is a large bias against the expression of two a chains.

Figure 6. The Response

of Transgenic

T Cells to Digoxin

(A) Resting anti-CD3 stimulated nontransgenic splenocytes (stippled bars) and anti-ldd&tJs activated transgenic splenocytes (solid bars) were assayed for their lymphokine response to solid-phase digoxinBSA as described in Experimental Procedures. Other stimuli used as controls were solid-phase antiCD3, F23.1, and anti-ld40-140. Supernatants were removed 24 hr after stimulation and tested for lymphokine production by the ability to stimulate an IL-2 and IL-2 responsive cell line, NK. The response is presented as mean counts per minute x 10m3of [3H]thymidine incorporated r standard deviation of quadruplicate cultures. rll-2, 100 U/ml, induced the NK cells to incorporate 63,000 cpm, while incorporation in the absence of any stimulus was 1,500 cpm. (8) Freshly isolated nontransgenic (stippled bars) and transgenic (solid bars) splenocytes were cultured in triplicate at lo7 cells/ml in the presence of the indicated solid-phase stimuli. Forty-eight hours later, the cultures were pulsed with 1 nCi of [3H]thymidine, and incorporated radioactivity was measured after 18 hr. The data are presented as means * standard deviation.

Digoxin

Reactivity

of Id+ T Cells

The transgenic mouse 9a34 expresses only the VDJH(40-140) and not the light chain. Experiments discussed below indicate that the lg heavy chain paired with a random lg lambda light chain does not show detectable

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Receptor

binding to digoxin in solution. Nonetheless, we tested the Id+ T cells for a possible response to digoxin. Since digoxin itself is relatively insoluble in aqueous medium, we used a soluble digoxin-BSA conjugate to test for antigenicity. Splenic T cells from a transgenic mouse were stimulated in culture with solid-phase anti-ld40-140, expanded in IL-2, then rested in the absence of 11-2. A line of nontransgenic splenic T cells was similarly derived by stimulation with solid-phase anti-CD8 These cells were assayed for lymphokine release in response to various stimuli, including solid-phase digoxin-bovine serum albumin (BSA; Figure 6A). Only the transgenic Id+ cells responded to digoxin-BSA or anti-ld4,--140 coated to plates, although both cell populations responded to solidphase anti-CD3 or anti-Vg8. The cultures indicated by “nil” in effect control for responsiveness to solid-phase BSA, since the culture cells are coated with BSA from the fetal calf serum. The magnitude of the response to digoxin-BSA was not changed by the addition of 5 x l@/ml irradiated syngeneic nontransgenic spleen cells (data not shown). We next determined the direct proliferative response of freshly isolated splenocytes to the same solid-phase stimuli, and the results are shown in Figure 66. Splenocytes from transgenic and nontransgenic mice responded to anti-CD3 and anti-VB8, whereas only splenocytes from the transgenic mice responded to anti-ld~00_140 or digoxinBSA. These data show that the transgenic VDJ+& chain confers upon mice a population of T cells expressing a digoxin-specific receptor., In a separate experiment, transgenie splenocytes were stimulated in culture with either solid-phase anti-CD9 anti-ld40-140ror digoxin-BSA, and 2 days later expanded by the addition of IL-2. The surface phenotype of these T cells was assessed by flow cytometry after staining with an$-ld40-140, and virtually all of Rhe digoxin- or anti-ldlo-140-stiniulated cells were Id+, as compared with about 10% of the anti-CD3-stimulated cells (data not shown). Discussion In this paper, we report the expression of a hybrid Ig-TCR gene in a line of transgenic mice. The protein product of this gene, an Ig,o-,,o VDJ&TCR C, fusion polypeptide, is expressed on the surface of approximately 3%~5% of thymic and peripheral T cells as detected by a purified rabbit aniiserum against the lg40-140 idiotype. ld40140 positive T cells were isolated on the fluorescence-activated cell sorter and expanded in culture by stimulation with the antiserum and IL-2. Expression of the idiotypic determinant appeared to be stable since T cells selected on the basis of the idiotype-positive phenotype could be maintained for mqnths by successive cycles of stimulation with solidphase anti-ld40-140 antibody and expansion in IL-2. The Hybrid Antigen Receptor ‘Several different lines of experimentation demonstrate that the hybrid protein can substitute for a native TCR a chain in forming a T cell receptor complex with TCR p chains and CD3 components. First, solid-phase anti-

idiotypic antibody specifically stimulates transgenic Id+ T cells but not nontransgenic T cells to release lymphokines. These data indicate that the anti-ld,lo-,40 antibody can bind (and cross-link) a cell-surface molecule capable of stimulating T cells. Second, modulatioli experiments using cross-linked antibodies showed that either anti-CD3 or anti-ld40-140 can down-regulate the expression of CD3, bo-+to, and TCR Vg8 on the surface of Id+ T cells, suggesting a physical association between these proteins. Third, immunoprecipitation with anti-CD3 olr F23.1 demonstrated coprecipitation of a 40 kd transgene-specific protein as part of a disulphide-bonded heterodimer on Id+ T cells. The fact that a VDJ&, fusion protein is not degraded but is expressed on the T cell surface in association with TCR fi chains has implications for studies of the comparative structure of immunoglobulins and T cell receptors. Several sequence analyses to date have predicted that the typical TCR will form a structure quite similar to immunoglobulin molecules (Beale and Coadwell, 1986; Novotny et al, 1986; Bougueleret and Claverie, 1987), and in particular, that V, and V, domains will interact to form a superdomain in much the same way as VH and VL domains (Davis and Bjorkman, 1988; Chothia et al., 1988). Thus, there are predicted to be many contact points between the a and p chains and very few between the variable and constant region domains. Kuwana et al., (1987) have shown that hybrid genes consisting of VH and VL exons shuffled onto the TCR 6, and CB regions in either combination can be expressed in the EL4 thymoma and can confer the antibody’s specificity to theise cells: These data suggest that interactions between the TCR constant regions and the Ig variable regions do not significantly affeet the configuration of the antigen bindinlg site. The fact th’at the VH(40-1’40) domain can repla&at IeaSt some V, dbmains to pair with a Vg domain suggests that the size and relativk orientation of the VH doindin must be quite similar to the V, domain. We note that V,, can :su’bstitute for VH in an antibody molecule (Gascoigne et ial., ,1987), and thus in two independent types of &nd’ly:ses,,ihe:two domains appear to have similar structuial prop&tie.%These results are evenmmorestriking ih light of the! fakt thafdifferent combinatibns of lg heavy and light chains have, different a+ociation rates!(de Preva! and Fou’gere&.i, 19%). We wpu’id ~expect:a:V~-V0 interaction to ,hdVe*a :su,Qst$ntially lower?rate thah any VH-VL association, sinde q, di)mains are ‘e\iolutionarily se&ted for :the abilif$ to co;irppi&x with VH doinaind,” bhereas‘VB dom’ains are halt: ,$$e‘c’uirently h&e r@evidbn:ce ‘that wduld !indicate ,&heth& a ‘V, domaih ~dould~sirb~titirte’for V, dr V,, or $heth& IVH could subhtute forjVp.’ In addition, except [for’the *iact that VP8 is, np@a’lly r+[esent&d, inlreh?ve no%$jrn$i/~ti &+rtaining ltd ‘ithe VB I&6el’tdiie iri thd’: Id+ :pdpti~laiti& iof P~;c&lls. Allelic Exclusion A s&cond !,inding of this work is that expression of the hybrid QDJH-C, Chain Can result in exclusion ,of endogenouS ‘TCR a gene rearrangement. The evidence to support ,this contention is first, that the anti-ld40-,40 is effektive’in modulating CD3 off the surface of Id+ T cells,

Cell 918

as effective as anti-CD3, and second, that a chain rearrangements are suppressed in Id+ cells. Either the endogenous a chains do not rearrange unless the expression of the transgene has been curtailed for any one of a number of reasons, or the endogenous a chain does rearrange (perhaps at a reduced rate not equal to zero) and the rate of association between V, and Vg is sufficiently higher than that of VH and VP such that no VH-VB pairs are expressed at the cell surface in the presence of competing V, chains. If the mechanism of allelic exclusion in this transgenic model is consistent with this latter hypothesis, then the only T cells that express the cell surface ld40-140determinant are those in which the endogenous a chains have not rearranged: those that express an endogenously encoded a chain have only intracellular expression of the VDJ&, polypeptide. If this allelic exclusion at the protein level exists, then the relatively strict allelic exclusion seen in these mice may not apply to naturally rearranging a chains. In fact, Bliithmann et al. (1988) have found that transgenic mice containing rearranged a and 8 chain genes have a diminished, though not entirely suppressed level of endogenous a chain rearrangements. The reasons why the VDJn-C, fusion protein appears to be expressed at the cell surface of only a small percentage of T cells are presently not clear, and there are a number of factors (not necessarily mutually exclusive) that could explain this phenomenon. It is possible that the antiserum used to detect the transgenic protein only binds to particular combinations of VDJn-C, and endogenous VP, and we are currently investigating this question using transgenic Id+ and Id- T cell clones. It is also possible that only a small proportion of T cells (or thymocytes) express the VDJ&, message; however, based on the average level of specific mRNA in the thymus (>0.2%, a normal level for a T cell receptor), this would indicate that a few cells express enormous levels of message and the rest express very little. If the antiserum detects all of VDJn-C, at the cell surface, the message is expressed uniformly in the T cell population, then the small number of expressing T cells could be due to differential associations between the chimeric protein and the endogenous TCR 8 chains. As stated above, some 8 chains may not associate well with the chimeric protein, or the rate of association may be sufficiently slow so that an a chain from an endogenously rearranged gene may completely eliminate the surface expression of the VDJ&,. Finally, many of the cells expressing the transgene may be eliminated by tolerance mechanisms. In fact, the transgenic mice used in these studies had only one-tenth the’number of thymocytes as nontransgenic littermates, similar to theefindings of Kisielow et al. (1988a) in a situation where ‘the, transgenie T cell receptor was reactive with the endogenous male antigen. The preliminary results of breeding experiments indicate that in 9a34 mice there is a large influence of MHC genes on the total number of cells in the thymus and on the,phenotype and number of ld40-140+cells in the thymus and lymph nodes. Antigen Reactivity of Id+ T Cells A third’finding is that the hybrid VDJ&,

chain can con-

fer on transgenic T cells a reactivity to solid-phase digoxin. Thus, a single chain with digoxin specificity is sufficient to generate a digoxin binding site, suggesting that at least some TCR Vis allow the VDJn(40-140) to fold correctly. This may be a consequence of the very high association constant of lg40-14r, for digoxin (5 x lOlO/M). The results of recent experiments indicate that VDJn(40-140) in combination with related V,‘s from any of three other digoxin-specific hybridomas generates immunoglobulins with approximately IO-fold lower affinity than lg40-140; whereas, in combination with one unrelated lambda light chain, no binding (affinity less than 105/M) was observed (Ft. Near and M. Mudgett-Hunter, unpublished data). Based on these data, we would predict that the association constant of VDJn(40-140) when combined.with an unselected VP would certainly be less than log/M, and most likely less than 105/M. Digoxin does not have to be processed and presented in association with MHC molecules since Id+ T cell hybridomas respond to solid-phase anti-ld40-140 (H. Kawasaki, unpublished data). The affinity of the Id+ T cells for digoxin thus appears to be high enough to allow the BSA-digoxin to cross-link surface TCR and thereby activate the T cells, in the same way as an anti-TCR antibody (Crispe et al., 1985). These data may indicate that the affinity requirement for the activation of Tcells via multimeric TCR-ligand interactions may be very different from the affinity requirement for the activation of B cells via the interaction of an lg receptor with soluble antigen. Although we have shown that essentially all digoxin-reactive T cells in the transgenic mice are Id+ as might be expected, we cannot say that all Id+ T cells are activated by digoxin. It is possible that certain VP’s interact with the VDJH(40-140) in such a way as to reduce the affinity of the binding site to a subthreshold level for activation. This question will also be studied using a panel of Id+ clones and hybridomas. Given the fact that Id+ T cells have an affinity for digoxin, and most do not express two receptors, we found it somewhat surprising that these T cells were found in the peripheral lymphoid organs. It has been shown that T cells have to express a TCR with affinity for self-MHC molecules in order to be positively selected and to egress from the thymus (Sha et al., 1988; Kisielow et al., 1988b). Certainly the transgenic a chain was not selected (either evolutionarily or within the animal) in the context of a receptor for MHC affinity, and yet if it mediates the positive selection of T cells, it apparently forms part of an MHCspecific receptor. We would be interested in determining whether this MHC specificity is independently conferred by the associated 8 chains,,or whether the specificity is dependent on the’ specific a8 heterodimer. Role in Leukemogenesis The VHJH-Ca fusion protein we constructed and characterized has a natural counterpart in the VH-J,C, gene, which can arise by chromosome 14 inversion or translocation in a variety of Tmjcell neoplasias (Denny et al., 1986; Mengle-Gaw et al., 1987; Russo et al., 1988). Our results provide #evidence that such ,a protein could be incorporated into a TCR cdmplex and could potentially generate

Expression 919

of an Ig-TCR Chimeric

Receptor

an antigen-driven proliferative signal for such T cells. We are unable to say whether such an aberrant receptor could play a role in the induction of neoplasias.

Experimental

Procedures

Gene Construction and DNA Probes The 40-140 VDJH gene segment in pBR322 (Near and Haber, 1989) was modified by insertion of the neomycin-resistance gene from pSV2’-neo (Southern and Berg, 1982). The murine TCR C, genomic clone (F. lvars, unpublished data) was kindly provided by Mark Davis (Stanford University). Details of the construction are available upon request. A genomic probe from the murine 3’ Js2 region was obtained from Dr. Y.-H. Chien (Stanford University). The TCR CI, probe used was 86T5 (Hedrick et al., 1984). Transgenic Mice pVDJ&, was digested with Sal1 and Pvul, and the resulting 17.5 kb fragment was isolated by electroelution from an agarose gel followed by binding and elution from a NENsorb column (New England Nuclear, Boston, MA). The DNA was resuspended in 1 m M Tris (pH 8.0), 0.1 m M EDTA at a concentration of 2 pglml, and injected into single-cell stage embryos of (C,BA/CaJ x BIO.BR)Fp mice, according to standard procedures (Hogan et al., 1986). Transgenic mice were identified by Southern analysis of tail DNA. The founder male 9a34 was backcrossed to BiO.BR mice. Antibodies and Antigens Anti-idiotypic serum was prepared by immunization of rabbits with 40-140 lg (1 mg) puiified by affinity chromatography on ouabainamini-Sepharose (Mudgett-Hunter et al., 1985). Protein A binding immu’noglobulin was isolated from the rabbit serum and adsorbed with lo* ‘nontransgenic mouse splenocytes per 5 mg of protein. The antibody was further purified by passage over a column of mouse gamma globulin coupled to activated aldehyde agarose (Calbiochem, La Jolla, CA): The flow-through from this column was used as an ‘anti-idiotypid’ reagent. Other antibodies were 2Cll (anti-CDS; Leo et al., 1987), 536.75 (anti-CD8; Ledbetter and Herzenberg, 1979), and F23.1 (anti-V,8; Staers et al., 1965). H28-710 is a hamster IgG MAb directed to an epitolje in the constant region of the TCR a chain (R. T. Kubo, M. Pigeon, S. M:!Hedrick, and M. L. B. Becker, unpublished data). This MAb binds strbn’gly with a chains from lysates prepared with NP-40 but it minimally immunoprecipitates polypeptides from lysates made in digitonin. Presumably, it binds preferentially to TCR a chains not associated with CDJ. Digoxin-BSA conjugate was prepared as described (MudgettHunter et al., 1985). The batch used in these experiments had on avera$iwo digoxin molecules per BSA molecule. It was used at a protein cohc&tration of 6 pglml. Flow Cytometric Analysis Lymphocyte suspensions were incubated at room temperature in rounti-bottomed 96-well plates (Costar, Van Nuys, CA) at a cell concentration of up to 5 .x 106/ml in PBS containing 1% FCS and 0.1% sod& azide for 60 min in 25 pglml of purified anti-idiotypic serum or undi(uted hybridoma supernatants. The cells were washed by centrifu@ti~h, then resuspended in the same buffer containing 10 Kg/ml of fluoroc&ome-conjugated second-layer antibodies (Caltag Laboratories.‘$& Francisco, CA), and incubated at room temperature for 30 min, fcillowed by washing. Fluorescence analysis was carried out on an OrthoCyto!luorograph 50-H or a Becton Dickinson FACScan. Data is ‘p&senied as histograms of cell number versus log fluorescence. RNAase Protection A frajment of the 40-140 VH gene was cloned into the vector pBS+ (Stratigene, l&i Jolla;*CA). The plasmid was linearized with EcoRl and radiolabeled bntisense RNA was synthesized with T3 RNA polymerase.!;Hybridiiation was for 20 hr at 52OC, and RNAase digestion was pdrfdrmed as’described for 30 min at 37°C (Zinn et al., 1983). RNA sa’mijles were electrophoresed on denaturing 8% polyacrylamide gels.

Cell Culture In initial experiments, idiotype-expressing cells were isolated from spleen and thymus of transgenic mice by sterile soirting on the OrthoCytofluorograph 50-H. The cells were activated by 48 hr of culture in EHAA medium supplemented with 10% fetal bovine serum, 2 m M glutamine, 100 U/ml of penicillin, 100 pglml of streptomycin, and 50 pM P-mercaptoethanol (referred to as supplemented EHIAA) with 10s irradiated syngeneic splenocyteslml, in the presence of 5 rig/ml of phorbol dibutyrate and 300 nglml of ionomycin (Calbiochem, La Jolla, CA), or solid-phase anti-ld40-140 antibody preadsorbed onto the plastic culture surface (100 pglml antibody in phosphate-buffered saline) for 3 hr at 37°C. The activated T cells were expanded in supplemented EHAA plus 100 U/ml of recombinant human IL-2 (Wang et al., 1984; a gift of Cetus Corp., Palo Alto, CA). Cells were rested in IL-2.free medium for 7-10 days, prior to restimulation with solid-phase anti-Id. The stimulatory effect of various solid-phase antibodies and antigens was quantitated in a lymphokine release assay. Tissue culture surfaces were coated by incubating for 3 hr at 37oC with the protein solution in PBS. Triplicate cultures of 5 x lo4 washed, resting T cells in flat-bottomed 96-well plates precoated with antibodies or digoxinBSA were incubated in supplemented EHAA for 24 hr at 37°C. Ten microliters of supernatant from each well was added to 90 pl of supplemented EHAA and 5000 washed NK cells (used 3 days after expansion in (L-2). These cultures were pulsed after 24 hr by addition of 1 Ci of 13H]thymidine, harvested 18 hr later, and incorporated radioactivity was measured by scintillation spectroscopy. The B cell myeloma P3X63-Ag8.653 was transfecteld by electroporation using a BTX-100 transfector (BTX, Inc., San Diego, CA) set at a voltage gradient of 2.7 kV/cm and a time-constant of 500 psec, at a cell concentration of 3 x 107/ml in a K-PBS buffer consisting of 30 m M ‘NaCI, 120 m M KCI, 8.1 m M Na2HP0.,, 1.5 m M KHzPOI, and 5 m M MgClp. Plasmid DNA for transfection was linearized with Pvul and used at 50 pglml. The cells were washed in serum-free EHAA medium, resuspended in ice-cold K-PBS containing the DNA., left on ice for 5 min, electroporated, left on ice a further 5 min, diluted IO-fold in serumfree EHAA medium, incubated 10 min at room temperature, and plated at lo6 cells/ml in supplemented EHAA. On day 2 of thie cu‘lture, theseldction agent G418 (Gibco-BRL, Burlington, Ontario)1 was added to a cbncentration of 0.4 mglml. Clones were visible on dcry 7 and were expanded’and screened for RNA expression. Modulation of T Cell Surface Antigens Activated T cells were incubated at a cell concentration of 106/ml in supplemented EHAA plus 100 U/ml of rlL-2 with saturating concentrations of primary antibody for 18 hr at 37oC in round-Ibottomed 96-well plates, washed by centrifugation, then incubated a flurther 3 hr in the same medium containing saturating concentrations of secondary antibody and washed once more. The cells were then prepared for flow cytometric analysis as described above. lmmunoprecipitation Actively growing T cells were metabolically labeled at 10’ cells/ml in methionine-free RPM1 medium containing 100 U/ml of rll-2 and 200 PCilml of [35S]methionine (Translabel, ICN, Costa Mesa, CA) for 5 hr. The’cells were washed and lysed in 1 ml/IO’ cells of 1% digitonin in 150 m M NaCI, 50 m M Tris-HCI (pH 7.6), and 1 m M phenylmethylsulphonylfluoride. One percent NP-40 was used in place of digitonin for precipitation with anti-C,. Surface labeling was performed by washing the cells five times in PBS, then incubating up to lo8 cells for 30 min at room temperature with lodobeads (Pierce, Rockford, IL) and 1 mCi of’Na”51 in 1 ml of PBS. Free Najz51 was removeld by washing the cells three times in PBS prior to lysis. The postnuclear supernatant was mixed at 4OC for 30 min with 25 WI/ml of fixed Cowan strain A StaphyloCOCCUSaureus (Calbiochem, La Jolla, CA), then precleared by successive,centrifugations at 1,000 x g for 5 min, 10,000 x g for 15 min, and 30,000 x g for 15 min. The precipitating antibodies were adsorbed onto protein A-Sepharose beads (Boehringer Marnnheim Biochemicals; Indianapolis, IN), which were then mixed at 4OlC for 30 min with IO6 Cell equivalents of postnuclear lysate of unlabeled P3X63 cells, in order to block sites of nonspecific adsorption. The beads were washed once in digitonin buffer, and mixed overnight at 4OC with labeled lysate (IO7 cell equivalents/sample). The beads were washed six times in 1 ml of digitonin or NP-40 buffer with mixing for 30 min at 4OC, then

eluted in SDS sample buffer at 90% with or without 100 m M dithiothreitol and electrophoresed on SDS-polyacrylamide gels. %-labeled samples were enhanced by treating the gel with Amplify (Amersham, UK) before drying and by autoradiography using Kodak XAR-5 film. Acknowledgments We would like to thank Ms. Judy Nordberg for her skill in flow cytometry. We also thank Dr. D. Cohen for reviewing this manuscript. This work was supported by National Science Foundation grant DCB8452023, and National Institutes of Health grant Al21372 to S. M. H. S. M. H. was supported by RCDA Al-00662. M. N. M. was supported by NIH grant PO1 HL-19259. R. K. was supported by USPHS grants Al-18785 and PO1 Al-22295 M. L. B. B. was a Fellow of the Leukemia Society of America and received support from the Poliomyelitis Research Foundation. J. K. was supported by an American Cancer Society, California Division, senior postdoctoral fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

May I, 1989; revised June 29, 1989.

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between J. Mol.

Expression 921

of an Ig-TCR Chimeric

Receptor

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