The EMBO Journal vol.6 no.9 pp.2753-2758, 1987
`
`Polymeric immunoglobulin M is secreted by transfectants of non-
`lymphoid cells in the absence of immunoglobulin J chain
`
`We were therefore interested in determining whether non-
`lymphoid cells could assemble and secrete IgM. Whereas IgM
`is secreted by transfected plasmacytoma cells (Neuberger, 1983;
`Ochi et al., 1983; Neuberger et al., 1984), the introduction of
`vectors driving expression of immunoglobulin cDNAs into yeast
`or Escherichia coli hosts has not resulted in effective antibody
`production - problems being encountered both with efficient
`assembly and with secretion (Cabilly et al., 1983; Boss et al.,
`1984; Wood et al., 1985). It has, however, been found that IgG
`was secreted by Xenopus oocytes that had been injected with im-
`munoglobulin mRNA (Colman et al., 1982), although it is not
`
`A p
`
`SV- HSVAI
`
`rLNpVIl
`
`Cl
`
`phsp 70
`HISp
`
`Hh/P
`
`B
`
`R
`
`Bg/Sa
`
`SP6-VA
`
`amp
`
`neo
`
`B p
`
`SV- HSVI,2
`
`L V
`r NP NP '1
`
`CR_
`
`3
`
`2
`
`phsp7O
`Sp X BgI5aSt
`
`R
`
`SP6-HSVNp
`
`4 tp
`=EL--
`- sv
`POLY gpt
`A
`
`g
`
`Ha/Sc
`
`amp
`
`500 bp
`Fig. 1. Structure of plasmids. (A) Structure of pSV-HSVXl. The thin line
`denotes the pSV2neo vector. Sequences of the immunoglobulin XI light
`chain gene are stippled, the Drosophila hsp7O promoter region (phsp7O) is
`hatched, the leader exon derived from the heavy chain variable region (LNP)
`is depicted by a filled box and the open box denotes a 60 nucleotide stretch
`of the 5'-untranslated region of tk. (B) Structure of pSV-HSVIL2. The
`various DNA segments are depicted as for pSV-HSVX1 except that the vec-
`tor (thin line) is pSV2gpt. The DNA encoding the A constant region is
`derived from a cDNA encoding the CI1I,2,3 and 4 domains as well as the
`secreted tailpiece (tp). The HS-Vjt2 transcription unit uses the SV40
`polyadenylation site (SV POLY A) of pSV2gpt. The transcription start sites
`are indicated by an arrow and the probes used for ribonuclease protection
`assays (SP6-Vx and SP6-HSVNp) are also indicated; the SP6-HSVNP probe
`lacks the LNP-VNP intron. Restriction sites are abbreviated: B, BamHI; Bg,
`BglII; H, HindIII; Ha, HaeII; Hh, HhzaI; P, PstI; Sa, Sau3AI; Sc, SacI; Sp,
`SphI; St, StuI; X, XhoI. Note that not all the sites for any one enzyme are
`shown - only those relevant to plasmid or probe construction are included.
`
`2753
`
`Antonino Cattaneo and Michael S.Neuberger
`Medical Research Council Laboratory of Molecular Biology, Hills Road,
`Cambridge CB2 2QH, UK
`Communicated by C.Milstein
`Plasmids were constructed in which expression of genes en-
`coding the heavy and light chains of a hapten-specific IgM
`antibody is under control of a heat shock promoter. Glioma,
`phaeochromocytoma and other non-lymphoid cell lines
`transfected with the plasmids were able to process and secrete
`immunoglobulin following heat induction. The glioma
`transfectants were studied in detail and were shown to secrete
`polymeric IgM in a yield similar to that obtained with a
`plasmacytoma. However, the glioma IgM was not associated
`with J chain and was largely composed of pentamers and hex-
`amers. Thus, neither J chain nor other lymphoid-specific pro-
`teins are required for assembly and secretion of polymeric
`IgM although the absence of J chain may encourage hexamer
`formation.
`Key words: immunoglobulin M/J chain/secretion/glioma
`
`Introduction
`Antibodies are secreted by plasma cells, which represent the ter-
`minal stage of B cell differentiation. Whereas a plasma cell
`secretes large quantities of pentameric immunoglobulin, an IgM-
`expressing B cell contains monomeric IgM in its membrane but
`does not normally secrete antibody. The differentiation of a B
`cell into a plasma cell is not only accompanied by a large in-
`crease in the steady-state level of immunoglobulin mRNA, but
`there is also a shift from the production of mRNA for the mem-
`brane form of the heavy chain (,) to the production of the
`secreted form (us). However, whilst B cells do not on the whole
`secrete IgM, many B cells and B cell lymphomas are found to
`contain A,s mRNA and intracellular Its polypeptide (Sibley et al.,
`1980; Vassalli et al., 1980; Sidman, 1981; Rubartelli et al.,
`1983). Thus, whilst the difference in the production of secreted
`IgM by B cells and plasma cells can be in part ascribed to regula-
`tion at the level of RNA processing, it is clear that post-
`translational processes are also implicated.
`Proteins involved in the assembly and secretion of the IgM
`pentamer could obviously play a role in this regulation. A
`lymphocyte-specific enzyme that may catalyse immunoglobulin
`assembly has been identified (Roth and Koshland, 1981) but a
`more popular candidate for fulfilling a regulatory function is im-
`munoglobulin J chain (see Koshland, 1985 for a review). Im-
`munoglobulin J chain is associated with secreted IgM and IgA
`(Halpern and Koshland, 1970; Mestecky et al., 1971) and is syn-
`thesized by plasma cells [regardless of heavy chain class express-
`ed (Kaji and Parkhouse, 1974 and 1975; Mosmann et al., 1978)]
`but not by B cells (Roth et al., 1979; Lamson and Koshland,
`1984). The onset of J chain expression roughly coincides with
`the switch from membrane IgM to secreted IgM production (Roth
`et al., 1979).
`
`© IRL Press Limited, Oxford, England
`
`Genzyme Ex. 1036, pg 905
`
`

`
`A.Cattaneo and M.S.Neuberger
`
`Table I. Antibody yields from transfected cells
`
`Cell line
`
`Cell type
`
`J[HSVi2]
`C6[HSVit2/HSVXI]
`PCl2[HSVlt2/HSVXl]
`CHO[HSVi2/HSVX1]
`HeLa[HSVA2/HSVX ]
`JW1I2I2a
`
`Plasmacytoma
`Glioma
`Phaeochromocytoma
`Fibroblast
`Epithelial
`Plasmacytoma
`
`Concentration of IgM
`(lAgIml)
`0.5
`0.6
`0.4
`0.01
`0.005
`3.5
`
`aThis cell line secretes IgM constitutively; all other lines show heat-inducible
`antibody production.
`The concentration of IgM was determined by radioimmunoassay on antigen-
`coated plates using purified JWI/2/2 IgM for calibration. Cells (3 x 10 /ml
`initial concentration) were incubated at 37°C for 3 days with 1 h heat
`shocks (42.5°C) every 24 h.
`
`possible to make a meaningful comparison of the efficiency of
`the secretion from injected oocytes to that from plasma cells.
`Here, we describe experiments to determine whether mammalian
`cell lines of non-lymphoid origin can assemble and secrete
`polymeric IgM and compare the efficiency of the process to that
`obtained using a plasmacytoma host.
`
`Results
`Inducible immunoglobulin genes
`Plasmids were constructed in which transcription of the coding
`regions for the immunoglobulin heavy and light chains is under
`control of the promoter of the Drosophila hsp70 heat-shock gene.
`The plasmid for heavy-chain expression, pSV-HSVit2 (Figure
`1B), includes the bulk of the cDNA for the immunoglobulin it
`polypeptide with the hsp70 promoter at the 5' end and the SV40
`polyadenylation signal at the 3' end. The transcription unit con-
`tains a single intron (between the regions encoding the bulk of
`the leader peptide and VHDJH) and is linked to a selective
`marker, gpt. The plasmid for light chain expression, pSV-HSVX)\
`(Figure IA), includes the expressed, genomic X1 gene of the
`mouse myeloma HOPC 2020 but with the promoter and leader
`exon replaced by the hsp7O promoter/VH-leader of plasmid
`pSV-HSVlt2. The HSVXI light chain transcription unit is link-
`ed to the neo selective marker. Association of the polypeptides
`encoded by the two plasmids should yield an IgM,XI antibody
`with specificity for the hapten 4-hydroxy-3-nitrophenacetyl (NP).
`The activity of plasmid pSV-HSV,t2 in different environments
`was determined by introducing it into a number of cell lines;
`stable transfectants were screened for the expression of im-
`munoglobulin gene mRNA and protein (Table I). As shown in
`Figure 2A, three different pools of HeLa cells transfected with
`pSV-HSVXI show inducible X gene transcription; the fourth pool
`Similarly, pSV-HSV/42
`transcribes the gene constitutively.
`transfectants of the mouse plasmacytoma J558L show inducible
`transcription of the introduced heavy-chain gene (Figure 2B); the
`size of the fragment obtained in the RNase protection assays in-
`dicates that the transcripts are initiated by the hsp7O promoter.
`It was confirmed by Northern blotting that the HSV,u2 and
`HSVX1 transcripts were indeed of the expected size (data not
`shown).
`Secretion of glycosylated IgM by non-lymphoid transfectants
`Analysis of cells transfected with pSV-HSVXl revealed that most
`of these transfectants secreted X1 light chains into the culture
`supernatant. Clones of the transfected rat phaeochromocytoma
`line PC12 and of the rat glioma C6 were analysed for X polypep-
`
`2754
`
`A
`
`r.
`cmX X -J
`C -
`
`'-
`
`,
`
`HeLa[HSVXI
`.-. + - ± -- + - +
`
`,
`
`'
`
`L
`
`HS X -.
`
`*
`
`a_
`
`..
`
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`
`..
`
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`
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`
`1:
`
`!1 + .-
`
`m
`
`HS/L
`
`-
`
`U
`
`gpt
`
`>
`
`lb
`
`Fig. 2. Analysis of immunoglobulin mRNA in transfectants. (A) Assay of XI
`mRNA in HeLa transfectants. Ribonuclease protection assays were carried
`out using the SP6-Vx probe and cytoplasmic RNA from four pools of HeLa
`cells stably transfected with pSV-HSVXI. RNA was prepared from cells
`before (-) and after (+) heat shock. RNA from the plasmacytoma J558L
`served as a control and an end-labelled HpaI digest of pBR322 provided
`size markers. The protected fragment (HSX) is the length of the entire V),
`exon. (B) Assay of HS-Vfi2 RNA in J558L transfectant J[HSV/42]. The
`ribonuclease protection assay was carried out on induced (+) and uninduced
`(-) cells using a probe for the 5'-end of the HS-VIL2 transcript as well as
`for gpt mRNA.
`
`tide synthesis. Whilst there was clonal variation in the amount
`of X expressed (some neo+ clones being negative for X1 expres-
`sion), it was clear from immunoprecipitation experiments that
`heat induction of many of the C6 and PC12 transfectants resulted
`in the secretion of X light chains (Figure 3A). Interestingly, the
`amount secreted by these transfectants is comparable to the
`amount of constitutive light chain secretion by the mouse
`plasmacytoma J558L (Figure 3A), despite the fact that J558L
`contains appreciably more X1 mRNA (not shown).
`In order to test whether the phaeochromocytoma and glioma
`lines were able to assemble complete immunoglobulin, we analys-
`ed biosynthetically labelled proteins of PC12 and C6 cells that
`had been transfected with both pSV-HSV/2 and pSV-HSVXl.
`The results (Figure 3B) show that heat shocking of both cell lines
`results in induction of the synthesis of it polypeptide. The it heavy
`chain is associated with XI light chain and is glycosylated as
`judged by the effect of the glycosylation inhibitor tunicamycin
`(Figure 3B).
`The C6 glioma transfectants were able to secrete the associated
`it and X polypeptides and the antibody secreted by these glioma
`transfectants was similar to that secreted by the plasmacytoma
`transfectant JWl/2/2 in that both antibodies bound the hapten
`NP (Figure 3D) and were recognized by anti-idiotypic antibodies
`
`Genzyme Ex. 1036, pg 906
`
`

`
`A
`
`+ +
`
`So
`
`CD
`
`"
`
`r1
`
`_2
`
`B
`
`-
`
`_
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`
`+
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`
`Polymeric IgM secretion from non-lymphoid cells
`
`D
`
`E
`
`-
`
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`
`_
`
`I+
`
`+ _1
`
`Anti-x
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`(Intracellular)
`
`Ant i- ,
`(Secreted)
`
`N
`
`>>rz I
`
`)SE
`
`Ac38 Ac 146
`Anti- idiotypes
`
`Fig. 3. Immunoglobulin secretion from transfected cells before (-) or after (+) heat-shock and in the absence or presence of tunicamycin (+Tm). (A) Light
`chain secretion by J558L and C6 and by pSV-HSVXl transfectants of C6 and PC12. Samples were immunoprecipitated from culture supernatants using anti-X
`antiserum. (B) Intracellular immunoglobulin in PC12 and C6 clones that had been transfected with both heavy and light chain expression plasmids. After
`biosynthetic labelling, cytoplasmic samples were immunoprecipitated with anti-p antiserum. (C) Secretion of IgM by C6[HSVX1/HSVyI2] transfectant and by
`constitutive IgM-secreting J558L transfectant, JW1I/2/2. After biosynthetic labelling, samples from the supernatant were precipitated with anti-i antiserum. The
`high molecular weight band in this gel that is indicated by an arrow is also precipitated from untransfected C6 cells by anti-ti antibodies and reflects
`serological cross-reaction (A.C., unpublished observations). The other arrow indicates the dye-front. (D) Comparison of IgM secretion by JWI/212 (con-
`stitutive plasmacytoma expression), C6[HSVXI/HSVA2] (heat-inducible glioma transfectant) and J[HSVA2] (which is a pSV-HSVIt2 transfectant of J558L and
`is therefore a heat-inducible IgM secreter). All samples were prepared following heat shock and analysed in parallel except that only half the JW1I2/2 sample
`was loaded on the gel and an incubation of J[HSVji2] cells in the absence of heat-shock was performed for the right hand lane. IgM from culture supernatants
`was purified by adsorption onto NIPcap-Sepharose and eluted with I mM NIPcapOH. (E) Immunoprecipitation of glioma IgM with anti-idiotypic antibodies.
`Immunoglobulin in the supernatant of biosynthetically labelled C6[HSVyt2/HSVXI] cells prior to (-) or following heat shock (+) was precipitated using
`monoclonal anti-idiotypic antibody Ac38 or Ac146 (Reth et al., 1979).
`
`(Figure 3E). Furthermore, the extent of glycosylation of the it
`polypeptide secreted by the two cell types appears similar as judg-
`ed by mobility in SDS/polyacrylamide gels.
`To compare the efficiency of antibody secretion by a glioma
`transfectant to that by a plasmacytoma, we used the C6 double
`transfectant C6[HSVAt2/HSVXl] and the J558L transfectant
`J[HSVfi2], which expresses an inducible /A gene from the
`transfected pSV-HSVi2 plasmid and constitutively transcribes
`its endogenous XI gene. In both transfectants, heat shocking
`leads to induction of i synthesis in at least 80 % of the cells as
`judged by cytoplasmic immunofluorescence. Following periodic
`heat pulses, the concentration of hapten-specific IgM in the culture
`supematants was estimated by radioimmunoassay. The results
`(Table I) confirm that the level of inducible secretion is similar
`a conclusion
`in the glioma and plasmacytoma transfectants
`which is in keeping with the results of biosynthetic labelling
`(Figure 3D). However, it is worth noting that the antibody yields
`obtained from the HeLa and CHO cell transfectants were con-
`siderably lower (Table I).
`IgM secreted by glioma transfectants is polymeric and does not
`contain J chain
`Secreted IgM is produced by plasma cells as a pentameric
`(fi2L2)5 structure (Miller and Metzger, 1965a,b; Parkhouse et
`al., 1970) that is linked by disulphide bonds and contains a single,
`covalently-linked molecule of J chain polypeptide per IgM pen-
`tamer (Mestecky et al., 1971). As it has been speculated (see
`Koshland, 1985) that J chain is necessary for IgM secretion and
`as J chain expression appears to be restricted to mature cells of
`the B lymphocyte lineage (Roth et al., 1979), it was important
`to ascertain whether the IgM secreted by the glioma transfec-
`tants did, in fact, contain immunoglobulin J chain.
`
`A
`
`-Trnt
`
`-rTTl
`
`-Tnm
`
`.1
`
`_m
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`1
`
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`
`T
`
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`
`)-
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`r
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`
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`+ Tm
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`Lc)
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`3:
`
`--
`
`><-c
`-3. _S-_
`I. o _
`
`__*q
`
`_MM4W -i - Tr-r
`
`Fig. 4. Analysis for the presence of J chain polypeptide. A rabbit antiserum
`against J chain was used to precipitate J chain from biosynthetically labelled
`cell extracts that had been made in the presence or absence of tunicamycin
`(Tm) and that had been pre-cleared with anti-light and anti-heavy chain an-
`tisera and protein A sepharose. Nevertheless, it will be seen that precipita-
`tion with the anti-J chain antiserum does bring down some residual
`immunoglobulin polypeptides. (A) Rat YO myeloma cells and cells of the
`C6[HSV,t2/HSVXI] transfectant that had been heat-induced 5 h previously
`were incubated in labelling medium for 5 h. (B) JWI/2/2 cells and cells of
`the J[HSV/A2] and C6[HSVit2/HSVXI] transfectants that had been heat-
`induced 7 h previously were incubated in labelling medium for 1 h.
`
`To determine whether there was any intracellular J chain in
`C6 cells, immunoprecipitation was carried with an anti-J chain
`antiserum using cytoplasmic extracts of biosynthetically labell-
`ed cells. The results (Figure 4A and 4B) show that J chain can
`be detected in extracts of the plasmacytoma transfectants but not
`in the C6 glioma sample. Various different labelling times were
`
`2755
`
`Genzyme Ex. 1036, pg 907
`
`

`
`A.Cattaneo and M.S.Neuberger
`
`A r,
`
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`0
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`H
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`Itm
`
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`1
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`2
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`red ciCI g'1
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`IgE x-
`
`IgG :-
`
`rioII-
`
`reducing
`
`Fig. 5. IgM secreted from C6 glioma transfectant is polymeric. (A) Analysis
`by sucrose gradient centrifugation. Supernatants (1 ml) from biosynthetically
`labelled C6[HSVtt2/HSVX1] (+---+), plasmacytoma IgM-secreting transfec-
`tant JW1/2/2 (o---o) or IgGI-secreting hybridoma P8.86.9 [gift of Thereza
`Imanishi] were loaded onto a gradient (15 ml) of 10-40% sucrose in PBS
`and subjected to centrifugation in a Beckman SW40 rotor at 40C for 18 h.
`Samples were collected, the refractive indices determined and anti-NP an-
`tibody measured by radioimmunoassay. The supematants from the C6 and
`J558L transfectants were analysed in parallel. (B,C) Analysis by
`polyacrylamide gel electrophoresis. Anti-NP antibody in the supematants of
`biosynthetically labelled JW1I2/2 (labelled as JW1) or of heat-induced
`C6[HSVst2/HSVXl] was purified on hapten sorbents and analysed either
`(left panel, B) after reduction on a 7.5% SDS/polyacrylamide gel or (right
`panel, C) unreduced on a 4% SDS/polyacrylamide gel made using
`N,N'-diallyltartardiamide to cross-link. The positions of the origin, IgM,
`IgE, IgG and it markers are indicated.
`
`used as it has been reported (Mosmann et al., 1978) that the half-
`life of J chain differs considerably in different cell lines. The
`detection of J chain polypeptide in the YO rat myeloma
`demonstrates that the anti-mouse J chain antiserum that was us-
`ed will indeed precipitate rat J chain - a point that needed con-
`
`2756
`
`Fig. 6. Electron micrographs of negatively stained IgM molecules purified
`on hapten sorbents from the supernatant of heat-induced C6[HSV,t2/HSVXlI
`transfectants. Different fields are shown in which both hexamers (H) and
`pentamers (P) are visible.
`
`firming as C6 is a glioma of rat origin. Not only was no J chain
`polypeptide detected in C6 cells, but it also appeared that these
`cells did not contain J chain niRNA as judged by primer exten-
`sion assays (data not shown). Furthermore, analysis of IgM
`in
`secreted by the plasmacytoma and C6 transfectants
`alkaline/urea polyacrylamide gels after reduction and alkylation
`revealed J chain to be present only in the plasmacytoma sample
`(data not shown).
`The sizes of the IgM secreted by the plasmacytoma and glioma
`transfectants were compared using both sucrose gradient cen-
`trifugation (Figure 5A) and electrophoresis through non-reducing
`SDS/polyacrylamide gels (Figure 5B). In both analyses, the IgM
`secreted by the glioma transfectant behaved exactly like the IgM
`secreted by the plasmacytoma and revealed itself to be a high
`molecular weight, covalently-associated aggregate. The resolution
`of the centrifugation and gel electrophoresis experiments was not,
`however, sufficient to demonstrate that the polymeric IgM
`secreted by the glioma transfectants was of exactly the same size
`as that produced by the plasmacytoma cells. We therefore resorted
`to an analysis of negatively stained protein samples in the elec-
`tron microscope (Figure 6). Comparison of the IgM samples
`prepared from plasmacytoma and glioma transfectants shows that
`whereas the IgM secreted by the plasmacytoma is almost ex-
`clusively pentameric, the sample prepared from the glioma
`transfectants is found to contain both cyclic hexamers and cyclic
`pentamers in approximately equal amounts. The sample prepared
`from C6 cells also contains some molecules which, at first sight,
`appear to be incompletely assembled structures. However, the
`same structures are seen in the plasmacytoma IgM sample and
`are most probably hexamers or pentamers that are not lying flat
`on the grid (see Feinstein and Munn, 1969). Analysis of IgM
`secreted from the heat-inducible plasmacytoma transfectant
`J[HSV,g2] revealed it to be exclusively pentameric, indicating
`that the presence of hexamers in the glioma sample is unlikely
`to be an artefact of heat shocking.
`
`Genzyme Ex. 1036, pg 908
`
`

`
`Finally, it is worth noting that pulse-chase experiments per-
`formed using biosynthetic labelling (not shown) revealed that the
`polymerization of the IgM molecules occurred prior to their secre-
`tion from the glioma or plasmacytoma cell.
`
`Discussion
`Several different non-lymphoid cell lines were transfected with
`immunoglobulin heavy and light chain genes whose expression
`was under control of a heat-shock promoter. Transfectants were
`obtained that showed inducible expression, demonstrating that
`non-lymphoid cells are capable of assembling, processing and
`secreting immunoglobulin. There was considerable variation in
`the yield of immunoglobulin obtained using different cell types
`as host. Whereas HeLa and CHO cells gave relatively poor yields,
`the amounts of antibody
`secreted by glioma and
`phaeochromocytoma transfectants were comparable to those ob-
`tained using a plasmacytoma host. The greater efficacy of C6
`glioma or PC12 as opposed to CHO or HeLa transfectants in
`antibody secretion does not appear to be due to a difference in
`the induction of immunoglobulin mRNA synthesis but rather may
`reflect the fact that the glioma and phaeochromocytoma are
`secretory cell types and may therefore be better equipped for the
`production of secreted immunoglobulin.
`The IgM secreted by the C6 transfectants is polymeric, not-
`withstanding the absence of immunoglobulin J chain. This was
`somewhat unexpected as it has been proposed that J chain is re-
`quired for the assembly and secretion of IgM (Koshland, 1985).
`However, assembly of polymeric IgM in the absence of J chain
`has previously been shown to occur both in vitro (Kownatzki,
`1973; Eskeland, 1974 and 1977) and in vivo (Stott, 1976)
`although in some cases the high molecular weight products have
`been shown to be non-covalently bonded (Wilde and Koshland,
`1978). In light of the results presented here, it is worth consider-
`ing what the role of J chain in plasma cells might be. It could
`be involved in catalysing the rate of IgM assembly - as has been
`suggested from in vitro experiments (A.Feinstein, personal com-
`munication) - or it could be involved in stabilizing the pen-
`tamers. Indeed, the IgM secreted by the glioma transfectants is
`part pentameric, part hexameric and it is conceivable that the
`presence of hexamers is a direct consequence of the absence of
`J chain. However, in this context, it is worth noting that whereas
`serum IgM - like the IgM secreted by the J558L transfectants
`- is pentameric,
`the IgM secreted by another mouse
`plasmacytoma (MOPC 104E) has been found to contain a very
`small proportion of hexamers, although J chain is expressed in
`this plasmacytoma (Parkhouse et al., 1970). It is interesting that
`the IgM antibody of Xenopus is exclusively hexameric (Metzger,
`1970; Parkhouse et al., 1970); it is not clear whether Xenopus
`IgM contains J chain although a polypeptide similar to it has been
`described in amphibia (Weinheimer et al., 1971).
`Therefore, in summary, it is clear that J chain is not required
`for the production of polymeric IgM in good yield from the
`glioma transfectants. It is, however, possible that the IgM secreted
`by these transfectants is deficient in some functional aspect. In-
`deed, evidence has been published that indicates a requirement
`for J chain in the selective transport of polymeric human IgM
`through secretory cells into exocrine fluids (Brandtzaeg and
`Prydz, 1984). The activity of the glioma IgM in epithelial
`transport and complement activation will be tested. Alternative-
`ly, it is possible that J chain may be involved in catalysing the
`production of IgM pentamers, as opposed to a pentamer/hex-
`amer mixture. This can be tested by introducing an inducible J
`
`Polymeric IgM secretion from non-lymphoid cells
`
`chain gene into both B cell lymphomas and into the glioma
`transfectants.
`Both PC12 and C6 cells are derived from cell types present
`in the nervous system. The fact that transfectants of both cell
`types are able to synthesize and secrete immunoglobulin means
`that it should be possible to engineer the production of specific
`antibodies in the central nervous system of transgenic organisms
`in order to perturb or modulate the activity of selected neuronal
`pathways.
`Materials and methods
`Plasmid construction
`Plasmid pSV-HSViA2 includes the HS-Vy2 transcription unit cloned into a
`derivative of pSV2gpt (Mulligan and Berg, 1981) in which the BamHI site has
`been converted to a SacI site by use of linkers. The HS-V/.t2 transcription unit
`was assembled in three parts. The promoter/transcription start region was ob-
`tained from plasmid pFl of Pelham (1982) as an EcoR-BgfIl fragment and con-
`tains the Drosophila hsp 70 promoter from an EcoRI site at the 5' end to the SalI
`site immediately 3' of the TATA where it
`is fused to the 60 nucleotide
`HaeIII-BgllI fragment of herpes simplex virus tk which includes the RNA cap
`site. The DNA encoding the leader region of the heavy chain polypeptide (LNP)
`was obtained from mouse VH gene V-47 (Neuberger, 1983) and extends from
`the Sau3AI site at the transcription start through the leader-VNP intron to a PstI
`site at codon +5 of VNP (Neuberger, 1983). The rest of the heavy chain coding
`region is derived from a cDNA clone for the heavy chain of an NP-specific an-
`tibody [pAB I1I (Bothwell et al., 1981)] and extends from the PstI site at codon
`+5 to a HaeIl site in the CQ 3'-untranslated region. After final assembly of pSV-
`HSVIs2, the Haell site at the 3' end of Cy is brought adjacent to the SacI site
`in the derivatized pSV2gpt vector. Plasmid pSV-HSVit2 is a cDNA derivative
`of pSV-HSV1tI (Mason, 1987).
`Plasmid pSV-HSVXI is based on a derivative of pSV2neo (Southern and Berg,
`1982) in which the HindlIl site has been destroyed by filling in and a new Hin-
`dIII site created by linker insertion in the BamHI site. The promoter/transcrip-
`tion start/leader region is the same as in pSV-HSVjt2 except (i) that the region
`5' of the promoter extends only as far as the SphI site, and (ii) the LNP-VNP
`intron is truncated at an HhaI site. This HhaI site in the LNP-VNP intron is fus-
`ed to a PstI site in the LX-V), intron of the expressed XI gene of the mouse
`plasmacytoma HOPC 2020 (Brack and Tonegawa, 1977), which provides the
`rest of the transcription unit.
`The probe for mapping HS-Vii2 transcripts was obtained by subcloning into
`pSP65 a fragment derived from an intronless derivative of pSV-HSVA2 that ex-
`tends from the StuI site in VNP back to the XhoI site in the hsp70 promoter. The
`probe for light chain expression was obtained by subcloning the BamHI-PstI
`Vx fragment of the HOPC 2020 light chain gene into plasmid pSP65.
`Cell lines and transfection
`Mouse plasmacytoma J558L (Oi et al., 1983), its IgM-secreting transfectant
`JW1/2/2 (Neuberger et al., 1984), rat myeloma YO (Galfre and Milstein, 1981),
`C6 rat glioma (Benda et al., 1968) and chinese hamster ovary (CHO) and HeLa
`cells were grown in DMEM containing 10% FCS whereas PC12 cells (Greene
`and Tischler, 1976) were grown in RPMI containing 10O% heat-inactivated horse
`serum and 5% FCS. The phenotype of the C6 glioma cells and its transfectants
`was confirmed by measuring receptors for nerve growth factor (NGF) (Cattaneo
`et al., 1983), fluorescent staining with antibody MC 192 (Chandler et al., 1984),
`immunoprecipitation of S100 protein (Labourdette and Mandel, 1978) and im-
`munofluorescent staining for glial fibrillar acidic protein (Raju et al., 1980). The
`PC12 cell transfectants were shown to respond to NGF. Transfection by
`spheroplast fusion was carried out as previously described (Neuberger, 1983)
`and electroporation (Neumann et al., 1982) was performed by giving four 2 kV
`pulses to a suspension of 2.5 x 107 cells in 0.5 ml PBS containing 20 Ag of
`supercoiled plasmid DNA. Transfection of J558L and C6 glioma was by
`spheroplast fusion, of PC12 cells by electroporation and of HeLa and CHO by
`calcium phosphate co-precipitation. For selection of neo transfectants, G418 was
`used at 0.5 mg/ml (PC12), 1 mg/ml (C6) or 2 mg/ml (other cells); gpt+ col-
`onies were selected as described by Mulligan and Berg (1981) for non-lymphoid
`cells and as previously described (Neuberger et al., 1984) for J558L.
`Analysis of RNA
`Total cytoplasmic RNA was prepared as previously described (Neuberger, 1983).
`For heat induction, RNA was extracted 4 h after the cells had been subjected
`to a 2 h shock at 42.5°C. Ribonuclease protection assays were performed as
`described by Melton et al. (1984).
`Analysis of protein
`For biosynthetic labelling of immunoglobulin, cells (5 x 106 in 1 ml) were heat-
`shocked at 42.5°C for 2 h, washed and resuspended in medium containing 5%
`
`2757
`
`Genzyme Ex. 1036, pg 909
`
`

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`Received on May 18, 1987
`Note added in proof
`The secretion of low levels of functional IgG from transfected COS and CHO
`cells has been recently reported [Weidle,U.H., Borgya,A., Mattes,R., Lenz,H.
`and Buckel,P. (1987) Gene, 51, 21-29].
`
`A.Cattaneo and M.S.Neuberger
`
`dialysed FCS, one-tenth the normal concentration of methionine and 50 jCi/ml
`L-[35 S]methionine (1220 Ci/mmol; Amersham International). After a 14-16 h
`incubation at 37°C, cells were collected by centrifugation, washed and lysed in
`0.2 ml PBS/0.5% Nonidet-P40/1 mM phenylmethylsulphonyl fluoride (PMSF).
`For analysis of J chain, the labelling period was shorter (see Figure 6 legend)
`and cells were lysed in 0.1 M Tris-HCI (pH 8.0)/0.1 M KCI/5 mM
`MgCl2/0.5% NP40/1 mM PMSF. When required, tunicamycin (8 pg/ml) was
`included during the heat-shock and labelling periods.
`Immunoprecipitations from culture supernatants or cytoplasmic extracts was
`carried out by incubation with purified rabbit anti-mouse At antiserum (kind gift
`of R.Sitia) or rat monoclonal antibody SM1/45 (kind gift of Y.Argon and
`M.R.Clark), goat anti-mouse X (Miles-Yeda), monoclonal anti-idiotype antibodies
`Ac38 and Ac146 [kind gift of M.Reth (Reth et al., 1979)] or rabbit anti-mouse
`J chain antiserum (kind gift of R.M.E.Parkhouse) followed by precipitation with
`protein A-Sepharose (Pharmacia). After washing with 50 mM Tris-HCI (pH
`7.6)/I M NaCl/0.25% NP40, samples were dissolved in reducing SDS sample
`buffer and analysed by electrophoresis through SDS/polyacrylamide gels (Laemmli,
`1970). Unreduced samples were analysed on 4% acrylamide/0.7%
`N,N'-diallyltartardiamide gels (Pearson et al., 1977). Purification on hapten
`sorbents was carried out using NIP-caproate linked to Sepharose as previously
`described (Neuberger et al., 1984).
`For analysis in the electron microscope (for which we are greatly indebted to
`John Finch), antibody that had been freshly affinity purified on hapten columns
`was concentrated to 1 mg/ml, layered onto carbon films and viewed by negativ