IMMUNOGLOBULIN PRODUCTION BY A HUMAN-MOUSE SOMATIC CELL HYBRID J. SCHWABER’ Department of Biophysics and Theoretical Biology and LaRabida-University of Chicago Institute, University of Chicago, Chicago, IL 60637, USA
SUMMARY The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobuIin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin.
Individual antibody-forming cells are limited to the production of a single molecular species of antibody. Myeloma cells are similarly restricted to the production of a monoclonal myeloma protein [l]. Examples of cells which synthesize or secrete more than one immunoglobulin are rare. A few myelomas which are biclonal have been reported [2, 31. These biclonal myeloma proteins appear to have shared idiotypes, possibly resulting from a ‘switch mechanism’ which controls the differentiation of IgM-producing cells into IgG-producing cells. Litwin et al. [4] have reported there is variation in the class of immunoglobulins ’ Present address: Immunology Division, Children’s Hospital Medical Center, Boston, MA 02115, USA.
in the intracellular, membrane-bound, and secreted pools of long-term lymphoblast cell lines. That is, cells which have membrane-bound IgM secrete IgG. Lymphoblasts may also express several classes of heavy chains on their plasma membranes [5]. Litwin et al. [5] argued that the restriction of immunoglobulin production by individual cells may accompany the differentiation of antibody-forming cells. The role of differentiation in the restriction of antibody formation may be studied in somatic cell hybrids. Cotton & Milstein [6] found that a hybrid between two myeloma cells secreted both parental myeloma proteins. I have previously reported the isolation of a somatic cell hybrid clone resulting from the fusion of TEPC-15 Exptl Cell Res 93 (1975)
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J. Schwaber
mouse myeloma cells with human periphera1 blood lymphocytes [7]. This clone of hybrid cells continued the production of myeloma protein and initiated the synthesis and secretion of human immunoglobulin. By fluorescent antibody methods, individual hybrid cells were shown to synthesize both mouse and human immunoglobulins. The hybrid cells secreted molecules cornposed of mouse alpha and light chains and human alpha, gamma, and light chains [8]. Some of these human and mouse components
were
assembled
into
hybrid
im-
munoglobulin molecules. These hybrid cells may be used to study the cellular restrictions of antibody synthesis and secretion. In this article, I report the examination of ten clones of hybrid cells resulting from the fusion of TEPC-15 mouse myeloma cells and human peripheral blood lymphocytes. Cells from each of the clones were examined for chromosome constitution, presence of mouse and human histocompatibility antigens, and secretion and synthesis of mouse and human parental immunoglobulins.
For
Purposes
of comPa~sonj
data
from clone H 1C11 [7] are included.
MATERIALS
AND METHODS
Human peripheral blood lymphocytes were obtained by sterile venipuncture from a healthy donor (J. S.). Heparinized whole blood was allowed to stand at room temperature for 4 to 6 h. The buffy coat cells were collected and washed three times with culture medium. They were incubated at 37°C for 1 h in plastic Petri dishes. The cells which failed to adhere to plastic (95 % lymphocytes) were used as the human parental cell. TEPC-I5 mouse myeloma cells were maintained by serial passage in BALB/cJ mice (Jackson Laboratories, Bar Harbor, Me). They secrete IgA (CQK~)with antibody activity for Pneumococcus C polysaccharide [9]. TEPC-I5 cells placed in culture did not initiate a cell line. However, the myeloma cells could be maintained in culture for periods up to 2 weeks, during which time they secreted myeloma protein. Sendai virus seed (Microbiological Associates, Exptl Cell Res 93 (I 975)
Bethesda, Md) was grown in 9-day embryonated chicken eggs according to the methods described by Klebe et al. [IO]. The virus was inactivated with P-propriolactone. Virus seed and inactivated virus were stored at -70°C. Human and mouse parental cells were fused with P-propriolactone inactivated Sendai virus. Parental TEPC-I5 mouse myeloma cells were placed in culture, and after 3 days non-adherent cells were removed by washing with culture medium. Only the myeloma cells which adhered to plastic were used for fusion. The medium was decanted from myeloma cell cultures, and human lymphocytes in ratios varying between I : 100 and 1 : 1000 (mouse/human) were added with 800 HAU of Sendai virus in serum-free medium. The cultures were kept at 4°C for 20 min. After a further 30 min of incubation at 37”C, culture medium with serum was added. The fusion cultures were then maintained in a 37”~ incubator with a continuous uow of 5% co,, 95 % air, with 99 % relative humidity. Hybrid cell cultures were grown in Dulbecco’s moditied Eagle medium supplemented with IO% fetal calf serum and 150 mM L-glutamine. Kanamycin and tylocine (anti-PPLO agent) were added intermittently. The medium. serum. and antibiotics were obtained from GIBCO (Grand Island, N.Y.). Hybrid cultures contained cells in suspension and attached to plastic. Clones of hybrid cells were isolated from cells attached to plastic. Cells in suspension were eliminated by washing the cultures with medium three times. Clones were isolated from subcultures which had been initiated by isolation of a single colony of hybrid cells, or by replating cells at low density. The clones were derived from apparent single cells in sub-
cu’tures. Metaphase chromosome spreads were prepared by a modification of standard techniques [I 11. Cells in the log phase of growth were arrested in metaphase with 0.6 mg/ml of colcemid for I h. The cells were incubated in 0.08 M KC1 hypotonic solution for 7 min and then fixed in three changes of I : 3 acetic acid/methanol at 4°C. Chilled cell preparations were dropped on microscope slides and dried at 40 to 50°C. To determine the modal number of chromosomes. chromosomes were stained with Giemsa stain diluted I : I with distilled water. For karvotyping, the chromosomes were Giemsa-banded a&o&i to the method of Sanchez et al. [12]. Slides were stained with a I : 80 dilution of Giemsa stain in 0.13 M phosphate buffer, pH 6.7, for 12 to I8 min. Giemsa-banded chromosome spreads were photographed with Kodak high-contrast copv 35 mm film using a Zeiss Photomicroscope I. The modal number of &omosomes was determined ty counting a minimum of 25 Giemsa-stained metaphase chromosome spreads of each cell type. Threz Giemsa-banded karyotypes were mounted from each hybrid clone. Seven additional chromosome spreads from each clone were analyzed by comparison with the mounted karyotypes. Individual chromosomes were identified according to the Paris Conference (1971) [I31 and the Committee on Standardized Genetic Nomenclature for Mice (1972) [ 141. Human and mouse parental histocompatibility antigens were assayed by an antibody-directed. complement-mediated, microcytotoxicity test [15].
Ig production Antiserum to mouse histocompatibility locus H-2d was raised by repeated immunization of C57B1/6J mice with BALBic spleen cells. Antiserum to human histocompatibilitv locus HL A7 (Cutten) was obtained via the ‘Histocdmpatibility Testing Service, NIH. Tests were performed in Falcon microtest tissue culture plates with 1000 to 2 000 cells in 1 ~l/well. The cells were incubated with 1 ~1 of serial two-fold dilutions of antiserum for 30 min at 37°C. They were incubated a further 30 min with 1 ~1 of guinea pig complement. The wells were then flooded with 0.25 % trypan blue in 0.15 M NaCl and incubated at room temperature for 10 min. The trypan blue was removed, the wells were flooded with PBS, and the test plates were read in an inverted microscope. Antiserum to human and mouse Ig was obtained as nreviouslv described 181.Human serum immunoelobuiin was fractionated from human serum by prezipitation with 50% (NH&SO,. An NZW rabbit was im.. munized biweekly (four times) with 1 ml of human serum immunoglobulin (2 to 3 mglml protein) with an equal volume of Freund’s complete adjuvant. The gamma globulin fraction of rabbit immune serum was obtained by precipitation with 50% (NH&SO+ The antiserum was absorbed once with TEPC-15 mouse myeloma protein and then repeatedly absorbed with TEPC-15 mouse myeloma cells. TEPC-15 mouse myeloma protein was isolated from the ascites fluid of TEPC-15-bearina mice bv precipitation with 50% (NH&SO, followed by chromatography on DEAE cellulose. The protein eluted with 0.1 M phosphate buffer, pH 6.5, was predominantly IgA with IgG and IgM present. An NZW rabbit was immunized biweekly (four times) with 1 ml of TEPC-15 myeloma protein (2 to 3 mg protein per ml) with an equal volume of FreundG complete-adjuvant. The gamma globulin fraction of rabbit immune serum, obtained by 50% (NH&SO4 precipitation, was absorbed once with human serum immunoglobulin and then repeatedly absorbed with human lymphocytes. The specificity of the antisera was tested by three methods: (1) Serial two-fold concentrations of antiserum to mouse Ig was incubated with ‘251-conjugated human immunoglobulin [8]. The antiserum was considered species specific when there was no direct immune precipitation. (2) A sample of the antimouse Ig was conjugated with fluorescein isothiocyanate (FITC) and the fluorescent antiserum was tested for reactivity with fixed preparations of human lymphocytes as described below. (3) iz51-conjugated human immunoglobulin with 1% bovine serum albumin (BSA) was incubated with anti-mouse Ig. Antiserum to BSA at equivalence was then added, and the immune precipitate was examined by direct radioactivity determination and by SDS gel electrophoresis (described below) for coprecipitation of human immunoglobulin. The same c&e&, using mouse immunoglobulin, mouse spleen cells, TEPC-15 mveloma protein, and TEPC-15 myeloma cells rather than human immunoglobulin and lymphocytes were used for antiserum to human Ig. Secretion of human and mouse immunoglobulin was determined by immune precipitation of 3H-leucinelabeled cell culture medium followed by electrophoresis in SDS-acrylamide gels. Five to lOxloB cells were washed twice with leucine-free medium and then
by a human-mouse
hybrid
345
incubated in leucine-free Eagle’s MEM with 20% dialysed fetal calf serum with 50 &i of 3H-leucine (3H-L-leucine-4,5, 38 Ci/mM, Amersham Searle) for 18 h. The culture medium was centrifuged at 150g for 10 min, and the supematant (cell-free) was used for analysis. The supernatants were incubated with antiserum to human or mouse Ig. Serial two-fold titrations of antisera were carried out to determine equivalence for each preparation of supernatant. Human serum immunoglobulin and TEPC-I5 myeloma protein were precipitated with the appropriate antiserum for analysis in parallel with hybrid cell protein. Supernatant culture media were also incubated with antiserum to BSA and examined for non-specific coprecipitation. The immune orecinitates were washed three times in 0.15 M NaCl and resuspended in 0.01 M phosphate buffer. uH 7.2. with 2 % sodium dodecvl sulfate (SDS). After overnight incubation at 37”C, they were heated to 90°C for 1 min to completely dissociate the immune complexes and then dialysed against electrophoresis buffer. The samples were made 8% in sucrose (w/v) and layered on top of the acrylamide gels. Electrophoresis followed the procedure of Maize1 [16] with 10 cm gels, 5 % in acrylamide, with 0.1% SDS. Electrophoresis buffer was 0.01 M phosphate buffer, pH 7.2, with 0.1% SDS. Samples were electrophoresed at 6 mA/gel. Sheep hemoglobin served as- the position marker: electrophoresis was stopped when the hemoglobin was 0.5 to 1.0 cm from the end of the gel. Hemoglobin, BSA, and rabbit IgG were used as references of known molecular weight. For optical density analysis, the gels were fixed and stained with Coomassie Blue, destained, and scanned at 520 nm in a Gilford spectrophotometer. For radioactivity determinations, 2 mm slices of gel were placed in scintillation vials with 1.0 ml of water and incubated at 37’C for 48 h. The radioactivity was determined in a Packard liquid scintillation counter with Bray’s solution [ 171. Double-label fluorescent antibody staining was used to determine the synthesis of human and mouse immunoglobulin by individual cells. Fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TMRITC) (Baltimore Biological Laboratories) were conjugated to antiserum prepared as described following the methods outlined by Goldman [18]. Briefly, 5 mlof0.15 M NaCl, 1.5 ml of0.5 M carbonate-bicarbonate buffer. oH 9.5. 1 ml acetone. and 5 ml of antiserum (20 mg&otein)‘ml were stirred with 3.0 mg of FITC or TMRITC at 4°C for 18 h. The conjugated antiserum was separated from unconjugated dye by chromatography on Sephadex G-25 in PBS followed by exhaustive dialysis against PBS. Cells to be examined were washed three times in PBS and placed on slides using a Shandon-Elliott cytocentrifuge. The slides were air-dried and fixed in acetone for 30 min. The slides were incubated sequentially with FITC-anti-mouse Ig and TMRITC-antihuman Ig at 37°C in a humid chamber for 30 min. Experiments in which the order of stamina was reversed were also performed. After staining, Ihe slides were rinsed in three changes of PBS for at least 20 min and mounted in 90 % glycerol, IO% PBS. Fluorescence microscopy was performed with a Zeiss microscope with an Osram HBO 200 W mercury lamp. For FITCantiserum, a BG-12 primary filter and an OG-1 secExprl Cell Res 93 (1975)
346
J. Schwaber
Fig. I. Karyotype from clone H4C2. The human chromosomes are labeled. The bottom row contains two chromosomes which resulted from centric fusion
of mouse chromosomes and chromosomes which could not be identified.
Eight to ten weeks after fusion, colonies of cells were seen in the fusion flasks. Hybrid cells from the 10 clones resembled large lymphocytes or plasma cells. IndividRESULTS ual cultures of all of the clones contained Cocultivation of TEPC-15 mouse myeloma cells attached to plastic and in suspension. cells and human peripheral blood lympho- Subculture of only the cells attached to cytes with Sendai virus resulted in the es- plastic yielded cultures which contained tablishment of in vitro cell lines. Since the cells attached to plastic and in suspension. parental cells had not been adapted to in Subculture of only the cells in suspension vitro culture, the selection for hybrid cells also yielded cultures which contained cells was growth in culture. Incubation of paren- attached to plastic and in suspension. Dital cells separately, with or without Sendai vision times measured in different cultures virus, did not result in cell lines. Varying from the same clone, in different clones, the ratio of parental cells between 1 : 1000 and in uncloned hybrid cells ranged from and 1 : 100 (mouse/human) did not result 20 to 40 h. The chromosome constitution of the in a significant increase in the number of parental cells and of the hybrid clones was hybrid clones. ondary filter were used. For TMRITC-antiserum, a 546 nm interference (primary) filter and a Wratten-25 secondary filter were used.
Exptl Cell Res 93 (1975)
Ig production
Table 1. Chromosome parental
distributions of the cells and the hybrid cell clones
Clone Human lymphocytes TEPC-15 Mouse myeloma HlCll H4CO H4C2 H4C3 HSCl HSC2 H5C3 H6Cl H6C2 H8Cl
Modal chromosome no. (range) 46 68 (68-73) 78 (74-82) 73 (73-77) 76 (71-79) 124 (122-125) 75 (72-80) 64 (61-69) 73 (71-76) 75 (68-79) 58 (54-60) 62 (56-59) 118 (115-129) 67 (62-69)
Mean human chromosome no. (range) 46 NT” 2 (O-3) 4 (3-6) 4 (3-4) 4 (2-5) 4 (3-6)
2 (2)
5 (3-5) 4 (3-5) 4 (3-4)
a Not tested by Giemsa banding.
determined by examination of metaphase chromosome spreads. The human and the mouse chromosome complement of the hybrid cells was determined by Giemsa banding. The modal number of chromosomes of the parental cells and each of the hybrid clones was determined. TEPC-15 mouse parental myeloma cells had a bimodal distribution of 68 and 78 chromosomes (table 1). All of the myeloma chromosomes were mouse acrocentrics. The human parental lymphocytes had the 46 chromosomes of a normal human male 46, XY karyotype. Five of the clones of hybrid cells had a modal number of chromosomes which was approximately the same as the myeloma parental cells (HlCl l-73, H4C2-75, H5Cl73, HSC2-75, and H8Cl-67). Three of the clones had a modal number of 10 fewer chromosomes than the myeloma parental cells (H4C3-64, H5C3-58, and H6Cl-62). Clone H6C2 had a modal number of 118, approximately twice the number of the myeloma parent. Clone H4CO was bimodal: 23-751810
by a humap-mouse
hybrid
347
70% of the cells had 76, and the other 30% had a mode of 124 chromosomes. On the basis of Giemsa banding, most of the human chromosomes had been excluded from the hybrid cells (fig. 2). The mean number of human chromosomes ranged from 2 (H5C3) to 5 (H6C2). The the human locus HL A7 did not lyse TEPCcell karyotypes were predominantly of the D, E, F, and G groups. There were some chromosomes which could not be identified as specific mouse or human parental chromosomes. The presence of the histocompatibility antigens of either human or mouse parental origin was tested. TEPC-15 mouse myeloma cells had the mouse histocompatibility antigen locus H-2d. Human parental lymphocytes carried the human locus HL A7. In control experiments, antiserum to the mouse locus H-2d did not lyse the human parental lymphocytes; antiserum to
Table 2. Results of the microcytotoxicity assay for the human (HL A7) and the mouse (H-2 “) parental histocompatibility antigens The percentages are cells which did not exclude trypan blue % Cytotoxicity with antiserum to Cell type
H-2d mo)
HL-A7 (92
TEPC-15 Human lymphocyte HlCll H4CO H4C2 H4C3 H5C1 H5C2 HSC3 H6Cl H6C2 H8Cl
60
3
2 47 70 55 56 83 78 85 55 40 60
52 40 5 45 52 4 8 42 2 15 4 Expri Cell Res 93 (1975)
348
J. Schwaber
All 10 of the hybrid clones had the mouse parental histocompatibility antigen, H-2d. Four of the clones (HlC 11, H4C2, H4C3, and H5C3) had the human parental antigen HL A7. The remaining six clones were not sensitive to lysis by antiserum directed against the human parental histocompatibility antigens. lmmunoglobulin secretion by the hybrid clones was determined by SDS-acrylamide gel electrophoresis of radioactive protein which precipitated following incubation with antiserum against mouse Ig or human Ig. The mouse parental myeloma protein was BALB/c IgA which migrated as peaks representing Lo, alpha*, and occasionally L in the SDS-gels (fig. 2). The dissociation of this molecule in SDS, due to the absence of H-L interchain disulfide bridges, served to characterize the TEPC-15 parental myeloma protein. Human serum immunoglobulin migrated as peaks representing L, H2 WpW, HL (I&), HL (IgA), and H,L, (IgM-monomer). The classes of human immunoglobulin were assigned by migration of protein which had been precipitated with class-specific anti3 serum. The L, and H, (alpha,) peaks are 100 j A L due to the dissociation in SDS of human IgA2 (Am+) which lacks H-L interchain disulfide bridges. The H2L2 IgM-monomer Fig. 2. Abscissa: cm. SDS-gel electrophoresis of mouse parental pro- peak was not always present and may tein secreted by hybrid clones. Hybrid cells were incubated with 3H-leucine, the supernatant was incubated have been the result of artefactual breakwith anti-mouse Ig, and the immune precipitates were down of IgM-pentamer. On the basis of applied to SDS-acrylamide gels. The TEPC-I5 mouse molecular size estimation in the SDS-gels, parental myeloma protein served as a reference. the peaks for H,L, (IgG) and H2L2 (IgA) could be distinguished only when both the human locus HL A7 did not lyse TEPC- classes were present in the same gel. The 15 parental myeloma cells. Cells incubated classes of immunoglobulin secreted by the alone, with antiserum, or with complement hybrid clones were determined by comparison of migration in the SDS-gels with the did not lyse. Cells from each of the 10 clones were reference TEPC-15 myeloma protein and tested for the human and the mouse paren- human serum immunoglobulin. tal histocompatibility antigen loci (table 2). Nine clones of hybrid cells secreted imExptl Cell Res 93 (1975)
Ig production
Fig. 3. Abscissa:
cm. SDS-gel electrophoresis of human parental protein secreted by hybrid clones. Radioactive hybrid culture medium was incubated with antihuman Ig, and the immune precipitates were electrophoresed in SDS-acrylamide gels. Human serum immunoglobulin served as a reference.
munoglobulin. Seven clones secreted mouse parental immunoglobulin which
by a human-mouse
hybrid
349
migrated as alpha, and Lz peaks in SDSgels (fig. 2). Two of these clones also secreted immunoglobulin precipitated by anti-mouse Ig which migrated as H2L2. Eight clones secreted human parental immunoglobulin (fig. 3). Each of the molecular species of immunoglobulin detected in human serum immunoglobulin was secreted by at least one clone. Six clones secreted immunoglobulin immunologically identified as human which migrated as H2 and Lz peaks in SDS-gels. This molecular species may be human IgA2 (Am+) or hybrid molecules containing human light chains and mouse alpha chains. The clones which were found to not secrete one or both parental species of immunoglobulin were re-examined. Protein from 25 ml of culture medium was precipitated with 50% (NH,),SOI and resuspended in 0.5 ml of PBS. It was incubated with serial two-fold increasing concentrations of antiserum to human Ig or mouse Ig. The initial concentration of antiserum was one-eighth the equivalence concentration used for clones which secreted immunoglobulin. No immunoglobulin was found in the re-examination. The results of experiments to detect secreted immunoglobulin can be briefly summarized. Six clones secreted both human and mouse parental immunoglobulin. Clones H5C2 and H6C2 secreted only human immunoglobulin. Clone H4C2 secreted only mouse immunoglobulin. Clone H8Cl did not secrete immunoglobulin. Re-examination of the clones which did not secrete immunoglobulin did not find immunoglobulin which was secreted in decreased quantity. Fluorescent antibody methods were used to detect immunoglobulin synthesized by individual cells. Cells from clone HlCl 1 were examined by sequential staining of Exptl Cell Res 93 (1975)
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J. Schwaber
Table 3. Parental human lymphocytes with FITC-anti-mouse
and TEPC-15 mouse myeloma cells were stained Ig and TMRITC-anti-human Ig
The percent cells staining with each of the fluorescent antisera alone and together are shown. Clone HlCll (*) was tested by sequential staining with fluorescein-conjugated antisera to human and mouse Ig. The results of experiments for Ig secretion are shown for comparison % Cells staining with FITC antimouse Ig Human lymphocytes TEPC mouse myeloma HlCll H4CO H4C2 H4C3 H5Cl H5C2 H5C3 H6C1 H6C2 H8Cl
>90 >90* >90 >90 >90 >90 90 >90 60 >90
TMRITC antihuman Ig 40 HO* >90 >90 >90 >90 >90 >90 >90 >90 >90
slides with FITC-antiserum, quenching the fluorescent dye between stainings [7]. Double-label staining with FITC-antimouse Ig and TMRITC-anti-human Ig was used with cells from all the other clones. Experiments were performed to determine the specificity of the fluorescent antisera. The human parental lymphocytes, incubated with FITC-anti-mouse Ig and/or FITC- and TMRITC-anti-human Ig, stained Ig. only with fluorescent-anti-human TEPC-15 mouse parental myeloma cells were incubated with FITC-anti-mouse Ig and FITC- and TMRITC-anti-human Ig. Only the FITC-anti-mouse Ig was bound to the myeloma cells. Mouse lymphocytes incubated with fluorescent anti-human Ig to determine cross reactivity with immunoglobulin other than IgA did not fluoresce. Prior incubation of mouse and human cells with appropriate non-fluorescent antiserum to Ig blocked the staining. Absorption of fluorescent antisera with immunoglobulin of the appropriate species also inhibited the Exprl Cell Res 93 (I 975)
FITC anti-mouse Ig & TMRITC anti-human Ig
>90* >90 >90 >90 >90 90 >90 60 >90
Secreted immunoglobulin Mouse
Human
-
+
t + + + + + +
+ t + + + + + + -
t
-
staining. Slides containing human lymphocytes and mouse spleen cells or human lymphocytes and TEPC-15 mouse myeloma cells were stained with FITC-antimouse Ig and TMRITC-anti-human Ig. Individual cells fluoresced in only one color. Finally, human lymphocytes were incubated with mouse immunoglobulin, and TEPC-I5 mouse myeloma cells were incubated with human immunoglobulin. The cells were washed and fixed on slides. Examination with FITC-anti-mouse Ig and TMRITC-anti-human Ig indicated that the heterologous immunoglobulin did not adsorb onto the cells. Individual cells from each hybrid clone were examined for cellular synthesis of human and mouse immunoglobulin (table 3). Eight of the clones of hybrid cells synthesized both human and mouse parental immunoglobulin. Most of these cells stained with the fluorescent antisera to human Ig and to mouse Ig. Greater than 90% of the cells from clone H6C2 stained with
Ig production by a human-mouse hybrid TMRITC-anti-human Ig, whereas only 60% stained with FITC-anti-mouse Ig. Cells from clone H5C2 stained with TMRITC-anti-human Ig but did not stain with FITC-anti-mouse Ig. The individual cells from eight clones of hybrid cells synthesized both human and mouse immunoglobulin. Sixty percent of the cells from clone H6C2 synthesized both parental species of immunoglobulin. The cells from clone H5C2 synthesized only human immunoglobulin. The results of experiments to determine the synthesis and secretion of immunoglobulin by individual cells can be briefly summarized. Clone HSCI did not secrete any immunoglobulin but was found to synthesize both parental species of immunoglobulin. Three clones secreted only one parental species of immunoglobulin but differed in their synthesis of parental immunoglobulin. Clone H5C2 secreted and synthesized only human immunoglobulin. Although clone H6C2 secreted only human immunoglobulin and clone H4C2 secreted only mouse immunoglobulin, cells from these clones synthesized both parental species of immunoglobulin. Cells from the remaining six clones secreted and synthesized both parental species of immunoglobulin.
DISCUSSION Ten clones of hybrid cells isolated from the fusion of human peripheral blood lymphocytes and TEPC-15 mouse myeloma cells were examined. Since neither parental cell was adapted to culture, the selection for hybrid cells was in vitro growth. The ability of the hybrid cells to grow in culture suggests that there may be complementation groups with respect to the
351
ability to grow in culture. The cells from some clones ceased dividing 2-8 months after isolation. The death of these clones may be characterized as senescence [19]. However, the death of the clones might have resulted from the exclusion of a property (gene product) required for in vitro growth of the hybrid cells. The 10 clones reported have not shown senescence. Examination of metaphase chromosome spreads indicated that the clones of in vitro cells resulted from the fusion of human and mouse cells. There was preferential exclusion of human chromosomes from the cells as has been reported for other human-mouse hybrids [20]. The mean number of human chromosomes ranged from two to five. Most of these chromosomes were from the D, E, F, and G groups. Three clones of hybrid cells had modal numbers of chromosomes which were approx. 10 less than the mouse parental myeloma cells. The loss of mouse parental chromosomes from these clones was not correlated with the loss of any mouse parental markers examined. Clone H6C2 had a modal number of 118 chromosomes. This clone may have resulted from a threeway fusion among two TEPC-15 mouse myeloma cells and one human lymphocyte, although reduplication of chromosomes cannot be excluded. Karyotypes from each of the clones included some chromosomes which could not be identified. The presence of mouse and human parental histocompatibility antigens was an indication of the hybrid character of the clones. All of the clones retained the mouse parental histocompatibility antigens. Only four of the clones also retained the human parental histocompatibility antigens. Because of the preferential exclusion of human chromosomes, the loss of human histocompatibility antigens was expected. The Exptl Ceil Res 93 (IWs)
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.I. Schwaber
human histocompatibility antigen locus has been associated with human chromosome C6 [21]. This chromosome was not present in karyotypes from the four clones which were sensitive to antiserum to HL A7. The human histocompatibility antigen locus may have been retained in these clones by translocation. Seven clones of hybrid cells secreted mouse parental immunoglobulin. All of these clones secreted the characteristic TEPC- 15 myeloma IgA , which dissociated into alpha2 and L2 subunits in SDS gels. Two of these clones also secreted a protein immunologically identified as mouse immunoglobulin which migrated as H,L, in SDS gels. Since BALB/c alpha chains lack the cysteine residue necessary for the H-L disulfide bridges, the H2L2 protein must contain either a new class of mouse heavy chain synthesized by the hybrid cells or human heavy chains assembled into hybrid molecules with mouse light chains. These alternative compositions of the H,L, molecules could not be distinguished. Eight clones of hybrid cells initiated the secretion of human parental immunoglobulin. Each of the molecular species of human serum immunoglobulin identified by SDS gel electrophoresis was secreted by one or more clones. Eight of the clones secreted human immunoglobulin of more than one class, which is in agreement with previous results [8]. Therefore, the initiation of human immunoglobulin secretion by human-mouse hybrids is not a classspecific event. Further, clones of hybrid cells are not limited to the production of a single class of immunogiobulin. Whether individual cells secreted more than one class of human immunoglobulin was not determined. Six clones secreted a molecular species of human immunoglobulin which migrated as Hz and LZ peaks in SDS Expt/ Cell Res 93 (1975)
gels. These molecules could be either human IgA2 (Am+) molecules or hybrid molecules containing mouse alpha chains and human light chains. The two clones which secreted only human immunoglobulin did not secrete molecules which dissociated into Hz and L, peaks in SDS gels. This suggests that the H2 and L2 peaks contained hybrid immunoglobulin molecules. Since Periman [22] reported that a myeloma-fibroblast hybrid secreted a greatly reduced quantity of myeloma protein, clones which apparently did not secrete immunoglobulin of one or both parental species were re-examined. The limiting step for detection of secreted immunoglobulin was precipitation with speciesspecific antisera. Therefore, 50-fold concentrated culture medium was used for reexamination. In this experiment, a reduced quantity of either parental species of immunoglobulin was not detected. Six clones of hybrid cells secreted both human and mouse parental species of immunoglobulin. Individual cells from these clones were examined by double-label fluorescent antibody staining. More than 90% of the cells from each of these clones fluoresced with the antisera directed against human Ig and against mouse Ig. These clones were homogeneous with respect to immunoglobulin synthesis: the individual cells synthesized both human and mouse immunoglobulin. Control experiments indicated that the fluorescent staining was specific: secreted immunoglobulin of one parental species did not adsorb to the surface of cells which produced only the other parental species of immunoglobulin. The individual cells from these clones synthesized both parental species of immunoglobulin. Presumably, these individual cells secreted both parental species of immunoglobulins.
Ig production
Three clones of hybrid cells secreted only one parental species of immunoglobulin. Examined with double-label fluorescent antibody, the cells from one clone secreted and synthesized only human parental immunoglobulin. This clone may have lost the structural gene coding for mouse immunoglobulin. Alternatively, the production of mouse immunoglobulin may have been suppressed in this clone. The remaining two clones synthesized both human and mouse parental species of immunoglobulin. One of these clones secreted only human parental immunoglobulin; the other clone secreted only mouse parental immunoglobulin. In these clones, there were no apparent differences in the pattern of staining with fluorescent antisera against the two parental species of Ig. Yet, the cells secreted only one parental species of immunoglobulin. This finding indicates that there was specific control of the immunoglobulin species which were secreted. Clone H8Cl did not secrete immunoglobulin. Individual cells from this clone synthesized both parental species of immunoglobulin as determined by doublelabel fluorescent antibody staining. Therefore, they were differentiated, immunoglobulin-forming cells. The pattern of staining with fluorescent antisera was similar to that of cells from other clones. The cells from this clone may have synthesized large quantities of one or both parental species of immunoglobulin but have had a secretion defect, similar to lymphocytes from some persons with agammaglobulinemia [23]. Alternatively, these cells may be the hybrid equivalent of small lymphocytes. They would be precursor antibody-secreting cells which synthesized immunoglobulin. With appropriate antigenic stimulation, they could initiate the secretion of immunoglobulin.
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353
The somatic cell hybrid clones examined, resulting from the fusion of human lymphocytes and mouse myeloma cells, synthesized and secreted immunoglobulin. The continued production of immunoglobulin by these cells is in agreement with production of immunoglobulin by other hybrids of lymphoid cells. A myeloma-lymphoma hybrid secreted only the parental myeloma protein [24]. However, a myeloma hybrid secreted both parental myeloma proteins [6]. In contrast, hybrids between myeloma cells and fibroblasts either did not secrete immunoglobulin [25] or secreted a greatly reduced quantity of immunoglobulin [22]. The distance in differentiation which separates the two parental cells would appear to determine whether differentiated properties are expressed. Fusion of myeloma cells with other lymphoid cells permits the continued production of immunoglobulin. The fusion of myeloma cells with fibroblasts or lymphoma cells did not result in the initiation of immunoglobulin by the non-Ig-forming parental cell. Similarly, fusion of lymphoblasts with tibroblasts resulted in the production of only the lymphoblast parental light chain [26]. The fusion of myeloma cells with lymphocytes resulted in the initiation of secretion of human immunoglobulin. Hybrids between two myeloma cells secreted only the immunoglobulin molecules produced before fusion [6]. The myelomalymphocyte hybrid reported, however, initiated the secretion of several molecular species of human immunoglobulin. The differentiation of plasma cells may result in the restriction of the genome, leaving only one integrated V-C gene accessible for transcription. It appears that small lymphocytes are not similarly restricted. Whether the repertoire of immunoglobulin molecules produced by the lymphoExptl Cell Res 93 (1975)
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J. Schwaber
cyte-myeloma hybrid cells is restricted to a homogeneous molecule of each class or subclass, has not been determined. The synthesis of human and mouse parental immunoglobulins in individual hybrid cells indicates that the limitations of immunoglobulin production by single cells proceeds from the restriction of transcription. Additionally, two clones of hybrid cells synthesized both parental immunoglobulins but secreted immunoglobulin of only one parental species. This finding is in agreement with the synthesis of IgG and IgM by lymphoblasts which secrete only IgG [4]. The limitations of immunoglobulin production appear to result from restriction of transcription and regulation of secretion. Finally, the hybrid between human lymphocytes and mouse myeloma cells produced human immunoglobulin despite the preferential exclusion of most of the human chromosomes. With random loss of most of the human genome, it would be expected that many clones would not have synthesized human immunoglobulin. The synthesis of human immunoglobulin by all of the clones examined may have been the result of the specific retention of activated genes in this hybrid. Alternatively, the immunoglobulin genes may have been linked to genes necessary for in vitro growth. This would have resulted in the examination of a particularly selected population of hybrid cells. This work has been supported by USPHS grant 5 TO1 GM 2037-04.
Exptl Cell Res 93 (1975)
REFERENCES 1. Hood, L & Prahl, J, Advances in immunol 14 (1971) 301. 2. Levin, A S, Fudenberg, H H, Hopper, J E, Wilson, S K & Nisonoff, A, Proc natl acad sci US 68 (1971) 68. 3. Penn, G M, Kunkel, H G & Grey, H M, Proc sot exp biol med 135 (1970) 660. 4. Litwin, S D & Cleve, H, Fed proc 32 (1973) 984 Abs. 5. Litwin, S D, Lin, P K, Hutteroth, T H & Cleve, H, Nature new biol246 (1973) 179. 6. Cotton, R G H & Milstein, C, Nature 244 (1973) 42. 7. Schwaber, J & Cohen, E P, Nature 244 (1973)444. 8. - Proc natl acad sci US 71 (1974) 2203, 9. Potter. M & Lieberman. R. J exp med 132 (1970) 737. IO. Klebe, R, Chen, T & Ruddle. F, J cell biol 45 (1970) 74. 11. Rothfels, K H & Siminovitch, L, Stain tech 33 (1958) 73. 12. Sanchez, 0, Escobar, J & Yunis, J, Lancet ii (1973) 269. 13. Paris conference (1971): Standardization in human genetics. Birth defects: Orig art ser, VIII, 7. The National Foundation, New York (1972). 14. Committee on standardized genetic nomenclature for mice. J hered 63 (1971) 69. 15. Terasaki, P & McClelland, J, Nature 204 (1964) 998. 16. Maizel, J, Fundamental techniques in virology (ed K Habel & N Salzman) p. 334. Academic Press, New York (1969). 17. Bray, G, Anal biochem 1 (1960) 279. 18. Goldman, M, Fluorescent antibody methods. Academic Press, New York (1968). 19. Orgel, L E, Nature 243 (1973) 441. 20. Miaeon. B R & Miller. C S. Science 162 (1968)
1063.
21. Van Someren, H, Westervald, A, Hagemeijer, A, Mess, J R, Merra Khan, P & Zaalber, 0, Proc natl acad sci US 71 (1974) 962. 22. Periman, P, Nature 228 (1970) 1087. ?1 Geha, R S, Schneeberger, E, Merler, E & Rosen, ~2. F S, New Engl j med 291 (1974) 1. 24. Mohit, B, Proc natl acad sci US 68 (1971) 3045. 25. Coflino, P, Knowles, B, Nathenson, S G & Scharff, M D, Nature new biol231 (1971) 84. 26. Orkin, S H, Buchanan, P, Yount, W J, Reisner, H & Littlefield, J W, Proc sot natl acad sci US 70 (1973) 2401. Received October 30, 1974 Revised version received December 18, 1974
SUMMARY The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobuIin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin.
Individual antibody-forming cells are limited to the production of a single molecular species of antibody. Myeloma cells are similarly restricted to the production of a monoclonal myeloma protein [l]. Examples of cells which synthesize or secrete more than one immunoglobulin are rare. A few myelomas which are biclonal have been reported [2, 31. These biclonal myeloma proteins appear to have shared idiotypes, possibly resulting from a ‘switch mechanism’ which controls the differentiation of IgM-producing cells into IgG-producing cells. Litwin et al. [4] have reported there is variation in the class of immunoglobulins ’ Present address: Immunology Division, Children’s Hospital Medical Center, Boston, MA 02115, USA.
in the intracellular, membrane-bound, and secreted pools of long-term lymphoblast cell lines. That is, cells which have membrane-bound IgM secrete IgG. Lymphoblasts may also express several classes of heavy chains on their plasma membranes [5]. Litwin et al. [5] argued that the restriction of immunoglobulin production by individual cells may accompany the differentiation of antibody-forming cells. The role of differentiation in the restriction of antibody formation may be studied in somatic cell hybrids. Cotton & Milstein [6] found that a hybrid between two myeloma cells secreted both parental myeloma proteins. I have previously reported the isolation of a somatic cell hybrid clone resulting from the fusion of TEPC-15 Exptl Cell Res 93 (1975)
344
J. Schwaber
mouse myeloma cells with human periphera1 blood lymphocytes [7]. This clone of hybrid cells continued the production of myeloma protein and initiated the synthesis and secretion of human immunoglobulin. By fluorescent antibody methods, individual hybrid cells were shown to synthesize both mouse and human immunoglobulins. The hybrid cells secreted molecules cornposed of mouse alpha and light chains and human alpha, gamma, and light chains [8]. Some of these human and mouse components
were
assembled
into
hybrid
im-
munoglobulin molecules. These hybrid cells may be used to study the cellular restrictions of antibody synthesis and secretion. In this article, I report the examination of ten clones of hybrid cells resulting from the fusion of TEPC-15 mouse myeloma cells and human peripheral blood lymphocytes. Cells from each of the clones were examined for chromosome constitution, presence of mouse and human histocompatibility antigens, and secretion and synthesis of mouse and human parental immunoglobulins.
For
Purposes
of comPa~sonj
data
from clone H 1C11 [7] are included.
MATERIALS
AND METHODS
Human peripheral blood lymphocytes were obtained by sterile venipuncture from a healthy donor (J. S.). Heparinized whole blood was allowed to stand at room temperature for 4 to 6 h. The buffy coat cells were collected and washed three times with culture medium. They were incubated at 37°C for 1 h in plastic Petri dishes. The cells which failed to adhere to plastic (95 % lymphocytes) were used as the human parental cell. TEPC-I5 mouse myeloma cells were maintained by serial passage in BALB/cJ mice (Jackson Laboratories, Bar Harbor, Me). They secrete IgA (CQK~)with antibody activity for Pneumococcus C polysaccharide [9]. TEPC-I5 cells placed in culture did not initiate a cell line. However, the myeloma cells could be maintained in culture for periods up to 2 weeks, during which time they secreted myeloma protein. Sendai virus seed (Microbiological Associates, Exptl Cell Res 93 (I 975)
Bethesda, Md) was grown in 9-day embryonated chicken eggs according to the methods described by Klebe et al. [IO]. The virus was inactivated with P-propriolactone. Virus seed and inactivated virus were stored at -70°C. Human and mouse parental cells were fused with P-propriolactone inactivated Sendai virus. Parental TEPC-I5 mouse myeloma cells were placed in culture, and after 3 days non-adherent cells were removed by washing with culture medium. Only the myeloma cells which adhered to plastic were used for fusion. The medium was decanted from myeloma cell cultures, and human lymphocytes in ratios varying between I : 100 and 1 : 1000 (mouse/human) were added with 800 HAU of Sendai virus in serum-free medium. The cultures were kept at 4°C for 20 min. After a further 30 min of incubation at 37”C, culture medium with serum was added. The fusion cultures were then maintained in a 37”~ incubator with a continuous uow of 5% co,, 95 % air, with 99 % relative humidity. Hybrid cell cultures were grown in Dulbecco’s moditied Eagle medium supplemented with IO% fetal calf serum and 150 mM L-glutamine. Kanamycin and tylocine (anti-PPLO agent) were added intermittently. The medium. serum. and antibiotics were obtained from GIBCO (Grand Island, N.Y.). Hybrid cultures contained cells in suspension and attached to plastic. Clones of hybrid cells were isolated from cells attached to plastic. Cells in suspension were eliminated by washing the cultures with medium three times. Clones were isolated from subcultures which had been initiated by isolation of a single colony of hybrid cells, or by replating cells at low density. The clones were derived from apparent single cells in sub-
cu’tures. Metaphase chromosome spreads were prepared by a modification of standard techniques [I 11. Cells in the log phase of growth were arrested in metaphase with 0.6 mg/ml of colcemid for I h. The cells were incubated in 0.08 M KC1 hypotonic solution for 7 min and then fixed in three changes of I : 3 acetic acid/methanol at 4°C. Chilled cell preparations were dropped on microscope slides and dried at 40 to 50°C. To determine the modal number of chromosomes. chromosomes were stained with Giemsa stain diluted I : I with distilled water. For karvotyping, the chromosomes were Giemsa-banded a&o&i to the method of Sanchez et al. [12]. Slides were stained with a I : 80 dilution of Giemsa stain in 0.13 M phosphate buffer, pH 6.7, for 12 to I8 min. Giemsa-banded chromosome spreads were photographed with Kodak high-contrast copv 35 mm film using a Zeiss Photomicroscope I. The modal number of &omosomes was determined ty counting a minimum of 25 Giemsa-stained metaphase chromosome spreads of each cell type. Threz Giemsa-banded karyotypes were mounted from each hybrid clone. Seven additional chromosome spreads from each clone were analyzed by comparison with the mounted karyotypes. Individual chromosomes were identified according to the Paris Conference (1971) [I31 and the Committee on Standardized Genetic Nomenclature for Mice (1972) [ 141. Human and mouse parental histocompatibility antigens were assayed by an antibody-directed. complement-mediated, microcytotoxicity test [15].
Ig production Antiserum to mouse histocompatibility locus H-2d was raised by repeated immunization of C57B1/6J mice with BALBic spleen cells. Antiserum to human histocompatibilitv locus HL A7 (Cutten) was obtained via the ‘Histocdmpatibility Testing Service, NIH. Tests were performed in Falcon microtest tissue culture plates with 1000 to 2 000 cells in 1 ~l/well. The cells were incubated with 1 ~1 of serial two-fold dilutions of antiserum for 30 min at 37°C. They were incubated a further 30 min with 1 ~1 of guinea pig complement. The wells were then flooded with 0.25 % trypan blue in 0.15 M NaCl and incubated at room temperature for 10 min. The trypan blue was removed, the wells were flooded with PBS, and the test plates were read in an inverted microscope. Antiserum to human and mouse Ig was obtained as nreviouslv described 181.Human serum immunoelobuiin was fractionated from human serum by prezipitation with 50% (NH&SO,. An NZW rabbit was im.. munized biweekly (four times) with 1 ml of human serum immunoglobulin (2 to 3 mglml protein) with an equal volume of Freund’s complete adjuvant. The gamma globulin fraction of rabbit immune serum was obtained by precipitation with 50% (NH&SO+ The antiserum was absorbed once with TEPC-15 mouse myeloma protein and then repeatedly absorbed with TEPC-15 mouse myeloma cells. TEPC-15 mouse myeloma protein was isolated from the ascites fluid of TEPC-15-bearina mice bv precipitation with 50% (NH&SO, followed by chromatography on DEAE cellulose. The protein eluted with 0.1 M phosphate buffer, pH 6.5, was predominantly IgA with IgG and IgM present. An NZW rabbit was immunized biweekly (four times) with 1 ml of TEPC-15 myeloma protein (2 to 3 mg protein per ml) with an equal volume of FreundG complete-adjuvant. The gamma globulin fraction of rabbit immune serum, obtained by 50% (NH&SO4 precipitation, was absorbed once with human serum immunoglobulin and then repeatedly absorbed with human lymphocytes. The specificity of the antisera was tested by three methods: (1) Serial two-fold concentrations of antiserum to mouse Ig was incubated with ‘251-conjugated human immunoglobulin [8]. The antiserum was considered species specific when there was no direct immune precipitation. (2) A sample of the antimouse Ig was conjugated with fluorescein isothiocyanate (FITC) and the fluorescent antiserum was tested for reactivity with fixed preparations of human lymphocytes as described below. (3) iz51-conjugated human immunoglobulin with 1% bovine serum albumin (BSA) was incubated with anti-mouse Ig. Antiserum to BSA at equivalence was then added, and the immune precipitate was examined by direct radioactivity determination and by SDS gel electrophoresis (described below) for coprecipitation of human immunoglobulin. The same c&e&, using mouse immunoglobulin, mouse spleen cells, TEPC-15 mveloma protein, and TEPC-15 myeloma cells rather than human immunoglobulin and lymphocytes were used for antiserum to human Ig. Secretion of human and mouse immunoglobulin was determined by immune precipitation of 3H-leucinelabeled cell culture medium followed by electrophoresis in SDS-acrylamide gels. Five to lOxloB cells were washed twice with leucine-free medium and then
by a human-mouse
hybrid
345
incubated in leucine-free Eagle’s MEM with 20% dialysed fetal calf serum with 50 &i of 3H-leucine (3H-L-leucine-4,5, 38 Ci/mM, Amersham Searle) for 18 h. The culture medium was centrifuged at 150g for 10 min, and the supematant (cell-free) was used for analysis. The supernatants were incubated with antiserum to human or mouse Ig. Serial two-fold titrations of antisera were carried out to determine equivalence for each preparation of supernatant. Human serum immunoglobulin and TEPC-I5 myeloma protein were precipitated with the appropriate antiserum for analysis in parallel with hybrid cell protein. Supernatant culture media were also incubated with antiserum to BSA and examined for non-specific coprecipitation. The immune orecinitates were washed three times in 0.15 M NaCl and resuspended in 0.01 M phosphate buffer. uH 7.2. with 2 % sodium dodecvl sulfate (SDS). After overnight incubation at 37”C, they were heated to 90°C for 1 min to completely dissociate the immune complexes and then dialysed against electrophoresis buffer. The samples were made 8% in sucrose (w/v) and layered on top of the acrylamide gels. Electrophoresis followed the procedure of Maize1 [16] with 10 cm gels, 5 % in acrylamide, with 0.1% SDS. Electrophoresis buffer was 0.01 M phosphate buffer, pH 7.2, with 0.1% SDS. Samples were electrophoresed at 6 mA/gel. Sheep hemoglobin served as- the position marker: electrophoresis was stopped when the hemoglobin was 0.5 to 1.0 cm from the end of the gel. Hemoglobin, BSA, and rabbit IgG were used as references of known molecular weight. For optical density analysis, the gels were fixed and stained with Coomassie Blue, destained, and scanned at 520 nm in a Gilford spectrophotometer. For radioactivity determinations, 2 mm slices of gel were placed in scintillation vials with 1.0 ml of water and incubated at 37’C for 48 h. The radioactivity was determined in a Packard liquid scintillation counter with Bray’s solution [ 171. Double-label fluorescent antibody staining was used to determine the synthesis of human and mouse immunoglobulin by individual cells. Fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TMRITC) (Baltimore Biological Laboratories) were conjugated to antiserum prepared as described following the methods outlined by Goldman [18]. Briefly, 5 mlof0.15 M NaCl, 1.5 ml of0.5 M carbonate-bicarbonate buffer. oH 9.5. 1 ml acetone. and 5 ml of antiserum (20 mg&otein)‘ml were stirred with 3.0 mg of FITC or TMRITC at 4°C for 18 h. The conjugated antiserum was separated from unconjugated dye by chromatography on Sephadex G-25 in PBS followed by exhaustive dialysis against PBS. Cells to be examined were washed three times in PBS and placed on slides using a Shandon-Elliott cytocentrifuge. The slides were air-dried and fixed in acetone for 30 min. The slides were incubated sequentially with FITC-anti-mouse Ig and TMRITC-antihuman Ig at 37°C in a humid chamber for 30 min. Experiments in which the order of stamina was reversed were also performed. After staining, Ihe slides were rinsed in three changes of PBS for at least 20 min and mounted in 90 % glycerol, IO% PBS. Fluorescence microscopy was performed with a Zeiss microscope with an Osram HBO 200 W mercury lamp. For FITCantiserum, a BG-12 primary filter and an OG-1 secExprl Cell Res 93 (1975)
346
J. Schwaber
Fig. I. Karyotype from clone H4C2. The human chromosomes are labeled. The bottom row contains two chromosomes which resulted from centric fusion
of mouse chromosomes and chromosomes which could not be identified.
Eight to ten weeks after fusion, colonies of cells were seen in the fusion flasks. Hybrid cells from the 10 clones resembled large lymphocytes or plasma cells. IndividRESULTS ual cultures of all of the clones contained Cocultivation of TEPC-15 mouse myeloma cells attached to plastic and in suspension. cells and human peripheral blood lympho- Subculture of only the cells attached to cytes with Sendai virus resulted in the es- plastic yielded cultures which contained tablishment of in vitro cell lines. Since the cells attached to plastic and in suspension. parental cells had not been adapted to in Subculture of only the cells in suspension vitro culture, the selection for hybrid cells also yielded cultures which contained cells was growth in culture. Incubation of paren- attached to plastic and in suspension. Dital cells separately, with or without Sendai vision times measured in different cultures virus, did not result in cell lines. Varying from the same clone, in different clones, the ratio of parental cells between 1 : 1000 and in uncloned hybrid cells ranged from and 1 : 100 (mouse/human) did not result 20 to 40 h. The chromosome constitution of the in a significant increase in the number of parental cells and of the hybrid clones was hybrid clones. ondary filter were used. For TMRITC-antiserum, a 546 nm interference (primary) filter and a Wratten-25 secondary filter were used.
Exptl Cell Res 93 (1975)
Ig production
Table 1. Chromosome parental
distributions of the cells and the hybrid cell clones
Clone Human lymphocytes TEPC-15 Mouse myeloma HlCll H4CO H4C2 H4C3 HSCl HSC2 H5C3 H6Cl H6C2 H8Cl
Modal chromosome no. (range) 46 68 (68-73) 78 (74-82) 73 (73-77) 76 (71-79) 124 (122-125) 75 (72-80) 64 (61-69) 73 (71-76) 75 (68-79) 58 (54-60) 62 (56-59) 118 (115-129) 67 (62-69)
Mean human chromosome no. (range) 46 NT” 2 (O-3) 4 (3-6) 4 (3-4) 4 (2-5) 4 (3-6)
2 (2)
5 (3-5) 4 (3-5) 4 (3-4)
a Not tested by Giemsa banding.
determined by examination of metaphase chromosome spreads. The human and the mouse chromosome complement of the hybrid cells was determined by Giemsa banding. The modal number of chromosomes of the parental cells and each of the hybrid clones was determined. TEPC-15 mouse parental myeloma cells had a bimodal distribution of 68 and 78 chromosomes (table 1). All of the myeloma chromosomes were mouse acrocentrics. The human parental lymphocytes had the 46 chromosomes of a normal human male 46, XY karyotype. Five of the clones of hybrid cells had a modal number of chromosomes which was approximately the same as the myeloma parental cells (HlCl l-73, H4C2-75, H5Cl73, HSC2-75, and H8Cl-67). Three of the clones had a modal number of 10 fewer chromosomes than the myeloma parental cells (H4C3-64, H5C3-58, and H6Cl-62). Clone H6C2 had a modal number of 118, approximately twice the number of the myeloma parent. Clone H4CO was bimodal: 23-751810
by a humap-mouse
hybrid
347
70% of the cells had 76, and the other 30% had a mode of 124 chromosomes. On the basis of Giemsa banding, most of the human chromosomes had been excluded from the hybrid cells (fig. 2). The mean number of human chromosomes ranged from 2 (H5C3) to 5 (H6C2). The the human locus HL A7 did not lyse TEPCcell karyotypes were predominantly of the D, E, F, and G groups. There were some chromosomes which could not be identified as specific mouse or human parental chromosomes. The presence of the histocompatibility antigens of either human or mouse parental origin was tested. TEPC-15 mouse myeloma cells had the mouse histocompatibility antigen locus H-2d. Human parental lymphocytes carried the human locus HL A7. In control experiments, antiserum to the mouse locus H-2d did not lyse the human parental lymphocytes; antiserum to
Table 2. Results of the microcytotoxicity assay for the human (HL A7) and the mouse (H-2 “) parental histocompatibility antigens The percentages are cells which did not exclude trypan blue % Cytotoxicity with antiserum to Cell type
H-2d mo)
HL-A7 (92
TEPC-15 Human lymphocyte HlCll H4CO H4C2 H4C3 H5C1 H5C2 HSC3 H6Cl H6C2 H8Cl
60
3
2 47 70 55 56 83 78 85 55 40 60
52 40 5 45 52 4 8 42 2 15 4 Expri Cell Res 93 (1975)
348
J. Schwaber
All 10 of the hybrid clones had the mouse parental histocompatibility antigen, H-2d. Four of the clones (HlC 11, H4C2, H4C3, and H5C3) had the human parental antigen HL A7. The remaining six clones were not sensitive to lysis by antiserum directed against the human parental histocompatibility antigens. lmmunoglobulin secretion by the hybrid clones was determined by SDS-acrylamide gel electrophoresis of radioactive protein which precipitated following incubation with antiserum against mouse Ig or human Ig. The mouse parental myeloma protein was BALB/c IgA which migrated as peaks representing Lo, alpha*, and occasionally L in the SDS-gels (fig. 2). The dissociation of this molecule in SDS, due to the absence of H-L interchain disulfide bridges, served to characterize the TEPC-15 parental myeloma protein. Human serum immunoglobulin migrated as peaks representing L, H2 WpW, HL (I&), HL (IgA), and H,L, (IgM-monomer). The classes of human immunoglobulin were assigned by migration of protein which had been precipitated with class-specific anti3 serum. The L, and H, (alpha,) peaks are 100 j A L due to the dissociation in SDS of human IgA2 (Am+) which lacks H-L interchain disulfide bridges. The H2L2 IgM-monomer Fig. 2. Abscissa: cm. SDS-gel electrophoresis of mouse parental pro- peak was not always present and may tein secreted by hybrid clones. Hybrid cells were incubated with 3H-leucine, the supernatant was incubated have been the result of artefactual breakwith anti-mouse Ig, and the immune precipitates were down of IgM-pentamer. On the basis of applied to SDS-acrylamide gels. The TEPC-I5 mouse molecular size estimation in the SDS-gels, parental myeloma protein served as a reference. the peaks for H,L, (IgG) and H2L2 (IgA) could be distinguished only when both the human locus HL A7 did not lyse TEPC- classes were present in the same gel. The 15 parental myeloma cells. Cells incubated classes of immunoglobulin secreted by the alone, with antiserum, or with complement hybrid clones were determined by comparison of migration in the SDS-gels with the did not lyse. Cells from each of the 10 clones were reference TEPC-15 myeloma protein and tested for the human and the mouse paren- human serum immunoglobulin. tal histocompatibility antigen loci (table 2). Nine clones of hybrid cells secreted imExptl Cell Res 93 (1975)
Ig production
Fig. 3. Abscissa:
cm. SDS-gel electrophoresis of human parental protein secreted by hybrid clones. Radioactive hybrid culture medium was incubated with antihuman Ig, and the immune precipitates were electrophoresed in SDS-acrylamide gels. Human serum immunoglobulin served as a reference.
munoglobulin. Seven clones secreted mouse parental immunoglobulin which
by a human-mouse
hybrid
349
migrated as alpha, and Lz peaks in SDSgels (fig. 2). Two of these clones also secreted immunoglobulin precipitated by anti-mouse Ig which migrated as H2L2. Eight clones secreted human parental immunoglobulin (fig. 3). Each of the molecular species of immunoglobulin detected in human serum immunoglobulin was secreted by at least one clone. Six clones secreted immunoglobulin immunologically identified as human which migrated as H2 and Lz peaks in SDS-gels. This molecular species may be human IgA2 (Am+) or hybrid molecules containing human light chains and mouse alpha chains. The clones which were found to not secrete one or both parental species of immunoglobulin were re-examined. Protein from 25 ml of culture medium was precipitated with 50% (NH,),SOI and resuspended in 0.5 ml of PBS. It was incubated with serial two-fold increasing concentrations of antiserum to human Ig or mouse Ig. The initial concentration of antiserum was one-eighth the equivalence concentration used for clones which secreted immunoglobulin. No immunoglobulin was found in the re-examination. The results of experiments to detect secreted immunoglobulin can be briefly summarized. Six clones secreted both human and mouse parental immunoglobulin. Clones H5C2 and H6C2 secreted only human immunoglobulin. Clone H4C2 secreted only mouse immunoglobulin. Clone H8Cl did not secrete immunoglobulin. Re-examination of the clones which did not secrete immunoglobulin did not find immunoglobulin which was secreted in decreased quantity. Fluorescent antibody methods were used to detect immunoglobulin synthesized by individual cells. Cells from clone HlCl 1 were examined by sequential staining of Exptl Cell Res 93 (1975)
350
J. Schwaber
Table 3. Parental human lymphocytes with FITC-anti-mouse
and TEPC-15 mouse myeloma cells were stained Ig and TMRITC-anti-human Ig
The percent cells staining with each of the fluorescent antisera alone and together are shown. Clone HlCll (*) was tested by sequential staining with fluorescein-conjugated antisera to human and mouse Ig. The results of experiments for Ig secretion are shown for comparison % Cells staining with FITC antimouse Ig Human lymphocytes TEPC mouse myeloma HlCll H4CO H4C2 H4C3 H5Cl H5C2 H5C3 H6C1 H6C2 H8Cl
>90 >90* >90 >90 >90 >90 90 >90 60 >90
TMRITC antihuman Ig 40 HO* >90 >90 >90 >90 >90 >90 >90 >90 >90
slides with FITC-antiserum, quenching the fluorescent dye between stainings [7]. Double-label staining with FITC-antimouse Ig and TMRITC-anti-human Ig was used with cells from all the other clones. Experiments were performed to determine the specificity of the fluorescent antisera. The human parental lymphocytes, incubated with FITC-anti-mouse Ig and/or FITC- and TMRITC-anti-human Ig, stained Ig. only with fluorescent-anti-human TEPC-15 mouse parental myeloma cells were incubated with FITC-anti-mouse Ig and FITC- and TMRITC-anti-human Ig. Only the FITC-anti-mouse Ig was bound to the myeloma cells. Mouse lymphocytes incubated with fluorescent anti-human Ig to determine cross reactivity with immunoglobulin other than IgA did not fluoresce. Prior incubation of mouse and human cells with appropriate non-fluorescent antiserum to Ig blocked the staining. Absorption of fluorescent antisera with immunoglobulin of the appropriate species also inhibited the Exprl Cell Res 93 (I 975)
FITC anti-mouse Ig & TMRITC anti-human Ig
>90* >90 >90 >90 >90 90 >90 60 >90
Secreted immunoglobulin Mouse
Human
-
+
t + + + + + +
+ t + + + + + + -
t
-
staining. Slides containing human lymphocytes and mouse spleen cells or human lymphocytes and TEPC-15 mouse myeloma cells were stained with FITC-antimouse Ig and TMRITC-anti-human Ig. Individual cells fluoresced in only one color. Finally, human lymphocytes were incubated with mouse immunoglobulin, and TEPC-I5 mouse myeloma cells were incubated with human immunoglobulin. The cells were washed and fixed on slides. Examination with FITC-anti-mouse Ig and TMRITC-anti-human Ig indicated that the heterologous immunoglobulin did not adsorb onto the cells. Individual cells from each hybrid clone were examined for cellular synthesis of human and mouse immunoglobulin (table 3). Eight of the clones of hybrid cells synthesized both human and mouse parental immunoglobulin. Most of these cells stained with the fluorescent antisera to human Ig and to mouse Ig. Greater than 90% of the cells from clone H6C2 stained with
Ig production by a human-mouse hybrid TMRITC-anti-human Ig, whereas only 60% stained with FITC-anti-mouse Ig. Cells from clone H5C2 stained with TMRITC-anti-human Ig but did not stain with FITC-anti-mouse Ig. The individual cells from eight clones of hybrid cells synthesized both human and mouse immunoglobulin. Sixty percent of the cells from clone H6C2 synthesized both parental species of immunoglobulin. The cells from clone H5C2 synthesized only human immunoglobulin. The results of experiments to determine the synthesis and secretion of immunoglobulin by individual cells can be briefly summarized. Clone HSCI did not secrete any immunoglobulin but was found to synthesize both parental species of immunoglobulin. Three clones secreted only one parental species of immunoglobulin but differed in their synthesis of parental immunoglobulin. Clone H5C2 secreted and synthesized only human immunoglobulin. Although clone H6C2 secreted only human immunoglobulin and clone H4C2 secreted only mouse immunoglobulin, cells from these clones synthesized both parental species of immunoglobulin. Cells from the remaining six clones secreted and synthesized both parental species of immunoglobulin.
DISCUSSION Ten clones of hybrid cells isolated from the fusion of human peripheral blood lymphocytes and TEPC-15 mouse myeloma cells were examined. Since neither parental cell was adapted to culture, the selection for hybrid cells was in vitro growth. The ability of the hybrid cells to grow in culture suggests that there may be complementation groups with respect to the
351
ability to grow in culture. The cells from some clones ceased dividing 2-8 months after isolation. The death of these clones may be characterized as senescence [19]. However, the death of the clones might have resulted from the exclusion of a property (gene product) required for in vitro growth of the hybrid cells. The 10 clones reported have not shown senescence. Examination of metaphase chromosome spreads indicated that the clones of in vitro cells resulted from the fusion of human and mouse cells. There was preferential exclusion of human chromosomes from the cells as has been reported for other human-mouse hybrids [20]. The mean number of human chromosomes ranged from two to five. Most of these chromosomes were from the D, E, F, and G groups. Three clones of hybrid cells had modal numbers of chromosomes which were approx. 10 less than the mouse parental myeloma cells. The loss of mouse parental chromosomes from these clones was not correlated with the loss of any mouse parental markers examined. Clone H6C2 had a modal number of 118 chromosomes. This clone may have resulted from a threeway fusion among two TEPC-15 mouse myeloma cells and one human lymphocyte, although reduplication of chromosomes cannot be excluded. Karyotypes from each of the clones included some chromosomes which could not be identified. The presence of mouse and human parental histocompatibility antigens was an indication of the hybrid character of the clones. All of the clones retained the mouse parental histocompatibility antigens. Only four of the clones also retained the human parental histocompatibility antigens. Because of the preferential exclusion of human chromosomes, the loss of human histocompatibility antigens was expected. The Exptl Ceil Res 93 (IWs)
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human histocompatibility antigen locus has been associated with human chromosome C6 [21]. This chromosome was not present in karyotypes from the four clones which were sensitive to antiserum to HL A7. The human histocompatibility antigen locus may have been retained in these clones by translocation. Seven clones of hybrid cells secreted mouse parental immunoglobulin. All of these clones secreted the characteristic TEPC- 15 myeloma IgA , which dissociated into alpha2 and L2 subunits in SDS gels. Two of these clones also secreted a protein immunologically identified as mouse immunoglobulin which migrated as H,L, in SDS gels. Since BALB/c alpha chains lack the cysteine residue necessary for the H-L disulfide bridges, the H2L2 protein must contain either a new class of mouse heavy chain synthesized by the hybrid cells or human heavy chains assembled into hybrid molecules with mouse light chains. These alternative compositions of the H,L, molecules could not be distinguished. Eight clones of hybrid cells initiated the secretion of human parental immunoglobulin. Each of the molecular species of human serum immunoglobulin identified by SDS gel electrophoresis was secreted by one or more clones. Eight of the clones secreted human immunoglobulin of more than one class, which is in agreement with previous results [8]. Therefore, the initiation of human immunoglobulin secretion by human-mouse hybrids is not a classspecific event. Further, clones of hybrid cells are not limited to the production of a single class of immunogiobulin. Whether individual cells secreted more than one class of human immunoglobulin was not determined. Six clones secreted a molecular species of human immunoglobulin which migrated as Hz and LZ peaks in SDS Expt/ Cell Res 93 (1975)
gels. These molecules could be either human IgA2 (Am+) molecules or hybrid molecules containing mouse alpha chains and human light chains. The two clones which secreted only human immunoglobulin did not secrete molecules which dissociated into Hz and L, peaks in SDS gels. This suggests that the H2 and L2 peaks contained hybrid immunoglobulin molecules. Since Periman [22] reported that a myeloma-fibroblast hybrid secreted a greatly reduced quantity of myeloma protein, clones which apparently did not secrete immunoglobulin of one or both parental species were re-examined. The limiting step for detection of secreted immunoglobulin was precipitation with speciesspecific antisera. Therefore, 50-fold concentrated culture medium was used for reexamination. In this experiment, a reduced quantity of either parental species of immunoglobulin was not detected. Six clones of hybrid cells secreted both human and mouse parental species of immunoglobulin. Individual cells from these clones were examined by double-label fluorescent antibody staining. More than 90% of the cells from each of these clones fluoresced with the antisera directed against human Ig and against mouse Ig. These clones were homogeneous with respect to immunoglobulin synthesis: the individual cells synthesized both human and mouse immunoglobulin. Control experiments indicated that the fluorescent staining was specific: secreted immunoglobulin of one parental species did not adsorb to the surface of cells which produced only the other parental species of immunoglobulin. The individual cells from these clones synthesized both parental species of immunoglobulin. Presumably, these individual cells secreted both parental species of immunoglobulins.
Ig production
Three clones of hybrid cells secreted only one parental species of immunoglobulin. Examined with double-label fluorescent antibody, the cells from one clone secreted and synthesized only human parental immunoglobulin. This clone may have lost the structural gene coding for mouse immunoglobulin. Alternatively, the production of mouse immunoglobulin may have been suppressed in this clone. The remaining two clones synthesized both human and mouse parental species of immunoglobulin. One of these clones secreted only human parental immunoglobulin; the other clone secreted only mouse parental immunoglobulin. In these clones, there were no apparent differences in the pattern of staining with fluorescent antisera against the two parental species of Ig. Yet, the cells secreted only one parental species of immunoglobulin. This finding indicates that there was specific control of the immunoglobulin species which were secreted. Clone H8Cl did not secrete immunoglobulin. Individual cells from this clone synthesized both parental species of immunoglobulin as determined by doublelabel fluorescent antibody staining. Therefore, they were differentiated, immunoglobulin-forming cells. The pattern of staining with fluorescent antisera was similar to that of cells from other clones. The cells from this clone may have synthesized large quantities of one or both parental species of immunoglobulin but have had a secretion defect, similar to lymphocytes from some persons with agammaglobulinemia [23]. Alternatively, these cells may be the hybrid equivalent of small lymphocytes. They would be precursor antibody-secreting cells which synthesized immunoglobulin. With appropriate antigenic stimulation, they could initiate the secretion of immunoglobulin.
by a human-mouse
hybrid
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The somatic cell hybrid clones examined, resulting from the fusion of human lymphocytes and mouse myeloma cells, synthesized and secreted immunoglobulin. The continued production of immunoglobulin by these cells is in agreement with production of immunoglobulin by other hybrids of lymphoid cells. A myeloma-lymphoma hybrid secreted only the parental myeloma protein [24]. However, a myeloma hybrid secreted both parental myeloma proteins [6]. In contrast, hybrids between myeloma cells and fibroblasts either did not secrete immunoglobulin [25] or secreted a greatly reduced quantity of immunoglobulin [22]. The distance in differentiation which separates the two parental cells would appear to determine whether differentiated properties are expressed. Fusion of myeloma cells with other lymphoid cells permits the continued production of immunoglobulin. The fusion of myeloma cells with fibroblasts or lymphoma cells did not result in the initiation of immunoglobulin by the non-Ig-forming parental cell. Similarly, fusion of lymphoblasts with tibroblasts resulted in the production of only the lymphoblast parental light chain [26]. The fusion of myeloma cells with lymphocytes resulted in the initiation of secretion of human immunoglobulin. Hybrids between two myeloma cells secreted only the immunoglobulin molecules produced before fusion [6]. The myelomalymphocyte hybrid reported, however, initiated the secretion of several molecular species of human immunoglobulin. The differentiation of plasma cells may result in the restriction of the genome, leaving only one integrated V-C gene accessible for transcription. It appears that small lymphocytes are not similarly restricted. Whether the repertoire of immunoglobulin molecules produced by the lymphoExptl Cell Res 93 (1975)
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cyte-myeloma hybrid cells is restricted to a homogeneous molecule of each class or subclass, has not been determined. The synthesis of human and mouse parental immunoglobulins in individual hybrid cells indicates that the limitations of immunoglobulin production by single cells proceeds from the restriction of transcription. Additionally, two clones of hybrid cells synthesized both parental immunoglobulins but secreted immunoglobulin of only one parental species. This finding is in agreement with the synthesis of IgG and IgM by lymphoblasts which secrete only IgG [4]. The limitations of immunoglobulin production appear to result from restriction of transcription and regulation of secretion. Finally, the hybrid between human lymphocytes and mouse myeloma cells produced human immunoglobulin despite the preferential exclusion of most of the human chromosomes. With random loss of most of the human genome, it would be expected that many clones would not have synthesized human immunoglobulin. The synthesis of human immunoglobulin by all of the clones examined may have been the result of the specific retention of activated genes in this hybrid. Alternatively, the immunoglobulin genes may have been linked to genes necessary for in vitro growth. This would have resulted in the examination of a particularly selected population of hybrid cells. This work has been supported by USPHS grant 5 TO1 GM 2037-04.
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REFERENCES 1. Hood, L & Prahl, J, Advances in immunol 14 (1971) 301. 2. Levin, A S, Fudenberg, H H, Hopper, J E, Wilson, S K & Nisonoff, A, Proc natl acad sci US 68 (1971) 68. 3. Penn, G M, Kunkel, H G & Grey, H M, Proc sot exp biol med 135 (1970) 660. 4. Litwin, S D & Cleve, H, Fed proc 32 (1973) 984 Abs. 5. Litwin, S D, Lin, P K, Hutteroth, T H & Cleve, H, Nature new biol246 (1973) 179. 6. Cotton, R G H & Milstein, C, Nature 244 (1973) 42. 7. Schwaber, J & Cohen, E P, Nature 244 (1973)444. 8. - Proc natl acad sci US 71 (1974) 2203, 9. Potter. M & Lieberman. R. J exp med 132 (1970) 737. IO. Klebe, R, Chen, T & Ruddle. F, J cell biol 45 (1970) 74. 11. Rothfels, K H & Siminovitch, L, Stain tech 33 (1958) 73. 12. Sanchez, 0, Escobar, J & Yunis, J, Lancet ii (1973) 269. 13. Paris conference (1971): Standardization in human genetics. Birth defects: Orig art ser, VIII, 7. The National Foundation, New York (1972). 14. Committee on standardized genetic nomenclature for mice. J hered 63 (1971) 69. 15. Terasaki, P & McClelland, J, Nature 204 (1964) 998. 16. Maizel, J, Fundamental techniques in virology (ed K Habel & N Salzman) p. 334. Academic Press, New York (1969). 17. Bray, G, Anal biochem 1 (1960) 279. 18. Goldman, M, Fluorescent antibody methods. Academic Press, New York (1968). 19. Orgel, L E, Nature 243 (1973) 441. 20. Miaeon. B R & Miller. C S. Science 162 (1968)
1063.
21. Van Someren, H, Westervald, A, Hagemeijer, A, Mess, J R, Merra Khan, P & Zaalber, 0, Proc natl acad sci US 71 (1974) 962. 22. Periman, P, Nature 228 (1970) 1087. ?1 Geha, R S, Schneeberger, E, Merler, E & Rosen, ~2. F S, New Engl j med 291 (1974) 1. 24. Mohit, B, Proc natl acad sci US 68 (1971) 3045. 25. Coflino, P, Knowles, B, Nathenson, S G & Scharff, M D, Nature new biol231 (1971) 84. 26. Orkin, S H, Buchanan, P, Yount, W J, Reisner, H & Littlefield, J W, Proc sot natl acad sci US 70 (1973) 2401. Received October 30, 1974 Revised version received December 18, 1974
