Establishment and characterization of a permanent murine hybridoma secreting monoclonal autoantibodies

CELLULARIiV~IvIUNOLOGY41, 188--194 (1978)

Establishment and Characterization of a Permanent Murine Hybridoma Secreting Monoclonal Autoantibodies JACQUELINE

M. PAGES AND ALAIN g. BUSSARD

Service d'fmmu~cologie CeUulaire, Institut PASTEUR, Paris, 75015 Received May 5, 1978

Hybridization between plasmocytoma cells from Balb/C mice (X63-Ag8) and peritoneal cells from NZB mice, which secrete, in large amounts autoantibodies directed against bromelin-treated mouse red blood cells, have been achieved. Clones of hybridoma secreting permanently autoantibodies were isolated. The biochemistry and the immunochemistryof these monoclonal antibodies have been studied. INTRODUCTION K6hler and Milstein (1, 2) have succeeded in creating permanent lines of hybrid cells that secrete homogeneous antibody, by fusing a myeloma cell with an immune cell of predetermined specificity. This methodology has opened a new era in irnmunology. In the B cell system, "hybridomas" have been established that secrete homogeneous antibodies against sheep red blood cells (1), TNP (2), histocompatibility antigens (3) and influenza virus (4) etc. One of the most interesting features of this methodology is that an individual antibody forming cell (AFC) can be isolated from an heterogeneous population of immunocytes and nmltiplied as a monoclonal AFC culture. In principle, a multicomponent system such as an immune cell population (from an immune lymphoid organ) could be resolved in its individual constitutents. We have attempted to do this analysis on the peritoneal cells from unimmunized mice--which, as shown previously (5), contain a large proportion of autoantibodies producers--by hybridizing these cells with a murine myeloma cell. Peritoneal cells (PC) from NZB mice produce after 4 to 5 days of culture, up to 10~ of plaques forming cells (PFC) against isologous erythrocytes treated with the proteolytic enzyme bromelin (MRBC(br)). A smaller proportion of PFC acts against sheep red blood cells (SRBC) (5, 6) this latter activity being attributed to a weak affinity of the same antibody for cross-reacting epitopes (5, 7). M A T E R I A L S AND METHODS Parent cells. The myeloma cell, X 63-AG 8, kindly provided by Dr. C. Milstein, is derived from M O P C / 2 1 , of Balb/C origin, and is a mutant resistant to 20 /~g/ml of 8-Azguanine. It does not grow in selective medium ( H A T ) (8). Electron-microscopy of these cells showed that they are middle-size lymphoblasts with

188 0008-8749/78/0411-0188502-00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Results of fusion experiment : CP 3 Eight positive wells/48 (by spot test) Population n ° : 8

12

17

18

19

20

23

24

Culture for 2 months with passage from HAT medium to DMM 20% FCS and test for PFC at different intervals Cloning and screening on MRBC(br) 17b 18•

Population No.

12b

Number of clones:

l

4

l

2

1

Anti-MRBC (br) activity Anti SRBC activity

+ 0

++ +

q-q+

8

23~

on SRBC 23b

1

1

+++ + +

+++ ++

6

8

All clones from this experiment are named CP 3, followed by the well number, and then by the number of the isolated clone i.e., CP 3/23/1. (a) Cloning in soft agar was followed by detection of antibody producing clones by an overlay of MRBC(br) in 1% agarose and complement. (b) For populations with low activity, cloning was done by collecting one single cell in the center of a plaque of hemolysis by micromanipulation and transfer of that cell in a culture well with DMM + 20% FCS. Anti-SRBC activity was estimated by counting plaques of hemolysis by the CMC technique. + is for 1 to 5000 plaques per million cells (ppm). + + is for 5000 to 50,000 ppm. + + + is for 50,000 to 800,000 ppm. few arrays of rough endoplasmic reticulum; their chromosomic formula is n = 63 with two metacentric chromosomes as m a r k e r s ; they secrete an I g G1 @) (9). T h e immunocyte comes from a population of peritoneal cells obtained by washing the peritoneal cavity of normal 8 months old female N Z B mice. P F C types, after 4 days of culture, are proplasmocytes ( 3 0 % ) and plaslnocytes ( 7 0 % ) as reported in a recent E M study (10). Their karyotype is the usual karyotype of the mouse with 40 acrocentric chromosomes. Cell fusion. T h e technique described by Galfre et al. (3) was used with P E G 1500 as fusing agent. I n all experiments 5 × 10 ~ X Z B peritoneal cells were added in each well to serve as feeder. Passage from selective H A T medium to H T and normal medium ( D M M plus 2 0 % F C S ) was made easier by using conditioned medium. T h e conditioned medium was obtained by g r o w i n g 2 × 10 r normal peritoneal cells in 20 ml D M M with 10% F C S for 5 days; the supernatant was centrifuged, filtered on millipore (0, 25 /,pore q~) and used at a final dilution of 1.5% in D M M with 20% F C S . Enzyme treatment of mouse red blood cells. Bromelin in treated mouse red blood cells were prepared as previously described (7). Detection of antibody secreted by the hybridoma. Spot tests, as described by K6hler and Milstein (2) were performed independently with M R B C / b r and S R B C . Detection of single antibody producing cells. T h e y were detected and counted by direct local hemolysis plaque assay in carboxymethyl-cellulose gel (7). Plaque inhibition by red cell stroma was done as described elsewhere (7).

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Clone plaques were developed on 8 to 12-days old colonies cloned in soft agar (1).

Analysis of secrcted p~'oducts. 2 × 106 cells were grown for 24 hr in Ieucine-free MEM supplemented with nucleosides (2'desoxycytidine, 2'desoxyguanosine, 2'desoxyadenosine, 2'desoxythymidine, final concentartion each 2 10-4 M) and 5 /~Ci/ ml of [14C]leucine (specific activity 310 mCi/mM). No FCS was added. Immunoelectrophoretic analysis (IEA) of culture media was performed before or after absorption by stroma of different erythroeytes (7). Isoelectrofocusing ( I E F ) (11) and sodium dodecylsulfate polyaerylamide slab gel etectrophoresis (SDS PAGE) (9) were performed on reduced or non reduced [14C] leucine labeled products. To identify the specific antibodies, aliquots of culture media were absorbed on MRBC/br or SRBC and, as negative control, on HRBC (horse red blood cells) before I E F and SDS PAGE runs. Phillips and Dresser technique (12) was used to localize the hemolytic IgM zone of the gel after I E F of nonreduced labeled products. RESULTS

AND

DISCUSSION

W e have succeeded in {using the two aforementioned types of cells and in establishing eight populations, from which 28 clones were isolated, continuously producing large amounts of Igl~[ hemolytic for MRBC(br), detectable by spot tests and standard local hemolysis in gel as shown in Diagram 1. The karyotypes of these different hybridoma are very much alike, Fig. 1 shows the karyotype of clone 23/3, with 110 chromosomes, among which the two acrocentric chromosomes from the X 63 can be seen. EiV[ observation of these hybridoma (Fig. 2) kindly performed by Pr D. Zagury, shows that the cells are extremely homogeneous, resembling classical lymphoblasts, poorly differentiated young cells. The doubling time of these cells, in exponentially growing populations, at 37 ° , varies from 14 to 20 hr according to the clone. The actively secreting hybridoma produce anti MRBC(br) antibodies (Diagram

FIG. 1. Karyotype of an hybridoma cell CP 3.23.3. n = 110 chromosomes. Arrows indicate the two metacentric chromosomesfrom the X 63-8 Ag parent.

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FIG. 2. Electron microscopy of CP 3 cell. Magnification : approximately X 4000. Arrows indicate rough endoplasmic reticulum.

1) (up to 80% of the population) while the anti SRBC activity is always lower and varies from one clone to another, never exceeding 5% of the anti MRBC(br) activity. Inhibition of plaque formation against MRBC(br) by stroma of the two types of erythrocytes showed that, while 5 and 10 MRBC(br) stroma per indicator RBC cause 97 and 99% inhibition respectively, 5 SRBC stroma inhibit only 2% and 10 stroma inhibit only 37%. Since the hybridoma ceils produce monoclonal antibodies, it is likely that lysis of SRBC results from cross reactivity of some epitopes of these erythrocytes with those MRBC(br) antigenic determinants against which the antibody, produced by the PC ancestor of the hybridoma, was directed. Stroma inhibition experiments and further absorption experiments are in agreement with the hypothesis that MRBC(br) epitopes are the main target for the secreted antibody which has less affinity for SRBC epitopes. This would explain the fact that SRBC plaques are always in smaller number than 3/IRBC(br) plaques; the hybridoma cell population, being homogeneous (by definition) there could be only differences in rate of secretion from one cell to another with no difference in antibody structure. Only strong secretors would produce SRBC plaques. IEA applied to the [14C]leucine labeled supernatants of several CP 3 clones, showed, with the use of specific antimouse IgM antiserum, that a mouse IgM was synthetized during the culture period. This Ig3/f was identified as a single radioactive band in front of the starting well, it was unaffected by absorption with horse red blood cells ( H R B C ) , it was reduced by absorption with SRBC, and it disappears almost completely by absorption with MRBC (br). I E F of IgM in acrylamide gel was found to be unreliable. Nevertheless, using the technique of Baumann et al. (11) with a highly porous gel, we were able to effect the migration of the IgM antimouse antibody samples in a pH gradient (pH 4 to pH 8.5).

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It was identified by hemolysis of M R B C ( b r ) in a gel of agarose, layered on top of the acrylamide slab, after focusing (Fig. 3). Radio-autography of the same I E F runs permitted the identification of the IgM antibody although not as a sharp band, with a mean isoelectric point pt-I = 6. W e have submitted the products of secretion of X 63-Ag 8 and of CP 3 different clones to chemical reduction prior to I E F or to SDS P A G E . The latter procedure permitted the separation of X 63 heavy chains from the NZB heavy chains. These chains labeled by [l*C]leucine can be seen in Fig. 4. The two K chains (from X 63 and N Z B ) were also separated. After absorption by M R B C ( b r ) of the supernatant from culture, the chain from the IgM antibody completely disappeared while there was only a reduction of the density of the K1 band from this IgM. This suggests that free light chains were secreted by hybridoma cells, as already specified by Milstein, and that they were not absorbed by the specific antigen. I P injections of 5 × 106 CP 3 cells into semi-allogenic recipients (Balb/C) were performed. After 3 weeks the sera of these mice and their peritoneal fluid contained high titers of anti M R B C ( b r ) hemolytic antibodies (between 27 to 212 final dilution for complete hemolysis of 8 × 10 G erythrocytes), though they had no ascites. These results differ from those of K6hler and Milstein (2) who found only low IgM anti SRBC antibody titer in the serum of mice injected with their hybridoma. In our experiments the hybrid cells could be recovered from the peritoneal cavity and cultured. They retained their plaque forming activity.

a

b

FIa. 3. Isoelectrofocusing (IEF) of a radioactively labeled supernatant of clone CP 3.23.3. culture. 1. Unabsorbed supernatant. 2. Supernatant absorbed with ttRBC. 3. Supernatant absorbed by SRBC. 4. Supernatant absorbed with MRBC(br). Vertical arrows indicate the direction of the migration, horizontal arrows indicate the starting zone. (a) Autoradiography. Note the disappearance of a radioactive zone in line 4 at pI-I = 6. (b) I-{emolysis in agarose. The MRBC(br) in agarose were layered on top of the acrylamide slab and incubated 2 hr at 37°C with complement. Note the reduction of hemolysis after absorption with SRBC (line No. 3) and its complete disappearance after absorption with MRBC(br) (line No. 4).

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1

2

3

4

1

2 3 4

M

G

-¢

K1

K2

a

b

FIG. 4. Sodium dodecyl sulfate (SDS) polyacryIamide gel electrophoresis (PAGE) of a radioactively labeled supernatant of clone CP. 3.23.3. (a) Non reduced supernatant. (1) Unabsorbed. 2. Absorbed by HRBC. 3. Absorbed by SRBC. 4. Absorbed by MRBC(br). M indicates the IgM from the PC component of the hybridoma. G. indicates the IgG from the myeloma (as identified by separate runs not shown here). One can note the disappearance of the IgM band in 4. (b) Supernatant reduced by 5% of 2-Mercaptoethanol. Lines 1, 2, 3, 4 as in la, ~ and "r indicate the two heavy chains of the two components of the hybridoma, K1 the Kappa chain from the PC and K= the Kappa chain from the myeloma cell components. Note the disappearance of F chain band and the reduction of the tc chain band in line 4 (adsorption on M R B C ( b r ) ) . W e feel that the capability of p r o d u c i n g large quantities of h o m o g e n e o u s antibodies d i r e c t e d against self-constituents m a y help to u n d e r s t a n d the n a t u r e a n d specificity of these self-antigens. I t m a y also lead to the p r e p a r a t i o n of specific antiidiotypes a n d to an u n d e r s t a n d i n g of the m e c h a n i s m s u n d e r l y i n g the regulation of a u t o - a n t i b o d y production.

ACKNOWLEDGMENTS We wish to thank Miss N. Bulsson and Mrs M. A. Vinit for their skillful assistance. This work has been supported by INSERM (C.277659) DGRST (C.7470618), and CNRS (ER 119).

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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

K6hler, G., and Milsteln, C., Nature 256, 495, 1975. K6hler, G., and Milstein, C., Eur. J. of Immun. 6, 511, 1975. Galfre, G., Howe, S. C., 1Viilstein, C., Butcher, G. W., Howard, .L C., Nature 266, 550, 1977. Koprowskl, H., Gerhard, W., and Croce, C., P.N.A.S. 74, 2985, 1977. Pag6s, 1. M., and Bussard, A. E., Nature 257, 316, 1975. Lord, E., and Dutton, R. W., J. Immunol. 115, 1199, 1975. Bussard, A. E., Vinlt, M. A., and Pag6s, J. M., Immunochem. 14, 1, 1977. Littlefield, J. W., Science 145, 709, 1964. Cowan, N. J., Seeher, D. S., and Milstein, C., J. Mol. Biol. 90, 691, 1974. Pag6s, J'. 1VL, Thiernesse, N., Bernard, J., Jeannesson, P., Arnaud, D., and Bussard, A. E., Cel. Immun. 35, 289, 1978. 11. Baumann, G., and Chrambach, A., Analyt. Biochem. 70, 32, 1976. 12. Phillips, J. M., and Dresser, D. W., Eur. J. Immun. 524, 1973.