Lymphocyte Hybridomas: Second Workshop on “Functional Properties of Tumors of T and B Lymphocytes”

269 Pages·1979·7.483 MB·English

by  D. E. Yelton, B. A. Diamond, S.-P. Kwan, M. D. Scharff (auth.), F. Melchers, M. Potter, N. L. Warner (eds.)

Lymphocyte Hybridomas
Second Workshop on "Functional Properties of Tumors of T and B
Lymphocytes"
Sponsored by the National Cancer Institute (NIH)
April 3-5, 1978 Bethesda, Maryland, U.S.A.
Edited by F. Me1chers, Basel
M. Potter, Bethesda N. Warner, Albuquerque
With 85 Figures
Springer-Verlag Berlin Heidelberg New York 1979
Editors
F. Melchers, Basel Institute for Immunology, 487 GrenzacherstraBe,
CH-4005 Basle, Switzerland
M. Potter, Division of Cancer Biology and Diagnosis, Laboratory of Cell Biology,
National Cancer Institute, Bethesda, MD 20014/USA
N. L. Warner, Dept. of Pathology, University of New Mexico, School of Medicine,
Albuquerque, N.M. 87131!USA
Reprint of Current Topics in Microbiology and Immunology Vol. 81
ISBN-13: 978-3-540-09670-2 e-ISBN-13: 978-3-642-67448-8
DOl: 1O .l 007/978-3-642-67448-8
Library of Congress Cataloging in Publication Data. Workshop on Functional Properties of Tumors of T and B
Lymphocytes, 2d, Bethesda, Md., 1978. Lymphocyte hybridomas. (Current topics in microbiology and immunology;
v. 81) Bibliography: p. 268. Includes index.!. Immunoglobulins-Congresses. 2. T cells-Tumors-Congresses.
3. B cells-Tumors-Congresses. 4. Cell hybridization-Congresses. I. Melchers, Fritz, 1936- II. Potter, Michael.
III. Warner, Noel Lawrence, 1934- IV. Title. V. Series. [DNLM: 1. B-Lymphocytes-Congresses. 2. T-Lympho
cytes-Congresses. 3. Neoplasms, Experimental-Immunology-Congresses. 4. Hybrid cells-Congresses. 5. Antigens,
Neoplasm-Congresses. 6. Antibodies, Neoplasm-Congresses. WI CU82K v. 81/QZ206 W925 1978L]. QR1.E6
vol. 81 [QRI86.7] 576'.08s [599'.02'9]
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned,
specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying
machine or similar means, and storage in data banks.
Under § 54 of the German Copyright Law where copies are made for other private use, a fee is payable to the
publisher, the amount of the fee to be determined by agreement with the publisher.
© Springer-Verlag Berlin Heidelberg 1979
The USe of registered names, trademarks, etc. in this publication, does not imply, even in the absence of a specific
statement, that such names are exempt from the relevant protective laws and regulations and therefore free for
general use.
2121/3321-543210
Table of Contents
F. Melchers, M. Potter, N.L. Warner: Preface . . . . . . . . . . IX
D.E. Yelton, B.A Diamond, S.-P. Kwan, M.D. Scharff: Fusion of Mouse
Myeloma and Spleen Cells. . . . . . . . . . . . . . .. 1
H. Koprowski, W. Gerhard, T. Wiktor, J. Martinis, M. Shander, e.M. Croce:
Anti-Viral and Anti-Tumor Antibodies Produced by Somatic Cell
Hybrids. . . . . . . . . . . . . . . . . . . . .. 8
T. Imanishi-Kari, M. Reth, G.J. Hammerling, K. Rajewsky: Analysis of
V Gene Expression in the Immune Response by Cell Fusion. . . . . 20
G. Buttin, G. LeGuern, L. Phalente, E.e.e. Lin, L. Medrano, P.A Caze
nave: Production of Hybrid Lines Secreting Monoclonal Anti-Idiotypic
Antibodies by Cell Fusion on Membrane Filters. . . . . . . . . 27
R. DiPauli, W.C. Raschke: A Hybridoma Secreting IgM Anti-Iglb Allo-
type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
D. Gottlieb, J. Greve: Monoclonal Antibodies Against Developing Chick
Brain and Muscle . . . . . . . . . . . . . . . . . . . 40
T. Springer, G. Galfre, D. Secher, C. Milstein: Monoclonal Xenogeneic
Antibodies to Mouse Leukocyte Antigens: Identification of Macro
phage-Specific and Other Differentiation Antigens . . . . . . . 45
B.B. Knowles, D.P. Aden, D. Solter: Monoclonal Antibody Detecting a
Stage-Specific Embryonic Antigen (SSEA -1) on Preimplantation Mouse
Embryos and Teratocarcinoma Cells . . . . . . . . . . . . 51
J.e. Howard, G.W. Butcher, G. Galfre, C. Milstein: Monoclonal Anti-Rat
MHC (H-l) Alloantibodies. . . . . . . . . . . . . . . 54
T.J. McKearn, M. Sarmiento, A Weiss, F.P. Stuart, F.W. Fitch: Selective
Suppression of Reactivity to Rat Histocompatibility Antigens by Hy
bridoma Antibodies. . . . . . . . . . . . . . . . . . 61
M.M. Trucco, J.W. Stocker, R Ceppellini: Monoclonal Antibodies to
Human Lymphocyte Membrane Antigens. . . . . . . . . . 66
W.e. Raschke: Expression of Murine IgM, IgD and Ia Molecules on Hybrids
of Murine LPS Blasts with a Syrian Hamster B Lymphoma. . . . . 70
RH. Kennett, K.A Denis, AS. Tung, N.R Klinman: Hybrid Plasma
cytoma Production: Fusions with Adult Spleen Cells, Monoclonal Spleen
Fragments, Neonatal Spleen Cells and Human Spleen Cells. . . . . 77
H. Hengartner, AL. Luzzati, M. Schreier: Fusion of in vitro Immunized
Lymphoid Cells with X63Ag8 . . . . . . . . . . . . . . 92
G.J. Hammerling, H. Lemke, U. Hammerling, e. Hohmann, R Wallich,
K. Rajewsky: Monoclonal Antibodies Against Murine Cell Surface Anti-
gens: Anti-H-2, Anti-Ia and Anti-T Cell Antibodies ........ 100
L. Claflin, K Williams: Mouse Myeloma - Spleen Cell Hybrids: Enhanced
Hybridization Frequencies and Rapid Screening Procedures . . . . 107
B. Clevinger, D. Hansburg, J. Davie: Murine Anti-a (1 ~3) Dextran Anti
body Production by Hybrid Cells . . . . . . . . . . . . . 110
V.T. Oi, P.P. Jones, J.W. Goding, L.A. Herzenberg, L.A. Herzenberg:
Properties of Monoclonal Antibodies to Mouse Ig Allotypes, H-2, and
Ia Antigens . . . . . . . . . . . . . . . . . . . . 115
J. Andersson, F. Melchers: The Antibody Repertoire of Hybrid Cell Lines
Obtained by Fusion of X63-AG8 Myeloma Cells with Mitogen-Acti-
vated B-Cell Blasts. . . . . . . . . . . . . . . 130
W.R. Geckeler, W.e. Raschke, R. DiPauli, M. Cohn: Mouse Al Hybrid-
omas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
RM.E. Parkhouse, G. Guamotta: Rapid Binding Test for Detection of
Alloantibodies to Lymphocyte Surface Antigens . . . . . 142
G. Kohler, M.J. Shulman: Cellular and Molecular Restrictions of the
Lymphocyte Fusion. . . . . . . . . . . . . . . . . . 143
RA. Goldsby, B.A. Osborne, D. Suri, A. Mandel, J. Williams, E. Grono
wicz, L.A. Herzenberg: Production of Specific Antibody Without
Specific Immunization. . . . . . . . . . . . . . . . . 149
VR Zurawski, Jr., S.E. Spedden, P.R. Black, E. Haber: Clones of Human
Lymphoblastoid Cell Lines Producing Antibody to Tetanus Toxoid. . 152
M. Steinitz, S. Koskimies, G. Klein, O. Makela: Establishment of Specific
Antibody Producing Human Lines by Antigen Preselection and EBV
Transformation . . . . . . . . . . . . . . . . . . . 156
R Levy, J. Dilley, L.A. Lampson: Human Normal and Leukemia Cell Sur-
face Antigens. Mouse Monoclonal Antibodies as Probes. . . . . . 164
R Levy, J. Dilley, K Sikora, R Kucherlapati: Mouse-Human Hybridomas.
The Conversion of Non-Secreting Human B Cells into Ig Secretors . . 170
R Laskov, KJ. Kimm, R Asofsky: Induction of IgM Secretion by Fusing
Murine B-Lymphoma with Myeloma Cells. . . . . . . . . . 173
M. Nabholz, R.D. Engers, D. Collavo, M. North: Cloned T-Cell Lines with
Specific Cytolytic Activity. . . . . . . . . . . . . . . . 176
R Griitzmann, G.J. Hammerling: Characterization and Functional Analysis
ofT Cell Hybrids. . . . . . . . . . . . . . . . . . . 188
G.M. Iverson, R.A. Goldsby, L.A. Herzenberg: Expression of Thy 1.2
Antigen on Hybrids ofB Cells and a TLymphoma. . . . . . . . 192
E. Simpson, S. Kontiainen, L.A. Herzenberg, E. Bohrer, A. Torano, P. Vogt,
P. Beverley, W. Fitzpatrick, M. Feldmann: T Cell Hybrids with T Cell
Functions . . . . . . . . . . . . . . . . . . .. 195
N.H. Ruddle: T Cell Hybrids with Specificity for Individual Antigens. . . 203
M. Taniguchi, J.F.A.P. Miller: Specific Suppressor T Cell Hybridomas. . 212
B.A. Osborne, RA. Goldsby, L.A. Herzenberg: Selective Expression of
Loci in the I-J Region on T Cell Hybrids. . . . . . . . . . . 217
R DiPauli, G. DiPauli: Attempts to Produce Cytolytically Active Cell
Hybrids Using EL4 and MLC Spleen. . . . . . . . . . . . 221
N. Saravia, RIo DeMars, F.J. Bollum, F.R. Bach: Phenotypic Expression
in T-Lymphocyte x "Fibroblast" Cell Hybrids . . . . . . . . . 224
L.A. Manson, E. Verastegui-Cerdan, R. Sporer: A Quantitative Disc Radio
immunoassay for Antibodies Directed Against Membrane-Associated
Antigens 232
J. Minna: Summary of Cloning of Differentiated Function Using Hybrid
Cells and Comparison of the Mouse and Human Gene Maps for Homo-
logous Markers 235
List of Contributors . 241
Indexed in ISR
Preface
F. Melchers, M. Potter, NL. Warner
HISTORICAL
Prior to 1964 many workers had experienced considerable difficulty in
establishing continuous lines of murine plasmacytoma in culture.
Pettingill and Sorenson in 1966 grew X5563 after a painstaking period
of adaptation. During a visit to the Salk Instiute in 1965 Leo Sachs
began experiments with K. Horibata,E.S. Lennox and M. Cohn that ulti
mateley convinced most workers that murine plasmacytomas could be
grown in vitro with ease. They adapted MOPC-21 * {called P3 at the Salk
Institute) to in vitro culture and showed the cells produced a large
amount of the IgG1 myeloma protein. Horibata, Lennox and Sachs did
not report the important finding though a manuscript was written and
accepted. The reason for the failure to publish the paper was that
the original line was not recovered from the freezer and knowing
there would be many requests for it the paper was withdrawn. As it
later turned out, the failure to recover the line may have been triv
ial, for Horibata and Harris (1970) soon re-established a continuous
line of P3 now named P3K. This line was distributed to other workers
including Cesar Milstein in Cambridge. Soon after the success with P3,
Scharff and colleagues at Albert Einstein established MPC-11*in cul
ture.
Up to now continuous growth of human myeloma cells in vitro has remained
a formidable problem. Success in this area of lymphocyte cultures
would certainly open the way for obtaining human lymphocyte hybridomas
producing human monoclonal antibodies - which may well revolutionize
their clinical applications.
Initially both P3 and MPC-11 murine cell lines were used extensively
to study Ig synthesis in vitro. The cloning efficiency of MPC-11
was very high (higher than 50 %)and this permitted Scharff to study
the stability of MPC-11 lines in regards to Ig synthesis in culture.
It also opened the way to using the plasmacytoma cells in mutagenesis
experiments. The high 3ate of loss of H chain synthesis and secre
tion ranging from 1x10 per cell per generation was a striking
finding. Non-secreting mutants were also found to occur in high
frequency. All of this work formed the background -for the drama tic
discoveries of Kohler and Milstein that are the basis for this
workshop.
Hybridization studies in other systems had not offered great promise
for immunologists. Most of these though involved fusion of lympho
cytes with cell types which were dissimilar in respect to their sta
tus of differentiation. Such hybrid cells of dissimilar parental
lines, in particular plasmacytoma-fibroblast hybrids, showed that Ig
synthesis was suppressed. Milstein nonetheless derived an 8-azaguani
nine resistant subline of P3, now termed P3/X63-Ag8. This line was
use1 first to study the fusion of two plasmacytoma cells. When this
revealed that the hybrid cells produced both myeloma proteins, Kohler
*both induced at the National Cancer Institute.
IX
and Milstein then went directly to the important and daring experi
ment of fusing a normal plasma cell with X63. They and all of us were
rewarded with a spectacular result: the hybrid of an anti-SRBC normal
plasma cell and X63 produced both the MOPC-21 myeloma protein and
SRBC antibody. Thus, the specific antibody production of a normal B
cell became immortal through hybridization with a neoplastic cell.
FUSIONS OF PLASMACYTOMAS WITH B-LYMPHOCYTES OR PLASMA CELLS
Cell Lines Used for Fusion
Table 1 summarizes the principal tumor lines that have been used to
date. In only a few instances have direct comparisons been made in a
single experiment, and in these cases it appears that similar fusion
frequencies and successes of establishing hybrids were obtained. In
the overall experience of many investigators it was again apparent
that X63, NS-1, and certain MPC-ll lines have been used successfully
with some variations in results. In view of the production of heavy
chains from myeloma parent lines, it might be desirable to fuse to a
non-secreting myeloma line such as NS-l, particularly in cases where
the myeloma protein and mixed molecules of myeloma and antibody pro
teins interfere with the specificity of the monoclonal antibody. Oth
er Ig-secreting or non-Ig-synthesizing myeloma lines should be
screened for this purpose.
Thus far, only mouse plasmacytoma lines are available. The develop
ment of human, primate, rat, and hamster Ig-secreting tumors would
greatly expand the numbers and types of useful hybridomas.
Table 1: Characteristics of tumor cell lines* used in fusion experiments
Complete Name Short Name Strain Characteristics
P3 x63 Ag8 X63 BALB/c Plasmacytoma (Y1, k)
P3-NSI-1-Ag4-1 NS-1 BALB/c Plasmacytoma (k, non-secreting)
MPC11-X45-6TG X45 BALB/c Plasmacytoma (Y2b, k)
BW5147 BW AKR Thymic Lymphosarcoma
(Ly-, 2-, 3-, 6+)
EL-4 EL-4 C57BL Thymic Lymphosarcoma
*The cell lines of these tumors are available from the Salk Institute Cell Distribu
tion Center, La Jolla, California.
Technology of Fusion
In general, little discussion or divergence of opinions was evident
in this conference on the actual procedures for fusion. Most investi
gators used polyethylene glycol (PEG) as fusing agent. PEG lots vary
considerably in degree of purity and suitability for in vitro use.
Each should be tested for toxicity with cell lines before use. The
optimal molecular weight of PEG for fusion was not clearly established.
Preparations of widely different molecular weights (between 1000 and
4000) were effective.
x
Several variations in the actual procedure of dispensing and locating
the cells in PEG were described, some of which certainly improved the
hybridoma success rate, and these are described in detail in the
articles.
Cell numbers used for fusion similarily were not a major issue, al
though variations in the frequencies of successful fusion events
yielding hybridomas appeared more dependent on cell numbers at lower
cell input. Most investigators dealing with this problem agreed that
under the present conditions for fusion - and independent of the fus
ing agent or the technique of locating myeloma cells and normal cells
next to each other - the best frequencies are one hybridoma in 2 to
5 x 104 normal cells. Single clones of normal B-cells, activated and
grown "in vitro" to approximately 103 cells are, therefore, too small
to be immortalized through fusion. Klinman et al., however, using the
spleen focus growth of single B-cell clones, reported successful
hybridoma production from such in "in vivo" gr01"n single clones.
Three specific points might be noted as the result of the discussions
at the workshop:
(i) It is not yet clear whether fusion occurs to a specific subpopula
tion of cells within the myeloma or lymphoma clone, although it is
clear that heterogeneity in cell cycle and cell differentiation -
states exist within tumor lines, indicating a potential for such re
striction.
(ii) Although the subject of the relative differentiation states be
tween normal and neoplastic cells is considered frequently throughout
this volume, no information is available on whether it is an absolute
requirement for obtaining functional hybrids expressing the desired
characteristic normal function of lymphocytes that the neoplastic tu
mor cell must belong to the same differentiation subpopulation as the
normal fusing cell. It is certainly clear that this similarity or
identity is not necessary for fusion itself to occur and for hybrids
to be clones although these hybrid lines mayor may not exhibit bio
logical functions.
(iii) The deSignation of a tumor line by a specific name does not
necessarily infer that all such tumor lines of identical name need be
equally suitable for fusion. Sublines, variants, mutants, and revert
ants of such lines will frequently occur, many of which may be quite
unsuitable. In an effort to minimize this problem, the available sub
lines that have been shown suitable for fusion are to be centered in
the Salk Institute Tumor Distribution Center for further distribution
to investigators. The moral in this area for newcomers to the field
is to "use a proven fusable tumor line" and to learn the tissue cul
ture rules for how to keep the line.
The Normal Target Cell of Fusion
It is perhaps on this issue, that the most debate and uncertainty
still exists. Cell fusions were successfully described where unfrac
tionated total spleen cell preparations were used.
Mitogen-induced polyclonal stimulation of the total B-cell repertoire
followed by cell fusion produced large numbers of Ig producing and
specific antibody forming clones. (See Andersson and Melchers, and
Goldsby et al., this vol.) This method also offers the potential to
immortalize the entire repertoire of antibody produclng cells, which
XI
could provide the means to analyze precisely the range of specifici
ties that are present in such a repertoire as derived from various
different genetic backgrounds. The exact differentiated type of the
normal target cell for fusion in the spleen has not been identified
in detail, although it seems likely that an activated B-lymphocyte in
its blast stage is a preferred partner of the myeloma cell lines in
the fusion reaction. Since the success of obtaining functional hy
bridomas would clearly be enhanced if the normal fusing population
were to be specifically pre-selected for specificity, it is to be
cautioned that this pre-selection may need to be not only for the re
quired antigen reactive specificity, but perhaps also for a specific
differentiation stage.
In attempting to analyze this problem, it was stressed in the dis
cussion that interpretations of the differentiation type of the nor
mal cell involved in the fusion, cannot be necessarily derived from
assessment of the functional state of the hybridoma product. This lat
ter status will clearly be complicated by unknown factors determining
gene expression (repression and de-repression) in the hybrid cell,
and its particular chromosomal complement. Direct answers to this
problem will only be obtained when isolated purified normal cell pop
ulations of different types are used for fusion.
The feasibility of hybridizing mouse myeloma cells with normal Ig pro
ducing cells of other species was discussed in several reports (see
Howard et al.; Levy et al.). Ig-secreting hybridomas were obtained
from fusions of mouse myelomas and rat Ig producing cells. Hybridomas
of human eLL cells and mouse myeloma cells were reported but these
did not secrete large quantities of the human Ig. The use of human
lymphoblastoid lines may be a fruitful source of human Ig producing
cells. Recent studies by Zurawski et al. and Steinitz et al. (this
vol.) indicate that human Ig producing cell lines can be established.
However fusion experiments have not yet been reported.
Use of Feeder Layers
Frequently throughout the conference discussion was raised on the ef
fect of the addition of other cell types either at the time of fusion
and immediately thereafter, or during the period of stabilization and
isolation of the specific clones. This problem clearly relates to oth
er tissue culture studies indicating a "feeder" effect from the addi
tion of certain cell types to those under culture. In general, feeder
cells can be used as monolayers, incorporated in a basal agar layer,
mixed in with the whole suspension, or used to prepare conditioned
media. It was evident that the effect of such feeder cells has not
been well characterized in this fusion system, and many diverse
opinions were expressed.
It is probably reasonable to conclude at this stage that the presence
of feeder cells will most likely be advantageous, and that where the
deliberate addition of such cells has not resulted in growth-support
ing effects may be because such cells are already present within the
normal fusing cell population.
Many examples were brought out in this conference, of approaches to
pre-selection which have improved the success of obtaining functional
hybridomas. These include:
(i) Biophysical separation procedures that select for either large
cells from polyclonal stimulation or for specifically enriched immune
cells.
XII