Table Of ContentCurrent Topics in
Microbiology
100 and Immunology
Editors
M. Cooper, Birmingham/Alabama· W. Henle, Philadelphia
P.H. Hofschneider, Martinsried· H. Koprowski, Philadelphia
F. Melchers, Basel· R. Rott, GieSen· H.G. Schweiger;
LadenburglHeidelberg· P.K. Vogt, Los Angeles
R. Zinkernagel, Zurich
T Cell Hybridomas
A Workshop at the Basel Institute
for Immunology
Organized and Edited by H. v. Boehmer, W. Haas,
G. Kohler, F. Melchers and J. Zeuthen
With the Collaboration of S. Buser-Boyd
With 52 Figures
Springer-Verlag
Berlin Heidelberg New York 1982
Editors
H. V. Boehmer, W. Haas
G. Kohler, F. Melchers
J. Zeuthen
Basel Institute for Immunology
Grenzacherstrasse 487
CH-4005 Basel
Switzerland
ISBN-13: 978-3-642-68588-0 e-ISBN-13: 978-3-642-68586-6
DOl: 10.1007/978-3-642-68586-6
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Table of Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . .. VII
K. Fischer Lindahl, A.A. Nordin, M.H. Schreier: Lectin-
Dependent Cytolytic and Cytolymic T Helper Clones and
Hybridomas . . . . . . . . . . . . . . . . . . . . . . . 1
Z. Eshhar, T. Waks, T . .oren, G. Berke, Y. Kaufmann:
Cytotoxic T Cell Hybridomas: Generation and
Characterization .................... 11
E.J. Culbert, S. Kontiainen, L.M. Douglas, M. Feldmann:
Definition of Function-Related Isotypic Markers on
TCells ........................ 19
M. Taniguchi, T. Tokuhisa, M. Kanno, T. Honjo: An
Antigen-Specific Suppressor T Cell Factor Controlled by
Two Genes in the Immunglobulin Heavy Chain Linkage
Group and in the I-J Subregion ofthe H-2 Complex '" 33
M.J. Taussig, G.L. Asherson, A. Holliman, N. Richardson,
G. Skibinski: Structural and Functional Studies on Antigen-
Specific Suppressor Factors from T Cells and T Cell
Hybrids. . . . . . . . . . . . . . . . . . . . . . . . .. 43
D.R. Webb, B.A. Araneo, C. Healy, J.A. Kapp, K. Krupen,
I. Nowowiejski, C.W. Pierce, C.M. Sorensen, S. Stein, K.J.
Wieder: Purification and Biochemical Analysis of Antigen-
Specific Suppressor Factors Isolated from T-Cell
Hybridomas . . . . . . . . . . . . . . . . . . . . . 53
M.E. Dorf, K. .okuda, M. Minami: Dissection of a
Suppressor Cell Cascade .............. 61
M. Cramer, R. Mierau, T. Takemori, G. Suzuki, T. Tada:
4-Hydroxy-3-nitro-phenylacetyl (NP)-Specific T Cell
Hybridomas . . . . . . . . . . . . . . . . . . . . . . 69
M. Suemura, K. Sugimura, A. Ishizaka, S. Kobatake,_
O. Shiho, H. Deguchi, S. Kishimoto, Y. Yamamura,
T. Kishimoto: Regulation of the IgE Response by IgE
Class-Specific Suppressor T Hybridomas . . . . . . . 79
P. Ricciardi-Castagnoli, F. Robbiati, E. Barbanti,
G. Doria, L. Adorini: Establishment of Functional,
Antigen-Specific T Cell Lines by RadLV -Induced
Transformation of Murine T Lymphocytes . . . . . 89
P. Lonai, E. Arman, S. Bitton-Grossfeld, 1. Grooten,
G. Hiimmerling: H-2-Restricted Helper Hybridomas: One
Locus or Two Control Dual Specificity? ......... 97
Z. Eshhar, T. Waks, H. Zinger, E. Mozes: T Cell Hybri-
domas Producing Antigen-Specific Factors Express Heavy-
Chain~Variable-Region Determinants ......... 103
S. Carel, C. Bron, G. Corradin: Cytochrome f. Specific
T Cell Hybrid . . . . . . . . . . . . . . . . . . . . .. 111
E. Heber-Katz, D. Hansburg, R.H. Schwartz: The Effect
of Antigen Presentation on the Fine Specificity of Anti-
Cytochrome f T Cell Hybridomas . . . . . . . . . . .. 117
A.M. Schmitt-Verhulst, M.A. Cooley, A. Guimezanes,
C. De Preval, F. Albert, M. Buferne: Analysis of the
Anti-Self + TNP Immune Response: T Cell Lines,
Clones and Hybridomas. . . . . . . . . . . . . . . . 125
M.H. Schreier, R. Tees, L. Nordin, A.A. Nordin: Func-
tional Characteristics of T Cell Hybridomas Obtained
by Fusion of TCGF-Dependent Helper T Cell Clones
with BW5147 . . . . . . . . . . . . . . . . . . . . . 135
K.-O. Gronvik, 1. Andersson, F. Melchers, 1. Zeuthen:
A Stable TCGF-Producing T Cell Hybridoma and its
Thioguanine-Resistant Variant Suitable as a Tool for the
Construction of New Functional T Hybridomas . . . .. 143
F. Melchers, 1. Zeuthen, W. Gerhard: Influenza Virus-
Specific Murine T Cell Hybridomas Which Recognize
Virus Hemagglutinin in Conjunction with H-2d and Display
Helper Functions for B Cells . . . . . . . . . . . . .. 153
1. Andersson, F. Melchers, 1. Zeuthen: A "Panreactive"
T Cell Line and T Cell Hybridoma: Their Function in
Helping B Cells . . . . . . . . . . . . . . . . . . . . 165
C. Corbel, 1. Andersson, F. Melchers, 1. Zeuthen: A
"Panreactive" T Cell Hybridoma Which Produces TCGF
Constitutively . . . . . . . . . . . . . . . . . . . . .. 173
A. Altman, R.D. Schreiber, D.H. Katz: Production of
Antigen-Nonspecific Immunoregulatory Lymphokines
by T Cell Hybridomas . . . . . . . . . . . . . . . . .. 179
E.C. DeFreitas, S. Vella, A. Linnenbach, C.M. Croce,
H. Koprowski: Human T Cell Hybridomas with Tetanus-
Toxoid-Specific Helper Activity ............ 191
K. Pfizenmaier, M. R611inghoff, H. Wagner: Functional
Analysis of Il-2 Produced by T-Cell Hybridomas: Il-2
Promotes T-Cell Growth But Does Not Mediate T-Cell
or B-Cell Maturation and Differentiation . . . . . . .. 203
S. Gillis, D. Mochizuki: Molecular Characterization of
Interleukin 2 Produced from Tumor Cell Lines and
T Cell Hybridomas . . . . . . . . . . . . . . . . . .. 211
J.W. Schrader, I. Clark-Lewis: The Use ofT Cell Hybri-
domas in the Biochemical and Biological Characterization
of Multiple Regulatory Factors Produced by T Cells. 221
C. Corbel: Different Factors Active in Lymphoid and
Hematopoietic Proliferation Produced by Single Clones
of Helper T Cell Hybridomas . . . . . . . . . . . . .. 231
N.H. Ruddle, B.S. Conta: Lymphotoxin and Immune (y)
Interferon Production by T Cell Lines and Hybrids . . .. 239
J.F.A.P. Miller, P.L. Mottram, W.R. Thomas, J. McKimm-
Breschkin, I.L. Walker, J. Gamble: Antigen Specificity
of Continuous T Cell Lines 249
List of Contributors . . . . . 257
Indexed in Current Contents
Preface
For more than ten years cell fusion techniques have been applied in
studies on various lymphocyte functions. Ig expression was first
studied in hybrids obtained by fusing myeloma cells with fibroblasts
(1) or lymphomas (2), both of which do not produce Ig, and with Ig-
producing myelomas (3) or human blood lymphocytes (4). Kohler and
Milstein (5) fused a myeloma with spleen cells from immunized mice.
Up to 10% of the hybrids obtained secreted antibodies specific for
the immunizing antigen. This suggested that plasma cells preferenti-
ally fused with the myeloma cells, a finding which was of enormous
practical value. It was found that both Band T lymphocytes could be
fused with the T cell tumor BW5147, which is however not permissive
for Ig synthesis (6). A very large number of T cell hybridomas were
generated by fusing BW5147 with cell populations containing in vivo
or in vitro activated cells (7). The hybrids showed no specific T
cell functions and binding assays for T cell receptors were not
available. In particular, no hybrids were obtained which expreS1ed
specific cytolytic activity that could be tested in short-term Cr-
release assays (8). However, the frustrations expressed about these
failures, published in January, 1978 (9), were relieved by Taniguchi
and Miller's publication a few months later of T cell hybridomas
producing antigen-specific suppressor factors (10). Unfortunately,
their hybrids rapidly lost factor production. Subsequently, many
laboratories generated murine and human T cell hybridomas which
produced antigen-specific molecules that suppressed or induced various
lymphocyte functions (this volume; 8,11). Many T cell hybridomas
could be induced to produce various lymphokines (this volume; 12).
Some T cell hybrids expressed antigen receptors which could be identi-
fied by binding antigen or antiidiotypic antibodies or antigen-
induced lymphokine production (this volume; 13). Indeed, T cell
hybridomas expressing specific lytic activity could also be generated
(this volume; 14). It is remarkable that BW5147 is permissive for
expression of all these T cell functions. Many more fusion experi-
ments will be required - including inter species fusions - to determine
the optimal conditions for fusion and expression of particular T cell
functions in hybrid cells. Although the problem of stable expression
of particular T cell functions in hybridomas has not yet been com-
pletely solved, several laboratories have generated sufficient numbers
of hybrid cells to allow purification and biochemical analysis of
antigen-specific T cell factors. Most advanced is the analysis of
suppressor factors (this volume).
T cell hybridomas are not the only source of monoclonal T cell pro-
ducts. First, several murine and human T cell tumors can be induced
to express normal T cell functions such as lymphokine production
(15). Second, transformed T cell lines expressing specific functions
can be obtained by infection of mice or cells in vitro with radiation
leukemia virus (16). Third, clones of all major T cell classes can
IX
now be grown continuously in tissue culture (17).
To help evaluate the potential of T cell hybridomas for understanding
the functioning of the immune system as well as for practical purposes,
the workshop on "T Cell Hybridomas: Sources of Specific Mediators in
the Immune System" was held January 27-29, 1982, at the Basel Insti-
tute for Immunology. The techniques used, the difficulties encoun-
tered and the present state of art were discussed, and most of this
is presented in the following papers.
References
1. Periman (1970) Nature 228: 1086; Coffino et al. (1971) Nature
New Biol 231: 87; Bevan et al. (1972) Prog Biophys Mol Biol 25:
131
2. Mohit (1971) Proc Natl Acad Sci USA 68: 3045
3. Cotton and Milstein (1973) Nature 244: 42
4. Schwaber and Cohen (1974) Proc Natl Acad Sci USA 71: 2203
5. Kohler and Milstein (1975) Nature 256: 495
6. Iverson et al. (1978) Curr Top Microbiol Immunol 81: 192
7. Hammerling (1977) Eur J Immunol 7: 743; Goldsby et al. (1977)
Nature 267: 707; Kohler et al. (1977) Eur J Immunol 7: 758
8. Curr Top Microbiol Immunol 81: 195-231 (1978)
9. Melchers (1978) Nature 271: 9
10. Taniguchi and Miller (1978) J Exp Med 148: 373
11. Research Monographs in Immunology, Vol. 3: Monoclonal Antibodies
and T Cell Hybridomas: Perspectives and Technical Advances,
G.J. Hammerling, U. Hammerling, J.F. Kearney, eds., Elsevier/
North Holland, 1982; Watanabe et al. (1978) J Immunol 121: -
2113; Hewitt and Liew (1979) Eur J Immunol 9: 572; Okada et al.
(1981) Proc Natl Acad Sci USA 78: 7717; Hiramatsu et al. (1981)
J Immunol 127: 1118; Irigoyen et al. (1981) J Exp Med 154: 1827
12. Harwell et al. (1981) J Exp Med 152: 893; Kappler et al. (1981)
J Exp Med 153: 1198
13. Kappler et al. (1981) J Exp Med 153: 1198
14. Nabholz et al. (1981) Nature 287: 437
15. Behring Institute Research Communications No. 57, Interleukin 2,
F.R. Seiler and H.G. Schwick, eds.; Lymphokines, Vol. 5, Mono-
clonal T Cells and Their Products, M. Feldmann and M. Schreier,
eds., Academic Press, New York, 1982
16. Ricciardi Castagnoli et al., this volume; Finn et al. (1979)
Proc Natl Acad Sci USA 76: 4033
17. Immunol Rev 54 (1981); Nabel et al. (1981) Cell 23: 19
Acknowledgments
The Basel Institute for Immunology, founded and supported by F.
Hoffmann-La Roche and Co., Ltd., and Springer-Verlag jointly sponsored
this workshop. Our sincere thanks are due for their support.
The work of B. Badovinac, J. Bron, M. Dekany, R. Rodrigues, J.-P.
Rudloff, G. Scialdone, and A. Wyss of the Basel Institute for Immunol-
ogy in managing the technical details of this workshop is much
appreciated.
x
We also thank Marga Botsch and Rick Mills of Springer-Verlag for
their assistance in the publication of the workshop proceedings.
We are grateful to all contributors to this book for meeting the
deadline for submission of their manuscripts, which greatly facili-
tated rapid publication. The articles appear essentially as sub-
mitted by the authors; no editing for style or content has been
done.
The Organizers
XI
Lectin-Dependent Cytolytic and Cytolymic T Helper Clones and
Hybridomas
K. Fischer Lindahl, A.A. Nordin, M.H. Schreier
A. Introduction
Any cytotoxic T lymphocyte (CTL) will kill P81S or EL4 target cells
on addition of a lectin such as phytohemagglutinin (PHA) or concanava-
lin A (Con A) (1,2), and lectin-dependent killing has been considered
a prerogative of CTL (3). However, lectin-dependent killers could be
found under conditions where no specific CTL were induced (4-6), and
nearly every T cell c~~ne stimulated by Con A and T cell growth
factor (TCGF) caused Cr-release in the presence of PHA (7). It
therefore seemed possible that any T cell can become a lectin-depen-
dent killer. To find out how cornmon this activity might be, we
tested a set of antigen-specific, I-A-restricted T helper cell lines,
clones and hybridomas (8-10).
B. Materials and Methods
Helper Cell Lines and Clones. All T cells were from CS7BL/6 mice.
The methods used to establish and clone these cells in vitro have
been publishEd together with a functional characterization (8-11).
All were I-A -restricted. They had been cultured serum-free for more
than a year by continued restimulation with antigen and irradiated
CS7BL/6 nu/nu spleen cells or in medium supplemented with TCGF.
Before testing, the lines were expanded for at least two weeks in
medium with TCGF in the absence of adherent cells and antigen.
Hybridomas. All hybridomas originated from two fusions of a chicken
egg albumin- (EA) specific clone, EA-14, with BWS147 and are described
elsewhere in this volume (12). They were cultured in RPMI 1640
supplemented with S% heat-inactivated fetal calf serum, glutamine,
pyruvate, 2-mercaptoethanol, and penicillin-streptomycin.
SlCr-Release Assay. A standard method (13) was used with 10 4 SlCr_
labeled target cells and effector-to-target ratios of 10:1, 3:1 and
1:1. Triplicates were set up in round-bottom microtiter plates in
200 ~l of RPMI 1640 with S% heat-inactivated fetal calf serum. PHA-P
(Difco) was added to a final concentration of 10 ~g/ml, Con A (Sigma)
to S ~g/ml (3 51 and the cultures were incubated for 3.S hours. The
experimental Cr-release is expressed as a percentage of the total
(measured in the presence of a detergent), and the spontaneous
release has been subtracted in the tables. Spleen cells stimulated
with Con A for three days or in allogeneic mixed lymphocyte cultures
