One cell–one antibody: prelude and
aftermath
G J V Nossal
This year marks the 50th anniversary of Burnet’s clonal selection theory. Here Gustav Nossal recounts his pioneering
work that supported Burnet’s theory and led to the death of the direct template hypothesis.
The date 21 October 2007 marks the 50th anniversary of the publication of Frank Macfarlane
Burnet’s clonal selection theory of antibody
formation1. The Walter and Eliza Hall Institute
of Medical Research will host an international
symposium in celebration. As I had the good
fortune to obtain the first evidence in favor of
the theory2, and as it was my fate to become
Burnet’s chief ambassador in its defense in
international fora, it is a pleasure to center this
essay on the heady events of 1957 and on the
slow but relentless march of clonal selection
toward general acceptance.
Medical and PhD studies
I am, as it were, an ‘accidental immunologist’. I wanted to be a doctor as far back as I
can remember and studied medicine at The
University of Sydney. Given that in those days
anyone who matriculated from high school
could enter medical school, and given further
that the Australian Government supported
university study for ex-servicemen and women,
we were 600 in first-year medicine in 1948. The
staff/student ratios were appallingly low; our
teachers struggled mightily, but the instruction left a lot to be desired. For third-year biochemistry and physiology, a few of us ‘clever’
kids took the matter into our own hands, each
studying selected topics in the library, then
giving the group a seminar on the latest data.
This fired my imagination for research, and
I took advantage of a program that allowed
students to take a year off and work in an academic department as research apprentices. In
G.J.V. Nossal is in the Department of Pathology, The
University of Melbourne, Victoria 3010, Australia.
e-mail: [email protected]
my case, I had a clear idea of what I wanted
to do. Fascinated by biochemistry in the preWatson-Crick era, I wanted to discover the
‘secrets of life’ by studying the multiplication
of the simplest forms of life, the viruses. In the
Bacteriology Department, a senior lecturer,
Patrick M. de Burgh, was investigating the
effects of the ectromelia virus on the metabolism of infected liver cells. I joined him and,
cognizant of the one-step growth curves of
bacteriophages, I charted the growth of ectromelia in the liver after intravenous infection,
recording the latent period and then successive waves of multiplication. Remarkably, when
liver cells were infected with up to 20 median
lethal doses of virus per cell, the viruses multiplied independently of each other3, showing
that “the fundamental virus-synthesising centres must be sought at a sub-cellular level,”3
antedating John Cairns’ demonstration of
vaccinia virus cytoplasmic factories by more
than a decade. Pat de Burgh arranged for me
to spend a week in Melbourne sitting at the feet
of Australia’s greatest virologist, F.M. Burnet,
and that experience at age 21 marked me for
life. I finished my medical course and hospital residency years, and went to Melbourne in
1957 to pursue a PhD degree. Imagine my disappointment to learn that Burnet had switched
his interests from virology to immunology, the
area he now wanted me to follow. How had this
come about?
Burnet’s clonal selection theory
Burnet had long had a theoretical interest in
immunology, prompted by some early studies on the formation of antibodies to bacterial toxins. He had noted several features not
explained by the then-fashionable ‘direct template’ theory of antibody formation4. These
NATURE IMMUNOLOGY VOLUME 8 NUMBER 10 OCTOBER 2007
The Walter and Eliza Hall Institute of Medical Research
© 2007 Nature Publishing Group http://www.nature.com/natureimmunology
E S S AY
Sir Gustav Nossal, successor to Sir Macfarlane
Burnet as Director of the Walter and Eliza Hall
Institute of Medical Research.
included the exponential rise in antibody titers
after immunization; the improvement in the
quality or affinity of antibody after repeated
immunization; the booster effect after secondary immunization; and, very significantly,
the phenomenon of immunological tolerance
that he had predicted on theoretical grounds5
and that Peter Medawar and associates proved
experimentally6. He had made attempts to
replace the direct template hypothesis with a
rather clumsy ‘indirect template’ theory, but
deep down he knew this was not quite right.
He was most impressed with Astrid Fagraeus’
work implicating the family of rapidly dividing
plasmablasts and plasma cells in antibody formation7. He was also intrigued with a case of a
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E S S AY
monoclonal gammopathy, macroglobulinemia,
that was accompanied by a high titer of autoantibodies8. Above all, he kept chewing over the
natural selection theory of antibody formation
promoted by Neils Jerne9, in which millions
of antibody specificities existed in the serum
before any immunization, with antigen serving
somehow to catalyze the production of much
more of the antibody than happened to fit the
antigen in question. So there was a picture of
antigen causing cell division, of autoimmune
clones gone wild, of antigen sometimes causing
tolerance, and of antibody formation perhaps
being selective rather than instructive.
Into this charged atmosphere burst David
Talmage. In a reflective essay covering many
topics, he wondered whether natural antibodies were actually cell surface receptors and
whether antigen selects cells for replication on
the basis of their having the right receptor10. He
did not come out and say that each cell has only
one kind of receptor. This prompted Burnet
to crystallize his ideas, and over one weekend in August 1957 Burnet drafted his short
paper for the Australian Journal of Science1. He
speculated that the small lymphocyte population of the body is actually a repertoire of
specificities, with each cell bearing on its surface just one kind of natural antibody. During
immunization, antigen selects cells with the
corresponding specificity for multiplication
and differentiation into antibody-secreting
status. This accounts for an exponential rise
in antibody concentrations and an increased
number of the ‘right’ cells to respond to a second immunization. As the immune response
progresses, somatic mutation of antibody
genes allows cells of higher affinity to arise
and to be selected by antigen, accounting for
affinity maturation. If a clonally individuated
cell encounters ‘its’ antigen while the system is
still immature, whether a ‘self ’ antigen or one
introduced artificially during embryonic life,
the cell, rather than being activated, is actually
killed, explaining natural self-tolerance as well
as artificially induced tolerance. Occasionally
the tolerance system goes wrong, allowing
‘forbidden clones’ to escape, resulting in autoimmune disease. The theory retains Jerne’s
random generator of antibody diversity and
Talmage’s idea of the cell as the unit of selection, but extends both ideas to cover the key
inadequacies of the direct template theory.
Antibody formation by single cells
At that time I was still scanning the virus literature and was intrigued by Marguerite Vogt’s
ability to grow poliovirus in isolated single
cells11. Also, I read a fair proportion of Joshua
Lederberg’s work, as he was expected at the Hall
Institute for a short sabbatical leave. He had
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cultured single bacteria in microdroplets. It
occurred to me that I could, in best Popperian
fashion, disprove clonal selection by showing
that one cell could simultaneously form two or
more antibodies. Lederberg duly arrived and
we discussed three possibly suitable sensitive
antibody titration techniques: immobilization of motile salmonella bacteria by antibody
to flagella; lysis of sheep erythrocytes in the
presence of antibody and complement; and
neutralization of bacteriophages. We chose
the first, largely because of Lederberg’s deep
knowledge of the H antigens of salmonella. We
faced a few hurdles, however. There were few
decent microscopes at the institute. We rustled
up an old Bausch and Lomb, still with vertical
rather than inclined eyepieces, and no darkfield condenser. I figured out that if I removed
the condenser and inserted a large coin in its
stead, permitting just an arcuate path of light
to come through, I could create a fuzzy but
adequate imitation of darkfield to visualize
swimming bacteria. There was no micromanipulator to move cells around, but one was
located in the Microbiology Department and
borrowed. Lederberg taught me the essentials
of micromanipulation and then unfortunately
had to go back to the United States.
By late 1957, my first few results trickled
through2. Rats were immunized with two
different strains of salmonella, and single
lymph node cells were suspended in hanging
microdroplets surrounded by mineral oil and
cultured for 4 hours. Then, ten or so motile
bacteria were instilled into each droplet, first
of one strain and then of the other, and were
monitored for immobilization. With a ‘good’
cell, the results were dramatic: immobilization
was nearly instantaneous. Over the ensuing
months, I improved the techniques considerably and obtained very clean results12. One cell
always formed only one antibody. This was a
first hint in the direction of clonal selection.
Using a phage-neutralization system, a group
headed by Mel Cohn and Ed Lennox obtained
contrary results13, which made me stick with
the problem longer than was really necessary.
The end result14 was that of 3,628 single cells
tested, only two seemed to secrete two antibodies, which could well have represented
monoclonal antibodies with a rare crossreactivity or perhaps binucleate plasma cells,
which are not uncommon. My confidence in
my own results was increased by results from
immunofluorescence studies (which of course
measure antibody content and not secretion)
that failed to find doubly specific cells15. My
colleague Olavi Mäkelä did indeed pursue the
problem using phage-neutralization techniques. He failed to find double producers16
but made the interesting observation that
when cells were tested with a cross-reacting
phage, each single cell gave its own unique
pattern of cross-neutralization, some even
making ‘heteroclitic’ antibodies that neutralized the cross-reactive phage better than the
immunizing one, foreshadowing the precise
qualities of monoclonal antibodies.
Death of the direct template theory
Selective theories made only slow headway,
so Gordon Ada and I sought to embark on a
test of the antigen content of single antibodyforming cells. Using flagellin heavily iodinated
with 125I, and relatively low antigen doses to
minimize background radioactivity, we micromanipulated single antibody-producing cells
to marked spots on a glass slide and analyzed
them by autoradiography. In circumstances
that would have detected as few as four antigen
molecules per cell, 216 single cells showed no
antigen whatever in them17. Hugh McDevitt et
al. reinforced this conclusion18. So there was
no template to copy!
Of course, studies of antibody-forming cells
did not directly address the characteristics of
the B lymphocyte that was their ancestor. The
discovery of David Naor and Dov Sulitzeanu19
that only very rare lymphocytes bound a given
antigen was critically important. The ‘hot antigen suicide’ experiment of Gordon Ada and
Pauline Byrt20, in which very highly radioactive antigen specifically abrogated the antibody-forming potential in an unimmunized
lymphocyte population, emphasized the probability of antibody receptors on clonally individuated cells. A further elegant experiment
was that of Martin Raff et al.21, which showed
that when an antigen was used to ‘cap’ the
immunoglobulin receptors of a B cell, there
was no detectable other immunoglobulin
left as a ring on the lymphocyte surface; thus,
all the immunoglobulin seemed to have the
same specificity. Final proof was slow in coming. The isolation of antigen-specific B cells
from unimmunized animals proved onerous,
despite the fluorescence-activated cell sorter.
Cloning single B cells in vitro to test such a
cell’s antibody-forming potential also required
methodological advances. When we finally
succeeded in both endeavors and showed that
the antibody secreted faithfully reflected the
original specificity22, our paper caused little
comment. By the time of the 1967 Cold Spring
Harbor Symposium on immunology, most
aspects of clonal selection had in fact been
accepted through the weight of the indirect
evidence.
‘One cell–one antibody’ had its most glorious aftermath in the work of Georges Köhler
and César Milstein23 and the revolution in
basic science and therapeutics ushered in
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E S S AY
by monoclonal antibodies. Furthermore, as
molecular biology demonstrated the somatic
‘minigene’ translocation events underlying
the generation of the B cell repertoire and the
post-antigenic hypermutations refining antibody specificity, it was satisfying to be able to
map these findings on to the original, insightful sketch1. That the T cell system also obeys
clonal selection rules, though with some noteworthy differences, is an extra bonus. Rarely
has one theoretical paper so comprehensively
fertilized a discipline or been so thoroughly
vindicated.
COMPETING FINANCIAL INTERESTS
The author delcares no competing financial interests.
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