Molecular Genetics of B Cell Development 
Michel C. Nussenzweig, M.D., Ph.D. — Assistant Investigator 
Dr. Nussenzweig is also Assistant Professor at the Rockefeller University. He received his undergraduate 
and his M.D. degrees from New York University and his Ph.D. degree from Rockefeller. He completed his 
residency and clinical fellowship at Massachusetts General Hospital and conducted postdoctoral research 
at the Harvard Medical School with Philip Leder. 
THIS laboratory is developing ways of under- 
standing the cells of the immune system at 
the molecular level, using the tools of molecular 
biology and genetics. 
The immune system is responsible for protect- 
ing vertebrates from both invasion by infectious 
organisms and deregulated growth of endoge- 
nous malignancies. In order to accomplish this 
task, the system must be able to distinguish self 
from non-self. Evolution has solved this problem 
in higher vertebrates by providing a network of 
cell types and humoral agents. It is the lympho- 
cytes — T cells and B cells — that direct the speci- 
ficity of immune responses. 
Although the mechanism of antigenic recogni- 
tion differs for these two cell types, the genera- 
tion of diversity in their surface receptors is 
achieved in a similar fashion. In both cases the 
business end of the receptor is created in individ- 
ual somatic cells by a series of genetic recombina- 
tions at a minimum of two loci. For example, the 
heavy- and light-chain immunoglobulin proteins 
that serve as the B cell receptor derive from two 
sets of rearranging genes. The same genes also 
direct the production of secreted antibodies that 
are an important component of the humoral im- 
mune system. Thus the regulation of T cell and B 
cell receptor rearrangements is a central feature 
of the generation of immune responses. 
The joining events that bring together the im- 
munoglobulin segments occur in an ordered and 
regulated fashion. In B lymphocytes, rearrange- 
ments begin at the heavy-chain locus with the re- 
combination of D and J segments. This is followed 
by the joining of DJ with one of 100-1,000 vari- 
able-region segments. After a functional immuno- 
globulin heavy-chain transcription unit is cre- 
ated, the light-chain genes undergo a similar set 
of rearrangements. 
One poorly understood aspect of these events 
is the ability of lymphocytes to limit themselves 
to the production of a single receptor. Since pro- 
ductive rearrangements could occur in two 
heavy-chain and four light-chain alleles, a single 
B lymphocyte could potentially make several 
types of receptors including hybrid molecules. 
The mechanism that ensures that only one recep- 
tor is produced is referred to as allelic exclusion. 
It is an important safeguard for the immune sys- 
tem, since production of multiple receptors by a 
lymphocyte would dilute the specificity of any 
given immune response. 
Much of the early work in the area of allelic 
exclusion was based on examining the status of 
immunoglobulin genes in transformed B cells. 
The transformed cells are frozen in one stage of 
lymphocyte development and for this reason 
offer only a static picture of important regulatory 
events. Unfortunately there is no in vitro system 
that faithfully reproduces regulated immunoglob- 
ulin gene rearrangements. In order to study how 
immunoglobulin genes can regulate allelic ex- 
clusion, we turned to transgenic mice. 
Our approach has been to introduce into the 
germline of mice human immunoglobulin genes 
that have been modified to direct the synthesis of 
either membrane-associated or secreted immuno- 
globulin heavy chains. We found that the expres- 
sion of human membrane-bound immunoglobu- 
lin M results in the exclusion of most endogenous 
mouse immunoglobulins. The secreted version of 
the same transgene had little effect. 
In order to examine the molecular basis for ex- 
clusion by membrane-associated IgM, we have de- 
veloped a sensitive PGR assay for heavy-chain 
gene recombination. This assay allows us for the 
first time to examine specific heavy-chain recom- 
bination events in pools of primary lymphoid 
cells. We are able to assess D to J segment recom- 
bination as well as V to DJ joining events for spe- 
cific families of variable regions. 
We find that the rearrangement of the endoge- 
nous heavy-chain locus is inhibited in transgenic 
mice that carry human membrane IgM. Gene rear- 
rangements are not affected in the control trans- 
genic mice that carry the secreted human IgM 
gene. Furthermore, not all gene segments are 
equally affected by the membrane transgene. The 
large families of variable-region genes that are in 
the distal part of the immunoglobulin locus are 
almost completely inhibited from undergoing re- 
arrangements, whereas the small families of more 
329 
