Lymphocyte Differentiation and Activation 
Alfred L. M. Bothwell, Ph.D. — Associate Investigator 
Dr. Bothwell is also Associate Professor of Immunobiology at Yale University Medical School. He received 
his A.B. degree in biology from Washington University and a Ph.D. degree in biology from Yale University, 
where he studied with Sid Altman. This was followed by postdoctoral work at the Cold Spring Harbor 
Laboratory and at the Massachusetts Institute of Technology with David Baltimore. 
THE cellular events that accompany lymphoid 
differentiation are highly regulated. This reg- 
ulation of lymphoid gene expression is central to 
the development of T and B cells and the ultimate 
function of mature lymphoid cells. We are inves- 
tigating several experimental systems: 
• The involvement of Ly-6 antigens in the de- 
velopment of T cell responsiveness, with a focus 
on such events as recognition of foreign antigen 
and processes associated with signal transduc- 
tion. 
• The potential effects of defective Ly-6 gene 
expression on development of autoimmunity. 
• T cell activation, which occurs as a result of 
specific recognition by the antigen-specific re- 
ceptor. We have developed an expression system 
that results in production of high levels of solu- 
ble T cell receptors (TCRs) for analysis of func- 
tion and biophysical properties. 
• Regulation of lymphoid-specific genes by 
RNA splicing. A factor that may regulate this pro- 
cess has been molecularly cloned and is being 
studied functionally. 
• Formation and development of memory B 
cells and plasma cell lineages. This is studied 
with an emphasis on commitment to a lineage 
and its development. 
The Ly-6 antigens were initially characterized 
as associated with activated T cells. The Ly-6A 
antigen is expressed on these cells and hemato- 
poietic stem cells as well. The antigen can also be 
used to induce activation, as monoclonal antibod- 
ies directed against Ly-6A activate T cells when 
crosslinked. Activation requires the presence of 
the TCR complex on the cell surface. 
To analyze this process functionally, we are us- 
ing site-directed mutagenesis. The protein is an- 
chored in the membrane not by a conventional 
transmembrane segment but by a glycosyl phos- 
phatidylinositol (GPI) lipid anchor. Mutants 
have been made with an altered anchor, and a 
transmembrane form has been created. These con- 
structs have shown that the GPI anchor is re- 
quired for function. 
Another form of mutation is the functional de- 
letion of the gene product. When the expression 
of Ly-6A is inhibited in a T cell clone by antisense 
RNA, the antigen recognition function of the cell 
is destroyed and survival of the clone in culture is 
dependent on certain lymphokines. These exper- 
iments reveal the importance of Ly-6 antigens to 
T cell function and have opened up new ap- 
proaches to the study of Ly-6 function. We have 
begun experiments to disrupt the endogenous 
gene in embryonic stem cells by homologous re- 
combination. These cells can be used to generate 
a mouse that can transmit the mutation in the 
germline. 
All inbred strains of mice tested display a com- 
mon pattern of Ly-6C expression except NOD, 
NZB, and ST. In these three strains, expression of 
Ly-6C is greatly reduced in the bone marrow and 
cannot be detected in the spleen or lymph nodes. 
NOD and NZB mice are well-studied models of 
autoimmune disease (insulin-dependent diabetes 
in the former case, systemic lupus erythematosus 
in the latter). These two strains of mice also ex- 
hibit a depressed syngeneic mixed-lymphocyte 
reaction (SMLR) , which, as a measure of the pro- 
liferative response of T cells to self-MHC class II 
determinants, represents an immunoregulatory 
cellular interaction. Analysis of recombinant 
inbred lines derived from NZB indicates cosegre- 
gation of the defect in Ly-6C expression and de- 
creased responsiveness in the SMLR. 
Cloning and sequencing of the affected Ly-6C 
gene segment from the NOD mouse indicates the 
presence of an interruption by sequences charac- 
teristic of retroposon insertion in the 5' flanking 
region of the Ly-6C gene. This disruption of the 
normal Ly-6C gene allows only a minute level of 
Ly-6C to be expressed, raising the possibility that 
the antigen is directly involved in regulatory T 
cell responses. Ly-6C might be necessary for 
some aspects of the normal development of the 
TCR repertoire. We have identified defects in he- 
matopoietic stem cell populations and, using a 
panel of antibodies, are attempting to identify 
and purify the cells that can transfer disease. 
The Ly-6C and Ly-6A genes are very inducible 
with interferons. Regions in the promoters of 
both that confer responsiveness to interferon 
have been identified but lack known consensus 
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