MECHANISM OF LYMPHOCYTE DIFFERENTIATION 
Alfred L. M. Bothwell, Ph.D., Associate Investigator 
There are three areas of research in Dr. Both- 
well's laboratory: 1) analysis of the function and 
structural properties of a family of T cell differentia- 
tion antigens, designated Ly-6; 2) molecular charac- 
terization of regulatory sequences controlling Ly-6 
gene transcription; and 3) study of the develop- 
ment of a model T cell-dependent immune re- 
sponse, especially with regard to the process of so- 
matic mutation and development of cell lineages. 
L Molecular Analysis of Structural and Functional 
Properties of Ly-6 Antigens. 
Several experiments have implicated Ly-6 anti- 
gens in lymphocyte activation. Mitogen stimulation 
of peripheral lymphocytes causes high levels of Ly-6 
expression of both B and T cells. Perhaps the most 
intriguing observation is that monoclonal anti-Ly-6 
antibodies can activate splenic T cells or antigen- 
specific T cell hybridomas to proliferate and release 
interleukin-2 (IL-2). Expression of the T cell recep- 
tor on the cell surface is necessary for activation by 
monoclonal antibodies. In addition, mutants of T 
cell hybridomas that have lost the normal levels of 
Ly-6 can no longer respond to the appropriate anti- 
gen. These results suggest that these antigens can 
either amplify or modify physiologic signals gener- 
ated by elements of the T cell receptor complex 
or are involved in a distinct pathway for T cell ac- 
tivation. 
Two of the Ly-6 antigens, Ly-6C and Ly-6A/E, have 
been characterized very extensively. The polypep- 
tides are —14-18 kDa and have extensive disulfide 
bonds. The proteins are anchored in the cellular 
membrane by a phosphatidylinositol (PI) lipid link- 
age. Dr. Bothwell's laboratory is attempting to elu- 
cidate the function of these molecules. A muta- 
tional analysis of the protein is being performed. 
Initially, the carboxyl terminus has been mutated to 
create a transmembrane form of this antigen. This 
analysis has revealed that there are at least two sig- 
nals involved in the biosynthesis of a Pl-linked pro- 
tein. During its biosynthesis, ~30 amino acids are 
removed from the carboxyl terminus and the PI 
linkage is attached. There is a minimal length of the 
carboxyl terminus that is required, and a second 
more complex signal is required near the point of 
proteolysis. 
A transmembrane form of the protein has been 
generated by appending the transmembrane and 
cytoplasmic tail of a class I antigen to the Ly-6 struc- 
ture. These constructs are being introduced into T 
cell clones to assess their effect on activation. An- 
other approach to alter normal Ly-6 function is to 
knock out the expression of the gene, using anti- 
sense transcripts or deletion of the gene by homol- 
ogous recombination. Both experiments are in 
progress. 
A potential human homologue of Ly-6 was identi- 
fied by others as a Pl-linked protein on erythrocytes 
and designated MEM-43 or CD59. This protein has 
also been studied as an inhibitor of complement 
function. It appears to inhibit the oligomerization 
of C9. In other experiments it has been character- 
ized as an antigen that is expressed on accessory 
cells and necessary for T cell activation via CD3. 
The cDNA was isolated and shown to have struc- 
tural homology to Ly-6 antigens. In contrast to mu- 
rine Ly-6, the human CD59 is found as a single 
gene on chromosome 11. It is possible that the mu- 
rine Ly-6 antigens have an analogous function in 
mice. The high level of Ly-6A in the kidney might 
function to protect cells from high levels of im- 
mune complexes. The possibility that Ly-6 may bind 
to murine C9 is being examined. 
II. Regulation of Ly-6 Gene Transcription. 
The Ly-6A/E and Ly-6C genes and their promoters 
are being analyzed in detail. These two genes are 
inducible with interferon-a,p and interferon-7, yet 
the DNA sequence of these promoters does not re- 
veal the consensus GA box found in all other inter- 
feron-a,(3-responsive genes. A deletion analysis of 
both of these promoters is being accomplished by 
deriving stably transformed clones of BALB/3T3 
cells. An attempt to analyze the interferon respon- 
siveness with transient transfections using chloram- 
phenicol acetyltransferase (CAT) vectors was unsuc- 
cessful. However, transient CAT assays have been 
used to identify other elements of the Ly-6A/E pro- 
moter. At present, the a,p as well as the 7 DNA ele- 
ments reside within —1.5 kb 5' of the start site for 
transcription. Deletions located about every 200 bp 
within these regions are being analyzed. 
Two autoimmune strains of mice, non-obese dia- 
betic (NOD) and NZB, have a common rearrange- 
ment in the promoter of the Ly-6C gene. This rear- 
rangement is not present in the progenitor of NOD, 
the NON strain. Genomic DNA clones of the NOD 
Continued 
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