Lymphocyte Surface Antigens in Health and Disease 
Benjamin D. Schwartz, M.D., Ph.D. — Investigator 
Dr. Schwartz is also Professor of Medicine and of Molecular Microbiology at Washington University School 
of Medicine, St. Louis, and Chief of the Division of Rheumatology at Jewish Hospital, Washington Univer- 
sity Medical Center. He received his B.A. degree in mathematics from Columbia College and his M.D. and 
Ph.D. degrees in immunology from Albert Einstein College of Medicine. Specializing in immunology, he 
was a postdoctoral research fellow with William Paul in the Laboratory of Immunology at the NIH. Dr. 
Schwartz is a member of the American Federation for Clinical Research, the American Society for Clinical 
Investigation, and the Association of American Physicians, and served as President of the American Fed- 
eration for Clinical Research. 
IN the initiation of an immune response against 
a foreign protein antigen, the antigen is taken 
up by a speciahzed antigen-presenting cell (APC) 
and broken down into small peptide fragments. 
These fragments are then thought to be bound by 
proteins of the immune system, namely major his- 
tocompatibility class II molecules. The complex 
of class II molecule and bound peptide fragment 
is presumably expressed on the APC surface, 
where it can be recognized by a specific T lym- 
phocyte. The recognition event activates the T 
lymphocyte, which then initiates the immune 
response. 
Despite a large body of circumstantial evi- 
dence from which the presence of class II mole- 
cule linked to a foreign peptide on the APC has 
been inferred, the existence of this complex on 
the surface of viable cells has not been directly 
demonstrated. 
We have been collaborating with Tom and Vi- 
vian Braciale in the Department of Pathology at 
Washington University School of Medicine to elu- 
cidate the immune response against influenza 
virus. A particular human T cell clone, termed 
VI, was demonstrated to recognize a synthetic 
peptide, HA 128-145 (corresponding to an 18- 
amino acid segment of the influenza viral hemag- 
glutinin protein) , only when that peptide is pre- 
sented on an APC bearing a particular class II 
molecule, HLA-DRwll. 
The recognition of this complex by the T cell 
clone indicated that the HA 128-145 peptide 
was immunogenic. We therefore used the HA to 
immunize a rabbit and raise an antiserum, anti- 
HA. The antiserum recognizes not only free HA 
peptide but also a complex of the peptide bound 
to HLA-DRwl 1 on the surface of an APC. 
The composition of this complex was proved 
in several ways. Anti-HA did not recognize a mu- 
tant APC that expressed no class II molecules 
after the cells were exposed to HA, demonstrating 
that the latter did not bind nonspecifically to 
APCs. Also, anti-HA did not bind to DRwl 1 -bear- 
ing APCs that had not been exposed to HA, nor to 
APCs bearing several DRs that had been exposed 
to the peptide. 
A second APC, known to express only half the 
amount of DRwl 1 as did the first APC bearing 
DRwl 1 , when exposed to HA bound only half the 
amount of anti-HA. This last result indicates that 
binding of anti-HA to complexed DRwll-HAs 
is commensurate with the level of DRwl 1 
expression. 
Further proof of the composition of the com- 
plex was achieved biochemically. The HA pep- 
tide was tagged with radioactive iodine and a 
photoreactive group, so that when exposed to ul- 
traviolet light, the photoreactive group on the 
peptide would form a covalent bond with any 
molecule to which the peptide had bound. 
DRwl 1 -bearing APCs were incubated with the 
modified peptide, exposed to ultraviolet light, 
and then lysed. The cell lysate was then reacted 
with either the anti-HA antibody or a monoclonal 
antibody that recognized the DRwl 1 molecules. 
Biochemical analysis of the molecules reactive 
with the antibodies demonstrated by several crite- 
ria that the molecules covalently bound to the 
radioactive iodinated peptide were indeed the 
DRwll molecules. Furthermore, the DRwl 1 
were the only molecules tagged with radioactive 
iodine, demonstrating that no other molecules of 
the APC were capable of binding peptide. 
A series of experiments was done with meta- 
bolic inhibitors to ascertain whether the HA 
128-145 peptide followed the proposed route 
of intracellular processing. These experiments 
demonstrated that metabolic inhibition, includ- 
ing poisoning and fixation of the APC, did not 
decrease the number of DRwll -HA complexes 
detected by anti-HA. These results therefore indi- 
cate that HA must be binding directly to cell sur- 
face DRwl 1 molecules. It is of interest, however, 
that a temperature of 4°C totally abrogated, and 
18°C significantly inhibited, the HA peptide's 
binding to DRwl 1 molecules. Collectively these 
389 
