Molecular Approaches to Lymphocyte Recognition 
and Differentiation 
Mark M. Davis, Ph.D. — Associate Investigator 
Dr. Davis is also Associate Professor of Microbiology and Immunology at Stanford University School of 
Medicine. He received his B.A. degree from the Johns Hopkins University and his Ph.D. degree in molecular 
biology from the California Institute of Technology. He held positions at the NIH as a postdoctoral fellow 
and Staff Fellow before joining the faculty at Stanford. 
WE have focused on several major areas in 
immunology that generally resolve into 
three problems: How do T cells recognize foreign 
entities? How are they selected in the thymus? 
How is lymphocyte differentiation controlled ge- 
netically? An additional goal is to refine and bet- 
ter integrate recombinant DNA technology with 
some of the other powerful techniques in immu- 
nology, as an approach to defining the function of 
unknown genes or poorly understood genes and 
their products. 
T Cell Recognition of Foreign Antigens 
The work of many investigators over the years 
has shown that T cells, through their antigen re- 
ceptor molecules, recognize fragments of foreign 
proteins (peptides) embedded in major histo- 
compatibility complex (MHC) molecules. This is 
in contrast to antibodies, which, while closely 
related to T cell antigen receptors, bind intact 
foreign proteins directly. Because of the consis- 
tently high concentration of sequence diversity 
in the V-J junctional region of T cell receptors 
(and other considerations), we have proposed 
that this is the important region for peptide recog- 
nition and that other V region-encoded residues 
might contact the surface of the MHC molecule. 
To test this possibility and learn about the mo- 
lecular dynamics of T cell receptor-peptide- 
MHC interactions in general, we have developed 
expression systems that allow us to produce ei- 
ther T cell receptor or MHC class II heterodimers 
in a solubilizable form. This involves replacing 
the normal membrane-spanning sequences of 
these polypeptides with a signal sequence for 
lipid linkage, such as employed normally by a 
number of cell surface proteins. Molecules ex- 
pressed in this fashion can then be conveniently 
cleaved from the surface of expressing cells with 
the enzyme phosphatidylinositol-specific phos- 
pholipase C. By utilizing high-density mamma- 
lian cell culture machines, we are able to make 
milligram quantities of a soluble T cell receptor 
and its cognate MHC molecule. We hope to dem- 
onstrate a direct interaction between these macro- 
molecules (with the appropriate peptide in the 
MHC component) and to measure the affinity 
with which this happens. This work is also in- 
tended to provide the raw material needed for 
structural studies, such as x-ray crystallography. 
We have already used the soluble MHC protein 
to show greatly enhanced uptake of antigenic 
peptides at low pH. This is important both in 
making significant quantities of a pure antigen- 
MHC complex and in understanding the biology 
of this type of MHC molecule (class II), which 
recycles through low pH endosomal compart- 
ments. Our current data suggest that the low pH 
triggers a specific comformational change in the 
MHC molecule, which allows it to bind new pep- 
tides more easily. 
Recent experiments relevant to the issue of 
whether the CDR3-equivalent regions of T cell 
receptors are responsible for peptide recognition 
take advantage of an immunological version of 
classical genetic second-site suppression analy- 
sis. In our experiments we change residues that 
are important for T cell recognition (and not 
MHC interaction) on a peptide, immunize mice, 
and then analyze the responding T cells that 
emerge with respect to their T cell receptor se- 
quences. To hold part of the original receptor 
constant, we immunize mice that are transgenic 
for either chain of the original T cell receptor. 
With this new approach, we have shown that of 
the two residues on the peptide that are most im- 
portant in the T cell response, one is governed by 
the CDR3 of the V„ polypeptide and the other, 
three amino acids downstream on the peptide se- 
quence, is specified by the CDR3 of the poly- 
peptide. Thus we have generated significant sup- 
port for the original hypothesis. 
T Cell Receptor Selection 
Another area of interest in T cell recognition 
involves the ways in which an organism might 
select for particular T cell receptor-bearing 
cells. The specific mechanisms and extent of 
such selections are just now becoming clear. To 
address these questions, we have made mice 
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