Molecular Approaches to T Lymphocyte 
Recognition and Differentiation 
Mark M. Davis, Ph.D. — Investigator 
Dr. Davis is also 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 NIH as a postdoctoral fellow and Staff Fellow 
before joining the staff at Stanford. He is the recipient of the Eli Lilly award in Microbiology 
and Immunology and a Gairdner Foundation award. 
WE have focused on several major areas in 
immunology that generally resolve into 
two questions: How do T cells recognize foreign 
entities? How is lymphocyte differentiation con- 
trolled, both in the thymus and in the periphery? 
An additional goal is to refine and better integrate 
recombinant DNA technology with other power- 
ful techniques in immunology, as an approach to 
defining the function of unknown genes or 
poorly understood genes and their products. 
Topology of T Cell Recognition 
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. In 
contrast, antibodies, although closely related to T 
cell antigen receptors (TCRs) , bind intact foreign 
particles directly. This suggests fundamental dif- 
ferences in the rules governing T cell recognition 
versus antibody-mediated recognition by B cells. 
Because of the consistently high concentration of 
sequence diversity in the V-J junctional region of 
TCRs (equivalent to the third complementation- 
determining region [CDR3] of immunoglobulins) 
as well as structural considerations, we have pro- 
posed that this is the important region for peptide 
recognition and that other V region-encoded resi- 
dues might contact the surface of the MHC 
molecule. 
To test this hypothesis, we recently developed 
an immunological version of the classical genetic 
technique of second-site suppression. In these 
experiments we change residues that are impor- 
tant for T cell recognition (and not MHC interac- 
tion) on a peptide, immunize mice, and then ana- 
lyze the responding T cells that emerge with 
respect to their TCR sequences. To hold part of 
the original receptor constant, we use mice that 
are transgenic for either chain of the original 
TCR. With this approach, we have shown that one 
of the two residues on the peptide that are impor- 
tant in the T cell response is governed by the 
CDR3 of the Va polypeptide, and that the other 
(three amino acids downstream on the peptide 
sequence) is specified by the CDR3 of the VjS 
polypeptide. Thus we have generated significant 
support for the original hypothesis. The results 
indicate that T cell recognition is a much more 
stylized event than antibody-antigen interactions. 
Kinetics of T Cell Recognition 
and Activation 
To learn more about the dynamics of TCR- 
peptide-MHC interactions, we have developed 
expression systems that allow us to produce ei- 
ther TCR or MHC class II heterodimers in a solu- 
ble form. This involves replacing the normal 
membrane-spanning sequences of these polypep- 
tides with a signal sequence for lipid linkage, 
such as employed normally by a number of cell 
surface proteins. Molecules expressed in this fash- 
ion can then be conveniently cleaved from the 
surface of expressing cells with the enzyme phos- 
phatidylinositol-specific phospholipase C. By 
utilizing high-density mammalian cell culture 
machines, we are able to make milligram quanti- 
ties of a soluble TCR and its cognate MHC mole- 
cule. This has provided us with the raw material 
to initiate structural studies such as x-ray crystal- 
lography and nuclear magnetic resonance analy- 
sis. We have also used the soluble MHC protein to 
show greatly enhanced uptake of antigenic pep- 
tides 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) that recycles 
through low-pH endosomal corripartments. Our 
current data suggest that the low pH triggers a 
specific conformational change in the MHC mole- 
cule, which allows it to bind new peptides more 
easily. 
Recently we have studied the kinetics of TCR 
peptide-MHC interactions, using soluble MHC- 
peptide complexes that competitively inhibit 
binding of labeled anti-TCR antibody fragments 
to TCRs. In several cases studied, we derive 
values for this interaction of 5 X 10~^ M, 1,000- 
to 10,000-fold weaker than antibodies to protein 
ligands of comparable size. These values are con- 
sistent with the scanning nature of T cell recogni- 
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