DEVELOPMENTAL BIOLOGY OF T LYMPHOCYTES 
Dennis Y.-D. Loh, M.D., Associate Investigator 
Thymus-derived lymphocytes (T cells) display 
three characteristic features. First, each T cell has a 
unique cell surface receptor, called the T cell re- 
ceptor (TCR), by which antigens are recognized. 
Second, unlike the case of immunoglobulin mole- 
cules, antigens can only be recognized in the con- 
text of the products of the major histocompatibility 
complex (MHC), a phenomenon known as self MHC 
restriction. Third, T cells whose TCR specificity is 
directed against self antigens present in the organ- 
ism must be rendered immunologically tolerant to 
avoid autoimmunity. 
The focus of Dr. Loh's laboratory is the study of 
the developmental process by which functional T 
cells are produced. These studies involve the eluci- 
dation of the molecular mechanism of positive and 
negative selection of the thymocytes that forms part 
of the basis of modern T cell biology. Recent inter- 
est is centered on the development of transgenic 
mouse models to study the cells, molecules, and sig- 
nals involved in determining T cell fate. 
TCR Transgenic Mouse Whose Receptor 
Is MHC Class I Restricted 
Previous work in Dr. Loh's laboratory, using 2C 
TCR transgenic mice, showed that thymocyte cell 
fate was determined by the interaction among the 
TCR, the co-receptor CDS on the thymocyte surface, 
and the specific MHC class I molecules displayed in 
the thymus. Moreover, mutational analysis of the 
MHC molecule responsible for positive selection, 
K'', showed that both the MHC-bound peptide and 
the amount of the available CDS molecules were of 
crucial importance. 
To fix the importance of the CDS molecule, the 
2C TCR transgenic mice were crossed with two 
kinds of CDS transgenic mice. One overexpressed 
the CDS constitutively, while the other had the CDS 
gene deleted in the germline. In these double- 
transgenic mice, it was shown clearly that an excess 
of CDS molecules on the thymocyte surface resulted 
in negative selection of the thymocytes (leading to 
programmed cell death), even in MHC backgrounds 
where positive selection (leading to cell survival 
and differentiation) was normally expected. On the 
other hand, no positive selection was observed 
when CDS molecules were altogether absent in the 
CDS"/ background. These results stress the impor- 
tance of the CDS as a co-receptor molecule that is 
crucial in determining cell fate. 
Two distinct functions are possible for the CDS 
molecule during selection. It can act as an adhesion 
molecule, binding to a molecule of MHC class I, or 
it can transmit an intracellular signal upon engage- 
ment. To decipher the relative contribution of each 
of these functions, Dr. Loh's laboratory has created a 
variety of CDS-mutant transgenic mice that are be- 
ing backcrossed now to the CD8~/~ background. 
These studies should allow for the unequivocal de- 
termination of the mechanism by which CDS deter- 
mines thymocyte cell fate. 
Transgenic TCR Mice Whose Receptor Is 
Directed Against a Specific Peptide Antigen 
To study the role of specific peptides in determin- 
ing thymocyte cell fate. Dr. Loh and his colleagues 
have utilized a TCR transgenic mouse whose recep- 
tor is directed against a specific peptide from 
chicken ovalbumin (cOVA). With this mouse 
model, they have shown that the in vivo administra- 
tion of the peptide leads to negative selection of the 
developing thymocytes by a process called apopto- 
sis or programmed cell death. Furthermore, to dis- 
sect the cellular and molecular requirements that 
lead to positive and negative selection, an in vitro 
model using the cOVA was developed. 
Experimentally, transgenic thymocytes will un- 
dergo apoptosis in a developmentally stage-specific 
manner when the appropriate antigenic peptide is 
presented. Surprisingly, the results showed that any 
antigen-presenting cell was capable of initiating 
thymocyte death so long as it had the correct MHC 
class II molecule capable of binding the cOVA pep- 
tide. These results clearly indicate that the propen- 
sity for programmed cell death upon antigen con- 
tact is an inherent property of the immature 
thymocytes and is not determined by the nature of 
the antigen-presenting cells. In contrast, mature T 
cells proliferate upon antigen contact. Thus there is 
a clear-cut distinction of cell fate upon antigen con- 
tact between the immature thymocytes that undergo 
cell death and the mature cells that proliferate and 
live. 
To start characterizing the variables that deter- 
mine these diametrically opposite cell fates, Dr. Loh 
and his colleagues initially analyzed the depen- 
dence of antigen stimulation on tyrosine kinase ac- 
tivity. Using a combination of tyrosine kinase inhibi- 
tors, genistein and herbimycin A, they showed, in 
the in vitro selection system, that programmed cell 
death of the immature thymocytes appeared to be 
independent of the tyrosine kinase activity, while 
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