T LYMPHOCYTE BIOLOGY AND GENETICS 
Robert R. Rich, M.D., Investigator 
Dr. Rich's laboratory studied three models of 
antigen recognition by T cells, focusing on struc- 
ture-function relationships of major histocompati- 
bility complex (MHC) -associated genes and gene 
products. 
L Maternally Transmitted Antigen (Mta). 
Mta is a murine cell surface antigen of broad tis- 
sue distribution with features of a class I MHC mol- 
ecule. It is identified by cytolytic T lymphocytes 
(CTL) and is unique in its maternal pattern of in- 
heritance. One gene required for Mta expression, 
Hmt, has been mapped telomeric to the Tla region 
of the H-2 complex and is thought to encode a class 
I polypeptide. Maternally inherited antigenic poly- 
morphism, however, is determined by a mitochon- 
drial gene, Mtf; two major genotypes, Mtf^ and 
Mtf^, determine expression of the major antigenic 
phenotypes, Mta'' and Mta'', respectively. Chloram- 
phenicol, a specific inhibitor of mitochondrial 
translation, was employed to show that Mta expres- 
sion reflected mitochondrial protein synthesis. Mta 
expression was also sensitive to treatment with 
monensin, which prevents protein movement 
through the Golgi apparatus. These data suggested 
that Mta expression requires a short-lived product 
of mitochondrial protein synthesis that may be 
transported through the Golgi apparatus to the cell 
surface. 
More recent studies investigated the structural 
requirements for a specific amino-terminal se- 
quence from the mitochondrial gene, NDl, as a 
possible candidate for the Mtf gene product. These 
studies, employing synthetic peptides added to cul- 
ture media, confirmed the finding of Dr. Kirsten 
Fischer-Lindahl (HHMI, University of Texas South- 
western Medical Center at Dallas) that antigenic 
polymorphism of Mta^ versus Mta'' was determined 
by substitutions at position 6 of this highly hydro- 
phobic peptide. The length of the peptide required 
for recognition by Mta-specific CTL clones ranged 
from 6 to 12 amino acids, with some clones recog- 
nizing an amino-terminal hexamer, whereas others 
required 8 or 12 amino acid peptides for optimal 
lysis by Mta-specific CTL. Formyl substitution was 
required on the amino-terminal methionine of the 
M//" peptide. Mitochondrial proteins, in contrast to 
proteins encoded by the nuclear genome, are initi- 
ated by formyl-methionine, perhaps as a remnant of 
their presumed prokaryotic ancestry Synthetic pep- 
tides initiated with nonsubstituted or acetylated 
methionine were not recognized by Mta-specific 
CTL. Inhibition studies suggested that the formyl 
group was required for effective interaction with 
the class I Hmt polypeptide in construction of the 
Mta antigen. These studies suggest possible novel 
functions of the Hmt gene product in transport of 
highly hydrophobic leader sequences of mitochon- 
drial gene products and/or in host defenses to fMet- 
initiated (i.e., bacterial) antigens. 
II. Structural and Functional Analysis of Human 
HLA Class II Genes and Antigens. 
Previous studies from Dr. Rich's laboratory dem- 
onstrated that HLA class Il-bearing cells from some 
DRl^ subjects did not stimulate DRl-restricted, 
trinitrophenyl-specific CD4^ T lymphocyte clones. 
Such cells were also unable to stimulate DRl-spe- 
cific alloreactive T cell clones. In general, nonstim- 
ulators were of the extended haplotype DR1,B14 
and/or expressed the HLA-associated gene defect 
for nonclassical 2 1-hydroxylase deficiency. Analysis 
of DR molecules from nonstimulators led to identi- 
fication of a class II MHC molecule with unique 
biochemical properties. Although immunoprecip- 
itated with anti-DR monoclonal antibodies, it mi- 
grated under reducing conditions in two-dimen- 
sional gels as a single molecular species of —50 
kDa. Surprisingly, several DRl^ individuals hetero- 
zygous for variant and wild-type DRl genes dis- 
played a nonstimulatory phenotype. Because an 
initial genomic analysis revealed a Bglil restriction 
polymorphism in the 3 -untranslated region of the 
DRA gene that was invariably associated with the 
biochemical abnormality, cDNAs for the DRA and 
DRB genes were amplified, cloned, and sequenced. 
Analysis of the DRA gene from variant DRl cell lines 
revealed only a silent nucleotide substitution in 
codon 77. In addition to a silent mutation in codon 
78, the DRB gene had coding mutations in two ad- 
jacent codons, 85 and 86, in which GTT(Val) 
GGT(Gly) was replaced by GCT(Ala) GTG(Val). 
From studies from a molecular model of class II 
MHC proteins, these variant residues are hypothe- 
sized to lie at the end of the DRB a-helix thought to 
be involved in antigen binding. In addition to af- 
fecting T cell recognition, these mutations may alter 
the avidity of the DRA and DRB polypeptides for 
Continued 
439 
