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Diversity of Class I HLA Molecules: Functional and
Evolutionary Interactions with T Cells
P. PARHAM,* R.J. BENJAMIN,*B.P. CHEN,*C. CLAYBERGER,tP.D. ENNIS,*A.M: KRENSKY,t
D.A. LAWLOR,*D.R. LITTMAN,$w A.M. NORMENT,:~H.T. ORR,**
R.D. SALTER,* AND J. ZEMMOUR*
*Department of Cell Biology and tDepartment of Pediatrics, Stanford University, Stanford, California 94305;
SDepartment of Microbiology and Immunology and w
Hughes Medical Institute, University of California
at San Francisco, California 94143; **Department of Laboratory Medicine and Pathology and the
Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455
In the past 5 years, the nature of major histocompatibility complex (MHC) glycoproteins, as well as the
mechanism by which they restrict antigen recognition
by T lymphocytes, has been clarified. These molecules
bind peptides within intracellular membrane compartments, forming complexes that are then brought to the
cell surface to be surveilled by antigen receptors of
circulating T lymphocytes. When a threshold of
molecular complementarity between T-cell receptor
(TCR) and an MHC/peptide complex occurs, the T
cell becomes activated, thus initiating an immune response. Through exploitation of distinct but poorly
understood pathways of intracellular traffic, class I
MHC molecules present peptides derived from endogenous proteins, whereas class II MHC molecules present peptides derived from exogenously synthesized
proteins. It is likely that these processes of peptide
presentation are central to thymic selection of the Tcell repertoire and to initiation, in the periphery, of
T-cell responses to foreign antigens (for review, see
Davis and Bjorkman 1988; Hedrick 1988; Marrack and
Kappler 1988; Long and Jacobson 1989; Townsend and
Bodmer 1989).
The essence of T-cell recognition is thus the interaction of an MHC molecule with a TCR. A consequence
of this functional interdependence is that genes encoding these two diverse families of genes evolve in concert. The evolutionary origins of the interaction probably lie with a nonvariable, nonimmunological ligandreceptor system that was subsequently adapted to immunological purpose. This argues for a fundamental,
intrinsic affinity between all functioning TCRs and
MHC molecules of a species, a concept originally developed for immunoglobulin and MHC molecules
(Jerne 1971). The similarities of class I and class II
MHC molecules provide compelling evidence for their
derivation from a common ancestral molecule that also
interacted with TCRs. That class I and class II molecules still interact with receptors derived from a common pool confirms this supposition and reveals that
genes encoding functional class I and class II genes do
not evolve in an independent fashion (Rupp et al.
1985).
Although MHC molecules and TCRs both interact
with a diversity of antigenic peptides, their adaptations
to the challenge of antigenic diversity are quite distinct.
Somatic gene rearrangements permit large numbers of
different and specific TCRs, each restricted to a small
number of cells, to be expressed. In contrast, the genes
encoding MHC molecules are somatically stable and
constitutively expressed by all cells of a given type.
Diversity in antigen presentation is, in part, accomplished by codominant expression of the products of
multiple class I and class II MHC genes. A second
critical factor is the relative degeneracy, a lack of
specificity, of the peptide-combining site of MHC molecules (Guillet et al. 1986). The kinetics of MHC peptide interactions are quite different from those observed for most antigen-antibody interactions (Buus et
al. 1987) and suggest that the MHC molecule may in
fact fold around the peptide in a manner that is, ironically, reminiscent of theories of antigenic instruction
proposed for immunoglobulins (Pauling 1940). The
unique feature of MHC genes lies not with diversity
within the individual but with diversity in populations
and species. Certain MHC genes are characterized by
large numbers of alleles and the absence of a dominant
"wild-type allele," features rarely found in eukaryotic
genes. The class I MHC genes in human populations
(HLA-A, B, C) have been particularly well characterized and provide examples par excellence of polymorphic MHC genes. Currently, some 20 alleles at the
A locus, 40 at the B locus, and 11 at the C locus have
been defined; however, this is a minimal estimate, since
the number of alleles continues to increase significantly
as more sensitive methods are brought to the analysis
(Dupont 1989).
Structural Analysis of Class I HLA-A,B,C
Polymorphism
To understand the nature of the diversity, its generation, its contribution to the functional interactions of
class I H L A molecules, and its evolutionary significance for the survival of populations requires structures
for many alleles and their products. This effort was
ColdSpringHarborSymposiaon QuantitativeBiology,VolumeLIV.9 1989 Cold Spring Harbor LaboratoryPress 0-87969-057-7/89$1.00
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PARHAM ET AL.
initiated in the early 1970s, and the first partial amino
acid sequences were presented at the previous "immunological" symposium in this series (Strominger et al.
1976). Since that time, there has been a continuing
acquisition of primary structural information using first
protein chemistry and then cloning of eDNA and genes
(for review, see Lew et al. 1986; Strachan 1987; Parham et al. 1989b).
Patterns of HLA-A,B,C Polymorphism
Human class I molecules consist of a polymorphic
MHC-encoded heavy chain, an invariant /32-microglobulin (/32-m), and a variable, and as yet poorly
characterized, bound peptide (Bjorkman et al. 1987a).
In the H L A gene complex there are 17-20 class I
heavy-chain genes, including various pseudogenes and
gene fragments. Only six genes (HLA-A, B, C, E, F,
and G) are known to make/3z-m-associated products,
and of these only HLA-A,B,C show the broad tissue
distribution characteristic of antigen-presenting molecules (Koller et al. 1989).
Over 50 HLA-A,B,C sequences have been determined and provide the data for analysis of diversity (for
review, see Parham et al. 1988, 1989a). Alleles of a
locus differ by 1-100 nucleotide substitutions that, although found throughout the sequence, are concentrated in exons 2 and 3, which encode the peptidebinding domains, a 1 and a 2. Alleles of different loci are
readily distinguished by 62 locus-specific substitutions,
mostly in exons 4-8 encoding the a 3, transmembrane,
and cytoplasmic domains. Many positions in the amino
acid sequence, perhaps as many as one third of the
total, exhibit polymorphism, but at most of the variable
positions there are only two or three different amino
acids, and one residue is usually dominant. Twenty
residues show high variability, exhibiting up to eight
different amino acids, and these are at positions in the
three-dimensional structure where the amino acid side
chain is hypothesized to contact bound peptide or TCR
(Bjorkman et al. 1987b; Parham et al. 1988). However,
such positions are not all variable, an effect that is more
vividly illustrated by class II H L A - D R molecules in
which the half of the peptide-binding groove that is
contributed by the a chain is totally invariant. The
helical region of the a 1 domain shows the greatest
variation and, by comparison, the helix of the a 2 domain is quite conserved. In contrast, the/3 strands of
the a 2 domain have greater variability than the corresponding strands of the al domain. Alignment and
comparison of allelic sequences reveal a characteristic
patchwork motif, due to the sharing of small clusters of
substitutions in many different combinations (Fig. 1).
This is most pronounced for exons 2 and 3 encoding the
a 1 and ot2 domains of HLA-B molecules (Fig. 1A).
Our general strategy has been to isolate and sequence genomic or eDNA clones encoding complete
HLA-A,B heavy chains. With recent refinements in
technology (Saiki et al. 1988), it has become feasible to
use the polymerase chain reaction (PCR) to obtain
full-length eDNA clones encoding HLA-A, B, C genes.
The strategy taken was to design oligonucleotide primers from sequences in the 5' and 3' untranslated regions
that flank the coding sequence and are relatively conserved between HLA-A, B, C alleles (Fig. 2A). These
primers were used to amplify single-stranded eDNA,
synthesized from total cellular R N A prepared from
human B-cell lines of known H L A type. The amplified
product, which contains a mixture of alleles, is subcloned into M13 sequencing vectors using SalI and
HindlII sites incorporated into the amplification primers. Analysis of over 100 randomly picked clones from
such "class I libraries" has shown them all to have
full-length class I H L A sequences.
Because the goal is to obtain clones faithfully encoding HLA-A,B,C molecules, which can then be expressed and used in functional studies, the errors arising from PCR amplification become of critical importance. To assess the nature and frequency of errors, an
initial experiment was designed to study H L A - A , B
genes for which considerable information was already
available but for which a complete determination of
their sequences would prove useful. These are the
HLA-A2 and HLA-B7 genes of the JY cell line, the
products of which may represent the most extensively
studied MHC molecules. Despite the wealth of information, including the crystallographic structure of
HLA-A2 (Bjorkman et al. 1987a,b), the sequences of
these proteins and the genes encoding them had yet to
be completed (Orr et ai. 1979a,b; Sood et al. 1985).
Clones were randomly picked from a PCR-amplified
JY class I library, and on the basis of preliminary
sequencing, were identified as encoding either HLAA2 or HLA-B7. Ten clones for each gene, five from
each orientation, were completely sequenced using
four pairs of oligonucleotide primers (Fig. 2).
Compilation of the ten HLA-A2 sequences gave a
clear consensus identical to the coding sequence of the
HLA-A2 gene obtained from the LCL-721 cell line
(Koller and Orr 1985). Sequences of four of the ten
eDNA clones are identical to this consensus and thus
represent faithful copies of the gene. Three of the
remaining clones have single nucleotide substitutions,
and one clone has three substitutions; however, none of
these "errors" were found in more than one clone
(Table 1). Two clones have hybrid sequences with a 5'
part derived from HLA-A2 and a 3' part from HLAB7. Although appearing as recombinations, these
clones presumably result from premature termination
of the polymerase on one cycle, with subsequent reannealing of the unfinished A2 strand to a B7 template
and completion of the strand in another cycle. The
"recombination" points in the two clones are different,
and one clone has an additional point substitution.
The pattern to emerge from the HLA-B7 analysis is
similar to that observed for HLA-A2. Three clones
have sequences identical to the consensus obtained
from all ten, with four clones having single nonidentical
substitutions, one a single base-pair deletion, and two
being "recombinants" with a 5' B7 sequence and a 3'
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A
exon
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exon
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Figure 1. Patchworkmotifin HLA-A, B sequences. The sequence relationshipsbetweenrepresentativeHLA-B (panel A) and
HLA-A (panelB) allelesare shown.Identicaland closelyrelatedpatternsof substitutionare representedby similarshading.Two
ChLA-A alleles,A108 and Ch25, are includedwith the H L A - A alleles. The Ch25 cDNA lacksexons6 and 7 and part of exon 5.
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532
PARHAM ET AL.
Table 1. Analysis of PCR Errors Using B7 and A2 from the JY Cell Line
Allele
Clone
Differences
fromconsensus
A2
1
2
6
8
9
20
23
25
28
29
685; G > A
none
none
none
none
361; A > G, "recombination" with B7; 561-602, 652; A > G
310; A > G , 538; T > C , 965; T > C
recombination with B7; 917-932, 1050; T > C
117; C > T
293; A > G
B7
3
4
7
10
12
21
22
24
26
27
none
none
188; A > G
218; C > T
111; C > T, "recombination" with A2; 874-909
recombination with A2, 653-734, 996; G > A
914; C > T
278; deletion
none
785; A > G
All misincorporationsare transitions.
A2 sequence. One recombinant has an additional point
substitution (Table 1). In contrast to the results with
HLA-A2, the consensus sequence obtained from the
HLA-B7 clones is not identical to the e D N A sequence
reported by Sood et al. (1985), since there are a total of
26 substitutions scattered throughout the coding sequence (Fig. 2B). It is likely that many, if not all, of the
substitutions in the e D N A sequence are the result of
sequencing errors. Comparison with over 50 HLAA, B, C alleles shows that 22 of the 26 substitutions that
distinguish the two B7 sequences are unique to the
e D N A sequence of Sood et al. (1985), a number of
unique substitutions significantly exceeding that found
in any other allele. Also unusual is the fact that a
majority of these substitutions (18 of 26) are silent, and
only two of the seven amino acid differences are found
in the extracellular domains. Comparison of other pairs
of alleles always gives a predominance of replacement
over silent changes that are mostly found in exons
encoding the extracellular domains (Parham et al.
1989a). Also of relevance is comparison of the B7
sequences with HLA-Bw42, a molecule found in black
populations and serologically related to HLA-B7. Previous comparison of the protein sequences indicated
that HLA-Bw42 resulted from homologous recombination between HLA-B7 and -B8 genes (Parham et al.
1988). This was supported by nucleotide identity in
exons 3 - 8 of the HLA-B8 and -Bw42 genes. It is now
confirmed by the identity in exons 1 and 2 between
HLA-Bw42 and the PCR-derived sequence for HLAB7 (Fig. 2B). Errors have been found in another sequence by this group (Srivastava et al. 1989), and in the
case of HLA-B7, an accumulation of erroneous silent
substitutions might have resulted from reliance on the
previously published protein sequence (Orr et al.
1979b) for interpretation of the sequencing gels.
k.
HLA-5P2
2S
I
I
I
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EXON 1
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aN
EXON 2
B
4S
3S
-"
6S
24N
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EXON 4
EXON 3
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A2.1
Aw24
Bw58
B27
Cwl
Cw2,1
Cw3
EXON 5
Hind
-CA-CCACCG .......
T ...........
-CA-CCACCG--"T
-CA-CCACC . . . . . . .
T .......
-CA-CCACC . . . . . . . .
T. . . . . .
TCA-CCACC ......
--T ............
TCA-CCACC-----G--T
. . . . . .
TCA-CCACC ........
T. . . . . . . . . .
k
v
5' Untranslated Region
Figure 2. (Continued on facing page.)
TGAT .....
----GGCAAGA-'T
TGAT. . . . .
GGCAAGA-TT
TGA . . . .
"---T-------GGCAAGA~T
TGA----T
TGCA~A~
TGA'
~
~T
TGCAGGA" TT
TGA
T
TGCAGGA*TT
TGA'T. . . . .
TGCAGGA*TT
TGA"
T ......
T G C A G G A ~T T
TGA"
T. . . .
TGCAGGA~
-GATG
GATG
"ATG
~
~
"ATG
"ATG
A
t
3 ' OH-CTGTCGACGGAACACTCCCTGACTCTTTCGAACCCC,-S
5'OB-GGGCGTCGACGGACTCAGAATCTCCCCAGACGCCGAG-3'OH
A
V
Coding Region
1098 - 1101 bp
/
V
3' Untranslated Region
' OH
L_
HLA-3P2
EXONS 6-8
Ill
I
I
12
6N
HLA-3P2
A2. I
A3
AW24
Bw58
B44
B7
CWl
Cw2. I
Cw3
Downloaded from symposium.cshlp.org on May 11, 2016 - Published by Cold Spring Harbor Laboratory Press
C
EXON
1
B7
PCR
Bw42
B8
B7 aDNA
EXON
30
ATGCTGGTCATGGCGCCCCGAACCGTCCTCCTGCTGCTCTCGGCGGCCCTGGCCCTGACCGAGACCTGGGCCG
. 73
g
2
30
B7 PCR
Bw42
B8
B7 eDNA
B7 PCR
Bw42
B8
B7 cDNA
B7 PCR
Bw42
B8
B7 cDNA
EXON
60
.........................................................................
.........................................................................
...........................................
g. . . ~
.
.. . - . . . . . . . . . . . . . . . . .
60
i00
GC TCCC~A~TCCATGAGGTATTTC
TACACC TCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCTCAGTGGGCTACGTGGACGACACCCAGTTCGT
....................................................................................................
.......................
g ..... g--a ..........................................................
-g ..........................
a ......
NNNNNN
g ........
...........................................................
130
160
GAGGT T~GACAGCGACGC~GCGAGT~cGAGAG~GAGCCGCGGGCGcCGTGGATAGAGcit~GAGGGG~CGGAGTATTGGGAC~GGAACACACAGATCTAC
....................................................................................................
200
..................................................................................................
....................................................................................................
230
260
AAGGCCCAGGCACAGACTGACCGAGAGAGCCTGCGGAACCTGCGCGGCTiCTACAACCAGAGCGAGGCCG
......................................................................
---a--a-ca
............................................................
........
a ...........................................................
t-
270
t-
3
30
60
GGTCTCACACCCTCCAGAGCATGTACGGCTGCGACGTGGGGCCGGACGG~CcTCCTcCGCGGGCATGiCCAGTACGCCTAcGACG~C~xGGATTACAT
....................................................................
a ...............................
....................................................................
a ...............................
.............
t ..............
t .......................................................................
i00
B7 PCR
Bw42
B8
B7 cDNA
130
160
~G~TGAACGAGGA~TGCG~T~TGGA~G~GCGGAC~CGCGGcT~GAT~ACCCAGCGCAAGTGGG~GCGG~CCGTGAGGCGGJ~CAGCGGAGA
.................................
g .................................................
t ..........
.................................
g .................................................
t ..........
..........................................
g--t--g ...................................................
200
B7 PCR
Bw42
B8
B7 cDNA
230
B7 PCR
Bw42
B8
B7 cDNA
EXON
276
GCCTACCTGGAGGGCGAGTGCGTGG~T~CTCCGC~AT~I2CTGGI~I~AiCGGGA~C~.Cl~I~CTGGI~CGCGCTG
...............
ac ............................................
c ............
...............
ac ............................................
c ............
...................................
a ........................................
*
gg-
4
B7 PCR
Bw42
B8
B7 cDNA
30
60
A~A~GA~A~A~GTGA~A~A~CATcTcTGACcATGAGGCCAcccTGAGGTGcTGGGcccTGGGTTTcTACccTGCGGAGATcAcACTGAc
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .c. . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .c. . . .
....... t ..............................................................
t ........
9
B7 PCR
Bw42
B8
B7 cDNA
.
130
.
.
160
i00
t ....................
.
.
.
200
CTGGCAGCGGGATGGCGAGGACC~%A~TC`~GAcACTG~CTTGTGGAGACC~ACCAGCi~GAGATAG~CCTTCCA~`i~GTGGGC~CTG~GGTGGTG
....................................................................................................
.............................................
- ......................................................
...................................................................................................
c
*
9
B7 PCR
Bw42
B8
EXON
260
gaG--gac---
.
230
260
276
CCTTCTGGAGA~AGCAGAGATACACATGCCATGTACAGCATGAGGGGCTGCCGI~.GCCCCTCACCCTGAGAT~,G
............................................................................
............................................................................
5
B7 PCR
Bw42
B8
B7 cDNA
30
60
AG~GTCTTC~CAGTCCACCGTCCC~ATCGTGGGCATTGTTGCTGGCCTGGCT~TCCTAGC~TTGTGGTCATCGGAGCTGTGGTCGCTGCTGTGATGTG
....................................................................................................
....................................................................................................
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .t. . . . . . . . . . . . . . . . .
c . . . . .c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i00
117
B7 PCR
Bw42
B8
B7 cDNA
EXON
6
B7 PCR
Bw42
B8
B7 cDNA
Tg~GAGGA~AGTTCI~
............
c ....
............
c ....
.................
EXON
7
9
GTGGAAAAGGAGGGAGC
TACTCTCAGGCTGC
33
GCAGCGACAGTGCCCAGGGCTCTGATGTGTC
.............................................................................
.............................................................................
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .a. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
TCTCACAC~
44
T TGA
Figure 2. HLA class I cDNA amplification and sequencing strategy. (A) HLA class I cDNA PCR product showing PCR primers
(large arrows), sequencing primers (small arrows), and exon boundaries (vertical lines). Arrowheads are at the 3' OH end of each
oligonucleotide primer and point in the direction of polymerase extension. Oligonucleotides 5P2, 2S, 3S, 4S, and 6S have sense
strand sequence; 3P2, 6N, 4N, 3N, and 2N have RNA strand sequence. (B) Design of HLA class I cDNA PCR primers and
alignment with representative HLA-A, B, and C sequences. Dashes indicate HLA-A,B,C nucleotide positions identical to
HLA-5P2 and complementary to HLA-3P2. (O) Single-base gaps introduced for improved alignment. (C) Consensus nucleotidecoding sequence of 10 PCR clones derived from HLA-B7 mRNA (B7-PCR) is compared to the exonic sequences of HLA-Bw42
and HLA-B8 genomic sequences and the B7 cDNA sequence of Sood et al. (1985). Dashes indicate identity. (*) Substitutions
found in the B7 cDNA sequence and no other HLA-B locus allele. Coding substitutions are double underlined.
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534
PARHAM ET AL.
In conclusion, this PCR-based method gives authentic full-length class I cDNA clones with a frequency of
about 30%. By comparison with conventional methods
for isolation of class I cDNA, the advantages are that a
full-length product is always obtained and that time is
saved in the preparation and screening of libraries. One
disadvantage is that multiple complete sequences must
be obtained in order to determine a consensus sequence and select a faithful clone. Another is the rather
high number of recombinant clones that could potentially confuse the identification of clones for previously
uncharacterized alleles. Further optimization of conditions may enable the frequency of recombinant products to be reduced. Point substitutions pose less of a
problem, and their frequency was low, a total of 15, all
transitions being found in the 21,960 bp sequenced.
Generation of HLA.A,B,C Polymorphism
Evolutionary change results from a two-step process.
First are the mutational events that create variation: in
this case, new HLA-A,B,C alleles. Second are the
selective events that can either eliminate new alleles or
increase their frequency in the population. These include positive selection, negative or purifying selection,
and neutral change through genetic drift. The conclusion to be drawn from comparison of sequences is that
the extraordinary polymorphism of HLA-A, B, C genes
does not stem from an unusually high rate of mutation
or from specific targeting of special mutagenetic mechanisms. The difference between MHC and other genes
is in selection for increased diversity that acts upon
genes encoding antigen-presenting molecules (Jaulin et
al. 1985; N'Guyen et al. 1985; Hughes and Nei 1988;
Parham et al. 1989a). It is possible, as discussed below,
that positive selection may also act to reduce polymorphism at MHC loci.
The evidence that the rate of mutation in HLAA, B, C genes is comparable to that in other genes is as
follows. No product of a new HLA-A,B,C mutation-where a child has a serological type not found in either
parent--has ever been detected. If these genes were
hypermutable, one would expect, in the course of the
many H L A typings of families, that such novel types
would have been found. Homo sapiens and Pan troglodytes (chimpanzees) are species that are estimated to
have diverged between 3.7 and 7.7 million years ago
(Sibley and Ahlquist 1984; Hasegawa et al. 1987).
Comparison of HLA-A, B, C alleles with their chimpanzee homologs (ChLA-A, B, C) thus provides an assessment of the new mutations that have accumulated
in each species during at least 3 million years of separate evolution. The result of such comparison is that (1)
a majority of the polymorphisms, including silent substitutions, are common to alleles of the two species, (2)
individual alleles of one species are more similar to
particular alleles in the other species than to the majority of alleles from either species (Fig. 3), and (3) no
species-specific features have been fixed in the alleles of
either species, although locus-specific features are
shared by the corresponding alleles of both species.
These observations show that much of the diversity in
contemporary HLA-A, B, C genes was already present
in the ancestral primate species that gave rise to
humans and chimpanzees and was subsequently passed
on to both species (Lawlor et al. 1988; Mayer et al.
1988). Since that time, the numbers of new polymorphisms accumulated, as most accurately assessed by silent
substitutions, seem comparable to those found in other
genes. These conclusions are reinforced by analysis of
low-frequency subtypes of HLA-A,B molecules that
are specific to particular human populations or races
and represent newer alleles that originated in Homo
sapiens. These alleles commonly differ from the more
frequent subtype by a single or a small number of
substitutions that can be accounted for by a single
mutagenetic event. An example shown in Figure 2C is
HLA-Bw42, which is specifically found in Africans and
their descendants and results from an intragenic recombination between HLA-B7 and -B8 alleles.
Comparison of related alleles also permits an assessment of the nature of genetic events that produce new
HLA-A, B, C alleles. The ultimate origin of all variation is, of course, in point mutations that occur in
individual alleles; these are subsequently assorted into
many different combinations by various recombinational mechanisms. In addition to single recombinations, as postulated in the formation of HLA-Bw42,
there is considerable evidence for events that convert a
short segment of sequence from one allele or gene into
the homologous sequence found in another.
A common feature of eukaryotic genes is homogeneity in sequence within species but divergence between species. For example, although /32-m is nonpolymorphic within humans, there are 96 nucleotide
substitutions in the coding region between human and
murine fl2-m (Suggs et al. 1981; Parnes and Seidman
1982). Although some of these species differences may
be adaptive, it is also likely that others, including the 51
silent substitutions, are neutral changes resulting from
genetic drift. The question then becomes why greater
amounts of neutral polymorphism in protein like/32-m
are not found more frequently within species. This
suggests that there are mechanisms that, in the absence
of selection for diversity, will tend to homogenize alleles. The accumulating evidence indicates that nonreciprocal gene conversion events are a common, and
possibly a general, feature of mammalian genes
(Smithies and Powers 1986; Dover and Strachan 1987)
that can act to homogenize or diversify a set of homologous sequences. In most genes, it may act as a mechanism for homogenization, as seems true for the 3' exons
(4-8) of HLA-A, B, C genes (Parham et al. 1989a). For
example, within exon 4 encoding the a 3 domain, there
are 13 silent (and 4 coding) substitutions that are invariant between alleles of a locus but differ between the
HLA-A, B, C loci. That such presumably neutral substitutions are fixed in a family of alleles with highly divergent 5' sequences is most readily explained in terms
Downloaded from symposium.cshlp.org on May 11, 2016 - Published by Cold Spring Harbor Laboratory Press
Bovine
At
A11e
A3.1
A30
Ch25
AI08
A126
A2.1
A69
A68.2
A68.1
A
A29
A32
A31
A33
Aw24
r[~
5.4LCL721"~
JY8
/ AR
12,
5.4LBF
5.4BB
/
.J
>E
[~
Bw42
B7
B8
Bw65
~
B14
Bw47
B27.1
B44.2
B44.1
ChLA-B1
Ch39
Bw58
B
B51
Bw52
B49
B13
. ~
B40*
BW60
Bw41
B18
..~ C
hhL74-B2
C
Ch18
Bw46
Cw2.1
Cw3
Ch ,
Cwl 1
Cwl
"~t
C
9
) G
C
h28
HLA-F
F
Figure 3. Tree based on parsimony depicting the relationships between human and chimpanzee class I alleles of the MHC.
Analysis of the complete nucleotide sequence for the coding region of 55 alleles was initially performed by the heuristic methods
of the PAUP (Swofford) program. Gaps were inserted in exon 5 as per Needleman and Wunsch (1970) to ensure optimal
alignment. This inexact search produced 32 most parsimonious trees with a length of 1256. The overall consistency index was
0.536. Discrepant branching orders were resolved by using more rigorous algorithms, branch and bound and aUtrees, with distinct
subsets of the alleles. Data were considered unordered, and bovine sequence BL3-6 served as the outgroup to root the tree.
Sequences and primary references are as in Mayer et al. (1988), Parham et al. (1989a), Trapani et al. (1989), and J. Zemmour et
al. (in prep.). We thank Bo Dupont and Soo Young Yang for unpublished results.
535
Downloaded from symposium.cshlp.org on May 11, 2016 - Published by Cold Spring Harbor Laboratory Press
536
PARHAM ET AL.
of homogenization by localized, homologous, nonreciprocal conversions between alleles.
In contrast, the 5' exons (1, 2, and 3) exhibit a
characteristic patchwork pattern of substitution that is
indicative of diversification again through the agency of
conversions and/or double recombinations (Fig. 1).
The evidence for positive selection for diversity in the
5' exons is the relative frequency of replacement to
silent substitutions, which is much greater than would
be expected if the substitutions were random (Jaulin et
al. 1985; N'Guyen et al. 1985; Parham et al. 1989a).
This difference is particularly striking if one differentiates between functional and nonfunctional positions
of diversity, as assigned from interpretation of the crystallographic structure of HLA-A2 (Bjorkman et al.
1987b; Hughes and Nei 1988). Furthermore, the frequency of the dinucleotide CpG within the 5' exons is
much greater than in the 3' exons and most other
eukaryotic genes. In the absence of sequence-specific
selection, CpG sequences tend to mutate through
specific methylation and deamination; thus, their preservation is also a marker of positive selection (Tykocinski and Max 1984).
In addition to conversion between alleles of the
HLA-A, B, or C loci, there is also evidence for conversions between alleles of different class I MHC loci; a
particularly striking example is provided by HLABw46, which has an a 1 helix donated by the linked
HLA-Cwll gene (Parham et al. 1989a,b). In contrast
to murine class I MHC genes (Nathenson et al. 1986),
where it has been a major source of diversification, this
mechanism has been a minor contributor to contemporary HLA-A,B,C diversity (Parham et al. 1988,
1989b). A consequence of the predominance of interallelic rather than intergenic conversion is that considerable divergence in both the conserved and polymorphic parts of the products of the HLA-A, B, and C
loci has occurred.
The divergence of structure in products of the HLAA, B, C loci may have functional implications. Differences in the a 1 and a 2 contribute specialization in the
types of peptide bound, whereas o/3 differences affect
the affinity for CD8. Extensive locus-specific differences in the transmembrane and cytoplasmic domains
may determine interactions with membrane and cytoplasmic components, perhaps providing signals that distinguish the intracellular trafficking or recycling of the
products of the different loci. These are critical and
outstanding questions to be addressed.
Do T-cell Responses Determine the Rise and Fall
of MHC Genes?
An individual's potential for T-cell responses is determined by the repertoire of peptides that can be
presented and the repertoire of TCRs that can respond.
The number of different MHC molecules expressed by
an individual has effects on both parameters.
Greater diversity in MHC molecules, and in particular of their peptide-binding sites, enables larger num-
bers of peptides to be presented; it is this effect that
creates the positive evolutionary selection for MHC
polymorphism and diversity. Multiple homologous,
codominantly expressed loci and extensive polymorphism at these loci both contribute to increased capacity
for antigen presentation.
In contrast to this simple effect upon antigen presentation, the influence of increased numbers of MHC
molecules on the T-cell repertoire is complex, involving
both positive and negative components (Marrack and
Kappler 1988). The receptors of immature T cells are
stringently selected on the basis of their interactions
with MHC/peptide complexes encountered in the
thymus. Although this process is poorly understood, it
is thought that cells having either too weak or too
strong an interaction with any self-MHC molecule are
eliminated, whereas receptors of positively selected
cells are of an intermediate affinity. Increasing the
diversity of MHC molecules in the thymus can therefore lead to diminution of the T-cell repertoire, a thesis
supported by the high frequency of alloreactive cells in
mature T-cell populations and by studies with transgenic mice (Kappler et al. 1987). Each additional class
I or class II molecule expressed in the thymus results in
the deletion of T cells that are potentially autoreactive
against that MHC molecule, but might otherwise have
been positively selected for interaction with another
MHC molecule. Thus, the necessity for self-tolerance
means that each individual MHC molecule has the
negative effect of reducing the repertoire of receptors
restricted to all other MHC molecules (Matzinger et al.
1984) (Fig. 4). That class I and class II MHC-restricted
T cells appear to draw their receptors from the same
pool (Rupp et al. 1985) suggests there will be evolutionary interplay between the numbers of expressed
class I and class II MHC products and the repertoires of
class-I-restricted (mostly cytotoxic) and class-II-restricted (mostly helper) T cells. Compromise between the
positive effects on antigen presentation and the negative effects on T-cell repertoire may explain why the
number of class I and class II loci expressed by any
individual in all species examined is limited. This burden of tolerance is not felt at the level of populations,
where the advantage of increased antigen presentation
can lead to greater and greater polymorphism in MHC
loci.
The relationships between T cells that are positively
or negatively selected by different MHC molecules suggest how evolutionary selection for advantageous immune responses, provided by particular MHC alleles or
loci, could result in the reduction of polymorphism,
expression, and functionality of other MHC alleles and
loci. Thus, as the antigenic environment changes, there
will be selection for certain MHC alleles and loci and
against others. The HLA-AR and HLA-C loci discussed in the next section both have features suggesting
that they were once selected for antigen presentation
but are now in varying states of decline. The alternative
to somatic deletion of the T-cell clone can thus become
evolutionary deletion of the MHC molecule. An exam-
Downloaded from symposium.cshlp.org on May 11, 2016 - Published by Cold Spring Harbor Laboratory Press
HLA-A,B,C: STRUCTURE, F U N C T I O N , AND E V O L U T I O N
537
HLA-AR: A Defunct Class I Antigen-presenting Gene
ell
Receptor Pool
Figure 4. Cartoon showing the total TCR pool and the subsets
of receptors affected by MHC molecules from two different
loci, A and B. For each MHC locus there is a set of receptors
that are negatively selected, and, as a consequence, the cells
expressing those receptors are deleted. In addition, there is a
set of receptors that are positively selected, and cells expressing those receptors enter the mature T-cell pool. Overlap
between the receptors that would be positively selected by one
MHC molecule and negatively selected by the other is indicated by shading. Since negative selection is dominant over
positive selection, both these populations would be deleted in
individuals expressing MHC A and B. If potential T-cell responses using receptors in either shaded area were particularly
advantageous, there would be selection for elimination of the
MHC locus responsible for the negative selection. Identical
arguments can be applied to individual alleles of either the
same or different loci.
pie of such a process in action is perhaps provided by
the striking deletion of clones expressing V~ 17 T-cell
receptors by I-E molecules in mice (Kappler et al.
1987). Elimination of I-E expression in certain H-2
haplotypes, and by a variety of different mutations
(Mathis et al. 1983), releases V~17-containing receptors from deletion and makes them available for positive thymic selection by other MHC molecules. The
differences in the numbers and polymorphism of expressed class I and class II loci in different species and
in different strains of mice provide evidence for the
dynamic nature of the MHC, adapting to the demands
of T-cell immunity imposed by everchanging antigenic
environments (Flavell et al. 1986).
Balancing of the repertoires selected by different
MHC molecules means that class I molecules will influence the class-II-restricted T-cell repertoire and vice
versa. Selection upon such interplay could result in
linkage disequilibrium, as frequently observed between
particular H L A alleles (Dupont 1989); it may also be
responsible for the Syrian hamster having monomorphic class I loci and polymorphic class II loci (Streilein
1987). In an environment such as that inhabited by the
Syrian hamster, in which viruses appear not to be a
problem, the reduction in class I polymorphism may be
in part a consequence of selection for greater diversity
in class-II-restricted responses. Evidence for the changing fortunes of MHC genes can also be found in the
H L A region.
H L A - A R (also called 12.4 and 5.4p) is closely linked
on chromosome 6 to H L A - A (Malissen et al. 1982;
Pontarotti et al. 1988; Koller et al. 1989), and the
sequences of H L A - A R alleles are most closely related
to H L A - A alleles (Fig. 3). For example, H L A - A R
alleles show identity to H L A - A at 47 of 62 locusspecific nucleotides that have been defined on the basis
of H L A - A , B, C sequences. It is therefore likely that
H L A - A and H L A - A R resulted from duplication of an
ancestral gene, and the descendant of that duplication
is now fixed in the human population. The remarkable
fact is that whereas all contemporary H L A - A alleles
are expressed and believed to function in antigen presentation, all H L A - A R alleles are pseudogenes. Six
H L A - A R alleles have been sequenced, and they all
have a phenylalanine encoded at position 164 instead of
the cysteine that forms the essential disulfide bond of
the a 2 domain (J. Zemmour et al., in prep.). In addition, five of the six alleles have a single nucleotide
deletion at codon 227 in the a 3 domain. This causes a
frameshift and premature termination at codon 272. If
it were not for these deleterious changes, the sequences
would appear to be those of functional class I H L A
molecules, indicating that they were previously under
selection for antigen presentation. For example, polymorphic substitutions are predominantly found in
exons 2 and 3. Thus, it is likely that the duplication that
led to H L A - A and H L A - A R was of a functioning allele
and that in the initial period of its history, H L A - A R
was a functioning locus. What sequence of events can
have led to its downfall?
The occurrence of identical deleterious mutations in
H L A - A R alleles by convergent evolution is improbable. It is more likely that each of the two mutations
was produced once and that fixation has occurred by
introduction of the mutation into other alleles through
nonreciprocal conversion and by propagation of the
mutant alleles. The fact that a deleterious mutation
became fixed suggests that a historic change in selection
took place, with the result that there was no longer any
advantage to be obtained from the presentation of
antigens by H L A - A R molecules, and that subsequently
there may have been positive selection for their elimination. Such positive selection could derive from advantageous immune responses mediated by T cells that
could only survive thymic selection in the absence of
H L A - A R proteins. The causative change in selection
upon the H L A - A R locus could have resulted from
alterations in the antigenic environment, from the development of new alleles at other MHC loci that more
effectively presented critical antigens, from changes in
the TCR gene repertoire, or from some combination of
these factors.
Once the positive functional selection on a class I
locus like H L A - A R has been removed, it will begin to
decline. Point mutations slowly accumulate; the action
of conversion events, in the absence of selection for
diversity, will be to homogenize and reduce poly-
Downloaded from symposium.cshlp.org on May 11, 2016 - Published by Cold Spring Harbor Laboratory Press
538
PARHAM ET AL.
60
70
80
PEYWDRETQI SKTNTQTDRESLRNLRGY
B7 ...... N---Y-AQA.............
B8 . . . . . . -~---~--------;--7-;~SB I 3 .........
818 ...... N .......... u ..........
Substitutions
}
HLA-B
36
HLA-A
28
B27 . 1 .......... C-AKA ..... D--T-LRBW58 ..... G--RNM-A SA--Y--N-- I ALR-
A1 .P .E .Y .W .D .Q .E .T .R .N V K AM H.S.Q.T.D.R.V.D.L G TA LN R.G.Y. . . . . t
A2 - .... G---K ....... H ..........
A3 . . . . . . . . . . . . .
Q ..............
A I 0 ..... R N ............
A N .......
A24
A32
..... E--GK .........
...................
Cwl
Cw2
PEYWDRETQKYKRQAQTDRVSLRKLRGY
.......................
N ....
....................
N .......
c~3 . . . . . . . . . . . .
BeWoCI
CI.9
CI.IO
5.4-LCLI
5 9 4-LCL2
P. . . . . . . . . .
]
N. . . .
H/A-C
.q
HLA-AR
4
................
A---N .......
......... N .......... N .......
.......................
N ....
PEYWDRNTQICKAQAQTERENLRIALRY
............................
...............
R ............
J~8 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HLAI2.4
5.4-LBF
5.4-BB
EN-RIALRES-RIALR-
-K ..........................
...............
R ............
......................
G .....
1
Figure 5. The amino acid sequence in the a helix of the a~
domain for six HLA-AR, HLA-C, HLA-A, and HLA-B
molecules is shown. This helix is the most variable region in
class I antigen-presenting molecules. Although HLA-AR
molecules are closely related to HLA-A through most of the
molecule, they show greatest similarity with certain HLA-B
alleles in this region.
morphism. This can be seen from comparison of the
H L A - A R and H L A - A loci. For example, the a helix of
the al domain, which is the most variable part of HLAA molecules, is quite homogeneous in H L A - A R (Fig.
5). The power of conversion events to homogenize
alleles is suggested by the presence of both deleterious
mutations in five of the six alleles. These mutations
presumably had independent origins in different alleles, and it is unlikely that their combination would
confer any selective advantage. Probably they have
been combined through selectively neutral conversion
events.
Nonclassical Class I Genes
These observations on the H L A - A R locus illustrate
that conservation of sequence or lack of polymorphism
in MHC alleles need not imply that the sequences are
under positive selection for function. In this case,
homogenization, or loss of diversity, may be the consequence of loss of function. What else might happen to
MHC genes that are no longer useful? In a situation
where selection is favoring the reduction of class I or
class II diversity expressed by an individual, there are
various mutational changes that could be advantageous, and not all would result in production of an obvious pseudogene like HLA-AR. Their critical feature
would be to remove the influence of an MHC gene
upon the repertoire of TCRs. One way to do this is to
select for mutations that alter the patterns of tissue
expression in such a way that expression on critical cells
in the thymus is lost. This could result in class I genes
with rather specific and different patterns of tissue
expression such as Tla, Qa antigens and HLA-F. A
second way is to prevent expression at the cell surface,
perhaps by sequestering the genes within the cell as
found for H L A - E and G or by secretion as occurs for
QIO (Chen et al. 1987; Robinson 1987; Koller et al.
1989). Thus, many nonclassical genes have properties
predicted for inactivating mutations of antigen-presenting loci. These properties, combined with the lack of
any known function for the products of nonclassical
class I genes and the lack of correspondence between
the nonclassical genes of different species (Rogers
1985), strongly suggest that these molecules are nonfunctional and are the products of genes in decline
(Klein and Figueroa 1986). Of course, this does not
prevent their participation in gene conversion events as
observed in the mouse (Nathenson et al. 1986), but this
function, as such, will be used infrequently and by a
very small minority of individuals.
HLA.C: A Declining Class I Gene?
Cellular interactions are dependent on the density of
the contributing cell-surface molecules, and another
possible mechanism for extinguishing the role of an
MHC gene in thymic selection is to reduce the level of
cell-surface expression. Perhaps this is what happened
to the HLA-C locus, the products of which are found at
the cell surface at about one tenth the level of H L A - A
and HLA-B molecules. Supporting the view that HLAC molecules are in decline is the reduced diversity,
especially in the a 1 helix (Fig. 5). H L A - C and HLA-B
alleles have closely related sequences and are clearly
the products of a gene duplication (Fig. 3). Despite this
common origin, the nature of polymorphism in contemporary HLA-B and C alleles is significantly different. HLA-B has many more alleles with greater diversification in sequence and with substitutions concentrated at positions directly involved in contacting bound
peptide and the TCR. HLA-C has fewer alleles differing by smaller numbers of substitutions, which are
frequently at positions distant from the combining site
groove. Reinforcing suspicions that HLA-C contributes
little to human immunity is the absence of evidence
showing antigen presentation by HLA-C to human T
cells. That HLA-C molecules are still capable of antigen presentation function has been demonstrated by
experiments with transgenic mice (Dill et al. 1988);
whether there are sufficient HLA-C-restricted T cells
for it to happen with any frequency in the human
immune response is another matter.
HLA-B Polymorphism Is Bigger, but Is It Better
Than That of HLA.A?
It is thus possible that the only human class I loci that
encode functional antigen-presenting molecules are
H L A - A and HLA-B. (However, even for these loci
the actual number of allelic products that have been
shown to present a peptide to a T cell is a minority).
Major differences in the patterns of polymorphism are
observed and suggest that the two loci are following
distinct evolutionary paths.
Downloaded from symposium.cshlp.org on May 11, 2016 - Published by Cold Spring Harbor Laboratory Press
HLA-A,B,C: STRUCTURE, FUNCTION, AND EVOLUTION
There are many more H L A - B than H L A - A alleles,
and they exhibit greater diversification of sequence in
the peptide-binding domains and greater homogeneity
of sequence in the a 3, transmembrane, and cytoplasmic
domains. Comparison of human and chimpanzee sequences reveals greater similarity between A than B
alleles in the two species (Parham et al. 1989a), showing that new polymorphisms have accumulated at a
higher rate in H L A - B compared to HLA-A. A final
difference is the existence of a high-frequency allele at
the A locus, HLA-A2, for which there is no equivalent
at the B locus. This, combined with the smaller number
of H L A - A alleles, increases homozygosity at the HLAA locus.
There are two ways of interpreting these observations. The first is to argue that decreased H L A - A
polymorphism and diversity are the result of negative
effects due to selection at another MHC locus, in a
manner analogous to that hypothesized for HLA-AR.
In this view, H L A - A is perceived as being in relative
decline, and the high frequency of HLA-A2 need not
be associated with any particular advantage. The second view is that the relative conservation of H L A - A
alleles is because their products are critical for immune
responses against prevalent and important pathogens.
In this view, H L A - A becomes a more optimized locus
and HLA-A2 a particularly advantageous allele. Adding to this interesting puzzle is the noticeable absence
of HLA-A2 in chimpanzees (Balner et al. 1978).
Binding of CD8 to Class I MHC Antigens
Two other molecules intimately involved in the function of MHC-restricted T cells are the CD4 and CD8
glycoproteins (for review, see Littman 1987; Parnes
539
1989). CD4 defines T cells reactive with class II MHC
molecules, and CD8 defines T cells reactive with class I
MHC molecules. The derivation of both class-I- and
class-II-restricted TCRs from a common pool suggests
that CD8 or CD4 expression is tightly coordinated to
the restriction specificity of mature T cells and that
these molecules play an essential role in thymic selection (for review, see Janeway 1988). In addition, the
inhibitory effects of monoclonal antibodies in in vitro
assays suggested CD4 and CD8 were important for
effector functions of T lymphocytes, a supposition that
has subsequently been demonstrated by transfection
experiments (Dembic et al. 1986). The molecular basis
for these functions has been hypothesized to lie with a
specific interaction between CD4 or CD8 and monomorphic sites of class I or class II MHC molecules,
respectively (Swain 1981). Such interactions could contribute to adhesion between T cells and antigenpresenting cells--both in the thymus and in the
periphery--and also to transmembrane signalling
events that determine T-cell function following antigen
recognition.
To test these ideas, an in vitro cell-binding assay to
detect specific interactions between CD8 and class I
MHC molecules was developed (Norment et al. 1988).
Its principle is the binding of cells in suspension, which
are radioactively labeled and express class I molecules,
to a monolayer of attached cells expressing CD8 (Fig.
6). Class I genes are transfected into CIR, a mutant
human B-cell line that expresses no endogenous HLAA,B molecules. (It does express low levels of class I
molecules, which are thought to be HLA-C; however,
they have no effect on the binding assay). Transfectants
of CHO cells, which have no endogenous expression of
CD8, are the source of CD8. Specific binding, which is
CD8 BindingAssay
Confluent
Monolayer
of CHOCells
Radioactive
B Cells
~
~T
400 x g
1 hr 37~
p~
l Wash
10x
Count
Radioactivity
r~
,
,
Detergent
Lysis
Figure 6. Cartoon of the cell-cell binding assay used to analyze the interaction between CD8 and class I MHC molecules
(Norment et al. 1988).
Downloaded from symposium.cshlp.org on May 11, 2016 - Published by Cold Spring Harbor Laboratory Press
PARHAM ET AL.
540
assessed by control experiments with untransfected
cells, is dependent on high levels of expression of the
transfected genes. This indicates that the affinity of
individual CD8 and class I MHC molecules is low and
that the specific binding between cells is dependent on
multipoint attachment. Background binding is relatively high and leads to signal to noise ratios of 3-10. In
addition to controls with untransfected cells, the
specific binding can also be assessed by using monoclonal antibodies directed against either class I MHC or
CD8 as inhibitors.
Comparison of a panel of CIR transfectants, each
expressing a different class ! MHC molecule, produced
two interesting findings (Salter et al. 1989). First, two
murine class I molecules (H-2K b and H-D P) bound
human CD8 as well as H L A - A , B molecules. Second,
two closely related H L A - A molecules, HLA-Aw68.1
and HLA-Aw68.2, do not bind CD8 in this assay. This
demonstration of polymorphism upsets the prevailing
paradigm that classical class I molecules have a monomorphic CD8-binding site. The question of differential
affinities of CD8 binding within the group of positive
binding molecules is also open, since the assay is only
A
qualitative and the significance of the quantitative differences seen in binding is uncertain.
The discovery that HLA-Aw68.1 does not bind CD8
was extremely fortunate, since this molecule has been
crystallized, and its structure has been determined. It
differs at only 13 residues in the three extracellular
domains from HLA-A2.1, the other MHC molecule for
which an X-ray structure is available (Bjorkman et al.
1985, 1987a). These two molecules thus provide an
excellent system with which to map the site of interaction between CD8 and the class I MHC molecule.
Site-directed mutagenesis of H L A - A 2 . 1 and H L A Aw68.1 genes has shown that the amino acid at position
245 in the a 3 domain is solely responsible for the difference in CD8 binding between HLA-A2.1 and H L A Aw68.1 molecules (Salter et al. 1989). This residue,
which is alanine in HLA-A2.1 and all other antigenpresenting class I molecules so far sequenced, is replaced by valine in HLA-Aw68.1 and the other nonbinding molecule, HLA-Aw68.2. These results, indicating that the a 3 domain is important for CD8 interaction, are supported by observations that a mutant
H-2D ~ molecule, with substitution of lysine for aspartic
B
Key
( ~ Mutation
affects CD8 binding
I I Mutation has no effect
* Interacts with ~2m
+ Conserved
between species ~
~,,,~2m
239 +
~,~,~=.
\
+ ~1
Cytolysis Assay
T~A 214 !~i~iii~iiiiiiii~i~;~i~i~;i~i!i!i~i~i~iii!iii!i~!iiiiiiiiiii~iiiii~i~
L~A 215 ?~i~i!~i~!:~!~!~!~!~!~)?~ii!ii~i!i!iii!i!!!~;!!~i~iiiii!iiii!ii~i!!1
I
T~A 216
~
IT
. 61+~~
*+"~ I.~+
F~'l+
*++
E~A222iii!ii!i:~iii!i~)iii~i~i~iii!i~i!iii~ii!ii!ii~i!ii~il
D-~A 2Z~
Q-~A 226
+
D~K227
D~A 227
E~A 229
t
T~1233
+
253
A-~V245
S~P 251
G~A 265
CD8 Binding Site
2o
40
60
8o ~oo ~2o
Lysis Relative to HLA-A2.1
Figure 7. Mutagenesis of the a 3 domain affects CD8 binding and T-cell recognition. (A) Approximate positioning of amino acids
214-253 comprising/3 strands 3, 4, and 5 of the a 3domain. This is viewed from the opposite side to the common representation as
depicted in Fig. 2a of Bjorkman et al. (1987a) and Fig. 5B of Bjorkman et al. (1987b). Positions at which point mutations of
HLA-A2.1 eliminate CD8 binding are circled and those that have no effect are in squares. Positions identified by Bjorkman et al.
(1987a) as contacting/32-m a r e indicated by asterisks, and the general positioning of/32-m is shown with arrows. Residues that are
conserved in class I molecules from different species are indicated by plus signs. (B) Lysis by a clone AMSH10 of alloreactive
HLA-A2-specificcytotoxic T cells of CIR cells transfected with mutant HLA-A2 molecules having substitutions shown at the left.
The lysis is given relative to target cells transfected with wild-type HLA-A2.1.
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HLA-A,B,C: STRUCTURE, FUNCTION, AND E V O L U T I O N
acid at position 227, cannot be recognized by CD8dependent cytotoxic T cells (CTL) (Connolly et al.
1988; Potter et al. 1989). Residue 245, which is on the
fifth strand of fl sheet of the a 3 domain, is within 10.5 ,~
of residue 227, which is found on the loop between the
third and fourth strands (Fig. 7A). This region of the a 3
domain is relatively exposed in the structure of HLAA2 and could be accessible for binding to a protein the
size of CD8.
To further investigate the class I/CD8 interaction,
additional point mutations in the a 3 domain have been
studied (Fig. 7A). As observed for H-2D d, the introduction of lysine at position 227 eliminates CD8 binding, as do other mutations at positions 223, 226, 229,
and 233 in this region of the a 3 domain. In contrast,
more distant mutations at positions 214, 216, 221,222,
232, 243, and 251 have no effect, as is also seen with
various mutations in residues of the a~ and a 2 domains.
Clustering of mutations that eliminate CD8 binding to a
region around the carboxy-terminal part of the loop,
between strands 3 and 4, indicates that this part of the
a a domain is involved in directly contacting the CD8
molecule. The sequence of the loop from residues 220 to
229 - - Asp. Gly. Glu. Asp. Gin. Thr. G In. Asp. Thr. Glu. - is noticeably acidic, and mutations that remove acidic
residues generally lead to loss of CD8 binding. The
importance of acidic residues is also suggested by a
mutant in which an additional acidic residue was introduced at position 221 and gave increased CD8 binding.
The effects observed in the CD8-binding assay can be
correlated with recognition and cytolysis by T cells.
Molecules that have lost CD8 binding are less efficiently recognized by CTL (Fig. 7B).
These results focus attention on the subtypes of
HLA-Aw68 and the function of these molecules in
antigen presentation. Experiments to prime alloreactive and antigen-specific CTL with HLA-Aw68 have
been negative. However, and as found for the H-2D d
mutant, CTL that are primed with a cross-reactive
molecule, such as HLA-A2 or HLA-Aw69, can in a
secondary response react with HLA-Aw68 (Gaston et
al. 1985). These results suggest that the loss in CD8
binding reduces the capacity of HLA-Aw68 to select
for T cells in the thymus. As such, HLA-Aw68 appears
to be a molecule that has lost, or is losing, function and
may provide another example of a mechanism that is
being used to reduce the polymorphism and functional
effect of an MHC locus, in this case HLA-A. Alternatively, the loss of CD8 binding may lead to a functional
advantage, perhaps the selection of higher-affinity T
cells, which have so far eluded detection. Answers to
these questions will come from further study of the
capacity of HLA-Aw68 to bind peptides and interact
with T cells.
The location of the CD8-binding site of class I MHC
molecules has been a topic for speculation. On the basis
of similarities between the immunoglobulin-like domains of CD8 and the variable regions of TCR, Davis
and Bjorkman (1988) proposed that CD8 interacts with
class I molecules in a manner analogous to the TCR
A
541
B
glI
Figure 8. Cartoon of hypothesized interactions between class
I MHC molecules, CD8 and the TCR. A and B are as depicted
by Davis and Bjorkman (1988). (A) Interaction between
TCR, a specific peptide (O), and the a 1 and a 2 domains of
class I MHC molecules. (B) The analogous interaction of the
V-like domains of CD8 with the al and/32 domains of class I
MHC, which has nonspecific peptide bound (9 C and D give
possible interactions if the a 3 domain of class I MHC molecules provides the binding site of CD8. (C) TCR and CD8
both binding to the complex of class I MHC and specific
peptide; (D) CD8 interacting with the complex of class I MHC
and nonspecific peptide.
(Fig. 8A,B). If this is true, then CD8 and a TCR could
not simultaneously interact with the same class I MHC
molecule (Fig. 8C). Our results with the CD8/class I
MHC cell-binding assay implicate a localized region of
the a 3 domain in the binding site of CD8 (Fig. 8D) and
argue against a direct involvement of the helices of the
a 1 and a 2 domains as hypothesized previously (Davis
and Bjorkman 1988). This leaves open the possibility of
simultaneous interaction of CD8 and TCR, with the
identical class I MHC molecule being the critical interaction that initiates T-cell responses (Fig. 8C).
CONCLUDING REMARKS
A characteristic of class I MHC molecules is their
diversity and polymorphism. Comparison of allelic sequences in humans and chimpanzees shows that diversity is accumulated gradually over the lifetimes of many
species and does not involve any unusual mechanisms
or rates of mutation that are specific to these genes.
Rather, it is the unique nature of the evolutionary
selection for advantageous T-cell immunity that is re-
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542
PARHAM ET AL.
sponsible for the diversity. Variation is found throughout the molecule and affects the interactions of class I
MHC molecules with antigenic peptides, TCRs, the
CD8 proteins, and possibly other cellular components.
Combined analysis of natural variants and site-directed
mutants is enabling the functional sites of class I molecules involved in these interactions to be mapped.
Within the individual, MHC diversity probably has
two opposing effects on T-cell immunity, which serve to
limit the numbers and polymorphism of MHC loci
expressed9 These are increased antigen presentation
due to diversity in peptide-binding domains (a I and a2)
and decreased TCR repertoire due to the establishment
of self-tolerance. The limiting effect of self-tolerance is
not imposed on populations, and diversity for antigen
presentation becomes the dominant effect and can lead
to extraordinary polymorphism. That MHC molecules
can both present antigens and delete T cells means that,
depending on the antigenic environment, there can be
evolutionary selection for the presence or elimination
of particular MHC alleles or loci. This can result in the
advantageous diversification, homogenization, or extinction of particular loci. Examples of different patterns of polymorphism at four human class I MHC loci
support this thesis. Since receptors restricted by all
MHC loci--class I and class I I - - a r e derived from the
same pool, polymorphisms at any one locus will affect
the functional potential at other loci. In essence, the
different MHC molecules are in competition with each
other. Thus, positive selection for responses that are
restricted by one MHC molecule, or set of molecules,
can result in the elimination of others. Such a dynamic
functional interplay between the products of different
MHC loci should result in the continuing, but gradual,
rise and fall of polymorphism and functionality at different MHC loci in response to changing antigenic
conditions. This can explain the remarkable diversity in
the numbers of MHC loci and their expression between
species and populations.
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Diversity of Class I HLA Molecules: Functional and
Evolutionary Interactions with T Cells
P. Parham, R.J. Benjamin, B.P. Chen, et al.
Cold Spring Harb Symp Quant Biol 1989 54: 529-543
Access the most recent version at doi:10.1101/SQB.1989.054.01.063
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