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580
D.
Snary,
C.J.
Barnstable, W.F. Bodmer and
M.J.
Oumpton Eur.
J.
ImmunoL 1977.8:
580-585
D.
Snary", C.J.
Barnstable',
W.F.
BOdmer' and M.J. Crumpton'
National Institute for Medical Research,
London' and Genetics Laboratory, Depart-
ment
of
Biochemistry,
Oxford'
Molecular structure
of
human histocompatibility
antigens: the
HLA-C
series"
The HLA-CW2 antigen of the B lymphoblastoid cell line BRI
8
is struc-
turally homologous
to
the HLA-A and
B
antigens as judged by various
criteria. Each antigen comprised a glycosylated polypeptide
of
43
000
rnole-
cular weight that is noncovalently associated with 02-microglobulin
(Pz
m).
Some small differences in molecular parameters were, however, revealed.
Thus, the
deoxycholate-solubilized
HLA-CW2 antigen sedimented at the same
rate as the HLA-A antigens but at a slightly faster rate than the HLA-B anti-
gens. This variation is apparently due to different amounts of bound deoxy-
cholate. Also, whereas essentially all
of
the HLA-A and B antigens and about
half of the HLA-CW2 antigen were adsorbed Rrongly by
Lens
culinaris
lectin-
Sepharose, the remaining HLA-CW2 antigen was bound much more weakly
and did not require sugar for elution. This difference reflects some structural
heterogeneity in the carbohydrate moiety
of
the HLA-CW2 antigen. The results
of
various studies suggest that the HLA-CW2 antigen
is
expressed to a lower
extent than the HLA-A
or
B antigens and that essentially all of the
P2m
of the
BRI
8
plasma membrane is associated with the HLA-A, B and C alloantigenic
polypeptides.
1.
Introduction
The expression of the classical transplantation antigens of
man is controlled by the major histocompatibility gene sys-
tem, the HLA complex. Initially two series of polymorphic
cell surface antigens, the HLA-A and B series, were defined.
These series of antigens have been extensively studied sero-
logically and biochemically
[
1,
21. Each series comprises 18
to
20
serologically defined variants which are expressed as if
controlled by alleles at the corresponding loci, A and B. More
recently, a third allelic series of antigens, HLAC (formerly
designated AJ), has been described
[
3,4]. Serological studies
of
families in which intra-HLA recombination has occurred
have placed the C locus between the A and
B
loci. The small
number of recombinants
so
far examined suggest a distance of
about 0.2 centimorgans between the B and C loci and about
0.6
centimorgans between the A and C loci [5].
The gross molecular structures of the HLA-A and B antigens
have been established
[6,
71. A comparison of their N-ter-
minal amino acid sequences clearly indicates that they arose
by gene duplication
[
8-
1
11.
They comprise a 43
000
mole-
cular weight glycosylated polypeptide chain which is nonco-
valently bound to a 12
000
molecular weight nonglycosylated
polypeptide, namely Pz-microglobulin
(&m).
The larger chain
carries the serologically detected polymorphism and is coded
[I
17251
*
This
work
was supported in part
by
a grant
from
the Medical Re-
search Council to the Genetics Laboratory.
0
Present address: Department
of
Immunochemistry, The Wellcome
Research Laboratories, Langley Court, Beckenham, Kent BR3
3BS,
GB
Correspondence: Michael
J.
Crumpton, National Institute for Medical
Research,
Mill
Hill,
London NW7
lAA,
GB
Abbreviations:
Am:
&Microglobulin
SDC:
Sodium deoxycholate
by a gene within the major histocompatibility complex on chro-
mosome 6
[
12, 131. In contrast, P2m is genetically unlinked
to the HLA-A and B genes, being coded by a gene
on
chro-
mosome 15
[
141.
Recent studies have, however, shown that
the genes are functionally related, since HLA-A and B antigen
biosynthesis and expression on the cell surface
is
dependent
on a functional
flZ
m
[
151. Much less information is available
regarding the structure of the HLA-C antigens, although it is
apparent from serological studies that the C series antigens
are also associated with P2m
[
161, and have a similar tissue
distribution to the HLA-A and B antigens, being present on
T
and B lymphocytes and platelets but not on erythrocytes
[
171. The present paper is concerned with the molecular pro-
perties of the HLA-C antigens and the comparison of their
structure with those of the HLA-A and B antigens.
2.
Materials and methods
2.1.
Lymphocyte plasma membrane
BRI
8,
a human lymphoblastoid B cell line (HLA-A1, A2,
B8, B13, CW2) grown by Searle Diagnostics (High Wycombe,
Bucks.,
UK)
was used as the source of HLA antigens. Plasma
membrane vesicles were prepared as previously described
[
181.
Briefly, viable cells (5 x 107/ml of
0.
I5
M
NaCl 10 mM Tris-HC1
buffer, pH 7.4) were broken using a Stansted Cell Disrupter
(model no.
A0
612 with disrupting valve no. 516; Stansted
Fluid Power, Stansted, Essex,
UK).
The plasma membrane
fraction was separated from the cell homogenate by differ-
ential centrifugation and purified by sucrose density gradient
centrifugation. The purified membrane was not contaminated
to a significant extent by other subcellular components as
judged by various biochemical and morphological criteria.
Plasma membrane (4 mg of protein/ml) was solubilized at
0
'C in 2
%
(w/v) sodium deoxycholate (SDC)
10
mM Tris-
HCl buffer, pH 8.2, and insoluble material was removed by
centrifuging at 100
000
x
g
for 1 h. Under these conditions,
the supernatant contained
90
%
of the membrane protein
and more than 85
%
of the HLA-A,
B
and
C
antigen acti-
vities.
Eur.
J.
Immunol. 1977.8:
580-585
Structure of HLA-C antigens 581
2.2.
Antisera
HLA-A,
B
and C antigens were detected using pregnancy
sera and antisera produced by planned immunization that
had been characterized relative to the specificities defined
by the 5th and 6th Histocompatibility Testing Workshops
[
1, 191. The source and specificity of the sera are shown in
Table
1.
Antisera which possessed more than one specificity
were rendered effectively monospecific by using appropriate
target cells. Allogeneic (no. P3302B, P2604B and P1530B)
and xenogeneic antisera against HLA-la antigens were de-
scribed previously
[
201.
The rabbit anti-human P2m serum employed for inhibition
assays was kindly donated by Dr. Howard Grey. The goat
antiserum against P2m was raised by immunization with a
purified preparation
of
human urinary Pzm which gave one
detectable band only
of
12
000
molecular weight on poly-
acrylamide gel electrophoresis in sodium dodecyl sulfate.
Each antiserum was judged to be monospecific on the basis
of its pattern
of
reactions with various human cell lines, in-
cluding its lack of reactivity with Daudi and erythrocytes
[
131, and its inhibition by an authentic sample
of
&m. In
addition, when tested on a panel
of
human-mouse somatic
cell hybrids they reacted only with those cells possessing
human chromosome 15.
Table
1.
Source
and
specificity of allogeneic antisera against
HLA-A,
Band
C
antigens
Serum Source Specificity')
Number
F1209D
F1014H
F3002X
F5221B
P1422B
PI
0408
P4375B
Name
DOWY
Bodmer
Al,
B8
Gillespie
Payne/Bodmer A1
Caminiti Bodmer/Payne/Hyland
A2,
A9
Yochum Cohen
(RF'MI)
A2
-
OPSb)
B8
-
OPS
B13
-
OPS
cw2
a)
As
defined
with
respect to cells typed in the Vth and
VIth
Histo-
b)
Oxford pregnancy
serum.
compatibility
Testing Workshops.
2.3.
Analytical methods
Stoke's radii were determined by gel filtration on columns
(85 x 1.6 cm)
of
Ultrogel AcA 34 (LKB Instruments Ltd.,
London), eluted with 0.5
%
SDC 10 mM Tris-HCI buffer,
pH 8.2, relative to the elution characteristics of water-soluble
globular proteins
of
known Stoke's radii (aldolase, 4.5 nm;
bovine serum albumin 3.55 nm; ovalbumin, 2.73 nm, soy-
bean trypsin inhibitor, 2.36 nm).
Sz~,w
values were estimated
on sucrose density gradients
of
8
to 20
%
(w/v) sucrose in
0.5
%
SDC
10
mM Tris-HCI buffer, pH 8.2, centrifuged in a
Beckman SW41 rotor at
38 000
rev/min for 24 h, relative to
the rate of sedimentation
of
standard water-soluble globular
proteins run at the same time (aldolase, 7.5
S;
bovine serum
albumin, 4.42
S;
ovalbumin, 3.55
S;
soybean trypsin inhibitor,
2.3
S).
BRI
8
plasma membrane solubilized in 2
%
SDC
10
mM
Tris-HCl buffer, pH 8.2
(0.5
ml containing 2 mg of membrane
protein) was used as the antigen source. HLA-A, B, C and la
antigens were located
on
gel filtration and sucrose density
gradients by measuring antigenicity. Molecular weights were
calculated from Stoke's radii and
S20,w
as described previous-
ly
Clycoproteins were specifically adsorbed to columns
(
15 x 1.5
cm)
of
Lens
culinnris
(lentil) lectin-Sepharose 4B
(1
mg
of
lectin/ml of gel sediment) in
0.5
%
SDC
10
mM Tris-HC1 buffer,
pH 8.2, and were subsequently eluted with 2
%
methyla-D-
mannopyranoside in
0.5
%
SDC
10
mM Tris-HC1 buffer, pH 8.2
[221-
Pzm-associated components
of
SDC-solubilized BRI
8
plasma
membrane (20 ml containing 16 mg
of
membrane protein)
were specifically depleted by adsorption
to
Columns (20 x
1.5 cm)
of
the immunoglobulin fraction
of
a goat anti-p2m
serum attached to Sepharose 4B
(10
mg
of
Ig/ml
of
gel sedi-
ment). Columns were eluted with 0.5
%
SDC 10 mM Tris-HCl
buffer, pH 8.2, at a flow rate of 15 ml/h.
2.4.
Antigenic activity
Antigenic activity was assessed quantitatively by inhibition
of fluorochromatic microcytotoxicity as described previously
[
18, 231. Peripheral blood lymphocytes, separated from fresh-
ly drawn defibrinated blood by flotation on Ficoll-Triosil
gradients, were used as target cells in assays for HLA-A, B
and C antigens and &m. HLA-Ia antigens were assayed using
cultured lymphoblastoid B cell lines. Rabbit complement for
use in the latter assay was selected for lack of naturally oc-
curring heterophile antibody to which the cell lines are par-
ticularly sensitive. Nonspecific lysis
of
target cells by SDC in
the antigen fractions was reduced by diluting the fractions
in
RPMI 1640 medium containing
20
%
(v/v) inactivated fetal
calf serum.
3.
Results
3.1.
Cell surface location
Subcellular fractionation
of
homogenates of BRI
8
lympho-
blastoid cells showed previously that HLA-A and B antigens
are located preferentially, if not exclusively,
on
the plasma
(cell surface) membrane
[
181. Similar studies have now re-
vealed that HLA-C antigens have an identical subcellular dis-
tribution with the HLA-A and B antigens. Thus, for example,
the purified plasma membrane fraction of BRI 8 cells con-
tained 42, 34 and 38
%
of
the HLA-A2, B8 and CW2 anti-
genic activities respectively,
of
the initial cell homogenate.
It was concluded that HLA-C antigens are also located almost
exclusively on the plasma membrane.
3.2.
Molecular size
The molecular size
of
the HLA-CW2 antigen was estimated from
measurements of its gel filtration and sedimentation behavior
in SDC. A purified preparation of BRI
8
plasma membrane
was used as the source of HLACW2 antigen. Fig.
1
shows
that HLA-CW2 antigen was eluted from a column of Ultro-
gel AcA 34 as a single, essentially symmetrical peak whose
position coincided with that for P2m antigenic activity and
with those found previously for HLA-A, B and Ia antigens
(e.g.
see Fig. 2, ref. [24]). This elution pattern is in marked
582
D.
Snary,
C.J. Barnstable, W.F. Bodmer and M.J. Crumpton
in
Figure
1.
Gel filtration on a column of Ultrogel AcA
34
of BRI 8
plasma membrane solubilized in
SDC.
The column was eluted with
0.5
%
SIX
10
mM Tris-HCI buffer, pH 8.2, and the eluate was moni-
tored for protein
(0,
E;;Znrn),
HLA-CW2 activity
(A)
and &m
(m).
HLA-A,
B
and la activities were coincident with those
shown
for
HLA-CW2 and &m. The apparent asymmetry of the antigen peaks
is
of questionable significance. Results obtained using various anti-
serum dilutions indicated that it was related to the sensitivity of the
assay and suggested that it may represent nonspecific activity rather
than aggregation.
contrast to the multiple peaks reported previously for the
HLA-A, B and C antigens of a crude membrane fraction from
human spleen that had been solubilized
in
Nonidet
P40
[
251.
The basis of this difference has not been unequivocally de-
fined, but it appears likely that it is related to the use
of
crude membrane and is due to two factors, namely degrada-
tion
[
251 and disulfide interchange
[
2
11.
Sucrose density gra-
dient centrifugation of SDC-solubilized BRI
8
plasma mem-
brane (Fig. 2) gave a single peak
of
HLA-CW2 activity with
Eur.
J.
Immunol.
1977.8:
580-585
an
S20,w
value
of
5.1
5.
This value was identical with that
estimated previously for HLA-A1 and A2 antigens [21], but
clearly differed from that of HLA-B8 antigen determined
at the same time (Fig. 2), and that reported previously for
HLA-B antigens
(S20,w
4.55)
[
211.
The Stoke's radii and S20,w values of the HLA-A, B and C
antigens and the molecular weights calculated from these
values are summarized in Table 2.
The
HLA-A and C anti-
gens possessed the same apparent molecular weight of
88
000
compared with 78
000
for the HLA-B antigens. As discussed
previously
[
2
I],
these values include bound SDC.
Table
2..
Molecular size of HLA-A,
B
and C antigens in SDC
Antigenic Stoke's Bound SDCa)
activity radius
S~O,~
Mol.
wt.
(g/g
of protein)
(nm)
Al,
A2
4.4
5.15 88000
0.6
B8,
813
4.4
4.55 18000
0.4
CW2
4.4
5.15 88000
0.6
a) Calculated from the difference between the mol. wt. determined
for the SDC-solubilized antigens and those estimated by poly-
aaylamide gel electrophoresis in sodium dodecyl sulfate for the
component polypeptide chains (one each of
43
000
and 12
000
moL wt.).
3.3.
Association with
&m
Antigens associated with P2m were depleted from a sample
of SDC-solubilized BRI
8
plasma membrane by passage
through a column of antibody against
S2m
attached to
Sepharose. The effluent was monitored for protein and P2m,
HLA-A2, CW2 and la antigenic activities. The results shown
in
Fig.
3
indicate that the majority of the membrane pro-
tein and the HLA-Ia antigenic activity emerged together and
very much earlier than the HLA-A2, and CW2 activities whose
position corresponded to saturation
of
the column by B2m.
Figure
2.
Sucrose density gradient centrifugation of
BRI
8 plasma
membrane solubilized in SDC. Fractions were collected from the
bottom of the gradient and were monitored for protein
(0,
E,&&rn),
HLA-CW2 activity
(A)
and HLA-B8 activity
(0).
am occupied a co-
incident position with the HLA-B8 and
CW2
antigens but
was
not de-
tected
in
the position of free
&m
at the top of the gradient (not
shown). The asymmetry of the leading edge of the HLA-B8 antigen
peak is of doubtful significance (see legend of Fig.
1).
Figure
3.
Immunoadsorption of
BRI
8 plasma membrane with anti-
body against
&m
A sample of SDC-solubilized plasma membrane
was
eluted from a column of the
lg
fraction of an anti-&m serum attached
to Sepharose 4B. The eluate was monitored for protein
(0,
Ei;Fn,rn),
and HLA-A2
(A),
HLA-CW2
(A),
HLA-la
(O),
and &m
(m)
antgenic
activities Although the elution of Pzm apparently preceded that of
the HLA-A,
B
and C antigens, this difference was related to the sensi-
tivity of the assay and was not significant.
Eur.
J.
Immunol. 1977.8:
580-585
Structure
of
HLA-C antigens
583
The retarded position of
HLA-CW2
activity clearly indicates
that the
HLA-CW2
antigen resembles the
HLA-A
and
B
anti-
gens in containing &m, whereas the lack of retardation of
the
HLA-Ia
antigen endorsed the previous conclusion that
this antigen is not associated with Pzm
[24].
3.4.
Association with carbohydrate
The results of various studies have shown that
L.
culinaris
lectin binds specifically most lymphocyte plasma membrane
glycoproteins including the majority of the
HLA-A,
B
and
la antigens
[
18,
22, 241.
Indeed, adsorption to lentil lectin
and subsequent elution with the specific sugar has been em-
ployed as a diagnostic criterion of association with carbo-
hydrate. The affinity chromatography of SDC-solubilized
BRI
8
plasma membrane on a column of lentil lectin-Sepha-
rose
is illustrated in Fig.
4.
The results showed that
12
%of
the membrane protein was bound and eluted with methyla-
D-mannopyranoside. The eluted glycoprotein fraction con-
tained
86
%
of the
HLA-A2
activity but only
50
%
of the
HLA-CW2
activity. The remaining
14
%
of the
HLA-A2
ac-
tivity was eluted together with the unretarded protein peak,
whereas, in contrast, the other
50
%
of the
HLA-CW2
antigen
emerged from the column just subsequent to the unretarded
protein, The latter result suggests that about half of the
HLA-
CW2
antigen had a much lower affinity for lentil lectin than
the remainder and that the less avid portion underwent suc-
cessive adsorption and elution. It was concluded that the
HLA-
CW2
antigen resembles the
HLA-A
and
B
antigens in being
glycosylated, but that its carbohydrate moiety
is
heterogene-
ous.
It has yet to be determined whether the lower affinity
for lentil lectin indicates incomplete addition of N-acetyl-
glucosamine and/or mannose residues, to which the lectin
1”
‘I
I
c
II
Figure
4.
Affinity chromatography of
BRI
8
plasma membrane
on
Lens
culinuris
lectin-Sepharose.
A
sample of SDC-solubiIized plas-
ma
membrane was eluted from a column of
L.
culinrvis
lectin-
Sepharose with
0.5
%
SDC
10
mM Tris-HCl buffer, pH
8.2.
At frae
tion no.
38
the adsorbed glycoprotems were eluted by washing the
column
with
2
%
methyl-cI-Dmannopyranoside
in
0.5
%
SDC
10
mM
Tris-HCI buffer,
pH
8.2.
The eluate was monitored for protein
(
(0,
Ei;rn,,,) and HLA-A2
(A),
HLA-CW2
(A)
and pzm
(m)
antigenic
activities
binds,
or
the addition of further sugar residues to the carbo-
hydrate chains that mask the
N-acetylglucosamine/mannose
units
(c.J
Thy-1 antigen from rat thymocytes and brain
[
261).
The absence of a discernible peak of &m associated with the
first peak of
HLA-CW2
activity most probably reflects the
low level of
HLA-CW2
antigen (see below).
3.5.
Relative content
of
HLA-A,
B
and
C
antigens
Estimation of the relative amounts of
HLA-A,
B
and
C
anti-
gens on
BRI
8
cells from inhibition data is not possible, since
different antisera, even of the same apparent specificity, re-
quire different amounts of antigen to cause a
50
%
reduction
in cytotoxic titer. This difference probably reflects variation
in the relative amounts of different antibody types (IgM and/
or IgG), their affinities and specificities.
A
comparison of the
distributions of
HLA-A,
B
and
C,
and &m activities on lentil
1ectinBepharose (Fig.
4)
suggests, however, that
HLA-CW2
antigen is expressed to a much lower extent
(<
10
%)
than
the
HLA-A
and
B
antigens. This suggestion is based on the
observation that all of the
HLACW2
activity is apparently
associated with Pzm (Fig.
3),
and on the assumption that all
of the &m of the glycoprotein fraction eluted from lentil
lectin-Sepharose is accounted for by the
HLA-A,
B
and
C
antigens. In this case, the level of PZm activity underlying
the first peak of
HLACW2
activity in
Fig.
4
reflects the
amount of
HLACW2
antigen and may be used to calculate
the amount of
HLACW2
antigen relative to the
HLA-A
and
B
antigens in the glycoprotein fraction.
3.6.
Distribution
of
Pzm
During the above experiments the following information was
collected on the distribution of Pzm. The &m activities of the
various subcellular fractions obtained during fractionation of
the
BRI
8
cell homogenate paralleled the content of
HLA-A,
B
and
C
antigens. No pool of free Pzm was detected
in
the cyto-
sol fraction. Free &m was also not detected in
the
SDC-soh-
bilized plasma membrane fraction by either gel filtration
or
sucrose density gradient centrifugation (Figs. 1 and
2).
The
latter results have an added significance since various studies
suggest that free P2m was much more readily detected than
an equivalent amount of the bound protein. Thus, a compari-
son of the capacities of free (human urinary) and bound
(BRI
8
plasma membrane) &m to inhibit the rabbit anti-&m
serum suggested that the free protein was a more effective
inhibitor (about 100-fold) than the bound polypeptide. (The
amount of bound Pzm added was based on the assumption
that it was all associated with the
HLA-A,
B
and
C
antigens
which collectively represented about 1
%
of the membrane
protein). This result is consistent with the observation that
heating
BRI
8
plasma membrane at
40
OC
for 15 min had a
minimal effect on the
HLA-A,
B
and Ia activities, but in-
creased the &m activity 3-fold (Fig.
5),
as would be expected
if some of the bound Pzm had been dissociated. These results
argue in support of the view that all
of
the &m of
BRI
8
cells
is bound to the polymorphic chains of the
HLA-A,
B
and
C
antigens. In contrast, the cytosol of human placenta
[27]
and
spleen (unpublished results) apparently contained some free
&m. It is not yet clear whether this variation reflects a differ-
ence in P2m metabolism in different cell types,
or
whether
the
lack of free Pzm is peculiar to
BRI
8
cells. Recent reports that
a lymphoblastoid cell line and a crude membrane fraction
584
D. Snary,
C.J.
Barnstable,
W.F.
Bodmer and M.J. aumpton Eur.
J.
Immunol. 1977.8:
580-585
from human spleen have free &m [25, 281 should be viewed
with caution since, as the authors pointed out, the techniques
used may well have promoted dissociation of the bound poly-
peptide.
Figure
5.
Effect
of
heating
BRI
8
plasma membrane on antigenicity.
The HLA-A2
(A),
HLA-la
(0)
and
&rn
(m)
antigenic activities were
determined on samples of
BRI
8
plasma membrane that had been
heated for
15
min at various temperatures am was assayed using
the rabbit antiserum donated by Dr. Grey, and HLA-A2 activity
was
measured using serum F3002X The HLA-la activity represents the
average
of
results obtained using various allogeneic and xenogeneic
antisera [20]. The results are expressed relative to the activity
of
a
control sample of membrane that had been stored at
0
OC.
Note that
membrane which had been heated at
40 OC
showed
a
>fold increase
in
&m
activity.
4.
Discussion
The current results indicate that the products of the HLAC
locus
are structurally homologous to the HLA-A and B gene
products. A similar interpretation was derived recently using
similar approaches to those described above
[
251 and, alter-
natively, by comparing maps of the tyrosine-containing pep-
tides of immune precipitated 1251-labeled HLA-A, B and C
antigens
[
291. The latter results are, however, difficult to
interpret for various reasons, including the presence of shared
peptides derived from the common &m component, the poor
resolution of one-dimensional separation techniques and the
coprecipitation of 1z51-labeled actin by indirect immunopre-
cipitation of cells that have
been
labeled by lactoperoxidase-
catalyzed iodination (B.H. Barber,
M.J.
Crumpton, D. Snary
and
F.S.
Walsh, unpublished observation).
In the present study, the homologous structure of the HLA-A,
B
and C antigens was established by the following studies. Im-
munoadsorption with anti-p2m clearly showed that essentially
all of the HLA-A,
B
and
C
antigenic activities of SDC-solu-
bilized BRI
8
plasma membrane were associated with
Pzm,
since no alloantigenic activity was detected prior to satura-
tion of the adsorbent with &m (Fig. 3). The immunoadsor-
bent also bound polypeptides of 12
000
(&m)
and 43
000
molecular weight only from a glycoprotein fraction of BRI
8
plasma membrane containing HLA-A, B, C and Ia antigens
[30].
Although only about half of the HLAC antigen was
adsorbed by lentil lectin and specifically eluted with sugar
compared with more than 85 %of the HLA-A and B anti-
gens, the remaining HLAC antigen was significantly retarded
relative to the nongfycosylated protein (Fig. 4). As a result,
the variation in HLA-C antigen behavior most probably re-
flects heterogeneity of carbohydrate structure (c.f. rat thy-
mocyte Thy-I antigen
[26]),
in which case all of the HLAC
antigen is glycosylated. The HLA-A, B and C antigens be-
haved in an identical manner on gel filtration, but the HLA-A
and C antigens sedimented at a faster rate than the HLA-B
antigens on sucrose density gradient centrifugation (Table 2).
The apparent closer structural similarity of HLAC antigen
to HLA-A than HLA-B antigen in SDC is interesting, since it
appears to be contrary to expectation from genetic recombi-
nation studies which have placed the C locus closer to the B
than the A locus
[S].
In this connection it
will
be of interest
to determine whether the amino acid sequence
of
the HLAC
polypeptide also resembles more closely that of the HLA-A
than the HLA-B antigen. The difference between the mole-
cular weight of the SDC-solubilized HLAC antigen (88
000)
calculated from gel filtration and sedimentation studies, and
the sum of the molecular weights of the polypeptide chains
(55
000)
most probably represents SDC bound to the non-
polar portion of the polymorphic chain that dips into the
membrane lipid bilayer [21]. In this case, the variation in sedi-
mention behavior of the SDC-solubilized HLAC and B anti-
gens reflects different amounts of bound detergent and sug-
gests that the HLAC 43
000
molecular weight chain is inter-
calated more extensively into the lipid bilayer than the HLA-B
polypeptide. Although the present work is based exclusively
upon the analysis of the HLACW2 antigen of BRI
8
lympho-
blastoid cells, similar interpretations have been derived for
the HLA-CW3 antigen from human spleen (251.
As
a result,
it appears likely that the overall conclusion is independent
of the source and specificity of the HLAC antigen. Similarly,
the HLA-A and B antigens of various lymphoblastoid cell-
lines and tissues behave in an identical manner with those of
BRI
8
cells.
It has not proved possible to determine the relative amounts
of HLA-A,
B
and
C
antigens on BRI
8
cells, although the re-
sults shown in Fig.
4
indicate the relative paucity of the HLAC
antigen. A lower level
of
HLA-C antigen may account for the
problems experienced in defining the serological specificities
of the C locus since the antigens would be more difficult to
detect. Another explanation for the poor definition
of
HLAC
antigens is that it is difficult to obtain antisera devoid
of
con-
taminating HLA-B antibodies. Since it has been suggested
that the polymorphism of the HLA-A, B and C antigens may
represent differential expression
of
one
of
a cluster
of
struc-
turalgenes [31], it is also possible that the high blank fre-
quency for the HLA-C locus may actually represent true
blanks in which no HLA-C antigen is expressed.
The structural relationships that are ultimately established
between the products of the HLA-A, B and C loci will be of
value in elucidating the evolution of these loci from a pre-
sumptive precursor gene(s). It remains to be determined
whether the small differences in molecular parameters be-
tween the HLA-A, B and C gene products that have been re-
vealed during this study are correlated with differences
in
biological function.
We express our gratitude
to
Julicr
G. Bodmer for the
HLA-A,
B,
C
and Ia typing sera,
to
Howard Grey
for
the
rabbit anrCp2rn
serum
and
to
PeterJ. Lachman for the
human
urinary
pzm
CLB.
ocknow
ledges the receipt
of
a MRC Research Studentship.
Received May 3,1977.
Eur.
J.
Immunol.
1977.
8
585-588
Human
MIF
tested
on
mouse bone marrow macrophages
585
5.
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Mouse bone marrow-cultured macrophage
as
indicator
cells
for mouse and human migration inhibitory factor
Max-Planck-lnstitut
fur
I
mmunbologie,
(MI
F)
Macrophages cultured from mouse bone marrow served as excellent indicator
cells for mouse as well as for human migration inhibitory factor (MIF) prepa-
rations. The tests were performed in the capillary system and showed high
reproducibility. Using mouse or human MIF-containing supernatants, we
found an inhibition
of
53
%
compared with the controls. Similar results were
obtained if concanavalin A-stimulated human peripheral blood lymphocytes
were mixed with mouse test macrophages in a ratio of
1
:5
or
1
:
10.
Freiburg/Brsg.
1.
Introduction
The macrophage migration inhibitory factor (MIF) is one
of
the most frequently tested lymphocyte mediators
[
11.
The
MIF
test is
used
to detect delayed-type hypersensitivity in
[I
17081
Correspondence:
Marie-Luise Lohmann-Matthes, Max-Planck-lnstitut
f*
Immunbiologie,
D7800
FreiburglBrsg., Postfach
1169,
FRG
Abbreviations:
MIF:
Migration inhibitory factor
PCS:
Fetal
calf
serum
DMSO:
Dimethylsulfoxide
Con
A
Concanavalin
A
research as well as in clinical applications.
To
be
effective, a
biological assay must
be
reproducible when carried out under
standardized conditions. For the MIF test, however, the avail-
ability
of
suitable macrophages is a major problem for its
routine application. Usually either guinea pig
or
mouse peri-
toneal macrophages elicited by peritoneal injection are used
as test cells. In the case
of
guinea pig macrophages it is rather
impractical to kill one animal for a single test. On the other
hand, the number of mouse peritoneal exudate cells obtained
from a single animal is limited even when the exudates are
in-
duced with thioglycollate. The main disadvantage of these
conventional sources is more fundamental. It is common ex-
