/. Embryo!, exp. Morph. Vol. 61, pp. 201-219, 1981
Printed in Great Britain © Company of Biologists Limited 1981
207
Expression of serologically detectable H-2 antigens
on mid-gestation mouse embryonic tissues
By KATRINA J. KIRKWOOD 1 AND W. D. BILLINGTON 1
From the Reproductive Immunology Group, Department of Pathology,
The Medical School, University of Bristol
SUMMARY
Mixed haemadsorption assays using antibody-coaled indicator sheep erythrocytes and
mouse alloantisera revealed that major histocompatibility complex (H-2) antigens were
expressed on cells of 24-72 h cultures of mid-gestation mouse embryonic skin, gut, lung,
limb-bud and heart but not of embryonic gonad or kidney. The precise time of detection of
H-2 antigen expression and the proportions of cells expressing these determinants depended
on inbred strain, specific haplotype, tissue of origin and antiserum batch employed. In all
tissues the proportion of cells expressing H-2 increased progressively from day 11-12 postcoitum onwards. Thefindingsare discussed with respect to hypotheses concerning the possible
role of major histocompatibility antigens in cellular recognition and interactions during
embryogenesis.
INTRODUCTION
The precise way in which the expression of antigens specified by the major
histocompatibility gene complex (MHC) develops on different tissues during
organogenesis is of considerable interest. Bodmer (1972) has suggested that the
MHC regions may contain a large number of genes, some of which control the
synthesis of either differentiation antigens or structures capable of recognizing
such antigens. It has also been shown that contact inhibition of movement
(which has long been thought to be involved in organogenesis) appears to be
under MHC control. If the cells of adult mouse kidney tubule explants are mismatched at either the K and/or D regions of the MHC, contact inhibition is
increased markedly (Curtis & Rooney, 1979). Other H-2-dependent phenomena
related to intercellular adhesion have also been reported (e.g. Bartlett & Edidin,
1978), although the aggregation experiments of McClay & Gooding (1978)
indicate that large genetic variability of H-2 antigen expression may not be
involved in controlling the in vitro sorting and reaggregation of cells according
to tissue type.
A further suggestion concerning the role of MHC antigens in organogenesis is
that they may act as sites through which organogenesis-directing proteins can
anchor to the cell surface (Ohno, 1977). It has also been postulated that H-2
1
Authors' address: Reproductive Immunology Group, Department of Pathology. The
Medical School, University of Bristol, Bristol BS8 1TD, U.K.
208
K. J. KIRKWOOD AND W. D. BILLINGTON
specificities involved in intercellular recognition during embryonic development
and morphogenesis might be selective and degenerate, with several variants of a
given H-2 molecule leading to the same result in terms of cell positioning or
interaction between neighbouring cells, with the consequence that a diversity of
H-2 expression will be found among the cells within a single mouse embryo
(Edelman, 1975).
A considerable amount of effort has been devoted to determining the ontogeny of histocompatibility antigen expression in murine embryonic and neonatal development (see reviews by Edidin, 1972 and Jenkinson & Billington,
1977). There is evidence from immunofluorescence (Palm, Heyner & Brinster,
1971; Muggleton-Harris & Johnson, 1976) and immunoperoxidase (Searle et ai,
1976) studies that preimplantation embryos express minor histocompatibility
antigens. The latter investigators also detected a transient expression of low
levels of H-2 on trophectoderm cells of the pre-implantation mouse blastocyst.
The inner cell mass (ICM), however, appears not to express cell surface H-2
antigens (Searle & Jenkinson, unpublished observations). Patthey & Edidin
(1973) carried out grafting experiments between congenic strains of mice and
suggested that H-2 expression by post-implantation embryonic tissues derived
from the inner cell mass begins at about 7 days/?.c.
More recent electron-microscope immunoperoxidase studies on the enzymically separated primitive endoderm and embryonic core of 7^-day egg cylinders
suggest that H-2 expression at this stage is limited to the endoderm cells (Searle
& Jenkinson, unpublished observations). Buc-Caron, Condamine & Jacob
(1978) found that whilst yolk sac and trophoblast-free preparations of whole
8-day-old mouse embryos failed to absorb out the activity of an anti-H-2 serum,
similar preparations from 9-day-old embryos were reactive in this respect. The
earliest stage at which H-2 antigens appear to have been demonstrated in the
few studies using specific embryonic tissues from the latter half of gestation
and with sera raised specifically to H-2 antigens (Klein, 1965; Worst & Fusenig,
1973) is in the 11-day p.c. developing otocyst, when H-2 was found on both
ectoderm- and mesoderm-derived cells (Prystowsky, Khan & Markovitz, 1978).
In view of the various hypotheses concerning the role of MHC gene products
in development it is clearly important to examine in more detail the temporal
expression of H-2 antigens during embryogenesis, using sensitive serological
techniques. To this end a series of experiments were carried out using a miniaturized version of the very sensitive mixed haemadsorption assay (MHA) to
examine the expression of H-2 and other histocompatibility antigens in cell
cultures of the organs of mid-gestation mouse foetuses.
H-2 antigens on mouse embryonic tissues
209
MATERIALS AND METHODS
(1) Animals
Mice from inbred strains A(H-2a), C57BL(H-2b), C57BL/10ScSn (H-2b) and
BIO. Br (H-2k) were used in these studies. Timed pregnancies were obtained by
identifying vaginal plugs in female mice that had been caged overnight with
males of proven fertility. The day of detection of the plug was designated day 0
post-coitum (p.c).
(2) Embryonic material
Pregnant females between 10 and 14 days p.c. were killed by cervical dislocation under ether anaesthesia. The uterus was removed under sterile conditions and placed in warm Dulbecco A and B phosphate-buffered saline (DPBS)
(Oxoid, Ltd). The foetuses were removed from the surrounding maternal and
extraembryonic tissues and a number of different organs were dissected from
each foetus. Similar organs were pooled, cleaned of surrounding connective
tissue as far as possible and placed in warm 0-04 % (w/v) trypsin (Difco Ltd) in
DPBS for 20 mins at 37 °C with periodic agitation to aid cell dispersal. Hepesbuffered culture medium (RPMl-1640 (Gibco Biocult) with added antibiotics
and 10 % foetal calf serum) was then added to each tube to neutralize the trypsin
activity and the test-tube contents were vigorously pipetted to release as many
cells as possible from the tissue lumps.
The trypsin sensitivity of different organs varied; heart, for example, being
much more resistant than lung. Organs from 14-day embryos were less readily
disaggregated than those from ll-day/?.c. embryos. Some tissue lumps usually
remained, but the small number of cells present in each organ pool made
filtration and repeated washing of the suspensions impracticable. After centrifugation of the suspensions at 1000 # for 5 mins the cells were resuspended in
volumes of bicarbonate-buffered RPMl-1640, varying from 0-3-1-5 ml according to the number of foetuses in the litter and the number of cells released from
the tissue by trypsinization. Forty/d aliquots were placed in immunofluorescence
(IF) wells and the plastic IF plates (Sterilin, Ltd.) were placed in sterile moist
airtight boxes gassed with 5 % CO 2 and air at 37 °C for 24 or 48 h. One or two
foetuses from each litter were minced and trypsinized after removal of liver and
amnion and cultured as fibroblasts. Some of the cell preparations of heart and
limb bud were resuspended in 1-2 ml RPM1 and cultured under standard conditions i n the larger wells of migration plates (MIF wells) (Sterilin Ltd).
(3) Sera for histocompatibility antigen detection
All sera produced in the laboratory were collected 7 days after the last injection and decomplemented by treatment at 56 °C for 30 min before use. All
sera were stored in small aliquots at - 2 0 °C to avoid repeated freezing and
thawing.
210
K. J. KIRKWOOD AND W. D. BILLINGTON
(A) Antisera recognizing both H-2 and non-H-2 antigenic determinants
(i) C57BL anti-A serum. This was prepared by giving adult male C57BL mice
two consecutive full-thickness skin grafts followed by multiple intraperitoneal
(i.p.) injections of A-strain spleen cells.
(ii) CBA anti-C57BL. Prepared as above.
(B) Antisera recognizing only H-2 determinants
(i) Three different H-2b anti-H-2k (ak) sera were used: akx and ak 2 were two
different vials of freeze-dried C57BL/10 anti-B10.Br serum obtained from
Searle Diagnostic (High Wycombe, Bucks). They were reconstituted with tripledistilled water on different dates. ak3 wasC57BL/10ScSn anti-B10.Br prepared
in this laboratory by giving males of the C57BL/10ScSn strain 6 i.p. injections
of spleen and lymph node cells from the congenic strain BIO .Br.
(ii) H-2k anti-H-2b (ab): Freeze-dried B10.Br anti-C57BL/10 (Searle
Diagnostic) was reconstituted with triple-distilled water.
Haplotype specificity tests were carried out with ak3 and ab. No anomalous
positive or negative results were observed.
(4) Mixed haemadsorption assays
Indicator cells: Indicator sheep red blood cells (iSRC) were prepared by
sequential incubation in mouse anti-SRC serum and rabbit anti-mouse immunoglobulin serum at concentrations which had previously been found to
give optimal haemadsorption in the MHA (Sellens, Jenkinson & Billington,
1978). The iSRC were used in the tests as a 2 % (v/v) suspension in DPBS supplemented with 0-2 % (w/v) bovine serum albumin.
Assays: The culture medium was removed from each IF well and the plate
incubated for 1 h at room temperature under 5 % CO 2 in air with various dilutions of antiserum in RPMI. A minimum of three wells was tested with each
tissue - one for anti-H-2 serum, one for anti-(H-2 + non-H-2), both at concentrations known to give extensive haemadsorption with tissues expressing appropriate
antigens, and a normal serum control. Wherever possible the anti-H-2 serum
was tested at a range of dilutions and a further control of the efficiency of washing procedures was provided by the use of a hyperimmune serum containing
activity directed against inappropriate antigens. After incubation, the antiserum
was removed and the cultures were washed through two to three changes of
300 ml of DPBS and left for 10 min. Twenty five /d iSRC were then placed in
each well and the cultures incubated for a further hour at room temperature.
After thorough washing (as above) to remove non-adherent iSRC the cultures
were read on an inverted microscope using phase-contrast illumination and
scored according to the proportion of cells binding iSRC, with a + + + , + + ,
+ , - °, - system similar to that devised by Hausman and Palm (1973). Tests on
.
++
-°
+++
+++
++
10
11
12
13
14
-°
-7+
-
.
.
.
Kidney
•
:
.
.
+
—
- °o
+o
+
-
Gonad
:
+
•
+ +t
+ ++t
.
+
+
+ ++tt
+ +t
+
+ ++ ++tt
+t
.
Gut
.
,
+
++
++
,
h++
,
+++
+++
++
Lung
.
+
+
+
+
+++
++
+
-
-
Skin
+
°4_
_
-°
ak2
.
_1_ _|_ _ | _
.
-
-
_I_ _1_ _ | _
+++
_
ak t
:
+++ +++
.
.
.
+ + + + ^ - + + -f
-°
_1_
+++
+
-° + + +
-
+++
.
ak3
Forelimb bud
.
_
4_
+
_
.
_i_
.
|.
^TQ
.
_L
+
I*
§
|
- ©
-
^
I
.
.
§
+ + + + +C?'
_I_
++
+
++
ak
XX XX .+ I
+
+ +
++
_J_ _1_ _|_
+++
+
-°
ak3
Heart
* + + + = > 75% cells binding iSRC after ak treatment; + + = 35-75%; + = 10-35%; - ° = 2-10%; - = < 2%. Each symbol
refers to the culture derived from the pooled organs from one litter and combinations of two symbols indicate cases where there was very patchy
iSRC adherence. ak3 was used unless indicated.
t Stomach and omenta included with dissected tissues.
o
Fibroblasts
Day
p.c.
Table 1. Ontogeny oj H-2 antigen expression in cultures oj embryonic tissues in the A-strain mouse*
212
K. J. KIRKWOOD AND W. D. BILLINGTON
Table 2. Ontogeny of H-2 antigen expression in cultures of embryonic C57BL/
WScSn tissues*
Day
p.c.
11
12
13
14
Fibroblasts
Kidney
+
+
.
+++
Gonad
Gut
.
.
.
.
++
-
+++
-
++
-
-
-°
+/-
.
Lung
+
+
Forelimb
bud
Heart
Skin
-
+/+
+
-
+
+
+ +
+t
-
+ +/-
-°t
++
*, t See footnotes to Table 1. ab was used instead of ak.
MIF well cultures were carried out in basically the same way, using the volumes
described by Sellens et al. (1978). All cultures were fixed for 40 min in absolute
alcohol and stained with Giemsa to form a permanent record.
In order to assess the development of antigen expression in vitro, cell cultures
of 11-day organs were grown for 48 or 72 h before assessment by the MHA.
RESULTS
Both anti-H-2 and anti-(H-2 + non-H-2) sera were used on cultures of 11-,
12-, 13- and 14-day p.c. cells from A-strain, C57BL and C57BL/10ScSn conceptuses in all instances.
Fibroblasts
In all the IF well cultures tested, the majority of adherent cells showed extensive
iSRC adherence after treatment with anti-(H-2 + non-H-2) sera. Only marginally
less iSRC adherence was observed after anti-H-2 serum treatment of 12- to
14-day p.c. cultures (Tables 1-3), but in two out of four tests on 11-day cells
very little iSRC adherence was observed (Tables 1, 2). When the cranial, middle
and tail pieces of 11-day foetuses were used separately for fibroblast preparation,
grown in MIF wells and treated with ak2 in MHAs, the highest proportion of
cells with adherent iSRC was found in cultures of cranial-region fibroblasts.
H-2 antigens on mouse embryonic tissues
213
Table 3. Ontogeny of H-2 antigen expression in cultures of embryonic
C57BL tissues*
Day
p.c. Fibroblasts Kidney Gonad
11
.
12
++
.
.
.
13
+++
-
14
+++
++
.
-
Lung
.
.
+/-
++
+
+
-
++
-
+++
++
•
+++
++ +/++ +
+++
++
.
+
.
+
Forelimb
bud
Heart
Gut
++*f
++f
Skin
.
.
*, t See footnotes to Table 1. ab was used instead of ak.
Kidney and Gonad
The primordial gonad and kidney from 12-day foetuses were cultured together,
but at 13 and 14 days/?.c. the two organs could be separated. Cultures of 13and 14-day kidney showed virtually no iSRC adherence after treatment with
anti-H-2 sera (Tables 1-3) and any adherence was limited to a few iSRC attached
to cells at the periphery of distinctly epithelioid patches of cells. When treated
with anti-(H-2 + non-H-2) sera the epithelioid patches showed no iSRC adherence but the majority of other cells had adherent iSRC. Anti-(H-2 + non-H-2)
serum treatment resulted in iSRC adhering to many of the cells in 12- to 14-day
p.c. gonad cultures, but only a small number of cells bound anti-H-2 serum and
then only in 30-40 % of the total of 24 cultures from all the three different
strains which were tested (Tables 1-3).
Gut
Anti-(H-2 + non-H-2) sera bound fairly consistently to most of the cells of
cultured foetal gut from all three strains but anti-H-2 binding showed more
variation between the strains. An increasing number of cells from A-strain
foetuses showed iSRC adherence after ak3 treatment from day 12 onwards
(Table 1), but ab-treated cells showed only light and patchy iSRC adherence
until day 14 p.c. (Tables 2, 3), when more of the cells bound ab, at least in the
C57BL/10ScSn tissues (Table 2). Attempts to analyse the patchiness of ab
labelling and the positivity of the A-strain tissue in terms of the amount of
mesentery remaining adherent to the gut and whether the stomach and its
omenta were included indicated that some but not all of the positivity was due
to such tissues.
214
K. J. KIRKWOOD AND W. D. BILLINGTON
Lung
The binding of anti-(H-2 + non-H-2) sera was always extensive and the
majority of cells became heavily labelled with iSRC. Almost as much binding
was seen with the anti-H-2 sera, the binding of which increased with the age of
the foetus. At day 12 there was some labelling in most of the cultures, but many
of the cells were iSRC free, while at day 14 most of the cells in each culture were
labelled with iSRC (Tables 1-3).
Skin
In all tests the skin was taken from the cranial region of the embryo and every
effort was made to ensure its freedom from underlying osteogenic and connective tissue, but at 13 and 14 days p. c. fragments of such underlying tissues as
blood vessels were impossible to exclude. Anti-(H-2 + non-H2) sera bound to
most of the cells in cultures of A-strain and C57BL/10ScSn skin from 11- to
14-day embryos and also to many C57BL cultures of the same age, although
slightly less consistently in these latter cultures. C57BL/10ScSn and C57BL
tissue contained ab-binding cells in increasingly large numbers at least from day
12 onwards (Tables 2, 3). There were ak3-binding cells in most A-strain cultures
but in lower numbers except on 13-day tissue, when there was a higher proportion
of ak3-binding cells than on either day 12 or day 14 (Table 1). Patchiness of
iSRC adherence was a feature of a significant number of skin cultures; there
were sheets of cells, some covered with iSRC, some completely free of iSRC and
some bounded by iSRC.
Forelimb bud
Anti-(H-2 + non-H-2) sera of appropriate specificity bound to almost all the
celJs of limb-bud cultures of all three strains and at all the ages examined. An
increasing number of cells bound anti-H-2 sera over the period 11-13 days/?.c,
and by 14 days p.c. there was fairly consistent binding of anti-H-2 by most of
the cells in each culture (Tables 1-3). With A-strain tissue (Table 1), some H-2
positive cells were observed with most 11- and 12-day material tested with ak2
and ak3, but akx did not reveal the presence of any H-2 positive cells until 13
days/?.c.
Heart
Most cultured heart cells from all ages and strains tested, bound anti(H-2 +non-H-2). A-strain cells (Table 1) bound ak3 to a greater extent than
C57BL and C57BL/10ScSn cells (Tables 3, 2) bound ab but, as with the limbbud cultures, ak2 and ak3 were bound to cultures of younger embryos than was
akx (Table 1).
H-2 antigens on mouse embryonic tissues
215
Table 4. Development of H-2 expression in 11-day p.c. A-strain tissues
cultured in vitro
Antiserum
Time in vitro
...
...
ak 3
ak«
24 h
48 h
24 h
72 h
Lung
-
-
ND
ND
Heart
+
+
+ +
-(+
+
+ +
-
+
+
-
-
—°
-
ND
Limb-bud
Fibroblasts
o
Skin
+
ND
o
-
ND
ND
ND = Not determined.
The development of H-2 expression in A-strain tissues in vitro
Eleven-day p.c. A-strain cultures were grown in IF wells for 48 h or in MIF
wells for 72 h before carrying out MHA's with ak3 and ak2 respectively. In IF
wells the number of cells binding anti-H-2 remained similar to that in cultures
derived from the same organs and tested after only 24 h in cultures of lung,
heart, limb bud and fibroblasts but H-2 positive cells disappeared from the
three cultures of skin which were tested (Table 4). After 72 h growing in MIF
wells, heart and limb-bud cells from 11-day embryos failed to bind ak2 but 72 h
cultures of 12-day tissues contained similar numbers of ak2-binding cells to the
24 h cultures.
DISCUSSION
The results obtained in this study suggest that the expression of serologically
detectable H-2 antigens does not begin simultaneously throughout the embryo.
It is not possible to define the precise time of onset of H-2 expression since it is
conceivable that antigenic status may alter from the in vivo state during the 24 h
culture period, however, whilst H-2 antigens can be detected on a few cultured
216
K. J. KIRKWOOD AND W. D. BILLINGTON
cells of some 11-day p.c. embryonic organs, many more cells from 12-day p.c.
embryos express H-2 both in cell cultures of minced whole foetuses and in
cultures of individual tissue types. By 13-14-days p.c. many cultured gut, lung,
skin and limb-bud cells express H-2 antigens detectable by MHA whilst kidney
and gonad cultures contain few, if any, H-2 positive cells. Although it is not
possible to identify precisely the cell types present in the cultures, the morphological and antigenic differences detected between cultures from different organs
and strains of mice indicate that distinct cell populations were being observed.
There appears to be no clear relationship between the germ-layer derivation
of an organ and its histocompatibility antigen expression. The cultures of gonad
used in this study may have included not only primordial germ cells but also
the developing mesodermal primordial gonads into which they migrate and the
developing kidney (of mesodermal origin) of embryos at 12 days p.c. Amnion
cells express relatively little H-2 (Dillon, 1979) and cultured cells of this tissue
are also thought to be largely mesodermal (Jetten, Jetten & Sherman, 1979).
However, a significant number of cells from the mesoderm-derived heart clearly
express H-2 antigens at this stage whilst some cells in cultures of endodermderived gut and some cells in cultures of ectoderm-derived skin do not express
these determinants.
The time of detection of H-2 expression is not only a characteristic of the tissue
of origin of the cells but is also dependent on the genotype of the mouse. A progressively increasing number of A-strain gut cells from embryos of 12 or more
days p.c. bound ak3, but C57BL and C57BL/10ScSn gut only showed consistent
ab-binding in cultures of 14-day p.c. tissue. This apparent difference was not
solely because of the inclusion of stomachs and omenta in some of the dissected
preparations, and seems most simply explained by postulating that H-2b is
switched on later, at least in one population of cells, than is H-2k. However,
such an explanation of this result must be considered in the light of the possible
expression of altered or unusual H-2 forms discussed below.
In the present experiments there was almost always some variability in the
extent of H-2 expression in similar cultures from embryos of the same age and
strain and although cultures of most organs express H-2 on some of the cells
from day 11 p.c. onwards, variation was observed between the different organ
types in the speed with which extensive H-2 expression is achieved. This gives
some measure of support to the predictions of Edelman's hypothesis. Other
factors, such as the rate of cell division, the rate of antigen modulation in different organs, antigen masking and the state of differentiation of the various organs
may also influence the detection of H-2 antigen expression in the MHA assay
(Lengerova,Pokorna, Viklicky & Zeleny, 1972; Hausman & Palm, 1973; Cikes &
Friberg, 1977). In addition, antigen expression might vary according to the
culture conditions employed. This is a possible explanation for the observation that although monolayers of 14^-day foetal kidney can be lysed by cytotoxic
lymphocytes the reaggregation of these cells in hanging-drop cultures is not
H-2 antigens on mouse embryonic tissues
111
impaired by the presence of hyperimmune lymphocytes (Szulman & Johnson,
1978).
The work of Ostrand-Rosenberg and her colleagues (1977) has shown that in
blastocyst-derived cell lines, although specificities representing products of both
maternal and paternal K and D regions were detectable on each cell line, only
some of the antigenic specificities associated with each of the K- and D-region
polypeptides were expressed. The examination of cultured A-strain heart and
limb bud in MIF wells with two different sera directed against H-2k products
suggested that the ontogeny of the expression of H-2 antigens might be more
complex than hitherto anticipated. All of the sera ak1? ak2 and ak3 were raised
by spleen cell injections into the same strain of mice and thus should potentially
recognize the same cell surface determinants. However, with ak2, cells expressing
H-2k determinants appeared in both heart and limb-bud cultures from 11- and
12- day embryos in greater numbers than with cultures incubated with ak3, but
with akx no H-2k-bearing cells were detected until tissue from 13-day p.cembryos was examined, when the majority of limb-bud cells and a few heart
cells bound the antiserum. Similar differences in binding pattern were also
observed in cultures of parietal yolk-sac endoderm and amnion cells (Dillon,
1979).
When two commercial H-2d anti-H-2k sera raised between B10.D2 (H-2d)
and B10.Br (H-2k) (Searle Diagnostic Ltd) were tested, H-2 expression was
detected on 14-day p.c. B10.Br embryonic fibroblasts but not on CBA/Ca or
A-strain fibroblasts, although these latter two strains showed reactivity with
H-2b a H-2k at this stage (V. Owen, unpublished observation). The H-2a
haplotype is a recombinant between Kk and Dd regions (Klein, 1975). The
positivity of both ab (recognizing specificities in the Dd region of the H-2a
haplotype) and ak l5 ak2 and ak3 on the A-strain fibroblasts thus indicates that
both the Dd and the Kk products are expressed on these cells. It is tempting to
speculate that differences between the ak antisera in the relative abundance of
antibodies to the diverse H-2k antigenic specificities may have been revealing
some incomplete expression of the H-2 antigen polypeptides at this stage in
embryonic development.
In a study on the development of H-2 expression on erythrocytes, Boubelik,
Lengerova, Bailey & Matousek (1975) found that the timing of expression in all
of eleven different strains of mice could be divided into two types with little or
no overlap between them. The antigens were detectable by haemagglutination
either at birth or not until 3 dayspost-partum. Such differences in H-2 expression
have not been detected clearly in the present study, although it should be noted
that the MHA assay is extremely sensitive and can detect much lower concentrations of antigen than a haemagglutination assay. There are several reports of
quantitative increase in histocompatibility antigen expression at or around the
time of birth (see Edidin, 1972), and it therefore seems possible that the experiments with erythrocytes in fact detected only a large genetically controlled
218
K. J. KIRKWOOD AND W. D. BILLINGTON
increase in H-2 expression at birth rather than the onset of H-2 antigenicity.
Boubelik and colleagues examined cells of only one type whereas the present
MHA studies included cells of a variety ef different types and developmental
potentials even within each organ. The study on erythrocytes might therefore
reveal independent genetically controlled maturation in the expression of H-2
antigens while the cultured cells might interact with one another and mutually
influence their expression of histocompatibility antigens.
Although it has generally been considered that the development of H-2 antigenicity is autonomous (see Edidin, 1972), the lack of development of antigen
expression in the longer term cultures of various organs (Table 4) could be taken
to indicate that the initial expression of H-2 is to some degree dependent upon
external factors, such as cellular interaction. This is implied in many of the
hypotheses postulating a role for H-2 antigens in the control of cell interactions
in differentiation and morphogenesis and suggests that correct intercellular
relations may be necessary for the normal development of histocompatibility
antigens by either the triggering or the suppression of their expression.
We are indebted to the Rockefeller Foundation for financial support and to the
Medical Research Council for a studentship to K.J.K. (formerly K. J. Dillon). Mrs V.
Owen and Miss Vanessa Merry provided excellent technical assistance.
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(Received 21 April 1980, revised 10 July 1980)