J. Embryol. cxp. Morph., Vol. 12, Part 2, pp. 309-316, June 1964
Printed in Great Britain
Transplantation antigens in the mouse embryo.
The fate of early embryo tissues transplanted
to adult hosts
by MICHAEL EDIDIN 1
From the Department of Zoology, University College, London
WITH ONE PLATE
THE EVIDENCE for and against the presence in cells of embryo and foetal mice of
transplantation antigens, the cell-bound substances provoking the rejection of
allogeneic tissue homografts, rests on two classes of experiments: those relying
upon serological techniques for the detection of the antigens, and those involving
some test of transplantation antigen activity in intact animals. Experiments of
the former class have indicated that there are no histocompatibility antigens
present on the cells of most newborn or late foetal mice, but that these antigens
quickly appear during the first 2 days of neo-natal life, rendering cells liable to
agglutination or lysis (Pizarro, Hoecker, Rubenstein & Ramus, 1961; Moller,
1961<2, 19616). However, Moller (1961a) was able to show that cells of newborn
mice resistant to cytotoxic isoantisera did absorb the antibodies of these sera,
suggesting that the tissues of newborn mice contain transplantation antigens,
but that these antigens are not present on cells in sufficient numbers to make
them susceptible to agglutination or lysis. This suggestion was borne out by
later experiments (Moller, 1963) in which transplantation antigens were
demonstrated in 13-day embryos by injecting lethally irradiated liver cells of
these embryos into allogeneic adults and measuring the injected animals'
production of humoral antibodies against the grafted tissues.
Experiments in intact animals have demonstrated transplantation antigens in
embryo tissue indirectly, in terms of grafted animals' subsequent responses to
adult tissue grafts of the embryo donor strain. These responses may either be
depressed, when embryo tissues are grafted to neo-natal hosts inducing tolerance (Billingham & Silvers, quoted by Medawar, 1959), or elevated when
1
Author's address: Section of Cell Biology, The Weizmann Institute of Science, Rehovoth,
Israel.
310
M. EDIDIN
embryo grafts are made to adult animals (Billingham, Brent & Medawar, 1956;
Haskova, 1959). In either case calculation of the age at which antigens appear
in the grafts is influenced by data on the minimum time required to stimulate
host immune systems. Thus, Billingham et al. point out that in their experiments 9 days elapsed between making the initial embryo transplants and the
examination of test grafts of adult skin. Since only 6 days' exposure to antigen
is necessary to activate the hosts' response to the first graft, they argue, up to
3 days of growth and differentiation of the transplanted embryo tissue might
have passed undetected by the test system, and the earliest time of appearance of
transplantation antigens that may be assigned from their data is 14£ to \5\ days
of gestation. The tissues of embryos of this age have also been shown by
Moller (1963) to promote accelerated rejection of adult skin grafts.
Embryo cells procuring tolerance when injected at the end of the tolerance
responsive period of neo-natal animals may be said to be certainly antigenic at
the time of injection only if there is no possibility of prolonging this period.
The experiments of Brent & Gowland (1961) and of Shapiro, Martinez, Smith &
Good (1961) show that the tolerance responsive period may be extended by
increasing the dose of injected cells with increasing host age; therefore, the
11-day embryo cells reported by Billingham & Silvers to procure tolerance may,
in fact, have done so by proliferating and developing histocompatibility
antigens after transplantation.
Direct demonstration of transplantation antigens of early mouse embryos has
been achieved by Simmons & Russell (1962). These workers transplanted
whole 7-day embryos to adult hosts pre-immunized against the embryo donor
strain. Embryo grafts examined 6 days after transplantation were undergoing a
homograft reaction and the portions remaining showed no signs of histodifferentiation. Since the grafts failed to differentiate and succumbed to a
homograft reaction it appears that 7-day mouse embryo do contain histocompatibility antigens. However, the criterion of differentiation offered as
security for the presence of antigens in the transplants at the time of grafting
must be carefully applied, for cell maturation and development of transplantation antigens might take place without obvious cyto- or histo-differentiation,
and the antigens provoking an immune response in a pre-immunized host on
the 6th day after grafting might not appear until the 3rd day after grafting.
Only daily samples of grafted tissues, examined to determine the time of onset
of the homograft reaction against embryo cells, could indicate the presence of
histocompatibility antigens on the tissues as grafted.
In the experiments to be described grafts of 9-day mouse embryo tissue were
made to pre-immunized hosts and sampled daily for histological examination
for the presence of a cellular infiltrate. Control grafts were made to nonimmunized allogeneic hosts and to syngeneic hosts immunized against a third
mouse strain. These grafts were also sampled daily for comparison with the
experimental series.
Transplantation antigens in the mouse embryo
311
MATERIALS AND METHODS
Allogeneic grafts were made between strains CBA, C57 and A, strains differing at a major histocompatibility locus, H-2. Experimental grafts to preimmunized animals were made between C57/A embryos and A adults injected
with 1 x 106 C57 adult lymphocytes 14 days before grafting, and between CBA/A
embryos and A hosts immunized in the manner described with CBA cells.
Control grafts to allogeneic hosts were in the same combinations, but were
made to non-immunized A strain adults. Syngeneic control grafts were made
between A/A embryos and A adults injected with 1 x 106 adult C57 lymphoid
cells 14 days before grafting. This series served as a specificity control as well
as a control for the development of tissue grafts.
Matings for embryos were made from randomly selected stock animals,
using mature males and 5- to 7-week-old females brought to oestrous by injecting gonadotropins following the schedule suggested by McLaren & Michie
(1959). The day of detection of copulation plugs in the mated females was
taken as day 0 of embryo development.
On day 9 of development pregnant females were killed by cervical dislocation,
their uteri removed with sterile precautions to 0 • 9 per cent. NaCl, and muscle
and decidua stripped from the embryos with blunt watchmaker's forceps. The
embryos were then transferred to Hanks solution buffered to pH 7-4 with
0-016 MTris-HCl.
Extra-embryonic membranes were stripped away with fine forceps and the
somite age determined in one quarter to one half of the embryos; embryos used
for grafts were in the range 17-22 somites. The material grafted was 'tail tip',
the last one or two somites of trunk, plus all posterior unsegmented material.
This unsegmented material may be expected to give rise to another 30 somites
of tail in normal development; it has great growth potential, and differentiates
a number of easily recognizable tissues, including epidermis and bone.
Excised tail tips were transplanted to the ventro-lateral aspect of an adult
host's kidney. This was exposed by making an incision ventrally and manipulating the small intestine and caecum out of the incision, on to sterile gauze pads
(where they remained, covered with gauze soaked in normal saline, during the
operation), exposing the kidney. The capsule was torn with fine forceps and
the grafts inserted into the hole with a fine pipette (Plate, Fig. A). The intestines
were then replaced, and the incision closed with two layers of 0000 silk sutures.
Host mortality after this procedure was nil.
Grafts were recovered at 24-hr, intervals in all series, beginning 24-30 hr.
after operation. Biopsied grafts, together with adjoining host kidney tissue,
were fixed in Carnoy-Chloroform or in Petrunkevitch's (1933) Fluid. Paraffin
sections of the graft were cut at 5-7 fx and stained in Ehrlich's haematoxylin and
eosin for examination.
21
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M. EDIDIN
RESULTS
Day 1—24 to 30 hr. after transplantation
Grafts of all three series, syngeneic control, allogeneic control and allogeneic
grafts to pre-immunized animals, present the same appearance. They are
moderately blood-engorged and many cells are in mitosis. Mesenchymal and
neural cells are intermingled and the grafts' margins are irregular and include
quantities of cell debris. Host fibroblasts and histiocytes and a few lymphocytes
margin the embryo tissue (Plate, Figs. B, C).
Day 2—48 to 52 hours after transplantation
Both the syngeneic and allogeneic controls are even more blood-engorged
than at 24 hr.; there are no signs of drainage developing from the grafts.
Mitoses continue, at a higher rate than at 24 hr., and accumulations of cell
debris may be found at the grafts' edges. Tissue organization is poor, though
epithelial cysts are evident. Fibroblasts make up most of the host reaction
tissue margining the transplants (Plate, Figs. D, F).
The internal topography of the experimental allogeneic graft is virtually the
same as that of the controls. However, the host's reaction tissue around this
transplants contains many lymphocytes, the cells of the homograft reaction, and
some of these lymphocytes are clearly infiltrating the graft body (Plate, Figs.
E,G).
Day 3
The syngeneic and allogeneic control grafts closely resemble one another.
They have begun to acquire drainage, and the greater part of their volume now
comprises cells, rather than fluids. There are fewer mitoses than in 2-day grafts
but a high overall level of mitosis is still evident. Cells of different types are now
well segregated and the first morphologic steps in the differentiation of cartilage
EXPLANATION OF PLATE
All figures are of paraffin sections of grafts, stained in Ehrlich's haematoxylin and eosin.
FIG. A. 9-day embryo tail tip beneath the kidney capsule immediately after grafting.
FIG. B. 24-hr, syngeneic graft (left).
FIG. C. 24-hr, allogeneic graft in a pre-immunized animal.
FIG. D. Centre of an allogeneic tail tip graft 50 hr. after transplantation. Note the cell
debris.
FIG. E. 48-hr, allogeneic tail tip graft to a pre-immunized animal.
FIG. F. Margin of a 48-hr, syngeneic graft. A few histiocytes border the embryo cells.
FIG. G. Lateral portion of a 48-hr allogeneic graft in a pre-immunized animal. The infiltrating round cells are apparently small lymphocytes.
FIG. H. 72-hr, allogeneic graft.
FIG. I. 72-hr, allogeneic graft margin.
FIG. J. 72-hr, allogeneic graft in a pre-immunized animal.
.1. F.mkryol. c.xp. Morph.
M. EDI DIN
Vol. 12, Part 2
(Facing page 312)
Transplantation antigens in the mouse embryo
313
have been taken. The grafts' margins are well defined; the host reaction at these
margins is more intense than at earlier stages, and includes numerous small
lymphocytes and histiocytes (Plate, Figs. H, I).
The allogeneic graft to a pre-immunized animal now differs from the controls
both internally and at its periphery. Internally the graft is still blood-engorged
and obviously has not developed a vascular drainage. Though a few mitoses are
seen the graft tissues are disorganized and their periphery is not as clearly
defined as that of the controls. The host reaction tissue to this graft consists
mainly of round cells, especially small lymphocytes, and these are vigorously
invading the graft's interior (Plate, Fig. J).
Day 4
Both control grafts still resemble one another. They are now free of extravasated blood and contain many capillaries and venules. Mitoses continue, at
least in some tissues, and the grafts' differentiation has proceeded beyond that
seen in the 72-hr, specimens. Host tissue margining the grafts is almost entirely
fibrous and fatty connective tissue. A few round cells are present in both grafts,
but at least some of these are histiocytes full of basophilic cell debris.
Invading lymphocytes and histiocytes have now penetrated to the centre of
the blood-engorged experimental allogeneic graft. There are also some plasma
cells to be found in the heavy round cell infiltrate in and around the remaining
embryo cells. These survivors appear no different from previous grafts, and a
few of them are in mitosis.
Day 5
The appearance of the control grafts is still one of good health and differentiation. Chondrogenetic foci are present in both grafts, and the host reaction tissue
at their margins is quite feeble, consisting mainly of mature fibroblasts and a
few histiocytes.
The allogeneic experimental graft also contains a small amount of cartilage,
but this is surrounded by a massive lymphocytic and histiocytic reaction. There
is no evidence of either mitotic activity or vascularization.
DISCUSSION
The fate of allogeneic grafts in pre-immunized animals contrasts markedly
with that of allogeneic and syngeneic grafts in normal animals. Transplants of
the former series differ from those of the latter in four important particulars.
These are:
1. Graft organization. The boundaries of aggregates of a single cell type are
well defined in syngeneic and allogeneic grafts in normal animals, as are the
perimeter boundaries of these grafts. Allogeneic grafts in pre-immunized animals
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M. EDIDIN
never organize in such fashion. A difference between grafts of the two series is
first evident around 72 hr. after transplantation.
2. Graft growth and differentiation. All transplants differentiate cartilage
within 4 or 5 days of grafting. However, allogeneic grafts in pre-immunized
animals synthesize much less matrix than do grafts of the other series.
Difference between the two are apparent 72 hr. after transplantation.
3. Graft vascularization. Allogeneic grafts in pre-immunized animals never
establish drainage connexions with host blood vessels and remain bloodengorged throughout their brief existence. Syngeneic grafts and allogeneic
grafts in normal animals show signs of vascularization 3 days after grafting.
4. Round cell infiltration. Not even syngeneic grafts are free of round cells
at their margins, and some of these cells actually penetrate syngeneic grafts
during the 3rd day after transplantation. Allogeneic grafts in pre-immunized
animals are infiltrated by lymphocytes as well as histiocytes within 48 hr. of
transplantation; the infiltration becomes progressively heavier during the
following 48 hr.
The absence of vascularization and the round cell infiltration seen in allogeneic grafts in pre-immunized animals are indications of a homograft reaction
against the embryo tissues, closely approximating the pattern of accelerated
rejection of adult tissue grafts. The observations point to the presence of transplantation antigens in the cells of 9-day mouse embryos, for the round cell
reaction against the transplants appears, if anything, slightly earlier than the
same reaction to adult tissue grafts—grafts that are antigenic at the time of
transplantation—in pre-immunized animals (cf. Waksman, 1960), and this
excludes the possibility that the transplantation antigens provoking the reaction
appear only after embryo tissues are transferred to their new environment. The
further development of these antigens might, perhaps, be better investigated in
vitro than in intact animals, but the results indicate that earlier embryos, which
are conveniently grafted but rather inconvenient material for serology, should
be tested for the presence of histocompatibility antigens. Elucidation of the
earliest time of appearance of the antigens, and of developmental changes in
their concentration might lead to further understanding of the role of these
substances in cell economy.
SUMMARY
1. The fate of 9-day mouse embryo tissue grafts to adult hosts has been
studied.
2. Grafts to syngeneic adults survive and differentiate the precursors of a
wide range of tissues.
3. Grafts to allogeneic non-immunized adults differentiate the same types of
cells as syngeneic grafts, and in the short period considered (5 days) do not seem
to be affected by a homograft reaction.
Transplantation antigens in the mouse embryo
315
4. Allogeneic grafts to pre-immunized animals are invaded by small lymphocytes within 48 hr. of transplantation. In addition the grafts never develop
vascular drainage connexions with their hosts and show less differentiation than
the controls.
5. The early host reaction to allogeneic grafts in pre-immunized animals is
taken to indicate that transplantation antigens are present on the cells of 9-day
embryos.
RESUME
Les antigenes de la transplantation chez Vembryon de souris. Le sort de tissus
de jeunes embryons greffes sur des hotes adultes
1. Le sort de tissus d'embryon de souris agees de 9 jours greffes sur des hotes
adultes a ete etudie.
2. Les greffes sur des adultes syngeneiques subsistent et differencient les
precurseurs d'une grande variete de tissus.
3. Les greffes sur des adultes allogeneiques non immunises differencient les
memes types de cellules que les greffes syngeneiques, et durant la breve periode
envisagee (5 jours), ne semblent pas affectees par une reaction d'homogreffe.
4. Les greffes allogeneiques sur des animaux pre-immunises sont envahies par
de petites lymphocytes dans les 48 heures qui suivent la transplantation. De
plus, les greffes ne developpent jamais de systeme de drainage vasculaire avec
leurs hotes et montrent moins de differenciation que les temoins.
5. La reaction native de l'hote aux greffes allogeneiques dans les animaux
pre-immunises est prise pour indiquer que les antigenes de la transplantation
sont presentes dans les cellules d'embryons ages de 9 jours.
ACKNOWLEDGEMENTS
I would like to thank Dr P. B. Medawar and Dr Leslie Brent for their interest in, and
encouragement of, this work. These experiments were conducted during the tenure of a
Pre-doctoral Fellowship of the National Science Foundation (U.S.A.), and is part of a
thesis submitted in fulfilment of the requirements for the degree of Ph.D. in the University
of London.
REFERENCES
R. E., BRENT, L. & MEDAWAR, P. B. (1956). Quantitative studies on tissue
transplantation immunity III. Actively acquired tolerance. Phil. Trans. (B), 239,357-414.
BRENT, L. & GOWLAND, G. (1961). Cellular dose and age of host in the induction of
tolerance. Nature, Lond. 192, 1265-7.
HASKOVA, V. (1959). Transplantation immunity and immunological tolerance and the study
of antigenicity of tissues and their derivatives. In Biological Problems of Grafting
(eds. F. Albert & P. B. Medawar), pp. 95-106. Oxford: Blackwell.
MCLAREN, ANNE & MICHIE, DONALD (1959). Superpregnancy in the mouse. I—Implantation and foetal mortality after induced superovulation in females of various ages. / .
exp. Biol. 36, 281-300.
MEDAWAR, P. B. (1959). Isoantigens. In Biological Problems of Grafting (eds. F. Albert &
P. B. Medawar), pp. 6-24. Oxford: Blackwell.
BILLINGHAM,
316
M. EDIDIN
G. (1961a). Studies on the development of the isoantigens of the H-2 system in
newborn mice. J. Immunol. 86, 56-68.
MOLLER, G. (19616). Demonstration of mouse isoantigens at the cellular level by the
fluorescent antibody technique. /. exp. Med. 114, 415-34.
MOLLER, G. (1963). Phenotypic expression of isoantigens of the H-2 system in embryonic
and newborn mice. /. Immunol. 90, 271-9.
PETRUNKEVITCH, A. (1933). New fixing fluids for general purposes. Science, 77, 117-18.
PIZARRO, OLGA, HOECKER, G., RUBENSTEIN, P. & RAMUS, ALICIA (1961). The distribution
in the tissues and the development of H-2 antigens of the mouse. Proc. nat. Acad. Sci.
Wash. 47, 1900-7
SHAPIRO, F., MARTINEZ, C , SMITH, JUNE M. & GOOD, R. A. (1961). Tolerance of skin
homografts induced in adult mice by multiple injections of homologous spleen cells.
Proc. soc. exp. Biol, N. Y. 106, 472-5.
SIMMONS, R. L. & RUSSELL, P. S. (1962). The antigenicity of mouse trophoblast. Ann. N. Y.
Acad. Sci. 99, 717-32.
WAKSMAN, B. (1960). A comparative histopathological study of delayed hypersensitivity
reactions. In Cellular Aspects ofImmunity (eds. G. E. W. Wolstenholme & M. O'Connor),
pp. 280-322. London: Churchill.
MOLLER,
(Manuscript received 10th December 1963)