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J. Embryol. exp. Morph. 76, 83-93 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
H-2 antigens as markers of cellular genotype in
chimaeric mice
ByB. A. J. PONDER 1 , M. M. WILKINSON 1 AND
MAUREEN WOOD2
From The Institute of Cancer Research, Haddow Laboratories
SUMMARY
We describe an immunohistochemical method, using monoclonal anti-H-2 antibodies, for
demonstrating H-2 antigens in cryostat sections of a variety of mouse tissues. Double staining
allows simultaneous demonstration of both cell populations in tissue sections from H-2b <->
H-2k aggregation chimaeras.
INTRODUCTION
Chimaeric mice are produced by aggregation of 4- to 8-cell embryos of two
different strains (Mintz, 1971). The aggregated embryos are placed into the
uterus of a pseudopregnant foster mother, where they develop normally to term.
The tissues of the resulting mice are a mosaic of cells derived from the component
Strains. Not all aggregations result in chimaeric offspring: in those which are
chimaeric, the proportions of cells derived from each strain may vary between
mice and between tissues in a single mouse. For many studies with these mice,
it would be useful to be able to distinguish the two cell populations in tissue
sections; but few satisfactory cellular markers have been found, and none which
is applicable to a wide range of tissues (McLaren, 1976). H-2 antigens are
potentially excellent candidates since they are thought to be widely distributed
on different cell types, they are highly polymorphic and the detailed H-2 type of
many inbred strains of mice is known. Until recently, however, consistent
demonstration of H-2 antigens in tissue sections was technically difficult, and the
potential of H-2 antigens as cellular markers remained unexplored.
Using monoclonal anti-H-2 antibodies directly conjugated to alkaline
phosphatase or peroxidase, we have demonstrated H-2 antigens in cryostat sections of mouse tissues beginning a few days after birth. A detailed description of
the distribution of H-2 antigens in these tissues and of technical aspects of the
immunohistochemical method will be reported separately (Ponder, Wilkinson,
Wood & Westwood, 1983). In this paper we show that H-2 antigens can be used
1
Author's address: Institute of Cancer Research, Haddow Laboratories, Clifton Avenue,
Sutton,
Surrey SM2 5PX, U.K.
2
Author's address: Medical Research Council Laboratories, Carshalton, Surrey, U.K.
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B. A. J. PONDER, M. M. WILKINSON AND M. WOOD
as cellular markers in chimaeric tissues. Because both cell populations can be
stained simultaneously, the risk of misclassification of unstained cells is considerably reduced compared with methods in which only one component is stained.
MATERIALS AND METHODS
Mice
CBA/Ca (H-2 ), C57BL/6 (H-2 ) and BALB/c (H-2d) mice were bred in the
animal facilities of the Institute of Cancer Research. Male and female mice 5- to
12-weeks old were used for studies of H-2 antigen distribution. CBA/CaLac <-»
C57BL/6JLac ( C B A ~ B 6 ) and B10.A/Lac~C57BL/10ScSnLac-cc (BIO.A
<•» BlOcc) aggregation chimaeras were made at the MRC Laboratories, Carshalton. The aggregated embryos were brought to term and reared by C57BL/
6JLac X DBA/2Lac Fi hybrid foster mothers. BIO.A/Lac and BlOcc are congenic strains differing only at the H-2 locus (H-2a and H-2b respectively) and in
the insertion into C57BL/10ScSnLac-cc of an albino coat colour marker. Although BIO.A is H-2a, it carries determinants at the H-2K locus which are
recognized by monoclonal anti-H-2k antibody 11.4-1 (see below). Chimaeras 6
to 30 weeks old were used for these experiments.
k
b
Antisera
Mouse monoclonal antisera to H-2 specificities were used. The anti-H-2k
antibody was clone 11.4-1 (anti-H-2K) (Oi etal. 1978). Cells of the 11.4-1 clone
were obtained from the American Type Culture Collection, Rockville,
Maryland, recloned, and injected into BALB/c mice to obtain ascites (Hudson
& Hay, 1980). The IgG2a monoclonal antibody was purified from the ascites by
elution from protein A sepharose (Ey, Prowse & Jenkin, 1978) and subsequently
conjugated directly with alkaline phosphatase or peroxidase (see below). The
11.4-1 antibody prepared in this way gave a staining pattern identical to another
H-2k antibody, clone 100-27-55 (Lemke, Hammerling, & Hammerling, 1979)
(which reacts with a public determinant of the H-2K region; obtained as a gift
from Dr G. Hammerling).
The anti-H-2b antibody, FT6a9 clone 4, was derived from a fusion carried out
by Dr P. Thorpe of this Institute and Dr P. Edwards of the Ludwig Institute for
Cancer Research (London Branch) against the EL4 lymphoma of C57BL origin.
Its anti-H-2b specificity was established by 125I-binding assays against spleen and
thymus cells from C57BL/10, CBA, BALB/c and B10.M mice (kindly carried
out by Dr D. Katz, ICRF Tumour Immunology Unit, London). The antibody
was shown to be subclass IgGi by agar gel immunodiffusion. After recloning and
characterization, the hybridoma cells were used to raise ascites in BALB/c mice,
and the antibody was subsequently purified and conjugated in the same way as
the anti-H-2k antibody. A second anti-H-2b antibody, clone 141-31 (anti-H-2b,
H-2 antigens as markers of chimaerism
85
reacts with a private determinant in the H-2D region) was obtained as lyophilized
ascites from Camon (Weisbaden, West Germany). It was used in some experiments and gave an identical staining pattern to FT6a9-4.
Preparation of conjugates
Alkaline phosphatase conjugates of monoclonal antibodies were prepared by
direct glutaraldehyde conjugation. 5mg of purified monoclonal antibody were
mixed with 5 mg of alkaline phosphatase (from calf intestine; previously dialysed
against 0-lM-sodium phosphate buffer pH6-8) in a total volume of lml in
0-lM-sodium phosphate buffer pH6-8 and 1 % glutaraldehyde (Analar) for 3h
at room temperature. The mixture was then dialysed at 4°C overnight against
two changes of 31 phosphate-buffered saline (PBS) pH7-5 containing 0-2%
sodium azide. We did not find it necessary to purify the conjugate from unconjugated protein. Initially we used alkaline phosphatase from Sigma (type VII,
from calf intestine, no. P4521): recently this has been unavailable and the
replacement (no. P5521) shows non-specific binding to intestinal mucins.
Boehringer 567744 is a satisfactory alternative.
Peroxidase conjugates were prepared by cross linking, using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)
(Carlsson et al. 1978). Optimal conditions were determined by experiment.
10 mg of horseradish peroxidase (Miles) was passed through a sephadex G25
column on 0-1 M-NaCl, 0-1 M-phosphate buffer, pH7-5; added to250/il of SPDP
solution (12mg/ml in absolute ethanol) and allowed to stand at room temperature for 40mins. The mixture was passed through a sephadex G25 column
in the same buffer, and the peroxidase-containing fractions stored at 4°C. 5mg
of monoclonal antibody in 0-lM-NaCl, 0-1 M-phosphate buffer pH7-5 were
passed down a sephadex G25 column as above, and the peak fractions made to
1 ml in the same buffer. 20 [A of SPDP solution were added with stirring, and the
mixture stood at room temperature for 60min. It was then passed through a
sephadex G25 column in 0-lM-sodium acetate buffer pH4-5 containing 0-1 MNaCl, 25mM-dithiothreitol; allowed to stand at room temperature for 60min;
passed through a final sephadex G25 column in 0-lM-NaCl, 0-1 M-phosphate
buffer pH 7-5; added to the peroxidase-SPDP from the first step; and the mixture
stood at room temperature overnight. Background staining was greatly reduced
in most cases by purification of the resulting conjugate on a sephadex G200
column.
Conjugates were stored at 4°C in 50mM-Tris pH8-0, 0-lM-NaCl, lmMMgCh buffer containing 5mg/ml bovine serum albumin and 0-1% sodium
azide.
Preparation of tissue sections
Mice were killed by cervical dislocation. Tissues were dissected out and immediately placed on cork discs in OCT (Tissue-Tek, Miles) than snap frozen in
86
B. A. J. PONDER, M. M. WILKINSON AND M. WOOD
isopentane in liquid nitrogen. Frozen blocks were stored over liquid nitrogen.
5 jum sections were cut on a Slee HR Mk II microtome and mounted on gelatincoated glass microscope slides. Sections were air dried for 30min at room temperature and either used at once or stored at — 30 °C for up to 1 week.
Fixation
Sections were fixed in 10 mM-periodate-lysine-1 % paraformaldehyde pH7-4
(PLP: McLean & Nakane, 1974) for 2min at room temperature. After fixation,
the sections were washed with PBS containing 0-5 % bovine serum albumin
(PBS-BSA), and incubated in 10% mouse serum (from CBA mice) for
15-30 min before application of the first antibody.
Antibody incubations
The sections were incubated with monoclonal antibody-enzyme conjugate
diluted 1: 50 to 1: 300 in 100 [A 10 % mouse serum in PBS (pH7-6) for 1 h at room
temperature in a moist staining tray. For double staining, the best results were
obtained when the conjugates were applied in succession, alkaline phosphatase
conjugate first. The sections were washed in PBS-BSA between incubations.
Staining reactions
For peroxidase, the substrate was 3'3' diamino benzidine (DAB) (Sigma) in
PBS pH 7-6. Where necessary, endogenous peroxidase was inhibited immediately after fixation by immersion of the sections in 0-1 % phenylhydrazine hydrochloride in PBS. The use of FbCh-containing mixtures for this purpose caused
bubble formation which damaged the sections of some tissues, notably spleen.
For alkaline phosphatase the substrate was naphthol AS-BI sodium salt
(Sigma) coupled to Brentamine Fast Red TR, in veronal acetate buffer pH9-2
(Ponder & Wilkinson, 1981). Endogenous tissue alkaline phosphatase, other
than in small intestine, was inhibited by addition of 1 mM-levamisole (Sigma) to
the substrate mixture (Ponder & Wilkinson, 1981). In double-staining experiments, development of the peroxidase colour reaction was done first.
Sections were counterstained with Mayer's haemalum, and mounted in XAM
(DAB stain) or polyvinylalcohol (Fast Red stain). Photomicrographs were taken
with a Leitz microscope using Ilford Pan-F (with a green filter) or Agfa 50L film.
Controls
All slides in each experiment carried comparable sections of both CBA
(H-2k) and C57BL (H-2b) tissue as controls for the specificity of the first antibody. The staining pattern was not acceptable as specific if any staining was
attributable to the first antibody on the control section. Blocking experiments
with purified H-2 antigen were not attempted. Non-specific staining due to
endogenous enzyme activity was controlled for by staining one section in each
H-2 antigens as markers of chimaerism
87
Table 1. Tissues of normal young adult CBA/Ca and C57BL/6 mice in which
strong immunohistochemical staining of H2 antigens is consistently obtained
Tissue type
Tissue type
Thymus (medulla)
Spleen
Lymph nodes
Peyer's patch
Bone marrow
Epithelia of
small intestine
colon
uterus
skin
salivary gland (ducts)
epididymis, seminal vesicle
prostate
mammary gland
Sinusoidal lining cells of liver
Vascular endothelium
Lung parenchyma*
Adipose tissue
* Diffuse staining of alveolar walls, not identified as localised to any one cell type.
experiment after incubation in parallel without addition of any specific reagents.
The results described here are each based on consistent observations in at least
four (for most tissues, many more) separate experiments using tissue from different mice.
RESULTS
Demonstration of H-2 antigens in normal mouse tissues
The tissues in which H-2 antigens were consistently demonstrated by this
technique in normal young adult CBA/Ca or C57BL/6 mice are shown in Table 1.
Fig. 1. Mosaic patches in cryostat sections of chimaeric tissues. A/jxn sections.
H2k = red, H2b = yellow-brown. Counterstain (A, B, C, F) haemalum.
A, B, C. C57B10a<-»C57B10ScSncc, 12 weeks old. Adjacent sections of proximal
colon. (x220). Staining: (A) H2k only; (B) H2b only; (C) H2b and H2k double stain.
The epithelium of any one crypt appears to be either H2b or H2k, never mixed. There
is a mixed H2b, H2k population of cells in the lymphoid follicle. Occasional endothelial
cells and lymphocytes can be seen between the crypts.
D. CBA/Ca~C57BL/6, 6 weeks old. Dorsal skin. (x400). Stained for H2b and
H2k. Two C57BL/6 patches (H2b; brown staining) can be seen either side of a CBA
hair follicle (H2k; red staining).
E. CBA/Ca <-»C57BL/6, 8 weeks old. Thymus: cortico-medullary junction.
(x 450). Stained for H2b and H2k. In this mouse, the maj ority of the cells in the thymus
were of C57BL/6 origin (H2b). Three probable dendritic cells of CBA origin are indicated by areas of red staining. The staining pattern in this tissue is not cell localized
(see text).
F. C57B10a«->C57B10ScSncc, 15 weeks old. Epididymis. (x500). Stained for
H2b and H2k. There is mosaicism in the epithelium for individual tubules.
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B. A. J. PONDER, M. M. WILKINSON AND M. WOOD
H-2 antigens were also demonstrated in the epithelium of the bronchus, biliary
and pancreatic ducts, glandular stomach, salivary gland acini, renal collecting
ducts, renal pelvis, bladder and thyroid follicles, but staining of these tissues was
sometimes weak or even absent in individual mice. Details of this variability and
of trials of different fixatives and embedding methods will be reported elsewhere
(Ponder etal. 1983). No specific H-2 staining was seen in frozen sections of whole
CBA or C57BL foetuses at 12 or 15 days post coitum. In 17-day foetuses there
was moderately strong staining of most of the cells in the developing thymus, but
of no other tissues.
H-2 antigens as cellular markers in chimaeric mice
Eight B6«*CBA and six B10.A«-»B10cc aggregation chimaeras have been
studied. Mosaic patches could be consistently demonstrated in cryostat sections
of each of the tissues listed in Table 1 except for mammary epithelium, which we
have so far not examined in chimaeras. Patches were also demonstrated in
bronchial epithelium, gall bladder epithelium, urinary bladder, salivary gland
acini and renal collecting ducts, but here the results were less consistent because
of variation in the intensity of H-2 staining, both from mouse to mouse and within
the same tissue. Examples of patches are shown in Figs 1 and 2. Both component
cell populations are positively identified.
The resolution of the present immunohistochemical method, using frozen
sections, can be judged from the Figures. Morphology is sufficient to delineate
the boundaries of patches clearly at low magnification: but in epithelia such as
in the skin, uterus, bronchus or epididymis assignment of the type of individual
cells at the interface between patches may be difficult. The morphology of lymphoid tissues, bone marrow and liver is less satisfactory, and assignment of the
H-2 type of individual cells in the finely intermingled populations in these tissues
is often impossible.
Detailed description of the shape, size and distribution of patches in different
tissues will require further analysis. One finding which we did not anticipate is
illustrated in Fig. 1. The epithelium of each intestinal crypt in our H-2b<-»H-2k
chimaeras appears to be composed entirely of cells of a single H-2 type. No
exception was seen in many thousands of crypts in 14 chimaeras.
Fig. 2. Mosaic patches in cryostat sections of chimaeric tissues. 4/im sections.
Double stained as in Fig. 1; photographed on Ilford Pan Ffilmwithout colour filters.
H2k (CBA component; red reaction product) appears black; H2b (C57BL/6 component; yellow-brown reaction product) appears grey. A, B: no counterstain; C:
lightly counterstained with haemalum.
A. CBA/Ca «*C57BL/6,6 weeks old. Liver, (x 100). The cells which are stained
are Kupffer cells.
B. CBA/Ca<-> C57BL/6, 8 weeks old. Non-pregnant uterus. (xl60).
C. CBA/Ca **C57BL/6, 6 weeks old. Salivary gland. (xl50). Patches of acini
of CBA origin appear black; a group of ducts of CBA origin is seen at bottom right.
H-2 antigens as markers of chimaerism
89
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B. A. J. PONDER, M. M. WILKINSON AND M. WOOD
DISCUSSION
We have shown that H-2 antigens can be used as cellular histological markers
in a wide variety of tissues from chimaeric mice. The results indicate that H-2
antigens satisfy, at least in part, most of the criteria required of an ideal cellular
marker. These are that the marker should be cell-autonomous, cell-localized,
easily demonstrated in tissue sections, ubiquitous in embryo and adult, stable
and developmentally neutral (McLaren, 1976; Oster-Granite & Gearhart,
1981). H-2 antigens have one advantage over existing markers, which is that both
component cell populations can be stained in the same tissue section. This
reduces the likelihood that unstained cells will be mistakenly assigned, which is
a possibility when only one component of the chimaera is positively identified.
Cell-autonomous expression (that is, expression in chimaeric tissue strictly
according to cellular genotype) might be expected of transplantation antigens,
but it is difficult to prove. We have some indirect evidence in that identical
mosaic patches are revealed in chimaeric colonic epithelium whether the marker
is H-2 antigen or the presence of binding sites for Dolichos biflorus agglutinin,
a polymorphism unrelated to H-2 haplotype in strain distribution (B. A. J.
Ponder & M. Wilkinson, unpublished results). This supports the view that each
of these markers is indeed cell-autonomous in chimaeric tissues.
The localization of H-2 antigens to the cell membrane is apparent both in Fig.
1 and in the immunoelectronmicroscopy studies of others (Van Ewijk, Rouse &
Weissman, 1980). The demarcation of adjacent patches in, for example, the
epithelium of the epididymis and of the uterus (Figs 1 and 2) suggests that there
is little extracellular shedding and spread of the antigens. A possible exception
may be in the thymic medulla, where we and others (Van Ewijk et al. 1980) find
a confluent pattern of staining which cannot be localized to a particular cell even
at the ultrastructural level.
The demonstration of H-2 antigens in tissue sections with our methods is
straightforward but has two major limitations. First, adjacent cells labelled by a
surface membrane marker are difficult to distinguish. Second, cellular detail is
lost in frozen sections. The problem of distinction between membrane markers
on adjacent cells may be improved by the use of double immunofluorescence
(see for example, Janossy et al. 1980). The problem of frozen section morphology
may be overcome if current attempts to raise antibodies against formalin-fixed
H-2 antigens are successful; or failing that, may be ameliorated by using the
techniques for immunoelectron microscopy mentioned above. Melnick, Jaskoll
& Marazita (1982) reported the demonstration of H-2 antigens in fixed, paraffinembedded sections of embryo mouse plate. In the absence of the control using
the same antibody on tissue of a different H-2 haplotype their results are difficult
to assess. Despite the use of the same 11.4-1 antibody we are unable to repeat
their finding.
Of the cellular markers so far described, only one seems likely to be truly
H-2 antigens as markers of chimaerism
91
ubiquitous: the satellite DNA polymorphism reported by Rossant, Vigh,
Siracusa & Chapman (1982). H-2 antigens seemed good candidates because
they have been reported present on the majority of adult tissues (Edidin, 1972)
as well as on many tissues of the embryo from midgestation onwards (Kirkwood
& Billington, 1981). These results were obtained using serological methods,
which are not only more sensitive than immunohistochemistry, but may be
misleading when they reflect the H-2 activity of a mixed population of cells in
a tissue sample. With the present immunohistochemical technique, the consistently positive tissues are restricted to the thymus in the embryo, and in the
adult to those listed in Table 1. Even so, this range of tissues compares well
with the restricted set for which markers were previously available (McLaren,
1976; Oster-Granite & Gearhart, 1981). Ironically, we were unable to demonstrate H-2 antigens in the central nervous system: the one tissue in which they
have previously been used as histological cellular markers in chimaeric tissue
(Dewey, Gervais & Mintz, 1976). Positive staining of endothelial cells in our
sections provided an internal control for the method. The discrepancy is
unexplained.
The stability of cellular markers is likely to be a particular problem when
chimaeras are used for studies of carcinogenesis. The inappropriate expression
of H-2 haplotypes in mouse transplantable tumours and cell lines has been
reported (Festenstein et al. 1979; Imamura & Martin, 1980). Our preliminary
findings in colonic epithelium of CBA/Ca and C57BL/6 mice treated with
dimethylhydrazine indicate that H-2 antigen expression is maintained on
dysplastic epithelium. We have no evidence of expression of H-2b determinants
on H-2k epithelium, or vice-versa.
Whether H-2 antigens are developmentally neutral is an interesting and unresolved question. Mismatch at H-2D or H-2K loci leads to increased inhibition
of movement between cell outgrowths confronted in culture (Curtis & Rooney,
1979; Bartlett & Edidin, 1978). From this experimental evidence and on theoretical grounds (Bodmer, 1972; Ohno, 1977) it has been argued that products of the
major histocompatibility complex may control interactions between nonimmune cells, and cell movements in organogenesis. The apparently normal
cooperation in chimaeric tissues of cells from pairs of strains similar to those
showing contact inhibition in the culture experiments seems to argue against an
in vivo counterpart of this in vitro phenomenon. Culture experiments using an
assay based on collection of single cells by aggregates of different H-2 haplotypes
also failed to show an effect of H-2 (McClay & Gooding, 1978). Nevertheless,
a subtle effect of H-2 type on cellular interactions in development might be
sought by a comparison of the patch size of the mosaic in tissues from chimaeras
of different strain combinations, an approach discussed by West (1976).
B. A. J. Ponder holds a Career Development Award from the Cancer Research Campaign,
who funded this work.
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B. A. J. PONDER, M. M. WILKINSON AND M. WOOD
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ROSSANT,
{Accepted 8 February 1983)
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