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346




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ORNA-p-346
Proceedings of the Xth Congress of the
International Society of Blood Transfusion
CLONE -703-2-
LACK OF EXPRESSION OF PARENTAL ISOANTIGEN(S) IN F, HYBRID MICE*
G. CUDKOWICZ AND J. H. STIMPFLINO
Biology Division, Oak Ridge National Laboratory, Ohk Ridge, Tenn., U.S.A.
and
The Jackson Laboratory, Bar Harbor, Maine, U.S.A.
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#Research sponsored jointly by the U.S. Atomic Energy Commission under contract
with the Union Carbide Corporation and by Research Grant CA-01329 of the National
Cancer Institute, U. S. Public Health Service.
Send proof and manuscript to: Dr. G. Cuidkowicz
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee, U. 8.A.
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INTRODUCTION
The growth of transplanted bone marrow cells of C57BL and C57BL/10 (3-2/a-2)
donor mice 18 deficient in (1) arredlated Fhybridsł,5, (11) R-2" beterozygous
backcross progeny mice, but not in H-2 bornozygous segregants”, and (111) Irradiatod
F bybrids from crosses between C57BL/10 and congenic mice differing at the K region
of H-2, but not in bybride from congenic parents differing at the D region of
A-2 or at other a loci. In contrast, transplanted hemopoietic cells of c3H
(H-2*/H-2*), A (H-2°/H-2), and C57BL/10.DE (H-2°/H-2°) donors grow equally well
in loogenic, F, bybrids, and/or segregating backcross progeny mice.303. Furthermore,
hybrid resistance to C57BL/10 marrow can be enhanced or abolished by prolonged
exposure of the resistant mice to C57BL/10 tissue antigens, whereas exposure to
18oantigens of C3A or C57BL/10.D? nice does not affect the hybrid's resistance
to C57BL/10 marrow st. Collectively, these data are consistent with the supposition
that genetic interaction in beterozygotos between alleles at the K region of
H-2 modified the expression of parental C57BL and C57BL/10 1soantigen (8) relevant
for the fate of transplanted hemopoietic cells. Such 18oantigens do not, however,
affect the fate of parental skin grafts, since these are usually tolerated by F.
bybride resistant to parental marrow grafts. In this study, we lovestigated the
growth pattera of marrow cells from a variety of inbred strain donors on transplantation
into irradiated F, bybrids, in an attempt to correlate bybrid :
manifested by deficient growth of parental marrow, with the H-2 phenotype of the
dunor and recipient mice.
lato irradiated
a an attempt to co
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EXPERIMENTAL
F, hybrid and parental strain mice of both sexes were exposed to 700 R
of 300 kv (peak) whole body x rays at the age of 12-15 weeks. A few hours later
they were injected by tail vein with 10° nucleated femoral marrow cells harvested
from 12-15 week old donors of the same sex and sus sended in 1 ml of Tyrode's
solution. The growth of the transplanted marrow cells was estimated 5 days
later by the extent of regenerative hyperplasia in the recipient spleen, as
measured by the incorporation of rad 10active 5-10do-3'-deoxyuridine- 1341743- SUAR)
into newly synthesized splenic desxyribonucleic acid (DNA). For this assay each
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recipient was given intraperitoneally 0.5 MC of - TUR, and seventeen bours
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later it was killed, its spleen removed, and tbe incorporated radioactivity measured
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by means of crystal scintillation counting. Details on the method and on the
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influence of variables such as sex and age of the animals, the magnitude of the
X ray exposure to recipients, and the time between grafting and 134 TUAR labeling,
were investigated previously. The splenio uptake of 13-TUAR was expressed in
percentage of the total radioactivity injected.
Of 16 different inbred strains of mice, 10 possessed marrow cells capable of
sustained proliferation in 18ogenic and in A-2 heterozygous F bybride (Table 1).
The 1-2 phenotype of these mice, as determined by bemagglutination, was A-2Ⓡ for
strain A, -2a for strains BALB/c and DBA/2, 8-2* for strains AKR, C30, C57BR,
and 101, -2 for strain DBA/1. The -2 type for strains FU and RFM 18 not
knowa, except that there are indications that the H-2 allele of RFM 18 similar to
8-26. The marrow of the other six strains o: mice, all homozygotes for 8-2,
falled to grow detectably in B-2 beterozygous P, recipients, although the cells
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were competent to grow in 18ogenic or in H-2° homozygous F recipients (Table 1).
Three of the H-2 strains were related to the C57BL strain, namely C57BL/6,
C57BL/10 and C57L; however, the strains LP and 129 were not. On the other hand,
C57BR, a moube strain related to C57BL, but A-2" in phenotype, did not possess
the trait of deficient growth of transplanted marrow in bybrids (Table I).
to find additional support for the correlation between deficient growth of
parental marrow in hybrids and the H-2° phenotype, marrow cells from 18ogenic-resistant
or congenic lines.° (1.6., lines identical with a given background strain except
for substitution of a chromosome segment carrying an allele at a single 8 locus)
were tested for growth in suitable F hybrids. The marrow of lines congenic with
C57BL/10 showed the deficient growth pattern characteristic of C57BL/10 marrow,
1f the allelic substitution occurred at one of the loci H-1, 1-3, or A-4 (Table II).
If however, the -2° allele of C57BL/10 was substituted by 8-2, 1-2 or 1-2
the narrow acquired the ability to grow without detectable impairment in F, bybrids,
irrespective of the C57BL/10 genetic background (Table II). Conversely, the marrow
of lines congenic with A, 038, or DBA/1 lost the ability of proliferating as well
in hybrids as in 1sogenic recipients, only when the A-2 allele was introduced,
irrespective of the genetic background of these strains (Table II).
(C38 X C57BL)F, mice are resistant to transplanted C57BL marrow and to marrow
grafts from other -2° strains (Table III). The resistance of such hybrids to
C57BL marrow can be abrogated by 4 injections of C57BL spleen cells given at weekly
intervals, but not by injected spleen ce.118 of the other parental strains,4. One
week after the last spleen cell injection (2 x 10' cells per injection, 1.p.)
groups of (c38 X C57BL)F, bybrids were irradiated and grafted with marrow cells
from C57BL donors and from donors of five other 8-2 strains to determine whether
or not the induced unresponsiveness toward C57BL marrow would extend to cells of
other H-2 strains toward which tbe hybrids were also resistant. Although the
treatment of the hybride with cz1 cells did not modify their resistance to 8-25
marrow cells, repeated injections of C57AL spleen collo induced unresponsiveness
toward all the tested 1-2 marrow grafts (Table III).
CONCLUSION
Marrow cells of parental donors failed to grow on transplantation into
irradiated F, hybrid recipients ir tbe donors were H-2° homo uygctes and if the
recipients were A-2° heterozygotes. This failure of growth could not be correlated
with any other known allele at the H-1, 1-2, H-3, or H-4 locus, or with any other
aspect of the donor's genetic background.
(C3H X C57BL)F, hybrids that had been
made unresponsive toward C57BL marrow grafts by pre-exposure to C57BL tissue
180antigens were similarly unresponsive, 1.e., nonresistant, toward marrow grafts
of other H-2°/8-2° donors. Tus indicated that the factor responsibe for bybrid
resistance to parental marrow was similar in all the A-2° /H-2° strains that were
investigated.
The findings support the interpretation that genetic laterection involving a
subunit of 8-2 (associated with the K region?) results in lack of expression in
F, heterozygotes of 1soantigen (s) of the 1-2 phenotype, affecting the survival
of homozygous parental, marrow grafts.
Table 1. Growth of parental marrow cells transplanted into F, hybrid recipient mice.
Donor
Recipient "
Splenio uptake of "LUAR (% $ staad, error)
Strain
H-2
Strain
H-2
A
ala
C57BL/10 x A
.
bla
0.49 .03
C57BL
b/b
C3H X C57BL
C57BL X C57L
k/b
b/b
0.02 $ .001
0.61 t .04
C57BL/6
6/6
C57BL/6 X DBA/2
C57BL X C57L
bld
b/b
0.02 .001
0.70 .05
C57BL/10 b/b
C57BL/10 X A
C57BL/10 X 129
0.03 = .002
0.78 5.07
6/6
C57L
b/b
C57L X A
C57BL X C57L
b/b
0.03 = .001
0.59 .03
LP
b/b
LP X C3H
LP X C57BL/10
b/b
0.03 £ .001
0.65 £ .03
129
b/b
C3H X 129
C57BL/10 X 129
k/b
b!b
0.03 = .001
0.70 .06
C57BL X BALB/C
bld
0.47 $.04
BALB/O
DBA/2
did
dla
C57BL/6 X DBA/2 . b/d
0.51 . .06
AKR
сзн
k/k
к/k
k/k
k/k
C57BL/10 X AKR b/k
C3H X C57BL kib
C57BR X C57BL/10 k/b
C57BL X 101 b/k
0.75 £ .03
0.55 + .04
0.59 .05
0.60 3.04
C57BR
101
DBA/1
gla
C57BL/10 X DBA/1 /9
0.67 £ .04
FU
C57BL/10 X FU
b/?
unknown
unknown
0.70 £ .07
0.53 .03
REM
RFM X C57BL/10
3/6
Ten - twenty mice of both sexes in each group. Donor colls vere also given to isogenic
recipients; the splenio uptake of ***IDUR in the latter was comparable to the uptake in
suscaptible Fj hybrids. :
Table II, The effect of allelic substitutions at single H loci on the growth pattern of
parental marrow grafts la F, hybrid mice heterozygous at the H-2 locus.
Background Strain
Growth Pattern of
- Congenic Strains
Allelio Substitution Designation
Marrow Grafts
C57BL/10
None
C57BL/10
Deficient
(H-1° H-26 1-34 Hungary
B10.BY and B10.129 (5M)
Deficient
H-20 and H-1
H-2*, H-2" and H-24
B10.A, 810.BR and B10.02
Optimal
H-36
B10.LP
Deficient
ii.
Handebo
B10.129(21M)
Deficient
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A (H-2
None
Optimal
H-23
A. SW
Optimal
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H-26
A.BY
Deficient
.me
C3H (H-19 H-2k)
None
сэн
Optimal
Hozó
C3H.K
Optimal
H-26
C3H. SW
Deficient
on
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DBA/1 (H-29)
DOWA (hazeny
None
DBA/1
Optimal
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D1.LP
Deficient
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Table III. Growth of trasplanted mirrow cells homozygous H-2° la (C3H X CS7BL)F,
hybrid vice pretruted with parental spleen cells.
Usuake of "
Donor Strain
Untrusted
(Resistant)
LODE (& stand, error) in recipient spleens
сэн spleens C57BL spleen
(Resistant)
(Nonresistant)
C57BL
A. BY
0.03 $ .001
0.05 = .01
0.07 $ .02
0.06 $.01
0.09 £ .04
0.04 + .01
C3H.SW
0.03 £ .001
0.06 $ .02
0.05 $ .009
0.04 $ .005
0.05 £ .01
0.03 $ .006
0.79 £ .05
0.58 £ .05
0.68 $ .07
0.73 $ .06
DI.LP
LP
0.65 £ .08
129
0.80 $ .06
Four lajections of 2 x 10' nucleated spleen cells were given latraperitoneally
at weekly Internals, Qae veek after the last injections groups of 7 • 10 alce
were irradiated and gnfted with 10° nocluted wrrow cells.
10
REFERENCES
1.
Cudkowicz, G. and Stimpfling, J. H.: Deficient growth of C57BL marrow cells
transplanted in F hybrid mice: association with the histo-
compatibility-2 locus. Iumunology, Lond., 1: 291-306 (1964).
2. Cudkowicz, G. and Stimpfling, J. B.: Hybrid resistance to parental marrow
sratts: association with the K region of H-2. Science 144:
1339-1340 (1964).
3. Cudkowicz, G. and Stimpfling, J. 8. : Inmunological basis for deficient growth
of C57BL/10 parental marrow in F bybrid mice. red. Proc. 23:
202 (1964).
4. Cudkowicz, G. and Stimpfling, J. H.: Induction of immunity and of unresponsiveness
to parental marrow grafts in adult F, hybrid mice. Nature, Lond.,
in pre68.
i 5. McCulloch, E. A. and 1111, J. E. : Repression of colony-forming ability of
C57BL hematopoietic celle transplanted lato non-18ologous bosts.
J. cell. comp. Physiol. 61: 301-308 (1963).
6.
Popp, R. A.:
Personal communication.
7. Snell, G. D.: Histocompatibility genes of mice. II. Production and analysis
of isogenic resistant lives. J. Nat. Cancer Inst. 21: 843-877
(1958).
8.
Saell, G. D. and Stevens, L. C. : Histocompatibility genes of mice. III. 8-1
and H-4, two histocompatibility loci in the first linkage group.
Imamology, Lond., 4: 366-379 (1961).
San
DATE FILMED
12/ 9 / 64




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