.
.
-TL11
LYW
)
.
+
Von
TE
tras
*
T27
UNCLASSIFIED
ORNL

1.
.
..
ar
RA
- L
yri-
.
-
..
42
T!
1

•
'.
P

M
.
SL
**
*
1
.
9
.
AS
PL
INT
.
S
.
.
CO2
4.
***
.
TAS
...
WE
.......
.
.
.
..
APR 27 1966
-LEGAL NOTICE ---
The report mu prepared a ma account of Govern.deat spoonored work. Nellber torr Valued
Batu, mar 0. Codmiasta, nor my paroog kung oo bedait of the Cabanatulon:
A. Maka wymtuary or produton, nproued or implied, wild rupect to the accu-
racy, completed., or wretainfo of the inforon os contatood in the report, or war the way
ol way latortention, appunta, pothod, or .ocu. daclosed la o report may not lalr logo
printly owad mau; or
B. Anunc. way liabilities will repect to the use of, or lor dumugo. Huta from the
une of usy taformation, appariaus, paibod, or procev. dixcloued to a report
Ao wrod in the above, person stof og betal of the Cantonboa" lacluceo way una.
ployer or coatructor of the Commission, or employee of such utractor, to the extent lidt
such maployee or coatractor of the common, or ecplayer of the roatractor prepares.
donabato, or provides acc. to, ay laformados purinuat to do employant or cootrace
with the Commiustoa, or wito employmrat mib such coats actor.
.
T
.
the Union Carbide Corporation.
*Research sponsored by the U. 5. Atomic Energy Commission under contract with
ARE ON FILE IN THE RECEIVING SECTION.
DE PUBLIC IS APPROVED. PRCUEDURES
SATENT CLEARANCE OSIND. REVCAN FO)
Oak Ridge, Tennessee
Biology Division, Oak Ridge National Laboratory
Gustavo Cudkowicz
IMPLICATIONS FOR BONE MARROW CHIMERAS*
HYBRID RESISTANCE TO PARENTAL HEMATOPOIETIC CELL GRAFTS:
ORN P1111
PENF-
Proceeding
?-19
Paris, France, Sept. 1-9, 1964.
on Allogeneic Bone Marrow Transplantation
Proceedings of the International Conference
MASTER

Running title: Hybrid Resistance to Parental Marrow Grafts.
Send proof to: Dr. Gustavo Cudkowicz
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37831
U.S.A.
INTRODUCTION
F, hybrids from crosses between inbred strains of mice are universal
acceptors of parental strain skin grafts, the only known exceptions having
arisen from unmatched sex-linked histocompatibility factors (2,3,18,20,35,
37).
This type of observetion and the results of more general investigations
on interstrain susceptibility to tissue grafts (4,22,29) and on distribution
of erythrocyte and nucleated cell isoantigens (1,19,32,33) in inbred strains
of mice are the basis for the widely accepted concept that the fute of
foreign tissue grafts 18 decided in this species by dissimilarities in the
products of codominant histocompatibility (or h) genes, the inheritance and
phenotypic expression of which are not complicated by genetic or immunological
interaccions (24, a review).
F, hybrid mice fail, however, to be universal acceptors of parental
strain hematopoietic cell grafts given after exposure of the prospective hybrid
recipients to conditioning doses of ionizing radiation. In fact, F, hybrid
mice of certain genetic constitutions may not support the optimal growth of
,
transplanted cells from bone marrow (11,15,23), spleen (5,23), or fetal liver
(23) of one of the parental strain donors, although they fully support the
growth of grafted skin tissues from donors of either parental strain (11).
The failure of bone marrow cells of inbred strains of mice to grow on transplantation
into irradiated F, recipients has been thoroughly investigated during the past
few years and has been ascribed to the lack in F, hybrids of one or more
parental isoantigens the expression of which requires homozygosity at the
relevant genetic locus (15). It has been firmly established, for example, that
marrow cells of donor mice which are homozygotes for the H-2 allele grow .
.
.
. . -
-
-
-
* - - -
-
-
-
-
-
(T-1)
deficiently in F, hybrid recipients if the latter are heterozygous at the
D region of the complex H-2 locus, irrespective of homozygosity or
heterozygosity at other regions of H-2 or at other I loci (12-15). On the
left side of Table 1, strains of mice H-?" in phenotype are listed,
possessing marrow cells which grow deficiently on transplantation into
appropriate F, recipients; on the right side of the same Table is a list of
mouse strains not H-2° in phenotype, possessing marrow cells which grow
optimally in F, recipients, that is, in a manner which is indistinguishable
from their growth on transplantation into isogenic recipients. Thus, the
grafting of marrow cells detected the occurrence of interaction in the
inheritance of a component of the H-2 locus, resulting, presumably, in lack
of expression in hematopoietic cells of parental isoantigens and, consequently,
in parent-hybrid incompatibility. Furthermore, experiments with other
hematopoietic cells, such as thymocytes or leukemia cells, indicated also that
certain sites of the complex H-2 locus control the synthesis of tissue specific.
1soantigens (7), not necessarily shared by skin tissue nor expressed on the
surface of mature red blood cells.
Another example of resistance by F, hybrids to grafted hematopoietic cells
has been recognized recently (9). The genetic locus which controls this
resistance has not yet been mapped, but is known to be associated with the sex
chromosomes. The properties of the sex-associated resistance are not known as
well as the properties of the H-2 controlled resistance. Therefore, in this
paper I will report only on the possible implications for marrow chimeras of
the latter type of resistance, particularly since it seems to be specific for
grafted hematopoietic cells.
The strength of the resistance of F, hybrids from crosses involving
several strains of mice has been estimated by determining the nwaber of grafted
parental H-2° donor cells necessary to override it and in repopulate
thereby the spleens of the recipients. The extent to Bhich 'strong' hybrid
resistance is weakened by exposure of the F, recipients to x rays has also
been established. From both studies, the strength of hybrid resistance to
parental strain marrow grafts appeared to be of the same order of magnitude
as the strength of the resistance of inbred mice to allogeneic marrow grafts.
Cimeras resulting from infusion of irradiated parental strain mice with
marrow or spleen cells from resistant F, hybrid donors do not display overt
signs of graft-versus-host, that is, hybrid-anti-parent, reactions, as would
be expected if the grafted F, cells or their competent descendents would
- reaction
mount a homograft against parental isoantigens. I have determined by a direct
method whether such a reaction was actually present in the hybrid-to-parent
chimeras, and found that although hematopoietic cells from resistant F,
donors repopulate the hematopoietic system of parental strain recipients, they
fail to transfer adoptively the resistance to recipiant mice H-2° in phenotype.
The F, cells do, however, transfer the resistance to isogenic and to parental
strain weight ont mice carrying the H-2 allele other than H-2. Presumably,
they become specifically unresponsive to parental isoantigens associated with
H-2 after transplantation into H-2° mice, owing to the excess of isoantigen
to Shich they are exposed. This may be the reason why the hybrid-to-parent
chimeras were not afflicted by a graft-versus-host reaction.
I wish to indicate that studies such as these may help to elucidate the
nature of the specific control mechanisms involved in the establishment and
growth of hematopoietic cell grafts in irradiated animals. Such studies have
shown, so far, that marrow transplantation is not complicated by hybrid
resistance, that is, by non codominant I factors, to a greater extent than
by histoincompatibilities due to the codominant I genes.
MATERIALS AND METHODS
Mice.
The strains of mice used, their origin and their relevant genotypes
(T-2)
are listed in Table 2. F, hybrid mice are designated by listing first the
female and then the male parental strain. The animal) were used at 12-15
weeks of age.
Assay for proliferative activity. The iododeoxyuridine-S-I (15-IVAR)
technique used to measure in vivo the proliferative activity of marrow cell:
suspensions, was similar to that described previously (11). Known numbers of
marrow cells were injected into the tail vein of x-irradiated recipient
132
animals. After an interval of 5 days, the mice were given intraperitoneally
0.5 uc of +SIUR and were killed 17 hrs. later. Their spleens were removed,
placed in a glass test tube, and counted in a well-type scintillator to
139
measure retained +S+I radioactivity. Injected marrow cells that had settled
in the spleen and were competent to proliferate produced DNA synthesizing
hematopoietic cells which become labelled upon exposure to 131IUAR, a specific
DNA precursor. The results of such measurements are expressed in terms of
percentage of the injected radioactivity retained in the spleen.
Preparation of cell suspensions. Marrow cells were obtained from femurs and
tibias by flushing the bones with Tyrode's solution. Spleen cells were
suspended by teasing the capsule of the organ with a needle and gently'shaking
the resulting spleen fragments in Tyrode's solution.
The cell suspensions were filtered through a 200 mesh stainless steel
screen, and nucleated cells were counted in an electronic particle counter, after
red blood cells were lysed.
4
Irradiation. Recipient mice were irradiated with 300 kv (peak) x rays,
HVL 0.5 mm Cu, at an exposure rate of approximately 70 R per minute.
During exposure, the mice were housed in partitioned revolving Lucite cages.
Exposure measurements were made under comparable conditions with a Victoreen
R meter.
RESULTS
Strength of hybrid resistance.
The following H-2 heterozygotes were
BIO X
used as recipients of marrow cell grafts from inbred H-2 parental strain
donor mice. C3H/Anf X C57BL; C3H x 129; C3H.SW X C3H; (B10.D2; and A X A.BY.
The hybri,ds were exposed to 700 or 900 R of x rays and injected a few hours
later with graded doses of nucleated marrow cells. Donor cells of the same
pool were also transplanted into similarly irradiated isogenic recipient mice
-
-
.
..
.
.
.
to assay the competence of the cells to proliferate in the absence of
.
9-
(F-1)
histoincompatibilities. To avoid sex-associated resistance, the donor and
recipient mice of all strain combinations were females. The promotiou of
splenic uptake of 15-IUAR by grafted marrow in isogenic recipients was
comparable for cells of strains C57BL, B10, 129, C3H.SW, and A.BY, and was,
furthermore, a linear function of the dose of grafted cells (Fig. 1).
Splenic uptake of 13-IUDR was also a linear function of donor cell dose in
resistant F, hybrid recipients; however, it was necessary to graft larger
numbers of cells than into iBogenic mice to colonize the recipient spleens,
as is indicated by the displacement to the right of the dose-response lines
(Fig. 1). It appears, therefore, that the spleen of F, hybrid mice can be
repopulated after radiation-induced hematopoietic depletion by an overdose of
parental marrow cells toward which the hybrids exhibit resistance.
The number of donor cells required to overcone the hybrid's resistance
varied markedly in the five strain combinations studied. Spleen repopulation
in B10 x B10.D2 hybrids required 15-to-20 times as many cells as spleen
repopulation in isogenic recipients, whereas lower multiples of the cell dose
inoculated into isogenic mice were adequate for repopulating the spleens of
the other, brids. It is noteworthy that differences in the strength of resistance
were detectable between hybrids sharing the same H-2 alleles, numely C3H.SW X .
C3H, C3H X 129, and C3H/Anf X C57Bt., all of which are H-2*/H-2° heterozygotes.
The A X A.BY mice were the weakest with respect to hybrid resistance, for
the number of A.BY marrow cells necessary for hybrid spleensrepopulatpanel
was only 2.5 times larger than the number of cells required by irradiated A.BY
hosts. The strength of the resistance seemed from these data to depend rather
on the genetic hokground of the strains entering the F, crosses than on the
H-2 alleles of the nybrids. In fact, tesistance was strongest in the hybrids : 1
whose parents shared the C57BL genetic background (e.g., B10 x B10.D2);
intermediate in the hybrids with one parent possessing the C3H genome (e.g.,
Ho
C3H/Anf X C57BL and C3H X 129); and weakest in the hybrids whose parents shared
the c3H or A genetic backgrounds (e.8., C3H.SW X C3H and A X A.BY).
One million nucleated marrow cells of strains B10, 129, C3H.SW, and A.BY
exposed
were transplanted into a series of allogeneic recipients to 800 R of x rays
to determine how the resistance of homozygous inbred allogeneic mice would
compare with hybrid resistance to H-2° marrow grafts. The results of these
assays, presented in Table 3, indicate that the proliferation in foreign hosts (T-3)
of all four types of donor cells followed the same general pattern, and that
the strength of the resistance varied according to recipient strain. The
growth of donor cells was unimpaired in the spleens of irradiated allogeneic
hosts/ H-2° in phenotype; deficient in the spleens of strain A and C3H
mice; and absent in the spleens of strain C57BR and DBA/2 mice (Table 3).
Differences in strength of resistance were noticeable between C3H and C57BR
mice, which share the H-25 allele. This pattern resembles the differences
in strength of hybrid resistance among H-2/H-2 hybrids, and suggests that
other factors in addition to H-2 influence the strength of resistance to
allogeneic marrow grafts.
DBA/2 mice and C3H X C57BL F, hybrids are strongly resistant to H-2°
parental marrow grafts (Table 3, Fig. 1). Mice of these strains were exposed
to graded doses of whole body X rays and grafted with 5 X 10° nucleated
C57BL marrow cells. This number of donor cells was sufficient to override the
resistance of DBA/2 and of the hybrid mice (Fig. 2). The extent of spleen (F-2)
colonization, measured by the 13-IUAR technique, was, however, dependent on . ,
the magnitude of the recipients exposed to radiation in the range of 600 to
1000 R of x rays. The progressive loss of resistance with increasing exposures
was greater in the DBA/2 mice than in the hybrids (Fig. 2), but the difference
was relatively small. Therefore, the effect of x irradiation on the strength
of resistance appeared to be similar in the two classes of mice.
Graft-versus-host reaction in hybrid-to-parent chimeras. Mice injected with
foreign spleen or marrow cells after exposure to lethal or sublethal irradiation
survive the acute phase of radiation injury, but frequently' die later because
of a wasting syndrome, attributed to an in vivo immune reaction by the
grafted cells against host 1soantigens (for review, see 21). To determine
whether or not such a reaction occurs in parental strain mice grafted with i
---------------------
--------
10.
hematopoietic cells of resistant F, bybrid donors, I modified an experimental
design used by Doria (17) to obtain direct evidence for graft-anti-host
activity in allogeneic chimeras. Fourty million C3H x B10 spleen cells were
infused into mice of either parental strain exposed to 850 R of x-rays, and
do isogenic F, mice exposed to 950 R of x rays. The chịmeras sựrvived
hematopoietic competence of
the supralethal dose of radiation, presumably, by virtue of the spleen cell
grafts and were not afflicted by the severe and acute wasting syndrome which

usually follows inoculation of allogeneic spleen cells into irradiated mice.
Thirty days after the primary irradiation and inoculation with F, spleen
cells, the surviving chimeras (about 80%) were re-exposed to 700 R of x rays
to be tested and classified for resistance or susceptibility to a standard
graft of 10° nucleated Blo marrow cells. Any reduction in the splenic
311
incorporation of 13-IVOR by the test-grafts in F,-to-blo chimeras, as compared
to isogenic Bl0-to-B10 control chimeras, should give a direct estimate of
.
1.
hybrid-anti-parent cr graft-anti-host reaction actually present in the chimeras.
,
137
On the other hand, any reduction in splenic incorporation of 13-IUR in F, -to-C3H
chimeras, as compared to C3H-to-C3H chimeras, should estimate whether or not
hybrid resistance was adoptively transferred. Hybrid-anti-parent reactions
cannot occur in the latter chimeras, since the H-2° associated parental
isoantigens are lacking in the H-2 c3H recipient mice. The results of this
experiment are presented in Table 4. B10 marrow cells promoted the uptake (T-4)
of 15-IUR in the spleens of isogenic B10-to-B10 and of allogeneic C3H-to-C3H
chimeras, but they did not so in the spleens of F,-to-F, chimeras. This
indicated that under the conditions of the experiment, the chimeric state did
not alter per se the resistance or susceptibility of recipient mice to Blo
marrow grafts. In fact, the proliferation of B10 marrow cells in the chimeras
was identical to the proliferation of Bio cells in Irradiated B10, C3H and
C3H X BlO mice, as reported. B10 marrow cells were able to promote
splenic uptake of 13AIUR in F,-to-Bl0 chimeras as if they were fully
susceptible, but BlO marrow cells were not able to do so in F,-to-C3H chimeras,
as if these animals were fully resistant.
To descriminate between different mechanisms which could lead to results
of this type, the experiment was repeated with the following modifications:
(i) chimeras were prepared by infusing 2 x 10' nucleated marrow cells instead
of spleen cells; furthermore, the test for resistance or susceptibility was
not done before 60 days had elapsed to allow complete transpopulation of
hemic cells to the donor type. Transpopulation was verified by determining
whether the peripheral blood hemoglobin of the chimeras was of donor type.
The method waployed was the one of Popp and Cosgrove (27); hemoglobin types
of the mouse strains involved are reported in Table 2. (ii) C3H x B10
donor marrow was given to parental strain mice, to c3H x B10.BY hybrids
(which posess & hemoglobin type different from the C3H x B10 donor mice but
.
,
Ara
are identical to them with respect to H-2 type and hybrid resistance) and, in
addition, to other inbred strain recipients. Of particular interest was the
use of C3H.SW recipient mice which share with one parent of the hybrid the
C3H genetic background, but poses in common with the other parent strain the
H-2° allele. The aim of this second experiment was to establish whether or
not results such as those seen in Table 4 could be obtained in marrow instead
e necífrient,
of spleen chimeras and attributed to repopulation by the F, cells; and if so,
whether or not differences in resistance among the chimeras were the consequence
of grafting the F, cells into mice homozygous H-2 as opposed to mice of other
H-2 constitutions. The results, presented in Table 5, clearly show that (T-5)
BlO cells grew deficiently in F,-to-C3H, F2-to-A, and F2-to-C3H X BLO.BY
chimeras, as if hybrid resistance was transferred adoptively by the F, marrow.
de winter's ancestros
n
tiendast minuten i
dillud
In contrast, blo marrow cells of the same pool grew without impairment in
F,-to-B10, F,-to-C3H.SW, and F,-to-129 chimeras, as if hybrid resistance was
not transferred adoptively to the latter recipients. The two classes of
chimeras seemed to correlate with the H-2 type of the recipients and 180
markedly with respect to resistance in spite of the fact that their erythron
was repopulated to the same extent by donof type F, hematopoietic cells.
The time sequence by which grafted C3H x BIO marrow (10' nucleated cells)
reestablished resistance in heavily irradiated (950 R) C3H X BlO.BY mice while
it failed to transfer resistance to irradiated (850 R) B10 mice is shown in
Fig. 3. Also included in the experiment (groups of parent-to-hybrid chimeras, (F-3)
i.e., irradiated (950 R) C3H X Bl0 mice given 10' nucleated Blo marrow cells,
to establish whether or not transpopulation to parental type hematopoietic
cells transfered adoptively susceptibility to B10 test-grafts. Groups of
the three types of chimeras were tested at intervals by reexposing them to
700 R of x rays and infecting them with 10° B10 marrow cells. The chimeras tested
for resistance 60 days after primary infusion of marrow possessed at that time
- Exclusively )
peripheral blood hemoglobin enty of donor type. F,-to-B10 and B10-to-F,
All
chimeras were susceptible at all chosen time interval
chimeras were
......ciwie
e ma
partially susceptible until 20 days of chimera age, but regained their resistance
at later intervals (Fig. 3).
I conclude from these experiments that hybrid resistance to parental marrow
grafts is adoptively transferred by F, marrow or spleen cells to mice that do
the a phenotype)
not posess the parental isoantigens associated with H-2, namelý to c3H, A, and
C3H x B10.BY mice, when they beer en functionat 031* BIO bomatopetettengrart.
Conversely, hybrid resistance is not detectably transferred to H-2 mice even .
where they bear a functional F, wybrid hematopoietic graft. Probably the
resistant F, hematopoietic and/or immune system becomes paralyzed or unresponsive
homasta
tinin
na
w
interne
momento
e tant
upon transplantation into H-2° mice, owing to the excess of parental
iboantigens in such recipients.
DISCUSSION
Assessment of the extent of hematopoietic cell proliferation in
spleens of a variety of irradiated 1-2 heterozygotes following trans-
plantation of parental strain H-25/11-25 marrow cells indicated that the
strength of hybrid resistance, measured by the number of cells necessary
to override it, varied among F, mice of different parentage. For example,
the resistance of H-2" /H-2 hybrids, such as C3H X 129 or c3H/Anf X C57BL,
differed by a factor of two or more from the resistance of 0311.SW X C3H
mice, which are also h-2°/H-2k in phenotype (Fig. 1). Furthermore,
11-2° /H-2 hybrids may also differ in strength of their resistance;
A X A.BY mice are weakly resistant toward A.BY marrow grafts (Fig. 1), but
B10.A X A.BY hybrids are strongly resistant (16), B10.A is a mouse line
congenic with B10, except that it carries the H-2° allele derived from
strain A istead of H-2 (33, 34). Thus, the two hybrids, A X A. BY and
B10.A X A.BY, are identical with::respect to their H-2 constitutiion,
yet they differ markedly with respect to the strength of their hybrid
resistance, Studies such as these provided evidence that, although
hybrid resistance is determined primarily by heterozygosity at the D
region of ll-2 (Table 1, and references 12, 13, 15), its strength
depends on other determinants contributed by by the genetic background
of the parental strains,
14
The generalization I would derive from the foregoing data,
but particularly from experiments involving Fy hybrids from congenic
parental strains, is that the genetic background of C57BL parental
mice endows F, hybrids with strong resistance. Hybrid resistance is
termed strong when it requires fox spleen repopulation by H-2°
homozygous marrow the grafting of 10 to 20 times as many cells as
required by nonresistant mice. On the other hand, the genome of C3H or
parental
A strain mice endows F, hybrids with weak resistance, that is, with one
which can be overridden by a two to fivefold increase in the number.
of grafted cells required to colonize the spleen of nonresistant
Isogenic recipients. Since hybrid resistance is related to genetic
interaction between alleles at the D region of H-2 (12, 13), it is
reasonable to postulate that the effects on strength of resistance
.
:
of varying the parental strains may be due to independent allelomorphic
modifier genes which may regulate the extent to which genetic interaotion
in H-2 heterozygotes leads to the suppression of isoantigens of the
H-2° phenotype.
Irrespective of the mechanism by which the differences in strength
of hybrid resistance were brought about, the range of values found were
of the same order of magnitude as the range of values found in allogeneio
inbred strain mice resistant to H-2 marrow grafts (Fig.1, Table 3). In
addition, the strength of hybrid resistance was evaluated by determining
the weakening effect of graded doses of whole body irradiation (Fig. 2).
Although hybrid resistance is known to be relatively insensitive to
radiation (11, 23), it was not substantially different in this respect
the
from the resistance of H-2 incompatible allogeneic host-donor combination
that has been studied (Fig. 2). The experiments demonstrated that
resistance phenomena in heterozygotes, due, preaumably, tr noncodominant
factors, do not constitute a greater impediment to marrow transplantation than
15
the resistance phenomena due to the classical,
to strong codominant I factors. Once the hybrid resistance is overridden
by an appropriate dose of grafted parental marrow cells and/or by a
supralethal dose of whole-body x radiation, the long-term persistence of the
marrow grafts is not deteruined by the H-2 associated resistance factor,
but by independent determinants (28). For example, C57BL mice possess
marrow which grows deficiently on transplantation into H-2 heterozygous hybrids
(Fig. 1) and which also regresses frequently in hybrid recipients whose
resistance has been overridden, as shown by Popp (25,26). The study of the
short- and long-term growth pattern marrow grafts from segregating F, mouse
progeny in F, recipients 'has clearly demonstrated the independence of the
two phenomena (28).
imadiated
Transplantation into irradiated resistant F, hybrids of appropriate
numbers of B10 marrow cells and transplantation into irradiated inbred strain
homozygotes of C3H x B10 F, marrow or spleen cells resulted in chimerism and
in complete transpopulation to donor-type erythropoiesis in 60 days.
Resistant hybrids repopulated by Bl0 cells became susceptible to Blo test grafts
of marrow (Fig. 3), whereas, resistant hybrids repopulated by C3H x B10 marrow
or spleen retained their resistance (Fig. 3, Tables 4-5). C3H and A strain
mice repopulated by C3H x B10 F, cells acquired resistance to Blo test grafts
(Tables 4-5). These experiments have demonstrated that susceptibility and
resistance to parental marrow grafts is transferred adoptively through,
hematopoietic cells.
Since hybrid resiętance is presumably directed toward 1soantigens or the
H-2 homozygous phenotype, it was expected that severe graft-versus=host
reactions would occur in Blo, C3H.SW, and 129 chimeras as their hematopoietic
system transpopulates to the C3H x B10 type. These chimeras, however, were
not detectably 111 and the direct assay for hybrid-anti-parent, i.e., graft-
versus-host, reactivity gave negative results (Tables 4 and 5). The
condition of these mice is similar to the condition of newborn parental strain
mice infused with F, hybrid spleen cells which fail to develop runt disease (5).
Thus, the F, marrow or spleen cells that were competent to transfer adoptively
hybrid resistance to C3H and A mice failed to do so specifically in H-2
mice. C3H.SW and C3H mice, for example, differ only by a chromosome segment
of linkage group IX containing the H-2 region, but they fell into two
distinct categories with respect to adoption of hybrid resistance (Table 5).
This particular finding strongly supported the conclusion that the H-2 allele
decided
of the host animal determines whether or not F,-to-parent chimeras met
become resistant to H-2 marrow grafts. It may also explain why no ill effects
due to graft-versus-host reactions are produced by functional F, spleen cell
grafts in
ozygous recipients.
The lack of adoptive transfer of hybrid resistance and of graft-versus-host
reactions in H-2 chimera mice in the face of functional hematopoietic grafts
fron resistant Abbott donors. is interpreted to mean that specific
unresponsiveness toward parental isoantigens by F, cells developed in such
chimeras. This is known to occur in long lived allogeneic marrow chimeras
TO
(8,36) owing to the excess of host isoantigens to which the grafted cells are
can be
ie, nonresistant
exposed. Furthermore, resistant hybrids are considerably rendered unresponsive,
to parental H-2 marrow grafts by exposing them to parental tissue isoantigens
during neonatal (10) or adult life (14,15). Hence, it is not surprising,
I have red
altogether, that the same process may foccur in F,-to-parent chimeras.
SUMMARY
F, hybrid mice that are heterozygous at the D region of the H-2
locus do not support optimal growth of transplanted parental marrow cells
when the donor 18 homozygous for the H-2 allele, although they fully
support the growth of parental skin grafts. Resistance to grafted parental
marrow, which is due to a single noncodominant gene, can be overriden either
by increasing the number of grafted donor marrow cells to several times the
number sufficient to repopulate the hematopoietic system of nonresistant
irradiated recipients, or by increasing the radiation exposure of the
resistant mice. The strength of the hybrid resistance, as measured by these.
two parameters, is of the same order of magnitude as the resistance to grafted
marrow found in some H-2 incompatible allogenic donor-host combinations. Once
the hybrid resistance is overridden by relatively marge marrow inocula, the
long-term persistence of these parental grafts is decided by independent
factor(s) not associated with H-2, as revealed by studies of appropriate
segregating mouse progeny (28).
Transplantation of marrow of spleen cells from resistant F, hybrid donors
into heavily irradiated isogenic mice and into parental strain mice carrying
an H-2 allele other than H-2° results in adoptive transfer of hybrid resistance.
On the other hand, no hybrid resistance is transferred, nor doel detectable
graft-versus-host reaction occur in parental strain recipients homozygous for
H-2 given F, hematopoietic cells. Presumably, the latter cells become
specifically unresponsive to parental isoantigen(s) associated with H-2° when
they are infused into H-2° mice.
The findings presented here indicate that marrow transplantation
experiments are not complicated by parent-F, hybrid incompatibility to
a greater extent than by histoincompatibilities due to the codominant H
genes.
-
.
-
..
..
.
.
.
.
va
r
L
-
L
.
-.
-
.
.
.
.
REFERERICES
1. Amos, D. B.: Isoantigens of Mouse Red Cells.
Ann. N. Y. Acad. Sci. 27: 69–82, 1962
2. Bailey, D. W.: Histocompatibility Associated with the X Chromosome
in mice.
Transplantation 1: 70-74, 1963.
3. Bailey, D. W.: Genetically Modified Survival Time of Grafts from Mice
bearing X-linked Histocompatibility.
Transplantation 2: 203-206, 1964.
4. Barnes, A. D. and Krohn, P. L.:
The Estimation of the Number of
Histocompatibility Genes Controlling the Successful
Transplantation of Normal Skin in Mice.
Proc. Roy. Soc., London, S.B.. 146: 505-526, 1957.
5. Billingham, R. E.: The Induction of Tolerance of Homologous Tissue
Grafts. In Transplantation of Tissues and Cells,
p. 87-106. Eds., Billingham, R. E. and Silvers, W. K.,
The Wistar Institute Press, Philadelphia, Pennsylvania,
1961.
6. Boyse, E. A.: The Fate of Mouse Spleen Cells Transplanted into Homologous
and F, Hybrid Hosts.
Immunology 2: 170-181, 1959.
7. Boyse, E. A., Old, L. J., and Luell, s: Genetic Determination of the
TL (Thymus-Leukemia) Antigen in the Mouse.
Nature 201: 779, 1964.
8. Cole, L. J. and Davis, W. E., Jr. Specific Homograft Tolerance in
Lymphoid Cells of Long-Lived Radiation Chimeras.
Proc. Nat. Acad. Sci., U.S. 47: 594-602, 1961.
9. Cudkowicz, G.: Sex-associated Hybrid Resistance to Parental Marrow
Grafts (Abstract).
Fed. Proc., in press.
10. Cudkowicz, G.: The Immunogenetic Basis of Hybrid Resistance to Parental
:
Marrow Grafts. In Proceedings of the Conference on
Isoantigens and Cell Interaction, Philadelphia,
Pennsylvania, 1965, in press.
11. Cudkowicz, G. and Stimpfling, J. H.: Deficient Growth of C57BL Marrow,
Cells Transplanted in F, Hybrid Mice. Association with
the Histocompatibility-2 Locus.
Immunology 1: 291-306, 1964.
.
Cudkowicz, G. and Stimpfling, J. H.: Resistance to Parental Marrow
Grafts: Association with the K Region of 11-21
Science 144: 1339-1340, 1964. .
13. Cudkowicz, G. and Stimpfling, J. H.: H-2 Region Controlling Hybrid
Resistance. Correction of Data.
Science 147: 1965.
14. Cudkowicz, G. and Stimpfling, J. H.: Induction of Immunity and of
Unresponsiveness to Parental Marrow Grafts in Adult
FHybrid Mice.
Nature 204: 450-453, 1964.
15. Cudkowicz, G. and Stimpfling, J. H.: Lack of Expression of Parental
Isoantigen(s) in F, Hybrid Mice.
Proc. Xth Congr. Int. Soc. Blood Transf., Stockholm,
1964, in press.
16. Cudkowicz, G. and Stimpfling, J. H.: unpublished observation.
17. Doria, G: Homograft Reaction in Mouse Radiation Chimeras. II. Direct
Evidence for Graft-anti-Host Activity.
18. Eichwald, E. J., Silmser, C. R. and Weissman, I.: Sex-linked Rejection
of Normal and Neoplastic Tissue: I. Distribution and
Specificity.
J. Natl. Cancer Inst. 20: 563-575, 1958.
2.9. Gorer, P. A.: Some Recent Data on the H-2 System of Mice. In Biological'i:
Problems of Grafting, p. 25-30. Eds., Albert, F. and
Medawar, P. B.,
Thomas, Springfield, Ill., 1959..
20. Hauschka, T. S. and Holdridge, B. A.: A Cytogenetic Approach to the
.
Y-Linked Histocompatibility Antigen of Mice.
Ann. N. Y. Acad. Sci. 101: 12-22, 1962.
21. Koller, P. C., Davies, A. J. S. and Doak, s. M. A.: Radiation Chimeres.
In Advances in Cancer Research, Vol. 6, p. 181-289.
Eds., Haddow, A. and Weinhouse, S., Academic Press,
New York, 1961.
22. Little, C. C.: The Genetics of Tumor Transplantation. In Biology of
the Laboratory Mouse. Ed., G. D. Snell, Blakiston Co.,
Philadelphia, 1941, p. 279-309.
23. McCulloch, E. A. and Till, J. E.: Repression of Colony-Forming
Ability of C57BL Hematopoietic Cells Transplanted into
Non-Isologous Hosts.
J. Cell. Comp. Physiol. 61: 301-308, 1963.
24.
Owen, R. D.:
Current status of Mammalian Immunogenetics.
J. Cell. Comp. Physiol. 59, Supplement 1: 73-87, 1960.
25. Popp, R. A.: Regression of Grafted Bone Marrow in Homologous Irradiated
Mouse Chimeras.
J. Natl. Cancer Inst. 26: 629-640, 1961.
.
.23
26. Popp, R. A.: Inheritance of Factors Affecting the Ability of Bone
Marrow of (C57BL X 101)F, Mice to Establish Permanent
Grafts in Irradiated F, Mice.
J. Natl. Cancer Inst. 33: 7-14, 1964.
27. Popp, R. A. and Cosgrove, G. E.: Solubility of Hemoglobin as Red Cell
Marker in Irradiated Mouse Chimeras.
Proc. Soc. Expti. Biol. Med. (N.Y.) 101: 754-758, 1959.
,
28. Popp, R. A. and Cudkowicz, G.: Independence of Deficient Early Growth
and Later Regression of (C57BL X 101)F, Marrow Grafts
in (C57BL X 101)F, Hybrid Mice.
29. Prehn, R. T. and Main, J. M.: Number of Mouse Histocompatibility Genes
.
,
Involved in Skin Grafting from Strain BALB/CAn to
Strain DBA/2.
J. Natl. Cancer Inst. 20: 207-209, 1958.
30. Snell, G. D.: Histocompatibility Genes of the Mouse. II. Introduction
and Analysis of Isogenic-Resistant Lines.
J. Natl. Cancer Inst. 21: 843-877, 1958.
31. Snell, G. D. and Stevens, L. C.: Histocompatibility Genes of the Mouse.
III. H-1 and H-4, Two Histocompatibility Loci in the First
Linkage Group.
Immunology 4: 366-379, 1961.
.
24
32. Spencer, R. A., Hauschka, T. S., Amos, D. B, and Ephrussi, B:
CO-Dominance of Isoantigens in Somatic Hybrids of
Murine Cells Grown in vitro.
J. Natl. Cancer Inst. 33: 893-903, 1964..
Immunogenetic Problems. In International Symposium
on Tissue Transplantation. pp. 37–54. Eds., Cristoffanini,
A. P. and Hoecker, G., Universidad de Chile, Santiago,
1962.
34. Stimpfling, J. H. and Richardson, A.: Immunogenetic Properties of
Cellular Isoantigens of the Mouse (MUS Musculus).
I. Recombination within the Histocompatibility-2 Locus.
35. Tuffrey, M. A. and Batchelor, J. R.: Tumor Specific Immunity Against
Murine Epitheliomas Induced with 9, 10-dimethyl-1,2-
benzanthracene.
Nature 204: 349-351, 1964. ·
Radiation Chimeras. In Mechanism of Immunological
Tolerance, p. 385-396. Eds., Hasek, M., Lengerova, A.,
and Vojtiskova, M., Academic Press, New York, 1962.
.
37. Zaalberg, O. B.: An Analysis of the Eichwald-Silmser Effect.
Transpl. Bull. 6: 433-435, 1959.
.
26
Table 1 - Growth of parental marrow from 16 Inbred strains and 13 isogenic-
resistant lines in H-2 heterozygous F, hybrid recipients.
Deficient growth
Optimal growth
Strains
H-2 type
Strains
H-2 type
C57BL
C57BL/6
B10.A #
C57BL/10
DBA/2
B10.BY
BALB/C
B10.02 #
B10.129(5M) +
B10.129(144) +
RFM
B10, 129(21M) +
СЭН
B10, LP +
C3H.K +
C57L
101.
129
AKR
C57BR
C3H, SM +
B10.BR #
A.BY #
DBA/1
D1.LP #
A.SW #
FU
unknown
* Data from Cudkowicz and Stimpflin
• Isogenic-reistant or
Cor congenic lines of
..
-
.-.-.-
..
mice are identical with a given background strain except for substitution of a
chromosome segment carrying a different allele at a single locus (30, 31).
+ Congenic lines differing at an H locus other than H-2 from their background
strain (30, 31).
# Congenio lines differing by an H-2 allele from their background strain (30, 33, 34).
Arri
N
WA
.
.
.
.
Table ? - List of strains
•
27
Strains.
Genotype *
Designation in
this paper
Origin
..
A/Sn
.........
11-28
1bD
our colonył
A.BY
A.BY
H-26
our colonyi
OUT
.
C3H/Anf
C3H/Anf
11-25
Cumberland View Farms
C3H1/HeSn
C3H
our colony+
C3H.SW
C3H, SW
our colony+
C57BL .
C57BL
Cumberlad View Farms
C57BL/10ScSn
B10
H-1°
H-2B
our colony+
B10. BY
B10.BY
H-10
11-26
our colony+
B10,D2-old
B10.02
Hapa
our colony+
C57BR/R1
C57 BR
H-24
W. L. and L. B. Russell's colony
DBA/2
DBA/2
H-20
Cumberland View Farms
129/R1
129
H-25
160
W.L, and L. B. Russell's colony
*
Only genes relevant to the context of this paper are listed.
+ Pedigreed breeders received in 1960-1962 from G. D. Snell
it
Pedigrecd breeders received in 1964 from G. D. Snell
Table 3 - Uptake of 134IVAR* in the spleens of allogeneic recipient mice grafted with marrow cells
from homozygous H-2 donors.
Recipient Strains'
Donor
Strains
B10
129
C3H.SW
:
А
СЗН
C57BR
DBA/2
B10
129
+1
0.845.09
0.27 + .10
0.71 = .07
0.70 = .05
0.75 +.06
0.70 + .06
0.85+ .08
0.79 +.06
0.78 + .05
0.73 + .07
0.77** .05
0.86+ .05
0.25 +.06
0.18 + .04
0.26 + .06
0.31 = .09
0.53 = .10
0.32 + .06
0.35 + .10
0.24 + .07
0.01 .001
0.02 + .01
0.01 + .003
0.01 + .002
0.06+ .01
0.03 + .01
0.09 = .04
0.03 + .01
C3H.SW
+1
A.BY
*Mean of % uptake + standard error of the mean.
*Exposed to 800 R of x rays and grafted with 10° nucleated femoral marrow cells (10-15 animals per group).
"OW
..........
.............................
...
........ com.com....
29;
Table 4 - Splenic uptake of 13-IUR in response to test grafts of Bl0 marrow cells in F,-to-parent
and in isogenic spleen chimeras*
Constitution of Chimeras
Uptake of 15-IUAR in recipient spleens
( + stand. error)
Spleen donor
Recipient
C3H X Blo
B10
(8)*
СЗН
C3H X BLO
0.84 + 0.07
0.09 + 0.04
0.07 + 0.01
0.92 + 0.08
0.60 + 0.07
(9)
(10)
(7)
B10
B10
сан
C3H
*30 days after primary irradiation and transplantation of 4 x 10' spleen cells, the mice were
reexposed to 700 R of x rays and tested for resistance or susceptibility to 100 grafted marrow
cells.
'Number of chimeras tested in parentheses.
. 302
Table 5 - Splenic uptake of +IUR in response to test grafts of B10 marrow cells in F,-to-parent,
Fy-to-allogeneic, and in isogenic marrow chimeras*
Constitution of Chimeras
Uptake of 13-IUDR in recipient spleens
Marrow donor
Recipient
(%+ stand. error)
C3H x B10
(10)"
B10
C3H.SW
(12)
(9)
..C3H
A
0.68
0.75 * .04
0.70 7.08
0.05 = .01
0.07 .02
0.03 = .005
0.71 = .06
0.437.05
0.35 = .02
C3H x B10.BY
(15)
(7)
(12)
(10)
( 8)
(16)
B10
B10
СЭН
сэн
*60 days after primary irradiation and transplantation of 2 X 10' marrow cells, the mice were
reexposed to 700 R of x rays and tested for resistance or susceptibility to 106 grafted marrow
cells.
*Number of chimeras tested in parentheses.
LEGENDS FOR FIGURES
Figure 1 - Mean splenic uptake of 131IUAR in relation to the number of
parental. marrow cells injected into x irradiated isogenic and F.
hybrid recipient mice (five mice per point).
.
.
Parental strain donor cells and
Isogenic recipients - 700 R
B10 cells
B10 x B10.D2 - 700 R
C3H/Anf X C57BL - 900 R
C57BL cells -
129 celle
C3H X 129 - 700 R
.
C3H.SW X C3H . 700 R
C3H. SW cells
A.BY cells
A X A.BY - 700 R
.
.
.
...,
Figure '2 - The effect of whole body x irradiation on resistance to
grafted C57BL marrow (5 X 10° nucleated cells). Growth of donor
cells, as judged by uptake of "SIUR in the spleeng of DBA/2 O
and C3H/Anf X C57BL F, y recipient mice. . .
о
C3H x B10- esito-B10 maine claimeras
сзн х ві0-таомиладоtо-сзн х вio.BY че са:
B10-esteemed to-C3H X BIO maine climten

Isplaltic U RTIKEL
TUAN (
.
.
.
.
--
--
--
-
.
.
-
-
- -
-
-
-
-
-
-
- -
-
,
,
104
}
-
.
.
-
-
-
-
.
.
.
.
.
+
--
-
.
..
.
.
1
6
6
-
--
-
-
-
-
----
--
7
7 8 9
8 9
105
-
-
.
L
1
.
"
.
TV,
1
-
-
-
.
.
.
. .
.
.
.
.
.
T.
....
.-.-.-
.
T
-
a
.
:
---
-
.........
!
!
.
.
.
1
2
.
--
...
:1
:
:.
-
-
-
.
-
-
•
1
-
-
-
-
.
!
1
-
-
-
17
.
1
....
NUMBER OF TRANSPLANTED MARROW CELLS! ...
.
+
+
-
.
.
1
-
-
-
X
+
.
1
-
>
--
.
-
-
-
=
+
..
--
--
-
.
-
.
.
.
1
.
.-
.
.-
.
.
.
-
1
.
.
.
.
.
.
.
-
--
--
1
-
-..-.-
.
-
-
-
-
-
-
-
1
--
.
--
-
0
.
-
.
-
-
-
..
.
-
1
-
-
-
-
-
-
-
-
-
-
-
.
.
11 10
.
.
I
'
'
:
:
!
1
+
-
+
1
0
.
-
-
..
...
-
-
.
..
-
1
...
.
.
.
-
-. .
-
1
+-+

var
10 X 10 TO THE ! INCH
KEUFFEL CSSER CO
358-12
KADI II U.S.A.
100MM
DOUTUI
INIDHINIRODA
STUDIIDII
LULLLLL'IIII
nOIMI 100mmDIRMDIRIURIMITmInm
CONDIDINOU ONOIDIDILIDINDINOODID
DUUUU01
CD
.
ITIONNTIT
(TNI MITITIT
LIMITTITUTO
UUUUUUUUUUU
UNUI LUL
UNTITTELITI
2011
UNIMIT
LIIIII
ILLU
V
LUUUUUDULI
IIIIIIII
VUUUUUU9000
RIIIIII
DRIDUD
TIITITITITITITIT
11
HIIIIII.RU
ODIULID
IIITITOITIINIT
CIUTILITIITTI
MIDII11I
HILUUUU
HIITTO
DIIVOOL
TIITTIIN
OI
MINI
UIDI
JITUTITITMOITTTTTTITUITTIITTITUITITATIITTTT
ITTIITTIINTITITUTIITTITTO
U
TUD
DUBO
.
LU
LUDWI
0
TA
NODOID
DITULI
UUUUUUUUU
لاندانان
C
ihontirminni
011
DWMDIITTITT
IIIIIIIIIIIIIII
IIII
QUIDO
10111
ILIN
ULDUT
GUIODONT
JOULIOUT
UUUUUUU
01DULTI
LUULUTUSID
UULUULIUUDIO
VII111UUUUUULUD
LLLLLLLL
DDUTINIONI
TUDIUULIOL
10MIIIIIIIIII
JUULUUUUUUUUUUUUUU
IIIIIIIIIIIIIIIIII
UUUUUUUUUUUUUU
i
WIWITITIMINNESOTOMIMMIT
TUTTI
T
LULIINILIITTTTHurttttttt
RITUULLIT
DENTITA
OOOOUU
NITI
LINIIIIIIIII
UUUUUUUUUIINID
ONDITII
C
AW202
biedtellit
UUUUUUUUUUUUU
LIITT
ITUNJITUD
LULUUNUUIILI
IIIIIII
DTDI
IIIIIIIII
DTD
IRIUMI
IRIDI111
IIIIIII
IIIIII
JIRIIIII
INO
JUTIRMIN
INOM
LUIIIIIIIIIIIIIIULLUTITUITITITIT
TITUT
TAVITI
UUDIOTOON
DVIII
L
COTIDIDUNT
DOLUUTUUDI
IIIIIIII
DOUIIIII
IIIT
DID
IDIODI
CUTITITI
INDIITIL
MIUTLIDIIL
IIIIIIIII
00011100101
1000INO
PIIIIII
OUI
Ouallall.de
INNTILI
ITU Tri
INUTI
IIIIIIIII
DIUTI
UDDIDIOIT
QUOLITOS
DINU
1011 1111
UUUUUU
LLLUITINITT
TITUTI
.
INNNNIITITUTIITTIMIT
CUIUIUTILID LIIDID
11
IIIIIIII
ch
: UNITI
JIRUDITOINTI
UUTUSIDULUITD
INDIANUODIDOLI
UUUUUIIULIDI
UUUULTAVITUDI
DUJIO
JUNDUBIO!
La
LI
TII
LUULUUIII
DOLIDOIO
UIUDDIN
DUDU
UUUUUUUDWID
UUUUU
TOOTMII
HRINO
TOUTOILU
DIMI
COUDOI
ULLOU
SIIII
i
11
DIT
TI1Naut
IN
TIIIIIIIULUI
ULLIIIIIIIIIIIIIIIIIIIIIIIIII
ITOU
UUUUUUUU
On
DIONI 1
UIDOOD
ULUWULUU
(TTTIIIIIIIII
IIIIIIIIIITEIT
LULUUUUUU
DUDUID
DI
OTO01
3
TITILITI
LIIIIIIIIIIIIIIIIIIIII
DITUTO
UL
IDITTITTTITTTTTTTTTTTTTTTTTT
CITITUUT
WWIIIIIIIIIIIIIIIIIIIIIIIIIIIDIL,
TITTITUTIILITIUITINUITTITUTITITIIIII
SIIIIIII
111110111
DVIDUUUUUU
LITTIIIIIIIIIIIITI
TOTTI
DIRITINIMO
IMOODID
HDIIN101011
UUDIO
MITIMITIIIITIT
0001

NIIDUDI0ODIUUUUUUUUU
UUUUUUUUUUUUUUUUU
WIDT
JO1III
NOINNOIIOONDIUUDIU
UUDIO
HUUUUUUUUU
RII
NOTITI
IIIIIIIIIIIIII
ODI11
010
IDIUI
IUUUUUURE
LUWUUUULUILII
LIT001
I1001
LUULUI
.
IIUDI
JUUON
INUT
MTITIIMIMIITTITUTTOMT
LULLII
DDIN
TIINNITTIIIIIIIIIIII
INILI
TIDIINITUTIMITIITTI
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIITITUIIIIIII
III
OII
IIIIIIIIIIIIIIIIIIIIIIIII
UIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIITT
IITTOUT
NIITITITITITUTI
INITIINTIN
CUIIIITITNOUTOUTILUUIIIIIIT
11
LO
ULU
JUNI
1
COLOU
:
DONUNU
CITI
T IITTLTTUTTO
TIITIUITTILIITUITIT
DIIDIITTITUT
TITIIMITUTIITTINTTM
TITIITITITUTIOTITION
QUIMITNIOTIIL
10MTIDIRODNOTONI
MINUTITITIVITTUITTITT
MTITI
DLOUDOUIJ
JUUUUUUUMID
TILIULUI
SUSTITI
VIII
ODUCIJITONO
GDOULU0DDOUDOU
WIDIIDIIDID
ULUDOUILO
UULIITTI
1
UUUUUUUUUUUUUU
DOUDOUNIDUD
BOULO
UUDIO
JULI 21
JUO
LUON
ODUJII
OULUI
OI
.
DET
DONIDO
SILI
2
ID0000
TOMTOITINTIINNI
OUTLULIITTITUIUTIUIIIIIIIIIIIIIIIIIII
1S
SUDU
WDIO
ITTITTTTTTTTTUITOIDUUNIITITI
TUTTIIIIIIIIIIIIIT
11
.
MITIIITMITTITUT
III IIIIIIIIIIIIII
LILIIIIIIIIIIIIIIIITI
011001000001IIIIIIIIIIIIIIINNIIIIIIIIIIIII
LLUITULUIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIITU
LIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
11
IIU.LIIIIIIIIIIIIIIIIIIIIIIIIIIIITTITTTIITTIT
0111
11
1
I
DIIDIITID
LULULU
TITIITOTOIITTIINTTUIUTIIIIIIIIIIIIIIIIIIIIIII)
UUDIILID
COINTITUITOTTOTT
LILUUIUUDII
JUODOJO
DI
LTD
TU
TUUIIIIIIIIIIIII
EHHERHEID
TONTTITIITIIIIIIIIUIIIIIIIIIIIIIIIIIIIITI
U
NIIDIINIUUUTTTTTTT
LIDT
IRI
UU
HIL010
LIIIIIIIIITTI
LUI
DIULUI
TU IUDUDI
UUDUINO
TITUT
TUNIT
1
.
COLOR
CLUJODIDO
DUDUTO
11
1
UUUUU
NIIDID
ONION
UIDUIT
:
IIIIIIIIIIIIIIILLTIDIUIIIIIII
1
1
1
DUUUU
OTTITI
TIT
IID
DITUUT
100 MOTO
111
UUUUUUUUUUIII10
UUUUUUUUUUUUUUUUUU
TIL0TUDII
MADE IN U.S.
-
---
-
--
KLUFFELLSSER Co.
O
.
.
"!
.
KA
10
11.
S
DATE FILMED
6 / 14 /65








-LEGAL NOTICE -
This report was prepared as an account of Government sponsored work. Neither the United
States, nor the Commission, nor any person acting on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or usefulness of the information contained in this report, or that the use
of any information, apparatus, method, or process disclosed in this report may not infringe
privately owned rights; or
B. Assumes any liabilities with respect to the use of, or for damages resulting from the
use of any information, apparatus, method, or process disclosed in this report.
As used in the above, "person acting on behalf of the Commission” includes any em-
ployee or contractor of the Commission, or employee of such contractor, to the extent that
such employee or contractor of the Commission, or employee of such contractor prepares,
disseminates, or provides access to, any information pursuant to his employment or contract
with the Commission, or his employment with such contractor.

orasistir wi
t
h
END

their