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"The Proliferation and Spread of
Neoplastic Cells" XXIst Annual
Symposium on Fundamental Cancer
Research, Houston, Texas
February 27 - March 1, 1967
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HYBRID RESISTANCE TO PARENTAL GRAFTS OF
RECEIVED BY DTIE OCT 2 1967
. HEMATOPOIETIC AND LYMPHOMA CELLS
Gustavo Cudkowicz, M.D.
...
.
Gustav
Department of Experimental Biology
Roswell Park Memorial Institute
New York State Department of Health
-
Buffalo, New York 14203 :
.
LEGAL NOTICE
. This report wo 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, completsdess, 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 on-
ployee or contractor of the Commission, or employee of such contractor, to the extent that
such saployee or contractor of the Commission, or employee of such contractor prepares,
diasominatos. or provides access to, any Information pursuant to his employment or contract
with the Commission, or his employment with such contractor.
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Cudkowicz i
SUMMARY
A gene locus restricting expected compatibility of grafted homozygous
parental hematopoietic cells in Fl hybride has been identifiod in linkage group
IX of the mouse and designated "Hybrid-histocompatibility-l" (Hh-1). The
gene differs from ordinary histocompatibility genes (H) in three respects: one of
its alleles comme not co-dominant; it influences specifically hematopoietic
grafts; it produces rejection by F1 heterozygotes and not necessarily by
allogeneic Hhul homozygotes. For example, marrow cells homozygous for
Hh-1 - an allele shared by strains C57BL, C57L, 129, and LP - are re-
jected by heterozygous Hh-1°/Hh-1° (C57BL x C3H)F1 hosts, but not by
allogeneic C3H mice homozygous for Hh-1°, an allele possessed by most
mouse strains. Other Hh genes or alleles generating this type of incom-
patibility occur and are being analyzed in strains DBA/2 and WB.
Resistance conferred by Hh genes to heterozygotes, called "hybrid resist-
ance, " can be viewed as a host control mechanism for the proliferation of
grafted hematopoietic cells. It results from non-humoral, cell-mediated,
specific destruction of the parental target cells. Hybrid resistance can be
abrogated either by whole-body preirradiation of recipients, or by admin-
· istration of killed Corynebacterium parvum (a maximal stimulant of the
.
reticu
endothelial system), or by induction of specific tolerance. Hybrids
of appropriate genotype become resistant to parental grafts at the age of
20 days. Their resistance, once established, applies also to cells of
Cudkowicz 2
C57BL/10 lymphomas homozygous for Hh-14. The marrow and lymphoma
cells are capable, however, of optimal growth in "susceptible' F1 hybrids
heterozygous at several 11 loci but not at the Hh-1 locus. Therefore, via-
bility of grafted parental cells is not adversely affected by the presence of
cellular alloantigens in the Fi host. It follows that immunogenetic and
physiological properties distinguish hybrid resistance from other causes
of deficient parental cell growth in Fi hosts, such as minor Halloantigens,'
stronger hybrid responses to tumor-specific or virus-associated antigens,
or donor cell hypersensitivity. .
The manner in which resistant hybrids reject parental marrow grafts
is indistinguishable from that in which inbred mice reject allogeneic mar-
· cow grafts. However, there are marked differences between rejection
patterns of marrow and of skin allografts.
Cudkowicz 3
... INTRODUCTION
During the last ten years transplantation biologists' have been confronted
repeatedly with the unexpected and unexplained observation that certain types
of parental tissue grafts from inbred mouse strains fail or survive poorly in
F1 hybrid hosts. The findings, although exceptional, are of considerable :
interest because they are at variance with one of the fundamental principles
of transplantation genetics, long regarded as universally valid. This "law'
states iisat F1 hybrid mice will fully accept tissue grafts from either of
their inbred parent strains, provided that the sexes of host and donor ani-
mals are roatched for sex-chromosome borne histocompatibility (H) genes.
A general review of the immunogenetic theory of transplantation and of the
known exceptions to it has been published recently by Snell and Stimpfling
(1966). The part of the theory which is at issue here and cannot account
for the findings in Fl hybrids, concerns the .co-dominant nature of all H
genes and the asserted lack of genetic interaction in the inheritance or ex-
pression of such genes in mice. In other words, if transplantation allo-
antigens responsible for graft rejection were the end products of strictly
co-dominant H genes, the antigens should be individually present and fully
expressed in every Fi heterozygote. Consequently, Fl mice should share'
all transplantation alloantigens with their inbred parents and be universal
acceptors of parental grafts.
Hauschka, Kvedar, Grinnell, and Amos (1956) were the first to report
that (C3H/St x Swiss)F1 mice are more refractory than C3H/St mice to low
Cudkowicz 4
.
ceil dosages of parental 6C3HED lymphosarcoma cells and to speculate
(Hauschka and Furth, 1957) on the possibility that Fl mice reacted immu-
nologically against the grafted parental cells. Snell (1958) reported that
strain-specific mouse lymphomas of C57BL/10 origin grow.poorly when
transplanted into F1 hybrids between C57BL/10 and unrelated strains.
Interestingly, hybrids from parents of certain congenic-resistant lines,
i. e., from parents sharing a common genome except for a short chromo-
some segment with differing alleles of H-2, are also refractory to small
numbers of grafted parental lymphoma cells (Snell and Stevens, 1961).
This can be taken as indication that heterozygosity at H-2 or at a closely .
.
linked gene locus might have been responsible for these hybrids' refractory
state. Later, several investigators have reported similar findings with
a variety of transplantable leukemias (Goldin and Humphreys, 1960;
Gorer, Tuffrey, and Batchelor, 1962; Glynn, Humphreys, Trivers, Bianco,
and Goldin, 1963; Hellström, 1963, 1966; Hellström and Hellström, 1965;
.
.
Huemer, 1965; Cudkowicz, 1967; Rossi and Friend, 1967; Sanford, 1967).
carcinomas, and sarcomas (Hellström, 1964, 1966; Uth, Robert, Michaud.
and Crestin, 1964; Oth and Burg, 1965, 1966; Oth, Donner, and Burg.
1967; Hellström and Hellström, 1965) of various C57BL sublines and of
other mouse strains. The tumors grow slower and in a smaller fraction
of Fi hosts than in host mice of the native parental strains. However, it
is noteworthy that not every tumor studied displayed deficient growth in
Fl mice (Wallace, 1965; Oth, Donner, and Burg. 1967; Suit, 1967), and
Cudkowicz 5.
--
-
-
that considerable heterogeneity existed among tumors with respect to quan- .
.
titative and qualitative characteristics of their growth potential in Fl hy-
brids. It is conceivable, therefore, that Fi mice may have resisted grafts
of such a variety of abnormal parental cell types for a number of different
-.
.-.
-
.
reasons, without necessarily contradicting in each case, or in the same
-.-
-.
.
-.-
.
IT
way, the established genetic laws of transplantation. For example, there
T
5
.
2
is the possibility that tumor cells possess "specific' or viral-induced trans-
plantation antigens (Prehn, 1965; Old and Boyse, 1965), or minor alio-
_XUXILI
antigens due to genetic divergence in the inbred strains of origin (Mihich,
1967). The Fl hybrids may be induced to elicit a more effective homograft
reaction than the inbred parental mice owing either to hybrid vigor (Glober-
son and Feldman, 1964; Oth, Donner, and Burg, 1967) or to reactivity to
'
a virus-associated antigen tolerated without measurable reactions by mice
'
T
FT
of a given inbred parental strain. Obviously, an immunogenetic analysis
is necessary to elucidate the mechanism of each case of parental graft
failure in Fl mice. In a few instances such analyses have been done and
the results were compatible with the prediction that Fl hybrids reacted
immunologically either against "specific" transplantation antigens of
chemically induced tumors or against minor alloantigens of tumors with a
long transplantation history (Glynn, Humphreys, Trivers, Bianco, and
Goldin, 1963; oth and Burg, 1965, 1966; Oth, Donner, and Burg, 1967;
Sanford, 1967). In most cases, however, it is not even known whether the
deficient growth of parental tumors resulted from host reactivity directed
Cudkowicz 6
against the grafted cells or, conversely, from an inherent (e.g., humorally
or nutritionally conditioned) inability of this grafts to survive in the Fi en- '
rus...
vironment, presumed immunologically neutral. We
.: During the time of these observations with neoplastic cells, quantitative
techniques were being developed to evaluate grafts of normal hematopoietic
cells in X-irradiated mice for purposes other than genetic (Till and McCulloch,
1961; Cudkowicz, Upton, Smith, Gosslee, and Hughes, 1964). · Upon intra-
venous infusion, hematopoietic donor cells with a potential for proliferation
and differentiation, such as the primitive precursors of erythroid, granu-
locytic, and lymphoid blood cells, seed the hematopoietic organs of the
host. The cellularity and mitotic rate of the parenchyma in these host organs
is depieted by exposure to whole body X-radiation, and the infused compe-
tent cells are thus stimulated to fill the vacancy. Histocompatible donor
stem cells give rise, within 5 to 10 days, to visible colonies of differen-
tiating cells. Some of the donor-derived cells are capable of synthesizing
hemoglobin and/or deoxyribonucleic acid (DNA) in preparation of mitosis
(Bennett and Cudkowicz, 1967; Fowler, Wu, Till, McCulloch, and Simino-
vitch, 1967); other cells become engaged in synthesis of gammaglobulins
(Herzenberg and Cole, 1966) or of specific antibodies upon exposure to
antigen (Kennedy, Siminovitch, Till, and McCulloch, 1965). In incompa-
tible hosts, primitive hematopoietic donor cells may be prevented from
expanding and differentiating and eventually rejected (Cudkowicz, 1965a,
1965b). Thus, the growth of a hematopoietic allograft, quantitated by the
measurable functions of differentiated henic cell populations descending
Cudkowicz 7
· from primitive transplanted cells, indicates the strength of host-donor
incompatibility. The methods have proved reproducible and sufficiently
accurate for immunogenetic studies. One of the earliest and most com-
spicuous observations was the failure of parental hematopoietic stefte in
Fí mice which invariably accepted parental skin grafts. When the hema-
topoietic cells to be grafted were taken from C57BL donors, erythropoiesis,
leukopoiesis and the production of immunocytes or antibody promoted by a
..
given number of donor cells, were all markedly reduced in Fl hybrids in
comparison with sogenic C57BL hosts (Boyse, 1959; Cudkowicz, 1961,
1965; Celada and Welshons, 1962; McCulloch and Till, 1963; Cudkowicz
and Stimpfling, 1964a; Goodman and Wheeler, 1966; Chaperon and Claman,
1967). This finding led to the discovery of a new class of genes, called
hybrid histocompatibility genes (Hh), restricting transfers of homozygous
hematopoietic cells, normal and neoplastic, into heterozygous recipient
mice. It is remarkable how the current experience with hybrid histoin-
compatibility follows the pattern of the earlier discoveries in transplantation
biology when tissue grafts were exchanged between unrelated homozygous
.strains of mice and their offspring to study classical histocompatibility.
genes. Much of the information about the genetice of normal tissue trans-
plantation was indeed derived from pioneering tumor transplantation studies,
but the fine analysis of the H and Hh genes, and of their control of histo-
compatibility, had to alvait studies with the more exacting.grafts of func-
.
tional and stable normal tissues.
.
Cudkowicz 8
HYBRID RESISTANCE TO PARENTAL HEMATOPOIETIC CELLS
CONTROLLED BY THE Hh-1 LOCUS
· Hybrid resistance was discovered in experiments on transplantation of
C57BL mouse marrow into (C57BL x C3H)F1 hybrids. The hematopoietic
cells of such transplants, although widely dispersed in the recipient ani-
mals, are easily distinguished from the cells of the irradiated hosts.
This is so because within giver time intervals DNA synthesis of pre-mitotic
cells in the hosts' hematopoietic organs is almost exclusively due to donor
cells. The data to be presented in the following were obtained by estimating,
5 days after transplantation, the relative size of an expanding hematopoietic
graft from incorporation values of the DNA precursor 5-iodo-2'-deoxyuridine
(labelled with 1311) in repopulated host spleens.
The heretofore generally accepted view that Fi heterozygous mice are
unable to recognize as foreign homozygous parental cells had been based
primarily on results with skin grafts. There were reasons for thinking
that this view may not hol:1 for different experimental conditions (Hauschka
and Furth, 1957; Owen, 1959) and the skepticism was justified by the re-
sults of marrow transplantation experiments. These showed, as exempli.
fied in Table 1, that relatively small grafts of about one million C57BL/10
(B10) marrow cells proliferate actively on trarsplantation into lethally or
sublethally irradiated isogenic and even into allogeneic C3H hosts, but
fail to do so in (B10°x C3H)F1 mice. The sexes of the donor, of the Fi
Cudkowicz 9
host, and of the parental mice entering the Fl crosses do not detectably
affect the outcome of such an experiment (McCulloch and Till, 1963;
Cudkowicz and Stimpfling. 1964a). In contrast to C57BL grafts, marrow
cells taken from C3H mice, the other parental strain, grow well in all
three types of irradiated hosts, i.e., in isogenic, Fi, and allogeneic
.
mice (Table 1). Similar observations were made in a large number of
other types of FI hybrids from outc ronses involving the B10 strain as one
parent (Figure 1). Occasionally an outcross yielded hybrids thit were sub-
ceptible to B10 marrow grafts, as for example (B10 x 129)F1 mice. Thus,
different inbred strains of nice could be classified with respect to the type
of hybrid, resistant or susceptible to B10 grafte, generated from out-
crosses with B10 (Cudkowicz, 1965b; Cudkowicz and Stimpfling, 1965).
The existence and consistency of variation within the species suggested
genetic control of hybrid resistance and provided the basis for the subse.
quent studies.
The possibility existed that during the five days of the experiments
grafted parental hematopoietic cells were not rejected but were prevented
from functioning in-Fi mice without actually losing their viability. This
possibility was ruled out by an experiment of periodic sampling of B10
marrow cells from the spleens of Fi hybrid recipients (Cudkowicz, 1965a).
It was also demonstrated that hybrid resistance was not weakened by sple-
nectomy preceding parental marrow transplantation; however, the resistance
Cudkowicz 10
of Fl hybrids can be overridden by increasing the number of inoculated
parental marrow cells five-to-ten fold. Strong resistance to marrow allo-
grafts between inbred mice of different strains can be overridden in the
samo way. (Cudkowicz, 1965a, 1965b).
One of the puzzling features of these observations was the lack of cor-
relation between the strength of the resistance elicited by Fl hybrid mice
and that eventually elicited by the second parental strain to B10 marrow
grafts. Some mouse strains, like C3H, DBA/1, and A, are less resist-
ant to the allogeneic B10 graft than the Fl hybrids are to the parental B10
graft; other strains, like DBA/2, are as resistant as the hybrids, or more,
to B10 grafts (Figure 1 and Cudkowicz, 1965b). Furthermore, both the
hybrid and the allogeneic recipient mice display resistance after having
been exposed to a dose of whole-body X-rays adequate to suppress or
greatly delay a primary homograft reaction against skin tissue (Brent and
Medawar, 1966). This resistance to marrow grafts cannot be transferred
passively by plasma or serum (Bennett and Cudkowicz, unpublished obser-
.
.
vations).
.
. The growth characteristics of C57BL marrow grafts under these condi-
tions have been demonstrated independently also by McCulloch and Till (1963)
.
and have been confirmed repeatedly (Cudkowicz, 1965a, 1965b; Goodman
and Wheeler, 1966). It becanie clear that understanding of hybrid resistance
' and of allogeneic resistance to marrow grafts was of importance in trans-
plantation biology, and that analysis of the causes might throw light on
Cudkowicz 11
hitherto unsuspected basic properties of the processes of foreign graft
recognition and rejection. The findings raised the possibility that the con- .
trol of histocompatibility is complicated by genetic interaction and by tis-
.
sue specificity, and that the rejection mechanisms for hematopoietic grafts
and for skin grafts are different.
Genetic studies of hybrid resistance were first conducted on F1 hybrids
in
ter
from outcrosses between B10 and fifteen unrelated strains of mice (Cudko-
wicz and Stimpfling., 1964a, 1965a). Hybrids from parents sharing with
B10 the H-2 allele were invariably susceptible, whereas F1 hybrids from
parents with different H-2 alleles and, therefore, H-2 heterozygotes, were
resistant to B10 marrow grafts. For this reason, backcross offspring mice,
obtained by mating resistant (B10 x C3H)F1 or (B10 x A)F1 hybrids with BIO
partners, were typed for the segregating H-2 alloantigens and tested for
resistance to Bló grafts. The results indicated unequivocally that the
manifestation of hybrid resistance was dependent upon heterozygosity at a
single autosomal locus which was either H-2 itself or closely associated
with it in linkage group IX (Cudkowicz and Stimpfling, 1964a). This con-
n ship inside
clusion was confirmed by independent experiments with F1 hybrid mice
born to B10 and congenic-resistant parents (Cudkowicz and Stimpfling,
1964b, 1965b). Such hybrids are essentially homozygous for most gends
of the B10 strain, but are heterozygous for one of several short chromo-
some segments containing either H-2 or another histocompatibility locus.
Again, hybrids were resistant only if the heterozygous portion of their
the signinni
heimsmeistari
..
genome included the H-2 region. Conversely, they were susceptible if the
heterozygous portion of their genome did not include H-2 and if they were
de
H-2° homozygotes. Figure 2 is a simplified genetic map of a part of linkage
group ix, about 2 crossover units long, including the complex H-2 region
and the thymus-leukemia antigen (Tla) locus. Only three of the five known
bubregions of H-2 and a few of the known genetic determinants of individual
alloantigens are indicated in the map, because these subregions adequately
define the mouse strains used.
Manifestation of hybrid resistance could have required heterozygosity
either over an extended portion of the H-2 region, or at a given site within
or near the region. Furthermore, the resistance could have been con-
trolled by one of the determinants of known H-2 alloantigens or by a closely
linked "private" gene. To test these possibilities special Fl hybrid mice
were necessary, heterozygous either at one of the subregions of H-2, or
at a neighboring gene locus such as Tla, while homozygous for the remaining
parts of the IXth chromosome segment illustrated in Figure 2. The strains
of parental mice required to produce such special F1 hybrids would have to
carry exceptional combinations of H-2 subregions or exceptional combina-
tions of H-2 and Tla alleles, resulting, possibly, from crossingover in
this particular chromosome segment. Stimpfling and Richardson (1965)
and Boyse, old, and Stockert (1966) described recombinant H-2 alleles and
exceptional combinations of H-2 and Tla alleles in offspring from H-2* /H-26
. heterozygotes in which H-2а was from strain A and H-2 from strains B10
..
Cudkowicz 13.
Cudkowicz 13
or C57BL6 (B6). The genetic material in linkage group IX of these recombi.
.
.
nant lines of mice derived in part from B10 of B6 and in part from A mice.
through crossingover. Marrow grafts from A strain donors, unlike grafts.
of Bio or B6 donors, grow without impairment in (B10 x AlF1 hybride. .
--
--
-
---
--.
.
--.
Hence, the A strain does not possess the allele determining hybrid resist..
--
ance, and recombinant genotypes resulting from exchanges between A and
.
::
B10 or B6 should be of great value in pinpointing the exact site of the hy
brid resistance.gene.'
Three crossover lines of mice congenic with B10 or with B6, were
kindly provided by Dr. J. H. Stimpfling. Dr. E. A. Boyse, and Dr. L. J.,
old. The mice were used in conjunction with B10 or B6 mice as parents
of hybrids and as donors of parental marrow grafts (Figure 3). They were
strain B10. A(2R)-H-24, possessing genetic material of strain A at the c -73
and' K subregions of H-2; strain B10. A(5R)-H-2. possessing genetic mate-
rial of strain A at the Tla locus and at the D and C subregions of H-2; and .
strain B6-TL+-H-2° possessing genes derived from strain A at the Tla .
locus only. The results of tests performed on hybrids for resistance or
Busceptibility to parental marrow grafts are represented schematically in
Figure 3. It is clear that manifestation of hybrid resistance depends upon
heterozygosity, in the D subregion of H-2." Heterozygosity or homozygosity
.. in other subregions of H-2 or at the Tla locus are irrelevant. On the other
hand, homozygosity for the D subregion of H-2° in donor mice, as occur.
ring in strains B10, B6, and B10. A[2R), confers the trait of deficient :
1.
Cudkowicz 14
growth of marrow grafts in heterozygous Fl recipienta..
Genetic determinants of three known alloantigens lie within the D sub-
region of H-2, i, e., components 2, 4, and 13 of H-2 (Figure 2 and review
article by Snell and Stimpfling, 1966). The determinants of antigeno 4 and
.
13 cannot be responsible for hybrid resistance because they are absent
from the H-2" and H-2h alleles of strains B10, B6, and B10. A(ZR). The
genetic determinant of antigen 2, although present in both the H-2and
- H-24 alleles, is not responsible for hybrid resistance since (B6 x WB)F1
(B10 x WB)F1, and (B10. A(2R) * WB]F1 hybrids, which are homozygotes
for component 2, are resistant to B6, B10, and B10. A(2R) marrow grafts
(Cudkowicz, unpublished observations). It follows that the genetic deter-
minant of hybrid resistance, which lies to the right of the Tla locus and
to the left of the C subregion of H-2, is a newly identified gene of linkage
group IX, designated tentatively hybrid histocompatibility-1 or Hh-1
(Figure 2). Two alleles of this gene are known so far, Hh-19 of the proto
:
type strain B10 and Hh-1 of the prototype strain C3H.
Table 2 gives a list of the inbred mouse strains that have been tested
for the growth pattern of their hematopoietic cells on transplantation into
suitable Fi recipients. The 12 strains listed as Hh-1a do not represent,
presumably, independent occurrences, because they all share the H-2
allele or the D subregion thereof. Mice of these strains possess hemato-
poietic cells growing deficiently on transplantation into Fl hybrid offspring
from crosses with any of the strains listed as Hh-10. The hematopoietic
cells of Hh-1° strains grow unimpaired in F1 hybrids, including those
.
.
.
. Cudkowicz 15
which are resistant to grafts from homozygous Hh-la parental donors.
'.
-
-
Strains listed as Hh-1° are heterogeneous with respect to their H-2 pheno-
type except for the absence of alloantigens controlled by the D subregion
of thé -2" allolo.
Other Hh genes or alleles restricting like Hh-12 the expected compati-
bility of homozygous parental hematopoietic grafts in heterozygous Fi hy-
brids have been recognized and are being analyzed in strains DBA/2 and .
WB. Although the general properties of hybrid resistance to DBA/2 and .
WB marrow grafts appear similar to those of hybrid resistance to B10
grafts, their genetic determination is dissimilar for hybrid resistance to
DBA/2 is sex-influenced in its expression, and hybrid resistance to WB is :
not dependent on heterozygosity in the D subregion of H-2 (Cudkowicz, ..
unpublished observations).
.
.
.
o
vival
...
.
Cudkowicz 16
MATURATION OF HYBRID RESISTANCE
· The capacity for primary rejection of normal skin and various neo-
plastic tissue allografts is well developed in mice at the time of birth or a
few days afterward (Boraker and Hildemann, 1965). In contrast, the
capacity for primary response to alloantigens and to xenogeneic antigens by
way of humoral antibody matures more slowly. The competence for pro-
duction of 7S-type antibodies does not mature in mice until 20 or more days
of age (Boraker and Hildemann, 1965; Bosma, Makinodan, and Walburg.:
1967). It was of interest, therefore, to establish the temporal relation-
ships of the appearance of hybrid resistance and of allogeneic resistance
:
to marrow grafts.
Infant (210 x B10. D2)Fi mice, genetically resistant, are phenotypically.
susceptible to grafted parental Bjo marrow cells at the ages of 16, 17, and
19 days, as illustrated in Figure 4. At 20 days of age, abruptly, the hybride
support less well then isogenic B10 mice the growth of transplanted B10
cells. It is only at 24 days of age that the hybrids become phenotypically
as resistant as adult hybrids. Irradiated infant B10. D2 and B10. A mice.
. .
are also.phenotypically susceptible to allogeneic B10 marrow grafts and
resistance
remain so until 20 days of age. Strong responsiveness to such grafts is
.
acquired one or two days later, as abruptly as it occurred in the hybrids ..
.
.
.
(Cudkowicz, unpublished observations). It appears, therefore, that resist-
ance to foreign marrow grafts in infant mice follows the developmental
maturation sequence described for 75 antibody types. Furthermore, pri-
-
-.
-
-
-
-.
-
-
Cudkowicz 17
مسكيمتصمیشنامج
mary reactivity to marrow allografts in irradiated infant mice becomes
.
demonstrable nruch later than reactivity to skin allografts in unirradiated
!
mice. More data are needed to fully assess the weakening effect of whole-
body Irradiation on tho rosponsivono.. of Infant mice to hematopolotia
allografts; radiation did not interfere, however, with an effective allo-
graft response in mice 21 days old or older. Thus, dissociation of skin
graft and of marrow graft compatibility in mice occurs in the two opposite
directions: (a) in adult F1 hybrids which accept parental skin grafts, but
reject parental marrow grafts from Hh-1a homozygous donors; (b) in
suckling mice which are capable of rejecting skin allografts, but accept
marrow grafts from the same, donors.
!
!
!
!!
!
!
!!!
......
!!..
!!!
!!
......4
.
.!!..».!!....
!
!
.!.!.!.
.
..
e
.
.
.
Cudkowicz 18
THE WEAKENING EFFECT OF PREIRRADIATION AND OF
CORYNEBACTERIUM PARVUM ON HYBRID RESISTANCE
.
The power of X radiation to weaken or abolish destructive host reactions
against skin and tumor allografts in mice is well known and explained by the
antiproliferative action of ionizing radiation (Brent and Medawar, 1966).
Skin allografts at first heal in, induce a primary immune response with
proliferation of lymphoid cells, and later are rejected. Irradiation of
prospective hosts interferes with the proliferation of lymphoid cells stimu-
, lated by the allografts. The course of events is different for blood-forming.
allografts. Irradiation of prospective hosts is required primarily to dis-
:
rupt homeostasis, to create a vacancy in the hematopoietic system witiba
maximal feed-back stimulation of hematopoiesis, and secondarily to
weaken transplantation immunity. Weak incompatibilities are, therefore,
meitenes
simply abolished by radiation and not detected. However, strong incom-
.
.
"g incom.
patitibilities cause prompt rejection of marrow grafts, within a day or
two after transplantation, long before the grafted stem cells had the oppor.
tunity to start proliferation and differentiation (Cudkowicz, 1965a). Early
and vigorous rejections occur in spite of lethal or supralethal doses of
X rays. The host animals are generally infused with marrow cells a few
hours after having been irradiated (Cudkowicz, 1965b). Clearly, radiation
is not as powerful in suppressing reactivity to marrow allografts as it is
in suppressing reactivity to skin allografts. This could reflect differences
.
. Cudkowicz 19
Cudkowicz 19
..
of rejection mechanisms, e.g., of the role played by cellular proliferation
in the allograft responses to skin and to hematopoietic target tissue.
It is known that doses of radiation as high or much higher than those
which affect tho proliferative activity of cells, can be harmless to other :
somatic cell functions. In particular, immunological functions such as
the normal lymphocyte transfer reaction (Brent and Medawar, 1966), the
synthesis of antibody (Miller and Cole, 1967), and the engulfing and degra-
dative capacities of phagocytes (Perkins, Nettesheim, and Norita, 1966)
are hardly affected by 1000 R of X rays. It can be argued that lethally
irradiated F1 hybrids or inbred strain mice are still capable of rejecting
parental or foreign marrow grafts because they are equipped with the rele
vant cell types, whose immunological functions have not been damaged by
:
radiation. If the argument is correct, the host cells which recognize
marrow allografte and carry out destructive reactions against them either
exist prior to irradiation, or are inducible afterwards without intervening.
cell divisions. Proliferation would be necessary, however, for the main-
tenance and replacement of this cell pool. Consequently, irradiation should
weaken or abolish reactivity to marrow allografts, provided that suitable
time intervals are allowed between exposure to radiation and grafting with
incompatible marrow. Experimental results presented in Table 3 verified
this prediction. Mice of the DBA/2 and of the (C57BL * DBA/2)F1 strains
. T3
strongly resisted C57BL marrow grafts given within a few hours to 4 days . !
after irradiation. The hybrids, in particular, displayed resistance after
an exposure as high as 1200 R of whole-body X rays. If resistant mice
Cudkowicz 20
were given a sublethal dose of radiation 5 to 7 days prior to a second ex-
posure and to grafting, C57BL marrow cells were not rejected and the
donor cells proliferated in the incompatible hosts. Thus,..600 R of X rays
were sufficient to weakon hybrid and allogeneic resistance to hematopoietic
grafts with a lag of about 5 days. Presumably this effect of radiation was
due to reduction of the proliferative activity amongst a cell population
turning over continuously and giving rise to the cells responsible for mar- .
row allograft rejection. This function, however, was not damaged by
radiation.
The manner in which whole-body exposure to X rays weakened hybrid
' resistance is reminiscent of the delayed radiation effect on phagocytosis.
This, in turn, suggests the involvement of the reticulo-endothelial sys-
tem (RES) in parental or allogeneic marrow graft rejection, since a
humoral mechanism has already been ruled out. Maximal stimulation of
the RES in mice can be obtained by intravenous administration of a single
dose of killed Corynebacterium parvum (Halpern,et al., 1964). This
organism provokes a complex of changes in the immunological reactivity
of the host attributable to intense proliferation of the lympho-reticular
tissues, both lymphocyte and mononuclear phagocyte elements being in. .
volved. Amongst various effects which have been recorded, the bacterium
produces in vivo a strong phagocytic response (Halpern et al., 1964), an
. adjuvant effect on antibody formation and on delayed hypersensitivity
(Neveu, Branellec, and Biozzi, 1964), an inhibitory effect on the growth of
Cudkowicz 21
transplanted tumors (Halpern, Biozzi, Stiffel, and Mouton, 1966; Woodruff
and Boak, 1966), and an inhibitory effect on graft-versus-host mortality
induced in non-irradiated Fi hybrids by large numbers of C57BL lymphoid
cells (Biozzi, Howard, Mouton, and Stiffel, 1965). The latter effect of
C. parvum and the pronounced influences on the immune system of mice
.
suggested that C. parvum might be a valuable tool to investigate the cellular
basis of hybrid and of allogeneic resistance. For example, if one of the
cute ..
lympho-reticular cell types produced in response to administration of the
organism were responsible for marrow graft rejection, the strength of
hybrid resistance should increase in paraliel.
Heat-killed cultures of C. parvum were obtained through the courtesy
of Dr. G. Biozzi. One half of a milligram (dry weight) of whole bacteria
were injected intravenously, as a suspension in saline, into inbred and Fi
hybrid mice. At varying time intervais, these mice were exposed to 700 R
of whole-body X rays and grafted with 10° nucleated marrow cells. Treat-
F5
ment with C. parvum weakened hybrid resistance (Figure 5) and allo-
geneic resistance (Bennett and Cudkowicz, unpublished observations) to
strongly incompatible marrow grafts with a lag period of about 6 days.
The time sequence of reticulo-endothelial stimulation, as measured by
the phagocytic index, paralleled the changes in hybrid and allogeneic
resistance. Both phagocytic index and hybrid resistance returned to nor-
mal values by the 20th day after bacterial administration (Figure 5). Thus,
the large number of lympho-reticular cells induced by C. parvum were
Cudkowicz 22
not deleterious to foreign hematopoietic grafts.. On the contrary, the in-
tensive proliferation which sustained the lympho-reticular hyperplasia
could have pre-empted a.cell production pathway from common primitive
precursor collø of the immune system to the mature cells roactivo to mar-
row allografts.
The precise mechanisms of the reported effects of radiation and of
· C. parvum on resistance to marrow grafts are far from being fully under
stood. These effects establish, however, an additional similarity between
i
the resistance elicited by hybrids and by inbred mice. Furthermore, they.
clearly indicate that resistance is the result of modifiable host reactions...
wirected against the graſts and not of inherent inability of the grafted cells
to survive in incompatible hosts.
biti
Cudkowicz 23
HYBRID RESISTANCE TO PARENTAL LYMPHOMAS
The deficient growth of certain transplantable parental tumors of the
hematopoietic system in Fl hybrids was described by several investigators
as a phenomenon of histoincompatibility not explained by known allograft i
reactions (Snell, 1958; Snell and Stevens, 1961; Gorer, Tuffrey; and
Batchelor, 1962; Hellström, 1963, 1966; Huemer, 1965). Most of the
studied tumors were lymphomas of C57BL origin. These homozygous
tumor cells could have expressed the trait controlled by the Hh-1a allele
-
--
-
:
like cells of the normal hematopoietic tissue of origin. Consequently, they
could have been subjected to hybrid resistance in appropriate Fi hosts...
To investigate this possibility, the growth of two unrelated radiation-
induced lymphomas of B10 origin, i. e., lymphomas S1033 and 31043,
was studied on transplantation into isogenic and Fl hybrid mice. The
-
-
-
-
two lymphomas, kindly provided by Dr. G. D. Snell, were chosen for their
transplantability, strain specificity, and for their known deficient growth
in Fl hybrids (Snell and Bunker, 1965). They were grafted by the intra-
venous route and the evidence for their take was death of the recipient.
. The experiments vere designed to verify whether the properties of
mineral water
Eskandhandelstahanan naman
hybrid resistance to parental marrow were applicable to the resistance of
talksha
M
hybrids to parental lymphomas. If so, it was expected that resistance is ·
lymphomas would occur only in Fi hybrids heterozygous at the D subregion
of H-2, irrespective of heterozygosity at other genetic sites. It was also
expected that pre-irradiated adult hybrids would lose their resistance and
a nta
24-1-
that infant hybrids would be temporarily susceptible to the parental lymphomas.
..
YO.
Le
Cudkowicz 24
The experimental results presented in Figure 6. confirm published
observations on the deficient growth of C57BL lymphomas in (C57BL x C3H)F,
hybride and illustrate the magnitude of this phenomenon for lymphomas S1033
and S1043. The data also demonstrate that sublethal irradiation completely
abolished hybrid resistance to both lymphomas. This occurred without a lag
instead,
period of 5 days, as was the case with hybrid resistance to marrow grafts
(Table 3). The discrepancy could be explained by the different kinetics of
proliferation of the two types of grafted cells soon after transplantation, i.e..
.
at the time of hematopoietic allograft rejection. Active proliferation of marrow
stem cells infused into irradiated recipients does not start until the second or
third day. Hence, marrow grafts offer a relatively small, non-expanding
target to the host's rejecting apparatus. Grafted lymphoma cells proliferate
actively from the time of transplantation and are more likely, for the increasing
size of the graft, to escape the host's rejecting apparatus impaired by . .
irradiation
Another series of hybrids, offspring from congenic-resistant parents,
were heterozygotes for subregions of H-2 (Figure 7). [B10 x B10. A(2R)]F 79
hybrids were homozygous for the D subregion and, consequently, also for
Hh-la; they were fully susceptible to the two parental lymphomas. In .
contrast, (B10 x B10. A)F, and (B10 x B10.A(5R)] F, hybrids, heterozygous
for the D subregion of H-2, were resistant. Hybrid resistance to parental
lymphomas was not demonstrable in appropriate hybrids until they were 21
days old (Figure 8). Hence, according to three criteria, genetic, host age,
and radiosensitivity, hybrid resistance controlled by the Hh-1 locus applies
.
.F8'
Cudkowicz 25
to B10 normal Kematopoietic cells and to lymphoma cells.
A very important aspect of hybrid resistance to lymphomas remains
to be established, i.e., whether or not the reported deficient growth in
hybrids of lymphomas from parental strains other than C57BL is due to
hybrid histocompatibility genes. The latter tumors were from strains A,
strains,
A. SW, and C3H, all of which were classified Hh-10, since their marrow cells
.
grew without impairment on transplantation into Fl recipients (Table 2). If
lymphomas originating in such strains express Hh or Hh-1 alleles not expressed
in normal tissues, the conclusion would be that the phenotypic expression of
these genes in neoplastic tissues differs from that in normal tissues. A
precedent for such an occurrence has been described by Boyse, old, and
Stockert (1966); the 'murine-thymus-leukemia antigen is specified by the struc-
tural gene Tla. The antigen is present in a proportion of lymphomas originating
in negative strains, i.e., in strains in which the antigen is absent from all
normal tissues.
..
Another interesting aspect of hybrid resistance to tumors which
remains to be firmly established is whether or not tumors originating from
tissues other than the hematopoietic ones express hybrid histocompatibility
.
.
.
.
: genes. It is known that among normal tissues the Hh-la allele finds expression
.
only in hematopoietic cells. Preliminary transplantation experiments with
adenocarcinoma 755 and with two chemically induced sarcomas of C57BL mice
suggest that the Hh-1a aliele is not expressed in these tumors (see qualifica-
tions to this statement in the open discussion following this paper at page ).
Cudkowicz 26
DISCUSSION
.. ... The properties of the hybrid histocompatibility-d system may be
summarized as follows: in C57BL and in a few other inbred mouse strains.
(Table 2) normal hematopoietic cells and lymphoma cells possess a tissue
specificity which is absent from hematopoietic cells of Fl hybrid mice such
as (C57BL x C3H)F>. The phenotypic expression of this specificity requires
homozygosity of its genetic determinant, i. e., of the Hh-1a allele. Its
presence in homozygous cells restricts transplantability into heterozygous
.
'.
recipients, e.g., into certain types of irradiated F1 hybrids which reject
parental marrow grafts. For this reason the specificity may be regarded as
alloantigenic; however, its presence does not restrict as effectively transfers
odae
of hematopoietic cells into homozygous allogeneic recipients, e.g., into
irradiated C3H, A, or DBA/1 strain mice which are unable to reject C57BL
marrow.grafts.
· The Hh-1a specificity is not found in normal epithelial cells, nor in
carcinomas, and sarcomas of C57BL origin (see qualifications in the open
discussion following this paper at page ), although the donor mice possess
the appropriate structural gene. Thus, normal and neoplastic cells of the
hematopoietic system are qualitatively distinguished from other somatic
:
:.
cells with respect to the expression of the Hh-la allele.
In a large number of other inbred mouse strains (Table 2) the Hh-la
specificity is not demonstrable in normal hematopoietic cells. The Hh-1
allele of the latter strains is designated Hh-1° and is identified by the lack
Cudkowicz 27
of restrictions in parent-to-hybrid marrow transfers. Consequently,
resistant F1 hybrids are Hh-1a/Hh-1° heterozygotes. The absence in the Fi
hybrids' hematopoietic cells of the Hh-1a specificity, and the hybrids' ability
to recognize and to mount an allograft reaction against cells carrying it,
.
22
are presumably the consequence of recessiveness of Hh-la or of genetic inter-
action between alleles at the Hh-1 locus. Interallelić interaction could result
either in suppression of the products of one or of both Hh-1 alleles, or in forma-
tion of new gene products called hybrid substances, or both. Hybrid resistance
to Hh-la parental grafts and hybrid susceptibility to Hh-1° parental grafts .
indicate that in heterozygotes the product of the H-1a allele is absent but not
that of the Hh-1° allele. The possibility that resistant hybrids possess also
:
WY
hybrid substances has not been investigated.
The failure of Hh-1° homozygous mice of the C3H, A, and DBA/1,
strains to reject Hh-1a homozygous allogeneic cells speaks against
recessiveness of Hh-1a and favors the interpretation of interallelic inter-
action at the Hh-1 locus. Unfortunately, all the allogeneic host-donor pairs
differing at the Hh-1 locus differ also at some other H locus. The tissue
incompatibility is, therefore, cumulative and the analysis of the Hh-1 effect
difficult. An explanation for the paradoxical susceptibility of certain, but
not all, Hh-i homozygotes may come from reaent. findings suggesting that the
outcome of marrow grafts in hybrids and in allogeneic inbred mice depends
“ot only on the antigenicity of the grafts (determined. by Hh or H genes), but
also on other genetic influences on the processes of alloantigen recognition
and/or allograft reactivity of the host animal. For example, strong "resistance"
Cudkowicz 28
or "reactivity'' genes against hematopoietic and tumor grafts seem to be
possessed by C57BL mice, but not by C3H, A, and DBA/1 mice (Snell,
Russell, Fekete, and Smith, 1954; Cudkowicz, 1965a, 1965b, and unpublished
observations).
·
. Several lines of indirect evidence strongly suggest that the specificity
of HH-1a homozygous hematopoietic cells is a transplantation antigen, although
with peculiar characteristics. The non-codominant mode of inheritance and
the phenotypic expression limited to hematopoietic cells distinguish this allo- .
'antigen from the classical transplantation antigens. However, genetically
resistant Fl hybrids can be rendered specifically tolerant to parental Hh-12..
marrow grafts by multiple injections of viable parental lymphoid cells, but
.
not by, multiple injections of parental thymocytes and non-lymphoid cells
(Cudkowicz and Stimpfling, 1964c, 1965a; Goodman and Wheeler, 1966). This
procedure is reminiscent of the induction of tolerance to classical transplanta-
tion antigens in adult mice, not only for the use of lymphoid cells (McKhann, 1964),
but also for the specificity of the genetic makeup of the tolerance-inducing
cells and for the specificity of the resulting tolerant state toward Hh-1a
marrow grafts. 'Hybrid resistance can, furthermore, be adoptively trans-
ferred by spleen and marrow cells to non-resistant animals, as can immunity
to allogeneic grafts (Cudkowicz, 1965a). 'Also, the weakening effects of X-
irradiation and of Corynebacterium parvum on hybrid resistance are consistent
with the view that hybrids. resist parental marrow grafts in a way which is
similar to that of inbred mice resisting allogeneic grafts. In fact, the effects
of these two agents on hybrid resistance and on allogeneic resistance are
...
.
.... Para
.
Cudkowicz 29
indistinguishable from each other. Identical is also the time of maturation
of hybrid and of allogeneic resistance to marrow grafts in infant mice.
The only major dissimilarity between classical transplantation antigens of
homozygoue hematopoietic cells and the specificity controlled by the Hh-la
allele is the failure of the latter specificity to induce consistently second-
.
set responses and to induce cytotoxic antibodies in resistant hybrids (Cudkowicz,
1964c, 1965a; Goodman and Wheeler, 1966). · It can be concluded, nevertheless,
that the allograft reaction elicited by resistant hybrids shares with the classical
primary reaction to hematopoietic allografts the essential properties of
specificity; mediation by cells of the lymphoreticular system; suppression
by radiation and Corynebacterium parvum; suppression by tolerance-inducing
procedures; and genetic control by a single gene.
.
i The deficient growth in vivo of parental antibody-forming cells and
of parental tumor cells grafted into F1 hybrids has been regarded in the past
as an inhibition phenomenon due to exhaustive hyperactivity or to hypersensitivity
of the grafted cells. The inhibition was thought to result from the cells' exposure
to the foreign environment of the Fi hybrid, in particular to the alloantigens
contributed by the hybrid' 8. second parental strain. It was assumed, therefore,
that the resistant hybrids were not playing an active role in these inhibition
phenomenon designated "hybrid effect" by Snell (1958), "allergic death!' by
Boyse (1959), and "allogeneic inhibition" by Hellström and Hellström (1965).
:
:: Clearly, hybrid resistance controlled by the Hh-1 locus does not fit such an
i
interpretation. It has been demonstrated conclusively that deficient growth
Cudkowicz 30
in hybrids was not due to inability of grafted parental cells to withstand the
Fi hybrid environment, since these parental cells were capable of growth
in infant hybrids and in adult hybrids in which the resistance had been
weakened or abolished by various procedures. Furthermore, normal and
neoplastic parental cells were capable of unimpaired growth in genetically
susceptible hybride, all of which were heterozygotes for one or more histo-
compatibility genes and possessed, therefore, alloantigens foreign to the
grafted cells. A number of other investigators have also contributed to the
unequivocal demonstration that transplantable antibody-forming cells
(Hattier, Schlesinger, and Amos, 1964), lymphoma cells (Glynn, Humphreys, '
.
Trivers, Bianco, and Goldin, 1963; Sanford, 1967), and sarcoma cells (Oth
and Burg, 1965) which fail to grow in Fi and allogeneic hosts are eliminated
by host controlled allograft reactions and not by "allergic death" or "allogeneic
inhibition" reactions.
In studying the hybrid resistance phenomenon it became apparent
that the mechanism of marrow graft rejection was similar in hybrid and in
allogeneic recipient mice grafted with Bl0 cells. However, as an intriguing
by-product of these studies, it was learned that striking differences existed
between the reactivity of mice to skin allografts and their manner of rejection,
and the reactivity to marrow allografts and their manner of rejection. The
major differences are summarized in Table 4; the observations are preliminary
in nature and, therefore, not yet fully understood. Under certain conditions,
-
mice were capable of rejecting one type of graft and not the other, as if the
Cudkowicz 31
effector mechanisms were different and independent. The general impression
iniowe
received from these studies is one of greater complexity, genetic and phy-
siological, in the host controls of hematopoietic graft proliferation than in
the controls of other tissue grafts. It is to be hoped that the special features
of hematopoietic cells will facilitate the understanding of the possible intrinsic
biological role of alloartigens - especially if they are tissue-specific in the
hemeostatic control of tissue growth.
Lihtne
- Wie
iscrimincovnée ici.
Cudkowicz 32
.
ACKNOWLEDGEMENTS
The author wishes to acknowledge the helpful advice of Dr. T.'s.
Hauschka in the preparation of the manuscript; the gift of congenic-
. resistant mouse breeders and of transplantable lymphomas $1033 and
$1043 from Dr. G. D. Snell and Dr. J. H. Stimpfling; the help and the
cooperation with Dr. 'J. H. Stimpfling, Dr. E. A. Boyse, and Dr. L. J.
Old in linkage experiments of the Tla, Hh-1 and H-2 loci; and the gift
of Corynebacterium parvum from Dr. G. Biozzi. This work was carried
out in part at the Biology Division, Oak Ridge National Laboratory, Oak
...
Ridge, Tennessee, with support of the U. S. Atomic Energy Commission
under contract with Union Carbide Corporation, and at Roswell Park
:
"{emorial Institute, Buffalo, New York, supported by research grant
6-66-RP-5 from the United Health Foundation of Western New York, and
by research contract PH-43-65-44 with the National Cancer Institute, U. S.
Public Health Service.
Cudkowicz 33
B6
C57BL/6
BIO
C57BL/10
C. parvum
Corynebacterium parvum
DNA
Deoxyribonucleic acid
H
Histocompatibility genes or loci
H-2
Histocompatibility-2
Hybrid histocompatibility genes of loci
HA
Hh-1
131JUDR
RES
Reticulo-endothelial system
Tla
Thymus-leukemia antigen gene or locus. :
Cudkowicz 34
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Miller, J. J., and L. J. Cole. 1967. The Radiation Resistance of Long-
Lived Lymphocytes and Plasma Cells in Mouse and Rat Lymph Nodes.
Journal of Immunology, 98: 982-990.
Neveu, T., A. Branellec, and G. Biozzi. 1964. Propriétés adjuvantes
de Corynebacterium parvum sur la production d'anticorps et sur
l'induction de l'hypersensibilité retardéé envers les protéines conjuguées.'
Annales de l'Institut Pasteur, 106: 771-777.
: old, L. J., and E. A. Boyse. 1965. Antigens of Tumors and Leukemias
Induced by Viruses. Federation Proceedings, 24: 1009-1017.
.: Oth, D., and C. Burg. 1965. Suppression par irradiation X du phenomène
de préférence syngénique présenté par deux sarcomes isologues
provoqués par le methylcholantrène et le dibenzanthracene. Comptes
Rend us des séances de la Société de Biologie, 159: 2231-2234.
Oth, D., and C. Burg. 1966. Resistance accrue d'hybrides Fl a l'egard
d'une deuxieme transplantation de tumeurs isologues parentales presentant
.
..
.
...
... -
.
Cudkowicz 40
le phenomene de preference syngenique. Comptes Rendus des Séances
de la Société de Biologie, 160: 342-345.
Oth, D., M. Donner, and C. Burg. 1967. Comparaison de l'immunite
. induite par plusieurs tumeurs isologues dans leurs souches d'origines
et chez des hybrides Fl. Comptes Rendus des Séances de la Société de
· Biologie, in press.
Oth, D., J. Robert, C. Michaud, and M. Crestin. 1964. Comportement :
anormal d'une tụmeur isologue de souris C3H transplantée chez des
hybrides Fi. Comptes Rendus des séances de la Société de Biologie, il
158: 841-844.
Owen, R. D. 1959. Genetic Aspects of Tissue Transplantation and Tolerance.
The Journal of Medical Education, 34: 366-383.
-.
-.-.-.-.-
Perkins, E. H., P. Netteshein, and T. Morita. 1966. Radioresistance of
the Engulfing and Degradative Capacities of Peritoneal Phagocytes to
Kiloroentgen X-Ray Doses. Journal of the Reticuloendothelial Society,
3: 71-82.
Popp, R. A., and G. Cudkowicz. 1965. Independence of Deficient Early
Growth and Later Regression of (C57BL x 101)F2 Marrow Grafts in
(C57BL x 101)F, Hybrid Miče. Transplantation, 3: 155-160.
Prehn, R. T. 1965. Cancer Antigens in Tumors Induced by Chemicals.
-
-
-
Federation Proceedings, 24: 1018-1022.
Rossi, G. B., and C. Friend. 1967. Inhibition of Spleen Colony Formation
of Cultured Leukemic Cells in Mice Immunized With Friend Virus
(Abstract). Federation Proceedings, 26: 314.
Cudkowicz 41
Sanford, B. H. 1967. Evidence for Immunological Resistance to a Parental
*Line Tumor by F1 Hybrid Hosts. Transplantation, 5: 557-560.
Snell, G. D. 1958. Histocompatibility Genes of the Mouse. II. Introduction
and Analysis of Isogenic Resistant Lines. Journal of the National
Cancer Institute, 21: 843-877.
Snell, G. D., and H. P. Bunker. 1965. Histocompatibility Genes of Mice.
V. Five New Histocompatibility Loci Identified by Congenic-Resistant
Lines on C57BL/10 Background. Transplantation, 3: 235-252.
Snell, G. D., E. Russell, E. Fekete, and P. Smith, 1954. Resistance of
Various Inbred Strains of Mice to Tumor Homoiotransplants, and its
Relation to the H-2 Allele Which Each Carries. Journal of the National
· Cancer Institute, 14: 485-491.
Snell, G. D., and L. C. Stevens. 1961. Histocompatibility Genas of Mice. :
III. H-1 and H-4, Two Histocompatibility Loci in the First Linkage
Group. Immunology, 4: 366-379. .
Snell, G. D., and J. H. Stimpfling. 1966. "Genetics of Tissue Transplantation,"
Biology of the Laboratory Mouse,' E. L. Green, Ed. New York, New York:
McGraw-Hill Book Company. Pp. 457-491.
1. Stimpfling. J. H., and A. Richardson. 1965. Recombination within the
Histocompatibility-2 Locus of the Mouse. Genetics, 51: 831-846.'
Suit, H. 1967. "Implications of Cell Proliferation Kinetic Studies for Radio-
therapy.." The Proliferation and Spread of Neoplastic Cells. (The .
University of Texas M. D. Anderson Hospital and Tumor Institute, 21st
.
Cudkowicz 42
Annual Symposium on Fundamental Cancer Research). Austin, Texas:
The University of Texas Press. Pp.
Till, J. E., and E. A. McCulloch. 1961. A Direct Measurement of the
Radiation Sensitivity of Normal Mouse Bone Marrow Colle. Radiation
Research, 14: 213-222.
Wallace, A. C. 1965. Growth of Mammary Tumors in F1 Hybrids. Nature,
207: 309.
Woodruff, M. F. A., and J. L. Boak. 1966. Inhibitory Effect of Injection of
Corynebacterium pårvum on the Growth of Tumor Transplants in Isogenic :
Hosts. British Journal of Cancer, 20: 345-355.'
..
Ta
r
y-n
ta
ut
.
.
.
. .
T
+
-
..
.
.
I
TABLE I
131
GROWTH OF MARROW GRAFTS ESTIMATED BY THE "SIUR* LABELLING TECHNIQUE
5 DAYS AFTER TRANSPLANTATION OF 100 CELLS INTO RECIPIENTS EXPOSED TO 700 - 800 R OF X-RAYS
DONOR STRAIN
RECIPIENTS
MEAN SPLENIC UPTAKE OF 15 IUDR (%) RELATIVE GROWTH
STRAIN
NO.
(WITH 95% CONFIDENCE LIMITS)
BIO
(0.50 - 0.59)
100
B10
(B10 X C3H)FI
55
0.54
0.02
0.45
(0.01 - 0.02)
(0.37 - 0.54)
С3Н
сзн
0.71
(0.64 - 0.78)
*100
сэн
(B10 X C3H)FI
103
*
0.88
(0.85 - 0.91),
123
3.
B10
0.57
(0.51 -0.63).
-
80
Cudkowicz 43
* 131IUDR = 5-Iodo-2'-deoxyuridine labelled with '
Cudkowicz 44
TABLE 2
· "THE STRAIN DISTRIBUTION OF Hh-1 AND H-2 ALLELES
Hh-la H-2". C57BL
C57BL/6
H h-1°
C57BL/10
C57L
LP
H-za Aim
B10. A*.
BALB/C
DBA/2
B10.02*
H-Z RFM :
B10. M
H-2". HTI* :
310. A(5R)**
H-2k C3H
129
C3H. SW*
A. BY*
DI. Lp*
H-28 HTGT
H-2" HTHT
.. B10. A(2R)**
C3H. K*
.
101
AKR
C57BR
B10. BR*
H-2* AKR. M*
B10. AKM*
H-29 DBA/1
H-2° A. Sw*
* CONGENIC-RESISTANT LINES
† CROSSOVER LINES WITH D REGION OF H-26..
3. CROSSOVER LINES WITH K REGION OF H-26
.
.-
.-. .
.
- -
- -
.
.
-
-
-
...
1:
.
TABLE 3
EFFECT OF PREVIOUS RADIATION INJURY ON HYBRID RESISTANCE TO C57BL
MARROW GRAFTS AFTER'A SECOND EXFOSURE TO RADIATION*
131
RECIPIENTS
MEAN SPLENIC UPTAKE OF "'IUDR (%) RELATIVE
(WITH 95% CONFIDENCE LIMITS) GROWTH
STRAIN
IRRADIATION
1st 2nd Interval
(R) (R) (days)
NO.
....
C57BL
.
· 0.52
1.45 - .61)
100
20
24
.
500
700
700
.
4-7
:
:
0.53
(.44-.63)
100
(C57BL x DBA/2)F1
10
-
700
.
11
-
1200
:. 11
500
700
0.02
0.03
0.06
0.23
0.45
6.01 - .03)
6.01 - .04)
6.03 - .12)
1.17- .31)
(31-.65)
11
500
700
11
500
700
DBA/2
il
700
..
.
0.04
0.23
6.02-.07)
6.15 - .36)
5
500
700
Cudkowicz 45
* 10° C57BL marrow cells transplanted within a few hours after the second exposure to radiation. Splenic
. uptake of "IUDR measured 5 days later.
TABLE 4
DIFFERENCES IN THE GENETICS AND IMMUNOLOGY OF H.STOCOMPATIBILITY TO HEMATOPOIETIC
GRAFTS IN IRRADIATED MICE AND TO OTHER TISSUE GRAFTS IN NON-IRRADIATED MCE
MARROW AND LYMPHOMA GRAFTS
SKIN, CARCINOMA, AND SARCOMA GRAFTS
NO SIMILAR GENETIC INTERACTIONS
MATURATION OF PRIMARY ALLOGRAFT
RESPONSIVENESS AT BIRTH
LATE REJECTION OF PRIMARY ALLOGRAFTS
GENETIC INTERACTIONS (INTERALLELIC, INTERGENIC)
AFFECT THE INHERITANCE OF, AND THE RESPONSIVE-
NESS TO TRANSPLANTATION ANTIGENS (E.G., HYBRID
RESISTANCE, RESISTANCE GENES).
MATURATION OF PRIMARY ALLOGRAFT RESPONSIVENESS
AT ~ 21 DAYS OF AGE
PROMPT REJECTION OF PRIMARY ALLOGRAFTS (WITHIN
48 HOURS) BEFORE THE HEALING-IN HAS TAKEN
PLACE
WHOLE-BODY X-IRRADIATION GIVEN A FEW HOURS
• BEFORE GRAFTING (500 - 1200 R) DOES NOT
· PREVENT PROMPT REJECTION OF PRIMARY
MARROW ALLOGRAFTS
CORYNEBACTERIUM PARVUM GIVEN 7 DAYS BEFORE
GRAFTING PREVENTS REJECTION OF PRIMARY
MARROW ALLOGRAFTS
(10 DAYS OR MORE) AFTER THE HEALING-
IN HAS TAKEN PLACE
WHOLE-BODY X-IRRADIATION GIVEN A
FEW HOURS BEFORE GRAFTING SUP-.
PRESSES OR DELAYS REJECTION OF
PRIMARY ALLOGRAFTS
CORYNEBACTERIUM PARVUM GIVEN
BEFORE GRAFTING STRENGTHENS
REJECTION OF PRIMARY ALLOGRAFTS
Cudkowicz 46
BIOXA
B10 X DBA/1
B10x129
BIO X DBA/2
RELATIVE GROWTH OF BIO
MARROW IN FI AND ALLOGENEIC
MICE


nonnut
F1 DBA/1 F1 A FI DBA/2 F1 129
RECIPIENT STRAIN
-
.
Figure 1. Resistance and susceptibility to B10 marrow grafts in
Fl hybrid and allogeneic mice. Growth of 106 marrow cells 5 days after
.
.
.
.
sonte.
'transplantation into groups of irradiated (700 R of X rays) recipients.
Growth is expressed in relative units, i.e., as percent of the graft's
..
growth in isogenic recipient mice. Ten or more mice in each group.
--
.
.
.-
.
-
.
.
-,
Hh-1
(2.4.13) (3.8)
K
:
.1.51
0.36:
Figure 2. Simplified map of the H-2 region in linkage group IX
of the mouse. Numbers in the lower row indicate map distances in cross-
over units. Numbers in the upper row, some of which in parentheses,
identify individual determinants of alloantigens; the sequence of the deter-
minants in parentheses is not known. Capital letters D, C, and K identify
subregions of H-2.
Gene symbols in italics: Tla - Thymus leukemia antigen
H-2
= Histocompatibility-2
Hh-1 = Hybrid histocompatibility-1
Ss · = Serum variant.
Nomenclature used according to Snell and Stimpfling (1966).
'... tla
HP2
DCK
STRAIN
CLASSIFICATION
:, OF HYBRID
35
B10
:
A
RESISTANT
B 10. A
RESISTANT
B10
B10.A(5R)
ses RESISTANT :
B10
B10. A (2R) SUSCEPTIBLE
B10. A (2R)
B10.A
RESISTANT
Video
Axis
B6
B6-TL+
i
SUSCEPTIBLE
o
Figure 3. Schematic representation of the relationship between
heterozygosity in restricted subregions of H-2 and manifestation of hybrid
- resistance to parental B10, B6, and B10. A(ZR) marrow grafts. The Fi.
hybrids were obtained by mating mouse pairs of congenic-resistant lines
:originated from crossovers within H-2 and between H-2 and Tla. The lines
congenic with Bl0 were described by Stimpfling and Richardson (1965); the
line congenic with B6 by Boyse, ord, and Stockert (1966). Adapted from
unpublished results of Cudkowicz, Stimpfling, Boyse, and Old.
0
RELATIVE GROWTH OF 010
MARROW IN (B10 x B10.D2) Fi.
MICE

0
تشملتمسخصصضضضحلفستنننعسنة
سمنضنننننننننننسنننشينغا
قصدتشنششتكتسعسعسننمنلنعنه
محصحضيمتنعسععمنغصتنخست
نغمممممعمسسسسسسسششتعل شمعة
نسيمتيسسسسسبيسئسنسسسسسخسمنة
0
نننننتتحسد
۴
۲
1
16 17 19 20 22 24.34
RECIPIENTS' AGE (DAYS )
Figure 4. Maturation of hybrid resistance. Growth of 5 x 105 B10
marrow cells 5 days after transplantation into groups of irradiated (600 -
700 R of x rays) infant F1 hybrid recipients. Growth is expressed in .
relative units, i. e. as percent of the graft's growth in groups of isogenic
infant recipients of the same age. Ten or more mice in each group.

RELATIVE GROWTH OF PARENTAL
MARROW IN FÍ MICE

.
4 6 7 14 16 18 20
TIME AFTER BACTERIAL INJECTION
(DAYS)
Figure 5. Weakening effect of Corynebacterium parvum on hybrid
resistance. Growth of 100 parental marrow cells 5 days after transplanta-
tion into irradiated (700 R of x rays) Fl hybrid recipients pre-treated with
heat-killed Corynebacterium parvum. Growth is expressed in relative units,
i. e., as percent of the graft's growth in similarly pre-treated isogenic ·
recipient inice. The dotted line indicates variations of phagocytic index,
adapted from Halpern et al. (1964)..
BIO
Strain combinations:
D B10
B10. A(2R)
C57BL
(B10 x C3H)F1...
(B10. A(2R) * B10. A)F
(C57BL * DBA/2)F1
.
OS1033 ( 100 CELLS.)
S 1043 ( 200 CELLS)
48/48 26/27
.
22/23
19/22
58/68
LETHAL
TUMOR TRES (%). - .



2/12 2/16
mis
ia
NO X-RAY 500R NO X-RAY 500R
STRAIN OF ORIGIN (B10) HYBRIDS (B10xC3H)F1
Figure 6. The radiosensitivity, of hybrid resistance to small inocula
of transplantable parental B10 lymphomas S1033 and 51043. The number
of lethal tumor takes and the total number of mice grafted are indicated
above the bars. Exposure to radiation and strains of recipients are :
indicated beneath the bars.
C S 1033 (100 CELLS)
S 1043 (200 CELLS)
DCK оск DCK REGIONS
OF H-2
31/32 40/40
13/22
LETHAL
TUMOR TAKES (%)


9/23 12/32
8/258
1
B10x B10.A (2R) B10x B10.A(5R) B10x. BIO. A
Figure 7. The relationship between heterozygosity in the D sub-
region of H-2 and manifestation of hybrid resistance to small inocula of:
parental lymphomas $1033 and 51043. The number of lethal tumor takes
the
and the total number of mice grafted are indicated above bars. Strains of
recipient hybrids are indicated beneath the bars.
.
1
ke
DS 1033 (100 CELLS)
W S1043 (200 CELLS)
. 21/22
18/22
15/19
:
LETHAL
TUMOR TAKES (%)

4/10
8/26
12/36
AGE (DAYS) 22-32 13-21
STRAIN B10x B10. A F1
ADULT 12-21
B 10 x B10.A(5R) F1
Figure 8. Maturation of hybrid resistance to small inocula of
parental lymphomas $1033 and 31043. The number of lethal tumor takes .
..
and the total number of infant mice grafted are indicated above the bars.
Age at the time of tumor inoculation and strains of recipient hybrids are
indicated beneath the bars.

END
DATE FILMED
11 / 28 / 67








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