immunogenetics 8: 13-26, 1979

Immunogenetics ~L'/1979 by Springer-Verlag New York Inc.

Original Investigations Natural Resistance of Irradiated 129-Strain Mice to Bone Marrow Allografls: Genetic Control by the H-2 K Region Gustavo Cudkowicz and John F. Warner Departments of Pathology and Microbiology, School of Medicine, State University of New York at Buffalo, Buffalo, New York 14214

Abstract. A major genetic determinant of natural resistance to bone marrow allografts, designated as Hh-3, was mapped to the H - 2 K region. This gene may code for or regulate the expression of cell surface structures selectively expressed on donor hemopoietic cells and recognized by naturally occurring cytotoxic effectors. Resistance was observed as failure of donor cell growth in the spleen of irradiated 129-strain (H-2 be) recipients of H-2 k bone marrow cells. The mapping was accomplished by substituting donor cells bearing k alleles throughout the H-2 complex with cells of recombinant mouse lines bearing k alleles at defined H-2 regions. The host antigraft reaction underlying resistance was abrogated by pretreating 129-strain mice with either rabbit antimouse lymphocyte serum or the antimacrophage agent silica. Grafting of H-2K k cells into mice ancestrally unrelated to 129 but sharing the H-2 bc or the similar H-2 b haplotype, and into H-2 b/k, H-2 k/he, and H-2 kid F 1 hybrids revealed that resistance was unique to 129 mice, since mice of the other strains, including F 1 hybrids, were susceptible to the grafts. Thus, Hh-3 incompatibility was a necessary but insufficient condition for the manifestation of allogeneic resistance; other genetic factors not associated with H-2 conferred responder status to 129-strain mice and nonresponder status to D1.LP, B10.129(6M), B10, B6, and possibly to F 1 hybrid mice. The possible relationships between allogeneic resistance to H-2 k marrow grafts, hybrid resistance to H-2 k lymphomas, and F a hybrid antiparental H-2 k cytotoxicity induced in vitro are discussed.

Introduction Shortly after exposing mice to lethal doses of irradiation, lymphoid cell replication is severely impaired so that the irradiated animals become incapable of eliciting conventional immune responses (Anderson and Warner 1976). For a time, however, irradiated mice maintain their capacity for natural cell-mediated cytotoxicity 0093-7711/79/0008/0013/$ 02.80

14

G. Cudkowiczand J. F. Warner

against parental or allogeneic bone marrow and lymphoma cells (Cudkowicz and Bennett 1971a, b, Kiessling et al. 1977, Hochman et al. 1978). The effectors of natural resistance are generated spontaneously in adult mice and, therefore, preexist treatments such as irradiation and challenge with normal or transformed cells of hemopoietic origin. Such effectors are thymus-independent cells that become functional relatively late in ontogeny; they differ from cells mediating induced immunity with regard to several other properties (Cudkowicz and Hochman 19791. Resistance of irradiated mice to bone marrow allografts involves the recognition of, and reaction against, target determinants controlled by the major histocompatibility complex (MHC). The H h - I gene(s) (Hemopoietic-histocompatibility-1) map within the H-2D region and control both allogeneic and hybrid resistance in most but not all strain combinations studied so far (Cudkowicz 1968, 1975a, 1978, Cudkowicz and Lotzov/t 1973). These genes may either code for or regulate the phenotypic expression of target determinants which are seemingly restricted to hemopoietic cells. Hh-1 is not inherited codominantly, unlike other genes of the MHC, since full expression of the gene products requires homozygosity (Popp and Cudkowicz 1965, Bennett 1972). Host responsiveness to Hh-1 determinants on allogeneic and parental bone marrow cells is regulated by a small number of It-like genes segregating independently of the M H C (Cudkowicz 1971, 1972; Cudkowicz and Lotzov/t 1973). The exclusive association of H h genes with H-2D may, therefore, merely reflect the prevalence of reactivity genes conferring responsiveness to Hh-1 products among the limited number of laboratory mouse strains studied. If this assumption is correct, investigations with additional mouse strains unrelated to the previously studied strains (i. e., with different reactivity genes) would detect H h genes associated with other MHC regions, e. g., H - 2 K or I regions. The basic symmetry of the M H C for genes specifying alloantigens suggests that H h genes also exist in or near the K end of the H-2 complex. Preliminary investigations provided evidence that 129-strain mice (H-21'c) are resistant to B10.A(2R) bone marrow cells bearing a recombinant H-2 haplotype with k alleles in the K through IE regions and b alleles in the D region (Cudkowicz and Lotzov/t 1973). The manifestation of this resistance to K-end-incompatible cells was critically dependent on using 129-strain recipients, since it was not observed in mice of other H-2 bc or H-2 b strains. The genetic analyses reported herein demonstrate that the gene(s) controlling target determinants for natural resistance of 129-strain mice to H-2kmarrow grafts reside within or near the K region, on the centrometric side. The critical importance of reactivity genes for this analysis is fully realized if one considers that mice of other strains sharing the H - 2 haplotype, but unrelated (Graft and Snell 1969, Klein 1975), were not resistant to H-2K k bone marrow cells.

Materials and Methods Mice. Ten to 20-week-oldinbred and F 1hybridanimals of both sexeswereemployed.Micewerebred in our own animal colony, except for the following strains: B10.129(6M)and C3H.OH (The Jackson Laboratory),CBA(M523)and A.TH(Dr. Marek B. Zaleski),and (B6 • C3H)F~,B6, C3H/He,and A/He (Mammalian Geneticsand Animal ProductionSection,Divisionof Cancer Treatment,National Cancer

Bone Marrow Graft Resistance Controlled by H-2K

15

Institute}. Sublines of 129-strain mice were either Rr or J. F, hybrid mice were designated by listing first the female and then the male parental strain.

Irradiation, transplantation,and grqf} evaluation.All procedures have been described previously (Bennett etal. 1968, Cudkowicz and Bennett 1971b). Briefly, prospective bone marrow graft recipients were exposed to lethal, total body irradiation a few hours before donor cell transfer. The radiation dose was 850 to 950 rads of 137Cs 7-rays delivered at the rate of 112 rads/min (Gammacell-20 Small Animal lrradiator, Atomic Energy of Canada, Ltd., Ottawa}. One-ml volumes of bone marrow cells suspended in Eagle's Minimal Essential Medium (Associated Biomedic Systems, Inc., Buffalo, New York) were injected into a lateral tail vein. Donor-recipient combinations were sex matched. Proliferation of donorderived cells was assessed 5 days later in the spleens of recipients by measuring the incorporation into splenic DN A of 125l-labeled 5-iodo-2'- deoxyuridine (1251UdRI, an analog of the deoxyribonucleotide precursor thymidine. Cell proliferation in the spleen at 5 days is predictive of repopulation of the lymphomyeloid complex. Eighteen h after i. p. injection of approximately 0.25 ltCi ~-~SIUdR(Amersham Searle Corporation, Arlington Heights, Illinois}, spleens were removed and the retention of radioactivit~r was determined by crystal scintillationspectrometry. The values of splenic ~2SlUdR uptake were expressed as the percent of retained radioactivity. Geometric means and standard errors (SEs) were calculated for each group of mice injected with aliquots of the same bone marrow preparation. The SEs were 10~'oof the mean uptake values. In each experiment, one group of irradiated recipient mice syngeneic with the donor was the positive control for uninhibited growth. Another group of irradiated mice not injected with ceils was the negative radiation control for background retention of ~2SlUdR (< 0.05~ splenic uptake).

Host pretreatments. In some experiments, prospective recipient mice were injected i. v. with either 0.3 ml of rabbit antimouse lymphocyte serum (ALS, Microbiological Associates, Bethesda, Maryland) or 2 mg of silica particles suspended in saline and exposed to ultrasonic waves (average size <5 #m, Steinkohlenberg Bauverein, Essen-Krey, Federal Republic of Germany). The pretreatment protocol for ALS was irradiation at time 0, antiserum 17 h later, and bone marrow transplants after 3 additional h. The protocol for silica was irradiation, silica 1 h later, and transplantation after 19 additional h. The radiation control groups were pretreated with ALS or silica, but not injected with cells. Results

Requirements Jbr demonstration of resistance A c c o r d i n g to p r e v i o u s studies, i r r a d i a t e d mice a c c e p t b o n e m a r r o w grafts f r o m allogeneic d o n o r s differing at m u l t i p l e n o n - H - 2 loci a n d / o r H-2 r e g i o n s o t h e r t h a n D. M o r e o v e r , i r r a d i a t e d mice of several C 5 7 B L lines (II-2 b) a n d of the D 1.LP s t r a i n (H-2 be) fail to reject b o n e m a r r o w grafts f r o m d o n o r s differing t h r o u g h o u t the M H C , such as H - 2 k grafts, p r e s u m a b l y b e c a u s e these mice are genetically d e t e r m i n e d p o o r r e s p o n d e r s . D 1 . L P a n d 129 mice share the H-2 bc h a p l o t y p e , b u t the l a t t e r do n o t accept H - 2 k grafts. 129-strain mice m a y differ f r o m the o t h e r strains s i m p l y b y b e i n g g o o d r e s p o n d e r s to M H C - c o n t r o l l e d target d e t e r m i n a n t s . I n this case, the r e c o g n i z e d a n t i g e n s of h e m o p o i e t i c stem cells c o u l d be specified b y either D - r e g i o n l i n k e d Hh-1 gene(s) or b y tth genes l i n k e d to o t h e r regions. T o d i s t i n g u i s h b e t w e e n these alternatives, a systematic s t u d y was m a d e u s i n g B6, B10, D l i P , a n d 129s t r a i n recipient mice, a n d d o n o r cells b e a r i n g k alleles at v a r i o u s M H C regions. A p r e l i m i n a r y search for o p t i m a l assay c o n d i t i o n s i n d i c a t e d t h a t 3 • 105 to 1 x 106, b o n e m a r r o w cells s h o u l d be grafted i n t o i r r a d i a t e d m i c e . T h e e x t e n t of splenic r e p o p u l a t i o n 5 days after cell transfer a l l o w e d u n e q u i v o c a l classification of allogeneic recipients i n t o three categories: (1) susceptible, w i t h splenic r e p o p u l a t i o n c o m p a r a b l e to t h a t of s y n g e n e i c c o n t r o l mice; (2) weakly resistant, with splenic r e p o p u l a t i o n o n e to t w o - t h i r d s t h a t of s y n g e n e i c c o n t r o l s ; a n d (3) resistant, w i t h

16

G. Cudkowicz and J. E. Warner

negligible r e p o p u l a t i o n so t h a t retention of 12SIUdR was c o m p a r a b l e to that of r a d i a t i o n controls. U n d e r the assay c o n d i t i o n s e m p l o y e d , splenic u p t a k e of a z s I U d R was a linear fnnction of the n u m b e r of t r a n s p l a n t e d b o n e m a r r o w cells (Bennett et al. 1968). F o r m a p p i n g target determinants, it is desirable to c o m p a r e susceptible a n d fully resistant h o s t - d o n o r c o m b i n a t i o n s . A l t h o u g h w e a k resistance m a y well be the result of M H C i n c o m p a t i b i l i t y , o t h e r factors such as sex a n d h o r m o n a l status, h e m o p o i e t i c m i c r o e n v i r o n m e n t , a n d genes influencing cellular differentiation m a y also cause s u b o p t i m a l proliferation of t r a n s p l a n t e d cells. Hence, small differences in splenic r e p o p u l a t i o n are difficult to interpret a n d m u s t be carefully a n a l y z e d before being exclusively a t t r i b u t e d to M H C products. M a p p i n 9 o f tar(let determinants to the H - 2 K region

Three series of experiments were p e r f o r m e d in which i r r a d i a t e d B6 or B10, D1.LP, a n d 129-strain mice were grafted with 106, 5 x 105, or 3 x 105 b o n e m a r r o w cells (Tables 1-3).H-2 b or H-2 bc cells injected into m i c e , o f the recipient strains verified susceptibility to allogeneic H - 2 - m a t c h e d bone m a r r o w cells (Table 1; see 129, D1.LP, a n d B10 donors). In o t h e r words, resistance was not o b s e r v e d in a n y of the recipients when foreign target d e t e r m i n a n t s could have been c o n t r o l l e d only by genes i n d e p e n d e n t of the M H C . The first set of experiments established that full M H C i n c o m p a t i b i l i t y has a different effect in B6, B10, a n d D 1 . L P host mice on the one hand, a n d in 129-strain hosts on the other, The i n c o m p a t i b i l i t y resulted in little, if any, d o n o r cell g r o w t h in 129-strain recipients, b u t in substantial, t h o u g h s u b o p t i m a l g r o w t h in recipients of

Table 1. Resistance of irradiated 129-strain mice and susceptibility of D1.LP or B10 mice to the growth of 106 transplanted bone marrow cells bearing k alleles at the K and l regions of H-2 Bone marrow donors* Strain

129 D1.LP B 10

B 10.BR Bi 0.A B10.A(2R) HTH B10.A(5R) B10.HTG

Percent uptake of 125IUdR in recipient spleenst

H-2 haplotype ~ 129 (H-2 be)

bbbbbbbbb bbbbbbbbb bbbbbbbbb kkkkkkkkk kkkkkdddd kkkkkdddb kkkkkdddb bbbkkdddd ddddddddb

D1.LP (H-2 be)

B10 (H-2 b)

F

M

F

M

F

M

0.73 0.65 0.73 0.13 0.08 0.09 ND 0.28 0.27

0.64 0.74 0.61 0.06 0.12 0.14 0.02 0.30 0.25

ND 0.69 0.66 0.24 0.23 0.55 0.48 0.24 0.28

ND 0.70 0.59 0.19 0.19 0.51 0.60 0.16 0.31

0.68 0.72 0.67 0.31 0.06 0.52 0.67 0.06 0.42

0.69 0.72 0.58 0.38 0.03 0.56 0.62 0.06 0.19

* Donor cells transplanted into syngeneic recipients measured uninhibited growth {0.45to 0.74~ splenic uptake of 125IUdR). Determined 5 days after transplantation. Geometric mean values of 6 to 12 mice: F=females; M=males: ND=not done. *K

I SGD regions. ABJEC

Bone Marrow Graft Resistance Controlled by H-2K

17

the other strains (Tables 1-3; see B 10.BR, C3H, a n d CBA donors). The i r r a d i a t i o n dose given prospective hosts provided o p t i m a l c o n d i t i o n s for the detection of natural, t h y m u s - i n d e p e n d e n t resistance (Rauchwerger etal. 1977). U n d e r these conditions B6, B10, a n d D1.LP mice were classified as weakly resistant or susceptible to H-2 k b o n e m a r r o w allografts, whereas 129-strain mice were unq u e s t i o n a b l y resistant. Since the donors were all H - 2 k a n d the H-2 type of recipients was similar, the difference in strength of resistance was a t t r i b u t e d to the degree of responsiveness conferred to each recipient strain by polymorphic, H - 2 i n d e p e n d e n t , / r - l i k e reactivity genes. A second series of grafts established that resistance of 129-strain mice was directed against target d e t e r m i n a n t s controlled by the K a n d / o r I A regions of H - 2 (Tables 1 a n d 2). Cells of B10.A, A, B10.A(2R), a n d H T H mice bear k alleles only at the K , I A , IB, Id, a n d I E regions, yet failed to grow, or grew deficiently, in 129-strain mice, as did cells bearing k alleles t h r o u g h o u t the M H C . Resistance to these cells was n o t influenced by d o n o r b a c k g r o u n d , since similar H - 2 haplotypes are associated with the A a n d B10 genomes. The n u m b e r of regions relevant for resistance was limited further by grafting cells of B10.A(4R) mice bearing k alleles only in K a n d I A : these cells also failed to grow in 129-strain mice (Table 2). Strong resistance could not have been directed against n o n - k allelic d e t e r m i n a n t s controlled by the I E , S, G, a n d D regions, since

Table 2. Resistanceof irradiated 129-strainmice and susceptibilityof D1.LP or B6 mice to the growth of

5 x 105 transplanted bone marrow cells bearing k alleles at the K and I regions of H-2 Bone marrow donors*

Percent uptake of ~-'5IUdRin recipient spleens~

Strain

129 (H-2be)

D1.LP (H-2~'c)

B6 (H-2h)

F

M

F

M

F

M

0.06 0.08 0.02 0.05 0.30 0.43 0.10 0.44 0.34 0.29 0.26 0.54 0.31

0.10 0.07 0.04 0.05 0.05 0.0l 0.03 0.48 0.22 0.31 0.24 0.52 ND

ND 0.16 ND 0.06 0.58 0.60 0.43 0.74 ND 0.24 0.71 0.78 0.44

0.26 0.21 0.l 2 ND 0.43 0.82 0.31 0.69 0.19 ND 0.43 0.72 0.59

0.31 0.4~~ 0.07 ND 0.60 0.75 0.49 0.07 0.19 ND 0.21 0.51 ND

0.53 0.57~ 0.01 0.02 0.79 ND 0.43 0.03 ND 0.25 0.25 0.28 0.01

B10.BR C3H B1 0 . A A B10.A(2R) HTH B10.A(4R) A.TL B10.AI5R) C3H.OH BI0.HTG HTG A.TH

H-2 haplotype~

kkkkkkkkk kkkkkkkkk kkkkkdddd kkkkkdddd kkkkkdddb kkkkkdddb kkbbbbbbb skkkkkkkd bbbkkdddd ddddddddk ddddddddb ddddddddb ssssssssd

* Donor cellstransplanted into syngeneicrecipientsmeasured uninhibited growth (0.52 to 1.02"..;splenic uptake of 125IUdR). t Determined 5 days after transplantation. Geomctric mean values of 5 to 10 mice: F=females; M =males; N D - n o t done. I K--SGD regions ABJEC BI0 instead of B6 recipients.

18

G. Cudkowicz and J. F. Warner

129-strain recipients were either weakly resistant to B10.A(5R), A.TH, and B 1 0 . H T G cells or susceptible to H T G cells that share d and b alleles at these regions with the former g r o u p of donors, i.e., with B10.A, A, B10.A(2R), B10.A(4R), and H T H mice (Tables 1-31. The weak resistance of 129-strain mice to B10.A(5R), A.TL, and A.TH grafts (Tables 2 and 3), as c o m p a r e d to their susceptibility to H-2 b grafts, might be due to a weak host response against H - 2 D d gene products. Likewise, weak resistance toward B10.HTG m a r r o w cells (Tables 1 and 2) might be due to a response against H - 2 K d antigens. It has been pointed out, however, that minor differences in repopulation of host spleens m a y be the result of m a n y factors in addition to M H C incompatibility. This genetic analysis is concerned with the strong and readily discernible resistance of 129-strain mice to H-2 k grafts. The major variable in determining resistance was the genetic background of recipient strains sharing the H - 2 bc haplotype. However, for certain d o n o r cells grafted into 129-strain mice (e. g., B10.A[2R) and HTH), the recipients' sex was an additional variable, since natural resistance was often stronger in males than in females, a frequent observation in previous studies (Cudkowicz and Rossi 1972). B6 and B10 mice were strongly resistant to B10.A, A, B 10.A(5R), A.TL, and A . T H cells, but this was due to reactivity of these recipient strains against H - 2 D d products (Cudkowicz and Lotzovfi 1973, Cudkowicz 1975a). Weak resistance of B10 and B6 mice was detected also against B10.HTG and H T G d o n o r cells as if there was a host response against products of d alleles at regions other than D. Again, such an interpretation needs to be qualified as explained above. A third series of grafts established that resistance to H-2 k grafts is controlled by the H - 2 K region or by H - 2 K plus I A (Tables 2 and 3). Cells of mouse strains B10.A(5R) (with k alleles in the I J and I E regions), C 3 H . O H (with the k allele in D),

Table 3. Resistance of irradiated 129-strain mice and susceptibility of D 1i P or B6 mice to the growth of 3 x 105 transplanted bone marrow cells bearing k alleles at the K and I regions of H-2 Bone marrow donors*

Percent uptake of lzSIUdR in recipient spleens*

Strain

129 (H-2 be)

C3H CBA CBA(M523) A.TL HTG

H-2 haplotype *

kkkkkkkkk kkkkkkkkk kakkkkkkkk w skkkkkkkd ddddddddb

D1.LP (H-2 hc)

B6 (H-2 b)

F

M

F

M

F

M

0.01 0.04 0.06 0.54 0.34

0.01 0.04 0.05 0.29 0.36

ND ND 0.26 0.44 0.53

0.37 0.17 0.22 0.33 0.54

0.29 0.31 0.33 0.07 0.35

ND ND 0.24 0.02 0.25

* Donor cells transplanted into syngeneicrecipients measured uninhibited growth (0.45 to 0.60~.0splenic uptake of l zSIUdR). Determined 5 days after transplantation. Geometric mean values of 4 to 8 mice: F=females; M =males; ND=not done. I K SGD regions. A BYE C

Mutation within the H-2K region (Blandova etal. 1975, Klein etal. 1975, 1976).

Bone Marrow Graft Resistance Controlled by H-2K

19

and A.TL (with k alleles in 1A t h r o u g h G) all grew in 129-strain mice. The A.TL cells were of particular interest, since they indicated that loss of the k allele in K alone ensured d o n o r cell proliferation, despite the presence of k alleles in seven other M H C regions. Since the importance of the K region was demonstrated indirectly via the selective loss of the k allele in this region, it was theoretically possible that Kand IA-region products must be recognized for the manifestation of resistance. CBA(M523) cells bearing the m u t a n t H - 2 K ka allele (Blandova et al. 1975, Klein et al. 1975, 1976) were indistinguishable from CBA cells with regard to proliferation in mice of the 129 and D1.LP strains (Table 3). Susceptibility to H - 2 k bone marrow grafts conferred by the BIO genetic background

The preceding experiments involving H - 2 K k bone m a r r o w grafts c o m p a r e d the strong resistance of 129-strain mice to (1) the weak resistance of D l i P mice that are H - 2 identical, but ancestrally unrelated; and (2) the susceptibility of B6 and B10 mice that are H - 2 b instead of H - 2 bc and, likewise, ancestrally unrelated. Additional experiments were required to exclude the possibility that resistance of 129-strain mice was due to minor differences between the H - 2 bc and H - 2 b haplotypes as opposed, or in addition, to b a c k g r o u n d reactivity genes. Such experiments were carried out even t h o u g h the H - 2 b and H - 2 bc haplotypes were not differentiated in a recent review (Klein et al. 1978). B10.A(4R) cells were transplanted into congenic B10.129(6M) mice that possess the B10 b a c k g r o u n d but are H - 2 bc (Snell et al. 1971): these mice were as susceptible to the B 10.A(4R) grafts as B6 or B10 recipients (Table 4). The same d o n o r cells were transplanted into (129 • B10)F 1 and reciprocal hybrids; these mice were also susceptible to B10.A(4R) grafts (Table 4). Thus, susceptibility resulted from the introduction of B10 b a c k g r o u n d genes into the genome of otherwise resistant mice and not from H - 2 haplotype differences between 129 and B10 or B6 mice. Moreover, only a single copy of B10 genes was required to render (129 x B10)F 1 hybrids susceptible. Unresponsiveness to H - 2 K k grafts was therefore, inherited by F 1 mice as a seemingly d o m i n a n t trait.

Table 4. Uninhibited growth ofB 10.A(4R)bone marrow cells in irradiated recipients of H-2 bor H-2 bctype sharing the B10 or B6 genetic background No. of cells grafted

5 x 105

1•

10 6

Recipientmice

Percent splenic uptake of 125IUdR*

Strain

H-2 type

F

M

B10.A(4R) B6 B10 B10.12916M) B10.AI4R) 129 x B10 B10 x 129

h4/h4 bib bib bc/bc h4/h4 bc/b b/bc

0175 0.49 0.63 0.54 0.53 0.87 0.89

0.68 0.43 0.61 ND 1.1 ND 0.59

* Determined 5 days after transplantation. Geometric mean values of 4 to 6 mice: F=females: M = males; ND = not done.

20

G. Cudkowicz and J. F. Warner

Abrogation of resistance by host pretreatments If i r r a d i a t e d 129-strain mice actively suppress the g r o w t h of H - 2 K k - i n c o m p a t i b l e bone m a r r o w cells, p r e t r e a t m e n t with A L S or silica particles should w e a k e n o r a b r o g a t e resistance. T h e t r e a t m e n t s were chosen because of their k n o w n effectiveness (Till et al. 1970, Lotzov/t a n d C u d k o w i c z 1974, Kiessling etal. 1977). B o t h t r e a t m e n t s a b r o g a t e d resistance of 129-strain mice against B10.A(4R) a n d B10.A bone m a r r o w grafts (Table 5). The substantial u p t a k e of I ~ 5 I U d R by spleens of treated mice was due to d o n o r cell proliferation r a t h e r t h a n to e n d o g e n o u s r e p o p u l a t i o n (secondary to A L S o r silica-induced splenic hyperplasia) for two reasons: (1) the agents were a d m i n i s t e r e d to 129-strain mice after i r r a d i a t i o n so that h y p e r p l a s i a could n o t have been induced; a n d (2) treated r a d i a t i o n controls, n o t given d o n o r cells, h a d negligible splenic u p t a k e of 125IUdR (Table 5).

Susceptibility o[' F 1 hybrids to parental H-2 k bone marrow grafts Cells of B10.BR, A K R , a n d C 3 H mice, b e a r i n g k alleles t h r o u g h o u t the M H C , a n d of B 10.A(4R) mice with k alleles in the K a n d I A regions only, were t r a n s p l a n t e d into H-2 k/b or H-2 kid F 1 h y b r i d mice of different p a r e n t a g e (Table 6). The t r a n s p l a n t e d p a r e n t a l cells grew w i t h o u t i m p a i r m e n t in all F l hybrids, as c o m p a r e d to i r r a d i a t e d syngeneic hosts. H y b r i d resistance to H-2 k cells could not be detected u n d e r sensitive assay conditions, i.e., t r a n s p l a n t a t i o n of as few as 5 x 105 bone m a r r o w cells into male hybrids. Cells of B 10.A(4R) mice are H-2D b a n d thus subject to h y b r i d resistance c o n t r o l l e d by the H h - I / H - 2 D region. As expected, these cells failed to grow in i r r a d i a t e d H-2D b heterozygotes such as (B6 • C 3 H t F 1, (B6 x A)F1, (B10 x B10.BR)F1, (B10.BR x 129)F1, a n d [ B I 0 . A x B10.A(2R)]F 1 h y b r i d mice (data n o t shown). Table 5. Abrogation of resistance to 5 x 105 H-2K-incompatible bone marrow cells by pretreatments of 129-strain recipients Donor cells

B10.A(4R) B10.A None

Host pretreatment

None ALS* Silica I None ALSf Silica ~ None ALSt Silica '

Percent splenic uptake of aeSlUDR* F

M

0.05 0.69 1.24 0.07 1.54 1.06 0.02 0.09 0.04

0.02 0.54 0.93 0.07 0.51 0.68 ND ND ND

* Determined 5 days after transplantation. Geometric mean values of 5 mice: F-females; M=males; ND=not done. t Mice were irradiated and 17 h later injected i.v. with 0.3 ml of rabbit antimouse lymphocyte serum. Bone marrow cells were transplanted after 3 additional h. TMice were irradiated and 1 h later injected i. v. with 2 mg of silica particles in suspension. Bone marrow cells were transplanted after 19 additional h.

Bone Marrow Graft Resistance Controlled by H-2K

21

Table 6. Uninhibited growth of H-2k-homozygoas bone marrow cells in heterozygous F 1 hybrid irradiated recipients Donors*

Recipients

Strain

No. of cells grafted

Strain

B 10.BR

1 x 106

B6 x C3H BI0 • B10,BR BI0.BR • 129 B6 x C3H BI0 x B10.BR B10.BR x 129 129 x 4R HTH x 129 AKR x DBA/2 B6 x AKR B6 x C3H AKR x DBA/2 B6 x AKR B6 x C3H B10 x C3H ; C3H x 129 C3H x A.BY AKR x DBA/2 B10.D2/n • B!0.BR B 10 x C3H ;

5 x l0 s

B10.A(4R) AKR

1 • 10 6 5 • 105 1 x 106

5 x 105

C3H

1 • 106

5 x l0 s

Percent splenic uptake of ~2sIUdRt

H-2 type b/k b/k k/bc b/k b/k k/bc bc/h4 h/bc k/d b/k b/k k/d b/k b/k b/k k/bc k/b k/d d/k b/k

M

F

0.82 0.50 1.14 0.46 0.5l 0.72 1.22 0.75 1.09 1.95 2.20 0.65 0.74 0.78 0.93 0.59 0.93 ND ND 0.26

0.64 0.59 0.71 0.46 0.35 1.05 0.86 0.63 0.76 ND 1.27 0.60 ND 0.56 0.77 ND 0.66 0.67 0.63 0.27

* Donor cells transplanted into syngeneic recipients measured uninhibited growth; in no experiment was there a significant difference between syngeneic and F 1 hybrid recipients. Determined 5 days after transplantation. Geometric mean values of 6 to 12 mice. F=females; M=males; N D = n o t done. Reciprocal C3H x BI0 and B6 x C3H F1 hybrids were tested. 125IUdR uptake values were not significantly different.

Discussion T h e o b j e c t i v e of o u r e x p e r i m e n t s was to identify the M H C r e g i o n r e s p o n s i b l e for n a t u r a l resistance o f i r r a d i a t e d 129-strain m i c e to a l l o g e n e i c H - 2 k b o n e m a r r o w grafts. P r e v i o u s studies in w h i c h h e m o p o i e t i c cells of H-2 b, H-2 a, a n d H-2 d d o n o r s w e r e grafted i n t o i r r a d i a t e d m i c e f r o m a l a r g e series of a l l o g e n e i c strains h a d i n d i c a t e d t h a t the D r e g i o n is of p r i m a r y i m p o r t a n c e in d e t e r m i n i n g a l l o g e n e i c resistance ( C u d k o w i c z a n d L o t z o v a 1973, C u d k o w i c z 1975a). O u r studies, in w h i c h a different set of h o s t a n d d o n o r strains w e r e e m p l o y e d , e s t a b l i s h t h a t the K r e g i o n , likewise, c o n t r o l s d e t e r m i n a n t s for a l l o g e n e i c resistance, b u t t h a t m o u s e strains r e s p o n s i v e to K - r e g i o n differences are r e l a t i v e l y i n f r e q u e n t . M i c e of t h e 129 s t r a i n w e r e g o o d r e s p o n d e r s to H - 2 k - p o s i t i v e cells, despite t o t a l b o d y i r r a d i a t i o n in t h e lethal d o s e range. T h e w e a k e n i n g effects o n r e s i s t a n c e of h o s t p r e t r e a t m e n t w i t h e i t h e r A L S o r the a n t i m a o r o p h a g e a g e n t silica verified t h a t t h e failure o f d o n o r cells was i n d e e d due to a h o s t a n t i g r a f t r e a c t i o n . T h e m a p p i n g of t a r g e t d e t e r m i n a n t s for r e s i s t a n c e w a s a c c o m p l i s h e d by r e c o m b i n a n t analysis. D o n o r cells b e a r i n g H-2 h a p l o t y p e s w i t h k alleles in d e f i n e d

22

G. Cudkowicz and J. F. Wariaer

MHC regions were transplanted into irradiated 129 recipients and their proliferation was compared with that of C3H, CBA, and BI0.BR cells bearing k alleles throughout the MHC. 129-strain mice were resistant to cells of recombinant donors having k alleles exclusively in the K and IA regions (Figure 1, upper section). The mapping was refined by grafting donor cells to which 129-strain mice were susceptible. These included cells of three recombinant strains with k alleles in different regions; collectively these cells possessed k allele products of every MHC region except K (Figure 1, lower section). Thus, only genes within H-2K, or adjacent to H-2K on the centromeric side, could have coded for or controlled the expression of relevant target cell surface structures. These were recognized by irradiated, nonsensitized 129-strain mice so as to elicit a cell-mediated response. The H-2 k recombinant strains employed as bone marrow donors had b, d, and s alleles at various MHC regions in place of k alleles of the original haplotype. Products of these non-k alleles failed to generate strong resistance in 129-strain mice and thus did not influence the outcome of H-2Kk-positive or negative grafts. Lack of, or poor immunogenicity of these allelic products was demonstrated by the fact that 129 mice were weakly resistant to two sets of grafts: (1) B10.HTG, HTG, and A.TH cells not possessing k alleles; (2) C3H.OH, B10.A(5R), and A.TL cells possessing k alleles at irrelevant regions, plus d, b, or s alleles in K and/or other regions (Figure 1, lower section). A mutation within the K region of the H - f i haplotype detected by histogenetic and serologic methods, and by in vitro-induced cell-mediated responses [-i.e., the CBA(M523) mutant originally described by Blandova et al. 1975 and then studied by Klein et al. 1975, 1976] failed to affect this resistance pattern. The relevant gene may not be involved in the CBA(M523)

~ N O R STRAINS

k ALLELES ~ H-2 REGI~S OF DONOR CELLS K I S G D

RESIS~NCE TO DONOR CELLS ~

B,O.BRC,,

CBA

129-STRAIN RECIPIENTS

TT

B,O.AB,O.A,2RIHTH

TTTTTT

TT +

TT

J

+

BIO.A(4R) SUSCEPTIBILITY TO DONOR CELLS BY 129-STRAIN RECIPIENTS BIO.HTG

C3H.OH

HTG

A.TH

I

I

J

T +

BIO.A(SR)

I

+

I

A.TL

Fig. 1. Summary of genetic requirements for natural resistance of irradiated 129-strain mice (H-2 b') to allogeneic H-2 k bone marrow grafts. Target determinants are coded for (or their expression is controlled) by the H-2K region, but the manifestation of resistance also requires appropriate reactivity genes present in the 129 strain genome, but absent from that of the D1.LP (H-2~'cL B10, and B6 (both H-2 ~) strains.

Bone Marrow Graft Resistance Controlled by H-2K

23

mutation; alternatively, the reactivity of 129 and D1.LP mice against the mutant H - 2 K ka products may not differ from their reactivity to H - 2 K k. Hence, control of target determinants by a gene associated with H - 2 K , tentatively designated as Hh-3, was independent of epistatic influences from other M H C genes. Weak resistance of 129-strain mice to marrow grafts bearing d alleles in the K or D regions was frequently observed in this study. Furthermore, B6 and B10 mice were weakly resistant to marrow cells bearing the k allele in the D region or the d allele in K. In all these instances, resistance might have resulted from feeble host responses directed against M H C gene products. However, additional host factors might also have contributed to the suboptimal donor cell growth. Further analysis is required to identify the contribution ofnonimmunogenetic influences in the strain combinations classified as weakly resistant. It should be noted, however, that weak resistance against cells bearing alleles other than k did not interfere with the mapping of genes controlling the strong resistance of 129-strain mice against H-2 k grafts. The genetic background associated with recombinant H - 2 haplotypes failed to influence the outcome of the grafts: B10.A(2R) and H T H cells, indistinguishable as to H-2 alleles but different as to background genes, grew deficiently in 129 mice. Likewise, B10.HTG and H T G cells which grew in 129-strain mice shared the H-2 haplotype, but not the genetic background. Finally, the outcome of grafts from several other B10 recombinant donors depended exclusively on the H - 2 K allele. Unlike the expression of Hh-3 target determinants, host reactivity was regulated by genes independent of the MHC. introduction of the H-2 bc haplotype either of LP mice into the DBA/1 genome or of 129-strain mice into the B10 genome produced the congenic strains D1.LP and B10.129(6M), respectively (Snell 1958, Snell et al. 1971). Both these strains were H-2 identical with 129 and thus met upon grafting with H-2 k bone marrow cells, one of the necessary conditions for resistance, i.e., host-donor differences at the H - 2 K region. However, this condition was not sufficient to ensure resistance in D1.LP and B10.129(6M) mice in which transplanted H-2 k cells were capable of growing. Other genes associated with 129, but not with DBA/1, B 10, or B6 mice were required for full manifestation of host reactivity. The genetic regulation of resistance was determinant-specific, since 129-strain mice were responders to H-2 k, but not to H-2 d and H-2 s grafts (Cudkowicz and Lotzovfi 1973, and unpublished observations). Moreover, responsiveness was directed against H-2K k but not H-2D k determinants. In contrast, D1.LP and B10 mice were poor responders to H-2 k, but strong responders to H-2 d and H-2 s grafts, their reactivity being selective for H-2D instead of H-2K-associated determinants. To some extent, resistance strength was modulated by hormonal in addition to genetic factors. Male mice were usually more resistant than females, and in some strain combinations of this and previous studies, sex-determined resistance differences were extreme (Cudkowicz and Rossi 1972). Responsiveness to allogeneic and parental marrow grafts is usually a polygenic, dominant trait (Cudkowicz 1971, 1972). It was therefore unexpected that (129 x B10)F 1 hybrids were susceptible to H-2K k marrow grafts. As the number of reactivity genes involved in this resistance system is unknown, intergenic suppressive interactions might have taken place. For these reasons, the hybrid's

24

G. Cudkowicz and J. F. Warner

susceptibility cannot be viewed simply as a case of recessive inheritance of reactivity genes. The Hh-1 gene has been defined in both parent-to-F 1 hybrid and allogeneic resistance systems (Cudkowicz 1968, Cudkowicz and Lotzovfi 1973). The Hh-3 incompatibility could only be defined in allogeneic bone marrow cell transfers, since cells of several H-2 k strains grew without impairment in a series of irradiated F 1 hybrid recipients that were H-2 b/k, H-2 k/bc, or H-2 k/~. One might postulate that Hh-1 is a compound locus with one set of components responsible for hybrid resistance (not fully expressed in the phenotype of heterozygotes) and another set responsible for allogeneic resistance. Accordingly, the Hh-3 locus would only possess the latter type of genetic determinants. There are, however, two experimental situations in which F a hybrid responder cells react against parental H-2 k targets. First, transplantable AKR lymphomas grow deficiently in heavily irradiated (Gallagher et al. 1976) as well as nonirradiated (Schmitt-Verhulst and Zatz 1977) hybrid recipients, in a manner reminiscent of hybrid resistance. However, host responses to tumor cell determinants other than Hh gene products cannot be excluded. Although it was not determined whether hybrid resistance to lymphomas was controlled by the MHC, this may be the case, since resistant hybrids were H-2 b/k or H-2 kid heterozygotes, and susceptible hybrids H-2 k/k homozygotes (Gallagher et al. 1976). Assuming that H-2 heterozygosity was essential, the MHC region relevant for hybrid resistance to AKR lymphomas remains to be identified. Second, F 1 hybrid antiparental H-2 k cell-mediated cytotoxicity was induced in vitro (Schmitt-Verhulst and Zatz 1977); the genetic control of target and stimulator determinants was mapped to the H - 2 K region and responder cells had to be heterozygous for H - 2 K k (Ishikawa and Dutton 1979, Warner and Cudkowicz 1979). The question then arises of whether Hh-3 is responsible for both allogeneic resistance in vivo and F 1 antiparent cytotoxicity in vitro. Several distinctive features of the in vivo anti-Hh response are also exhibited by in vitro-induced F1 antiparental cytotoxicity, notably the close genetic association of target determinants with H - 2 D or H - 2 K , the critical dependence of the response on macrophage-like cells, the late maturation of responsiveness, and the concomitant induction of specific unresponsiveness (Shearer and Cudkowicz 1975, Shearer etal. 1977). These correlations exist, even though in vivo effectors are thymusindependent cells (Cudkowicz and Bennett 1971a, Cudkowicz 1975b), whereas in vitro effectors are Thy-l-positive lymphocytes {Shearer etal. 1977). Several possibilities might explain the correlations and inconsistencies of the F~ antiparental models: (1) Recognition of one set of Hh products by non-T cells and of another set of closely linked but distinct Hh gene products by T lymphocytes; (2) dual recognition in vitro by T lymphocytes ofHh gene products in association with serologically defined H-2D or H-2K specifities, as opposed to simple recognition in vivo of Hh antigens by non-T cells; and (3) recognition by derepressed responder cells of autologous MHC-coded, serologically defined antigens whose expression may be modified by gene dose effect and/or allelic interaction (Cudkowicz et al. 1979). Acknowled{lements. The authors thank Dr. Marek B. Zaleski for the gift of A.TH and CBA(M5231 mice. This work was supported by research grants AM-13969 and CA-12844 from the National Institutes of Health, Bethesda, Maryland.

Bone Marrow Graft Resistance Controlled by H-2K

25

References

Anderson, R. E. and Warner, N. L. : Ionizing radiation and the immune response. Adt,. hnmunol. 24 : 215335, 1976 Bennett, M.: Rejection of marrow allografts. Importance of H-2 homozygosity of donor cells. Transplantion 14: 289-298, 1972 Bennett, M., Cudkowicz, G., Foster, R. S. Jr., and Metcalf, D.: Hemopoietic progenitor cells of W anemic mice studied in vivo and in vitro. J. Cell. Physiol. 71: 211-226, 1968 Blandova, Z., Mnatsakanyan, Y. A., and Egorov, I. K. : Study of H-2 mutations in mice, VI. A new K -end mutant, lmmunogenetics 2:291-295, 1975 Cudkowicz, G.: Hybrid resistance to parental grafts of hemopoietic and lymphoma cells, b~: The Prolijeration and Spread of Neoplastic Cells, pp. 661-691, Williams and Wilkins, Baltimore, 1968 Cudkowicz, G.: Genetic control of bone marrow graft rejection. I. Determinant-specific difference of reactivity in two pairs of inbred mouse strains. J. Exp. Med. 134: 281-293, 1971 Cudkowicz, G. : In H. O. McDevitt and M. Landy (eds.): Genetic Control of Immune Responsiveness, pp. 323-330, Academic Press, New York, 1972 Cudkowicz, G.: Genetic control of resistance to allogeneic and xenogeneic bone marrow grafts in mice. Transplant. Proc. 7: 155-159, 1975a Cudkowicz, G.: Rejection of bone marrow allografts by irradiated athymic nude mice. Proc. Am. Assoc'. Cancer Res. 16: 170, 1975b Cudkowicz, G.: Natural resistance to foreign hemopoietic and leukemia cells. In G. Cudkowicz, M. Landy, and G. Shearer (eds.): Natural Resistance Systems Against Foreign Cells, Tumors, and Microbes, pp. 3-30, Academic Press, New York, 1978 Cudkowicz, G. and Bennett, M.: Peculiar immunobiology of bone marrow allografts. I. Graft rejection by irradiated responder mice. J. Exp. Med. 134: 83-102, 1971a Cudkowicz, G. and Bennett, M.: Peculiar immunobiology of bone marrow allografts. II. Rejection of parental grafts by resistant F 1 hybrid mice. J. Exp. Med. 134: 1513-1528, 1971b Cudkowicz, G. and Hochman, P. S.: Do natural killer cells engage in regulated reactions against self to ensure homeostasis? Immunol. Rev. 44, in press, 1979 Cudkowicz, G. and Lotzovfi, E.: Hemopoietic cell-defined components of the major histocompatibility complex of mice: Identification of responsive and unresponsive recipients for bone marrow transplants. Transplant. Proc. 5: 1399-1405, 1973 Cudkowicz, G., Nakano, K., and Nakamura, I. : Autoreactivity specific for routine antigens controlled by the H-2D region. In F. Milgrom and B. Albini (eds.): Autoimmunity, S. Karger, Basel, 1979 Cudkowicz, G. and Rossi, G. B.: Hybrid resistance to parental DBA/2 grafts: Independence from the H-2 locus. I. Studies with normal hematopoietic cells. J. Natl. Ca~cer Inst. 4~: 131-139, 1972 Gallagher, M. T., Lotzova, E., and Trentin, J. J.: Genetic resistance to'marrow transplantation as a leukemia defense mechanism. Biomedicine 25: l-3, 1976 Graft, R. J. and Snell, G. D.: Histocompatibility genes of mice. IX. The distribution of the alleles of the non-H-2 histocompatibility loci. Tra~zsplantation 8: 861-876, 1969 Hochman, P. S., Cudkowicz, G., and Dausset, J.: Decline of natural killer cell activity in sublethally irradiated mice. J. Natl. Cancer Inst. 01 : 265-268, 1978 lshikawa, H. and Dutton, R. W.: Primary in vitro cytotoxic response of F 1 T lymphocytes against parental tumors. J. Immwlol., in press, 1979 Kiessling, R., Hochman, P. S., Haller, O., Shearer, G. M., Wigzell, H., and Cudkowicz, G. : Evidence for a similar or common mechanism for natural killer cell activity and resistance to hemopoietic grafts. Eur. J. lmmunol. 7: 655-663, 1977 Klein, J.: Bioloqy ql" the Mouse Histocompatibility-2 Conlplex. Springer-Verlag, New York, 1975 Klein, J., Flaherty, L., Van de Berg, J. L., and Shreftler, D. C.: H-2 haplotypes, genes, regions, and antigens: First listing. Immunooenetics 6: 489-512, 1978 Klein, J., Forman, J., Hauptfeld, V., and Egorov, I. K.: Immunogenetic analysis of H-2 mutations. III. Genetic mapping and involvement in immune reactions of the H-2 k~mutation. J. Immunol. 115 : 716718, 1975 Klein, J., Hauptfeld, M., Geib, R., and Hammerberg, C.: Immunogenetic analysis of H-2 mutations. V.Serological analysis of mutations H-2 d~, H-2 s", and H-2 k". Transplantation 22: 572-582, 1976

26

G. Cudkowicz and J. F. Warner

Lotzovfi, E. and Cudkowicz, G.: Abrogation of resistance to bone marrow grafts by silica particles: Prevention of the silica effect by the macrophage stabilizer poly-2-vinylpyridine N-oxide. J. Immunol. 113: 798-803, 1974 Popp, R. A. and Cudkowicz, G. : independence of deficient early growth and later regression of (C57BL x 101)F 2 marrow grafts in (C57BL x 101IF1 hybrid mice. Transplantation 3: 155-160, 1965 Rauchwerger, J. M., Gallagher, M. T., Monie, H. J., and Trentin, J. J.: Relative radioresistance of xenogeneic and hybrid resistance to bone marrow transplantation. Transplantation 23: 158-160, 1977 Schmitt-Verhulst, A. M. and Zatz, M. M.: F 1 resistance to AKR lymphoma cells in vivo and in vitro. J. Innnunol. 11~: 330-333, 1977 Shearer, G. M. and Cudkowicz, G.: Induction of F~ hybrid antiparent cytotoxic effector cells: An in vitro model for hemopoietic histocompatibility. Science 190: 890-893, 1975 Shearer, G. M., Cudkowicz, G.. Schmitt-Verhulst, A. M., Rehn, T. G., Waksal, H., and Evans, P. D.: F 1 hybrid antiparental cell-mediated lympholysis: A comparison with bone marrow graft rejection and with cell-mediated lympholysis to alloantigens. Cohl Spring Harbor Syrup. Quanr. Biol. 41 : 51 l 518, 1977 Snell, G. D.: Histocompatibility genes of the mouse. I1. Production and analysis of isogenic resistant lines. J. Natl. Cancer Inst. 21: 843-877, 1958 Snell, G. D., Graft, R. J., and Cherry, M.: Histocompatibility genes of mice. XI. Evidence establishing a new histocompatibility locus, H-12, and new H-2 allele, H-2 h'. Transplantation 11: 525-530, 1971 Till, J. E., Wilson, S., and McCnlloch, E. A.: Repression of colony formation reversed by antiserum to mouse thymocytes. Science 169: 1327-1329, 1970 Warner, J. F. and Cudkowicz, G.: F 1 hybrid anti-parental H-2 k cell-mediated lympholysis. I. Stimulator and target determinants controlled by the H-2K region. J. lmmunol. 122: 575-58I, 1979 Received October 10, 1978: revised version received November 11, 1978