HAMASHIMA, YOSHIHIRO

IKEHARA, SUSUMU

INOUE, SHUJI

NAKAMURA, TAKAO

OO, MAUNG MAUNG

Kyoto University Research Information Repository

Title The Fate of Liver Allografts in Radiation Bone MarrowChimeras in MiceAuthor(s)NAKAMURA, TAKAO; IKEHARA, SUSUMU; INOUE,SHUJI; OO, MAUNG MAUNG; HAMASHIMA,YOSHIHIROCitation日本外科宝函 (1985), 54(2): 59-66Issue Date1985-03-01URL http://hdl.handle.net/2433/208687RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityArch Jpn Chir 5412), 59～6. ¥Ia川1‘198ヨ原 著The Fate of Liver Allografts in Radiation Bone Marrow Chimeras in Mice T AKAO .:-; AKAMURA，メL冶UMUIKI<.HARA, SHUJI b:ol・E、:¥l.;.u;-;c :¥!AU:¥G Oo and YりちHI HIRO Hc¥MASHl"vlA Department of l'athol川；v.F山 ultvof :¥I日licine.k、りtoUniver,ity I Director: Prof. 1 lr.YosHIHIRC〕 HミMλ川l!MA)Received for Puhlication, Dec. 7, 1984. Summary We attempted to 引 tablisha new methodology of tolerance induction in liver allografo When liver tissue of BALB/c (H 2<l) or ( '57BL/6J (H 2h) mice were minced and grafted under the kidney capsules of C3H/Hc.¥: (H 2k) miceヲ itwas rejected. However、whenC3H/He.¥: mice were irradiated and reconstituted with BALB/c nu/nu bone marrow cells、theya付じptedboth bone marrow donor-type (BALB/c) and host (thymus) type (< '3H/He.¥:) Jiyer tissue. A州 aysfor both mixed-lymphocyte reaction (:¥ILR) and induction of cytotoxic T-lymphocytes (CTL) revealed that the newly developed T cells are tolerant to bone marrow donor-type and host (thymus）ーtypemajor histocompatibility complex (:¥IHC) determinant;;. Ba州 lon these data. we propose th乱tliver allografts combined with bone marrow transplantation should be considered as ,1 viable therapy for humans. Introduction ~inc、ぞ 1963, when STARZL described the first succe州 fulorthotopic liver transplantation in man, more than 500 patients have been treated in this way3,i3,i41 In al species, liver allografts are rejected less aggressively than allografts of other organs、becauseI region antigens are less prominent in the liver than in other organ4,10,1s1 In man. how「ver,aggressive destructive rejection of a liver allograft will usually occur if no immunosuppression is induced. It would be of benefit to patients if rejection could be controlled without giving immunosuppressive agent、We have previously reported that fully allogeneic chimer川 inmice an、cptboth thymm-t¥・pe and bone marrow-type skin6>. In addition町 W ぞ haverecently found that allogeneic bone marrow transplantation can treat autoimmune diseases in :¥IRL/l and B ¥:SB mice羽・itho以 showingraft一versus一hostreaction （』1アHR）’ providedthat bone marrow cells of young nu/nu mi【、eor Key words: Liver transplantation. Bone marrow transplantation, Maiot histocompatibility complex. Graft versus-host reaction, Tolerance. 索引語：肝臓移植，骨髄移植，主要組織適合l立伝子複合体，移植片対宿主反応，寛容．l’l<'l'l1t‘idrln川白 Departmentof Pathology、hwultyof :¥[edicine. Kyoto Univcr,iり汁仏、o,Kvotn. 606 Jλp:tn 60 日外宝第54巻第2号（昭和60年3月）T cel depleted bone marrow cells are used8•9>. Furthermore、wehave found that the newly-developed T cells are tolerant to both bone marrow donor type and host-type major histocom-patibility complex (:¥I Hぐ） determinants 9). These data prompted us to examine the fate of liver allografts in radiation bone marrow chimeras in mice. Small animals have been of limited use as a model for liver transplantation because the surgery has been technically di伍cult. In rats, :¥I ITO et al. discovered that splenic pulp is the most suitable location for long-term survival of isolated hepatocytes11>. In the present study, we attempted to graft liver tissue minced to rice-grain size under the kidney capsules of mice. We show that C3H/He'.¥ mice reconstituted with BALB/c nu/nu bone marrow cells accept both BALB/c bone marrow donor-type and C3H/He '.¥host勾pelivers. Materials and民1ethods.J1川河als: Inbred C3H/He:¥ (H-2k）司 BALB/c(H-2<l), and C57BL/6J (H-2h) mice were used under standard laboratory conditions. BALB/c nu/nu mice were obtained from the Central Institute for IうxperimentalAnimal只， Tokyo, and maintained under specific pathogen-free condi-tions in our facilities. 7'1’ill/.＼アla1/al/f11of bone押zarrowcells and lz・ver: Two mo old C3H/HeN mice were exposed to 850 rad from a 60Co source and subsequently injected with 1 to 2×107 bone marrow cels obtained from 2 mo old BALB/c nu/nu mice. Two or three months after bone marrow trans-plantation, the mice were anesthetized with pentobarbital (0.05 mg/g body weight, Pitman-Moore, N .J.). Livers taken from 1 to 4 wk old C3H/HeN, BALB/c, or C57BL/6J mice were minced to about rice-grain size in RP:¥11-1640 medium (:.Jisui Seiyaku Co., Ltd., Tokyo, Japan) with 0.01 % collagenase (Type I. Sigma Chemical Co., USA). The liver tissue was grafted under the left kidney capsdles of the C3H/Hel'¥ mice. Two months later, the mice were sacrificed, and the engrafted liYげ tissuewas examined macroscopically and microscopically. Cell siアaratirm: The spleens were aseptically removed, minced, and gently passed through a fine mesh stainless-steel sieve into phosphate-buffered saline. J/zλγd-ii'mphrnylt reactz・onινLR): :¥!LR was examined by measuring the incorporation of 0.5 fL℃i aH thymidine （＇.＼ぞW England :¥ uclear, Boston, l' SA) into D :¥A. Triplicate cultures were set up in 96 well round bottom microtiter trays (Corning <;lass Works 25850). Each well contained 2×105 responder cells and l×105 stimulator cells in a total volume of 0.2 ml RPMI-1640 medium that wa,., supplemented with 2 m:¥I L-glutamine, penicillin (100 I LT/ml), strepto-myげl(100 μ,g/ml）司 5%heat-inactivated human serum喝 and2 mercaptoethanol (2-ME: 5× 10 5 :¥I. Wako Pure Chemical Industries, Tokyo). Stimulator cells were treated with 50 μ,g/ml mitomycin C for 30 min at 37°C. The cultures were incubated for 96 hr in a humidified atmos-phere of 5'/0 C＜）ょinair. 3H thymidine was present during the last 4 hr of the culturing period目The number of 3H incorporated into trichloroacetic acid-insoluble material was measured by a liquid scintillation counter. Cv！日／川1Ci~v assay: Responder cells (7.5×1Q6) and mitomycinぐ（50μ,g/ml)-treated stimulator cells (2.5×1Q6) were cocultured in RPl¥TI-1640 medium containing 10% he川 inactivatedhuman LIVER ALLOGRAFTS IK ¥IH、上： 61 serum, supplemented with 5×io-5 '.¥! 2'.¥IE, penicillin, and 刈reptomycin. Cultures were incubated for 5 days at 37°C in 5% C02 incubator. P815 (H-2<l), EL-4 (H-2b), and X5563 (H-2k) were used as target cels. They were labelled with 100 μCi .'¥ a2 [51ぐr]04(l¥iew England Nuclear, Boston, USA) by means of incubation for 1 hr at 37°C. Labelled cells were washed three times. These cells (5×104) were mixed with effector cells in 200μ1 of RP:¥Il-1640 medium in round-bottomed micro-plates and incubated at 37°C in 5°,0 CO" for 4 hr. Using the Titert即日upernatantCollection System (Flow Lab., Irvine, Scotland), supernatant was harvested in order to determine released radioactivity. ト.；pecificlysis was calculated according to the following formula: . Experimental release-Spontaneou、release% Specific lysis - ~ ；＼［ぷi両日瓦両面E Spontaneous release ×100 Histl少athology: The left kidney with engrafted liver tissue was obtained at autopsy and prepared for light-microscopical observation. Results C3H/HeN (H-V) mice reconstituted with BALB/c nu/nu (H-2d) bone marrow cells survived more than 8 mo without showing graft-versus-host reaction (GVHR). Using anti-H 2k and anti-H 2d serum plus complement, we confirmed that more than 95% of spleen cells from the chimeras were donor-derived. The mice possessed normal numbers of Thy-1+ cells in the spleen, and the spleen cells significantly responded to PHA, Con A, and LPS (data not shown). The fate of the engrafted liver tissue is summarized in Table 1. .'.¥on-treated C3H/HeN mice rejected allogeneic liver tissue of BALB/c (6/6) or C57BL/6J mice (5/5). In contrast, C3H/HeN mice reconstituted with BALB/c nu/nu bone marrow cells rejected third-party C57BL/6J liver tissue (5/5), whereas they accepted both BALB/c bone marrow donor-type (8/10) and C3H/He::¥ host-type (3/5) liver tissue. As shown in Fig. 1-A, liver cells and central veins of BALB/c mice were found under the kidney ca psulcヘofC3H/He::¥ mice. However, due to rejection, liver cells of Table 1. Fate of allografted liver tisue in C3H/l!じNmice rec川 1叫itutedwith hone marrow cels of BALB/c nu/nu mic 孔louse Liver donor ？くり examined No. accepted C3H/HeNIH-2k) C3H/lle :¥ 10 8 C3H/HeK C57BL/6J(H-2b) 5 。C3H/He:¥' BALB/c (H-2<l) 6 。／ C3H/Hel¥; with BALB/c nu/nu B.\l.• C3H/He:¥' 5 3 ／ C3H〆/HeN with BALB/c nu/nu B. ¥[ C57BL/6J 5 。C3H/HeN with BALB/c nu/nu B.I¥I. BALB/c 10 8 a C3H/HeN (H-2k) mice were irradiated (850 rad) and reconstituted with bone marro" cels (2×107) of BALB/c nu/nu (H-2d) mice. Two or three months after bone mar-row transplantation, livers taken from C3H/HeN, BALB/c, or C57BL/6J mice were minced and grafted under the kidney capsules of the C3l!/HeN mice. 62 日外宝第54巻第2号（昭和60年3月）Fi邑 lA.Fig. lA. Hi,topathological findings in allografted liver tissue of 6 mo old C3H/HeN mice reconstituted with hone marrow cells of young BALB/c n叫nu( <2 mo) mice. A. l¥on-reiection of the liver tissue from a BALB/c (bone marrow donor-type) ouse grafted under the kidney capsule of a C3H/HeN mouse which had been irradiated and reconstituted with Lone marrow cel完ofBALB/c mice （×200). C3H/HeN (H-2k) BALB/c (H-2りC3H/He iwith BALB/c nu/nu B.M. and C3H/HeN liver C3H/He ~with BALB/c nu/nu B.M. and C57BL/6J liverl C3H/He ~with BALB/c nu/nu B.M. and BALB/c liver Stimulation Index 4 10 12 Fig. 2. Allotolerance expressed Lv T cells after bone marrow transplantation. :¥lixcd-lymphocyte reaction C¥l LR I r円 eelsthat C3H/HeN mice reconstituted ¥¥ith BALB/c nu/nu l>one marrow cells 礼rl'tokr;int to both bone marrow donor-type (Jl;¥LB/rl and host (thvmu叶－tvpe(('311/l!l'"ii ¥!HC til'lerni日川nts~Ii川LIVER ALLOGRAFTS IN :¥IICE Fi邑.lB. Fig. lB. Rejection of the liver tissue from a C57BL/6J (third party) mouseιrafted under the kidney capsule of a C3H/HeN mouse which had been irradiated and recon stituted with bone marrow cells of BALB/c nu/nu mice. The liver ti州 UC W川replaced by五broustissue containing rnultinucleated giant cells ( X 200) Table 2. Generation of cytotoxic T-lymphocytes from C3H/I I℃＇＼ mice 1econstituted with bone marrow cells of BALB/c nu/nu mice• % spec1品crel仁川Jfrom target cells Spleen cel Li、仁rdonor E/Tb (m凶 n↓ s.d.)rat10 X5563 (H-2k) EL-4 (II 2b) P815 (H-2d) C3H/HeN(lf-2k) 4/1 。 25. 7士0.4 40.9土 6.3C57BL/6.T(H-2b) 4/1 54. 7±10.1 。 94.4←23.9 BALB/c(H-2d) 4/1 71.9±15.4 66. 9 : 9. 1 。C31!/l!c'.'i with BALB/c C3H/Ilc:¥" 2/ 1 。 22.0土7.5 。/ nu/nu B.i¥I.b C3H/HeN with BALB/c C57BL/6J 211 。 7.6土0.3 5. 7土 8.-t nu/nu B. :¥!. C3ll/Ifr:¥ with BALB/c BALB/c 2/1 。 :21. 0士1.6 0. 3, 3. 4 nu/nu B.M. a Responder cells (7.5×1()6) and rnitornycin C (50 μg/rnl）ーtreatedstimulator c、1'(2.5×105）れじれcocultured in RP:¥II-1640 medium containing 10% heat-inactivated human serum, supplemented with 100 μg/ml streptomycin, 100 I. l' ./ml penicillin, and 5 x l 0-5:¥I 2-mercaptoethanol. After 5 days of co-culture in九 humidifiedCOz incuhator, the cells 、rere（＿υII円廿cl.and their cytotoxic activity was determined by 51 Cr release assay as described prn・iou、l、川b E仔ector/targetcel ratio. c C3H/HeN(H-2k) mice were irradiated and reconstituted with bone marrow cells of BALB/c nu/ nu mice. 63 64 日外宝第54巻第2号（昭和60年3月）C57BL/6J mice were replaced by fibrous tissue containing multinucleated giant cells (Fig. 1-B). It is well known that precursor T cells of donor bone marrow migrate into the host thymus and then differentiate into mature T cels. In order to verify whether or not newly-developed T cells are tolerant to both host勺アpeand donor-type MHC determinantsぅMLRand CTL assays were performed. Fig. 2 shows that spleen cells of chimeras signi自cantlyrespond to the thrid party cels, whereas they do not respond to either bone marrow donor type or host-type MHC determinants. As shown in Table 2, the assay for induction of CTL also revealed that the T cels are tolerant to donor-type as well as host-type :¥IHC determinants. Discussion It has been reported that liver allografts in al species are rejected less aggressively than allografts of other organs•> In the present study we demonstrated that C3H/HeN (H-2k) mice reject allogeneic liver tissue of BALB/c (H-2d) and C57BL/6J (H-Zb) mice. By contrast, when C3H/HぞN mice were irradiated and reconstituted with BALB/c nu/nu (H-2d) bone marrow cels, the C3H/He~ mice accepted both BALB/c bone marrow donor-type and C3H/HeN host (thymus)-type liver tissue, but rejected third party C57BL/6J liver tissue (Table 1). Using assays for both MLR (Fig. 2) and induction of CTL (Table 2), we clearly demonstrated that the newly-developed T cells are tolerant to both donor-type and host-type :¥IHC determinants. SLAVIN et al. have also reported that radiation bone marrow chimeras accept donor-type as well as host-type skin 12>. Thus, it is likely that donor stem c、els(or precursor T cells) migrate into the host thymus and acquire self-tolerance during the differentiation in the thymus1>. lmmunosuppressive agents such as azathioprine, steroid hormones, and anti-lymphocyte globulin2> have been used in order to prevent allograft rejection. However, these agents have cytotoxic effects on lymphocytes, especially T cels. Therefore, most patients die of infection. A new immunosuppressive drug, Cyclosporin A (Cy A), has been shown to be eflective in pro-longing kidney, heart, pancreas, and liver allograft survival in man3> as well a日animals5>.However, it has been reported that CyA has toxic eflects on kidney and liver. In the present study, we showed that liver allografts combined with bone marrow transplantation have no side eflects. We have previously reported that MHC-across bone marrow transplantation can treat autoimmune diseases in mice without GVHR, provided that T cells contained in bone marrow are entirely depleted町. Goon et al. reported that more than 20 diseases otherwise fatal for man can be treated by bone marrow transplantation from HLA-matched or mismatched donors7>. Based on our experiments、wethink that organ allografts combined with bone marrow transplantation will become a viable therapy for humans. Acknowledgment ¥Ve thank Dr. Y. ¥'UKAIVA for performin宮irradiationstudies. ¥'" also thank ;¥Ir. K. TSUCHIDA, 1¥lr. T. 0BATA, 1¥Js. K. KITAML'RA, Ms. M. ASANO and :¥Is. ;¥]VINT MYINT THAN for their expert technical asistance, and :¥Is. S Kt'RIMOTO and ¥ls. K. S111~11zu for their help in the preparation of the manuscript LIVER ALLOGRAFTS IN MIC、E 65 References 1) Aizawa民SadoT, et al: Immunology of fully H 2 incompatible bone marrow chimeras induced in specific-pathogen-free mice: evidence for generation of donor-and host-H-2 restricted helper and cytotoxic T cels. J Immunol 127: 2426-2431, 1981. 2) Bentley FR, Sutherland DER, et al: Synergistic effect of posttransplant total lymphoid irradiation and pharmacologic immunosuppression with low-dose anti-lymphocyte globulin or cyclosporine on prolongation of rat heart allograft survival. Transplant Proc 15: 671-673, 1983. 3) Calne RY: Recent advances in clinical transplantation of the liver and pancreas. Transplant Proc 15: 1263 1268, 1983. 4) Davis HS, Taylor JF, et al: Difference between pig tissues in the expression of major transplantation antigens: Possible relevance for organ allografts. J exp Med 143: 987 992, 1976. 5) Flye MW, Rodgers G, et al: Prevention of fatal rejection of SLA-mismatched orthotopic liver allografts in inbred miniature swine by cyclosporin-A. Transplant Proc 15: 1269-1271, 1983. 6) Furukawa F, Ikehara S, et al: Fate of engrafted skin in thymic chimeras. Micro biol Immu口ol28: 1071ー1076, 1984. 7) Good RA, Kapoor N, et al: Bone marrow transplantation← An expanding approach to treatment of many diseases. Cell Immunol 82: 36 54, 1983. 8) Ikehara S. Pahwa RN, et al: Functional T cells in athymic nude mice. Proc Natl Acad Sci 81: 886 888, 1984 9) Ikehara S, Good RA, Nakamura T, et al: Rationale of bone marrow transplantation for the treatment of autoimmune diseases. Proc Natl Acad Sci: in press. 10) Kamada N and Caine RY: Orthotopic liver transplantation in the rat. Transpl川1tation28: 17-50, 1979. 11) Mito M, Ebata H, et al: Studies on ectopic liver utilizing hepatocyte transplantation into the rat spleen. Transplant Proc 11: 585-591, 1979. 12) Slavin S, Strober S, et al: Induction of spec1凸ctissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts. J Exp Med 146: 34-48, 1977. 13) Starzl TE, Marchioro TL, et al: Homotransplantation of the liver in humans. Surg Gynecol Obstet 117: 659-676, 1963. 14) Starzl TE, Koep LJ, et al: Liver transplantation-1978. Transplant Proc 11: 240-246, 1979. 15) Zimmermann FA, Knoll PP, et al: The fate of orthotopic liver allografts in di仔erentrat strain comLinations. Transplant Proc 15: 1272 1275, 1983. 66 日外宝第54巻第2号（昭和60年3月）和文抄録マウスにおける同種肝移植法の確立一一骨髄移植による免疫学的寛容を利用して一京都大学医学部第2病理学教室（主任：泌島義博教授）中村敬夫，池原 進，井上秀治， MaungMaung Oo，漬島義博同種肝移植の際の拒絶反応を抑制するため，免疫学的寛容を誘導する試みとして骨髄移横を併用した．免疫学的機構の解析には， 7 ウスが一番適しているが，7 ウスで血管縫合により肝を移植することが困難であるため腎被膜下に細切した肝を移植した．C3H/HeN(H-2k) ＜ ウスに C57BL6j(H-2b) やBALH Cirト2d)＜ ウスの肝を移植しでも拒絶された．しかしながら，放射線照射し BALB/Cnu/nu(H-2d) 7 ウスの骨髄を移植した C3H,llcNマウスでは骨髄don er-typeの BALB/Cマウスの肝も， hosttypeのC:ill 1-frN ＿，，.ウスの肝も拒絶されなかった． リンパ球混合培養法（MLR）や killerT細胞の誘導で解析すると，新しく分化して来たT細胞は骨髄doner匂peのみならず， host-type の組織適合抗原IC対して免疫学的寛容が誘導されていることが判明した．人では HLAを一致させた肝を入手することが困難なととや，免疫抑制剤を長期間投与すると肝や腎IC毒性を有すること，また免疫抑制の結果感染による死亡が増加しているととから肝移植と同種骨髄移植の併用療法は今後の臓器移植に新しい道を拓くものと考えられる．

マウスにおける同種肝移植法の確立 : 骨髄移植による免疫学的寛容を利用して

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マウスにおける同種肝移植法の確立 : 骨髄移植による免疫学的寛容を利用して

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