The Homotransplantation of Kidneys and of Fetal Liver and Spleen After Total Body Irradiation (Annals of Surgery, Vol. 152, No. 3, September 1960)

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Reprinted from ASYALS OF SLRGERY,Vol. 155,’No. 3, September, 1960 Copyright 0 1960 by J. B. Lippincott Company e- The Honiotraiisplantation of Kidneys and of Fetal Liver and Spleen after Total Body Irradiation * From the Deportment of Surgerg and the Surgical Research Laborotories, Medical College of Virginia, Richmond, Virginia lieved to be a consequence of antibody formation by the host against the antigens of the transplanted foreign tissue. If a sufficiently large dose of total body radiation antibody formation and hematopoiesis can be suppressed and bone marrow homotransplants will take successfully. The repopulation of the hematopoietic system by the grafted tissue leads to the production of an antigenic mosaic pattern which is characteristic of the donor rather than the recipient. Radiated mice with bone marrow homo- @ transplants will therefore accept skin homografts from members of the donor train.^'^ 95 The use of adult bone marrow transnlants after r:icliation has led to the appearance in a significant percentage of animals of the “runt syndrome” in lvhich failure to grow and eventual death appear to be related to a reaction of the mature graft against the host.8*g5 Uphoff loo found that if fetal hematopoietic tissues (liver and spleen) were used instead of adult marrow the runt syndrome did not appear. This is presumably because the fetal tissue, by virtue of its immunologic immaturity, was able to acquire a tolerance to the host’s antigens and thus did not react against the is administered to mice 6 , 2 S , 659 56, 66, 8 0 , 96 Presented before the American Surgical Association, White Sulphur Springs, West Virginia, This work has been supported in part by a contract with the Atomic Encrgy Commission ’AT (40 1) 2439 and by USPH grant A-2350. The rejection of homografted kidneys in the dog is accompanied by the appearance of Ijmphocytes and plasma cells in the interstitial spaces of the cortex, edema, tubular destruction, and cessation of urine formation.31,43,84 It has been postulated by Simonsen 8 4 and Dempster 31 that the round cells which appear in the interstitial spaces are of donor origin and represent a reaction of the donor against the host. In the present study an answer was sought to three questions: 1 ) whether infusions of homologous fetal liver and spleen cells would permit indefinite survival after lethal doses of total body irradiation (TBR) in the dog, 2 ) whether irradiation of the host prior to renal 1iomotrr;nsplantntion homograft rejection pJienqnw+,, and 3) graft rejection plienomena. q y vir f ue ob the answers to questions 2 bnd 2 a fotjrth question would be answered, nkmelj whether the lymphocytes and las@ celis seep in of donor origin, repreQenti a Tactiqn of the graft against the /hos$s chime8 by Simonsen and Demps+r, of of sost origin, representing a reactibn of the recipient against the graft. the interstitial spaces oP the bansplant ivere A total of 71 unrklated mongre1:dogs were used for the experiments. They were dewormed, immunized against canine distemper and splenectomized. The animals

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355 HUXIE, ET AL. were divided into six groups as follows: 1) 28 dogs received 600 r total body irradiation (TBR) without transplantation of hematopoietic tissue, and served as controls; 2 ) 16 dogs received GOO r TBR followed within 94 hours by an infusion of fetal hematopoietic tissue (liver and spleen) from one or more donors unrelated to the recipient; 3 ) five dogs received renal homoceived 1,000, 1,200, or 1,500 r TBR 24 hours before transplantation; 4 ) 14 dogs received renal homotransplants from nonirradiated donors 24 hours after receiving 1,000, 1,200, or 1,500 r TBR; 5 ) seven dogs received renal homotransplants from irradiated donors (600, 1,000, or 1,500 r TBR) 24 hours after receiving 1,000, 1,200, or 1,500 r TBR; 6 ) one dog received a renal homotransplant without radiation of either host or donor. The transplant functioned five days, and was removed on the seventh day. On the thirteenth day the animal received 1,500 r TBR, and on the same day a secondary homotransplant was carried out using the remaining kidney of the original donor. The animal was sacrificed after 24 hours. As controls for the renal honiotransplantation experiments we utilizcd data from our previous experiences with over 150 renal homotransplants in nonirrudiatcd Total body radiation was administered from one of two sources. The first source dogs were placed one meter from the source and were held in a special cnge so that they could be radiated on both sides. The rate of radiation was 37.8 r per minute. The second source wasa multiple cobalt-60 machine specifically designed for TBR in which the cobalt is contained in the \vaIIs of a cylinder which coinplet,-ly surrounds the animal. This radiation sotlrce was made available to us at the Niival XIcdical Research Institute of the National Naval hIec1ical Center.* The rate of radiation was 32 r per minute and radiation was uniform throughout the entire field in which the animal was confined. In the early eaperiments animaIs were placed under pentothal anesthesia during irradiation with the 1000 KVP machine, but not during irradiation with the cobalt source. In the later esperiments anesthesia was not used during irradiation with the x-ray source either. The animals were maintained on dailjr injections of penicillin and I-V fluids after irradiat'ion. The fetal hematopoietic tissue was prepared by remojing the liver and spleen asceptically from a fetus n~hichin turn had been removed by cesarian section. The gallbladder, major bile ducts, and vessels of the hilum were removed. The parenchymal cells were then scraped from the stroma with the back of a scalpel. The cells were passed through a tea strainer with an aperture size of approximately 1,000 microns. In the earlier experiments the cells were suspended in an equal volume of isotonic saline and were injected slo\vly into be less satisfxtory than intravenous. All work was done \vith sterile glxsvare in an ice bath. In the later e\periiiients the cells were suspended in an eqiid volume of hondogous dog serum, \vhich \\'as also kept in an ice bath. The cells n-ere not injected all at one time, but were injected in se\,eral aliquots. The portion of cells to be injected at the time of preparation \vas kept in the ice bath except for that sinall amount which was in the syringe at any given time. The remainder of the liver-spleen cell suspension \vas stored in the deep freeze at of 30 per cent gl!-cerol in sa I 'me. In some animals cleath occurred during the injection of the liver-spleen cell suspension. That this was not due entirely to ' IYitIi the Iielp and coiipcration of Captain F. IY. Chambers, XISC, USN.

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'VolumeSumber 1513 . HOAIOTRANSPLAKTATIOS OF KIDNEYS 356 TABLE1. Total Body Irradialiotz and Renal embolic phenomena was born out by the fact that a Waring blender homogenate of liver in which particulate matter had been centrifuged down and the cell free supernatant portion injected proved to be lethal in a dose range (4 Gm. of liver per kg. of dog) which was similar to that seen with ndiole-cell suspension injections. The lethal dose of liver cell suspension fell into a fairly well defined range but sometimes very small doses caused rapid demise of the animal. In order to inject a sufficient number of cells, injections were given S ~ OWI ~and a period of several hours was 1,200 r to host During the injection the animals were mon- (1) KT-312-primary transplant functioned 5 days day 7 kidney removed day 13 1,500 r TBR to host clay 13 secondary kidney transplant F--received fetal hematopoietic tissue. R-radiation of host aftcr transplantation. The figures in parenthescs indicate the period of survival ol the animal in days; or, in thc case of radiation of the donor, the time when the kidney transplant vas removed after it had ceased functioning. The only esception to this is KT-46, which was still functioning itored on an EKG machine. It was found that before death inversion and peaking of pression occurred. In some cases the aniinal would survive if the injection was stopped hen EKG changes first became evident, but other animals died even if the injection The average last trimester-dog fetus yields roughly 4.0 x 1 0 ' O nucleated cells per liver. Cell counts were done on all injected in addition the liver cells were weighed before the serum n a s added in order that the lethal dose could be calculated in t e r m of Grams of liver per kilogram of body weight. An animal was sometimes given an injection of fetal liver and spleen from a single fetus and at other times from seI-eral fetuses of the same uterus. Renal homotransplants were carried out as described by Hume and Egdah1,43 the transplant being placed in the' renal fossa with the vessels anastomosed to the host renal artery and vein. A polyethylene catheter was placed in the ureter and the ureter was brought out through the skin. Daily collections of urine \yere made. The dogs \yere ipaintained until death or until death appeared imminent. The renal homotransplant was taken from an animal who was not related to the host, to the pregnant

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35 7 HUXIE, ET AL: animal donating the fetuses, or to the tered to a male host the female leukocyte tags were searched for by the method of In some cases fetal liver and spleen injections were given to irradiated animals receiving kidney transplants, but in most cases no hematopoietic cell injections were made, the transplants being carried out primarily for the purpose of determining the dose of irradiation necessary to prevent the renal homograft reaction during the period for which the animal survived. The nonirradiated host animals receiving irradiated kidney transplants were sacrificed after renal transplant function had ceasedwhich, with a single exception, fell within the period of time the irradiated animals In two instances in the group in which both host and donor were irradiated, the irradiation of the donor kidney was accomplished immediately after transplant at'ion all other cases the host and donor were A. Total Body Irradiation with and without the Infusion of Fetal HematoFIG. 1. A female leukocyte in the peripheral smear of an irradiated ninle host receiving cells . from the liver and spleen of a single female fetus Annals. of Surgery FIG. 2. Liver and hematopoietic cells in the lung of a dog who was irradiated and infused with fetal liver and spleen cells 8 days before. poietic Tissue. All 28 splenectomized dogs receiving 600 r total body irradiation died within a period of seven to 17 days. Of the 16 dogs receiving infusions of fetal liver and spleen following 600 r total body irradiation, four aniinals are surviving 12, 16, 18 and 22 months later. They appear in normal health. One male animal receiving female cells had persistent female leukoThese have since disappeared from the Following an intlavenous injection of fetal liver and spleen cells, some of the injected cells are caught up in the capillary bed of the lungs, as s11oir.n in Figure 2. Hon.ever many of the cells make their way to the bone niarron., ~ h i c hultimately becomes the site of the most active proliferation. The course of the cells is easy to trace for some time after the infusion because the hematopoietic cells are accoikpanied by easily recognizable liver parenchymal cells. In a single clinical case, we injected fetal human liver intravenously and reco\-ered healthy hepatic cells in the recipient's bone marrow ten days later. B. Total Body Irradiation plus Kidney Transplantation. The results of these experiments are listed in Table 1. They may be summarized as follo ~ s : 1. Zrradintion of the donor: In four of five cases in which the donor animal was

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Saw l fic rd on 6ih day The transplanted kidney is on the right and the host’s own kidney on the left, The donor received plant functioned for 6 days and was removed largement of the kidney, and the edeina and ischemia of the cortes which plant rejection pattern. irradiated prior to transplantation with doses of 1,000, 1,200 or 1,500 r, a typical transplant rejection pattern was seen. After a few days of good function, urinary flow of the transplant ceased, or declined very sharply, usually a day or two before the removal time shown on the chart. The renal ,@ artery and vein were patent at the time of removal. All transplants from donors who had received 1,500 r sho\ved prompt cessation of function and a typical transplant rejection pattern. A representative gross picture is shown in Figure 3, and a microscopic view of the same kidney (KT 47) in Figure 4. A photomicrograph of a normal dog kidney and of a renal homotransplant between nonirradiated dogs is shown in Figures 5 and 6 for comparison. In one instance (KT 46) in which the donor received 1200 r the transplant appeared normal at seven view of the transplant s1ion.n in Figure 3. There is tremendous lymphoid struction of the tubular elements, and plugging of a small artery of the cortex seen in cross sec-

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a ’ Sumber 3 HO\IOTRASSPLASTATIOS OF KIDXEYS 360 The transplanted kidney is on the right and the host’s own kidney on the left. The host received plant ‘was removed while still functioning 8 days after transplantation. It appears normal grossly, in marked contrast to the transplant in Figure 3. both the host and donor received 600 r TBR the renal transplants showed a homograft reaction. When doses of 1,000, 1,200, or 1,500 were administered to both host and donor, the transplants continued to function and appeared normal grossly and niicroscopically. Fetal liver and spleen was administered to two cases (KT 7 and KT 11) immediately following transplantation and irradiation. These kidneys appeared normal as well. 4. Irradiation of the 12ost after primary brit before secondary transplantation: In one case, a primary renal homotransplant was carried out between two nonirradiated dogs. The transplant. ceased functioning on the fifth day and was removed on th? seventh day. A severe homograft reaction was present. Six days after removal of the transplant, the animal was given 1,500 r TBR, and a secondary renal transplant was carried out employing the other kidney of the original donor. This was removed 24 hours later while still functioning. A photomicrograph of the primary transplant is shown in Figure 9, and that of the secondFIG. 8. (left) hlicroscopic view of the transplant shown in Figure 7. The kidney appears normal that of a typical transplant reaction. The kidney functioned for 5 days and was removed on the 7th day

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HUhlE, ET AL. Annals of Surgery FIG. 10. (left) Dog KT-312. hIicroscopic view of the secondary transplant removed after 24 hours in the host. Six days after the primary transplant had been removed the host was given 1,500 r TBR, and the secondary transplant was done on the same day. The appearance of the kidney is nornial ( X 100). FIG.11. (right) Llicroscopic view of a …