JOURNAL OF NUCLEAB MEDICINE 7:416-423, 1966

Homotransplantation Studies in Dogs Following Selective Radioisotopic Lymphatic Ablation1

H. S. Winchell, M. Pollycove, W. D. Loughman, V. Richards, L. Kim, and J. H. Lawrence

Berkeley, California In previous communications we described a technique for relatively selec tive radiation of lymphatic structures using internally administered Y-90 chelated with diethylene triamine penta-acetic acid—DTPA (1,2,3). This technique results in radiation doses to lymph nodes approximately two and one-half times greater than the radiation dose to bone marrow. A second method outlined in this paper results in further selectivity in radiating lymphatic tissue. Since the homograft rejection response appears to be a function of cells residing in lym phatic structures, we used these techniques in preparing dogs for tissue homo- grafting.

MATERIALS AND METHODS All dogs were mongrels of unspecified type between the ages of six months and two years. In most cases the irradiated animals were males, while the donors of the tissue homotransplant were unrelated females. Following irradia tion they were given daily injections of penicillin with streptomycin (600,000 units penicillin, 0.75 gm streptomycin) for a four week period. All counts were performed on venous blood. Duplicated counts of platelets were made by phase contrast microscopy and those of WBC by routine tech niques. At post mortem examination, tissues were fixed in Bouin's solution, em bedded in paraffin or celloidin and stained with hematoxylin and eosin or Pollak's trichrome. The Yttrium-90-DTPA intravenous urine recycling procedure was the same as that previously described (1,3). Intralymphatic injection of colloidal chromic :|-P phosphate (Abbott ) was performed on each of the four legs of the dog after the lymphatic channels had been rendered visable by the intracutaneous and subcutaneous injection of Evans* blue dye into the foot pad. The mesenteric

'University of California, Doner Laboratory, Berkeley 4, California.

416 HOMOTRANSPLANTATION STUDIES IN DOGS 417 nodes were injected directly with colloidal chromium :i-P phosphate at the time of laparotomy using a number 27 needle. X-irradiation of the anterior medias tinum was performed using a supervoltage x-ray machine set at 250 Kvp, 15 milliamps, with an absorber of 0.6 mm tin, 0.25 mm copper and 1.0 mm alumi num. Tube to target distance was 50 centimeters. The field was 15 X 20 cm and extended from above the xyphoid to the cricoid cartilage and laterally to include the medial one-half of both lung fields. Radiation flux rate was approximately 40 r per minute. The radiation was given in two exposures of 1000 r each (air dose). Splenectomy was performed in a routine manner. Renal homotransplanta- tions were performed utilizing an end-to-end anastomosis of the renal artery with either the hypogastric or common iliac artery, and an end-to-side anastomo sis of the renal vein with the common iliac vein. The ureter was implanted in the bladder through a submucosal tunnel. Surgical transplantation of the spleen was performed with vascular anastomosis similar to that outlined for the kidney. Bone marrow was obtained utilizing multiple aspirations with Bierman needles from the femoral, tibial, iliac and humoral bone marrow sites. Spleen cell suspensions were made by chopping the donor spleen into fine particles and then passing them through graded steel mesh filters and through a nylon mesh filter prior to its intraperitoneal administration. The animals receiving the col loidal chromic :i-P phosphate intralymphatic injections and splenectomy, re ceived Y-90-DTPA radiation as a single dose, without subsequent urinary re cycling. Dosimetry estimates were obtained using the data previously pub lished (3) and the plasma Y-90-DTPA clearance curves obtained on these dogs. "Clubbed neutrophiles" were scored in the male dogs receiving female bone marrow according to the technique described by Porter (4). Karyotypes were obtained on peripheral blood leukocytes following short-term culture by the tech nique of Loughman (5), which is a modification of the technique used in humans by Hastings et al (6). All samples containing an isotope were appropriately diluted and plated on stainless steel planchéisand counted in a Nuclear-Chicago gas flow counter fitted with a micromil window of 150 ¿ig/cm-surface density.

RESULTS Figure 1. demonstrates changes in peripheral blood cell values which occur in animals given lethal doses Y-90-DTPA by the intravenous urine re cycling technique (3), with and without subsequent treatment with bone marrow homotransplants. The ordinate of the graph represents the mean value of the given parameter expressed as per cent of its mean value on the day of radia tion. The broken lines represent mean values obtained on seven dogs who had lethal doses of Y-90-DTPA but no marrow transplant ( 1); the solid lines repre sent values on 14 male dogs given lethal doses of Y-90-DTPA and subsequently given homologous bone marrow from unrelated female donors. In the lethally irradiated animals, which did not receive marrow, the lymphocyte and granulo- cyte values fell to low levels between one and two weeks following radiation. The mean time of death of these dogs was the fourteenth day following radia- 418 WINCHELL, POLLYCOVE, LOUGHMAN, RICHARDS, KIM, LAWRENCE tion with a range of eleven to twenty-four days. Between the 14th and 18th days following radiation the animals in this group had the following ranges of blood values, expressed as a percentage of their preirradiation values: Granu- locytes 0.7^-5.0%; lymphocytes Q.Q%-14%;platelets ll%-30% reticulocytes O.Of-1.2%. Dogs given homologous bone marrow showed depressions in mean peripheral lymphocyte values comparable to that obtained in the lethally irradiated dogs not receiving bone marrow, however, the mean granulocyte levels of these dogs remained generally within the normal range as compared to the marked de pression seen in animals not receiving bone marrow transplants. Between the 14th and 18th days following radiation the animals in this group had the fol lowing ranges of blood values, expressed as a percentage of their preirradiation values: Granulocytes 20%-705%;lymphocytes 0.0%-46£;platelets 12^-79%; reticu locytes 0.0%-1700%.We did not see the marked rebound granulocytosis seen by others following successful marrow transplantation (7 ). Figure 2 demonstrates changes in the peripheral blood platelet and reticu- locyte levels of these animals. In the animals given homologous bone marrow, there was a rise in peripheral reticulocytes (solid lines) following bone marrow transplantation as compared to zero values in the lethally irradiated nontrans- planted group (broken lines). Following radiation, platelet levels fell in animals

200 LYMPHOCYTES

100

O 400 \ GRANULOCYTES Y90 + Horn, bone marrow (14 dogs) 300 Y90 No bone marrow (7 dogs) o> o. 200 O) u_ 0) o. 100

10 20 30 40 50 60 Days after irradiation

MUB-3737

Fig. 1. Peripheral blood lymphocyte and granulocyte changes following Y-90 DTPA irradiation in control dogs (broken lines), and in dogs given homologous bone marrow (solid lines) (ordinate expressed as per cent of value on day of radiation). HOMOTRANSPLANTATION STUDIES IN DOGS 419 given homologous bone marrow (solid lines) but not to the low level reached in the lethally irradiated nontransplanted group (broken lines). In the 14 animals given lethal radiation plus homologous bone marrow, 11 survived over 25 days. Of these 11, three died in the 25-60 day period following radiation; one secondary to surgery, one of gastrointestinal bleeding, and one of a syndrome resembling "secondary disease" of the mouse. Of the eight animals surviving over 60 days following radiation, five lived over one and one-half years. In the long term survivors five renal homotransplants were attempted. Two of these were technical failures, two appeared to be rejected and one renal homo- transplant survived 36 days following the homotransplant procedure. Several of the male dogs given lethal doses of radiation and homologous marrow transplantation from female donors had increased numbers of "female type" clubbed neutrophiles in the peripheral blood 12 to 25 days following bone marrow homotransplantation. The finding of these "clubbed" neutrophiles in the peripheral blood of irradiated animals is difficult to interpret relative to the "success" of marrow homotransplantation in our experiments. We have noted an increase in nuclear projections on male neutrophiles which could have been mis taken for female neutrophiles in animals given sub-lethal doses of radiation with out transplantation of homologous bone marrow (8).

1500

+ Hom. bone marrow (14 dogs)

20 30 40 Days after irradiation

MUB-3738 Fig. 2. Peripheral blood reticulocyte and platelet changes following Y-90 DTPA irradia tion in control dogs (broken lines), and in dogs given homologous bone marrow (solid lines). (Ordinate expressed as per cent of value on day of radiation.) 420 WINCHELL, POLLYCOVE, LOUGHMAN, RICHARDS, KIM, LAWRENCE

In two of the long term survivors, peripheral blood leukocyte cultures ob tained one-to-two years following the radiation procedure failed to show dividing cells of the donor sex karyotype. One animal, not represented on this graph, received a lethal dose of Y-90- DTPA radiation and in addition had splenectomy and 2000 r of x-rays delivered to the area of its thymus. This animal subsequently received homologous bone marrow and a renal homotransplant which functioned for 34 days. Evidence that the Y-90-DTPA radiation suppressed the homograft rejection response may be summarized as follows; homologous bone marrow transplanta tion saved the lives of lethally irradiated animals and some of these animals developed findings suggestive of "secondary disease"; renal homografts had prolonged survival in some irradiated animals. That the suppression of the homo- graft rejection response was incomplete or temporary was evidenced by eventual rejection of renal homografts. The failure of long term bone marrow homograft survivors to show cells of donor karyotype in peripheral blood leukocyte cultures was further evidence against permanent chimerism in these animals. In an attempt to improve our selectivity of lymphatic ablation, we developed another technique which involved the injection of colloidal chromic phosphate containing :!-P into lymphatic channels afferent to lymph node chains, as well as direct injection of the radio-colloid into mesenteric lymph nodes at laparotomy, combined with splenectomy, thymic irradiation, and sublethal single intravenous doses of Y-90-DTPA. The results obtained on four animals irradiated in this manner are summarized in Figure 3. The various hématologieparameters are expressed as the mean value of the per cent of preirradiation blood level on the ordinate. Between the 14th and 18th days following radiation, the animals in this group had the following ranges of blood values, expressed as a percentage of their preirradiation values: Granulocytes 78^-315%; lymphocytes 5.0$-43?; plate lets 103%-236£;reticulocytes 150^-3300%. It is clear that the peripheral blood lymphocytes are reduced to low levels following this treatment and remain de pressed for prolonged periods of time. The granulocytes are not concomitantly depressed and remain generally in the normal range until approximately one month after the administration of chromic phosphate :i-P at which time there was a definite depression in peripheral blood granulocytes. We attribute this to either the contamination of the colloidal :!-P phosphate with soluble :!L'Pphosphate or to small sized colloid particle uptake by marrow. In Figure 4 we see that the platelets and reticulocytes were maintained at near normal levels until one month following irradiation, at which time the platelets became depressed in a manner similar to that seen with the granulocytes. Again we attribute this to the small marrow uptake of the :|-P phosphate. One of these animals died at eight days and another at twenty-one days following the irradiation while two survived forty-five and seventy-three days. The cause of death in the animals living over one month could not be determined at post mortem examination. At the time of death, the peripheral lymphocyte values of the dogs which survived forty-five and seventy-three days were markedly depressed and most of the lymph nodes throughout the body were acellular and fibrotic. HOMOTRANSPLANTATION STUDIES IN DOGS 421

Five other dogs underwent a preparative procedure, similar to that per formed on the dogs summarized in Figures 3 and 4, following which renal homotransplantation was attempted without concomitant bone marrow trans plantation. Three of these animals survived over one month following irradiation. In one of these dogs, the transplanted kidney appeared to be intact for 40 days following transplantation, the animal dying of systemic infection following ex ploratory surgery. One of these three dogs had a renal transplant which was viable 30 days following surgery. However, the kidney had impaired function and the animal died of uremia 41 days following irradiation. The third animal was sacrificed 34 days after irradiation at which time it appeared histologically that it was rejecting the renal transplant. It was clear from these results that the techniques described resulted in selective lymphatic ablation with concomitant prolongations in homograft sur vival. It was equally clear that the animals were immunologically crippled and died of infections which they normally might have aborted. Despite the danger of inducing a severe secondary disease in these animals, we attempted to replace damaged lymphatic structures in the irradiated animals by homotransplantation of lymphatic tissue in order to restore the hosts' im munologie competence for antigens other than those of the homograft donor. Seven animals had lymphatic irradiation using the techniques mentioned above

Y90, CrP"04, Splenectomy, 200 LYMPHOCYTES Thymic irradiation (4 dogs)

10 20 30 40 50 60 70 90 100 Days after irradiation

MUB-3742

Fig. 3. Peripheral blood lymphocyte and granulocyte change in dogs given a single intravenous injection of Y-90 DTPA, intralymphatic collodial Cr P-32 O4, splenectomy and 2000 r of x-rays to the region of the thymus. (Ordinate expressed as per cent of value on day of radiation). 422 WINCHELL, POLLYCOVE, LOUGHMAN, RICHARDS, KIM, LAWRENCE and subsequently were given spleen homotransplants. Three of these animals had transplantation of the solid organ by anastomosis of the donor splenic vessels to the vessels of the recipient. The remaining four animals had an intraperitoneal infusion of a cell suspension of homologous spleen. The level of peripheral blood lymphocytes in these animals was somewhat higher than the control group which did not have spleen Homotransplantation. However, a considerable increase in peripheral blood lymphocytes was not noted. In each case the source of the splenic lymphocyte donor was of a different sex than the recipient. In two cases short term culture of peripheral blood leukocytes showed rare cells of donor karyotype at the ninth and fourteenth day after transplant. Subsequent cultures were failures, or showed only recipient cells. In one case, cells from nodules found attached to the peritoneal wall were cultured as monolayers. Cells of donor origin were found in one such culture obtained 29 days after spleen cell infusion. Subsequent cultures showed only recipient cells. Five of these dogs died 43 to 67 days following irradiation either of infection or of causes which could not be determined at the time of death. It was clear that the animals in this series were also immunologically crippled and could not withstand infec tion. No evidence was obtained for a permanent "take" of the homologous spleen cells.

3000 Y90,CrPJX»32 Splenectomy,

2000 Thymic irradiation (4 dogs)

~5 > 1000 o

10 20 30 40 50 60 70 80 90 100 Days after irradiation

MUB-3743 Fig. 4. Peripheral blood reticulocyte and platelet changes in dogs given a single intra venous injection of Y-90 DTPA, intralymphatic colloidal Cr P-32 O4, splenectomy and 2000 r of x-rays to the region of the thymus. (Ordinate expressed as per cent of value on day of radiation. ) HOMOTRANSPLANTATION STUDIES IN DOGS 423

CONCLUSIONS The results suggest that selective lymphatic ablation using the techniques described produces generalized suppression of the hosts' immunologie compe tence. The animals thus treated are less able to resist infection as well as being less able to reject homografts than untreated animals. In the cases where homol ogous bone marrow saved the lives of lethally irradiated animals, it appeared that long term survival of the animal was accompanied by eventual return of host immune response and eventual rejection of homologous tissue.

REFERENCES 1. WINCHELL,H. S., POLLYCOVE,M., ANDERSEN,W., LAWRENCE,J. H.: Relatively Selective Beta Irradiation of Lymphatic Structures in the Dog Using Y-90-DTPA, Blood, 23:321-336, 1964. 2. WINCHELL, H. S., POLLYCOVE,M., LOUGHMAN,VV. D., LAWRENCE,J. H.: Autologous Bone Marrow Transplantation Studies in Dogs Irradiated by Y-90-DTPA Urine-Recycling Technique, Blood, 23:44-52, 1964. 3. WINCHELL, H. S., POLLYCOVE,M., LOUGHMAN,W. D., LAWRENCE,J. H.: A Method for Maintenance of Curie Quantities of Y-90-DTPA in the Human Body for Defined Time Periods: Technique and Dosimetry. /. Nucí.Med. 5:16-26, 1964. 4. PORTER,K. A.: Use of the Sex Difference in Morphology of Polymorphonuclear Leuko cytes to Indicate Survival of Marrow Homotransplants. Transplantation Bulletin, 4:129, 1957. 5. LOUGHMAN,W. D.: In preparation. 6. HASTINGS,J., FREEDMAN,S., RENDON,O., COOPER,H. L., HIRSCHORN,K.: Culture of Human White Cells Using Differential Leukocyte Separation. Nature, 192:1214-1215, 1961. 7. THOMAS,E. D., ASHLEY,C. A., LOCHTE,A. L., JAHETZKI,A., SEHLER,O. D., FERREBEE, J. W.: Homografts of Bone Marrow in Dogs After Lethal Total Body Radiation. Blood, 14:720- 736, 1959. 8. WINCHELL,H. S.: Selective Beta Irradiation of the Lymphatic System Using Internally Administered Y-90 DTPA: Kinetics, Dosimetry and Biological Evaluation. U.C.R.L. Report No. 9755. Lawrence Radiation Laboratory, Berkeley, California, June, 1961.

ACKNOWLEDGMENT This work was supported in part by research grant number CA-01440-13 from the National Cancer Institute of the United States Public Health Service.