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ARMED SERVICES TECHNICAL INFORMMON AGENCYARLINGTON HALL STATIONARLINGTON 12, VIRGINIA

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216 039 Pnd other

(1) Titlo of Study: Tissue Transplantation: Homograft antigens,ionizing radiation and other factors influencinghomnograft survival.

S(2) Period covere'd by Report: 1 June 1961 - 31 I-lay 1962

S(3) Responsible Investigators: Gu, tave J. Darwiin, 11.D.J. Mackie Corson, ri.D.Lewis T. ;:ann, Jr., Ph.D.

Harvard Medical School

C 25 Shattuck StreetBoston 15, 1iass.

4) Contract Eumber: DA-49-193-14D-2061

(5) Supported by: United States Army Miedical Research a-i DevelopmentCommand, Department of the Army, Washing-ton 25, D.C.

(5) Security classification: Non'e.

Distribution: "C.ua1ifid requestors :ay obtain copies of this reportfrom ASTIA."

Table of Contents1. Introducticn.2. Transplantation of the !adney in Lan,3. Transplantation of the ,Idney in tht Dog.4. Transplantation of the Skin in Lan.5. Transplantation of the Liver in the Dog.6. Transplantation of the Spleen in the Dog.7. Fractions of Tissues and Cells Active AntigenicalJy in

a Homologous Systen.8. 7mmunologic Capacities of Tissues.9. Synthetic Polypeptides as Anti-"ens.

10. SzmSary.11. Publication:'.

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1. IntroductionThis pro-ivess report, concerns itself vith -Yorl: of the Brigham Department

of Patholojy in its la oratori,-o at th- iarvard L.odical School and with theDepartments of iedicine, Surgery and iiadiology in the Brigham Hospital. Themajor research effort of ti.e Departnent of Pathology relates to transplanta-tion of tissues and organs. In designinr a research program which has impli-cations for the riilitar:-, considerable breadth of activity has been necessarybecause :f the com'lexity of the field of transplantation. Within the De-partment of Patholor, the disciplines of immunology, genetics, histochemistryand biochernistry are repr'esentcd as wall as pathologic anatomy. Other disci-plines represented in this hospital-wide pro-Taam, include physiology, pharma-coloc-, surer-Y, and radiation biology. The list of participants in thesestudies is a large one. In addition to the principle investigators mentioned,there are o-her members of the ?athology Department who contribute to thesestudies. The• names of these investigators and those who represent theDepartments of ; edicine, Surgery and Radiolo,,7, are listed in the publications(please see prar-ph 11).

The ulta.-.ate air, of ti.e prooram is to achieve successful transplantationof tissues and organs and to so ncdify the recipient, that indefinite sur-vival may be achieved. The pursuit of these aims has lad toea number ofobservations c7 bzsic and practicnl im.ortance. These are described brieflyin the succeeding paragraphs.

2. Transp!antation of the Kidne.v in :,anThe best result achieved thus f:,r is in the case of the fraternal twins,

JR and AR. It is now over 40 months since UR donated a kidney to JR. Thisuas done following an exposure of JR to tuo doses of ionizing radiation for

a total of 450 r. Evidence of incompatability was shoirx by AR's rejectionof JRis skdn grafts. The early rejection pattern noted in the renal isograftdid not occur until;Xoproxitately 10 months after transplantation. At thistine the pattern vas well established and presumably reversnd by administra-tion of periodic small doses of total body irradiation, and cortisone. %bsubsequent antibody -depressing measures have bein used. The recipient JR, iscurrently in good health and survives writh the renal isogra:rt as his onlyrenal tissue.

Protocols sirilar to the one just menticned have been used in subsequentcases here and also by the groups in Paris. Although prolonged survival withfiunction has been obtained in some cases, none has approached the resultmentioned above. In our owni experience, we have observed in periodicbiopsies of the k"dney homograft, a .ariety of patterns which we can interpretas accelerated rejection. These patterns differ from the primary rejection,although there appear to be gradations between the classical pximary rejectionof a kidney hovioraft as observed in the dog, and the accelerated rejectionpattern as we have seen it in man and as it can be induced by previoussensitization of the canine recil-ient.

Total body irradiation is known to suppress the primary immune response buthas little effect on the secondary response. This has been shown in man and theexperimental animals. OtO the other hand, 6-mercaptopurJne and related anti-metabolites have bean shoim to alter the secondary or anamnestic response.Since most of our potential recipients have received blood transfusions, andtherefore have been sensitized because of the presence of antigens in leuko-cytes, it is plain that we can expect a rejection pattern which is differentfrom the primary one. The expectation that 6-mercaptopurine might providea better recipient under these circtmstances, has been borne out by recentstudies. Us have observed the early phases of rejection reversed by adcainis-tration of 6-mercaptopurine. In our current case, the patient is now in theeighth post-transplant week having been given 6-mercaptopurine, followed by

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Imuran (5457-322) and occasional actinormycin C. This represents the longestsurvival obtained in nan .,-ith c, enical modification of the recipient.

It is lilhcly tIt ve are a ;roachin- a state c: tolerance rather thanone of adaptation. It aeelcars from studies in the dog" that a delicatebalance has been achieved, and that this balance may be disturbed by theaddition of antigen in the form of a skin graft. The course of a skin graftfrom the kidney donor will provide valuable information. It has been shownto alter the course of a kidney homograft at the same time that it shows apattern of delayed rejection. If adaptation were to occur, then the coursewould be one of a primary rejection. If sensitization has been induced, thenrejectior: of a skin graft would be accelerated. If tolerance Lf a high degreehas been induced, then there should be markedly prolonged survival of the skringraft from ti e kidney donor when placed upon the modified recipient.

3. Transplantation of the Nidney in the DopTE'6ee studies have been conducted with Dr. Joseph E. ianrray and his staff.

Dr. G. .. lexandre and Dr. K. *!ilsson are currently active in this program.The maj~r experience diarin,? tf`.- year has been writh a combination of 6-mercaptopurint given initially a.id follored by oral doses of Imuran andperiodic administration of actinomycin C. The mean survival time of function.of the renal homograft has been 58 days with 3 dogs showL.- survival of thegraft for over 3 months. In these subjects, the homograft is the only renaltissue, the recipient's own kidneys having been removed at the time of trans-plantation. Of special interest in these lon, survivors, is the effect of askin graft from the donor on the function of the graft as mentioned above.Although biopsies of the kidney graft have not been done at this time, funwtionbecomes Lmpaired. This can be reversed by the administration of actincmycin C.In the few subjects so studied, there has been prolongei survival of the skingraft indicatLng possibly th3 combination of some level of tolerance and theeffect of the continued administration oi Imuran.

In more recent experiments, Inuran has been combined with azaserine.Imuran blocks incorporation of preformed purines into nucleic acids and aza-serine acts earlier in purine synthesis. In the first 8 dogs studied with thisplan of chemical modification, 4 of 8 dogs have Lhd survival of the kidneyhomograft for over 60 days. There is indication that cortisone may enhancethe effect of actinomycin C in reversing rejection and this wiln be pursuedfurther. Since ac'inomycin D is more potent than actinomycin C, later experi-ments may 1nvclve the use of this cytotoxic agent.

Other studies have been directed toward identifying the features ofrejection when the canine recipient has been sensitized by antigens from thedonor. In one group of experiments conducted with Dr. Brownell Wheeler, crosscirculation was used as a means of sensitization. Nine days after a 1-hourcross-circulation, crossed kidney horo.orafts vrere performed. None functionedfor more than 1 day and these showed patterns which included interstitial edema,focal infiltration writh mononuclear cells and degeneration of tubular epithelium.In an experiment conducted with Dr. Oilason, a potential recipient of a kidneygraft received spleen cells from 6 donor dor-s. Following this a kidney homograftwas carried out and this showed a pattern of accelerated rejection. This wastaken as e-'.dence for a sharing of transplantation antigens among dogs. Instudies with Dr. B. Altman, it was clear that sensitization of the recipientthrough skin or a previous kidney homoIraft resulted in accelerated rejectionwhich had its basis in humoral components of the blood and that this sensiti-zation bore no relationship to the dog's blood group antigens.

Attempts will be made to modify rejection as it occurs under thececircumstances.

4. Transnlantation of ihe Shin in ian.Our laboratory hlis acsistcd in extensivc studies of the transplantation of

tiie slain in ma.-.. . t. fe3turcs of tio coursf of th) autograft in mani:ere ,letailcd, It ucz .lain fLo~n this, that certain alterations unlessunder_'stood, night bc regarded as evidence of rejection as seen in homografts.The homograft iiý man also wag studied, and here it was sheon that varyingnumbers of transplantation antigens are shared armong individuals. The featuresof eoch tirpe of rejection follow a fairly standard pattern and by histologicmeans it is possible to identify exactly, orimary rejection, accelerated re-jection and also "white graft" rejection. These observations are important indetermining how closely individuals may be related one to another. For example,if two indivpiduols arc considered as potential donor and recipient of a kidneyhomograft, if the s!:in of one is placed on a.i indifferent recipient, and a 3kingraft from the other is placed on the indifferent recipient two weeks after thefirst Craft, then, if 'white giaft' rejection occurs, this identifies the twoindividuals as having a close genetic relationship one to another. If thesecond graft follows a course of ;rirary rcjection, then the two individualsare genetically disparate.

5. Trarsplantation of the Liver in the DoZIn these studies, 35 dogs were used as subjects for hcmotransplantation,

27 for autotransplantation and 13 as sham transplants. The third group wasadded because there was initially poor survival after autotransplantation.So.-ie of the difficulties .,rit,- the surrical procedures have been described innrcziou~s rc%,crtz . t6chaiMuc iproved, •o did survival and it became possibleto identify features of rejection of the liver homotransplant. The hepatecto-nized dog seldom survives more tLan 2A hours. The liver homotransplant provestemporarily life-sustai, inn as a funcdiornal organ. Failure of the transplantrelates to the infiltration of cells from the recipient with a pattern resemblingthat seen in the kidney homograft. Anatomiz findings in homografts and auto-grafts are similar for the first 3 days after transplantation. Following this,monor.aclear cells appear in the liver homograft. It was not possible to iden-tify any lesions representative of a graft-versus-host reaction. These weresought because of the immunologic potential of the liver. Further studies ofthe liver homograft in tLe dog ray be undertaken after chemical modificationof the canine recipient of a renal homograft has been improved further.

6. Transplantation of the Spleen in the Don,.These studies were urdertaken Tith a view to establishing a splenic homo-

graft which, if it were tolerated, vould make this recipient a host for otherorgans from the same donor. Although it is possible to establish a state ofmutual tolerance between donor and recipient when dealing with puie mouse lines,whether this was Possible in a large manmal had to be determined. In the un-modified recipient of a splenic homograft, there appeared to be two phases,the first representing a graft-versus-host reaction of possibly 2 days duration,this being folloued by rejection of the canine splenic horiograft and charac-terized by the entrance of cells from the recipient. Rejection -usually pro-gressed rapidly so that by day 7 - 9, the homograft was completely necrotic.It was not possible to identify a specific vascular component in the rejectionprocess. h'hen the recipient was modified by nitrogen mustard, there was pro-lonjed survival of the spleen archit turally. However, few of the recipientssurvived long enough to determine the nature of the terminal process. A protocolfor akiii-istration of nitrogen mustard which led to prolonged suppression of thelymph nodes and marrow had been devised. Despite this, few of the recipientssurvived into the second week and only one survived into the fourth post-transplant week. However, it was possible to I denti t•y fol3licular architecturefor much longer periods in the homo~raft when the recipient had received

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nitrogen mustard. The possibility exists that there was a continuation of agraft-versus-host reaction when the recipient was given nitrogen mustardbecause it aO:)earc ' tLat there u_-s proloi.7,ed suppression .f b -1 marr'-ii and].:.'..ph nodes in tih•.u• subjects ieceiving nitrogen r n ar, e,,., Lso a 3plenic

--,c~rift. Here aain, it may be posji.:le 1.o prolon; suzvival of both graftand recipient throuh modification ii h m:ntimatabolitez . rather than withprimary cytotoxic agoots. 11hen more ,,-, ,r. ;ncc has been gained writh imlrnanand azascrinVc, I rther ex:erifrents wilh A., -anine splenic hoiograft .!ay beunderltaken.

7. Fractioiis of Tissues and Cells Active Anticenically in a Homologous SystemNcwcr methods of fractionation of mouse spleen cells have been used. Anti-

roieicity of these fractions in a homologous system has been compared with thatof w:I 'le snleen cells. With separation of constituents from sucrose suspensionsat various speedo, it has been found that antigenic activity is found in com-ponents which must be al.!ost entirely cyto)lasmic in origin. Chemical analysesof active fractions still show that major components are protein and lipid,with a minor fraction consisting of carbohydrate. Cells of other organs willbe tested for antigenicity after fractionation to determiae relative anti-genicity among organs. It has been determined already, that lymph node cellsare Uhe most antigenic when whole cells are used in the assay. Less antigenicoare spleen cells, thymus cells and bone marrow cells. Preparations of highstability and antigenicity wrill be tested for their ability to induce tolerancein the neonatal mouse and in the modified adult mouse.

8. Ir•:unologic Capacities of TissuesMe of the mouse kidney bed has continued in evaluation of graft-versus-

host reactions. In these experiments, parent tissue is the donor and the F-1hybrid is tLe recipient. Under these circumstances, there can be only agraft-versus-host reaction, because the recipient possesses all of the antigensrepresented in the parent tissues. As already reported, tie have no. evidencethat trie kidney as a hmoograft can react against the host. On the other hand,when solenic tissue is the homograft, there is reactivity in the folliclessuggesting that even as a tissue, spleen is capable of reacting against thehost. '1hen the parent as donor is sensitized by F-1 cells, then the graft-versus-host reaction is even more marked.

9. Synthetic Polyeptides as AntipensThis workhas been carried out by Dr. Gill in the laboratories of Dr* Paul

Doty at Harvard University. Some phases of the work have been done in ourcontrac-o-upported laboratory. The synthetic polypeptides serve well as modelsystems for an understanding of the chemical basis of antigenicity, and iden-tification of sucir determinants will have a bearing on problems of transplan-tation. Of. particular interest is the antibody formed to synthetic polypeptides.Antibodies to a variety of synthetic polypeptides can be studied for theircytotoxicity against immmalian cells. We have already observed that certainof the polypeptides have antigenic resemblanoes to substances which the rabbitencounters in nature. This was noted with a polypeptide with the followingamino acid composition, G56L38T6.

It is important to determine which high molecular weight polypeptideslack antigenicity and therefore may serve as blood protein substitutes, Poly-peptides tith such properties may be identified through isotope labeling anddetermination of their pattern of eliminat 'n by the experimental animal.

1.0. SumiaryIn the paragraphs above are outlined the various approaches which have been

used to seek a modification of the host in order that there may be prolonged

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functional survival of tissues and organs as hcmografts. The most encouragingapproaches thus far constitute modification of the host trith an analog of6-mercaptopuriiie and the usu of azaserine. •he Ih' 5t recent roeult& 4n manhave offeved hope of possibly achieving. a state o_ mutual tolerance hetweenthe kidney hoiuc)graft and the host. Chemical modification of the host appearsto offer more promise than suppression of the immune response through agentssuch as ionizing radiatien and nitrogen mustard. Basic information on immuneroopionso has been g'ained from these stvdies. The investigation of syntheticp'olypeptides of known ardno acid composition may provide information onantigenic determinants in wm-mralian cells and also offer information on thecharacteristics of antioodies as they are elicited in response to antigens ofknowm constitution.

11. List of PublicationsCouch, 1I.P., LcBride, R.A ., Dammin, G.J., and ',urray, J.E.: Observations on the

naturc of thc enlargcment, the regeneration of the nerves, and the function ofthe canine renal autorraft. Drit. J. Exper. Path. 42: 106, 1961.

Darein, 0.41 : Clinical Use of Radiation in Organ Transp-lantation. Joint Committeeon Atoiric ELergy, 87th Congress, p. 439, 1961.

Darmin, G.J., -heeler, H.B., hontague, A.C.U., Deily, J.3,, Jr., Greenberg, J.D.and Loore, F.D.: The splenic homograft: its course in the unmodified and modi-fied canine recipient. Ann. !I. Y. Acad. Sci., in press (1962).

Friedman, Z.A., Retan, J.*!., ilarshall, D.C., Henry, L., and Merrill, J.P.:Accele-rated skin graft rejection in humans preimmunized with leukocytes.Fed. Proc. 20: 33, 1961.

Friediman, L.A., iRetan, J.".., Zarshall, D.C., Henry, L., and Merrill, J.P.:Accele-rated skin 7raft rcjection in humans preiomunized irith homologous peripheralleuI:ocytes. J. Clin. Invest. 40, 2162, 1961.

GillpT.J.III, and Daxrini, G.J.: Studies on synthetic polypeptide antigens. IV.The metabolic fate of the antigen. Biochin. liophys. Acta 56: 314, 1962.

Henry, L., liarshall, D.C., Friedkman, E.A., and Dammin, G.J.: Histological andclinical correlations in skin homograft rejection in man. Fed.Proc.20:35,1961.

Henry, L., ilarshall, D.C., Friedman, E.A., Dammin, C.J., and iierrill, J.P.: Therejection of skin horngrafts in the normal human subject. II. Histologicalfindings. J. Chin. Invest. 41: 420, 1962.

Henry, L., iiarshall, D.C., Friedman, L.A., Coldstein, D.P., and Dammin, G.J.: Ahistological study of the human skin autograft. kn. J. Path. 39: 317, 1961.

Mann, L.T., Jr., Corso-, J.H., and Darnin, G.J.: TIomotransplant7-ntigens. Pre-paration of active cellular fractions by a nodified method; properties andattempted dose response. Nature 193: 168, 1962.

Iiarshall, D.C., Friedman, E.A., Coldsrein, D.P., Henry, L., and Merrill, J.P.:Clinical criteria for evaluating first set, accelerated, and white graft re-Jection in human skin homografts. Surgical Forum 12: 469, 1961.

Marshal!, D.C., Friedman, E.A., Goldstein, D.P., Henri, L., and Merrill, J.P.:The rejection of skin homografts in the normal human subject. Part I. Clini-cal observations. J. Clin. invest. 41: 4?1, 1962.

Montague, A.C.I., Greenberg, J.B., Daminf, G.J., and Noore, F.D.: The effect ofnitrogen mustard in altering the histocompatibility rejection sequence insplenic homotransplantation in the dog. J. Surgical Res. 2: 130, 1962.

liontague, A.C.h., Halgrimson, C., Dixon, J., Dammin, G.J., anQ Hoore, D.F.: Themaintenance of prolonged reticulo-endothelial system depression in dogs usingnitrogen mustard. J. Surgical Res. 2: 124, 1962.

Wheeler, H.B., Balankura, 0., Pendower, J.E., Greenberg, J.B., Damm.n, G.J., andMoore, F.D.: The homograft response to whole organ transplantation of thecanine spleen. J. Surgical Res. 2: 124, 1962.

ABSTRACT

AD NO. ACCESIO1 NO.1. Preparing Institution.: - -- Harvard imedical School, Bfoston 15, Mass.2. Title of lieport: Tissue Trvnsplantation: Honiograft antigens,

ionizing ri,'Lation and other factors in-fluencing hcmnograft smui~ral.

3. Principal Investigators: G. J. Djriln, J. ri. Corson, and L. T. Mann

4. 11unbcr of pageo, and date: 5 pagez, 1 June 19625. Controct Numlber: DA-49-1934-ID-20 6 16. Supportcd by: United States Army Medical Research and

Development CommandDepartment of the Army, !Washington 25, D.C.

Surxmrized in this abuitract 4s work done independently by the principal investi-gatars and also that conducted with representatives of other departments in thetissue/or-an transpLantation program (sur'ery - Drs. F. D. Mcore, J. E. Murray, andR. Vilsoi; medicine - Drs. J. P. Lerrill a.;d E. Hager; radiology - Dr. J. B. Dealy).

!;odifications have been ni2de in the method for extracting antigens from mousespleen cells. The essential constituents of this active fraction are protein andlipid, as previcusly reported. The new:er procedure for preparation of the antigenicfraction is simpler and provides a fraction ,:hich retains antigenicity longer onstorage.

In further study cf the canine splenic homograft, it was concluded that an earlygraft versus hcst reaction uas present for a period probably not exceeding 48 hours.This was followed prcmptly by a progreszive increase in cellularity of the splenichomograft. Suppression of the host's marrow and lymphoid tissues with nitrogen mus-tard prevented the lyn-hocytic and plasmocytic infiltration of the splenic homograftand prolonged its survival. Few dogs survived long enough to determine preciselyv howthe host wras modified, but it did appear that there might be a prolongqtion of thegraft versus host res,'onse induced by the nitrogen mustard. It was plain that theaim of attainin6 a state of mutual tolerance had not baen achieved, and for thisreason no further experiments of this design have been carried out.

The best survival achieved thus far for the canine renal Lomograft has folloveda program consisting of the initial administration of 6 mercaptopurine, with M157-3,22given as a daily dose, writh periodic addition of Actinomycin C. ffith this program,more than half of the do-s survived mors than 30 days, with the renal homograft asthe only renal tissue in the recipient. A similar program has been fol]Lowed in manfor the first time and has resulted in the lonc'est survival obtained thus far withchemical treatment only.

The characteristics of synthetic polypeptides as nntigers have been definedfurther. Amino acid content as well as sequence are important determinants of anti-genicity. This is borne out also by examinatirn of the inhibitory effec's onantigen-antibody combination by amino acids and dipeptides.

r --

APSTRACT

AD NO. ACCZOSS101! NOi I0.________ 1_,_1. Pruparing Initut on: Harvard i~edical School, Boston 15, Mass.2. Title of :Keport: Tissue 'r.nsplantation: Honogaft antigens,

ionizin•g radiation and othc~r factors in-fluencing hciaotýraft surviva-L.

3. Prinripal Investigators: G. j. DoarLcLn, J. r:. Corson, and L. T. Mann4. ih!ubtr of paecs, and date: 5 pages, 1 June 19625. Contract iitsiber: DA-49-193-il-20616. Supported by: United States Army Medical Research and

Development CommandDepartment of the Army, Washington 25, D.C.

Summarized -n this abstract is w:ork done independently by the principal investi-gators, and also that conducted with representatives of other departments in thetissue/orr~an transplantation prorfram (sur7ery - Drs. F. D. M.core, J. E. Murray, andR. Wilson; medicine - Drs. J. F. i.errill a.,d E. Hlager; radiology - Dr. J. B. Dealy).

liodifications have been made in the method for extracting antigens from mousespleen cells. The essential constituents of this a.ztive fracticn are protein andlipid, as previcusly reported. The no:-cr procedure for preparation of the antigenicfraction is simpler and provides a fraction uhich retains antigenicity longer onstora ge.

in further study of the canine splenic horcgraft, it was concluded that an earlygraft versus host reaction was present for a period probably not exceeding 48 hours.This was follow;ed promptly by a progressive increase in cellularity of the splenichomograft. Supprcssion of the host's marrow and lymphoid tissues with nitrogen mus-tard prevented the lyrvhocytic and plasmocytic infiltration of the splenic homograftand prolonged its survival. Few doas survived long enough to determine precisely howthe host was modified, but it did appear that there might be a prolongation of thegraft versus host res-onse induced by the nitrogen mustard. It was plain that theaim of attainin; a sta`. of nutual tolerance had not been achieved, aad for thisreason no further experiments of this design have been carried out.

The best sturvival achieved thus far for the canine renal homograft has folloemda piogram consisting of the initial administration of 6 mercaptopurine, with M157-322given as a daily dose, •iith oeriodic addition of Actinomycin C. With this program,more than half of the do-s survived rore than 30 days, with the renal homograft asthe only renal tissue in the recipient. A similar program has been followed in manfor the first time and bas resulted in the lonrest survival obtained thus far withchemical treatment only.

The characteristics of synthetic polypeptides as antigens have been definedfurther. Amino acid content as well as sequence arc importint determinants of anti-genicity. This is borne out also by examinati~n of the inhibitory effects onantigen-antibody combination by amino acids and dipeptides.

B S U u iC T

AD MO. qCCýS3Sj~i! HIU.1. Pr•i,•-ring Inoti.tutirn: Ha-rvard i,odical School, Eoston 15, Mass.2. Title of :-.eport: Tisceo Tr, nnlntation: homogcraft antigens,

ionizing ra-iation and other factors in-fluencing hcmograft survival.

3, ?=rincipfl investigators: G. j. D)xr.;'.Ln, J. v.. Corson, and L. T. Hann•. .�iub..r of r , and date,: 5 pagcs, 1 June 19625. Contr-4ct hnaiber: DA -4V-193-41 D-2O616. Sup-lrtcd by: United Statý,s Army Nedical Research and

Deyelonlnent Coma ndDepartment of the Army, k!ashington 25, D.C.

Surnarized in this abstract is work done independently by the principal investi-r•ators, and also that ccnducted ,ith reprcsentdtives of other departments in thetissue/cr[•n troqsclantaticn prczran (sur-ery - Drs. F. D. 1core, J. E. iurray, andP. U'ilso:; :rodicine - Drs. J. 7. *.crrill a.:.d Z. Eager; radioloy - Dr. J. B. Dealy).

:2odificaticns have been nade an tr.e rethod for extracting antigens from mousespleen cells. The essential constituents of this active fraction arp protein andli:'id, as previcusly 'reported. The he-:er procedure for preparation of the antigenicfraction is simr ',r &Ad provides a fraction •.iiich retains antigenicity longer onstorage.

in further study cf the canine splenic hcncgraft, it was concluded that an earlygraft versus hcst reaction vas p'c sent f-r a !eriod nrobably not exceeding 48 hours.This was fcllowed prcmptly by a progressive increase in cellularity of the splenichomograft. Supprcssionr of the host's marrow and lynphcid tissues with nitrogen mus-tard prevented the lyr-hocy-dc a."d 'las'rocytic infiltration of the splenic homograftand prolonged its survival. FAw dcgs survived long enough to determine precisely howthe host ,as .:)dified, but -t did a!)near that there might be a prolongation of thegraft versus hcst res icnse i-nducec by the nitrogen mustard. It was plain that theaim of attainin; a state of mutual tolerance had not been achieved, and for thisreason no further experiments of this desig:n have been carried out.

The best survival achieved thus for fcr the canine rtal honograft has followeda prGgran consisting of the initial administration of 6 mercaptopurine, writh 5157-322given as a daily dose, trich periodic additicn of Actinoycin C. WIith this program,more than half of the do-s surviived more than 30 days, with the renal homograft asthe only renal tissue in the recipient. A similar program has been followed in manfor the first time and has resulted i' tie lon"est survival obtained thus far withchemical treatment only.

The ,uharacteristics of synthetic poly eptides as amizenrs have been definedfurther. Anino acid content as wel as seqvence arc -mportant determinants of anti-genicity. This is borne out also by exan*nati,ýn cf the inhibitory effects onantigen-antibody combination by a:ino acids and aipeptides.

ABSi~ CT

1. Prep~ring InSTttin H'vrdiEd~l _ScI,ool7, Joston 15, iMiass.2. Title ci' :wlport: i'izsue Trz-nopl~intation: Homogiraft antigens,

ionizin.g ra:liat=,r an~d othcr factors in-fluencing homo graft sur'.'i-ral_

3. Princip,:l Inves-tigators: G. j. Danmin, J. 1_.1. Corson, and L. T. Hann4. :hunbt.r of' pagcýs, and dattý: 5 pages, 1 June 1962

5. ontract 1izubcr: DA-4'9-l934iD-2o616. Sup~nortod by: United States Army Medical Research and

Develo~':rent ComrmandDepartr~c-nt of the Army, Washington 25, D.C.

Summarized in this abstract is w-.ork done independently by the principal investi-rators, and also that conducted wi- th reprcsentatives of other departments in thetissue/'wpan trans planta tion program (sux'~cry - Drs. F. D. !.:Core, J . E. :*:urray, andR. Uilso:i; mcdicine - Drs. J. P. ILerrill a-,d E. 1'agor; radiology - Dr. J. B. Dealy).

l:odifications have been rade in the nl"etod for extractingr antigens from mousespleen cells. The essential constituents cif thiE Lctive traction are protein andlipid, as previcbsly reported. The ne7:er procedure for preparation of the antigenicfraction is simpler and provides a fraction i~hich retains antigenicity longer onstora ge.

in further study of' th-e canine splenic homograft, it was concluded that an earlygraft versus best react-ion was present for a period probably rot exceeding 48 hours.This was followied promptly by a prog;rescive increase in cellualarity of the splenichor~ograft. Stupprcssion of the 'host's mnarrot, and lymphoid tissues with nitrogen mus-tard prevented the lrm:-:hocytLic and plasmocyrtic infiltrition of the splenic homograftand prolonged its survival. Few dogs survived long enough to determine precisely howthe host was modified, but it did apnear that there night be a prolongation of thegraft versus host resoonse induced byr the nitrogen mustard. It was plain that theaim of attainin;ý a state of mutual tolerance had not been achieved, and for thisreason no further ex,)erinents of this design have been carried out.

T~he best s,.r-vi vl achievedC th1-Us far foýr t!-, caziiiie ruial honograft has folloteda program consisting of the initial administration of 6 mercaptopurine, with MY'57-322given as a daily dose, with r-eriodic addition of Actinorrycin C. With this program,more than half of the do'-s survived rore tlhan 30 days, with tbe renal homograft asthe only renal tissue in the recipient. A similar program has been followed in manfor the first time and has resultr-d -n thle lonr'est survival obtained thus far withchemical treatment only.

The characteristics of synthetic in~lyneptides as antigens have been definedfurther. Amino acid content as well as sequence arc important determinants of anti-genicity. This is borne out also by examirnati'n of the inhibitory effects onantigen-antibody combination by anino acids and dipeptides.

A1STRACT

AD A0O. ACCMSIUI! i,1O.1. Preparing Institution: Harvard ' edical School, Boston 15, Mass.2. Title of ýicport: Tizzue orcnopllantation: Hom• graft antigens,

ionizingri rniatLon and other -ýactors in-fluencing homoGraft survival.

3. Principal Investigators: G. J. Laniin, J. ,*. Corson, and L. T. Mann4. Dubc.r of paýes, and datL: 5 pages, 1 June 1962

5. Contract NumIber: DA-49-193-iD-206l6. Supported by: United States Army Medical Research and

Development CommandDepartment of the Army, Washington 25, D.C.

Surinarized in .this abstract is work done independently by the principal investi-g'tors, and also that conducted writh representatives of other departments in thetissue/orphan tranopiantatin prograr (sjr-ery - Drs. F. D. oore, J. E. Murray, andR. Wilson; medicine - Drs. J. 0. Lerrill a.d E. Hager; radiology - Dr. J. B. Dealy).

i odificatic.is have been made in the meth.od for extracting antigens from mouse*pleen cells. The essential constituents o' this active fraction arp protein and-ipid, as previcusly reported. The neý:er procedure for preparation of the antigenic

fraction is simpler and provides a fraction !i.ich retains antigenicity longer onstorage.

In further study cf the canine splenic hoiicgraft, it was concluded that an earlygraft versus host reaction ijas present for a period probably not exceeding 48 hours.This was followed promptly by a progressive increase in cellularity of the splenichomograft. Supprcssion of the host's marrowr a.:d lymphoid tissues with nitrogen mus-tard prevented the lyn.;hocyiic and plasn~ocytic infiltration of the splenic homograftand prolonged its survival. Fewr dogs survived long enough to determine precisely howthe host was modified, but it did appear that there might be a prolongation of thegraft versus host res,'onse induced by tne nitrogen mustard. It was plain that theaim of attaining a state of nutual tolerance Lad not been achieved, and for thisreason no further exleriments of this desigr have been carried out.

The best survival achieved thus far for tie ca -ine rei,al homograft has folloemda program consisting of the initial administration of 6 mercaptopurine, with 3157-322given as a daily dose, !Iith reriodic addition of Actinomycin C. Iith this program,more than half of the do-s survived rore tlhan 30 days, with the renal homograft asthe only renal tissue in the recipient. A similar program has been followed in manfor the first time and has resulted in tie lontest survi-val obtained thus far withchemical treatment only.

The characteristics of synthetic polypeptides as antigens hcve been definedfurther. Amino acid content as well as seqvence arc important determinants of anti-genicity. This is bcrne out also by exaninaticn of the inhibitory effects onantigen-antibody combination by amino acids and dipeptides.

ABSTRALCT

AD NO. ACCiESSIOI NO.1. Preparing Inrtitution: Harvard iedical School, Boston 1 Mass.2. Title of ýZeport: Tissue Trcnsplantation: Fomogi'aft antigens,

ionizing raiation and other factors in-fluencing homograft survival.

3. Principal Investigators: G. j. Damn'in, J. i'i. Corson, and L. T. Mann4•. l lnbur of pages, and date: 5 pages, 1 June 19625. Contract Numaber: DA-49-1l344D-206l6. Supported by: United States Army Medical Research and

Developm.,nt CommandDepartm•nt of the Army, lVashington 25, D.C.

Sum, Larized in this abstract is work done Lidependently by the principal investi-gators, and also that conducted with representotiwv of other departments in thetissue/organ transplantation program (sur-ery - Drs. F. D. 1oore, J. E. Murray, andR. Wilson; medicine - Drs. J. P. Lerrill a.;d E. Eager; radiology - Dr. J. B. Dealy).

S•l'odifications have been made in the method for extracting antigens from mousespleen cells. Thp essential constimnents of this active fraction arc protein andlipid, as previcusly reported. The ne-,,er procedure for preparation of the antigenicfraction is simpler and provides a fraction i:hich retains antigenicity longer onstore ge.

in further study cf the canine solenic homcgraft, it was concluded that an earlygraft versus hcst reaction •ias present for a period probably not e::ceeding 48 hours.This was followced promptly by a progressive increase in cellularity of the splenichomograft. Supprcssion of the host's marro;u and lymnphoid tissues with nitrogen mus-tard prevented the lyno-hocytic and plasrocytic infiltration of the splenic homograftand prolonged its survival. Few dogs survived long enough to dutermine precisely howthe host was modified, but it did appear that there might be a prolongation of thegraft versus host response induced by the nitrogen mustard. It was plain that theaim of attaining a state of mutual tolerance had not been achieved, and for thisreason no further ex,•eriments of this desitn have been carried out.

The best survival achieved thus far for t!e canine renal homograft has followeda program consisting of the initia. admintstration of 6 mercaptopurine, with H57-322given as a daily dose, with periodic additior. of Actinomycin C. WIith this program,more than half of the do-s survived rore tian "1 days, with the renal homograft asthe only renal tissue in the recipient. A sinmir program has been followed in manfor C• first time and has resulted in the lon,•eat survival obtained thus far withche•ia! treatment only.

'he characteristics of synthetic pnlypeptides as antigens have been definedfurther. Amino acid content as well as beqvence arc importaat determinants of anti-genicity. This is borne out also by examinaticn (f the inhibitory effects onantigen-antibody combination by amino acids and dipeptides.

ABSTRACT

AD NO. ACC.-S ,SIW NO. _______

1. Preparing Institution: Harvurd Ledical School, Boston 15, Mass.

2. Title of leport: Tissue Trznsplantation: Fomofraft antigens,ionizing rad4iation and other factors in-fluencing homograft survival.

3. Principal Investigators: G. J. Danmin, J. il. Corson, and L. T. Mann

h. 1!unb(.r of pages, and dat,: 5 pages, 1 June 19625. Contract iumber: DA-49-19341D-20616. Supported by: United States Army. Medical Research and

Development Comma ndDepartment of the Army, Washington 25, D.C.

Summarized in this abstract is work done independently by the principal investi-gators, and also that conducted with representatives of other departments in thetissue/organ transplantation program (surgery - Drs. F. D. oCore, J. E. ,urray, andR. Wilson; medicine - Dr-. J. i. Lerrill a.;d E. Hager; radiology - Dr. J. B. Dealy).

iodifications ha-ve been made in the metl.od for extracting antigens from mousespleen uells. The essential constituents of this active fraction are protein andlipid, as previcusly reported. The ne':er procedure for preparation of the antigenicfraction is sitpler and provides a fraction ilhich retains antigenicity longer onstorage.

in further study cf the canine splenic homcgraft, it was concluded that an earlygraft versus host reaction was present for a period probably not exceeding 48 hours.This was folloced promptly oy a progressive increase in cellularity of the splenichomograft. Suppression of the ',ost's marrow and lynphoid tissues with nitrogen mus-tard prevented the lyn, hocyiic and plasmocytic infiltration of the splenLc homograftand prolonged its survival. Few dogs survived long enough to determine precisely howthe host was modified, but it did appear that there might be a prolongation of thegraft versus host resionse induced by the nitrogen mustard. It was plain that theaim of attaining a state of mutual tolerance had not been achieved, and for thisreason no further exjceriments of this design have been carried out.

The best su.vivai achieved thus far for the canine renal homograft has followeda program consisting of tho initial administration of 6 mercaptopurine, with BW57-322given as a daily dose, uitch Periodic addition of Actinornycin C. With this program,more than half of the do-s survived more than 30 days, with the renal homograft asthe only renal tissue in the recipient. A similar program has been followed in manfor the first time and has resulted in the longest survival obtained thus far withchemical treatment only.

The characteristics of synthetic polypeptides as antigens beve been definedfurther. Amino acid content as well as scquence arc important determinants of anti-genicity. This is borne out also by exam.naticn of the inhibitory effects cnantigen-antibody combination by amino acids and dipeptides.