humoral and cellular factors in homograft and isograft...

Immunity against Sarcoma Cells*

EVA KLEIN AND HANS OLOF SJoGREN

(In@titu1efor Tumor Biology and Institutefor Ce21Ratearch, Karolinska In.,tiiutet Medical School, Stockholm, Sweden)

SUMMARY

Mouse sarcoma cells derived from recent methyicholanthrene-induced tumors ofknown genotypes were inoculated into isologous or homologous hosts after incubationwith serum or lymph node cells from different sources. Isoantiserum enhanced the outgrowth of the tumors in genetically incompatible, homologous systems, whereas inisologous systems it had no effect. Lymph node cells of preimmunized homologous hostswere inhibitory in both homologous and isologous systems. Homologous lymph nodecells, taken from untreated mice, showed variable effects, depending on the systemused.

Based on this experience, obtained with various experimental designs, all of whichwere concerned with some aspect of the homograft reaction, similar studies were carriedout with completely isologous systems. Primary or first or second passage sarcomaswere exposed to lymph node cells of isologous mice, pretreated with heavily irradiatedcells of the same tumor, or of the primary autologous host. Lymph node cells derivedfrom autologous, or untreated or pretreated isologous, hosts were inhibitory in a number of cases.

Homograft immunity is probably mediated bycellular as well as by humoral factor8 (for reviewsee [@81).Depending on the experimental systemand on the type of homotransplanted tissue, theformer or the latter may appear dominating. Thedetails of the reactions involved are still notclear. This is probably owing to the complexityof the phenomena observed and the large numberof variable factors influencing the results in anygiven experimental system. As an example, humoral antibodies may result either in immunityor in enhancement in the same tumor-host system,depending on their dose (7, 11).

Goner demonstrated that lymphoma cells weresensitive to humoral antibodies in â€œneutralizationâ€•experiments (4), involving the exposure of the cellsto isoantiserum before inoculation. Kidd obtainedan analogous â€œneutralizingâ€•effect in similar experiments, in which sensitized lymph node cellswere used rather than antiserum (1@).

In the present investigation an analogous neu

â€˜Thiswork was supported by research grants from theSwedish Cancer Society, Lotten Bohmans Fund, and by grantC-4747 from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service.

Received for publication October 12, 1959.

tralization system was employed to compare homograft immunity and the isologous immunitywhich can be induced against methyleholanthreneinduced sarcomas in their own isologous hosts(3, 18). In particular, the question was studiedwhether the reaction is mediated by the humoralor the cellular pathways, or both.

MATERIALS AND METHODS

The mice used were derived from the strainsA/Sn, A.SW, A.CA, A.BYâ€”four isogenic resistant(IR) lines developed by Snell (fl)â€”, DBA/@/K1,C57BL/KI, and C3H/Kl. The mouse strains aremaintained by continuous, single-line, brother tosister mating in our laboratory. The sublinesA.SW, A.CA, and A.BY have an A strain background but differ with respect to the allele theycanny at the histocompatibiity-@ (H-s) locus. Inorder to maintain the common genetic backgroundas closely identical as possible in all four lines,the brother-sister breeding scheme was interruptedafter three or five generations by a backcrossingto the A strain in all the three JR sublines. Thehomozygosity of the strains used was recentlystudied with the help of skin transplants. It wasshown that all the 24 isografts within the strains

452

Humoral and Cellular Factors in Homograft and Isograft

on May 31, 2018. © 1960 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

KI@IN A.NDSJoGRENâ€”HOmOgraft and Isograft Immunity 453

C57BL, CSH, DBA/2, and 30 of 32 within theA strain survived the entire length of the observation period (more than 150 days). On the othen hand, almost all the 18 A.SW, A.CA, andA.BY isografts were rejected after a more orless prolonged survival period, indicating thatthese strains still retain a residual hetenozygosis,which does not affect the H-2 locus, however.Tumor transplantation with a number of tumorsof recent origin failed to reveal the nonhomogeneity of these strains even when preimmunized animals were used (15). These results suggest thata large number of genes may exist that are oflittle, if any, significance for the establishmentand progressive growth of tumor grafts but whichnevertheless lead to the ultimate rejection of skingrafts.

Animals 2â€”Smonths old of both sexes wereused for the experiments. A standard diet in pelletform and drinking water were available ad libitum.

One estrogen-induced lymphoma and severalmethylcholanthrene (MC)-induced sarcomas denived from the above mentioned strains and theirF1 hybrids were used. The MC was dissolvedin 0.1 ml. trioctanoin and deposited in doses varying between 0.02 and 0.5 mg. into the right thighof 1-month-old mice. Developing tumors whichshowed no signs of ulceration were removed underaseptic conditions. No tumors were carried seriallyover extended periods. Tumors of the first transfergeneration were usually frozen in several paralleltubes and maintained in the tumor bank (14).This enabled us to use them in their earliesttransfer generations even under extended timeperiods. In studies on the isologous immunityof the sarcomas, only the primary tumors and theirfirst and second transplants were used.

Tumor cell suspensions were prepared in Ringer's solution which contained 100 I.U. penicillinand 100 ;@g. streptomycin per ml. The tissuewas forced through a 60-mesh stainless steel screenand was diluted 1 :5 on a volume basis. Eitherthis suspension was used directly or it was further diluted and filtered through two layers ofgauze, and the number of eosin-unstained cellswas counted (21). Aliquots containing knownnumbers of unstained cells were inoculated.

For transfer of lymph node cells the axillarand inguinal lymph nodes draining the side ofthe immunizing inoculations were dissected out,trimmed free of fat, and washed in Ringer's solution. The cell suspensions were prepared by thesame: method as used for tumor tissue. Usuallythe yield of eosin-unstained single cells was veryhigh, of the order of 60â€”70per cent.

Serum was obtained from the mice by punctureof the retroorbital sinus with a glass capillary.

For obtaining immunized lymph node cells orantisera, the mice were immunized by inoculationof untreated or preirradiated (15,000 r) tumortissue, as specified in the different experiments.

In the case of isograft immunity, the preirradiatedtissue was also stored in the frozen tumor bankbefore challenge. This procedure was adopted tobe able to use the primary tumor itself as animmunizing challenge repeatedly, at several times.Irradiation of the tumor tissue was performedeither in the suspension form or with small solidtissue pieces (to avoid the presence of saline incases of subsequent frozen preservation) in a small

.â€”.GroupA

S-SGroup B

&-4 Group C@ /4

. 0â€”0 Group

.___Days after inoculation

CUART 1.â€”Growth of 1.5 X 1O@MSWB sarcoma cells inisologous A X A.SW F1 hybrid hosts alone (Group A), or afterincubation with normal homologousserum (GruupB), isoantiserum (Group C), and lymph node cells derived from isoimmime A X C3H F1mice (Group D). The numbers marked withasterisks denote the number of mice killed by progressivelygrowing tumor as related to the total number inoculated.

plastic irradiation chamber submerged in an icebath. X-rays were generated at 185 kv., 15 ma.,and filtered by 1 mm. Al. The dose rate was 419r/min at a focus target distance of 33.5 cm.

Incubation of tumor cells with serum or lymphnode cells was carried out at 370 C. in a waterbath for 1 hour. Subsequently the mixtures wereinoculated subcutaneously. The inoculated micewere inspected and palpated at regular intervalsto ascertain the latency period of tumor development. Developing tumors were regularly measuredby caliper. Three different tumor diameters weremeasured, and the geometric mean was calculated.

RESULTS

HoMowcious SYSTEMS

Isotrantplantaiion of tumor cell8 after exposureto homologous serum or lymph node celk.â€”The10th transplant generation of MSWB MC-induced

EE

4)

4)

E00

C04)

20.0

6.0

12.0

8.0

4.0



454 Cancer Research Vol. 20, May 1960

sarcoma (A X A.SW origin) was brought intosuspension. It contained 1.5 X 10@eosin-unstainedcells/mi. Antiserum was collected from 2 A XC311 F@ hybrids that had received bilaterallysix challenges of MSWB cells at 7â€”10days' intervals, the last challenge having been given 6days previously. The axillar and inguinal lymphnodes of the same mice were removed and broughtinto suspension, and the cells were washed withRinger's solution. Control serum was collectedfrom untreated A X CSH F1 mice. Three aliquotsof MSWB sarcoma cell suspension were mixedwith equal volumes of normal serum, immune

by using F1 hosts as the recipients of mixed inoculacontaining cells derived from a tumor that originated in one of the parental strains and lymphnode cells from the other parental strain. Morespecifically, 1.6 X 10@MSA sarcoma cells (derivedfrom strain A, 7th transplant generation) andlymph node cells of DBA/2 oniginâ€”1.4 X 10@untreated and 0.9 X 10@ preimmunized againststrain A tissueâ€”were mixed, incubated, and inoculated subcutaneously to A X DBA/2 F1 mice.The result is illustrated in Chart 2. Both untreatedand preimmunized lymph node cells exerted aninhibiting effect which was somewhat more pronounced with the preimmunized cells.

A similar experiment was performed with lymphoma LNSA (A x A.SW origin). Incubation andmixed inoculation with strain A lymph node cells,

4/'20.0 @Gr@upA

16.0 â€˜@Group B

h-4GroupC *

I@ o..o:ro@J4/:

Days after inoculation

CHART 3.â€”GrOWth of incubated inocula containing 5,400LNSA lymphoma cells (of A X A.SW F1 hybrid origin), alone(Group A) or in mixture with 8.6 X 10' isologous lymph nodecells (Group B), 3.2 X 10' lymph node cells from untreated Amice (Group C), and 4.3 X 10. lymph node cellsfrom A micepreimmunized against A.SW tissue (Group D), respectively.The numbers marked with asterisks denote the number of micekilled by progressively growing tumor as related to the totalnumber inoculated.

either from untreated mice or from mice preimmunized against A.SW tissue, delayed the outgrowth of the tumor in isologous hosts to approximately the same extent. Incubation with isologousA X A.SW F1 lymph node cells had no detectableeffect. These results are illustrated in Chart 3.

Homotransplantation of tumor cells exposed toserum or lymph node cells i@ologous with the host.â€”

a) Preimmunization with viable inocula: Chart4 illustrates the growth and regression of theMSWB sarcoma in incompatible A X C3H F1hosts after the cells had been pretreated withserum or with lymph node cells from A X C3HF1 mice, untreated or preimmunized, respectively,against strain A.SW tissue. The same tumor,

EE

4)

4)

E0@0

C04@

EE

a,a,Ea

V

C04)


CHART 2.â€”Growth of 1.6 X 10' MSA sarcoma cells (ofstrain A origin) in A X DBA/2 F1 hybrid hosts, alone (GroupA), or after incubation with 1.4 X 10' lymph node cells ofuntreated DBA/2 mice (Group B), or 0.9 X 10' lymph nodecells of DBA/2 animals preimmunized against strain A tissue(Group C). The numbers marked with asterisks denote thenumber of mice killed by progressively growing tumor as related to the total number inoculated.

serum, and immune lymph node cells, respectively.All three mixtures and a sample of the unmixedsarcoma cells were incubated at 37Â°C. for 1 hour.Subsequently, each suspension was inoculated subcutaneously into the right flank of 4 isologous(A x A.SW F1) mice in volumes correspondingto a count of 1.5 X 10@eosin-unstained sarcomacells before incubation. The growth curves ofthe tumors are illustrated in Chant 1. Neithernormal non immune serum had any detectableeffect on tumor growth. Treatment with immunelymph node cells resulted in a prolonged latencyperiod and a reduced incidence of takes, indicating a certain inactivation of the inoculum.

In the experiment described above, the lymphnode cells had been derived from genetically different, homologous hosts and were incompatiblewith the recipients; their survival and functionafter inoculation must have been terminated rather soon by the homograft reaction (17). In anotherexperiment this homograft reaction was avoided serum, and lymph node material was used as in



KLEIN AND SJÃ¶IRENâ€”Homograft and Isograft Immunity 455

homologous host-tumor systems too. The enhancing effect of the treatment with nonimmunizedlymphocytes was seen less regularly but couldnevertheless be confirmed in two other experimentswith MSWB and LNSA tumor cells, respectively.

b) Preimmunization with irradiated inocula: A1:5 suspension of MSWB sarcoma cells was divided into two parts. One was left untreated,and the other was irradiated with 15,000 r. Eachsuspension was inoculated in 0.1-mi. volumes totwo A X DBA/2 F1 hybrids subcutaneously onboth sides. The mice were killed 8 days later, together with two untreated A X DBA/2 F1 hybridsand serum as well as axillar and inguinal lymphnodes were collected. The lymph node cells weresuspended in 0.2-mi. volume and mixed with 0.2

-Group A%'

S-4Group B@/4

hâ€”4GroupC%'

0-0 Group D%'

D-0Group E44'

Aâ€”AGroup F%'


Cnaur 5.â€”Temporary growth and regression of theMSWB sarcoma in homologous A X DBA/2 F1 hybrids afterexposure to serum or lymph node cells taken from untreatedA X DBA/2 mice (Groups A and B, respectively), or after exposure to serum orlymph node cells taken from A X DBA/Q F,hybrids preimmunized with a single challenge of living MSWBcells (Groups C and D, respectively), or after exposure to serum or lymph node cells taken from A X DBA/2 F1 hybridspreiminunized with a single challenge of heavily irradiatedMSWB cells (Groups E and F, respectively). The numbersmarked with asterisks denote the number of mice with temporarygrowing tumor as related to the total number inoculated.

ml. MSWB cell suspension containing 2 X 10@eosin-unstained cells. Another aliquot of MSWBcell suspension was mixed with 0.2 ml. serum.After incubation each suspension was divided into4 parts and inoculated into four A X DBA/2Fi mice. The resulting tumor growth curves areshown in Chart 5. It appears that lymph nodecells of preiminunized mice inhibited the temporary growth of the tumor. The results were thesame whether the lymph node cells were taken

from animals pretreated with viable (Group D) orwith heavily irradiated (Group F) tumor cells.Lymph node cells taken from untreated animalshad no certain effect (Group B). Serum from untreated or pre-immunized mice was ineffective(Groups A, C, and E).

the experiment described in the previous paragraph and illustrated in Chant 1, but the hosttumor relationship was different (homologous instead of isologous). Also, in order to permit atemporary outgrowth of the homografted tumortissue during the observation period, a large doseof tumor cells was inoculated after incubatingan unfiltered tumor suspension (1 :5) rather thana fine suspension of counted cells. Volumes of0.4 ml. tumor suspension were incubated withequal volumes of serum or lymph node cells.After incubation each suspension was inoculatedsubcutaneously into four A X C3H F1 mice. Unmixed MSWB inocula gave rise to a temporaryoutgrowth of tumors which all regressed within 14days (Group A). Treatment with normal A XCSH F1 serum did not influence tumor growth(Group B). Treatment with isoantiseruin resultedin a clear enhancing effect (Group D), reminiscentof the findings of Kaliss (10, 11). The enhanced

Caawr 4.â€”Homotranspiantationof the MSWB sarcoma(of A X A.SW F1 origin) to A X @8HF1 hybrids, alone (GroupA) or after exposure to serum isologous with the recipient host,derived from untreated (Group B) or preimmunized (againstA.SW tissue) A X CSH F1 hybrids (Group D), or after exposureto lymph node cells derived from the same two sources(Groups C and E, respectively). The numbers marked withasterisks denote the number of mice killed by progressivelygrowing tumor as related to the total number inoculated.

tumors grew progressively and finally killed allmice. Treatment with normal A X C3H F1 lymphnode cells resulted also in a certain enhancement(Group C), but this was less marked, three out offour tumors regressing after a temporary outgrowth. Incubation with the immune lymph nodecells completely inactivated the inoculum (GroupE). The enhancing effect of antiserum and normallymph node cells was specific for this tumor-hostsystem. When the same mixtures were testedin A.SW X C3H F1 mice, no enhancement wasobtained. Analogous enhancing effects with specific isoantisenu.m could be obtained in other

EE

a)

a,E0

V

C04?

2.0

8.0

4.0-

8.0-

4.0

0

-.Group A hâ€”@GroupC

o-oGroup DIâ€”SGroup BEE

4)

a,E0

V

C0a,

12 16

4 8 12 16 20 24 28 32Days after inoculation




TOTALLY ISOL000TJS SYSTEMS

The experiments described in the previous chap.-ters were essentially designed to study variousconstituents of the homograft reaction. The effectof isoantiserum and isoimmunized lymph nodecells taken from genetically foreign (homologous)animals was assessed on tumors growing in homologous or isologous hosts. These experimentsdo not give any information with regard to possibleimmunological reactions against tumor-specificcomponents in the tumor's own autologous orisologous hosts. It appeared to be of interestto extend our studies to such systems too, by exposing tumor cells to serum on lymph node cellsfrom genetically identical, isologous animals, andinoculating the mixtures into fully isologous hosts.

Since heavily irradiated cells can be used tobuild up immunity against recently induced methylcholanthrene-induced sarcomas in their ownisologous host strain (20), a series of experimentswas carried out to determine whether lymph nodecells of such animals would show any inhibitingeffect upon incubation with the sarcoma cellsin vitro. These experiments were all carried outwith tumor cells derived from the primary methylcholanthrene-induced sarcomas or from their firsttwo transplant generations.

The experimental design and the main resultsobtained are shown in Table 1. The data shownin this table show the situation 13 days afterinoculation. Considering, in addition, the finalend result of each experiment, including detailedgrowth curves, the results can be evaluated asfollows:

1. Serum from the primary tumor-bearing animal, tested with two tumors, had no detectableeffect.

2. Lymph node cells from the primary tumorbearing animal, tested with four tumors, showedan inhibiting effect with one tumor.

3. Lymph node cells from untreated isologousmice showed an inhibitory effect with four tumorsin six out of ten experiments.

4. Lymph node cells from isologous mice pretreated with irradiated tumor cells (2-5 times)showed inhibition with two of five tumors tested.

5. Intravenous or intraperitoneal injection oflymph node cells from the primary tumor-bearingmouse or isologous mice preimmunized with irradiated tumor cells had no detectable effect onthe tumor growth.

The following experiments may serve to illustrate the findings in detail:

One A.SW mouse which received 0.02 mg. MCdissolved in 0.1 ml. trioctanoin on January 20,1958, carried a tumor on November 6th. The

mouse was killed, the tumor removed (designation:MWF), and the regional lymph nodes and serumwere collected. Part of the tumor was used forthe first experiment (see below), while anotherpart was irradiated with 15,000 r. One part ofthe irradiated tissue was used for the first immunizing challenge; another part was distributedinto small pyrex tubes and frozen at â€”79Â°C.according to our usual procedure of frozen preservation (14). A third untreated sample was transplanted into two A.SW mice.

In the first experiment aliquots of 0.4 ml. ofa cell suspension, prepared from the original pnimary tumor by diluting it with Ringer's solution1 :5, was mixed with 0.4 ml. serum from an untreated A.SW X C3H F1 mouse, 0.4 ml. serumfrom the primary tumor-bearing mouse, 0.2 ml.lymph node suspension from an untreated A.SWx C3H F1 mouse, on 0.2 ml. lymph node suspension prepared from the regional inguinal and axillarlymph nodes from the primary tumor-bearingmouse. After 1 hour's incubation 0.1 ml. of theunmixed tumor cell suspension and correspondingamounts of each mixture were inoculated subcutaneously into groups of four A.SW X C3HF1 mice, and tumor development was followedby caliper measurements. The results (Chart 6)show an inhibition with isologous (Group C) orautologous (Group B) lymph node cells, morepronounced in the latter group. Serum had noeffect (Groups D and E).

The second experiment was performed withcells derived from the first transfer of the sametumor. Incubation was carried out in the usualway. The control group received 0.05 ml. tumorsuspension subcutaneously and consisted of fourA.SW X C3H F1 mice (Group A in Chart 7).The following mixtures were used: tumor cellsuspension and lymph node cells from an untreated A.SW mouse (Group C), tumor cell suspension and lymph node cells from the tumor-bearingmouse (first transplant) (Group B), tumor cellsuspension and lymph node cells from isologousmice pretreated four times with irradiated tumorcell suspension (Group D). The growth curvesof Chart 7 indicate a somewhat prolonged latencyperiod in tumor outgrowth in all the groups inwhich the tumor cells were incubated with lymphnode cells.

The experiment performed with cells derivedfrom the second transfer of the same tumor isillustrated in Chart 8. The control group (GroupA), consisting of five A.SW X DBA/2 F1 mice,received 3 X 10@eosin-unstained tumor cells. Thesame amount of tumor cells was mixed with 10@lymph node cells from untreated (Group B) and



.@ ES@aa

.8a

@0,

. 4.@ES54)

4?.4

E@*

@0-.@ =4) 4â€”â€˜@ a,

a) a) @a,

=4 ::@0 4), .ot.-@)@râ€¢a,@54)

.@E@â€˜

@@ â€¢0@@,

@)@ a, 4)0 0

@0 @0@ a,@4).0

[email protected]

E@s@

a,,) 4)@ â€¢)a,4)4)0 0.

4â€” 4.+a,.o a,.a a,.a a,a,a,4)@:5

@

@ .:@@

* @.

â€”4)

* 4â€”* 4-

4))

54)0 0

40

.4a a,..

8

a8

8@0

S..@

-1Iâ€¢0

.5

I

I

cc

I

0a

@ 2V 5S 0

@ =8 8

@ V

S

.@ .@@

a

.8 .@ â€¢8

-i@

0S

V

S

a

.80

aS

.8

.8

.88

8a

Ia

a, 0

.0 1.

â€Õ

.0x

@:@

x@ a, V a.V

.@@

S8

S.0

S .@

.5

:@@

V

.@ @.

@ .@â€ã.@ @j

@ . @,.c

@x@

9@4) 4. S 4-=@ 4?-

a

V

@.0a@ aa..0 8

if!Ip.@@

@ -

.@ I@@

4.. V V@ I

â€¢8Ss @V

@ .5 .@.@ ga@ .a

:@ @X8

aVV O@â€ã@@ . . .0. 4)

@ S@@ .@

I@@ I:!I I I .1

.@@@ I@

axx.@0a,a,z .i,@@

*@

.8

a,

a,

4)

.4)

a,

4)4) _4)@ â€”0

â€œa,@

a,aa a,@o *4)

;@@

4)04) 4)

4?@a,â€¢@ 4),

4)@a,4) 4)

0

â€˜0

VS. â€˜0

.Ee.@

II!V

Q.ES@S

x@: xâ€• xÂ°'4.@ 4.@

@@@ U,

Q c) Q

â€˜0 â€˜0 â€˜0 4)4a) 0 0

@ Â© a

4-â€ÕQ

Cl).@

0

â€˜0

0

Q4-â€˜0@: Ã§@

cc Qâ€˜0 â€˜0 â€˜0

V V4?@

.@@

0 0 0@ z z

a4

a

I.

*

4.

a4

4)@

44.0a

a44.

0



12 16 20 24


with 3 x 1o@cells from isologous pretreated (4times with irradiated sarcoma cells) A.SW animals(Group C). The resulting growth curves indicateinhibition with both untreated and preimmunizedisologous lymph node cells.

Chart 9 illustrates another experiment witha tumor induced in a C57BL mouse (tumor designation MC57C). One part of the original sarcomawas transplanted to isologous recipients, anotherpant was irradiated with 15,000 r and used as animmunizing challenge in both isologous C57BL

:@2::: :: :

I ___________Days after inoculation

CHART 6.â€”Growth of primary methylcholanthrene-inducedsarcoma (MWF) upon transplantation into isologous hosts,alone (Group A) or subsequent to in vitro exposure to lymphnode cells (Groups B and C) or serum (Groups D and E) fromthe primary tumor-bearing host and from untreated isologousmice, respectively. The numbers marked with asterisks denotethe number of mice killed by progressively growing tumor asrelated to the total number inoculated.

EE

a,

a,E0V

C0a,

Caa.wr 7.â€”Growth of MWF sarcoma of the first transfergeneration upon further transplantation into isologous hosts,alone (Group A) or subsequent to in vitro exposure to lymphnode cells from the tumor-bearing host (Group B), untreatedisologous mice (Group C), or animals which had received fourinoculations of heavily irradiated cells from the primary tumor(Group D). The numbers marked with asterisks denote thenumber of mice killed by progressively growing tumor as rejated to the total number inoculated.

and homologous A.CA mice. Small pieces of theirradiated tumor were also preserved in the frozentumor bank for 7 days, after which they wereused for a second immunizing challenge. Tumortissue from the first transfer was used for the actualexperiment which included the groups shown inthe legend of Chant 9. The results indicate thatI.P. injection of lymph node cells from untreated(Group B) or preimmunized hosts (Group C)had no effect on tumor growth. On the otherhand, in vitro exposure to lymph node cells exhibited various degrees of inactivation. The inhibiting effect of these cells increased in the following order: cells from homologous preimmunizedmice (Group H), isologous untreated mice (GroupE), homologous untreated mice (Group G), andisologous preimmunized mice (Group F).

EE

4)a,E0

V

C0a,

.â€”. Group A

-UGroupB


CHAur8.â€”Growthof S X 1O@cellsfrom the second transplantation generation of the MWF sarcoma upon transplantstion into isologous hosts, alone (Group A) or subsequent to invitro exposure to 1O@lymph node cells from untreated isologousmice (Group B) or S X lO@cells from animals which had received four inoculations of heavily irradiated cells from theprimary tumor (Group C). The numbers marked with asterisksdenote the number of mice killed by progressively growing tumor as related to the total number inoculated.

4/4*4/*

4/*DISCUSSION

Mitchison has shown that a heightened resistance to a homologous tumor graft can be passivelytransferred by cells from the lymphoid tissue ofimmunized mice. Isoantiserum was ineffective(16). The continued function of the transplantedlymphoid cells in the recipient host after transferwas demonstrated by Bilhingham, Brent, andMedawar (2), and they have coined the termâ€œadoptiveimmunityâ€• for this phenomenon. Kidd(12) has found that the growth of a lymphomawas inhibited after in vitro incubation with mincedlymph nodes derived from preimmunizeci animalsof a foreign strain. Neither serum from the sameimmune mouse nor lymph nodes of untreatedanimals showed any inhibiting effect. On the otherhand, in Gorer's experimentslymphoma cells couldbe neutralized by isoantiserum in vitro, and pas

24'

12Days after inoculation



.4â€”I.

Â°@?

@8

U00VU

â€”V

S S4?@

@ .0 .0

@ .@@

@ ._ 5

.5 5Gaor@HOSTLTupE

NODECSLL*Da,@ved

fromAmountROUtc@@o:@1?:1ini8.A*3AXC57BL

FiB3

AXC57BLF,Untreated C57BL107Intraperitoneally

tothehostC3

AXC57BLF,â€œPreinununisedâ€•C57BL1.9X10'Intraperitoneally

tothehostDt6

CS7BLE6

C57BLUntreatedC57BL8X10'Intermixed

and Incabated with the tumor cells prior to in

oculationF6

C57BLâ€œPreimmunizedâ€•C57BL8X10'Intermixed

and Incabated with the tumor cells prior to in

oculationU6

C57BLUntreated A.CA7X10Intmnixed and incubated with the tuourcellspriortoin

oculationH6

C57BLPreimmuninedA.CA7X10'Intmnixed

and incabated with the tunor aellspriortoInoculation

KLEIN AND SJÃ¶GEi@â€”Homograft and Isograft Immunity 459

sive transfer of homograft immunity with isoantiserum was also possible in his system (4, 6).It seems that isoantiserum can neutralize leukemias and lymphomas with relative ease, whereasit is considerably more difficult to obtain a positiveeffect with such tissues as skin or transplantedcarcinomas and sarcomas. The in vitro cytotoxictest of Gorer and O'Gorman (8), which involvesthe exposure of cell suspensions to isoantiserumand complement, has recently been applied byHellstrÃ¶m (9) to a series of different tumors, andhis results show a high and regular susceptibilityin five lymphomas and a considerable resistancein five transplanted sarcomas.

EE

a,0

E0V

Caa,

I

aa0z

a0aa

aa0

z

V4)

4))

Vâ€¢0

0S

.55.E

4-.

-â€˜-C4))0

.5â€¢0

4-'.0.5

.4-

cl@zSE0

.55.

S

.5

I

I

a

.8Cl)

4 8 12 6 20 24 28 32Days after inoculation

I

a0

.8

4?

4) .0

P4.4

ES

* Unincubated control. f IncubatedcontroL @S.0

CLI:Catwr 9.â€”Growthof MC&7C,a MC-inducedsarcoma, in

isologous hosts. Diluted suspension (1: 5) inoculated subcutaneously.

054-)

@?

05

4-'05

.4.40

0

a0

.â€”0

.0

4)C?

4)

-g

I.

a0

.0

S0

.0

.5S

I

a.8

aa0z

.0

I 8C? .0

4)C?

Ia.0a.a

a.8

aa0z

.0

.0 0

a..ES00

SV

V0 5

.4-) 4J

aU

Sa0

aS0z




In our experiments sarcoma tissue was exposeddirectly to antiserum or to lymph node cells.The results are schematically summarized in Table2. In the homologous system, preimmunized lymphnode cells always exerted an inhibitory effecton the tumor cells, and in some cases lymphnode cells of previously untreated animals foreignto the tumor showed a similar effect too. Theprobable immunological nature of this reactionis suggested by the fact that only immunogenetically competent lymph node cells did react; nonimmunized lymph node cells from one parentalstrain did inhibit an F1 lymphoma, but no effectwas observed with isologous F1 lymph node cells.

The preimmunized lymph node cells were always inhibitory, whether compatible with therecipient host or not. This is in accordance withthe results of Mitchison (17) ; immunologicallyactivated spleen cells (antigen: Salmonella typhi[H]) transferred to homologous hosts producedantibodies for about 5 days before they succumbedto the homograft reaction.

Whenever the recipient host was isologous withthe tumor cells, the only effects observed were theregular inhibition by the preimmunized lymphnode cells and the occasional inhibition by untreated, immunogenetically competent lymphnode cells. Isoantiserum or serum from untreatedanimals had no detectable effect. The situationwas different in those experiments in which thetumor cells were inoculated into genetically incompatible hosts. In these cases untreated tumorinocula showed a temporary growth, followed byregression. Pretreatment with hyperimmune serum resulted in enhancement; normal homologousserum had no effect. In the view of Kaliss, thephenomenon of immunological enhancement isdue to the presence of specific humoral antibodiesin the host : in his experiments these were elicitedeither by an active immunization with homogenized or lyophilized tissue from the strain of miceto which the test tumor was indigenous, or bypassive transfer of hetero- or isoantisera. According to Kaliss, the survival of the tumor despitethe hostile responses of the host was due to somealteration of the tumor cells induced by theircontact with the antiserum (10, 11).

While exposure to preimmunized lymph nodecells had a regular inhibitory effect even in thehomologous system, lymph node cells from untreated animals isologous with the recipient (andtherefore homologous to the homotranspiantedtumor) sometimes exerted a weak enhancement.This might be attributed to variable quantitiesof isoantibodies produced by the lymph nodecells; for some tumors the dose of antibody is

extremely critical. Small quantities of antibodiesmay produce enhancement and large quantitiesinhibition (7, 11).

The experience obtained in the experimentshitherto discussed is entirely limited to variousaspects of the homograft reaction. Recently, ithas been discovered that methylcholanthreneinduced sarcomas can provoke a state of immunityin isologous animals (3, 18). No similar immunization could be brought about with spontaneousmammary carcinomas. The critical experimentsof Prehn and Main have largely excluded thepossibility that the effect could be due to residualheterozygosis in the inbred strains used or, inother words, to just another case of homograftimmunity. They strongly suggest that the sarcomas contain specific and individually differentnew antigens. RÃ©vÃ©sz(20) has confirmed the findings of Prehn and Main in experiments involvingtransplantation to isologous hosts. Mammary carcinomas and estrogen-induced and spontaneouslymphomas give negative results, thus furtherpointing towards the peculiarity of the phenomenon for methylcholanthrene-induced sarcomas. G.Klein and 0. SjÃ¶gren' have recently shownthat animals with primary methylcholanthrene-inducedtumors could be made resistent against their ownautologous neoplastic cells, thus finally excludingthe possibility of an artifact due to a possibleimmunogenetic heterogeneity of the mouse strainsused.

Another line of work suggestive of the occurrence of tumor-specific antigens (X-factor) hasbeen carried out on various transplanted lymphomas by Gorer and Amos (5) and Amos andDay (1). They could demonstrate that a passivetransfer of immunity was possible by hyperisoimmune serum in compatible mice. It was necessaryto use the lymphoma to the immunizing inoculations ; isoantiserum prepared against liver or othernormal tissues could not protect. They could notbuild up an active immunity in an isologoushost, however, and neither could RÃ©vÃ©sz,as mentioned above, by using irradiated lymphoma cells(20) although the same procedure was efficientwith the sarcomas.

Nothing is known about the way in whichthe autologous immunity against methylcholanthrene-induced sarcomas is being mediated. Itis not necessarily of a nature similar to the homograft reaction. The test procedure employed withthe homograft systems was therefore applied tofully isologous systems of this type. Heavily irradiated cells were used to build up immunity,a procedure found efficient in previous studies

â€˜G.Klein and 0. Sjdgren, to be published.



KLEIN AND SJÃ¶GRENâ€”JJomograft and Isograft Immunity 461

on different systems (13, 19). It turned out thatthe serum of the primary autologous host had nodetectable effect. On the other hand, tumor cellsexposed to lymph node cells were often delayedin their outgrowth after inoculation. Inhibitoryeffect could be observed with lymph node cellsderived from the primary autologous hosts in whichthe tumor had originally been induced and whichwas otherwise untreated, and with lymph nodesfrom preimmunized and untreated isologous hostsas well.

Intravenously or intraperitoneally injectedlymph node cells from isologous, preimmunizedhosts had no detectable effect on tumor growth.It cannot be decided whether this was due to thecomparative weakness of the isologous immunityor to an unsatisfactory dosage of lymph nodecells. This point is the subject of further study.

In analogy with the homograft system, theinhibitory effect of lymph node cells is most probably due to the immunological activity of thesecells against the tumor, although other possibleinterpretations cannot be entirely excluded. Inthis system, the tumor cells are being assayedin recipients which are themselves capable ofdeveloping a certain response against the sarcoma,and it would be conceivable that a high numberof isologous lymph node cells in the inoculumcould influence this response in some indirect way.

REFERENCES

1. AMos, D. B., and DAY, E. D. Passive Immunity againstFour Mouse Leukoses by Means of Isoimmune Sera.Ann. N.Y. Acad. Sc., 64:851-58, 1957.

2. BILLINGTIAM,R. E.; Bumer, L.; and MEDAWAR,P. B.Quantitative Studies on Tissue Transplantation Immunity. II. The Origin, Strength and Duration of Activelyand Adoptively Acquired Immunity. Proc. Roy. Soc. London, s.B, 143:58â€”80,1954.

3. For.ny, E. J. Antigenic Properties of Methylcholanthreneinduced Tumors in Mice of the Strain of Origin. CancerResearch, 13:835â€”37,1953.

4. Gounu, P. A. Some Recent Work on Tumor Immunity.Adv. CancerResearch,4:149-86,1956.

5. GORER,P. A. andAMos,D. B. PassiveImmunity in Miceagainst the CS7BL Leukosis E.L.4 by Means of Isoimmune Serum. Cancer Research, 16:338â€”43, 1956.

6. Gonna, P. A., and BOYSE, E. A. Some Reactions Observedwith Reticulo-endothelial Cells in Mice. Biological Problema of Grafting. Universitet de Liege, pp. 192-206, 1959.

7. Gonna, P. A., and K@&uss, N. The Effect of Isoantibodiesin Vivo on Three Different Transplantable Neoplasms inMice. Cancer Research, 19:824â€”SO,1959.

8. Gonna, P. A., and O'Goius.&N, P. The Cytotoxic Activityof Isoantibodies in Mice. Transpi. Bull., 3: 142â€”43,1956.

9. HELLSTRtSM,K. E. Cytotoxic Effect of Isoantibodies onMouse Tumor Cells in Vitro. Transpl. Bull., 6:411â€”16,1959.

10. KALISS, N. The Survival of Homografts in Mice Protreated with Antisera to Mouse Tissue. Ann. New YorkAced. Sc., 64:977â€”90,1957.

11. . Immunological Enhancement of Tumor Homografts in Mice. A Review. Cancer Research, 18:992-1003,1958.

12. KIDD, J. G. Experimental Necrobiosisâ€”a Venture inCytopathology. Proc. Inst. Med. Chicago, 18:50-60, 1950.

13. Kinni, E. Isoantigenicity of X-Ray Inactivated Implantsof a Homotranspiantable and a Non-homotransplatableMouse Sarcoma. Transpl. Bull., 6:420â€”24,1959.

14. Kiniw, G., Rs@vi@sz,L.; and Ki.anr, E. Experiences with aFrozen Tumor Bank. Transpi. Bull., 4:31â€”33, 1957.

15. LuiDan, 0., and Ki.mr, E. Skin and Tumor Grafting inCoisogenic Resistant Lines of Mice and Their Hybrids.J. Nat. Cancer Inst. (in press).

16. MITCHISON,N. A. Passive Transfer of TransplantationImmunity. Proc. Roy. Soc. London, s.B, 142:72â€”87, 1954.

17. . Adoptive Transfer of Immune Reactions by Cells.J. Cell. & Comp. Physiol., 50 (Suppi. 1):247-64, 1957.

18. PREEN, R. T., and Mtmr, J. M. Immunity to Methylcholanthrene-induced Sarcomas. J. Nat. Cancer Inst., 18:769â€”78,1957.

19. Rihr@sz, L. Effect of X-irradiation on the Growth of theEhrlich Ascites Tumor. I. Nat. Cancer Inst., 15:1691-1701,1955.

20. . Detection of Antigenic Differences in IsologousHost-Tumor Systems by Pretreatment with HeavilyIrradiatedTumor Cells. CancerResearch, 20:448-51, 1960.

21. Scmtnx, R. A Method of Counting the Viable Cells inNormal and in Malignant Cell Suspensions. Am. J. Cancer,28:389â€”92,1936.

22. SNss@, G. D. Isogenic Resistant (IR) Lines of Mice.Transpl. Bull., 2:6-8, 1955.

23. . The Homograft Reaction. Ann. Rev. Microbiol.11:439â€”58, 1957.



1960;20:452-461. Cancer Res Eva Klein and Hans Olof Sjögren Immunity against Sarcoma CellsHumoral and Cellular Factors in Homograft and Isograft

Updated version

http://cancerres.aacrjournals.org/content/20/4/452

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/20/4/452To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



humoral and cellular factors in homograft and isograft...

Documents