[CANCER RESEARCH 35, 409-414, February 1975]

SUMMARY

Attempted nonspecific immunotherapy led to acceleration rather than retardation of tumor growth. Mice giveninjections of Freund's adjuvant were more susceptible totransplanted syngeneic Gross virus-induced leukemic cellswhen Freund's complete adjuvant was administered i.p. 0 to7 days before or I day after tumor; thereafter, the adjuvanthad no effect.

Two serum-mediated phenomena were demonstrated invitro: (a) sera from mice immunized with Freund's completeadjuvant and tumor facilitated killing of tumor cells byperitoneal exudate cells from nonimmune mice; (b) serafrom all mice with progressivetumor blocked the cytotoxicity of a xenogeneictumor-specific serum.

Certain sera produced both effects. However, sera thateither blocked or facilitated tumor killing in vitro had noeffect on the growth in vivo of transplanted tumor cells.

INTRODUCTION

Nonspecific stimulation of the RES3 with agents such asBCG, endotoxin, and zymosan increases antibody formation, phagocytic activity of the RES, and bacteriocidalactivity of macrophages (2, 3, 7, 10). FCA increasesbothdelayed hypersensitivity and antibody formation and causesmarked hyperplasia of the RES. FCA, BCG, and bacteriallipopolysaccharides have been reported to protect animalsto some extent against subsequent challenge with varioustumors, including L1210 leukemia, methylcholanthreneinduced tumors, and Sarcoma 180 of mice, and L2C guineapig leukemia (4, 11, 14, 16). BCG has been reported toprolong the duration of remission in children with acutelymphocytic leukemia (12).

1 Supported in part by Cancer Research Funds of the University of

California; The California Institute for Cancer Research; AmericanCancer Society, California Division Special Grant 568; National AmericanCancer Society Grant E-45 I B; National Cancer Institute Grant CA 08930;and the Mark Richardson Memorial Fund. Results included here werepresented in part at the I5th Annual Meeting of the American Society ofHematology. Hollywood, Fla., December 1972.

2 Address reprint requests here.3 The abbreviations used are: RES, reticuloendothelial system; BCG,

Bacillus Calmette-Guérin; FCA, Freund's complete adjuvant; LC, leukemic cell; HBSS, Hanks' balanced salt solution; FIA, Freund's incomplete adjuvant; PE, peritoneal exudate; [3H]TdR, tritiated thymidine; C',guinea pig complement; RLS, rabbit antiserum specific for leukemic cells.

Received April 13, 1973: accepted November 5, 1974.

The studies reported here and other studies (I, 13, 21, 22)have shown that FCA treatment may promote, rather thanprotect against, tumor growth. Two serum “factors―thatmight alter host reactivity to tumor were found in micegiven injections of FCA and LC's.

MATERIALS AND METHODS

Animals. The mice used in this study were C3H/eB/FeJ(hereafter called C3H) adult mice, 2 to 5 months of age,produced by 5 years of single-line and brother-sister breeding in our laboratory. The original mice were provided byDr. E. Hayes.

Cell Cultures. C3H Gross virus-induced LC's were used:their characteristics and culture have been described previously (9). Cells were washed 3 times with HBSS and thenumber of viable cells was determined by counting in thepresence of 1.0% eosin, prior to i.p. inoculation.

Animal Inoculation. FCA or FIA (Difco Laboratories,Detroit, Mich.), 0.2 ml, or control 0.9% NaCI solution wasinjected i.p. or s.c. at various intervals before or afterinoculation of l0@ to l0@ viable LC's. In I series ofexperiments LC's were incubated with various murine seraprior to inoculation. Mice were observed3 times weekly todocument the onset oftumor and time ofdeath. Some micewere given 4 injections of Gross virus prepared from LCculture supernatants as described previously (6), and theirsera were tested in the growth inhibition assay. Sera frommice inoculated with irradiated LC's were used in some ofthe experiments. The cells had been irradiated with 6 MeVelectrons using a Varian 6 MV linear accelerator (totaldose, 3,500 to 10,000 rads) and then were administeredeither as I or 4 weekly injections of 1 x 106 LC's.

Growth Inhibition Test. The test has been described (6).Briefly, PE cells were incubated in vitro with target LC's,and subsequently incorporation of [3H]TdR by the targetcells was measured. Cultures of 2 ml were prepared,containing approximately 4 x 106 PE cells and 2 x l0@LC's; 2 @zCi[3H]TdR (specific activity, 5.0 Ci/mM; Amersham/Searle, High Wycombe, England) were added after54 hr. The cultures were harvested 18 hr later, andincorporation of [3H]TdR into acid-insoluble material wasassessed. As shown previously in our laboratory, [3HJTdRincorporation at this time is due to LC's rather than PE cells(6).

To evaluate the effect ofsera in this system, 2 x l0@ LC's

FEBRUARY 1975 409

The Role of Serum Factors in the Acceleration by Freund's CompleteAdjuvant of the Growth of Transplanted Murine Leukemic Cells'

Patricia E. Byfield, Jerry Z. Finklestein, Karen 1. Tithe,Connie Hsi, and David T. Imagawa

Department of Pediatrics,2 UCLA School of Medicine, Harbor General Hpspital Campus. Torrance, California 90509 [P. E. B., J. Z. F., K. L. T.,C. H.. D. T. I.], and Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California 90024 [P. E. B.. D. T. I.]

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P. E. Byfie!d et a!.

in 0.1 ml HBSS and 0.2 ml of sterile murine serum weremixed and incubated for I hr at 37° in a 5% CO2atmosphere, and then 4 x 106 PE cells in 0.2 ml HBSS and1.5 ml of medium were added. [3H]TdR was added 2 dayslater and tritium incorporation was assayed after 18 hr. asabove.

PE cells were separated into adherent and nonadherentfractions by incubation in plastic tissue culture tubes at 37°for 30 mm. The supernatant was then decanted into FalconPetri dishes and the precedure was repeated twice. Cellsadherent to the sides of the 1st tube were termed “adherentcells―and those cells present in the final suspension werecalled “nonadherent cells―(15).

Microcytotoxicity Assay. Sera were tested for bothcytotoxicity and blocking properties using an adaptation ofthe Terasaki microcytotoxicity technique (19, 20). Under 1

@lof mineral oil the following were pipetted as appropriate:2 M1murine serum or rabbit anti-LC serum, inactivated at56°for 1.5 hr: 2 M1 LC's (106 viable cells/mi); and 1@undiluted C' (Grand Island Biological Co., Grand Island,N. Y.), which had been absorbed with agarose (Behringwerke AG, Marburg-Lahn, Germany). One volume of0.25% eosin was then added and unstained viable cells wereenumerated. The cytotoxicity index was defined as [Livingcells (control) —living cells (experimental)J/living cells(control) and an index of 0. 15 or more was consideredsignificant ( I8). Several sera were tested with rabbit complement as well as C': the cytotoxic titer of the sera was thesame with each type of complement.

Blocking Assay. New Zealand white rabbits were givens.c. injections of 106 LC's in FCA and were boosted 13 timesi.p. and i.v. with 106to l0@LC's. The rabbit serum was thenabsorbed to remove species-specific antibodies in vitro withwashed mouse spleen cells ( 1 spleen per ml serum, 6 times)or, after an initial absorption in vitro, sera were absorbed invivo by injection of I ml antiserum into the peritoneal cayity of a normal mouse and harvesting 6 hr later. The absorbed sera were leukemia specific; in the standard cytotoxicity assay they killed LC's to a titer of 1:32 to 1:128but had no effect on C3H thymocytes at 1:4.

The ability of murine sera to block killing of LC's byRLS was tested by preincubating the murine serum withleukemia-specific RLS, I @czlof each, for 30 mm at 37°.The blocking index (8) was defined as follows:

[% R (NMS) - % R (ES)]/% R (NMS)

where % R (NMS) is percentage of reduction of viabilitywith RLS, NMS, and C', and % R (ES) is percentage ofreduction of viability with RLS, experimental serum, andC'.

An index of 0.40 was considered positive. Murine seraknown to block the Ieukemia-specific, absorbed RLS didnot block the cytotoxic action of unabsorbed rabbit antiserum to LC's, although this serum was diluted to give thesame cytotoxic strength as the absorbed serum. Thisindicates that the blocking effect was not due to anticomplementary effects of the murine serum.

RESU LTS

Effect of Freund's Adjuvant on Tumor Growth. FCA i.p.caused an earlier onset and increased incidence of fataldisease after inoculation of syngeneic leukemic cells. Thiswas true for a 1000-fold range of LC doses (Chart I ). Thetime of FCA administration relative to LC inoculation wascritical. FCA accelerated the course of the disease wheninoculated I to 7 days before, with, or I day after LC's. Themost pronounced acceleration was produced when FCApreceded LC's by 3 to 4 days. The effect was not observed inanimals given FCA 2 to 5 days after injection of viableLC's, nor was it observed when FCA was administered 3 or7 weeks prior to tumor inoculation. Increased tumorformation was found in I experiment when FCA wasadministered 10 weeks prior to challenge.

Growth Inhibition. PE cells from immunized mice did not,as a rule, significantly inhibit [3H]TdR incorporation intoLC's (6). However, when LC's were preincubated with serafrom mice previously inoculated with LC's plus FCA, PEcells almost abolished [3H]TdR uptake (Table I). Thiseffect was observed with sera from animals that hadpreviously received either viable or X-ray-treated LC'salone or with FCA or FIA. Sera from nonleukemic mice,including those that had received inoculations ofGross virusor of FCA alone, did not cause PE cells to inhibit [3H]TdRincorporation by LC's.

The immune serum enhanced the PE cell-mediatedinhibition of LC growth when added to cultures undiluted(Tables 2 and 3) but had no effect at I: 10 or I: 100 dilution.When LC's were incubated with the serum, the serum wasremoved, and the cells were washed once before addition ofPE cells, the killing was nearly as great as when serum wasadded directly to the cultures. After the 3rd incubation of 2x l0@LC's with 0.5 ml serum, the activity ofthe serum wasnot depleted. A representative experiment of this type isshown in Table 2.

When the growth inhibition experiment was performedusing PE cells from DBA/2 mice and the syngeneicleukemic cell line Ll210, the immune serum had no effect(data not shown). Cells adherent to plastic (primarilymacrophages) were as effective as unfractionated PE cells ininhibiting growth of LC's in the presence of “immune―serum, while nonadherent cells (primarily lymphocytes) hadno cytotoxic effect under similar conditions (Table 3).

Microcytotoxicity Sera from the following mice weretested for complement-dependent cytotoxicity: normalmice; pregnant mice: mice given inoculations ofGross virus:and mice given inoculations of fragmented, X-irradiated, orliving LC's with or without adjuvant, including both tumorbearing mice and those that survived challenge with tumor.Only 9 of 56 different sera tested proved to be cytotoxic; 2 ofthe 9 sera were from normal mice. In no case did thecytotoxicity index of these sera exceeed 0.50 at a 1:4dilution.

Antiserum from a rabbit immunized against mouse LC'swas absorbed to remove antibodies against antigens ofnormal mice. Various batches ofthe absorbed serum (RLS)had titers of 1:32 to 1:128 (final dilution) tested against

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Treatment ofserum donorsNo.

ofexperiments[3H]TdR

uptake (cpm)Significance

(p)ExperimentalControlFCA

+LC's103,10032,790<0.001FCA

alone223,79028,430NSa

Growth of Murine Leukemia Accelerated by Adjuvant

I.cnz<Ui

00

U)U)Uia:>a.;:x<Ui @j1:3

<:3WO0<

zUiLIa:Uia.W>

-I:3

0

U)0WU)C')Uia:a.

Ui

II-

Ui0

Chart I. Time ofdeath in animals that have been given inoculations of l0@(A), lO' (B). l0 (C), and l0@(D) viable LC's and with FCA on the sameday or I to 7 days prior to the administration of LC's. Ten to 30 mice constitute each experimental group. Points, days when a further increase in animaldeath was noted.

DAYSAFTER INOCULATION OF LEUKEMIC CELLS

DAYSAFTER INOCULATION OF LEUKEMIC CELLS DAYSAFTER INOCULATION OF LEUKEMIC CELLS

Table I

Growth inhibition testIncorporation of [3H]TdR by LC's on Day 3 of culture in the presence

of PE cells from normal mice and serum from mice given injections of FCA+ 106 LC's or of FCA alone (control). The PE cells inhibit DNA synthesis

by LC's only in concert with serum from mice immunized with LC's.Significance was evaluated by Student's t test.

mice had progressive tumor (Table 4). Sera from mice thathad not received FCA but had progressive tumor were alsofound to block the cytotoxicity.

Blocking strength was estimated by adding murine serumto various concentrations of cytotoxic RLS and was expressed as the number of “cytotoxicunits―blocked (a unitbeing defined as the amount of antibody in the last well toshow cytotoxicity when no murine serum was added). Therewas no detectable increase in the quantity ofblocking factoras a result of FCA injection. The results of I experiment areshown in Table 5. Untreated mice (Group A) showed no lessblocking factor than did FCA-treated mice bearing eitheradvanced (Group B) or undetectable (Group C) tumors.Tumors grew more rapidly in FCA-treated mice.

Test of in Vivo Activity. Several experiments were done todetermine the relevance, if any, of the 2 in vitro phenomenato events in the whole mouse. For these we chose a serumpool that was active in both systems; it inhibited the actionof RLS and complement and it caused normal PE cells tobecome cytotoxic. LC's were incubated with 0.5 ml of serumfor 1 hr at 37°and both cells and serum were inoculatedi.p. Neither a significant protective nor enhancing effect

a NS, not significantly different from control.

LC's, but they were not cytotoxic to thymus cells. Murinesera were added to RLS to test for a blocking or “neutralizing―effect. Since a few pools of normal sera had someblocking activity, only individual sera were tested. Theblocking sera were not anticomplementary (see “Materialsand Methods―).Sera from mice given injections of FCA +106 LC's blocked the cytotoxic effect of RLS only if the

FEBRUARY 1975 411

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Immune serumNormaiserumcpm%

inhibitioncpm%inhibitionPE

cells incubated withLC's after serum incubationI

316,714 13,09284 8014,690 34,1578562LC's

+ unabsorbed serum6.463901 2, I2886Cultures

without PE cellsLC's + unabsorbedserum103,83699,620LC's

+ 3 times-absorbedserum66,83289,773

Serum donorsNo. ofsera testedMean blockingindexProgressive

tumor682.3Tumorfree612.6

GroupPE cellsMean

incorpora

tion (cpm)Significancecompared to

Group4Murine

serumaddedtoLCcultures1ImmuneUnfraction

ated676+2ImmuneAdherent786+3Immune

ControlNonadherent96,569—4NoneNone37,571—5ImmuneNone68.488—6NormalUnfraction

ated50.843—

GroupDetectabletumorFCA injectionDays

frombleedingto deathCytotoxic

(unitsblocked)AYesNo252BYesYes41CNoYes161

P. E. Byfield et a!.

Table 2A bsorption of the cytotoxicity-enhancing factor to and by target cells

A representative experiment in which LC's were incubated for 30 mm at 37°in normal orimmune murine serum. The serum was removed by centrifugation and mixed with fresh LC's, whilethe serum-treated LC's were used as target cells. The absorption was repeated 3 times. LC's fromthe 1st and 3rd incubations were used as targets. The 3 times-absorbed serum was added to freshLC's. LC's without serum incorporated 91,637 cpm; PE cells alone incorporated < 500 cpm.

Table 3

Growth inhibition by PE cellsfrom normal mice in concert with immuneserum

LC's were grown alone or in the presence of PE cells and serum frommice that were either normal or immunized with FCA + 10' LC's.Adherent PE cells were separated by incubation in glass tubes for 30 mmand gentle washingaway of remainingcells. The nonadherent cells wereincubated twice in plastic dishes and residual adherent cells were discarded.Incorporation of [3H]TdR was assessed between 54 and 72 hr. Serum alonehad no effect on incorporation by LC's. and PE cells alone incorporated<500 cpm. Statistical significance was assessed by Student's t test; p <0.05 was considered significant.

Table 4

Ability of murine sera to block killing of LC's by a leukemia-specificxenogeneic serum

Sera from mice given injections of FCA + LC's were preincubated for30 mm at 37°with RLS, and then target LC's and C' wereadded.Viabilitywas determined after I hr at 37°.Sera from mice with progressive tumorinhibited the cytotoxic effect of RLS, while sera of mice withoutdemonstrable disease did not. The groups were significantly differ byStudent's t test (p < 0.005).

Table 5

Semiquantitative assessment ofblockingfactor in mice given injections oftumor with or without pretreatment with FCA and tumor cells

Mice, previously immunized with 5 weekly injections of 10' irradiatedLC's, were given i.p. injections of l0@irradiated lymphoma cells I day afteri.p. injection of 0.2 ml FCA. They were bled 29 days after injection of thecells and the sera from groups with and without obvious tumor werepooled. Sera were tested as indicated in the text. lfquantitative differencesin blocking factor were related to increased growth of tumor. Group Band/or Group C should have shown greater concentrations of blockingfactor than did Group A, but they did not.

could be demonstrated in comparison with normal serum.These experiments used l0@or 106 LC's i.p., and the serawere used undiluted and at dilutions of 1:10 and 1:100.

In certain experiments, mice were given injections 3 dayspreviously of glycogen, which was used to induce the PEcells used in vitro, and LC's were injected with normal orimmune serum, more closely to simulate the in vitroexperiments. This procedure did not lead to protection ofmice by immune serum.

DISCUSSION

It is apparent that, under defined experimental conditions, pretreatment with FCA may either protect against

growth of transplanted tumors (4, 11, 14, 16) or facilitatetumor formation (I, 13, 21, 22). The mechanisms of theseactions are unknown.

We have demonstrated in vitro 2 serum factors oractivities that might be expected to inhibit and facilitatetumor growth, respectively. One factor caused in vitrokilling of tumor cells by PE cells from nonimmune mice.The sera have no inhibitory effect alone, but in combination

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Growth of Murine Leukemia Accelerated by Adjuvant

with PE cells they can cause nearly complete inhibition ofLC growth. The effect is unlike that shown by Perlmann andHolm (17) in 2 ways: the serum is effective only at highconcentrations, and the cells that mediate the effect areadherent to plastic (presumably macrophages) rather thannonadherent (lymphocytes?).

The factor in immune serum that enhances PE cellinhibition of LC growth resembles an opsonin specific forthe tumor cells. It is heat stable and can be absorbed to thesurface of LC's.

The PE cells show a spectrum of activity: occasionalpreparations are cytotoxic without serum addition, someare inactive even in the presence of immune serum, but mostpreparations inhibit LC growth only in the presence ofserum from mice immunized with FCA + LC's. Appar

ently, this factor augments the natural ability of adherentPE cells to kill or damage syngeneic tumor cells, possibly byvirtue of opsonic activity of specific antibody or antigenantibody complexes.

An alternative mechanism might be by a nonspecific“macrophage-activatingeffect―(5). This seemsunlikely tobe the only factor for 2 reasons: (a) the active principlecould be absorbed to the surface of LC's and was notremoved by l washing (Table 2): and (b) the immune serumcould not be shown to cause DBA/2 PE cells to inhibit thegrowth of (syngeneic) L I2 10 cells. The effect may be similarto the phenomenon described by Evans and Alexander (5),who showed that immune spleen cells can “arm―macrophages (presumably by cytophilic antibody) and that thesemacrophages, if activated specifically or nonspecifically(e.g., by glycogen in our experiments), can kill allogeneictumor cells. At present, we regard the mechanism of action

of the “PEcell cytostasis-enhancing factor― as unproven.

In order to search for a serum factor that might facilitatetumor growth, we used a leukemia-specific xenogeneiccytotoxic serum (neither lymphocyte-mediated tumor celldestruction nor cytotoxic antibody had been demonstratedin mice bearing this syngeneic tumor; see Ref. 6 and“Results―). A rabbit antiserum against C3H murine leu

kemic cells, absorbed extensively to remove all activityagainst C3H thymocytes, was used in a complementdependent cytotoxic test system. Tumor antigen or antigenantibody complexes would be expected to neutralize leukemia-specific cytotoxic antibodies, while enhancing antibody might block their action competitively or by sterichindrance.

Sera from 9 of 12 animals with progressive tumor,regardless of immunization with irradiated tumor cellsand/or adjuvant, inhibited tumor cell killing by antileukemic serum. The activity was not demonstrably increased in mice immunized with killed LC's in FCA,although the mice were more susceptible to tumor inductionby LC's. The blocking activity appeared to correlate withthe fate of the mouse, but it may well be a result of thetumor (e.g., antigen shed by the tumor or antigen-antibodycomplexes) rather than a contributory factor to tumorgrowth. We are reluctant to ascribe an important causal roleto it because the sera did not show enhancing activity invivo, although our test conditions, 1 injection of 0.2 mlserum, may be too stringent to demonstrate an effect.

We suspected that FCA given with leukemic cells mightpromote formation of enhancing antibodies, but the evidence presented here does not support that hypothesis.Block ing factor(s) appeared whenever progressive tumorwas present, regardless of immunization. In contrast, thefactor that facilitated killing of tumor cells by PE cellsappeared only in mice given FCA plus LC's. Since micegiven FCA plus living LC's almost invariably died ofprogressive tumor, these mice had both serum factors.

Experiments designed to demonstrate an in vivo effect ofthe 2 serum factors showed no significant effect; neitherretardation nor acceleration of tumor growth was observedin mice given injections of these sera. It appears that FCAalters the host response by either a physiological effect, e.g.,on the activity of RES, or by an effect on cell-mediatedimmunity. It may be that the 2 activities, blocking andarming, coexist in the serum of tumor-bearing mice andtend to offset each other. On the other hand, both may beirrelevant in vivo.

Since the detrimental effect of FCA on the course of atransplantable tumor occurs only when FCA is injectedbefore or with tumor cells, it may not be relevant to thetreatment of autochthonous tumors. However, the recurrence of demonstrable disease in animal and human systemsafter an induced remission might well mimic this experimental situation. If so, the experiments reported heresuggest that immunotherapeutic stimulation of the RESmust be approached with caution.

ACKNOWLEDGMENTS

We thank E. H. Smith for editorial assistance and Dr. John Byfield forperforming the irradiation of leukemic cells.

REFERENCES

I. Alford,T. C. Hollinshead,A., and Heubner, R. J. ParadoxicalEffectof Freund's Complete Adjuvant upon Transplantation Efficiency ofAdenovirus-induced Tumor Cells. J. Gen. Virol., 5: 541-543, 1969.

2. Benacerraf, B., and Sebestyen, M. M. Effect of Bacterial Endotoxinson the Reticuloendothelial System. Federation Proc., 16: 860-67,1957.

3. Biozzi, G.. Stiffel, C., Halpern, B. N., and Mouton, D. Recherches surIa Méchanisme de l'Immunité Non-Spécifique Produite par lesMycobactéries. Rev. Franc. Etudes Clin. Biol., 5: 876-90, 1960.

4. Ellman, L.. and Green, I. The Role ofComplete Freund's Adjuvant inthe Immunoprotection of L2C Guinea Pig Leukemia. Proc. Soc. Exptl.Biol. Med., 138: 566-570, 1971.

5. Evans, R., and Alexander, P. Mechanism of Immunologically SpecificKilling ofTumour Cells by Macrophages. Nature, 236: 168-170, 1972.

6. Finklestein,J. Z., Byfield,J., Tittle, K., and Imagawa,D.T. StudiesofInhibition of Tumor Cell DNA Synthesis by Immune Cells in GrossVirus Induced Leukemia. Rev. Europ@ene Etudes Clin. Biol., 17:287-292,1972.

7. Halpern. B. N., Biozzi. G., Stiffel, C., and Mouton. D., Correlationentre I' ActivitC Phagocytaire du Systètime RCculoendothelial et laProduction d'Anticorps Antibactériens. C. R. Soc. Biol., 152: 758-61, l958.

8. Hellström, I., Hellström, K. E., Evans, C. A., Heppner, G. E., Pierce,G. E., and Yang, P. P. 5. Serum-mediated Protection of NeoplasticCells from Inhibition by Lymphocytes Immune to Their Tumorspecific Antigens. Proc. Nail Acad. Sci. U. S., 62: 362-368, 1969.

FEBRUARY 1975 413

on July 21, 2021. © 1975 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

P. E. Byfle!d ci a!.

9. Imagawa. D. T., Issa, H.. and Nakai, M. Cultivation of GrossVirus-Induced Murine Thymic Lymphoma Cells in Vitro. CancerRes., 28: 2017-2026, 1968.

10. Jenkin, C., and Benacerraf, B. In Vitro Studies on the Interactionbetween Mouse Peritoneal Macrophages and Strains of Salmonellaand Escherichiacoli. J. Exptl. Med., 112:403-417, 1960.

l I. Mathe, G. ImmunotherapieActivede Ia LeucemieL12l0 AppliquCeaprès Ia Greffe Tumoral. Rev. Franc. Etudes Clin. Biol., 13. 88 I - 883,1968.

12. Mathe. G.. Amid, J. L., Schwarzenberg, L.. Schneider. M.. Cattan,A.. Schlumberger, J. R., Hayat, M., and Dc Vassal, T. ActiveImmunotherapy for Acute Lymphoblastic Leukemia. Lancet. 1:697-699,1969.

13. Meltzer. M. S., and Leonard, E. J. Enhanced Tumor Growth inAnimals Pretreated with Complete Freund's Adjuvant. J. NatI.Cancer Inst.,50:209-218,1973.

14. Mizuno, D., Yoshioka,0., Akamatu, M., and Kataoka, T. AntitumorEffect of Intracutaneous Injection of Bacterial Lipopolysaccharide.Cancer Res.. 28: 1531 1537, 1968.

15. Mosier, D. E. A Requirement for Two Cell Types for AntibodyFormation in Vitro. Science, 158: 1573-1575, 1967.

16. Old, L. J.. Benacerraf, B., Clarke, D. A.. Carswell. E. A.. andStockert, E. The Role of the Reticuloendothelial System in the HostReaction to Neoplasia. Cancer Res.. 21: 1281 1301. 1961.

17. Perlmann, P., and HoIm, G. Cytotoxic Effects of Lymphoid Cells InVitro. Advan. Immunol., ll.@ 117 185, 1969.

18. Slettenmark, B., and Klein, E. Cytotoxic and Neutralization Testswith Serum and Lymph Node Cells of Isologous Mice with InducedResistance against Gross Lymphomas. Cancer Res., 22: 947 954,1962.

19. Terasaki, P. 1.,and McClelland,J. D. MicrodropletAssayof HumanSerum Cytotoxins. Nature. 204: 998- 1000, 1964.

20. Terasaki, P. I.. Vredovoe, D. L., and Mickey, M. R. Serotyping forHomotransplantation: X. Survival of 196 Grafted Kidneys Subsequentto Typing. Transplantation. 5. 1057@ 1070, 1967.

21. Wong. J. K. Y.. Hart, M. L.. and Carnes, W. H. Enhancement ofTransplanted Rad LV Induced Lymphomas in C57BL Mice byFreund's Adjuvant. Proc. Am. Assoc. Cancer Res., 13: 42. 1972.

22. Zola, H. Modulation of the Immune Response to TransplantationAntigens. I. Effects of Immunization using Adjuvants on the ImmuneResponse of Mice to a Tumor Allograft. Clin. Exptl. Immunol.. 11.

585 593,1972.

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1975;35:409-414. Cancer Res   Patricia E. Byfield, Jerry Z. Finklestein, Karen L. Tittle, et al.   Leukemic CellsComplete Adjuvant of the Growth of Transplanted Murine The Role of Serum Factors in the Acceleration by Freund's

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