[CANCER RESEARCH 43,4670-4675, October 1983]

Mechanisms of the Potentiation of Specific Antitumor Immunity byIntratumor Injection of Corynebacterium parvum^

Hiroshi Miyata,2 Kunisuke Himeno, and Kikuo Nomoto

Department of Immunology, Institute of Cancer Research, School of Medicine, Kyushu University, Fukuoka, Japan

ABSTRACT

Meth A fibrosarcoma-bearing BALB/c mice given ¡ntratumoral

injections of 0.5 mg of Corynebacterium parvum showed a highlyand tumor-specific transplantation antigen specifically potenti

ated concomitant immunity to a subsequent tumor challenge.This potentiated antitumor immunity could be locally transferredin the Winn assay to normal recipients with whole draining lymphnode cells from the tumor-bearing mice, but the potentiated

effect disappeared when adherent cells were removed fromthese cells. Moreover, the potentiated cytostatic effect on tumorcells was detected in the peritoneal macrophages but not in thenonadherent draining lymph node cells in in vitro tests. On theother hand, nonadherent draining lymph node cells from thetumor-bearing mice, when mixed with C. parvum-induced mac

rophages, exhibited a specifically potentiated antitumor effect.In addition, this effect was completely abolished by treatment ofthe draining lymph node cells with anti-Thy-1 and complement.

Thus, the potentiated antitumor effect following intratumor injection of C. parvum may be ascribed to the collaboration ofspecifically sensitized T-lymphocytes with C. para/m-activated

macrophages.

INTRODUCTION

Injection of Corynebacterium parvum i.t.3 has been found to

inhibit tumor growth in experimental animals (10-12). This effect

may be explained by several mechanisms, one of which is anonimmune direct cytotoxicity by nonspecifically C. parvum-

activated macrophages (9,14). Woodruff ef al. (21,22), however,reported that the antitumor effect of local injection of C. parvumwas T-cell-dependent, since such a treatment of mice markedly

inhibited tumor growth in intact mice but did not exert antitumoreffect in T-cell-deprived or athymic nude mice. Furthermore, it

was shown that s.c. injection of C. parvum mixed with irradiatedtumor cells caused a strong and specific cell-mediated antitumor

immunity (1), and that this immunity could be passively transferred systemically to normal recipient mice with lymphoid cellsfrom immune donors (16,19). In spite of these obvious findings,mechanisms of an antitumor effect caused by i.t. injection of C.parvum, especially at the cellular level, have been poorly understood. A highly and specifically potentiated antitumor immunitycaused by i.t. injection of C. parvum in tumor-bearing mice

appears to be attributable to the collaboration of specifically

1Supported in part by Grant-in-Aid from the Ministry of Education, Science, and

Culture, Japan.2To whom requests for reprints should be addressed, at Department of Immu

nology, Institute of Cancer Research, School of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka City, Japan, 812.

3The abbreviations used are: i.t., intratumor; PEC, peritoneal exúdate cells;[12Sl]ldUrd, 5-[1Z5l]iodo-2'-deoxyuridine; MMC, mitomycin C; TSTA, tumor-specific

transplantation antigens.Received April 19,1982; accepted July 14, 1983.

sensitized T-lymphocytes with C. parvum-activated macro

phages as suggested by the results in the present study.

MATERIALS AND METHODS

Animals. Male mice of an inbred BALB/c strain were purchased fromShizuoka Union for Experimental Animals, Japan. They were used in ourexperiments when 8 weeks of age.

Tumors. Two different lines of the 3-methylcholanthrene-induced fi

brosarcoma of BALB/c origin, Meth A and Meth 1, were used for theseexperiments. These tumors were antigenically distinct (18). They weremaintained in an ascites form by weekly passage. Unless otherwisestated, mice were usually inoculated s.c. with 107 Meth A tumor cells in

the right flank, and the day of inoculation was referred to as Day 0.Tumor growth in vivo was measured as 2 diameters at the right angle.

C. parvum. A suspension of heat-killed and formalin-treated C.parvum

(IM 1585) containing organisms (2 mg/ml dry weight) was obtained fromInstitute Merieux, Lyon, France, or Research Laboratories, Kissei, Mat-

sumoto City, Japan. Mice were usually given i.t. injections of 0.25 ml(0.5 mg) of this suspension on Day 3. Tumor growth was not affected atall by injecting the same volume of 0.9% NaCI solution i.t.

Concomitant Immunity. Mice inoculated s.c. in the back with 10'Meth A cells on Day 0 were subsequently rechallenged s.c. with 107

Meth A and Meth 1 cells each in the right and the left flank, respectively,on Day 10. The growth of each rechallenge tumor was measured 5, 7,and 10 days after rechallenge.

Nonadherent Lymph Node or Peritoneal Cells. Nonadherent lymphnode or peritoneal cells were recovered from whole lymph node orperitoneal cells by removing glass-adherent cells after incubation indishes at 37°for 30 min or 2 hr, respectively. The procedure of adherence

was repeated 2 or 3 times. The ratio of lymphocytes in the recoveredcells from whole lymph node cells or peritoneal cells was more thanabout 99 or 96%, respectively, as determined by morphological andfunctional criteria (8).

In Vivo Neutralization Test. According to the method of the Winnassay (20), 2 x 106 Meth A cells were fixed with 8 x 106 whole or

nonadherent draining lymph node cells and inoculated s.c. into normalrecipient mice. Then, tumor growth was measured. In another experiment, in order to observe collaborative effects by sensitized lymphocytesand macrophages in vivo, 2 x 106 Meth A cells were mixed with 2 x 106nonadherent immune lymph node cells and 2 x 106 C. pa/vu/n-induced

PECs. After s.c. inoculation of this mixture into normal recipients, thetumor growth was measured.

Culture Medium. Roswell Park Memorial Institute 1640 medium,supplemented with 10% heat-inactivated fetal calf serum, 50 mM A/-2-hydroxyethylpiperazine-A/'-2-ethanesulfonic acid buffer, NaHCO3 (2 mg/

ml), penicillin (50 units/ml), and streptomycin (50 /ig/ml), was used.Cytostatic Assay. Nonadherent lymph node or peritoneal cells (5 x

105) or whole peritoneal cells (2 x 105) were seeded into flat-bottomed

microplates (N-1480; Nunclon). In the case of whole peritoneal cells, cells

were subsequently incubated for 6 hr on these plates, after whichnonadherent cells were removed by 3 washes with medium. To ascertainthe actual number of adherent cells that remained after washing, culturemedium containing 30 mM lidocaine was added into several wells ofadherent cells prepared in the same way and incubated for 1 hr at roomtemperature. After incubation, cells were recovered by vigorous pipetting

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Antitumor Effect of C. parvum

and were morphologically enumerated. Then, varying numbers of tumorcells were added into each well. These mixtures in 0.2 ml of mediumwere incubated for 24 hr and pulsed with 0.4 /iCi of [125l]ldUrd for the

last 6 hr of incubation. After incubation, the tumor cells in each well wereharvested by an automatic cell harvester (Mash II; Labo Science, Japan),and the incorporation of [125l]ldUrd into tumor cells were counted. Lymphnode cells or peritoneal cells alone scarcely incorporated [125l]ldllrd.

Immune Lymph Node Cells. Cells were obtained from the right axillarylymph node of Meth A or Meth 1 tumor-bearing mice that were inoculated

with tumor cells at the right flank 10 days earlier. The lymph node wasabout 10 times larger in size than that from normal mice.

C. pam/m-induced Peritoneal Cells. Peritoneal exúdate cells were

obtained from mice given i.p. injections of 1.0 mg of C. parvum 7 daysearlier. About 80% of the harvested PEC were macrophages as determined by morphological criteria.

MMC-treated Meth A Cells. Meth A cells (3 x io'/mi) were treated

with MMC at a concentration of 75 /¿g/mlat 37°for 45 min and washed

3 times with medium.In Vitro Generation of Cytostatic Macrophages. Normal peritoneal

cells or C. pa/vum-induced PEC included about 40 to 80% of macro

phages, respectively, as determined by morphological criteria. So, 4 x106 normal peritoneal cells or 2 x 106 C. para/m-induced PECs were

seeded into 2.0-cm plastic dishes (Falcon 3001). Macrophages wereenriched by 6-hr subsequent adherence of these cells at 37°and removal

of nonadherent cells by 3 washes with medium. Between these 2 groupsof peritoneal cells, there was no significant difference in the actual numberof adherent cells that remained after washing (1.3 to 1.5 x 10* cells/

dish), as enumerated by the same method that was demonstrated in thecolumn of cytostatic assay. Then, 5 x 105 Meth A or Meth 1 cells and 4x 106 nonadherent normal or immune lymph node cells in 2.0 ml were

added and incubated for 48 hr. Growth inhibition of tumor cells wasdetermined by counting the number of viable tumor cells at the end ofthe incubation using the trypan blue exclusion method. In this cytostaticassay, growth inhibition was not determined by the method of countingthe incorporation of [125l]ldUrd, taking into consideration the possibility

of the incorporation of the radioactivity by lymph node cells and macrophages resulting from their mutual interactions during the 48-hr incubation. In another experiment, 107 immune lymph node cells from Meth A-bearing mice and 5 x 10e MMC-treated Meth A cells in 2.0 ml were

cultured for 24 hr. Supematants obtained from these cultures werediluted twice with medium. Then, enriched macrophages prepared asmentioned above were incubated in 2.0 ml of the diluted supematantsfor 24 hr. After incubation, the supematants were removed, and 5 x 10s

Meth A or Meth 1 cells in 2.0 ml were added and cultured for another48 hr. Growth inhibition of tumor cells was determined as mentionedabove.

Treatment of Cells with Anti-Thy-1.2 Antibody. Anti-Thy-1.2 mono

clonal IgM antibodies (Clone F705) were purchased from Olac, Ltd.,Bicester Oxon, England. This antibody had a cytotoxic titer 1:500,000against mouse thymocytes. Nonadherent immune lymph node cells weretreated at 2 x 107/ml with a 1:2500 dilution of the antibody for 45 minat 10°.The cells were then washed and incubated for 30 min at 37°in

the same volume of a 1:12 dilution of rabbit complement (Low-Tox-M

Rabbit Complement; Cedartane, Ltd., Hornby, Ontario, Canada). Afterbeing washed twice in culture medium, the viable cells were adjusted.

Statistics. The significance of differences between experimental andcontrol groups was analyzed using Student's f test; p < 0.05 was taken

as significant.

RESULTS

Potentiation of Concomitant Immunity by i.t. Injection of C.parvum. In Meth A-bearing mice given i.t. injectionsof C. parvum

on Day 3, although the tumor was not regressed, the tumorgrowth was significantly inhibited as shown in Chart 1. Micewere then rechallenged with Meth A and Meth 1 cells at differentsites simultaneously on Day 10. While the growth of Meth A

500-1

100-

300•

200-

100-

10

Daysaftertumorinoculation

20

Chart 1. Effect of i.t. injection of C. parvum on the Meth A tumor growth. C.parvum (0.5 mg) was injected i.t. on Day 3.0, control mice; •,C. pa/vum-injectedmice. Points, means of 8 mice; bars, S.D.

tumor as the rechallenge was considerably inhibited in tumor-bearing mice, the tumor growth in tumor-bearing mice treated

with C. parvum was completely inhibited (Chart 2A). The growthof Meth A tumor as the rechallenge in non-tumor-bearing mice

was hardly affected by s.c. treatment with C. parvum alone 7days earlier. The growth of Meth 1 tumor was the same in allgroups of tumor-bearing and non-tumor-bearing mice, regardless

of treatment with or without C.parvum (Chart 2B).Identification of a Cell Population Responsible for the C.

pa/vum-potentiated Antitumor Immunity. Antitumor activity of

cells obtained from the lymph nodes draining the site of tumorinoculation was investigated by means of Winn's neutralization

assay. While the whole draining lymph node cells obtained fromMeth A tumor-bearing mice on Day 10 inhibited considerably thetumor growth in the assay, those obtained from Meth A tumor-bearing mice treated with C. parvum inhibited completely thetumor growth in the assay at the same effector to target cellratio (Chart ZA). As shown in Chart 3B, however, the activity ofthe whole draining cells obtained from Meth A tumor-bearing

mice treated with C. parvum to inhibit completely tumor growthin the assay became undetectable when nonadherent cells wereused as effector cells. In other words, i.t. injection of C. parvumdid not augment antitumor activity of nonadherent lymph nodecells in tumor-bearing mice.

Antitumor activity of effector cells was observed by in vitroassay of cytostasis. Nonadherent draining lymph node and non-adherent peritoneal cells from Meth A tumor-bearing mice treated

or untreated with C. parvum did not exhibit cytostatic activity toMeth A cells at effectortarget cell ratios of 100:1 to 100:4 (dataare not shown). In contrast, as shown in Table 1, adherentperitoneal cells obtained from Meth A tumor-bearing mice treated

with C. parvum exhibited a significantly potentiated cytostaticactivity against Meth A and also against Meth 1 cells compared

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H. Miyata et al.

200 n

200 1

100 •

B, Growth of Metti I

57 10Daysaftersecondarytumorinoculation

Chart 2. Enhancement of concomitant immunity to Meth A tumor cells and itsspecificity. Mice were inoculated s.c. with 107 Meth A cells in the back on Day 0,given i.t. injections of 0.5 mg of C. parvum on Day 3 and then rechallenged s.c.with IO7 Meth A and Meth 1 cells at the right and the left flank, respectively, on

Day 10. A, growth of rechallenged Meth A in mice. B, growth of rechallenged Meth1 in mice. O, normal control; •,inoculated s.c. with 0.5 mg of C. parvum alone 7days before the day of rechallenge; D, primary tumor bearers; •primary tumorbearers given i.t. injections of C. parvum. Points, means of 8 mice; bars, S.D.

with that of adherent peritoneal cells from Meth A tumor-bearing

mice. If a small amount of nonadherent cells was included inadherent cell preparations, the activity seems to be attributableto a macrophage cell population, since nonadherent peritonealcells did not exhibit the activity. Treatment with C. parvum alonein normal mice did not induce cytostatic activity of nonadherentlymph node, nonadherent peritoneal cells, or adherent peritonealcells.

Enhanced Inhibition of Tumor Growth by a Collaboration ofImmune Lymph Node Cells with C. panrum-induced Macro

phages. The following experiments were carried out to ascertainthe plausible mechanism that specific antitumor immunity potentiated by i.t. injection of C. parvum might be expressed by acollaboration of immune T-lymphocytes with nonspecifically C.parvum-activated macrophages. The Winn assay was carriedout with cell mixtures containing nonadherent lymph nodecells:PEC:Meth A cells as 1:1:1. As shown in Chart 4, tumorgrowth showed the same curve when normal lymph node cellswere mixed with normal or C. pa/vum-induced PEC. Tumor

growth was inhibited appreciably with a mixture of immune lymphnode cells from Meth A tumor bearers and normal PEC comparedwith that of normal lymph node cells and normal or C. parvum-

induced PEC. When the mixture of immune lymph node cells andC. parvum-induced PEC was used as effector cells, inhibition oftumor growth was augmented further. When immune lymph nodecells were treated with anti-Thy-1.2 and complement before theaddition to C. parvum-induced PEC, their activity of augmenting

antitumor effect was completely abolished (Chart 5).

100 -,

50 -

ier

A, Whole

100n

50 -

B, Nonadherent

57 10

DaysaftertumorInoculationChart 3. Tumor growth in mice receiving Meth A cells with whole or nonadherent

lymph node cells. Meth A cells (2 x 10*) were mixed with 8 x 10" whole or

nonadherent draining lymph node cells from donor mice; O, normal control; •,inoculated s.c. with 0.5 mg of C. parvum alone 7 days before; D, 10-day Meth Atumor bearers; •10-day Meth A tumor bearers given i.t. injections of 0.5 mg ofC. parvum on Day 3 and then inoculated s.c. into normal recipient mice. Points,means of 8 mice; bars, S.D. ", significantly different from that of mixtures containinglymph node cells from tumor bearers at p < 0.01 ; *", significantly different from

that of mixtures containing lymph node cells from tumor bearers at p < 0.001.

In vitro assay of cytostatic activity was carried out with mixtures of nonadherent lymph node cells and peritoneal macrophages (Table 2). Growth of Meth A or Meth 1 cells wasconsiderably inhibited by normal or C. parvum-induced macro

phages alone, but not at all by normal or immune lymph nodecells alone. When Meth A cells were cultured with immune lymphnode cells from Meth A bearers and C. parvum-induced macrophages, tumor growth during the 48-hr incubation was com

pletely inhibited. However, the complete inhibition of tumorgrowth was not observed when Meth 1 cells were cultured withimmune lymph node cells from Meth A bearers and C. parvum-induced macrophages. Again in this experimental system, reciprocal specificity tests were done. Immune lymph node cells from

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Antitumor Effect of C. para/m

Table 1Cytostatic activity of peritoneal macrophages from Meth A tumor-bearing mice

[125l]ldUrd uptake (cpm)

Adherent cells* in 2 x Meth A at following Effector targetcell ratio

Meth 1 at following Effectortargetcell ratio

mice"Normal

Treated with C. parvumalone"

Tumor bearers8

Tumor bearers treatedwith C. parvum'10:129,199

±28,891 ±14,514

±770 ±816e

,037758

10210:240,899

±1,53343,627 ±2,1494,687

± 6641,371 ± 26510:121,503±1

20,932 ±15,321

±856 ±,051

,132563

30110:230,982

±1,83631 ,883±1,2316,1

53 ± 7822,01 5 ± 265

"The actual number of adherent cells in 4 groups was almost alike (7.4 to 7.7 x lO'/well)."Each group consisted of 10 to 20 mice.°Mean ±S.D. of 6 identically prepared wells.dMice were given s.c. injections of 0.5 mg of C. parvum 7 days before.8Mice were inoculated s.c. with 107 Meth A cells 10 days before.'Mice were inoculated s.c. with 107 Meth A cells 10 days before and subsequently were inoculated i.t. with

0.5 mg of C. parvum 3 days after tumor inoculation.

75 I

~ 50-

25 -

75 -i

5 7 10

Daysaftertumorinoculation

Chart 4. Enhanced inhibition of tumor growth in mice receiving Meth A cellswith nonadherent immune lymph node cells and C. parvum-induced PEC. Meth Atumor cells (2x10*) were mixed with: 0,2 x 106 nonadherent normal lymph nodecells and 2 x 10* normal peritoneal cells; ». normal lymph node cells and C.parvu/77-induced PEC; D, immune lymph node cells and normal peritoneal cells; •immune lymph node cells and C. parvum-induced PEC, then inoculated s.c. intonormal recipient mice. Points, means of 8 mice; bars, S.D. *, significantly different

from that of mixtures containing immune lymph node cells and normal peritonealcells at p < 0.05. *", significantly different from that of mixtures containing immune

lymph node cells and normal peritoneal cells at p < 0.001.

Meth 1 tumor bearers augmented cytostatic activity of C. par-vom-induced macrophages when cultured with Meth 1 cells but

not when cultured with Meth A cells. Therefore, the augmentationof cytostatic activity of C. para/m-induced macrophages by

immune lymph node cells was antigen specific.As shown in Table 3, the cytostatic activity of C. parvum-

induced macrophages was also augmented by pre-incubating

them with supematants from cultures of immune lymph nodecells and MMC-treated tumor cells. However, the expression ofaugmented cytostatic activity by C. para/m-induced macrophages was not antigen-specific, since C. parvum-induced macrophages pre-incubated with supematants from cultures of MethA-immune lymph node cells and MMC-treated Meth A cells

- 50 -

25 -

Doys after tumor InoculationChart 5. Tumor growth in mice receiving 2x10* Meth A cells mixed with: 0,2

x 10" nonadherent normal lymph node cells plus 2x10* normal peritoneal cells;•,nonadherent immune lymph node cells plus C. parvum-induced PEC; U, immunecells treated with complement alone plus C. parvum-induced PEC; and •,immunecells treated with anti-Thy-1.2 and complement plus C. parvum-induced PEC.

Points, means of 8 mice; bars, S.D.

exhibited significantly augmented cytostatic activity against Meth1 cells as well as Meth A cells.

DISCUSSION

In our present study, i.t. injection of C. parvum into the primarytumor augmented TSTA-specific resistance to a subsequent

tumor challenge. Analytical studies with in vivo neutralizationassay and in vitro cytostatic assay support the mechanism thatan augmented specific resistance is attributable to the collaboration of TSTA-specific immune T-lymphocytes and C. parvum-

activated macrophages. In these experimental systems, finaleffector cells of augmented resistance were macrophages butnot TSTA-specific immune lymphocytes. This type of cooperative

effect by immune lymphocytes and macrophages was reportedalso by Evans and Alexander (3-7), who showed in the in vitro

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H. Miyata et al.

Table 2

Enhanced inhibition of tumor growth and its specificity in culture of Meth A orMetti 1 cells incubated with immune lymph node cells and C. parvum-induced

macrophages

Nonadherentlymph node cells

(4 x10")NormalImmune

(MethA)cImmune(Meth1)"NormalImmune

(MethA)Immune(Meth1)NormalImmune

(MethA)Immune(Meth 1)No.

of viable tumor cells after 48-hrincubation3 (x10s)MacrophagesNormal*C.

parvumNormalNormalNormalC.

parvumC.parvumC.

parvumMeth

A35.7±2.8"37.1

±3.135.4±2.335.2±3.021.7

±1.89.7±1.121.

5±3.518.9±1.320.8±2.510.3

±0.52.9±0.3911.5±1.2Meth

134.3

±3.036.3±2.335.0

±1.934.7±2.621

.3±2.110.2±1.822.3

±4.021.0±1.720.5±1.310.8±0.510.7

±0.84.2±1,0"

aCells (5 x 105) were cultured for 48 hr in dishes with nonadherent lymph node

cells and macrophages as indicated."Mean ±S.D. of 6 identically prepared cultures.c Nonadherent immune lymph node cells against Meth A tumor cells."Nonadherent immune lymph node cells against Meth 1 tumor cells.8Adherent cells in 4 x 10* normal peritoneal cells.'Adherent cells in 2 x 10°C. parvum-induced PEC.9Significantly different from that of normal lymph node plus C. pa/vum-macro

phages group at p < 0.001.Significantly different from that of normal lymph node plus C. pa/vum-macro-

phages group at p < 0.01.

Table3Enhanced inhibition of tumor growth by C. parvum-induced macrophages thatpre-incubated with supernatants from cultures of nonadherent Meth A-immune

lymph node cells and MMC-treated Meth A cells

No. of viable tumor cells after 48-hr incubation* (x10s)

MacrophagesNormal0

C. parvum0

Normal (preincubatedfC. parvum (preincubated)Meth

A37.3±5.2"

25.7 ±3.214.3 + 1.825.3 ±2.7

5.8 ±0.8'Methl32.5

±3.426.2 ±2.312.3 ±2.128.2 ±3.7

7.1 ±1.59

aCells (5 x 105) were cultured for 48 hr in dishes with each macrophage as

indicated."Mean ±S.D. of 6 identically prepared cultures.cAdherent cells in 4 x 106 normal peritoneal cells.aAdherent cells in 2 x 106 C. parvum-induced PEC.8Macrophages were pre-incubated for 24 hr with supernatants.

Significantly different from that of C. pa/vum-macrophages group at p < 0.001.'Significantly different from that of C. pa/vum-macrophages group at p < 0.01.

tests that immune lymphocytes interacted with tumor cells andsubsequently released factors that conferred specific recognitionproperties on macrophages to exert specific antitumor effects.In our present experimental system, however, a final expressionphase of augmented antitumor effect by macrophages was notantigen-specific (Tables 1 and 3), although a recognition phaseby immune lymphocytes was antigen-specific (Table 2), as dem

onstrated in the in vitro cytostatic assays. Therefore, immunelymphocytes in our study appear to be comparable to sensitizedlymphocytes responsible for delayed-type hypersensitivity and

may contribute to the generation of cytostatic macrophages bylymphokines as reported by Tornita ef a/. (17).

It should be emphasized here that the antitumor activity ofnonadherent lymph node cells from C. para/m-treated tumor-

bearing mice was at almost the same level as the antitumoractivity of such cells from nontreated tumor-bearing mice asshown by the Winn assay (Chart 3B). So the potentiated antitu-

mor activity by C. parvum treatment seems likely to be attributable to the functional elevation of macrophages rather than that

of TSTA-specific T-lymphocytes. On the other hand, it has been

shown that macrophages induced by sterile irritants were moreresponsive to lymphokines than were macrophages from resident cell populations of normal mice (15). Macrophages treatedwith C. parvum seem likely to be functionally activated by lymphokines to exert potentiated antitumor effect more efficientlythan are normal macrophages. Then, it may be possible tosupport the following explanation as the mechanism of antitumorimmunity augmented by i.t. injection of C. parvum. (a) Macrophages accumulating into the primary tumor focus are activatednonspecifically by C. parvum, and suppress the tumor growth tosome extent in collaboration with TSTA-specific immune T-lymphocytes. (b) Macrophages activated by C. parvum accumulate efficiently into the focus of rechallenged tumor under theinfluence of lymphokines released from TSTA-specific immuneT-lymphocytes. These macrophages exert potentiated antitumoreffect efficiently in collaboration with TSTA-specific T-lymphocytes.

In the experiments demonstrating the collaborative effect ofTSTA-specific immune lymphocytes with C. para/m-activated

macrophages (Tables 2 and 3; Charts 4 and 5), PEC from C.parvum-stimulated peritoneal cavities of normal mice were used.

One may doubt that such cells are equivalent to cells producedin response to the i.t. injection of C. parvum in the initial experiment. However, we observed in preliminary experiments that theimmune lymphocytes had not yet been induced in tumor-bearingmice at the time when C. parvum was injected i.t. (3 days aftertumor inoculation). It seems that i.t. injection of C. parvum at thistime induces the nonspecifically activated macrophages, beingfunctionally equivalent to C. pa/vum-induced macrophages fromnormal mice at the site of C. parvum injection in tumor-bearingmice. The effector functions being assayed with C. parvum-induced PEC in Table 2 and Chart 4 seem to actually contributeto the antitumor resistance present in the concomitant immune,C. parvt/m-treated mice.

Dye et al. (2) reported that a s.c. injection of syngeneic tumorcells (P815) admixed with C. parvum induced a systemic T-cell-mediated immunity to TSTA. In such a system, the onset oftumor regression was preceded by much greater generation ofT-cells capable of specifically lysing P 815 target cells in theconventional 51Cr release assay (13). In our study, cytotoxic T-cell activity to Meth A cells could not be detected by 51Crrelease

assay with nonadherent lymph node cells obtained from tumor-bearing mice treated or untreated with C. parvum (data are notshown). Reasons for these different cellular effector functionsenhanced by C. parvum treatment in tumor-bearing mice are not

clear at present.In the Winn assay, nonadherent lymph node cells of tumor-

bearing mice treated or untreated with C. parvum exhibitedantitumor effect to some extent, although these cells did notshow antitumor activity in in vitro assays for cytotoxicity andcytostasis. In the Winn assay, macrophages of the normal recipients may contribute to the expression of antitumor immunity byTSTA-specific immune lymphocytes.

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OCTOBER 1983 4675

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1983;43:4670-4675. Cancer Res   Hiroshi Miyata, Kunisuke Himeno and Kikuo Nomoto 

Corynebacterium parvumby Intratumor Injection of Mechanisms of the Potentiation of Specific Antitumor Immunity

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