[CANCER RESEARCH 48, 4163-4167, August 1, 1988]

Adoptive Immunotherapy by Pantropic Killer Cells Recovered fromOK-432-injected Tumor Sites in Mice1

Motoo Saito,2 Masaki Nanjo, Motoyuki Kataoka, Yoichiro Moriya, Yutaka Sugawara, Takeshi Yoshida, and

Nakao IshidaCentral Research Laboratories, Chugai Pharmaceutical Co., 3-41-8 Takada, Toshima-ku, Tokyo 171 [M. S., M. N., M. K., Y. M., Y. S.J; Tokyo Institute forImmunopharmacology, Takada, Toshimaku, Tokyo 171 [M. S., T. Y.]; and Tohoku University, 2-1 Seiryo-machi, Sendai 980 [N. I.]; Japan

ABSTRACT

A murine malignant ascites model with BAMC-1 tumors was established previously, which was cured completely by five consecutive i.p.injections of OK-432. We have found that peritoneal mononuclear cellsfrom these animals contained antitumor effector cells which could destroynonspecifically a variety of tumor cells in vitro. They were tentativelycalled pantropic killer cells (PKCs). The present study was essentiallydesigned to show the antitumor effectiveness of the PKCs in vivo by theuse of an adoptive immunotherapy model. The growth of BAMC-1 tumorstransplanted s.c. 5 days earlier was significantly suppressed by passivetransfer of 5 x 10e to 2 x 1117PKCs induced by injection of OK-432 intoBAMC-1 bearing donor mice, while more than 1 x IIIs immune spleencells from the same donors treated with OK-432 were required to achievethe similar effects. Furthermore, if the tumor-bearing recipients werepretreated with 180 mg/kg of cyclophosphamide l h before the adoptivetransfer, even 5 x 10* PKCs could induce complete regression of the

tumors transplanted 5 days earlier. This protocol made it possible evento achieve the complete regression of larger tumors (9-10 mm in diameter) in recipients transplanted 12 days earlier. The PKCs were, asexpected, able to cure not only BAMC-1-bearing animals but also Meth-A-bearing mice. As effector cells for adoptive immunotherapy, therefore,the PKCs induced by OK-432 seem to be as effective as, if not betterthan, lymphokine-activated killer cells expanded in vitro by culturingtumor infiltrating lymphocytes with interleukin-2. Although the study onsurface markers of PKCs did not unequivocally discriminate these fromlymphokine-activated killer cells, the present findings are consideredsignificant indicating that a potent biological response modifier such asOK-432 can induce pantropic killer cells which are extremely effectivein destroying various tumor cells in vivo. One of the advantages of OK-432 therapy over lymphokine-activated killer cell therapy, therefore, isthat the former does not require the tedious and time-consuming in vitroprocedures which are essential for the latter.

INTRODUCTION

The immunotherapy of cancer patients by various BRMs3

has been widely performed, especially in Japan. For example,OK-432, a streptococcal preparation, has been shown to beeffective for treatment of cancer patients for the last 10 years,proving itself to be one of the most potent BRMs (1-3). Theexact mechanism for antitumor effects by BRMs, however, hasnot been fully disclosed. Above all, the important question hasbeen to clarify the nature of the effector cells induced by BRMs.Thus, OK-432 has been shown to augment NK activity (4, 5),to induce cytotoxic macrophages (6) and cytotoxic polymor-phonuclear cells (7), and eventually cytotoxic T-cells (8).

Received 11/9/87; revised 3/23/88; accepted 4/29/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1The data in this article were partly presented at the Satellite Symposium of

Japanese Society for Cancer Therapy at Matsue, Japan, October 1986.2To whom requests for reprints should be addressed.3The abbreviations used are: BRM, biological response modifier; PKCs, pan-

tropic killer cells; LAK, lymphokine activated killer; NK, natural killer; PECs,peritoneal exúdate cells; PACs, plastic adherent cells; PNACs, plastic non-adherent cells; TICs, tumor infiltrating cells; CY, cyclophosphamide; NWNACs,nylon wool nonadherent cells; TIL, tumor-infiltrating lymphocyte; IL2, interleukin-2; CTL, cytotoxic T-lymphocyte.

We reported previously that more than 80% of mice bearingmalignant ascites (syngeneic BAMC-1) could be cured by i.p.administration of OK-432 (9). The results also indicated thatmononuclear cells obtained from the peritoneal cavity of thesemice could nonspecifically kill a variety of tumor cells includingNK-sensitive and NK-resistant cell lines in vitro, indicating

pantropic nature of the killer cells. Extending these findings,the present series of experiments were designed to show the invivo effectiveness of such pantropic killer cells in adoptiveimmunotherapy of solid tumor-bearing mice. The results to bereported here clearly indicate that a surprisingly small numberof the effector cells recovered from the tumor sites where OK-432 was injected, are able to regress the growth of both specific(BAMC-1) and nonspecific (Meth-A) tumors in the recipientswhen they are adoptively transferred.

MATERIALS AND METHODS

Animals. Specific pathogen-free BALB/c female mice at 6-8 weeksold were purchased from Charles River/Japan (Tokyo). BAMC-1,methylcholanthrene-induced fibrosarcoma, was established in our laboratory and maintained in ascitic form by weekly passages in BALB/cmice. The lethal dose of BAMC-1 was IO3cells when injected i.p. and10s cells when injected s.c. in mice. Meth-A fibrosarcoma was also

maintained in ascitic form by weekly passages in the same strain ofmice.

Preparation of Killer Cells. OK-432, a lyophilized streptococcal preparation (Picibanil; Chugai Pharmaceutical Co., Ltd., Tokyo), was suspended in saline solution at a concentration of l mg/ml. 0.1 ml of thesuspension was injected i.p., 2,4,6, 8, and 10 days after i.p. inoculationof 10s cells of BAMC-1 in BALB/c mice. PECs were obtained by

washing the peritoneal cavity of mice 2 days after the last injection ofOK-432 namely 12 days after the tumor inoculation. The PECs wereseparated into two populations, PACs and PNACs, based on theiradhesivity to the surface of plastic culture dishes after l h incubationat 37°C.Usually, PAC represented 75% and PNAC, 25% of the PECs.

In some experiments, the PNACs were further applied to a nylon woolcolumn to obtain NWNACs. The PNACs were found capable of killingnot only syngeneic BAMC-1 tumor cells but also various tumor targetcells, including NK-sensitive, NK-resistant, syngeneic, and allogeneicmurine tumor cells, and in addition, xenogeneic human tumor cells,when examined by 4-h "Cr-release assay (9). These pantropic killer

cells were tested for in vivoantitumor activity using an adoptive transferassay (10).

Treatment of Effector Cells with Monoclonal Antibodies. In order todeplete a specific population of the cells, the PECs were collected inminimum essential medium from tumor-bearing mice treated with OK-432 and washed once with the medium, finally suspended in serum-freeRPMI 1640 medium at a concentration of 2 x IO7 cells/ml. Onemilliliter of the cell suspension was incubated for 30 min at 37°Cwith

either anti-Thyl.2 monoclonal antibody (Olac, clone F7D5) at 1/500dilution, or anti-asialo-GMi antibody (Wako Chem. Ind., Tokyo) at1/200 dilution. Then, the mixture was added with guinea pig complement (Cedertene Labs, Ltd., Ontario, Canada) at 1/10 dilution andincubated for 45 min at 37"C. The treated cells were washed twice with

minimum essential medium and the viability was assessed by a trypanblue dye exclusion test. Cell concentrations were adjusted appropriatelyprior to the use for Winn type assay and for adoptive transfer.

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Adoptive Transfer. Recipient BALB/c mice were inoculated s.c. with1 x 10' BAMC-1 tumor cells on Day 0. On Day 5, when tumor sizesreached 4-6 mm in diameter, the mice were injected with 180 mg/kgof cyclophosphamide (endoxan, Shionogi Pharmaceutical Co., Osaka)¡.p.,and l h later injected i.v. with the peritoneal cells or their subpop-

ulations.Winn Type Neutralization Assay. The PECs or their subpopulations

(1 x IO7cells) mixed with 1 x 10'' tumor cells were injected s.c. in the

flank of syngeneic mice, as described previously (6). Tumor diameterswere measured with a caliper 3 weeks after inoculation of the cellmixtures or tumor cells alone.

RESULTS

Adoptive Immunotherapy of Transplanted Solid Tumors byTICs induced by OK-432. Adoptive transfer of the antitumoractivity was performed according to the protocol shown in Fig.1, except the first experiment shown in Fig. 2, where therecipient did not receive the pretreatment with CY. Briefly,BALB/c mice were injected i.p. with 1 x 10s BAMC-1 cells,and administered i.p. with 0.1 mg of OK-432 on 2, 4, 6, 8, and

BALB/c mouse

BAMC-1O/ Ixl0s/mouse, sc inoculation

5 days

BALB/c mouse

BAMCIIx IO5/mouse, ip inoculation

1hr prior— Cydophosphomide

I80mg/Kg,„V.¿¿P^1^ ^F^"~^I

^-^-i =~\

day2-U

4"6-8-

10-12

daysaftePeritoneal

E^M—

OK-432*—

Olmg/mouse•-

ipinjection^Mr

inoculation

xudate Celteadoptive iv transfer (PEC)

Fig. 1. A typical protocol for adoptive immunotherapy. Most of the experiments in the present study were performed according to this protocol except thefirst experiment shown in Fig. 2 where the tumor-bearing recipients did notreceive a cyclophosphamide injection. In some experiments, spleen cells insteadof PECs were used for i.v. injection.

15

- 10E

•S 5

O 5 IO 20 30Doys after inoculation

Fig. 2. Adoptive immunotherapy of BAMC-1 fibrosarcoma. Sx 10* (D) or 2x 10' (A) of peritoneal exúdatecells were transferred i.v. to the recipient mice as

shown in Fig. 1. However, cyclophosphamide was not given to the recipient priorto the cell transfer. Control mice (O) did not receive any transfer of the cells. Sixmice per group were used. Tumor size was expressed as (a x i)1/2, where a and brepresent the longest and the shortest diameters of a tumor, respectively.

10 days after the tumor injection. While all the untreated micewould die within 14 days after inoculation of this number ofBAMC-1 cells, more than 80% of the mice treated with OK-432 survived (9). The survived mice were shown to specificallyreject the BAMC-1 cells when they were reinoculated 90 daysafter the OK-432 treatment (data not shown).

In an adoptive transfer experiment, peritoneal exúdate cellswere obtained 2 days after the last injection of OK-432, andthey were transferred to the solid tumor-bearing recipient miceprepared as described in "Materials and Methods." As shownin Fig. 2, 5 x IO6and 2 x IO7 PECs could suppress in a dose-

dependent fashion the tumor growth in mice transplanted with1 x IO6 BAMC-1 cells. The successful treatment of tumors

achieved by these relatively small numbers of effector cells is incontrast to the previous finding that lymphoid cells obtainedfrom nontumor sites (e.g., spleen) of the OK-432-treated micecould not suppress the tumor growth in recipients unless morethan 1 x IO8cells were transferred (11). This seems to indicate

that the antitumor effector cell population is enriched in tumorinfiltrating cells (TICs) after the injection of OK-432.

To confirm this point further, the same number of TICs wereinjected i.v. l h after the treatment with 180 mg/kg of cyclophosphamide (CY) of the tumor-bearing mice, since it has beenshown that such a suboptimal dose of CY significantly enhancedtherapeutic effects of OK-432 (12), and also the effect of adoptive immunotherapy probably due to elimination of suppressorcells (10, 13). As shown in Fig. 3, tumors in the recipients werecompletely regressed after treatments with CY followed byadoptive transfer of the effector cells, while almost negligibledegree of the tumor growth suppression was observed in micetreated with the same amount of CY alone. Regression of thetumors was achieved in a dose-dependent fashion of the transferred TICs. Thus 5x10* cells could not only inhibit growth

of the tumors, but also achieve the complete regression in allanimals within 21 days after the tumor inoculation, while 2 x107 cells resulted in complete regression of tumors in all mice

within 10 days after the inoculation. Similar results were obtained in each of the three experiments repeated. This again

15

EE

IO

I 5

O 5 IO 20 30Days after inoculation

Fig. 3. Adoptive immunotherapy of BAMC-1 fibrosarcoma with cyclophosphamide (180 mg/kg) and PECs obtained from tumor-bearing mice injected withOK-432. The protocol is shown in Fig. 1 as follows: O, an untreated controlgroup of mice; •,animals treated with cyclophosphamide alone; D, animalspretreated with cyclophosphamide (180 mg/kg) and transferred with 5 x 10*PECs; A, mice pretreated with CY and transferred with 2 x 10' PECs; A, micepretreated with CY then transferred with 5x10' PECs.

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indicates the enrichment of effector cells in TICs due to theOK-432 treatment, since the combined treatment by CY andadoptive transfer with the non-TICs (i.e., immune spleen cells)

from the same donor could not achieve such regression of thetumor, but only partial growth inhibition (11). It should benoted here that the mice cured by the adoptive immunotherapycould also specifically reject the BAMC-1 tumor cells whenthey were reinoculated. Thus, the surviving mice cured ofBAMC-1 tumors by adoptive transfer with the TICs could rejectonly BAMC-1 tumors but not Meth-A tumors when the tumorswere transplanted 90 days after the adoptive transfer (Fig. 4).Similarly, the survivors of Meth-A tumors by the adoptiveimmunotherapy could reject only Meth-A tumor cells but notBAMC-1 cells when rechallenged with the tumor 90 days later

(data not shown).OK-432 May Increase the Number of Effector Cells in TICs.

The above results are already suggestive of the preferentialeffect of OK-432 on the antitumor effector population in theTICs. To confirm this notion further, next examined was thecapability of the PECs from nontumor-bearing mice treatedwith OK-432 to transfer the antitumor activity into the CY-pretreated, tumor-bearing mice. These donor mice were dosedwith OK-432 according to the same schedule as used for treatingtumor-bearing mice. As shown in Fig. 5, these PECs were noteffective at all, while PECs from the tumor-bearing mice treatedwith OK-432 was shown again to effectively suppress the tumorgrowth in the recipients.

Effect of TICs on Advanced Tumors. The PECs from thetumor-bearing mice treated with OK-432 was further shown tobe effective for treatment of the larger and well-grown tumors.Thus tumors with 9-10 mm in diameter on Day 12 after thes.c. inoculation of 10s BAMC-1 cells were regressed completely

within 3 weeks in all the mice pretreated with CY and followedby the transfer with 1 x IO7TICs.

Characterization of Effector Cells in TICs. As shown in Fig.6, plastic nonadherent cells could transfer adoptively the tumorsuppressive activity. The PNACs were further incubated in anylon wool column as described in "Materials and Methods."

NWNACs were still able to passively transfer the antitumoractivity into tumor-bearing recipients. When the TICs were

20 -

BAMC-1 Meth-A

(3/3)

(9/9)

15 20 25BAMC-11x106 s.c

f 5 10Meth-AIxlO6 Sx.

15 20 25Days

Fig. 4. Tumor specificity of the immunity acquired by surviving mice after theadoptive immunotherapy. The mice (A) cured of BAMC-1 tumors by adoptiveimmunotherapy with TICs were reinoculated s.c. with I x 1U"cells of BAMC-1or with 1x10* cells of Meth-A, 90 days after the first inoculation. As controls(O), untreated BALB/c female mice were inoculated with 1 x 10' cells of eitherBAMC-1 or Meth-A.

10

20 30Days after inoculation

Fig. 5. Failure of adoptive immunotherapy with PECs from normal miceinjected with OK-432. The tumor-bearing recipient mice were treated with CY(180 mg/kg) and l h later i.v. transferred with PECs from nontumor-bearingmice (•)or with those from tumor-bearing mice (A) treated with OK-432. •.mice treated with CY alone; O, untreated control.

treated either with anti-Thyl antibody and complement, oranti-asialo-GMi and complement, the in vivo activity of theTICs was completely abolished. However, the treatment withanti-Lytl.2 and complement, or with ami I.>t2.2 and complement, did not abolish the antitumor activity of the TICs. Thesecharacteristics of the effector cells in vivo coincide well withthose previously defined in the in vitro killing assays (9).

Since the cytotoxic cells in in vitro assays exerted their effectsnonspecifically on target cells, we next examined target cellspecificity by the use of a Winn-type assay as described in"Materials and Methods." PNACs from the TICs were foundto suppress the growth of both Meth-A and BAMC-1 cellsequally, although the adherent population at a high concentration was also able to neutralize them as shown in Table 1.These results indicate that, in addition to adherent cells, non-adherent cells in the TICs show strong pantropic killer cellactivities.

The lack of specificity of the effector cells was further confirmed by adoptively transferring the effector cells into eitherBAMC-1 (specific syngeneic tumor)-bearing or Meth-A (syn-geneic tumor with different antigenicities)-bearing mice. Asshown in Fig. 7, both types of tumors in the recipients disappeared after the adoptive transfer of the effector cells obtainedfrom BAMC-1-bearing mice treated with OK-432.

Specific Killer Cell Induction in Peritoneal Cavity of CuredMice. Since the above data (e.g., Fig. 4) suggested the specifickiller lymphocytes seemed to replace nonadherent pantropickiller cells, we have next attempted to obtain some directevidence to support this contention. Either BAMC-1 or Meth-A tumor cells with or without OK-432 was injected i.p. intomice 90 days after the successful adoptive immunotherapy withthe pantropic killer cells as described above. The killing activityof the PECs against BAMC-1 or Meth-A was assayed in vitroaccording to the method described in "Materials and Methods."As shown in Table 2, BAMC-1-specific killer cells were obtained when the animals were challenged with either BAMC-1cells alone or BAMC-1 with OK-432. Meth-A-specific killercells never appeared in these animals which once had highlyefficient pantropic killer cells, even after the rechallenge withMeth-A with or without OK-432 as shown.

DISCUSSION

Rosenberg et al. (14-16) reported that advanced cancer patients who could not be effectively treated with any known

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10

Fig. 6. Antitumor activity of subpopula- 15lions of the PECs obtained from tumor-bearing donor mice treated with OK-432. A, activity of PNACs (A) and NWNACs (A) in tumor-bearing mice pretreated with CY. See "Materials and Methods" for details on cell prepa

rations, (•)mice treated CY alone and untreated control mice (O). B, activity of a Thy 1positive subpopulation of PECs. Mice treatedwith CY and transferred with 2 X 10' PECs

(A) and mice with CY and transferred with thesame number of PECs incubated with anti-Thy1 and complement (•).».mice treated withCY alone; O, untreated control mice. C, activity of an asialo-GMi-positive subpopulation ofPECs. A, mice treated with CY and transferred 05 10with 2 x IO7 of PECs; •.mice treated with DaysCY and transferred with the same number ofPECs preincubated with anti-asialo-GMi andcomplement. •.mice treated with CY alone;O, untreated control mice.

15

20 30after inoculation

20 30after inoculation

05 10Days after

20 30inoculation

Table 1 Results of Winn-type neutralization assay of two different syngeneictumor cells with PECs

Table 2 Tumorspecificity of the killer cells induced in the mice cured of initialtumor inoculation by adoptive immunotherapy

Population ofTumorPECsBAMC-1C

NoneUnfractionatedNonadherentAdherentMeth-A17

NoneUnfractionatedNonadherentAdherentE/T"101010101010%

Inhibitionof tumor

take*0

(0/7)"100(7/7)100(7/7)100

(7/7)0

(0/7)72(5/7)100(7/7)100(7/7)

" Effector/target cell ratio.* The results were evaluated 3 weeks after tumor implantation.'1x10* cells/mouse, s.c.J Numbers in parentheses, free/total.

0 5 IO 20 30Days after inoculation

20 SOafter inoculation

Fig. 7. No target tumor specificity in adoptive immunotherapy by PKCs inPECs from tumor-bearing mice treated with OK-432. PKCs obtained fromBAMC-1-bearing mice were adoptively transferred to either BAMC-1-bearingrecipients (Fig. (vl ). or Meth-A-bearing recipients (Fig. 6B). A, mice treated withCY and transferred with 2 x IO7 PECs; O, mice treated with CY alone; •,

untreated control mice.

therapeutic measures were still beneficially treated by the adoptive transfer of LAK cells grown in vitro in the presence of IL2.Our previous study has revealed that LAK-like cells were responsible for tumor cell killings in mice bearing BAMC-1ascites, as shown by in vitro assays as well as in vivo Winn-typeneutralization assays (9). Extending this observation, our present study showed that nonspecific broad spectrum killer cellsobtained from tumor sites (e.g., peritoneal cavity) of the OK-432-treated mice were highly effective in destroying solid targettumors, when adoptively transferred into tumor-bearing syngeneic mice. Interestingly, these cells were equally effectiveagainst syngeneic nonspecific tumors too. Thus, the TICs ob-

Cured mice injectedwith"None

ThyoglycolateBAMC-1 (1 x IO5,i.p.)Meth-A (1 x IO5, i.p.)OK-432 (0.1 mg,i.p.)BAMC-1 + OK-432Meth-A + OK-432BAMC-12.6

±0.22.6 ±0.48.9 ±1.30.7 ±0.31.1 ±0.3

11.3 ±2.81.8 ±0.2%

Cytotoxicity*Meth-A0±0

0±00±00±0

2.4 ±0.33.0 ±0.5

0±0YAC-16.9

±1.136.8 ±6.212.7+1.7

0±051.6 ±8.151.4 ±7.341.9 ±6.7

" The mice cured of BAMC-1 tumor by adoptive immunotherapy were injected

i.p. with various agents to induce PECs, 90 days after the first inoculation oftumors. PECs were harvested 4 days after the injections and were used for effectorcells in the 4-h 5lCr-release assay.

*E:T = 50:1.

(ained from BAMC-1-bearing mice treated with OK-432 waseffective not only for BAMC-1 tumors but also for Meth-A-tumors, which have been known to possess different tumorantigens from BAMC-1 tumors. In addition, it is notable thata surprisingly small number (106-107) of the cells, when trans

ferred adoptively, were sufficient to suppress tumor growth incontrast to a large number (more than Id8) of the cells required

for successful adoptive immunotherapy when the transferringcells were obtained from Meth-A-specific immune animals (10)or from nontumor sites in tumor-bearing mice treated with OK-432 (11). Therefore, we tentatively call these effector cells inTICs "pantropic killer cells," as mentioned above. Although

the TICs contained a certain number of adherent cells (mostlymacrophages) which could also show antitumor activity inWinn-type neutralization assay as shown in Table 1, it isunlikely that these macrophages made a significant contributionto the PKC activity, since the PKC activity detectable by thisadoptive transfer system was abolished by the treatment withanti-Thy 1 antibody and complement (Fig. 6).

Interestingly, the curative effect achieved by the PKC issomewhat similar to that achieved by the LAK cells obtainedwith the expansion of TIL which was reported recently byRosenberg et al. (13). The PKC, however, is different from theTIL-derived LAK in that apparently the former does not requireIL2 for its exertion of antitumor effect. Instead, the PKC wasinduced in a tumor growing site (namely peritoneal cavity) byinjection of OK-432. It is possible that OK-432 acting on TILsor other lymphocytes may have induced a large amount of IL2which might help the TIL to "transform" to the PKC in vivo.

However, it has been rather rare to detect any amount of IL2in the peritoneal cavity after injection of OK-432 (17). At thepresent time, therefore, the PKC is considered to represent adifferent population from the LAK. Further studies are required

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to define these populations by obtaining specific surface markers, as a monoclonal antibody against a specific marker forhuman LAK was recently reported (18).

Previously many investigators have reported that OK-432could facilitate the induction of the specific antitumor activity(8) through the induction of CTL, and augment the nonspecificantitumor activities of various kinds of effector cells includingNK cells and macrophages (4-6). However, the characteristicsof PKCs, namely that they are plastic-nonadherent, Thyl-positive, and asialo-GMi -positive, seem to exclude most of thesecandidates. The data presented here may support the contentionthat PKCs may represent a new type of nonspecific killer cells,or a subtype of the previously described effector cells such as"activated" NK cells (19, 20). Enrichment and/or cloning of

the PKCs may be a prerequisite for further characterization ofthis most effective killer cell in vivo ever described.

To make the effector cell mechanisms in the OK-432-treatedanimals more complicated, it was found that the surviving micecured by adoptive transfer with the PKC could reject onlyantigenically specific tumors when the tumor cells were transplanted 90 days after the adoptive transfer (Fig. 7). This resultclearly suggests that the processes of tumor cell killing bynonspecific killers such as the PKC may facilitate the inductionof a specific killer population such as killer F colIs (8), and thatthe enhanced activity of the PKC or other nonspecific killersmay be relatively short lived. This sequence of events is also inaccord with our previous in vitro analysis of the cytotoxic cellswhich appeared after the OK-432 treatment (9). In those studies, sequential change in the recruited I -cell populations fromLAK-like cells to specific killer cells were speculated (9, 21).Merluzzi et al. (21) have reported that LAK cells were inducedbefore the induction of CTL. Kumagai et al. (22) have alsoreported that asialo-GMi-positive cells (NK cells) were requiredbefore the induction of CTL. In the present study, it is unclearif PKC induced the CTL or if they differentiated to CTL.

In conclusion, the present study showed that the OK-432treatment could induce highly effective nonspecific killer cellsconcentrated more in tumor-growing sites than in noniuniorsites. Adoptive immunotherapy by these cells (PKCs) has clearlyindicated that the PKC is at least as effective as (if not morethan) the TIL-derived LAK in destroying tumor cells in vivo.Although the definitive identities of PKC and TIL-derived LAKremain to be determined, the OK-432 therapy has a clearadvantage over the LAK therapy to achieve a similar therapeuticeffect since the latter requires tedious and time-consuming invitro procedures.

REFERENCES

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2. Torisu, M.. Katano, M., Kimura, Y., Itoh, 11.,and Takesue, M. New approachto management of malignant ascites with a streptococcal preparation, OK-432. I. Improvement of host immunity and prolongation of survival. Surgery(St. Louis), 93: 357-364, 1983.

3. Watanabe. Y., and Iwa, T. Clinical value of immunotherapy for lung cancerby the streptococcal preparation, OK-432. Cancer (Phila.), 53: 248-253,1984.

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5. Uchida, A., and Micksche, M. Lysis of fresh tumor cells by autologousperipheral blood and pleural effusion lymphocytes activated by OK-432. J.Nati. Cancer Inst., 71: 673-680, 1983.

6. Saito, M., Nanjo, M., Aonuma, E., Noda, T., Nakadate, I., Ebina, T., andIshida, N. Activated macrophages are responsible for the tumor-inhibitoryeffect in mice receiving intravenous injection of OK-432. Int. J. Cancer, 33:271-276, 1984.

7. Watabe, S., Sendo, F., Kimura, S., and Arai, S. Activation of cytotoxicpolymorphonuclear leukocytes by in vivo administration of a streptococcalpreparation, OK-432. J. Nati. Cancer Inst., 72: 1365-1370, 1984.

8. Mojo, IL, and Hashimoto, Y. Cytotoxic cells induced in tumor-bearing ratsby a streptococcal preparation (OK-432). Gann, 72:692-699, 1981.

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10. North, R. J. Cyclophosphamide-facilitated adoptive immunotherapy of anestablished tumor depends on elimination of tumor-induced suppressor Tcells. J. Exp. Med., 55: 1063-1074,1982.

11. Tominaga, T., Yoshida, Y., Watanabe, H., Saito, M., Kataoka, M„Moriya,Y., Sugawara, Y., and Kosaki, G. Adoptive immunotherapy using spleen cellsfrom OK-432-treated tumor-bearing mice against BAMC-I solid tumor inmice. In: T. Tobe (ed.), New Application of OK-432, pp. 141-151. Tokyo:Excerpta Medica, 1986.

12. Hattori, T., Kaibara, N., and Iguchi, K. Experimental and clinical investigations on combination anticancer therapy with a hemolytic streptococcalpreparation (OK-432). Jpn. J. Cancer Clin., 19: 929-934, 1973.

13. Rosenberg, S. A., Paul, S., and Lafreniere, R. A new approach to the adoptiveimmunotherapy of cancer with tumor-infiltrating lymphocytes. Science(Wash. DC), 233: 1318-1321, 1986.

14. Rosenberg, S. A. The adoptive immunotherapy of cancer: accomplishmentsand prospects. Cancer Treat. Rep., 68: 233-255, 1984.

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1988;48:4163-4167. Cancer Res   Motoo Saito, Masaki Nanjo, Motoyuki Kataoka, et al.   from OK-432-injected Tumor Sites in MiceAdoptive Immunotherapy by Pantropic Killer Cells Recovered

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