some characteristics of the in vivo antitumor immunity exhibited by mice cured of a large mopc-315...

Cancer Immunol Immunother (1987) 25:215-224 ancer mmunol9gy

tlSmmunotherapy © Springer-Verlag 1987

Some characteristics of the in vivo antitumor immunity exhibited by mice cured of a large MOPC-315 tumor by a low dose of melphalan*

Edward Barker** and Margalit B. Mokyr

Department of Microbiology and Immunology, University of Illinois at Chicago, Health Sciences Center Chicago, Illinois 60612, USA

Summary. BALB/c mice cured of a large MOPC-315 or MOPC-104E plasmacytoma following treatment with a low dose (2.5 mg/kg) of melphalan (L-PAM) were resistant to challenge with the other plasmacytoma but to a much lesser extent than to challenge with the autochthonous plasmacytoma. The resistance of the L-PAM-cured MOPC-315-tumor bearers to challenge with MOPC-104E tumor cells was increased when the MOPC-104E tumor cells were admixed with MOPC-315 tumor cells prior to their inoculation. This enhanced resistance to MOPC-104E cells was due to elimination of the MOPC-104E tumor cells through an innocent bystander killing effect since it did not render the mice more resistant to a subsequent challenge with MOPC-104E tumor cells alone. Adminis- tration of carrageenan to L-PAM-cured MOPC-315-tumor bearers 1 day after the challenge with the mixture of MOPC-104E and MOPC-315 tumor cells drastically reduced the ability of the mice to resist the tumor challenge. All of the tumors that developed in such mice were of MOPC-104E origin only (as judged by the binding specificity of the myeloma proteins secreted by the tumor cells as well as that present on their surface) even though (a) the tumor inoculum used consisted of up to 10-fold more MOPC-315 than MOPC-104E tumor cells and (b) the MOPC-315 tumor cells divide more rapidly. The same protocol of carrageenan treatment did not reduce the ability of normal BALB/c mice to develop in vivo a primary cell- mediated cytotoxic response nor a primary antibody response indicating that it has no effect on the initiation of an immune response. Therefore, it is conceivable that carrageenan treatment reduced the ability of L-PAM-cured MOPC-315-tumor bearers to reject a challenge with MOPC-315 and MOPC-104E tumor cells by interfering at the effector stage. The ability of the L-PAM-cured MOPC- 315-tumor bearers to reject the MOPC-315 cells present in the challenge mixture was reduced when the mice were treated with anti-Thy 1.2 antibody but not with carrageenan, indicating that T-cells independent from carrageenan- sensitive effector cells are required for the rejection of the

* Supported by Research Grant IM-435 from the American Can- cer Society and Research Grant CA-35761 from the National Cancer Institute ** In partial fulfillment of the requirements for the Doctor of Phi- losophy degree Offprint requests to: M. B. Mokyr

MOPC-315 tumor cells. Thus, at least two different effector mechanisms participate in the rejection of a challenge composed of MOPC-315 and MOPC-104E tumor cells by L-PAM-cured MOPC-315-tumor bearers. One of the mechanisms is dependent on the action of carrageenan- sensitive effector cells while the other does not require carrageenan-sensitive effector cells but is T-cell-dependent.

Introduction

The therapeutic efficacy of anticancer drugs can be facili- tated by antitumor immunity [14, 21, 22, 28]. Consequent- ly, situations should exist in which a low dose of drug that is not immunosuppressive would be as effective as a high dose of drug that is immunosuppressive for the treatment of tumor bearers even though the low dose of drug exerts a much weaker direct tumoricidal/tumoristatic effect than the high dose of drug. Indeed, a low dose (___ 15 mg/kg) of cyclophosphamide (CY) has been shown to be as effective as a high dose (> 200 mg/kg) of CY in the treatment of mice bearing a large MOPC-315 [8, 9] or MOPC-104E tumor [13]. Moreover, several investigators have reported that a lower dose of drug can be even more effective than a high dose of drug in treatment of tumor bearers. For exam- ple, Mathe et al. [15] have shown that administration of 134 mg/kg CY was curative for 44% of mice bearing the L1210 leukemia while a dose of 403 mg/kg was curative for just 5% of the mice. Similarly, Hilgard et al. [10] have shown that a dose of 3 mg/kg CY was curative for all rats bearing the L522 tumor while a dose of 20-100 mg/kg was not curative for any of the tumor-bearing rats.

The tumor burden eliminated by the immune system following low dose chemotherapy of tumor bearers can be quite large. Antitumor immunity has been claimed by Hil- gard et al. [10] to be the primary mechanism responsible for the eradication of a day 5, L522 tumor following low dose CY therapy. The basis for their claim was the observation that the L522 tumor cells were relatively resistant to the direct toxic effects of the drug and its active metabo- lites. Antitumor immunity was also shown to play a major role in the eradication of a large MOPC-315 tumor following low dose CY or melphalan (L-phenylalanine mustard, L-PAM) therapy. Specifically, a low dose of either drug did not exert sufficient tumoricidal/tumoristatic activity even for the eradication of a nonpalpable tumor yet it was

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capable in cooperation with host T-cell-dependent antitumor immunity to bring about the cure of most mice bearing a 20-25 mm tumor [3, 8, 9, 20].

As a consequence of the difference in the immunosuppressive activity of the low and the high dose of drug, tumor-bearing rodents cured by a low dose of drug were substantially more resistant to subsequent tumor challenge than were tumor bearers cured by the high dose of drug. Specifically, all mice cured of a large MOPC-315 tumor by a low dose of CY or L-PAM were resistant to a subsequent tumor challenge with 1000 times the minimal lethal tumor dose for 100% of normal mice (MLTD100) administered 1 to 60 days after the chemotherapy while, none of the mice cured of a large MOPC-315 tumor by a high dose of either drug were resistant to such a challenge at the above time intervals after the chemotherapy [16]. Approximately 70% of the MOPC-315-tumor bearers cured by a high dose of L-PAM were resistant to a challenge with a 10-fold smaller tumor inoculum (i.e., challenge with 100-fold the MLTDI00 of MOPC-315 tumor cells) [1, 2]. Thus, low dose chemotherapy not only alleviates many of the deleterious side-effects associated with high dose chemotherapy [26] but may also offer the important advantage of dealing with progression of dormant tumor foci.

The present study was undertaken to elucidate some of the characteristics of the in vivo antitumor immunity exhibited by mice cured of a large plasmacytoma by a low dose of L-PAM. Particular emphasis was placed on determining whether mice cured of a large MOPC-315 or MOPC-104E tumor are resistant to challenge only with the autochthonous tumor or also to challenge with the other plasmacytoma. In addition, we gained some insight into the nature of the in vivo antitumor immunity which is important in the resistance of the L-PAM-cured MOPC-315-tumor bearers to a subsequent tumor challenge.

Materials and methods

Tumors. Two plasmacytomas of BALB/c origin, the MOPC-315 and the MOPC-104E were used. The plasma cell tumors were maintained by serial passage in female BALB/c mice (6 to 8 weeks old; Goodwin Institute for Cancer Research, Plantation, Fla.). In the case of MOPC- 315, the tumor was maintained by s.c. inoculation and a single cell suspension was prepared by mechanical disrup- tion between glass slides [18]. For the preparation of MOPC-315-tumor bearers for chemotherapy, the mice were inoculated s.c. with 1 x 10 6 MOPC-315, a dose which is 1000-fold greater than the MLTD~00, and leads to the appearance of palpable tumors in 4.2_+ 1.7 days. Unless the mice are treated with chemotherapy, they die within 17 days after tumor inoculation. In the case of MOPC-104E, the tumor was maintained in an ascitic form. For the preparation of MOPC-104E-tumor bearers for L-PAM therapy, the mice were inoculated s.c. with 1 x 10 6 MOPC- 104E cells, a dose which is 100-fold greater than the MLTD~00, and leads to the appearance of palpable tumors in 8.7 + 1.4 days. In addition to plasmacytomas, we employed the EL4 thymoma of C57B1/6 origin. The EL4 tumor cells were maintained in culture in Roswell Park Memorial Institute Tissue Culture Medium 1640 supplemented with 10% fetal calf serum (Grand Island Biological Co., Grand Island, NY).

Chemotherapy. A stock solution of L-PAM (Burroughs Wellcome Co., Triangle Park, NC) was prepared by dis- solving the powder in a solvent mixture composed of one volume of acid-alcohol (95% ethanol and 2 N HC1 at a ratio of 5/1) and one volume of propylene glycol (Sigma Chemical Co., St. Louis, Mo.) buffer (45 ml propylene glycol diluted to 100ml with distilled H20 and K z H P O 4

added to a final concentration of 2% w/v). A stock solution of I0 mg/ml L-PAM was freshly prepared just prior to injection and was further diluted with Dulbecco's phosphate-buffered saline pH 7.2 (Grand Island Biological Co.) to the desired concentration. A dose of 2.5 mg/kg L-PAM was immediately administered i.p. to BALB/c mice bearing a large (>20ram) MOPC-315 or MOPC- 104E tumor. This dose of drug is the lowest dose of L-PAM which is curative for at least 90% of such tumor- bearing mice.

Tumor challenge. In experiments in which the L-PAM- cured tumor bearers were challenged with either MOPC- 315 or MOPC-104E tumor cells, the challenge inoculum consisted of 1 x 105 or 1 x 10 6 MOPC-315 or 1 x 10 6 MOPC- 104E tumor cells. In experiments in which the mice were inoculated with both MOPC-315 and MOPC-104E tumor cells either admixed or in separate inocula, 1 x 10 6 MOPC- 315 and 1 x 105 or 5 x 105 MOPC-104E cells were employed. These tumor inocula when administered to normal mice on opposite flanks, led to the appearance of a palpable tumor at the site of MOPC-315 inoculation in 5.1+1.0 days whereas it took 11.5+3.0 days for the appearance of a palpable tumor at the site of MOPC-104E tumor inoculation. By the time a palpable tumor iaodule appeared on the MOPC-104E tumor site, the MOPC-315 tumor nodule was at least 20 mm in diameter. When the MOPC-315 and MOPC-104E tumor cells were admixed prior to inoculation into normal mice the kinetics o f tumor appearance and progression were essentially the same as that observed for MOPC-315 tumor cells alone.

Titer of antibody with specificity for trinitrophenyl or dextran. To determine the type of myeloma protein(s) secreted by tumors that developed in mice that had been challenged with a mixture of MOPC-315 and MOPC-104E cells, the titer of their serum hemagglutinating activity against sheep red blood cells (SRBC) to which trinitrophenol (TNP) or dextran (DEX) was coupled was assessed. Coupling TNP to SRBC was done according to the method of Hannestad et al: [7] with slight modifications [19]. Briefly, 50mg 2,4,6-trinitrobenzenesulfonic acid (Eastman Kodak Co., Rochester, NY) was reacted with 1 ml packed SRBC in modified barbital buffer, pH 7.3 to 7.4. Coupling of DEX to SRBC was done according to the method Hiramoto et al. [11]. Briefly, 1 ml of 14% SRBC in borate-saline buffer pH 8.6 was incubated with 1 mg of periodate oxidized dextran B-1355 (available to us through the generosity of Dr M. E. Slodki from the USDA-Agricultural Research Ser- vice, Peoria, Ill.) at 37°C for 6 h. The SRBC were then washed with borate-saline buffer to remove unbound DEX. The hemagglutination assay was done by admixing 0.05 ml of 1% TNP-SRBC or DEX-SRBC with 0.05 ml of serum diluted serially in Dulbecco's phosphate-buffered saline pH 7.2 (Grand Island Biological Co.) in 96-well round-bottom microtiter plates (Dynatech Laboratories Inc., Alexandria Va.). The plates were then left at room

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temperature for 18 h, at the end of which the hemagglutinating titer was determined.

Enumeration of cells rosettable with TNP-SRBC or DEX- SRBC. To determine the type of immunoglobulin expressed on the surface of cells obtained from the primary tumor nodule of mice inoculated with a challenge consist- ing of a mixture of MOPC-315 and MOPC-104E tumor cells, we determined the ability of the cells to form rosettes with TNP-SRBC and/or DEX-SRBC. This was done by admixing 2.5 × l 0 6 tumor cells suspended in 0.25 ml Mini- mal Essential Medium supplemented with 10% fetal bo- vine serum with 0.25 ml of 1% TNP-SRBC or DEX-SRBC. To promote cell-to-cell contact, the mixtures were centri- fuged at 250 g for 15 s and then allowed to stand at 4 ° C for 30 min. At the end of the incubation period, the pellets were resuspended, and the percentage of rosettable cells determined.

Depletion of T-cells. To deplete T-cells, we employed monoclonal anti-Thy 1.2 which was the supernatant of hybri- doma 30-H12 originally produced by Ledbetter and Her- zenberg [12]. The effectiveness of this antibody in deplet- ing T-cells was illustrated by the ability of the antibody in the presence of low toxicity rabbit complement to elimi- nate virtually all T-cells. Specifically, incubation of spleen cells with monoclonal anti-Thy 1.2 plus complement reduced the number of cells stained positively with fluores- cein-conjugated monoclonal anti-Thy 1.2 antibodies from 31% to less than 2% as determined after analysis of 1 × 10 4

viable cells in a flow microfluorometer (Epics V, Coulter Electronics Inc., Hialeah, Fla.). In addition, treatment of spleen cells with monoclonal antibodies plus complement abolished their ability to generate a T-cell-dependent antitumor immune response and to proliferate in response to a T-cell mitogen (phytohemagglutinin) while the response to a B-cell mitogen (lipopolysaccharide) remained normal.

Carrageenan treatment. Carrageenan (Sigma Co.) was dis- solved in 0.85% NaC1 solution to a concentration of 2 mg/ ml using the double boiler technique [25]. Briefly, the tube containing the carrageenan and saline was placed in a beaker containing boiling water. The stock solution was sterilized by filtration and administered to the mice i.p. The carrageenan treatment was always initiated on day 1 after antigenic stimulation and consisted of either the daily administration of 1 mg/mouse for a period of 8 days or the administration of 2 mg/mouse every other day for a total of 4 injections.

Anti-EL4 cell-mediated cytotoxic response. BALB/c mice were injected s.c. with 1 × l 0 6 EL4 cells. On day 10 after tumor inoculation the mice were sacrificed and their spleen cells assayed for their anti-EL4 lytic activity using the 3.5 h 51Cr release assay [18]. This was done by incubating 2.5 x 104 or 5.0 × l 0 4 51Cr-labeled EL4 tumor cells with various numbers of spleen cells to achieve an effector/target cell ratio of 25/1, 100/1, and 200/1. The percentage of specific 5~Cr release was calculated using the following formula:

Percent of specific 51Cr release = (T-C)/(M-C) x 100, where T is the percentage of release with test spleen cells, C is the percentage of spontaneous release by the target cells alone which ranged between 15% and 25%, and M is percentage of maximal 51Cr release obtained by 3 cycles of

freezing and thawing, which ranged between 82% and 92%. The experiment was performed twice, each time employing 3 to 4 mice per group and processing the spleen of each mouse separately. The results presented represent the mean 51Cr release _+ SE for triplicate samples.

Anti-SRBC antibody response. BALB/c mice were injected i.p. with 3 × l 0 9 SRBC. Six days later, the mice were sacrificed and their spleen cells assayed for the content of plaque-forming cells using a modification of the Jerne plaque assay. Briefly, 1 × 106, 5 × 105, or 2.5 × 105 spleen cells in 0.1 ml were admixed with 0.1 ml of 10% SRBC and 0.05 ml of guinea pig complement (Grand Island Biologi- cal Co.) that was prediluted 1:2. This mixture was added to an equal volume of 1% agar (Sea Kern, FMC Corpora- tion Marine Colloids Div., Rockland, Me.) and then placed in a Petri dish. Three separate samples were placed in each Petri dish and each sample was covered with a 20 × 30 mm microscope cover glass (Erie Scientific, Ports- mouth, NH). The plates were then incubated for 2 h at 37 ° C prior to the determination of the number of plaques formed under each cover glass. The experiment was performed twice, each time employing 3 to 4 mice per group and processing the spleen of each mouse separately. Each determination was done in triplicate.

Statistics. The test of independence using G-statistics was employed to evaluate the significance of observed differ- ences between experimental and control groups.

Results

Ability of L-PAM-cured MOPC-315-tumor bearers to reject a lethal challenge with MOPC-IO4E tumor cells

We have shown previously that mice cured of a large MOPC-315 tumo r following low dose L-PAM therapy are resistant to a subsequent challenge with the autochthonous tumor [16]. Experiments were performed to determine whether such L-PAM-cured MOPC-315-tumor bearers are also resistant to a challenge with a different syngeneic plasma cell tumor, the MOPC-104E. Specifically, mice that rejected a 22-ram MOPC-315 tumor on the right flank following therapy with 2.5 mg/kg L-PAM were challenged on the contralateral flank with either MOPC-315 or MOPC-104E tumor cells. The MOPC-104E challenge inoculum consisted of 100-fold the MLTDI00 (i.e., 1 × 10 6)

while the MOPC-315 challenge inoculum consisted of either 100 or 1000-fold the MLTD100 (i.e., 1 × 105 or 1 × 10 6,

respectively) (Table 1). As expected, all mice cured of a large MOPC-315 tumor by low dose L-PAM therapy were resistant to challenge with 100-fold or even 1000-fold the MLTD100 of MOPC-315 cells [16]. In addition, one-third (i. e., 8/24) of the L-PAM-cured MOPC-315-tumor bearers were resistant to challenge with 100-fold the MLTDI00 of MOPC-104E tumor cells. Thus, mice cured of a large MOPC-315 tumor following low dose L-PAM therapy were resistant to challenge with a different syngeneic plasmacytoma, but to a much lesser extent than to challenge with the autochthonous plasmacytoma.

Ability of L-PAM-cured MOPC-104E-tumor bearers to reject a lethal challenge with MOPC-315 tumor cells

Initially we established that most ( -90%) mice bearing a large MOPC-104E tumor can be cured with 2.5 mg/kg

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Table !. Ability of L-phenylalanine mustard (L-PAM)-cured MOPC-315-tumor bearers to resist a challenge with MOPC-315 or MOPC-104E tumor cells

Tumor cells used Number of tumor % Resistance c for challenge" cells inoculated b

MOPC-104E 1 × 106 33% (8/24) MOPC-315 1 × 106 100% (26/26) MOPC-315 1 x 105 100% (10/10)

" Challenge was administered 10-20 days after treatment of mice bearing a 22-mm MOPC-315 tumor with a curative low dose of L-PAM (2.5 mg/kg) b Minimum lethal tumor dose for all inoculated normal BALB/c mice is 1 x 103 for MOPC-315 and 1 x 104 for MOPC-104E cells ° Percent of mice resistant to tumor cell challenge. Numbers in parentheses represent the number of mice without a tumor/total mice inoculated

Table 2. Ability of L-PAM-cured MOPC-104E-tumor bearers to resist a challenge with MOPC-104E or MOPC-315 tumor cells

Tumor cells used Number of tumor % Resistance ° for challenge" cells inoculated b

MOPC-104E 1 × 106 92% (20/22) MOPC-315 1 × 106 0% (0/9) MOPC-315 1 x 105 30% (3/10)

a Challenge was administered 20-25 days after the treatment of mice bearing a 20- to 22-mm MOPC-104E tumor with a curative low dose of L-PAM (2.5 mg/kg) b Minimum lethal tumor dose for all inoculated normal BALB/c mice is 1 x 103 for MOPC-315 and 1 x l04 for MOPC-104E cells ° Percent of mice resistant to tumor challenge. Numbers in parentheses represent the number of mice without a tumor/total mice inoculated

L-PAM and that most of the cured mice are resistant to a challenge with the autochthonous tumor. Subsequently we evaluated whether such L-PAM-cured MOPC-104E-tumor bearers are resistant to challenge with MOPC-315 tumor cells. Specifically, mice that rejected a 20 to 22-ram MOPC-104E tumor on the right flank following therapy with 2.5 m g / k g L-PAM were challenged on the left flank with either MOPC-104E or MOPC-315 tumor cells (Table 2). The MOPC-104E challenge inoculum consisted of 100-fold the MLTDI00 (i.e., 1 x 106) while the MOPC- 315 challenge inoculum consisted of either 100 or 1000-fold the MLTDI00 (i.e., 1 x 105 or 1 x 106, respectively). Almost all (20/22) of the L-PAM-cured MOPC-104E- tumor bearers were resistant to challenge with 100-fold the MLTDj00 of MOPC-104E tumor cells. In addition, 30% (3/10) of the L-PAM-cured MOPC-104E- tumor bearers were resistant to challenge with 100-fold the MLTDI00 of MOPC-315 while none were resistant to challenge with 1000-fold the MLTDa00 of MOPC-315 cells. Thus, mice cured of a large MOPC-104E tumor fol lowing low dose L- PAM therapy were resistant to challenge with a different syngeneic plasmacytoma, but to a lesser extent than to challenge with the autochthonous tumor.

Effect o f MOPC-315 tumor cells on the ability o f L -PAM- cured MOPC-315-tumor bearers to resist a challenge with MOPC-IO4E tumor cells

Experiments were performed to determine whether the immunity triggered in L-PAM-cured MOPC-315- tumor bearers by a MOPC-315 tumor challenge can be. exploited to facilitate the rejection of MOPC-104E tumor cells. For this purpose, mice that had been cured of a large MOPC-315 tumor fol lowing low dose L-PAM therapy as well as resisted a subsequent challenge with MOPC-315 tumor cells were rechallenged with either a mixture o f MOPC-104E and MOPC-315 tumor cells or with separate inocula of MOPC-104E and MOPC-315 tumor cells administered on contralateral flanks. The tumor challenge consisted of 1 x l06 MOPC-315 cells and 1 x 105 or 5 x 105 MOPC-104E ceils, i.e., a dose representing 1000-fold the MLTD~00 of MOPC-315 and a dose representing 10- or 50-fold the MLTDI00 of MOPC-104E (Table 3). When the MOPC-315 and MOPC-104E tumor cells were administered to L-PAM-cured MOPC-315-tumor bearers in separate inocula and on opposite flanks, only 31% of the mice (21/67)

resisted the challenge with the MOPC-104E tumor cells. On the other hand, when the MOPC-315 and MOPC-104E tumor cells were administered as a mixture, 74% o f the mice (49/66) resisted the combined tumor challenge. Thus, when MOPC-104E tumor cells were admixed with MOPC- 315 tumor cells prior t o their inoculat ion into L-PAM- cured MOPC-315-tumor bearers, the immunity triggered by the MOPC-315 tumor cells could be exploited to facilitate the rejection of the MOPC-104E tumor cells.

The nature o f the tumors that developed in L-PAM-cured MOPC-315-tumor bearers which did not resist the challenge with the mixture o f MOPC-315 and MOPC-IO4E tumor cells Experiments were performed to determine whether the tumors that developed in L-PAM-cured MOPC-315-tumor bearers which did not resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells, are of MOPC- 315 a n d / o r MOPC-104E origin. This was done initially by determining the binding specificity of the myeloma proteins secreted by the tumors taking advantage of the fact that MOPC-315 tumor cells secrete antibodies specific for T N P while MOPC-104E tumor cells secrete antibodies

Table 3. Ability of L-PAM-cured MOPC-315-tumor bearers to resist a challenge with MOPC-315 and MOPC-104E tumor cells administered at the same site or on contralateral flanks

Composition of tumor challenge a administered on:

% Resistance b

Left flank Right flank Left flank Right flank

MOPC-315 + MOPC-104E c - 74% (49/66) d -

MOPC-104E MOPC-315 31% (21/67) 100% (67/67)

Challenge consisted of 1 x l06 MOPC-315 and 1 x 105 or 5x l0 s MOPC-104E tumor cells h Percent of mice resistant to tumor cell challenge. Numbers in parentheses represent number of mice without a tumor/total number of mice inoculated c Administered as a mixture d p _<0.001 versus mice challenged with MOPC-104E alone on the left flank

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Table 4. The binding specificity of the myeloma proteins secreted by the tumors which developed in L-PAM-cured MOPC-315- tumor bearers that did not resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells

Mouse Titer of serum hemagglutinating activity a,b,c against number

Trinitrophenol sheep red Dextran blood cells (TNP-SRBC) (DEX)-SRBC

l < 2 512 2 <2 128 3 < 2 4,096 4 <2 32,768 5 <2 16,384 6 < 2 4,096 7 < 2 2,048 8 < 2 4,096

a Serum was obtained from mice bearing a 15- to 20-mm tumor b Titer of hemagglutinating activity for serum from mice bearing a 15- to 20-ram MOPC-315 tumor ranged between 2,560 and 65,536 against TNP-SRBC and was < 2 against DEX-SRBC c Titer of hemagglutinating activity for serum from mice bearing a 15- to 20-mm MOPC-104E tumor ranged between 1,024 and 65,536 against DEX-SRBC and was < 2 against TNP-SRBC

specific for DEX. Specifically, some of the L-PAM-cured MOPC-315-tumor bearers that developed a tumor following a challenge with a mixture of MOPC-315 and MOPC- 104E tumor ceils were bled when their s.c. tumor nodule reached 15 to 20 mm in diameter, and the titer of the serum agglutinating activity against TNP-SRBC and DEX- SRBC was determined (Table 4). As a control, the titer of serum hemagglutinating activity with TNP-SRBC and with DEX-SRBC of mice bearing a 15- to 20-mm tumor of either MOPC-315 or MOPC-104E origin was evaluated (Table 4). As expected, serum from MOPC-315-tumor bearers agglutinated only TNP-SRBC and not DEX- SRBC while serum from MOPC-104E-tumor bearers agglutinated only DEX-SRBC and not TNP-SRBC. Sera from all L-PAM-cured MOPC-315-tumor bearers that developed a tumor following a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells agglutinated only DEX-SRBC but not TNP-SRBC. In other words, serum from all L-PAM-cured MOPC-315-tumor bearers that did not resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells contained a high titer of myeloma proteins secreted by MOPC-104E tumor cells with no detectable levels of myeloma proteins secreted by MOPC- 315 tumor cells (Table 4).

Next, the binding specificity of the immunoglobul ins expressed on the surface of tumor cells derived from the primary tumor nodule of L-PAM-cured MOPC-315-tumor bearers that did not resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells was determined. This was done by determining the percentage of cells that formed rosettes with TNP-SRBC a n d / o r DEX-SRBC. As a control we determined the percentage of TNP- and DEX-rosettable cells among the cells derived from the primary tumor nodule of mice bearing a large MOPC-315 or MOPC-104E tumor. The tumor cells present within the s. c. tumor nodule of L-PAM-cured MOPC-315-tumor bearers that did not resist a challenge with a mixture of MOPC- 315 and MOPC-104E tumor cells did not possess surface immunoglobul in specific for MOPC-315 cells as evident

by the inability of such cells to form rosettes with TNP- SRBC under conditions in which approximately 80% of the cells from the primary tumor nodule of MOPC-315-tumor bearers did so. On the other hand, approximately 90% of the tumor cells present within the s.c. tumor nodule of L-PAM-cured MOPC-315-tumor bearers that did not resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells formed rosettes with DEX- SRBC which was comparable to the percentage of DEX- rosettable cells in the primary tumor nodule of mice bearing a large MOPC-104E tumor. Thus, the tumors that developed in L-PAM-cured MOPC-315-tumor bearers that did not resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells were of MOPC-104E origin only.

Effect of resisting a challenge with a mixture of MOPC-315 and MOPC-IO4E tumor cells on the ability of L-PAM-cured MOPC-315-tumor bearers to resist a subsequent challenge with MOPC-IO4E tumor cells alone

Experiments were performed to determine whether as a result of rejecting the MOPC-104E tumor cells present in a mixture with MOPC-315 tumor cells, the L-PAM-cured MOPC-315-tumor bearers developed enhanced anti- MOPC-104E tumor immunity. Specifically, the resistance to challenge with MOPC-104E tumor cells of L-PAM- cured MOPC-315-tumor bearers that resisted a previous challenge with a mixture of MOPC-315 and MOPC- 104E tumor cells was compared to that of L-PAM-cured MOPC-315-tumor bearers that resisted a previous challenge with MOPC-315 tumor cells alone. The MOPC-104E tumor challenge consisted of 5 x 105 cells and was administered to the L-PAM-cured MOPC-315-tumor bearers 60 days after their previous challenge (Table 5). Approxi- mately 15% of the L-PAM-cured MOPC-315-tumor bearers that resisted a challenge with MOPC-315 were resistant to challenge with 5 x 105 MOPC-104E tumor cells. Essen- tially the same magnitude of resistance to challenge with MOPC-104E tumor cells was displayed by L-PAM-cured MOPC-315-tumor bearers that resisted a challenge with the mixture of MOPC-315 and MOPC-104E tumor cells (i. e., 17%). Thus, the rejection of MOPC-104E tumor cells admixed with MOPC-315 tumor cells by L-PAM-cured MOPC-315-tumor bearers did not enhance the anti- MOPC-104E immuni ty of the mice.

Table 5. Resistance to challenge with MOPC-104E tumor cells of L-PAM-cured MOPC-315-tumor bearers that rejected a previous challenge with either MOPC-315 cells alone or with a mixture of MOPC-315 and MOPC- 104E cells

Previous tumor % Resistance to Time of appearance challenge challenge with of palpable tumors consisted of MO PC- 104Ea (days) b, o

MOPC-315 + MOPC-104E 17% (4/23) 9.1 +0.3

MOPC-315 15% (4/26) 11.1 ___0.9

Percent of mice resistant to challenge with 5 x 105 MOPC-104E cells. Numbers in parentheses represent number of mice without a tumor/total mice inoculated b Calculated only for mice that developed tumors during the 60-day observation period and presented as mean + SD c All normal mice injected with 5 x 105 MOPC-104E cells developed palpable tumors in 9.5 +2.5 days

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The role of T-cells in the resistance of L-PAM-cured MOPC- 315-tumor bearers to challenge with a mixture of MOPC-315 and MOPC-IO4E tumor cells

Experiments were performed to determine whether treatment of L-PAM-cured MOPC-315-tumor bearers with anti-Thy 1.2 ant ibody interferes with the ability of the mice to reject a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells. For this purpose, L-PAM-cured MOPC-315-tumor bearers were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells and then di- vided into two groups, one of which was treated with monoclonal anti-Thy 1.2 ant ibody 6, 48, and 96 h after the tumor challenge, while the other group received saline (Table 6). While 85% of the saline-treated L-PAM-cured MOPC-315-tumor bearers rejected the challenge with the mixture of MOPC-315 and MOPC-104E tumor cells, only 20% of the anti-Thy 1.2 antibody-treated mice were able to do so. In addition, the time of appearance of a palpable tumor in the anti-Thy 1.2 antibody-treated L-PAM-cured MOPC-315-tumor bearers was similar to the time of appearance of a palpable tumor in normal mice injected with a different aliquot of the same batch of tumor cells. Thus, treatment with anti-Thy 1.2 ant ibody drastically reduced the ability of L-PAM-cured MOPC-315-tumor bearers to resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells indicat ing the importance of T-cells in the rejection of the mixed challenge by L-PAM- cured MOPC-315-tumor bearers.

Next, the origin of the tumors that developed following treatment with anti-Thy 1.2 ant ibody in the L-PAM- cured MOPC-315-tumor bearers challenged with the mixture of MOPC-315 and MOPC-104E tumor cells was in- vestigated by determining the titer of serum hemagglutinat ing activity against TNP-SRBC and DEX-SRBC (Table 7). The sera of all seven mice evaluated for this purpose contained significant levels of hemagglutinat ing activity against TNP-SRBC and at the same time the serum of only one of the mice contained significant hemagglutinat ing activity against DEX-SRBC. Thus, all the tumors that developed as a result of treatment with anti-Thy 1.2 ant ibody in the L-PAM-cured MOPC-315-tumor bearers

Table 6. Effect of treatment with monoclonal anti-Thy 1.2 antibody on the ability of L-PAM-cured MOPC-315-tumor bearers to resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells ~

Treatment of % Resistance b Time of appearance challenged mice of palpable tumors

(days) c. d

Saline 85% (17/20) 24.3 + 3.5 Anti-Thy 1.2 e 20% (4/20) f 4.6+ 1.3

Consisted of 1 x l06 MOPC-315 and 1 x 105 MOPC-104E tumor cells b Percent of mice resistant to tumor challenge. Numbers in parentheses represent the number of mice without a tumor/total mice inoculated c Calculated only for mice that developed tumors during the 60-day observation period and is presented as mean + SD d All normal mice inoculated with the tumor mixture developed a palpable tumor in 4 to 5 days. e Administered 6, 48, and 96 h after tumor challenge r p <0.001 versus mice treated with saline

Table 7. The binding specificity of the myeloma proteins secreted by the tumors that developed following treatment with monoclonal anti-Thy 1.2 antibody ~' in L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells

Mouse number Titer of serum b hemagglutinating activityd against

TNP-SRBC DEX-SRBC

1 65,536 < 2 2 32,768 <2 3 16,384 <2 4 512 <2 5 16,384 <2 6 65,536 <2 7 32 1,024

a Anti-Thy 1.2 antibody was administered 6, 48, and 96 h after tumor challenge b Serum was obtained from mice bearing a 15- to 20-mm tumor ° Titer of hemagglutinating activity for serum from mice bearing a 15- to 20-mm MOPC-315 tumor ranged between 2,560 and 65,536 against TNP-SRBC and was <2 against DEX-SRBC d Titer of hemagglutinating activity for serum from mice bearing a 15- to 20-mm MOPC-104E tumor ranged between 1,024 and 65,536 against DEX-SRBC and was < 2 against TNP-SRBC

challenged with a mixture of MOPC-315 and MOPC-104E secreted myeloma protein specific for MOPC-315 tumor cells illustrating the importance of T-cell-dependent antitumor immuni ty in controll ing the progression of the MOPC-315 tumor cells in L-PAM-cured MOPC-315-tumor bearers.

The effect of carrageenan treatment on the ability of L-PAM-cured MOPC-315-tumor bearers to res&t a challenge with a mixture of MOPC-315 and MOPC-IO4E tumor cells

Experiments were performed to determine whether treatment of L-PAM-cured MOPC-315-tumor bearers with carrageenan interferes with the ability of the mice to reject a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells. Specifically, L-PAM-cured MOPC-315-tumor bearers were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells and subsequently some of the mice were treated with carrageenan while the remaining mice were treated with saline. The carrageenan treatment was always initiated 1 day after tumor challenge and consisted in one group of daily injections of 1 mg/mouse for a period of 8 days while in another group it consisted of 2 rag/mouse every other day for a period of 7 days (Table 8). The protocols of carrageenan treatment used were found not to interfere with the inductive stage of an immune response. Specifically, treatment of BALB/c mice with carrageenan starting 1 day after tumor inoculat ion did not reduce their ability to develop in vivo a primary splenic anti-EL4 cell-mediated cytotoxic response as judged from the magnitude of anti-EL4 cytotoxicity exerted by such spleen cells in the 4 h 51Cr release assay (29.7% + 1.6% vs 33.3% + 5.3% 5~Cr release with spleen cells from carrageenan-treated vs untreated EL4-immunized BALB/c mice). Similarly, treatment of BALB/c mice with carrageenan starting 1 day after SRBC administrat ion did not reduce their ability to develop in vivo a primary splen-

Table 8. Effect of carrageenan treatment on the ability of L-PAM-cured MOPC-315-tumor bearers to resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells a

Treatment Dose ofcarra- % Resistance b Time of appear- of mice geenan adminis- ance of palpable

tered (rag) tumor (days) °, d

Saline - 78% (25/32) 24.3 _ 3.0 Carrageenan 2 e 46% (13/28)g 11.8+_5.1 Carrageenan 1 r 36% (4/11)g 13.9 +_ 5.8

a Consisted of 1 x 106 MOPC-315 and 1 x 105 MOPC-104E tumor cells b Percent of mice resistant to tumor challenge. Numbers in parentheses represent the number of mice without a tumor/total mice inoculated c Calculated only for mice that developed tumors during the 60-day observation period and is presented as mean +- SD d All normal mice inoculated with the tumor mixture developed a palpable tumor in 4 to 5 days e Administered 1, 3, 5, and 7 days after tumor challenge f Administered daily from day 1 to day 8 after tumor challenge

P <0.025 >0.01 versus mice treated with saline

ic anti-SRBC ant ibody response as judged from the number of plaque-forming cells present per 1 x 106 splenic cells (164+9 vs 106+7 plaque-forming cells with spleen cells from carrageenan-treated vs untreated SRBC-immunized BALB/c mice).

As can be seen from Table 8, treatment of L-PAM- cured MOPC-315-tumor bearers with carrageenan via either of the two protocols substantially reduced the ability of the mice to reject a challenge with a mixture of MOPC- 315 and MOPC-104E tumor cells. Specifically, while 78% (25/32) of the saline-treated MOPC-315-tumor bearers were resistant to challenge with a mixture of MOPC-315 and MOPC-104E tumor cells, only 46% or fewer of the L-PAM-cured MOPC-315-tumor bearers retained the ability to reject the challenge after treatment with carrageenan (P <0.025 >0.01). In addition, the time of appearance of a palpable tumor nodule in the L-PAM-cured MOPC- 315-tumor bearers that did not resist the challenge with the tumor cell mixture was significantly shorter in mice treated with carrageenan than in mice treated with saline. In fact, the time of appearance of a palpable tumor in the carra-

g e e n a n - t r e a t e d L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells did not differ from the time of appearance of a palpable tumor in normal mice injected with a different aliquot of the same batch of tumor cell mixture. Thus, carrageenan-sensitive cells contributed to the ability of L-PAM-cured MOPC-315-tumor bearers to resist a challenge with a mixture of MOPC-315 and MOPC-104E tumor cells.

The nature o f the tumors that developed in L-PAM-cured MOPC-315-tumor bearers challenged with a mixture o f MOPC-315 and MOPC-IO4E tumor cells upon treatment with carrageenan

Experiments were performed to determine whether the tumors that developed following carrageenan treatment of L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells are of MOPC-315 a n d / o r MOPC-104E origin. For this purpose, the b inding specificity of the myeloma

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Table 9. The binding specificity of the myeloma proteins secreted by the tumors that developed as a result of carrageenan treatment a in L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC- 104E tumor cells

Mouse number

Titer of serum hemagglutinating activity b, o, d with

TNP-SRBC DEX-SRBC

1 <2 32,768 2 < 2 1,024 3 <2 32,768 4 < 2 2,048 5 <2 8,192 6 < 2 1,024

a Carrageenan (2 mg/mouse) was administered on days 1, 3, 5, and 7 after tumor challenge b Serum was obtained from mice bearing a 15- to 20-mm tumor c Titer of hemagglutinating activity for serum from mice bearing a 15- to 20-mm MOPC-315 tumor ranged between 2,560 and 65,536 against TNP-SRBC and was <2 against DEX-SRBC

Titer of hemagglutinating activity for serum from mice bearing a 15- to 20-mm MOPC-104E tumor ranged between 1,024 and 65,536 against DEX-SRBC and was < 2 against TNP-SRBC

proteins secreted by tumors that developed in the carrageenan-treated mice was determined. Specifically, when the tumors had reached 15 to 20 mm in diameter, 6 mice were bled and the titer of their serum hemagglutinating activity against TNP-SRBC and DEX-SRBC was determined (Table 9). Serum from all the L-PAM-cured MOPC- 315-tumor bearers that developed a tumor when challenged with a mixture of MOPC-315 and MOPC-104E tumor cells followed by carrageenan treatment, exhibited a substantial agglutinating titer only against DEX-SRBC and not against TNP-SRBC. Thus, the tumors that developed as a result of carrageenan treatment in L-PAM-cured MOPC-315- tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells appeared to secrete only myeloma protein specific for MOPC-104E and not for MOPC-315 tumor cells.

Next, the b inding specificity of the immunoglobul ins expressed on the surface of cells derived from the primary tumor nodule of mice that developed a tumor following carrageenan treatment of L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells was determined. Cells derived from the primary tumor nodule of such mice failed to form rosettes with TNP-SRBC under condit ions in which at least 75% of the cells derived from the primary tumor nodule of MOPC-315-tumor bearers did so. At the same time, more than 90% (92%+ 1%) of the cells derived from the primary tumor nodule of the carrageenan-treated mice formed rosettes with DEX-SRBC, a percentage which was comparable to the percentage of DEX-rosettable cells in the primary tumor nodule of MOPC-104E-tu- mot bearers. Thus, the tumors that developed following carrageenan treatment in L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells were of MOPC-104E origin only, indicating that carrageenan-sensitive cells are required for the eradication of the MOPC-104E but not of the MOPC-315 tumor cells present in the challenge mixture.

222

Discussion The results presented illustrate that mice cured of a large MOPC-315 or MOPC-104E plasmacytoma following low dose L-PAM therapy are resistant to challenge with a different syngeneic plasmacytoma but to a much lesser extent than to challenge with the autochthonous plasmacytoma. The antitumor immunity triggered by the autochthonous plasmacytoma could be exploited to enhance the resistance of the L-PAM-cured MOPC-315-tumor bearers to the other plasmacytoma. However, L-PAM-cured MOPC- 315-tumor bearers that rejected a challenge with MOPC- 104E admixed with MOPC-315 tumor cells did not acquire enhanced anti-MOPC-104E immunity. The rejection of the MOPC-104E in the mixture with MOPC-315 tumor cells was mediated, in part, in the L-PAM-cured MOPC-315-tumor bearers through the action of carrageenan-sensitive effector cells. On the other hand, the rejection of the MOPC-315 tumor cells in the mixture with MOPC-104E tumor cells was mediated in the L-PAM-cured MOPC- 315-tumor bearers through a mechanism independent of carrageenan-sensitive effector cells but dependent on T- cells.

We have recently shown that spleen cells from L-PAM- cured MOPC-315-tumor bearers exhibit enhanced antitumor immune reactivity against plasmacytoma-shared antigens as evident by the ability of the spleen cells to develop a secondary type antitumor cytotoxic response following in vitro immunization with MOPC-315 tumor cells as well as in response to in vitro immunization with a different plasma cell tumor such as MOPC-104E [17]. The results presented here extend our previous observations by illustrating that in vivo L-PAM-cured MOPC-315-tumor bearers exhibited immunity not only against the autochthonous plasmacytoma but also against a different plasmacytoma. In addition, we report the results of a reciprocal experiment in which mice cured of a different plasmacytoma, the MOPC-104E, following low dose L-PAM therapy were resistant to a challenge not only with the autochthonous tumor but also with MOPC-315 tumor cells. The magnitude of antiplasmacytoma lytic activity exerted by spleen cells from L-PAM-cured MOPC-315-tumor bearers following in vitro immunization with MOPC-104E tumor cells was essentially the same as that exerted by the spleen cells following in vitro immunization with MOPC-315 [17]. In vivo, however, the L-PAM-cured tumor bearers were substantially more resistant to challenge with the autochthonous tumor than to challenge with the other plasmacytoma. The discrepancy between the in vitro and in vivo observations is not surprising. The splenic antitumor immunity developing in vitro under optimal immunization conditions might not represent what happens in the spleen following a s.c. tumor inoculation. Moreover, the immunity in the spleen might bear no relevance to the ability of the mice to reject a s.c. tumor challenge. Finally, the antitumor immunity measured in vitro was mediated by cytotoxic T-lymphocytes which might not be the cells important in vivo in the eradication of the tumor challenge [6]. Thus, in order to obtain information regarding the specificity of the antitumor immunity exhibited by L-PAM-cured MOPC- 315-tumor bearers, as well as to assess the magnitude of the antitumor immunity effective only against the autochthonous tumor (e.g., directed against antigens unique to the immunizing plasmacytoma) vs that effective against a panel of plasmacytomas (e.g., directed against antigens

shared by the different plasmacytomas), it was necessary to evaluate these parameters in vivo.

The resistance of L-PAM-cured MOPC-315-tumor bearers to challenge with MOPC-104E tumor cells was enhanced when the MOPC-104E tumor cells were admixed with MOPC-315 tumor cells prior to their inoculation. In this situation, the immunity triggered by the MOPC-315 tumor cells was exploited in the rejection of the MOPC- 104E cells. However, the immunity was not always sufficient to handle the combined tumor burden and in approximately 25% of the mice a tumor developed. The tumor that developed in such mice was always of MOPC- 104E origin with no traces of MOPC-315 tumor cells. In other words, in situations in which the antitumor immunity was not sufficient to eradicate the entire combined mixture of tumor cells, the MOPC-315 cells were always eliminated yet enough MOPC-104E tumor cells remained to establish lethal tumors. The first explanation that comes to mind is that in these situations the limiting factor is the immunity directed against plasmacytoma-shared antigens and consequently not all MOPC-104E nor MOPC-315 tumor cells are eliminated by this immunity. However, the remaining MOPC-315 tumor cells can still be eliminated by the immunity directed against MOPC-315 unique antigens whereas the remaining MOPC-104E tumor cells are free to grow and establish a lethal tumor. Under this scen- ario, it would be expected that mice that rejected a mixed challenge with MOPC-315 and MOPC-104E tumor cells would display enhanced immunity against the plasmacytoma-shared antigens and consequently will be more effective in eradicating a subsequent challenge with MOPC- 104E tumor cells when administered alone. In contrast to this expectation, rejection of MOPC-104E tumor cells by L-PAM-cured MOPC-315-tumor bearers when administered in a mixture with MOPC-315 tumor cells did not enhance the resistance of the mice to a subsequent tumor challenge with MOPC-104E tumor cells alone. These results indicate that the enhanced rejection of the MOPC- 104E when administered to L-PAM-cured MOPC-315-tumor bearers in a mixture with MOPC-315 tumor cells is due to elimination of the MOPC-104E tumor cells through an innocent bystander killing mechanism. The cell(s) responsible for exerting the innocent bystander killing effect have not as yet been identified. We are currently entertain- ing the possibility that T-cells of L-PAM-cured MOPC- 315-tumor bearers produce, upon stimulation with MOP@ 315 tumor cells, lymphotoxin-like factors that have a nonspecific lytic effect [4, 24] and/or that the T-cells of such mice upon restimulation with MOPC-315 tumor cells recruit and/or activate other nonspecific immune mechanisms which in turn exert an innocent bystander killing effect [5].

Administration of carrageenan to L-PAM-cured MOPC-315-tumor bearers reduced the ability of the mice to reject MOPC-104E tumor cells present in admixture with MOPC-315 tumor cells. These results illustrate the importance of carrageenan-sensitive cells in the rejection of MOPC-104E tumor cells. In view of the selective cyto- pathic effects of carrageenan on macrophages [27], it is conceivable that the carrageenan-sensitive cells which con- tribute to the eradication of MOPC-104E tumor cells present in a mixture with MOPC-315 are macrophages. The carrageenan treatment most likely interfered at the effector stage of tumor rejection since the same protocol of carra-

223

geenan treatment that reduced the ability of L-PAM-cured MOPC-315-tumor bearers to reject the combined challenge did not reduce the ability of normal mice to develop a primary cell-mediated cytotoxic response nor a primary antibody response. Moreover, the protocol of carrageenan treatment employed in our study has been shown by other investigators to be effective in impairing the expression of existing delayed-type hypersensitivity responses [25] as well as the ability of immune spleen cells to confer system- ic antitumor immunity upon administration to tumor- bearing mice [23]. Thus, a mechanism responsible for the eradication of MOPC-104E tumor cells admixed with MOPC-315 tumor cells by L-PAM-cured MOPC-315-tumor bearers is most likely mediated via effector macrophages. It should be pointed out however, that although carrageenan drastically reduced the ability of the L-PAM- cured MOPC-315 tumor bearers to reject a challenge with MOPC-104E admixed with MOPC-315, a significant percentage of the mice retained the ability to reject the mixed challenge. Thus, the rejection of the MOPC-104E tumor cells administered to L-PAM-cured MOPC-315-tumor bearers in a mixture with MOPC-315 appeared to be mediated through at least two mechanisms one of which is carrageenan-sensitive while the other is carrageenan-resistant.

All tumors that developed following treatment with anti-Thy 1.2 antibody in the L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells were of MOPC- 315 origin as judged by the binding specificity of the myeloma protein secreted by the tumor cells as well as pos- sessed on their surface. Serum from only one out of the seven mice that developed a tumor following anti-Thy 1.2 antibody treatment of L-PAM-cured MOPC-315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells also secreted myeloma proteins specific for MOPC-104E cells. The secretion of myeloma protein specific for MOPC-104E tumor cells by one of the tumors also secreting myeloma protein specific for MOPC-315 tumor cells was not necessarily the result of the anti-Thy 1.2 antibody treatment since approximately 25% of the saline-treated L-PAM-cured MOPC-315-tumor bearers were unable to reject the MOPC-104E present in the admixture with MOPC-315 tumor cells (Table 3). Thus, if we take into consideration the frequency of MOPC- 104E tumor progression in L-PAM-cured MOPC-315-tumor bearers challenged with a mixture of MOPC-315 and MOPC-104E tumor cells it appears that treatment with anti-Thy 1.2 antibody did not reduce the ability of the mice to resist the challenge with MOPC-104E tumor cells. The fact that MOPC-104E tumor cells were not detected in virtually any of the tumors that developed as a result of anti- Thy 1.2 antibody treatment of L-PAM-cured MOPC- 315-tumor bearers that were challenged with a mixture of MOPC-315 and MOPC-104E tumor cells does not necessarily mean that T-cells did not play any role in the rejection of MOPC-104E tumor cells within the challenge mixture. The composition of the challenge mixture used fa- vored the progression of MOPC-315 tumor cells over that of MOPC-104E tumor cells under conditions in which the immunity to the challenge breaks. Specifically, the tumor inoculum consisted of 1 × 1 0 6 MOPC-315 and just 0.1 × 106-0.5 × 106 MOPC-104E tumor cells (i.e., 1000-fold the MLTD100 of MOPC-315 and only 10 to 50 fold the MLTDI00 of MOPC-104E) and when inoculated into nor-

mal mice it led to the development of tumors that appeared to be exclusively of MOPC-315 origin when the primary tumor nodule reached 15 to 20 mm in diameter. Moreover, even if the same number of MOPC-104E and MOPC-315 tumor cells were to be administered, the MOPC-315 cells would grow much faster as expected based on the observations that inoculation of 1 × 10 6

MOPC-315 cells into normal mice led to the appearance of palpable tumor nodules within 5 days whereas a period of at least 8 days was required for the appearance of a palpable tumor nodule following the inoculation of 1 × 106 MOPC-104E tumor cells into normal mice. In order to get information as to the role of T-cells in the rejection of MOPC-104E tumor cells administered in a mixture with MOPC-315 tumor cells to L-PAM-cured MOPC-315-tumor bearers it will be necessary to select conditions under which the progression of MOPC-315 tumor cells is retarded (e.g., by treating the MOPC-315 tumor cells with mitomycin C) so that the progression of MOPC-104E can be monitored.

The failure of carrageenan treatment to reduce the ability of L-PAM-cured MOPC-315-tumor bearers to resist a challenge with MOPC-315 tumor cells when admixed with MOPC-104E tumor cells did not necessarily mean that the activity of the carrageenan-sensitive effector cells was specific for MOPC-104E cells nor that MOPC-315 tumor cells were resistant to the action of the carrageenan- sensitive effector cells. The most likely reason for this observation is that the immunity mediated by the carrageenan-sensitive effector cells is not necessary for the eradication of MOPC-315 tumor cells since the immunity that is mediated by T-cells and is independent of the contribution of carrageenan-sensitive effector cells is sufficient for the eradication of the MOPC-315 tumor inoculum. In fact, we have recently observed that all L-PAM-cured MOPC- 315-tumor bearers were able to reject a challenge with a 10-fold higher dose of MOPC-315 tumor cells than that employed here (i.e., 10,000 the MLTD100), and when carrageenan was administered to such mice it caused the appearance of tumors in three out of seven mice studied which increased in size until the tumors reached up to 15 mm in diameter and then regressed completely (data not shown).

Taken together, the studies presented here illustrate that mice cured of a large MOPC-315 tumor by a low dose of L-PAM exhibit more than one type of immunity simultaneously. In fact, our studies suggest that at least three different types of immunity participate in the rejection of the mixed tumor challenge. First, the L-PAM-cured MOPC-315-tumor bearers exhibit immunity to what appears to be MOPC-315 unique antigens. Second, the L-PAM-cured MOPC-315-tumor bearers exhibit immunity against antigens shared by different plasmacytomas which can be triggered efficiently by MOPC-104E tumor cells also when administered alone. Third, immunity mediated through an innocent bystander killing mechanism, which does not lead to enhanced resistance of the mice to a subsequent challenge with the same tumor. These three types of antitumor immune responses may act simultaneously thereby endowing the L-PAM-treated MOPC-315-tumor bearers with a very potent mechanism to eradicate MOPC- 315 tumor cells. The synergistic effect of these three antitumor immune mechanisms may be the reason why therapy with a low dose of L-PAM, the tumoricidal activity of

224

which is not sufficient for the eradication of even a nonpalpable MOPC-315 tumor, can eradicate a large ( > 2 0 r a m in diameter) MOPC-315 tumor and extensive metastases [3].

Acknowledgements. We wish to thank Dr. Shlomo Ben-Efraim for his critical review of the manuscript. We also wish to thank Mary M. Bartik for her excellent technical assistance.

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Received May 4, 1987/Accepted July 16, 1987

some characteristics of the in vivo antitumor immunity exhibited by mice cured of a large mopc-315...

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