Rabbit lymphoid cells differentiated with respect to alpha-, gamma-, and mu- heavy polypeptide chains and to allotypic markers Aa1 and Aa2.

Lymphoid cells present in spleen and lymph nodes of hyperimmune rabbits were found to be differentiated with respect to the class of immunoglobulin heavy chain which they contained. The relative proportions of cells containing the various heavy chains were as follows: alpha-chain (5 to 8%), micro-chain (14 to 21%), and gamma-chain (71 to 81%). The allotypic markers Aa1 and Aa2, found on heavy chains, were also found to be separately localized in cells of Aa(1)/Aa(2) heterozygous rabbits. The ratio of cells in spleen and lymph nodes containing the Aa1 marker to those containing the Aa2 marker varied with individual rabbits; the range was 53 to 88% Aa1 versus 12 to 47% Aa2.

Eression of allelic immunoglobulin in homozygous rabbits injected with RNA extract.

Ribonucleic acid extracts obtained from lymph nodes of immunized rabbits homozygous for the b(4)or b(5)allele of light chain immunoglobulin allotypes were injected intravenously into nonimmunized rabbits homozygous for the alternate allele. Approximately 30 percent of the plaque-forming cells in the spleen yielded plaques with immunoglobulin M antibody possessing the allotype of the RNA donor. The allotype of the RNA donor was also found in the IgG immunoglobulin of lymphoid cell lysates as well as in the IgG isolated from the serum. These results suggest that the injected RNA has an informational role in the in vivo synthesis of immunoglobulins by host lymphoid cells.

Unexpected male choosiness for mates in a spider.

Sexual selection theory traditionally considers choosiness for mates to be negatively related to intra-sexual competition. Males were classically considered to be the competing, but not the choosy, sex. However, evidence of male choosiness is now accumulating. Male choosiness is expected to increase with an individual's competitive ability, and to decrease as intra-sexual competition increases. However, such predictions have never been tested in field conditions. Here, we explore male mate choice in a spider by studying size-assortative pairing in two natural sites that strongly differ in the level of male-male competition. Unexpectedly, our results demonstrate that mate choice shifts from opportunism to high selectivity as competition between males increases. Males experiencing weak competition did not exhibit size-related mating preferences. By contrast, when competition was intense we found strong size-assortative pairing due to male choice: while larger, more competitive males preferentially paired with larger, more fecund females, smaller males chose smaller females. Thus, we show that mating preferences of males vary with their competitive ability. The distinct preferences exhibited by males of different sizes seem to be an adaptive response to the lower reproductive opportunities arising from increased competition between males.

Antitumor immunity in strain 2 guinea pigs immunized with potassium chloride extracts of L2C tumor cells.

Extracts of L2C tumor cells stimulated in vitro production of macrophage migration inhibitory factor (MIF) in peritoneal exudate cells from guinea pigs immunized with L2C tumor cells. Guinea pigs immunized with extracts of L2C tumor cells that were active in vitro (in the MIF assay) were completely resistant to challenge with viable tumor cells given 2 weeks later. Furthermore, guinea pigs immunized with extracts of L2C tumor cells within 1 hour after challenge with viable L2C tumor cells survived substantially longer than did nonimmunized controls. The immunoprotective and immunotherapeutic effects seen in guinea pigs given injections of viable L2C tumor cells were obtained with extracts of L2C tumor cells but not with extracts of another guinea pig tumor (line 10 hepatoma) or with extracts of normal guinea pig lymphoid cells.

Opposite effects of different strains or batches of same strain of BCG on in vitro generation of syngeneic and allogeneic antitumor cytotoxicity.

Pretreatment of mice with three batches of BCG Phipps strain 10 days before in vitro immunization of their spleen cells with syngeneic or allogeneic tumor cells augmented the levels of antitumor cytotoxicity (compared to the levels exhibited by in vitro immunized spleen cells from normal mice), whereas pretreatment with another batch of BCG Phipps strain or with a batch of BCG Tice strain suppressed antitumor cytotoxicity. The suppressive effects of these BCG vaccines could not be attributed to route of administration, dose of BCG, or percentage of colony-forming units in an inoculum. The effect of the interval between BCG pretreatment and in vitro immunization on the generation of antitumor cytotoxicity was evaluated; one BCG batch was of special interest, inasmuch as augmented cytotoxicity was obtained when the interval was short and suppressed cytotoxicity was obtained when the interval was long.

Protection against MOPC-315 plasmacytoma by immunization with C-type particle preparations.

Mice were given injections of C-type particles extracted from the ascitic fluid of plasmacytoma-bearing mice. These particles, extracted from MOPC-315 tumor-bearing mice and injected into BALB/c mice, protected them against challenge with MOPC-315 tumor cells. The protection was dependent upon tumor cell dose; 66% survival was observed with a lethal dose of tumor cells. No protection was observed against challenge with another plasmacytoma (S13). Attempts to protect mice against S-13 plasmacytoma by immunizing them with C-type particles originating from S-13 tumor-bearing mice were unsuccessful.

Immune RNA-mediated transfer of tumor antigen responsiveness to unresponsive peritoneal exudate cells from tumor-bearing animals.

Peritoneal exudate cells (PEC) from mice inoculated 5 to 7 days previously with 1 X 10(6) MOPC-315 plasmacytoma cells exhibit in vitro migration-inhibitory factor reactivity to soluble tumor-associated antigens. By 10 to 14 days of tumor growth, PEC from MOPC-315-bearing mice did not elicit migration-inhibitory factor when stimulated with MOPC-315 tumor-associated antigens but were still capable of migration-inhibitory factor production when stimulated with nontumor antigens. RNA-rich extracts prepared from 5- and 6-day postgrafting tumor bearers were capable of transferring tumor antigen reactivity to both normal PEC and PEC from unresponsive MOPC-315-bearing mice. On the other hand, RNA from unresponsive tumor bearers was incapable of transferring tumor antigen reactivity to normal mouse cells.

Some advantages of curing mice bearing a large subcutaneous MOPC-315 tumor with a low dose rather than a high dose of cyclophosphamide.

Mice bearing a large s.c. MOPC-315 tumor can be cured by a dose of cyclophosphamide (CY) ranging from 15 to 200 mg/kg. However, the low (15 mg/kg) and the high (200 mg/kg) doses mediate tumor eradication via different mechanisms. Tumor eradication by the low dose of drug requires the cooperation of the toxic effect of the drug and T-cell-dependent antitumor immunity. On the other hand, tumor eradication by the high dose of drug does not require the participation of antitumor immunity but depends primarily on the tumoricidal activity of the drug. Spleen cells from tumor-bearing mice treated with the low dose of CY exhibit an augmented antitumor immune potential, whereas spleen cells from tumor-bearing mice treated with the high dose of CY exhibit suppressed antitumor immune potential. More importantly, tumor-bearing mice treated with the low dose of drug are able to reject a challenge with 300 times the minimal lethal tumor dose given 1, 6, or 31 days after CY therapy, whereas mice treated with the high dose of drug are unable to reject such a challenge given within the same time intervals after CY therapy. Moreover, when mice bearing a large tumor are treated with the high dose of CY and subsequently challenged again with tumor cells to establish a Day 4 nonpalpable tumor, this tumor is less responsive to cure by combined chemoimmunotherapy than is a Day 4 nonpalpable tumor established in normal mice. Thus, although the high dose of CY can cure most mice bearing a large-size MOPC-315 tumor, it not only does not result in antitumor immunity, but it actually reduces the effectiveness of chemoimmunotherapy for a second tumor challenge. In contrast, mice cured with the low dose of CY exhibit long-lasting potent antitumor immunity.

Enhancement of the effectiveness of Lyt-2+ T-cells for adoptive chemoimmunotherapy by short-term exposure of tumor-bearer spleen cells to polyethylene glycol and/or melphalan.

Uncultured tumor-infiltrated spleen cells (TISpC) from mice bearing large (20-22 mm) s.c. MOPC-315 plasmacytomas were previously shown to be ineffective in bringing about the cure of mice bearing a nonpalpable (Day 4) tumor that had been treated with a subcurative dose (10 mg/kg) of cyclophosphamide (i.e., adoptive chemoimmunotherapy, ACIT) (M. B. Mokyr, J. C. D. Hengst, and S. Dray, Cancer Res., 42:974-979, 1982). Here we show that TISpC cultured for 5 days in the presence of inactivated MOPC-315 stimulator cells acquire some effectiveness in curing mice by ACIT, and this effectiveness is greatly enhanced if polyethylene glycol 6000 (PEG) is also added to the culture. The Lyt 2+ T-cells, and not the L3T4+ T-cells, are responsible for the effectiveness of the cultured TISpC in ACIT. In fact, the L3T4+ T-cells are apparently not required even during culture of TISpC for the generation of Lyt 2+ T-cells effective in ACIT. Although the TISpC cultured with MOPC-315 cells and PEG contained approximately twice as many Lyt 2+ cells as did TISpC cultured without PEG, the increase in the activity of the former cells is not due simply to the increase in the percentage of Lyt 2+ cells, but is most likely due to an increase in the percentage and/or activity of Lyt 2+ cells with specificity for MOPC-315-associated antigens. The effectiveness of TISpC cultured with MOPC-315 stimulator cells and PEG in ACIT can be enhanced even further by pretreatment of these cells with the immunomodulating agent melphalan (0.5 nmol/ml) prior to culture initiation. Thus, the above methods of culture render ineffective lymphoid cells effective in ACIT and are suitable for evaluation in protocols for human cancer therapy.

Enumeration and identification of human leukemic lymphocytes by their natural binding of bacteria.

The recently described property of bacteria to bind to human lymphocytes was used to distinguish between normal and chronic leukemic lymphocyte (CLL) populations. Strains of the following bacteria were used in this study: Arizona hinshawii, Escherichia coli strains 1 and 2, Bacillus globigii, Brucella melitensis, Corynebacterium diphtheriae strains 1 and 2, Corynebacterium xerosis, Sarcina lutea, Staphylococcus aureus, and Staphylococcus epidermidis. For identification of immunoglobulin-bearing lymphocytes, a strain of E. coli that did not bind to human lymphocytes was coated with anti-human light-chain antibody. Labeling of lymphocytes with bacteria was promoted by centrifugation. In the eight CLL patients studied, in which greater than 90% of the lymphocytes were leukemic cells, 52 to 77% were labeled by anti-human light-chain antibody-E. coli, 80 to 93% were labeled by Br. melitensis, and 78 to 95% were labeled by E. coli 1 compared to 11 to 24, 11 to 22, and 30 to 44%, respectively, in normal individuals, Thus, Br. melitensis, E. coli 1, and the anti-human light-chain antibody-E. coli may have diagnostic value for CLL. The percentage of the lymphocyte population that bound each of the other bacteria varied from patient to patient. Preliminary results obtained by studying the pattern of binding of E. coli 2, B. globigii, Sa. lutea, or S. aureus by leukemic lymphocytes suggest that categories of CLL patients may be distinguished by this method.

Complete and apparently specif local tumor regression using syngeneic or xenogeneic "tumor-immune" RNA extracts.

Syngeneic and xenogeneic RNA-rich extracts of lymphoid tissues were used in an immunotherapeutic regimen to treat strain 2 guinea pigs that were given intradermal injections of a uniformly lethal dose (1 x 10(6)) of line 10 diethylnitrosamine-induced transplantable hepatoma cells. When 1 X 10(7) syngeneic nonsensitive peritoneal exudate cells, 2.5 mg RNA from line 10-immune strain 2 guinea pigs or line 10-immune Rhesus monkeys, and 1.0 mg of a line-10 tumor-specific antigen preparation were injected s.c. under the tumor cells injected 5 days previously, complete local tumor regression in all treated animals was observed. If either nonsensitive peritoneal exudate cells, RNA, or line 10 tumor-specific antigen was omitted, or if Escherichia coli RNA or RNA from animals sensitized to a different tumor (line 1) was used, little or no tumor regression was observed, suggesting that the action of the RNA may have resulted in an antitumor response specific for the noplasm being treated. The long-term tumor-free survival of all treated animals indicates that the action of the RNA is systemic, since metastases are known to occur frequently by the time our therapeutic regimen was given. Also, in testing the biological activity of the "tumor-immune" RNA in the in vitro cell-migration-inhibition assay, both the syngeneic and xenogeneic RNA extracts could transfer tumor-specific immunological sensitivity, as demonstrated by the elaboration of migration-inhibitory factor by the RNA-treated nonsensitive peritonial exudate cells in the presence of the line 10 tumor-specific antigen.

Mode of action of polyethylene glycol 6000 in potentiating the in vitro generation of antitumor cytotoxicity by MOPC-315 tumor bearer spleen cells.

Some of the possible mechanisms by which polyethylene glycol (PEG) augments the ability of MOPC-315 tumor bearer spleen cells to mediate in vitro antitumor cytotoxicity were evaluated. The level of antitumor cytotoxicity obtained in 5-day cultures of tumor bearer spleen cell suspensions correlated inversely with the percentage of Trinitrophenol (TNP)-rosettable cells (presumably metastatic tumor cells) present in the spleen. The kinetics of decrease in the percentage of TNP-rosettable cells coincided with the appearance of antitumor cytotoxicity. In addition, PEG was shown to interfere with the ability of viable tumor cells to suppress the in vitro generation of antitumor cytotoxicity in normal spleen cells cultured with mitomycin C-treated tumor cells. However, the decrease in the content of TNP-rosettable cells and the concurrent increase in the level of antitumor cytotoxicity were not due to direct cytotoxic and/or cytostatic effects of PEG on tumor cells. Spleen cells cultured in the presence of PEG had an increased rate of [3H]thymidine incorporation and proliferation compared to spleen cells cultured in the absence of PEG. However, the PEG-induced decrease in the percentage of TNP-rosettable cells either preceded or occurred at the same time that the PEG-induced increase in spleen cell number was observed. Therefore, spleen cell proliferation can at best explain only partially the PEG-induced decrease in the content of TNP-rosettable cells, and other mechanisms for the decrease must be considered.

Effect of polyethylene glycol 6000 on the generation of antitumor cytotoxicity in MOPC-315 tumor bearer spleen cells cultured in the presence or absence of inactivated stimulator tumor cells.

Noncytotoxic spleen cells from BALB/c mice bearing 15- to 26-mm (but not 29-mm) s.c. MOPC-315 tumors that were cultured in medium containing 2% polyethylene glycol 6000 (PEG) developed substantial levels of anti-MOPC-315 cytotoxicity as assayed by 51Cr release. The level of cytotoxicity obtained increased with progression of tumor growth. Addition of mitomycin C-treated stimulator tumor cells and PEG to the culture of tumor bearer spleen cells resulted in augmentation of antitumor cytotoxicity to a level that was greater than the sum of the levels of cytotoxicity exhibited by spleen cells cultured in the presence of either mitomycin C-treated tumor cells or PEG. Maximal levels developed when the spleen cells were cultured for 5 to 6 days in 2% PEG at a responder/stimulator cell ratio of 15/1 or 30/1. Tumor bearer spleen cells that were cultured in PEG with or without added MOPC-315 stimulator cells exhibited strong anti-MOPC-315 cytotoxicity but were virtually noncytotoxic to allogeneic EL4 and syngeneic blast cells. Furthermore, these spleen cells were far superior to spleen cells cultured without PEG in mediating in in vivo antitumor activity in the local adoptive transfer assay. Thus, tumor bearer spleen cells cultured in the presence of PEG might be useful in immunotherapeutic regimens requiring histocompatible cells with augmented antitumor cytotoxicity but devoid of reactivity against normal cells.

Cooperation between cyclophosphamide tumoricidal activity and host antitumor immunity in the cure of mice bearing large MOPC-315 tumors.

A single i.p. injection of cyclophosphamide (CY), 15 mg/kg, was shown previously to be curative if administered to BALB/c mice 10 to 16 days post-MOPC-315 tumor inoculation when the tumors reached 20 to 25 mm (large tumors) but not if administered to mice four days post-tumor inoculation when their tumors were nonpalpable. Here we show that the curative effect of CY, 15 mg/kg, for mice bearing large tumors was not due solely to the tumoricidal activity of the drug, because three or four days after therapy, when the CY had been cleared from the circulation, viable proliferative tumor cells were present in the primary s.c. tumor site. During tumor regression, the tumors became heavily infiltrated by mononuclear cells. Following therapy, mice bearing large tumors exhibited an active antitumor response, as illustrated by their ability to reject a tumor challenge with 350-fold the minimum lethal tumor dose given as early as 24 hr posttherapy. That the curative effect of CY, 15 mg/kg, for mice bearing large tumors required the presence of T-cell-dependent antitumor immunity (cellular and/or humoral), was indicated by the fact that tumor regression was abrogated by treatment of the tumor bearers with anti-thymocyte serum. Thus, CY drug tumoricidal activity and host antitumor immunity cooperated in the eradication of large MOPC-315 tumors.

Generation of anti-MOPC-315 cytotoxicity in uneducated or in vitro educated spleen cells from normal or MOPC-315 tumor-bearing mice pretreated in vivo with Bacillus Calmette-Guérin.

Cultured spleen cells from normal or MOPC-315 tumor-bearing BALB/c mice that were pretreated in vivo with Bacillus Calmette-Guérin (BCG) exhibited in vitro cytotoxicity against MOPC-315 plasmacytoma. In vitro education of BALB/c spleen cells from normal or tumor-bearing mice by cocultivation with mitomycin C-treated MOPC-315 stimulator cells also resulted in antitumor cytotoxicity. The combination of BCG pretreatment of donor mice with the in vitro education of their spleen cells resulted in a level of anti-MOPC-315 cytotoxicity that was greater than the sum of the levels of cytotoxicity exhibited by spleen cells subjected to either process alone. The levels of cytotoxicity exhibited by educated or uneducated spleen cells from BCG-pretreated mice were dependent on the dose of BCG used and on the time interval between in vivo pretreatment and the initiation of in vitro culture. Thus, our findings suggest that educated spleen cells from tumor-bearing hosts that were pretreated with BCG might be useful in immunotherapeutic regimens requiring histocompatible cells with augmented antitumor cytotoxicity.

Role of antitumor immunity in cyclophosphamide-induced rejection of subcutaneous nonpalpable MOPC-315 tumors.

Previously, we had reported that a single i.p. injection of 15 mg cyclophosphamide (CY) per kg cured most mice bearing large MOPC-315 tumors (20 to 25 mm; Day 12 to Day 16 tumors) but rarely cured mice bearing nonpalpable tumors (Day 4 tumors). Also, mice that were not cured if treated with CY, 15 mg/kg, when they had nonpalpable tumors could not be cured if treated again with CY, 15 mg/kg, when they had large tumors (14). Here, we show that CY therapy with 15 mg/kg at early stages of tumor growth did not lead to alteration in the biology of the tumor so as to cause an increased resistance to CY-tumoricidal effects, increased resistance to immune lysis, and/or decreased immunogenicity. Treatment of nonpalpable tumor bearers with CY, 15 mg/kg, prior to in vitro immunization of their spleen cells did not reduce the ability of the spleen cells to generate antitumor cytotoxicity in vitro. However, the level of antitumor cytotoxicity generated was lower than that exhibited by in vitro-immunized spleen cells from mice treated with CY, 15 mg/kg, when they had large tumors. With CY, 15 mg/kg, mice bearing nonpalpable tumors could be cured in two ways: (a) by treating a mouse bearing a nonpalpable tumor in the presence of a contralateral large tumor; (b) by adoptive transfer of immune spleen cells given 1 day post-CY therapy. Both procedures resulted in higher levels of antitumor immunity which was apparently responsible for the cure of the mice in cooperation with CY. Thus, the ineffectiveness of CY therapy with 15 mg/kg at early stages of tumor growth correlated with the presence of relatively low levels of host antitumor immunity.

Rosetting of antibody-sensitized tumor cells with anti-immunoglobulin antibody-coated erythrocytes by a new method for detecting antigens on cells.

Tumor cells were treated with rabbit antibody to tumor-associated cell surface antigens and tested with erythrocytes coated with antibody specific for the sensitizing rabbit immunoglobulin. The sensitized tumor cells formed rosettes with the indicator cells. By this method, we confirmed that line 1 and line 10 hepatoma cells (from two tumors independently induced by diethylnitrosamine in strain 2 guinea pigs) bear antigens not present on normal liver cells. We also confirmed that line 1 and line 10 cells bear antigenically different tumor-associated cell surface antigens. This method appears simpler than other serological methods for detecting tumor-associated cell surface antigens on tumor cells. Also, this method may be a general one for detecting and enumerating cells bearing surface antigens.

Ability of cyclophosphamide in the absence of cross-linking activity to exert the immunomodulatory effect required for the cure of mice bearing a large MOPC-315 tumor.

We have previously shown that mice bearing a late-stage, large primary MOPC-315 plasmacytoma and extensive metastases can be cured by a low dose of the bifunctional alkylating drug, cyclophosphamide (BiCY) (J.C.D. Hengst et al., Cancer Res., 40: 2135-2141, 1980). Here we show that therapy with the monofunctional form of cyclophosphamide (MoCY) can also cure such mice. However, a dose of at least 150 mg of MoCY per kg is required to approximate the curative effectiveness of the lowest curative dose of BiCY, i.e., 15 mg/kg. This need for a 10-fold higher dose of MoCY is due, at least in part, to the 10-fold lower direct tumoricidal and/or tumoristatic activity of MoCY compared to BiCY. Consequently, a 10-fold higher dose of MoCY is required to directly reduce the tumor burden to the level reduced by 15 mg of BiCY per kg. Other than dose, the therapy of the mice with 150 mg of MoCY per kg was similar in its essential features to that shown previously for therapy with 15 mg of BiCY per kg (J.C.D. Hengst et al., Cancer Res., 40: 2135-2141, 1980; J.C.D. Hengst et al., Cancer Res., 41:2163-2167, 1981; Q-W. Ye et al., Cancer Immunol. Immunother., 16:162-169, 1984; Q-W. Ye and M.B. Mokyr, Cancer Res., 44: 3873-3879, 1984; M.B. Mokyr and S. Dray, Cancer Res., 43: 3112-3119, 1983), namely: (a) the drug does not directly eradicate all tumor cells; (b) host T-cell-dependent antitumor immunity is also required for the curative effect; (c) the therapy of tumor bearers leads to the rapid appearance of an augmented antitumor immune potential in their hitherto immunosuppressed spleen; and (d) the cured mice are resistant to a subsequent challenge with at least 300-fold the minimal lethal tumor dose. Thus, cross-linking is not an essential property for the immunomodulatory activity of BiCY nor for its direct antitumor effect. However, in the presence of cross-linking activity, a much lower dose of drug is effective.

Lysis of antigenically unrelated tumor cells mediated by Lyt 2+ splenic T-cells from melphalan-cured MOPC-315 tumor bearers.

We have previously shown that mice cured of a large MOPC-315 tumor following low-dose melphalan (L-phenylalanine mustard, L-PAM) therapy can exert, upon challenge with MOPC-315 tumor cells, an antitumor effect against innocent bystander tumor cells present within the same tumor site (Barker, E., and Mokyr, M.B. Cancer Immunol. Immunother., 25: 215-224, 1987). Here we show that T-cells are important for the MOPC-315-induced rejection of MOPC-104E tumor cells present within the same site. To further characterize the innocent bystander killing activity exerted by L-PAM-cured MOPC-315 tumor bearers upon stimulation with MOPC-315 tumor cells, we established the in vitro conditions under which lymphoid cells from L-PAM-cured MOPC-315 tumor bearers can exert an antitumor effect against innocent bystanders. Specifically, we established that spleen cells from mice that just completed the rejection of a large MOPC-315 tumor following low-dose L-PAM therapy can, upon stimulation with MOPC-315 tumor cells, bring about the killing of antigenically unrelated tumor cells in a 12-h 51Cr release assay. The magnitude of lysis of EL4 and WEHI 22.1 tumor cells by MOPC-315 in vitro-immunized (IVI) spleen cells from L-PAM-cured MOPC-315 tumor bearers can be substantially enhanced upon reexposure of the spleen cells to MOPC-315-associated antigens during the 12-h 51Cr release assay. The lysis of innocent bystander tumor cells by these MOPC-315-IVI spleen cells was found to be mediated by T-cells of the Lyt 2 and not the L3T4 phenotype. These Lyt 2+ T-cells did not appear to mediate their lytic activity for innocent bystander tumor cells via effector macrophages, since a drastic reduction in macrophage frequency among the MOPC-315-IVI spleen cells just prior to assessing the lytic activity of the spleen cells did not reduce, but actually enhanced, the magnitude of EL4 lysis. In addition, a Lyt 2+ T-cell clone derived from mice cured of a large MOPC-315 tumor by a low dose of drug was capable, upon stimulation with MOPC-315 tumor cells, of exerting a potent lytic effect against EL4 and WEHI 22.1 tumor cells in the 12-h 51Cr release assay. Thus, Lyt 2+ T-cells independent of effector macrophages are responsible for lysis of innocent bystander tumor cells by MOPC-315-IVI spleen cells from L-PAM-cured MOPC-315 tumor bearers.

Advantages of adoptive chemoimmunotherapy with polyethylene glycol-cultured, antigen-activated, tumor-infiltrated spleen cells for the complete eradication of lethal MOPC-315 plasmacytomas.

The incorporation of polyethylene glycol-6000 (PEG) into the culture media of tumor-infiltrated spleen cells (TISpC) and MOPC-315 stimulator tumor cells at a responder to stimulator cell ratio of 30/1 had been shown to lead to the appearance of CD8+ T-cells that were effective in adoptive chemoimmunotherapy (ACIT) of mice bearing a barely palpable MOPC-315 tumor (J. A. Wise, M. B. Mokyr, and S. Dray, Cancer Res., 49:3613-3619, 1989). Here we show that in the presence of substantially fewer added stimulator tumor cells (responder to stimulator cell ratio, 100/1), the inclusion of PEG in the cultures of TISpC also enhanced the appearance of cells that were highly effective in curing such mice by ACIT. Moreover, these PEG-cultured TISpC were more effective in ACIT than TISpC cultured in the presence of an optimal concentration of recombinant interleukin-2 (60 IU/ml). The potency of the tumor-eradicating activity of the PEG-cultured TISpC in ACIT was further illustrated by their ability to cause the complete regression of a large (20-22 mm) s.c. MOPC-315 tumor in conjunction with a dose of drug that by itself did not cause tumor regression. PEG-cultured TISpC that were effective against MOPC-315 tumor cells in an antigen-specific manner. In fact, PEG-cultured TISpC were more effective than recombinant interleukin-2-cultured TISpC, not only in ACIT, but also in their ability to lyse MOPC-315 tumor cells in vitro. Thus, a direct specific lytic activity against the tumor by cytotoxic T-lymphocytes is the apparent mechanism through which the complete regression of the large tumor burden is brought about by the PEG-cultured TISpC. Finally, we suggest that the incorporation of PEG to render ineffective lymphoid cells effective in ACIT may offer some advantages compared with the incorporation of recombinant interleukin-2 and may be suitable for protocols to generate human cytotoxic cells for cancer therapy when there are relatively low numbers of available tumor cells.