Effect of chemotherapy and immunotherapy on tumor-specific immunity in melanoma.

The effects of chemotherapy, with nitrosoureas or dimethyl-triazeno-imidazole-carboxamide (DTIC), or immunotherapy with Bacillus Calmette-Guérin (BCG), on cell-mediated immunity (CMI), and serum blocking factor (BF) to melanoma cells were studied in 23 patients. Studies were performed with autologous or allogenic melanoma target cells obtained from recent biopsy, in 16 mm diameter plastic wells. Assays for lymphocyte-mediated cytotoxicity and BF were performed at weekly intervals over the course of 3-4 mo, with some studies extending beyond 3 yr. The specificity of cytotoxicity was good with these methods. Nine patients given nitrosoureas, predominantly methyl-chloroethyl-cyclohexyl-nitrosourea, showed a transient decline in CMI from 42.2 to 14% 3 wk after administration of a single dose of the agent, with a rapid recovery within 1 week. 10 patients given 5-day courses of DTIC at 3-wk intervals showed no decline in CMI after two courses, and 7 of the 10 had no decline even after three courses. Three of the four patients who achieved a remission lost BF previously present: BF reappeared in both patients studied during a subsequent relapse. BCG intradermally or intralesionally elevated CMI within 2 mo after initiation of therapy, but despite continuation of the injections CMI returned to base line in all but two of the nine patients studied. These results indicate that chemotherapy for melanoma with nitrosoureas or DTIC at these schedules is not profoundly immunosuppressive towards tumor-specific immunity, as measured by our procedures. Putative immunotherapy with BCG at these schedules was likewise only transiently stimulatory.

Stimulation of lymphoid cells by components of BCG.

BCG was fractionated into a delipidated mycobacterial cell fraction (DMC) and lipid by exhaustive chloroform-methanol extraction. The effects of these fractions were tested on mouse spleen cells, nonadherent spleen cells (lymphocytes), thymus cells, and adherent spleen cells (macrophages) in vitro and were compared with effects of the whole bacilli and a methanol-extraction residue (MER). Tritiated thymidine incorporation into spleen cells, purified spleen lymphocytes, and thymus cells was measured as an indicator of activity on these cells; lymphocyte-activating factor (LAF) production was used to measure activation of macrophages. DMC and MER were at least equivalent to, and often exceeded, whole BCG in their stimulation of spleen cells and spleen lymphocytes. DMC was a poor thymic mitogen in contrast to MER, which was as strong as BCG in this regard. Lipid was far less effective a mitogen for all cells tested, and failed to augment the effectiveness of DMC on thymus cells when both were present in the incubation mixture. LAF production was significantly increased by whole BCG (18-fold above controls), whereas each fraction increased production threefold to sixfold. These in vitro results seemed to reflect the known in vivo activity of BCG and its components and suggest further antitumor applications.

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.

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.

Cyclophosphamide-induced appearance of immunopotentiating T-cells in the spleens of mice bearing a large MOPC-315 tumor.

Following low-dose cyclophosphamide (CY) therapy (15 mg/kg) of mice bearing a large MOPC-315 tumor, the suppressive activity of their Sephadex G-10-adherent spleen cells (primarily macrophages) is overcome. Accordingly, when Sephadex G-10-adherent spleen cells from CY-treated tumor-bearing mice are added to the in vitro immunization culture of normal spleen cells, they do not suppress but actually bring about the generation of an augmented level of antitumor cytotoxicity. The ability to enhance the generation of antitumor cytotoxicity appears in the Sephadex G-10-adherent spleen cell population by Day 5 post-CY therapy of tumor-bearing mice and persists for at least 55 days; no such immunopotentiation is observed following administration of a low dose of CY to normal mice. In order for the immunopotentiating cells from CY-treated tumor-bearing mice to be effective in enhancing the generation of antitumor cytotoxicity, they must be added to the immunization culture of normal spleen cells no later than Day 3 (out of the 5 days) post-culture initiation. The CY-induced immunopotentiating activity resides in the T-cells, as is evident from the following observations. The immunopotentiating activity was abolished when the Sephadex G-10-adherent spleen cell population from CY-treated tumor-bearing mice was depleted of T-cells by anti-Thy 1.2 plus complement but not when this adherent spleen cell population was depleted of macrophages by carbonyl iron and magnet. Moreover, the immunopotentiating activity was also present in a population of CY-treated tumor-bearer spleen cells highly enriched for T-cells by passage through nylon wool columns. Thus, low-dose CY therapy overcomes the immunosuppressive activity of macrophages and induces the appearance of T-cell-mediated immunopotentiating activity, thereby leading to the development of an augmented level of antitumor cytotoxicity that can cooperate effectively with the tumoricidal activity of CY in the eradication of a late-stage, large s.c. tumor and extensive metastases.

Some characteristics of the cyclophosphamide-induced immunopotentiating cells in the spleen of mice bearing a large MOPC-315 tumor.

We have shown previously (Ye, Q-W., and Mokyr, M. B. Cancer Res., 44: 3873-3879, 1984) that, following low-dose cyclophosphamide (CY) therapy (15 mg/kg) of mice bearing a large s.c. MOPC-315 tumor and extensive metastases, T-cell-dependent immunopotentiating activity appears in their hitherto immunosuppressive Sephadex G-10-adherent spleen cell population. Here we show that the CY-induced immunopotentiating T-cells express the Lyt 1, Lyt 2, and L3T4 phenotypes. The phenotype of the immunopotentiating T-cells was deduced from our observations that depletion of Lyt 1+, Lyt 2+, or L3T4+ cells from the Sephadex G-10-adherent spleen cell population of CY-treated tumor bearers abolished the ability of the adherent cells to enhance the generation of antitumor cytotoxicity when added to the in vitro immunization culture of normal spleen cells. Moreover, admixture of a Sephadex G-10-adherent cell population depleted of Lyt 2+ cells with a Sephadex G-10-adherent cell population depleted of L3T4+ cells failed to restore the immunopotentiating activity, indicating that T-cells that are apparently expressing simultaneously the Lyt 2 and L3T4 antigens are required for the exertion of the CY-induced immunopotentiating activity. The CY-induced immunopotentiating T-cells from MOPC-315 tumor bearers brought about the appearance of enhanced antitumor cytotoxicity not only against the MOPC-315 tumor cells, but also against two other syngeneic plasmacytomas, with surface immunoglobulin of a different class and antigenic specificity than the MOPC-315 tumor cells, as well as against a variant MOPC-315 tumor line which lacks surface immunoglobulin. The CY-induced immunopotentiating T-cells did not enhance the appearance of antitumor cytotoxicity against a syngeneic (WEHI 22.1) or an allogeneic (EL4) tumor of T-cell origin nor against the natural killer-sensitive YAC-1 cells. Thus, L3T4+, Lyt2+ T-cells from CY-treated MOPC-315 tumor bearers enhance the generation of antitumor cytotoxicity that is directed against plasmacytoma shared antigens other than immunoglobulins.

Importance of Lyt-2+ T-cells in the resistance of melphalan-cured MOPC-315 tumor bearers to a challenge with MOPC-315 tumor cells.

We have previously shown that mice bearing a large MOPC-315 tumor can be cured by a low dose of melphalan (L-phenylalanine mustard; L-PAM) if T-cell-dependent antitumor immunity also aids in tumor eradication (S. Ben-Efraim et al., Cancer Immunol. Immunother., 15: 101-107, 1983). Here we describe the phenotype of the T-cells that are responsible for the potent antitumor immunity exhibited by the L-PAM cured MOPC-315 tumor bearers. Initially, we identified the subset of T-cells responsible for the ability of spleen cells from L-PAM-cured MOPC-315 tumor bearers to neutralize tumor growth in the local adoptive transfer assay. The tumor-neutralizing activity of spleen cells from L-PAM-cured tumor bearers was drastically reduced when the spleen cells were depleted either in vitro or in vivo of Lyt-2+ cells. On the other hand, the tumor-neutralizing activity of spleen cells from L-PAM-cured MOPC-315 tumor bearers was not reduced, but actually enhanced, when the spleen cells were depleted either in vitro or in vivo of L3T4+ cells. The Lyt-2+ T-cells, and not the L3T4+ T-cells, were also found to be important for the ability of the intact L-PAM-cured MOPC-315 tumor bearers to reject a challenge with MOPC-315 tumor cells. Specifically, treatment of L-PAM-cured MOPC-315 tumor bearers with monoclonal anti-Lyt-2.2 antibody drastically reduced the ability of the mice to reject the tumor challenge. In contrast, treatment of the L-PAM-cured MOPC-315 tumor bearers with monoclonal anti-L3T4 antibody did not interfere with the ability of the mice to reject the tumor challenge, yet the same protocol of anti-L3T4 antibody treatment abolished the ability of splenic T-cells to provide help for the generation of a primary T-cell-dependent antibody response. The resistance of the L-PAM-cured MOPC-315 tumor bearers to challenge with MOPC-315 tumor cells was also dependent on the participation of carrageenan-sensitive effector cells, most likely macrophages, in tumor eradication. However, the immunity mediated by the T-cells, independent of carrageenan-sensitive effector cells, was extremely powerful and sufficient for the rejection of a tumor challenge with at least 300-fold, and possibly even 3000-fold, the minimal lethal dose of MOPC-315 tumor cells for 100% of normal mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Importance of tumor-specific cytotoxic CD8+ T-cells in eradication of a large subcutaneous MOPC-315 tumor following low-dose melphalan therapy.

We have previously demonstrated that depletion of CD8+ T-cells by the use of a monoclonal anti-Lyt-2.2 antibody abolishes the curative effectiveness of low-dose melphalan (L-phenylalanine mustard; L-PAM) therapy for BALB/c mice bearing a large (greater than or equal to 20 mm) s.c. MOPC-315 tumor and extensive metastases (Mokyr et al., Cancer Res., 49: 4597-4606, 1989). Here we show that as a consequence of low-dose L-PAM therapy, CD8+ T-cells accumulate in the s.c. tumor nodules of MOPC-315 tumor bearers. Specifically, an 80-fold increase in the number of CD8+ T-cells was seen within 5 days after the chemotherapy. Treatment of MOPC-315 tumor bearers with low-dose L-PAM in conjunction with monoclonal anti-Thy-1.2 or anti-Lyt-2.2 antibody, in contrast to treatment with monoclonal anti-L3T4 antibody, prevented the appearance of the massive CD8+ T-cell infiltrate in the s.c. tumor nodules. Fresh CD8+ T-cells derived from s.c. MOPC-315 tumor nodules that were regressing as a consequence of low-dose L-PAM therapy exhibited a potent direct lytic activity against the MOPC-315 plasmacytoma in a short-term in vitro assay. The specificity of the lytic activity exhibited by the CD8+ T-cells was illustrated not only by the inability of the CD8+ T-cells to lyse two antigenically unrelated thymomas (the WEHI 22.1 and the EL-4) and a natural killer-sensitive lymphoma (the YAC-1), but also by their relatively weak lytic activity against an antigenically related plasmacytoma (the MOPC-104E). Thus, CD8+ T-cells that infiltrate the s.c. tumor nodules of MOPC-315 tumor bearers following low-dose L-PAM therapy most likely exploit a CTL-type lytic mechanism to eradicate at least part of the large tumor burden not eliminated by the direct antitumor effects of the drug.

Analysis and reversal of the inhibition of cytophilic antibody receptors produced by antibody.

Administration of hyperimmune antibody to leukemia L1210 to allogeneic mice inhibited the development of macrophage-mediated immunity to L1210 in those hosts. In contrast to immunized mice, animals pretreated with antibody showed rapid activation of their peritoneal macrophages, followed by their disappearance and the inability of the residual peritoneal monocytic cells to attach L1210 cells even in the presence of proved cytophilic antibody to L1210. The inhibitory activity of the antibody, which resided entirely in its IgG2 fraction, was manifested only when the specific antigen (L1210 cells) was also injected within 2 days. Pretreatment with antibody to a different leukemia, EL4, failed to inhibit the monocytic uptake of L1210, but it did inhibit uptake of EL4 by monocytes if injected with its homologous antigen. Restoration of the functional capacity of macrophages was accomplished by injecting 1 X 10-7 bone marrow cells i.v. into "suppressed" mice, but 1.5 X 10-7 thymocytes failed to correct the defect. Significantly, thymocytes antagonized the restorative capability of bone marrow cells when they were injected concomitantly. These results indicate that specific inhibition of cytophilic antibody receptors on monocytes could be accomplished through a direct mechanism involving activation and exhaustion of macrophages and an indirect mechanism, perhaps mediated through "suppressor" thymus-derived cells. Although enhancement of the growth of leukemia cells did not occur, several parallels exist in mice with enhanced growth of different tumors. This inhibiotry phenomenon may thus represent another instance of "blocking" in tumor immunity, where the target of suppressive antibody-antigen is the macrophage as well as the lymphocyte.

Interleukin 2 requirement for the in vitro generation of antitumor cytotoxicity by thymocytes from melphalan-cured MOPC-315 tumor bearers.

We have previously shown that enhanced antitumor cytotoxicity is generated when thymocytes from melphalan (L-phenylalanine mustard; L-PAM)-treated MOPC-315 tumor bearers, but not thymocytes from normal mice, are added to the immunization culture of syngeneic normal spleen cells and MOPC-315 tumor cells (Bartik et al., Cancer Res., 47: 4848-4855, 1987). Here we show that normal spleen cells produce, upon stimulation with MOPC-315 tumor cells, helper-like factors which are sufficient for thymocytes from L-PAM-treated MOPC-315 tumor bearers, but not for thymocytes from normal mice, to develop antitumor cytotoxicity in response to stimulation with MOPC-315 tumor cells. Since one of the helper-like factors produced by in vitro-immunized spleen cells is interleukin 2 (IL-2), we assessed the exogenous IL-2 requirements for the development of anti-MOPC-315 cytotoxicity in thymocytes from L-PAM-treated MOPC-315 tumor bearers, relative to thymocytes from normal mice. Thymocytes from L-PAM-treated MOPC-315 tumor bearers were found to require a 10-fold lower concentration of recombinant IL-2 (rIL-2) than thymocytes from normal mice in order to develop antitumor cytotoxicity in response to stimulation with MOPC-315 tumor cells. The concentration of rIL-2 required for the development of anti-MOPC-315 cytotoxicity by thymocytes from L-PAM-treated MOPC-315 tumor bearers was also 10-fold lower than the concentration of rIL-2 required by thymocytes from untreated MOPC-315 tumor bearers or thymocytes from L-PAM-treated normal mice. In addition, at any concentration of rIL-2 employed, thymocytes from L-PAM-treated MOPC-315 tumor bearers developed a higher level of anti-MOPC-315 cytotoxicity than did thymocytes from normal mice, L-PAM-treated normal mice, or untreated MOPC-315 tumor bearers. The enhanced antitumor cytotoxicity exhibited by thymocytes from L-PAM-treated MOPC-315 tumor bearers, following in vitro stimulation with MOPC-315 tumor cells plus rIL-2, was evident not only against MOPC-315 tumor cells but also against other syngeneic plasmacytomas but not an allogeneic thymoma. In addition, thymocytes from L-PAM-treated MOPC-315 tumor bearers required less rIL-2 than thymocytes from normal mice to develop antitumor cytotoxicity in response to stimulation with MOPC-315-associated antigens but not in response to stimulation with an allogeneic antigenically unrelated thymoma (EL4).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

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.

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.

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.

Melphalan-induced enhancement of antitumor immune reactivity in thymocytes of adult BALB/c mice bearing a large MOPC-315 tumor.

At no stage of tumor growth are thymocytes from MOPC-315 tumor bearers capable of bringing about the generation of enhanced antitumor cytotoxicity when added to immunization cultures of syngeneic normal spleen cells and "autochthonous" tumor cells. However, by Day 7 after low-dose melphalan [L-PAM (L-phenylalanine mustard)] therapy of mice bearing a large (greater than or equal to 20 mm) s.c. MOPC-315 tumor, their thymocytes exhibit such activity and it persists for at least 17 additional days. The ability of thymocytes from L-PAM-treated MOPC-315 tumor bearers to bring about the generation of enhanced antitumor cytotoxicity when added to immunization cultures of normal spleen cells and MOPC-315 tumor cells is evident over a 10-fold range of responder/stimulator cell ratios, and requires the presence of the thymocytes within the first day after initiation of the 5-day immunization cultures. In addition, immunization cultures containing normal spleen cells and thymocytes from L-PAM-treated MOPC-315 tumor bearers exhibit enhanced antitumor cytotoxicity by Day 4 after culture initiation that persists for at least 3 additional days. Thymocytes from L-PAM-treated MOPC-315 tumor bearers are able to bring about the generation of enhanced antitumor cytotoxicity only in response to stimulation with autochthonous tumor cells but not in response to stimulation with unrelated allogeneic EL4 tumor cells. The apparent specificity of the enhanced antitumor immune reactivity of thymocytes from L-PAM-treated MOPC-315 tumor bearers is not the result of extensive metastasis of tumor cells to the thymus. In fact, no tumor cells were found in the thymuses of MOPC-315 tumor bearers with methods that can detect 1 X 10(3) tumor cells, indicating that if MOPC-315 tumor cells metastasize at all into the thymus, the thymuses of mice bearing a large MOPC-315 tumor contain fewer than 1 X 10(3) tumor cells. Thus, thymocytes from mice which are engaged in the eradication of a large MOPC-315 tumor display enhanced antitumor immunity in response to stimulation with the autochthonous tumor cells. Such thymocytes may prove important to the outcome of low-dose L-PAM therapy for mice bearing a large MOPC-315 tumor, since the low-dose chemotherapy requires the contribution of T-cell-dependent antitumor immunity for its therapeutic effectiveness.

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.

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.