Enhanced expansion of the thymic CD8+ cell subset as a potential mechanism for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers

Summary We have previously shown that thymocytes from low-dose melphalan (l-phenylalanine mustard)-treated MOPC-315-tumor-bearing mice (melphalan TuB) are able to generate an enhanced level of anti-MOPC-315 cytotoxicity, as compared to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated or low-dose melphalan-treated normal mice, upon in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2). Here we show that the generation of enhanced anti-MOPC-315 cytotoxicity by melphalan TuB thymocytes depends on the ability of the thymocytes to proliferate. In addition, the ability of melphalan TuB thymocytes to generate an enhanced level of anti-MOPC-315 cytotoxicity correlated with their ability to proliferate more readily than thymocytes from untreated tumor-bearing mice and thymocytes from untreated or melphalan-treated normal mice in response to stimulation with MOPC-315 tumor cells plus a low concentration of rIL-2. Moreover, although fresh melphalan TuB thymocytes do not contain a higher percentage of phenotypically mature cells (i.e., CD4^–/CD8⁺ or CD4⁺/CD8^–) than do thymocytes from normal mice or untreated tumor-bearing mice, after a 5-day culture with both MOPC-315 tumor cells and a low concentration of rIL-2, cultures of thymocytes from melphalan TuB contained a much higher percentage of CD4^–/CD8⁺ (but not CD4⁺/CD8^–) cells than did cultures of thymocytes from the other two sources. Since CD4^–/CD8⁺ cells were previously shown to be responsible for the exertion of antitumor cytotoxicity by thymocytes stimulated with MOPC-315 in vitro, our results indicate that the enhanced antitumor cytotoxicity exerted by melphalan TuB thymocytes following in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of rIL-2 is due, at least in part, to an expansion of the pool of CD4^–/CD8⁺ effector cells.Supported by research grant CA-35 761 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy degreeSupported by career development award CA-01 350 from the National Cancer Institute     

Characterization of the exogenous interleukin-2 requirements for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers

We have shown previously that thymocytes from MOPC-315-tumor-bearing mice treated with low-dose melphalan (l-phenylalanine mustard) (l-PAM TuB mice) are superior to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated normal mice or normal mice treated with low-dose melphalan in their ability to generate an antitumor cytotoxic response following 5-day in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2) [Mokyr MB, Bartik MM, Ahn M-C (1989) Cancer Res 49; 870]. Here we characterize the rIL-2 requirements for the generation of enhanced antitumor cytotoxicity byl-PAM TuB thymocytes relative to normal thymocytes upon in vitro stimulation with MOPC-315 tumor cells. Specifically, we show that delaying the addition of a low concentration of rIL-2 to 5-day in vitro stimulation cultures of thymocytes resulted in a progressive decline in the generation of antitumor cytotoxicity by both normal andl-PAM TuB thymocytes. However, even when rIL-2 was added on day 2 after culture initiation, thymocytes froml-PAM TuB mice generated a more potent antitumor cytotoxicity than did thymocytes from normal mice. In addition, when rIL-2 was added at the time of culture initiation, replacement of the conditioned medium with fresh medium lacking rIL-2 on day 3 of the 5-day in vitro stimulation culture period eliminated the ability of normal thymocytes, and reduced (but did not eliminate) the ability ofl-PAM TuB thymocytes, to generate a significant level of antitumor cytotoxicity. A low concentration of fresh rIL-2 was sufficient to restore completely the generation of antitumor cytotoxicity by normal orl-PAM TuB thymocytes when added to the stimulation cultures immediately after the removal of the rIL-2-containing conditioned medium. The same low concentration of rIL-2 was also sufficient for restoring the generation of antitumor cytotoxicity by cultures ofl-PAM TuB thymocytes, but not normal thymocytes, from which the rIL-2-containing medium was removed 1 day earlier. At the same time, conditioned medium from stimulation cultures ofl-PAM TuB thymocytes was not superior to conditioned medium from stimulation cultures of normal thymocytes in supporting the generation of antitumor cytotoxicity by either normal orl-PAM TuB thymocytes. Thus, the enhanced lytic activity generated byl-PAM TuB thymocytes, relative to normal thymocytes, upon stimulation with MOPC-315 tumor cells and a low concentration of rIL-2, does not appear to be the result of enhanced production of helper-like factors byl-PAM TuB thymocytes.Supported by research grant CA-35 761 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy degreeSupported by Career Development Award CA-01350 from the National Cancer Institute     

Eradication of a large MOPC-315 tumor in athymic nude mice by chemoimmunotherapy with Lyt2+ splenic T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor

Summary We have previously shown that spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose (2.5 mg/kg) melphalan (l-phenylalanine mustard) therapy are effective in preventing tumor progression upon adoptive transfer into BALB/c mice bearing a barely palpable tumor that had been treated with a subcurative dose of melphalan [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that such spleen cells in conjunction with a subcurative dose of drug (adoptive chemoimmunotherapy, ACIT) can cause the complete regression of a large (15–20 mm) s.c. MOPC-315 tumor in a large percentage of T-cell-deficient (athymic nude) tumor-bearing mice. Spleen cells that were effective in ACIT of athymic nude mice displayed in vitro a substantial direct lytic activity against MOPC-315 tumor cells, and the lytic activity was greatly enhanced when the spleen cells were cultured for 5 days with or without mitomycin-C-treated MOPC-315 stimulator tumor cells. The cells responsible for the therapeutic effectiveness of the spleen cells in ACIT of athymic nude mice, as well as the cells responsible for the direct in vitro anti-MOPC-315 lytic activity of the spleen cells, were of the Lyt 2 and not the L3T4 phenotype. Most of the athymic nude mice that completely eradicated a large MOPC-315 tumor as a consequence of ACIT were capable of rejecting a challenge with 30–100 times the minimal lethal tumor dose for 100% of normal BALB/c mice administered more than 1 month after the ACIT. The ability of these athymic nude mice to resist the tumor challenge was associated with the presence of a greatly elevated percentage of cells expressing T cell surface markers in their spleens. Thus, it is conceivable that splenic Lyt 2⁺ T cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor mediate their therapeutic effectiveness in ACIT of athymic nude mice bearing a large MOPC-315 tumor, at least in part, through direct cytotoxicity for MOPC-315 tumor cells. In addition, eradication of a large MOPC-315 tumor through cooperation between antitumor immunity and melphalan toxicity endues the athymic nude mice with an elevated percentage of T cells in their secondary lymphoid organs, and these T cells are probably responsible for the long-lasting protective antitumor immunity exhibited by these mice.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01350 from the National Cancer Institute     

Phorbol ester-induced enhancement in lytic activity of CD8+ splenic T cells from low-dose melphalan-treated MOPC-315-tumor bearers

Summary We have previously shown that while spleen cells from untreated mice bearing a large MOPC-315 tumor are not cytotoxic in vitro for MOPC-315 tumor cells, spleen cells obtained from such mice on day 7 after low-dose melphalan (l-phenylalanine mustard);l-PAM therapy exert a substantial anti-MOPC-315 cytotoxicity [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that this anti-MOPC-315 lytic activity is evident by day 5, and peaks on day 7 after the low-dose chemotherapy, at a time when the mice are actively engaged in tumor eradication. Short-term exposure of spleen cells from mice bearing a MOPC-315 tumor and treated with low-dosel-PAM (l-PAM TuB mice) to phorbol 12-myristate 13-acetate (PMA) was found to enhance greatly the ability of these spleen cells to lyse MOPC-315 tumor cells. The highest level of anti-MOPC-315 cytotoxicity was obtained when spleen cells from tumor-bearing mice that had received chemotherapy 7 days earlier were exposed to PMA at a concentration of 1–10 ng/ml. The exertion of the enhanced anti-MOPC-315 lytic activity byl-PAM TuB spleen cells exposed to PMA was found to require CD8⁺, but not CD4⁺, T cells. The apparent specificity of the lytic activity exerted by the PMA-stimulatedl-PAM TuB spleen cells was illustrated not only by the inability of the spleen cells to lyse an allogeneic, antigenically unrelated thymoma (EL4), but also by their relatively weak lytic activity for two antigenically related syngeneic plasmacytomas. In addition, when EL4 target cells were admixed with MOPC-315 tumor cells, the lytic activity triggered in thel-PAM TuB spleen cells by the MOPC-315 tumor cells plus PMA was not effective in lysing the antigenically unrelated target cells. Moreover, even in the presence of the calcium-specific ionophore, ionomycin,l-PAM TuB spleen cells exposed to PMA were unable to lyse the EL4 target cells. Thus, fresh CD8⁺ splenic T cells froml-PAM TuB mice that are in the process of eradicating a large MOPC-315 tumor as a consequence of low-dosel-PAM therapy can be triggered with PMA to exert enhanced lytic activity against MOPC-315 tumor cells. Since the curative effectiveness of low-dose chemotherapy for MOPC-315 tumor-bearing mice requires the participation of CD8⁺ T cells that exploit a cytotoxic T lymphocyte type lytic activity for tumor eradication, it is feasible that in some situations PMA-like stimulants could be used to augment the antitumor cytotoxic activity of the CD8⁺ T cells, which in turn could improve the therapeutic outcome of low-dose chemotherapy.Supported by research grant IM-435A from the American Cancer Society and research grant B-8806 from the Bane EstateIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of Career Development Award CA-01 350 from the National Cancer Institute     

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-immune spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon (IFN) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFN. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.Supported by research grant IM-435 from the American Cancer Society and CA54413 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of career development award CA-01350 from the National Cancer Institute     

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma

We have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard (l-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dosel-PAM therapy (l-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was mimicked by the membranepenetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The -adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal orl-PAM TuB spleen cells. Since the curative effectiveness of low-dosel-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8⁺ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.     

Specificity of the generation and expression of enhanced anti-plasmacytoma immunity by spleen cells from melphalan-treated MOPC-315 tumor bearers

Summary We have shown previously that low-dose melphalan (L-PAM) therapy of mice bearing a large MOPC-315 plasmacytoma enables their hitherto immunosuppressed spleen cells to exert potent anti-MOPC-315 cytotoxicity following in vitro immunization with MOPC-315 tumor cells [3]. Here we show that, following in vitro immunization with MOPC-315 tumor cells, spleen cells from such L-PAM-treated MOPC-315 tumor bearers exhibited enhanced T-cell-mediated cytotoxicity not only against the MOPC-315 tumor, but also against another plasmacytoma (MOPC-104E) possessing surface immunoglobulin (SIg) of a different idiotype than the MOPC-315 cells, as well as against a variant of the MOPC-315 tumor which does not produce nor possess SIg (SIg^–MOPC-315). The enhanced cytotoxicity was directed against target antigens which are not expressed on the surface of the syngeneic WEHI 22.1 thymoma or the natural killer-sensitive YAC-1 cells. Plasmacytoma shared antigens, other than immunoglobulins, were able to stimulate spleen cells from L-PAM-cured MOPC-315 tumor bearers to generate in vitro a secondary type anti-plasmacytoma cytotoxic response. L-PAM-cured MOPC-315 tumor bearers exhibited in vivo immunity against SIg^–MOPC-315 tumor cells, which was sufficiently triggered by the SIg^– cells to bring about the rejection of a challenge of at least 100-fold the minimal lethal tumor dose of the SIg^–MOPC-315 cells. Thus, SIg^– MOPC-315 tumor cells present among SIg⁺ tumor cells in the parental MOPC-315 tumor inoculum [9, 26] can be eradicated in the L-PAM-treated MOPC-315 tumor bearers by the immune response to SIg⁺ tumor cells as well as by the immune response to SIg^– tumor cells themselves.Supported by Research Grant CA-35761 from the National Cancer Institute and Research Grant IM-435 from the American Cancer SocietyIn partial fulfillment of the requirements for the Doctor of Philosophy degree.     

Deficiency in immunocompetence of mice cured from large MOPC-315 plasmacytomas by melphalan therapy

Summary Mice cured from large MOPC-315 tumors by a single dose of melphalan, 7.5 mg/kg, were examined for up to 60 days after the drug treatment (71 days after the tumor inoculation) for their ability to respond to mitogenic stimulation, specific and nonspecific antigenic stimulation and for their susceptibility to inoculation with an unrelated tumor, L10 lymphoma. The response of spleen cells from cured mice to mitogenic stimulation by phytohemagglutinin or concanavalin A was slightly depressed at an early stage after the drug treatment. The allogeneic response against C57BL spleen cells and the antibody response against sheep red blood cells (SRBC) of spleen cells from cured mice remained below normal levels during the whole observation period. The deficiency in response to antigenic stimulation was found to be due to impairment in T-cell function. Cured mice were also deficient in their response to SRBC immunization (antibody and delayed-type hypersensitivity responses) and were more susceptible to inoculation with an unrelated tumor, L10 lymphoma, than normal, noninoculated mice. On the other hand, spleen cells of cured mice developed a highly specific cytotoxic response against target MOPC-315 tumor cells and the cured mice were resistant to challenge with an otherwise highly tumorigenic dose of MOPC-315. Thus, cured mice remained deficient for a long period of time in their response to MOPC-315-unrelated antigens but, at the same time, they showed a potent specific antitumor immunity potential in vivo and in vitro.Presented in part at the Ninth European Immunology Meeting, September 14–17, 1988, Rome, ItalyThe contribution of S. Shoval is in partial fullfillment of a PhD Thesis     

B7-2 expression on tumor cells is important for the acquisition of cytotoxic T lymphocyte activity by spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers via a mechanism that requires either B7-1 or B7-2 expression on host antigen-presenting cells

 We have previously shown that B7-2 (CD86) and, to a lesser extent, B7-1 (CD80) contribute to the curative effectiveness of low-dose melphalan (l-phenylalanine mustard) for mice bearing a large MOPC-315 tumor under conditions that lead to the acquisition of potent cytotoxic T lymphocyte (CTL) activity at the tumor site. Since B7-1 and B7-2 are expressed on both tumor cells and host antigen-presenting cells (APC), the current studies were undertaken to examine the relative importance of each costimulatory molecule on tumor cells and on host APC for the acquisition of anti-MOPC-315 CTL activity. Utilizing an in vitro system for the acquisition of CTL activity, we found that B7 expression on host APC is important for the development of CTL activity in stimulation cultures of spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers, although the expression of either B7-1 or B7-2 is sufficient. In addition, we found that B7-2, which is expressed at high levels on stimulator tumor cells, but not B7-1, which is expressed at much lower levels, is also important for the acquisition of CTL activity. However, the vast majority of the CTL activity acquired in vitro in response to stimulation with the B7-2-expressing MOPC-315 tumor cells was found to depend on B7-expressing host APC. Thus, it is likely that B7-2, which is expressed at high levels on MOPC-315 tumor cells, promotes the rapid lysis of MOPC-315 stimulator tumor cells, thereby making tumor-associated antigens more readily available for efficient presentation by B7-expressing host APC which, in turn, stimulate the acquisition of CTL activity by spleen cells from low-dose-melphalan-treated MOPC-315 tumor bearers. Received: 2 September 1999 / Accepted: 19 November 1999     

Effect of low-dose cyclophosphamide therapy on specific and nonspecific T cell-dependent immune responses of spleen cells from mice bearing large MOPC-315 plasmacytomas

Summary Some T cell-dependent immune parameters were examined in mice bearing a large MOPC-315 plasmacytoma before and after treatment with a low dose (15 mg/kg) of CY. Prior to CY therapy, spleen cells from mice bearing a large MOPC-315 tumor were depressed in their ability to generate an in vitro cytotoxic response to the MOPC-315 tumor, to a different syngeneic plasmacytoma, MOPC-104E, and to an allogeneic thymoma, EL4. The spleen cells of these mice were also depressed in their ability to proliferate in response to the T cell mitogen PHA. Following CY therapy, the spleen cells generated an enhanced anti-MOPC-315 cytotoxic response by day 2, and the level of this response continued to increase so that by day 7, it was greatly enhanced and was much greater than the response of normal spleen cells. The recovery of the cytotoxic responsiveness to the antigenically related MOPC-104E tumor after CY therapy followed a similar pattern. In contrast, the spleen cells of these animals remained depressed in their cytotoxic response to the antigenically unrelated EL4 thymoma for at least 11 days after CY therapy. Although the anti-EL4 response recovered by day 14, the level of antitumor cytotoxicity generated did not exceed that generated by normal spleen cells. The PHA response remained greatly depressed in CY-treated MOPC-315 tumor bearers, even 14 days after the chemotherapy. Thus, at a time following low-dose CY therapy, when potent T cell-dependent antiplasmacytoma immunity had completed the eradication of a large MOPC-315 tumor burden not eliminated through the direct effect of the drug, the T cell-dependent response to an unrelated tumor and to PHA remained depressed.Supported by United Public Health Service Research Grant #CA-30088 and by Training Grant #CA-09318 from the National Cancer InstitutePresented in part at the 77th annual meeting of the American Association of Cancer Research, May 7–10, 1986In partial fulfillment of the requirements of the Graduate College for the Doctor of Philosophy degree Abbreviations used: CY, cyclophosphamide; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PHA, phytohemagglutinin     

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

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. Administration 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 carrageenansensitive effector cells are required for the rejection of the 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 carrageenansensitive effector cells while the other does not require carrageenan-sensitive effector cells but is T-cell-dependent.Supported by Research Grant IM-435 from the American Cancer Society and Research Grant CA-35761 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy degree     

Low-dose-melphalan-induced up-regulation of type-1 cytokine expression in the s.c. tumor nodule of MOPC-315 tumor bearers and the role of interferon γ in the therapeutic outcome

We have previously shown the importance of endogenous tumor necrosis factor (TNF) production for the curative effectiveness of low-dose melphalan (L-phenylalanine mustard) for mice bearing a large MOPC-315 tumor. In the current study we demonstrate that low-dose melphalan is actually associated with enhanced expression of mRNA for TNFα in the s.c. tumor nodule. Moreover, the expression of mRNA for interferon γ (IFNγ) and interleukin-12 (IL-12; p40) is also elevated at the tumor site. However, while elevation in the expression of mRNA for TNFα and IFNγ is evident within 24 h after the chemotherapy, elevation in the expression of mRNA for IL-12(p40) is first evident 72 h after the chemotherapy. Moreover, neutralizing anti-IFNγ mAb, like neutralizing anti-TNF mAb but not neutralizing anti-IL-12 mAb, reduced the curative effectiveness of low-dose melphalan for MOPC-315 tumor bearers. Studies into the mechanism through which IFNγ mediates its antitumor effect in low-dose-melphalan-treated MOPC-315 tumor-bearing mice revealed that MOPC-315 tumor cells, which are not sensitive to the direct antitumor effects of TNF, display some sensitivity to the antiproliferative activity of high concentrations of IFNγ. However, unlike TNFα, IFNγ is unable to promote the generation of anti-MOPC-315 cytotoxic T lymphocyte activity and, in fact, exerts an inhibitory activity on CTL generation. Taken together, our studies illustrate that low-dose melphalan therapy of MOPC-315 tumor bearers is associated with the rapid elevation in the expression of mRNA for IFNγ and TNF, two cytokines which are important for the curative effectiveness of low-dose melphalan, and which mediate their antitumor effect, in part, through distinct mechanisms.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.     

Two tumor models of curative adoptive chemoimmunotherapy using tumor-infiltrated spleen cells with potent antitumor cytotoxicity stimulated by antigen-sharing tumors

Previously we have established curative protocols for adoptive chemoimmunotherapy (ACIT) of mice bearing different plasmacytomas that are known to bear cross-reacting antigens: (a) the cure of mice bearing an early-stage, nonpalpable MOPC-315 tumor by a very low dose of cyclophosphamide (10 mg/kg) and cultured MOPC-315-tumor-infiltrated (TI) spleen cells (25×10⁶) and (b) the cure of mice bearing a late-stage, relatively drug-resistant, highly metastatic RPC-5 tumor with cyclophosphamide (100 mg/kg) and cultured RPC-5 TI spleen cells (25×10⁶–50×10⁶). In both models, the spleen cells were obtained from mice bearing a late-stage tumor and were cultured for 5 days in the presence of polyethyleneglycol 6000 and autochthonous tumor cells as a source of tumor antigen. Here we show that RPC-5 tumor cells could substitute for MOPC-315 tumor cells in the 5-day culture of MOPC-315 TI spleen cells so that they became curative in ACIT for mice bearing an early-stage MOPC-315 tumor. Similarly, MOPC-315 tumor cells could substitute for RPC-5 tumor cells in the 5-day culture of RPC-5 TI spleen cells so that they became curative in ACIT of mice bearing a late-stage RPC-5 tumor. In addition, RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were effective in curing all mice bearing an early-stage MOPC-315 tumor by ACIT. However, MOPC-315 TI spleen cells whether cultured with MOPC-315 or RPC-5 tumor cells, were much less effective than cultured RPC-5 TI spleen cells in curing mice bearing a late-stage RPC-5 tumor by ACIT (although the survival of these mice was extended significantly). Interestingly, whereas RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were as effective as MOPC-315 TI spleen cells cultured under the same conditions in lysing MOPC-315 tumor cells in vitro, MOPC-315 TI spleen cells that had been cultured with either MOPC-315 or RPC-5 tumor cells exerted a much weaker in vitro cytotoxic T lymphocyte activity against RPC-5 tumor cells than did RPC-5 TI spleen cells that had been cultured under the same conditions.Work was supported by research grant CA-30088 from the National Cancer Institute and IM-435 from the American Cancer Society. M. B. M. was supported by Career Development Award CA-01350 from the National Cancer InstituteThis work is in partial fulfillment of the requirements for the Ph. D. degree     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

Summary We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.This paper was presented in part at the annual meeting of the American Association of Immunologists, Chicago, Illinois, 10–15 April 1983     

Melphalan-induced enhancement of tumor cell immunostimulatory capacity as a mechanism for the appearance of potent antitumor immunity in the spleen of mice bearing a large metastatic MOPC-315 tumor

Summary Exposure of MOPC-315 cells from the primary tumor nodule to a low concentration (0.5 nmol/ml) of melphalan (L-phenylalanine mustard; L-PAM) rendered the tumor cells capable of bringing about the generation of a potent primary antitumor cytotoxic response. Accordingly, the level of antitumor cytotoxicity generated by normal spleen cells immunized in vitro with L-PAM-treated tumor cells was at least five-fold greater than the level generated in response to untreated tumor cells. The marked superiority of L-PAM-treated tumor cells over untreated tumor cells in bringing about the generation of antitumor cytotoxicity was evident over a wide range of responder to stimulator cell ratios. The higher level of antitumor cytotoxicity exhibited by normal spleen cells immunized with L-PAM-treated tumor cells as compared with untreated tumor cells was not merely the result of direct drug-mediated tumoricidal activity, thereby reducing the number of tumor cells present which can act as cold target cell inhibitors during the ⁵¹Cr release assay. This is apparent from the observation that the level of antitumor cytotoxicity generated in response to a given percentage of stimulator tumor cells pretreated with 0.5 nmol L-PAM/ml, a drug concentration associated with retention of 60% tumor cell proliferative capacity, is substantially greater than that generated in response to less than half that percentage of untreated stimulator tumor cells. Moreover, stimulator tumor cells exposed to a fully antiproliferative concentration of L-PAM brought about the generation of a higher level of antitumor cytotoxicity than stimulator tumor cells exposed to mitomycin C at a concentration which inhibited the proliferation of the tumor cells to the same extent as the L-PAM. A low concentration of L-PAM which was effective in rendering isolated tumor cells from the primary tumor nodule capable of bringing about the generation of antitumor cytotoxicity was also effective in inducing the appearance of potent antitumor immune potential in tumor bearer splenic cells containing metastatic tumor cells. Thus, metastatic tumor cells within the spleen of tumor-bearing mice may have the potential to act as in situ stimulators for the generation of potent antitumor immunity which brings about the eradication of a large, metastatic tumorigenic load remaining after clearance of the drug from the circulation.Supported by United States Public Health Service Research Grants CA-30088 and CA-35761 from the National Cancer InstituteIn partial fulfillment of the requirements of the Graduate College for the Doctor of Philosophy degree for RCBSBE was visiting Fulbright Professor from the Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel, and was supported by the Council for International Exchange of Scholars     

The difference between 5-fluorouracil and melphalan in their ability to promote antitumor immune response against MOPC-315 plasmacytoma

Summary The anticancer chemotherapeutic drugs melphalan (L-phenylalanine mustard; L-PAM), 5-fluorouracil (5-FU), methotrexate (MTX), and daunorubicin (DAU) were tested for their toxic activity against MOPC-315 tumor cells in vitro. L-PAM, 5-FU, and DAU had a marked toxic effect whereas MTX did not affect the rate of thymidine incorporation in the tumor cells. L-PAM (7.5 mg/kg) induced permanent regression of large s.c. MOPC-315 plasmacytoma tumors, 5-FU (200–250 mg/kg) induced transient regression of MOPC-315 tumors with reappearance starting on the 6th day after the 5-FU injection and DAU (5 mg/kg) was not effective. L-PAM treatment restored the cytotoxic potential of spleen cells of tumor-bearing mice against target MOPC-315 tumor cells whereas spleen cells from tumor-bearing mice treated with 5-FU were unable to mount a cytotoxic response.L-PAM and 5-FU were also assayed for their effect in vitro on induction of suppressor T cells by ConA. L-PAM treatment in vitro markedly reduced the induction of suppressor T cells by ConA whereas 5-FU had no effect. It is suggested that anticancer chemotherapeutic drugs can be classified in immunopromoting (L-PAM as prototype) and nonimmunopromoting (5-FU as protoype) on the basis of their effect in vivo on established tumors and their effect on induction of suppressor T cells by ConA.     

Expression of idiotype 315 (Id315) and I-A antigens in MOPC-315 tumor cells grown in vivo

MOPC-315 murine myeloma grown intraperitoneally as ascites cells in BALB/cJ mice were removed at successive days after transplantation. They were stained for Id³¹⁵ and I-A markers by indirect immunofluorescence techniques by means of FITC conjugates. Flow cytometry (FCM) measurements on cell surface markers were correlated with the phase of the cell cycle by quantitating cellular DNA of cells stained with propidium iodide. Variations in cell size due to cell growth were determined by low-angle light scattering. FCM data on the two cell surface markers were normalized for unit cell surface and cell volume. Cells grew rapidly in the early days (5–7) of tumor growth. No significant variation in the expression of surface markers was observed during this period. Parallel with a slowdown in cell growth, the expression of Id³¹⁵ increased about threefold between the 7th and 9th days. The increase in the Id³¹⁵ marker was dependent on the cell cycle, with G₁ cells having the highest density. No cell cycle dependence was observed for the I-A marker.     

Norepinephrine-mediated inhibition of antitumor cytotoxic T lymphocyte generation involves a beta-adrenergic receptor mechanism and decreased TNF-alpha gene expression.

We have previously shown that norepinephrine (NE) inhibits the in vitro generation of anti-MOPC-315 CTL activity by spleen cells from BALB/c mice rejecting a large MOPC-315 tumor as a consequence of low-dose melphalan (l -phenylalanine mustard (l -PAM)) treatment (l -PAM TuB spleen cells). Since TNF-alpha plays a key role in the generation of antitumor CTL activity in this system, we determined whether NE mediates this inhibition through inhibition of TNF-alpha production. Here, we show that NE inhibits the production of TNF-alpha protein and mRNA by l -PAM TuB spleen cells stimulated in vitro with mitomycin C-treated tumor cells. Flow cytometric analysis of intracellular expression of TNF-alpha revealed substantial NE-mediated decreases in the percentages of TNF-alpha+ cells among CD4+ and CD8+ T cells and F4/80+ activated macrophages. NE inhibition of CTL generation was largely overcome by addition of TNF-alpha to the stimulation cultures. When the beta-adrenergic antagonist propranolol was added to the stimulation cultures of l -PAM TuB spleen cells at a concentration that prevented NE-induced cAMP elevation, the NE-mediated decrease in TNF-alpha mRNA and NE-mediated inhibition of CTL generation were reversed. Collectively, these results suggest that NE inhibits antitumor CTL generation, at least in part, by inhibiting TNF-alpha synthesis through a mechanism(s) involving beta-adrenergic receptor signaling.