Role of Interleukin-2 Activated MHC-Nonrestricted Lymphocytes in Antileukemia Activity and Therapy

Although the application of biological therapy for solid tumors with cytokines and adoptively transferred activated lymphocytes has received substantial attention, this approach has not been actively explored in treatment of hematopoietic neoplasms. This review will address the possibilities of interleukin-2 (IL-2) and IL-2 activated natural killer (NK) cells and T cells in antileukemia reactivity and therapy. The new approaches to optimal activation and generation of oncolytic cells, selective propagation of lymphocyte subsets, and the role of adhesion molecules in antileukemia cytotoxicity will also be addressed. We trust that this article will be conducive to the development of new directions in leukemia research and treatment.     

Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin 2

Monoclonal antibodies (mAB) with tumor specificity are able to enhance the immunological specificity of interleukin 2 (IL-2)-activated lymphokine activated killer (LAK) cells. Antibodies may also be used to broaden the range of tumor types susceptible to immune mediated cytotoxicity by the activated LAK cells. In these studies, mAB with relative tumor specificity were used to target immunologically activated effector cells in an in vitro antibody dependent cell mediated cytotoxicity (ADCC) assay. The mAB included: 3F8 and 14.G2a, which are both specific for neuroblastoma and melanoma and recognize ganglioside GD2, and mAB ING-1, a mouse-human chimeric antibody with constant regions from human IgG1 and kappa chains and variable regions from a mouse mAB that binds to a broad range of human adenocarcinomas. Each of these mAB was able to mediate ADCC with fresh effector cells and antibody binding targets. When peripheral blood mononuclear cells were obtained from cancer patients prior to and following in vivo therapy with interleukin 2, a significant increase was noted in ADCC activity by peripheral blood mononuclear cells obtained following IL-2 therapy. Inclusion of IL-2 in the medium during the cytotoxic assay with mAB further boosted ADCC. The total activity seen was often greater than the sum of the independent LAK activity and standard ADCC activity. The cells responsible for this ADCC had the CD16+ Fc receptor. Combining IL-2 with mAB in clinical tumor therapy may lead to a wider range of tumor types being responsive to immunotherapy and may also enhance the efficacy of therapy by specifically targeting activated effector cells to tumor cells recognized by mAB. Our results provide strong support for the testing of these hypotheses in clinical trials by combining in vivo treatment with IL-2 and mAB able to mediate ADCC.     

Targeting Hsp90 by 17-AAG in leukemia cells: mechanisms for synergistic and antagonistic drug combinations with arsenic trioxide and Ara-C.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new anticancer agent currently in clinical trials. The ability of 17-AAG to abrogate the function of heat-shock protein Hsp90 and modulate cellular sensitivity to anticancer agents has prompted recent research to use this compound in drug combination therapy. Here we report that 17-AAG has striking opposite effects on the activity of arsenic trioxide (ATO) and ara-C. Combination of 17-AAG with ATO exhibited a synergistic effect in leukemia cells, whereas coincubation of 17-AAG and ara-C showed antagonistic activity. Mechanistic studies revealed that ATO exerted cytotoxic action by reactive oxygen species generation, and activated Akt survival pathway. 17-AAG abrogated Akt activation and enhanced the activity of ATO. In contrast, treatment of leukemia cells with 17-AAG caused a G1 arrest, a decrease in DNA synthesis and reduced ara-C incorporation into DNA, leading to antagonism. The ability of 17-AAG to enhance the antileukemia activity of ATO was further demonstrated in primary leukemia cells isolated from patients with acute myeloid leukemia and chronic lymphocytic leukemia, including cells from refractory patients. Our data suggest that combination of 17-AAG and ATO may be an effective therapeutic regimen. Caution should be exercised in using 17-AAG together with ara-C, as their combination effects are schedule dependent.     

mda-7/IL-24 induces apoptosis in human GBC-SD gallbladder carcinoma cells via mitochondrial apoptotic pathway

mda-7/IL-24 has tumor-suppressor activity in a broad spectrum of human cancer cells. However, the therapeutic effect of the recombinant human IL-24 protein on human gallbladder carcinoma has rarely been explored. In this study, we used a human gallbladder carcinoma cell line (GBC-SD) to explore the effect of adenovirus-mediated IL-24 (Ad-IL24) gene therapy on GBC-SD cells. We show that Ad-IL24 treatment of GBC-SD cells in vitro conspicuously induced apoptosis of GBC-SD cells. We also demonstrate that the in vivo treatment of GBC tumor-bearing athymic nude mice intratumorally injected with Ad-IL24 significantly suppressed GBC growth. To further explore the mechanism that mda-7/IL-24 utilized in tumor cell apoptosis, we examined molecules and pathways involved in apoptotic regulation and found that Ad-IL24 induced the down-regulation of anti-apoptotic gene Bcl-2 and the release of cytochrome c, which subsequently activated caspase-9, caspase-3 and PARP to induce apoptosis. In summary, adenovirus (AdV)-mediated IL-24 overexpression exerted potent antitumor activity via stimulating mitochondrial apoptotic pathway in GBC-SD. Therefore, mda-7/IL-24 has the potential to serve as a tool for targeted gene therapy in the treatment of gallbladder cancer.     

Receptor-mediated cancer therapy--tumoricidal cytokines, adoptive therapy of LAK, TIL]

Three modes of receptor-mediated cancer therapy were reviewed presenting our own data. Employment of tumoricidal cytokines (IFN, TNF, LT) to this type of therapy has been expected to be the most promising approach. However, preclinical and clinical results so far obtained, revealed that they were useful only for the very limited diseases including renal cancer or some hematological malignancies. Second approach is to utilize growth factors conjugated with toxin or carzinostatin which are readily internalized into tumor cells. In this context, transferrin-neocarzinostatin was examined in our laboratory both in vitro and in vivo for its anticancer activity and was found to suppress tumor growth more significantly than neocarzinostatin alone on the basis of molar ratio. Thus this approach may be worthy to be clinically investigated. Adoptive therapy of lymphokine activated killer (LAK) or tumor infiltrating lymphocyte (TIL) may also be categorized into receptor mediated cancer treatment since both are activated by signals through IL-2 receptor. Although clinical evaluation is still on going, the therapy appears to be effective only when effector cells are administered locally to tumors.     

Prospects for Clinical Cancer Treatment Using Interleukin-2

Even if many of these immunologic approaches to anticancer therapy show the desired effect, with a greater percentage of patients having antitumor responses (hopefully without need for ICU level toxicity), it may be unrealistic to hope that these biological treatments will significantly prolong the survival of most patients with large progressively growing tumors. It is possible that the large, yet finite, number of cells with LAK activity that can be activated endogenously, or infused into a patient, may only be able to destroy a finite number of neoplastic cells. If so, the smaller the number of neoplastic cells at the time of in vivo LAK induction therapy, the more likely the chance for effective eradication of all tumor cells. Experimental animal studies support this postulate (46). Clinical regimens utilizing IL-2 therapy, alone or combined with other agents, with documented immunologic and antitumor activity, will need to be tested in a large number of patients in randomized adjuvant trials. This testing will probably require the involvement of large cooperative oncology trials groups, in order to determine the potential prolongation of survival by any adjuvant IL-2 approach. A vast number of regimens could presently be suggested for combining these separate approaches in groupwide trials (especially when issues of dose, route, and scheduling are considered). Trials in murine models must continue rapidly and be interpreted with caution in generating regimens to be tested clinically. Further treatment improvements, to be identified in Phase I and Phase II clinical trials, are still required to allow large groupwide randomized Phase III trials to test tolerable and effective IL-2-containing regimens likely to significantly prolong survival.     

Interleukin-8 increases vascular endothelial growth factor and neuropilin expression and stimulates ERK activation in human pancreatic cancer

Interleukin-8 (IL-8) is associated with tumorigenesis by promoting angiogenesis and metastasis. Although up-regulation of IL-8 is indicated in many cancers, its function in pancreatic cancer has not been well characterized. In this study we examined the expression of IL-8 on pancreatic cancer cells and clinical tissue specimens, and investigated the effect of exogenous IL-8 on gene expression, and signaling in human pancreatic cancer cells. We found that pancreatic cancer cells expressed higher amount of IL-8 mRNA than normal human pancreatic ductal epithelium cells. IL-8 mRNA was also substantially overexpressed in 11 of 14 (79%) clinical pancreatic-adenocarcinoma samples compared with that in their surrounding normal tissues. Exogenous IL-8 up-regulated the expression of vascular endothelial growth factor₁₆₅, and neuropilin (NRP)-2 in BxPC-3 cells, one of human pancreatic cancer cell lines. IL-8 expression was inducible by hypoxia mimicking reagent cobalt chloride. In addition, IL-8 activated extracellular signal-regulated kinase (ERK)1/2 signaling pathway in BxPC-3 cells. Our studies suggest that IL-8 might be a malignant factor in human pancreatic cancer by induction of vascular endothelial growth factor and NRP-2 expression and ERK activation. Targeting IL-8 along with other antiangiogenesis therapy could be an effective treatment for this malignancy. ( Cancer Sci 2008, 99: 733–737)     

Bexarotene and DAB389IL-2 (Denileukin Diftitox,ONTAK) in Treatment of Cutaneous T-Cell Lymphomas: Algorithms

Mycosis fungoides (MF), CD4⁺ epidermotropic cutaneous T-cell lymphoma (CTCL), often arises as indolent, inflammatory, chronic, persistent patches and plaques. Conservative and sequential topical therapy patients have the same survival as patients treated with aggressive chemotherapy. Hence, until curative therapy is found, therapies that keep MF in check and prevent progression to more advanced lymphoma may be desirable alternatives and may preserve quality of life. Stage IA patients with stable disease have a very favorable prognosis and often initially receive psoriasis-type therapy. Bexarotene gel, a new topical retinoid X receptor retinoid will resolve MF lesions, reducing dermal T-cell infiltrates when used as a single agent. However, it may be even more effective when combined with topical steroids, with phototherapy (ultraviolet B and psoralen-ultraviolet A), or even with oral bexarotene. The gel may also provide a safe adjunctive therapy for individual lesions that are refractory to other agents, including keratodermas. When more than 10% of the body is involved with CTCL or when adenopathy is present (> stage IB), systemic therapy is indicated. Bexarotene capsules have the advantage of easy oral administration and are extremely effective both for early-stage patients with long-standing extensive plaques and for late-stage patients with Sézary syndrome or large-cell transformation. Monitoring of white blood cell count, lipids, and thyroid function is required. Bexarotene should be tested in combination with interferon-alfa or other therapies such as photopheresis, psoralen-ultraviolet A, and methotrexate and for maintenance after total body skin electron beam. DAB₃₈₉IL-2 is targeted to CD25⁺, the interleukin-2 receptor on activated T cells as measured by the expression of CD25. DAB₃₈₉IL-2 has given complete or partial remission in 30% of highly refractory patients with extensive plaques and disfiguring tumors. Because it is effective in killing T cells that surround dermal vessels, cytokine release may occur and result in capillary leak syndrome. Hence, it will be reserved for more advanced and refractory patients and will require intravenous administration and monitoring. The use of oral bexarotene first to reduce dermal infiltrates prior to DAB₃₈₉IL-2 administration might reduce subsequent side effects imparted by this therapy. With three new highly effective agents in the armamentarium for the treatment of CTCL, new combination treatment algorithms can be tested to achieve maximal benefit and quality of life for these patients.     

Alpha-interferon activated cytotoxic lymphocytes in hairy cell leukemia patients: evaluation of cytotoxic events

To further define the mechanisms responsible for the alpha-interferon (alpha-IFN) efficacy in the treatment of hairy cell leukemia (HCL), experiments were carried out to specify the cytotoxic events taking place following this type of therapy. Although an increased natural killer (NK) activity was demonstrable after alpha-IFN treatment, evidence has been provided that hairy cells were not specifically lysed either by fresh autologous/allogenic NK lymphocytes or by lymphokine activated killer (LAK) cells. This property could not be induced in vitro by alpha-IFN or by interleukin-2 (IL-2). Our data favour the hypothesis that the increase of NK cell activity observed following alpha-IFN therapy has not a direct antineoplastic effect but is likely to be of relevance for a non-specific enhancement of the host immune system. In alpha-IFN treated HCL this latter property may account for the better resistance to infections which usually represents the major cause of mortality in these patients.     

Lymphokine-activated killer activity induced by in vivo interleukin 2 therapy: predominant role for lymphocytes with increased expression of CD2 and leu19 antigens but negative expression of CD16 antigens

The phenotype and function of lymphocytes from cancer patients treated with repetitive weekly cycles of continuous i.v. infusions of recombinant interleukin 2 (IL-2) were examined. Peripheral blood lymphocytes (PBL) obtained after IL-2 therapy showed an increased percentage of cells bearing the CD16 and leu19 markers which are associated with natural killer cells. These PBL mediated significantly increased levels of IL-2-dependent lymphokine-activated killer (LAK) activity against the Daudi cell line. Depletion of CD16+ cells from PBL obtained after in vivo IL-2 caused only slight inhibition of their LAK activity or their proliferative response to IL-2 in vitro. This indicates that CD16+ cells are involved but play only a minor role in these responses. In contrast, depletion of leu19+ cells, from PBL activated in vivo with IL-2, virtually abrogated their LAK activity and their proliferative response to IL-2. Two-color flow cytometry studies showed that a leu19+/CD16- population was expanded by in vivo IL-2 therapy and was responsible for the majority of LAK activity by in vivo-activated PBL. Moreover, this CD16- population showed an increased density of leu19 and CD2 (E rosette receptor) antigens when compared to the resting PBL obtained prior to IL-2 treatment. These data show that the predominant population mediating in vitro LAK activity, induced by in vivo IL-2 therapy, consists of activated natural killer cells with a high density of leu19 and CD2 antigens but negative for the CD16 antigen.     

Interleukin-2 and cancer therapy

In summary, this meeting provided a useful overview of state-of-the-art research on IL-2. Significant gains have been made toward understanding the molecular biology and function of IL-2 and its receptors, but much remains to be learned about the intracellular signaling pathway by which IL-2 transduces its biological effects. Likewise, the nature of the lymphocyte subpopulation that responds to IL-2 requires detailed study, since it is now clear that T cells as well as LGLs can mediate non-MHC-restricted killing of tumor cells. Cell trafficking and effector cell activity within the tumor are other important issues to address. We also need to define the mechanism(s) by which IL-2-activated lymphoid cells mediate their antitumor effect in vivo; it is still far from clear whether these cells directly lyse the tumor cells, interrupt the tumor's vascular supply through endothelial cell damage, or produce other cytokines which then activate other cellular pathways. Preclinical data indicate that IL-2, with or without adoptive cellular therapy or in combination with other cytokines or drugs, can cause tumor regression. The success with animal model systems, however, has not always translated to success in the clinical setting-disappointingly, the majority of cancer patients still fail to respond to this treatment. It is hopeful the reasons for this will become apparent as more is learned about the molecular and cellular mechanisms involved in this "new" form of immunotherapy.     

Phase 1 clinical trial of recombinant interleukin-2: a comparison of bolus and continuous intravenous infusion

The toxicologic, biologic, and clinical effects of recombinant interleukin-2 (IL-2) were tested in 25 patients with cancer. Escalating doses from 10(3) to 10(7) U/m2 per day were given by either daily bolus injection (BI) or continuous infusion (CI) for 7 consecutive days. Dose-limiting toxicities included a decline in performance status, systolic hypotension, and fever, which reversed promptly with discontinuation of therapy. The maximum tolerated dose of IL-2 by BI for 7 days was 3 X 10(6) U/m2 per day and for CI was 10(6) U/m2 per day. Significant changes in the number, phenotype, and function of circulating peripheral blood lymphocytes occurred at doses greater than or equal to 10(6) U/m2 per day by both administration schedules. With the initiation of therapy, a decline in the number of circulating peripheral blood lymphocytes (PBL) was seen in patients treated by either BI or CI. Additionally, the in vitro cytotoxic activity of these PBL against K562 was markedly decreased. Within 24 h of completing BI or CI, a rebound increase in the number of circulating PBL was seen. The phenotype of the circulating PBL after completion of treatment showed a significant (p greater than or equal to 0.05) increase in the numbers of OKT-3+, OKT-8+, OKT-10+, OK-Ia+, OKM1+, and OKT-11+ for patients treated by CI. Those patients treated by BI had a significant increase in Ia+ and OKT10+ cells. At IL-2 doses greater than or equal to 10(5) U/m2 per day, the PBL obtained following treatment with rIL-2 demonstrated in vitro cytotoxic capacity against K562 target cells that was significantly enhanced over pretreatment levels. This study demonstrates that IL-2 can be given by CI or BI in a non-ICU setting with acceptable dose-dependent toxicity. Upon completion of treatment an increase in the number of activated cells could be detected. Although no clinical responses occurred, the generation of endogenous activated PBL capable of enhanced cytotoxicity is encouraging. Future studies will explore the use of multiple courses of treatment with IL-2 to determine if therapeutic efficacy can be accomplished.     

Vaccine with beta-defensin 2-transduced leukemic cells activates innate and adaptive immunity to elicit potent antileukemia responses

Murine beta-defensin 2 (MBD2) is a small antimicrobial peptide of the innate immune system. Recent study showed that MBD2 could not only recruit immature dendritic cells but also activate them by Toll-like receptor 4 and thus may provide a critical link between the innate immune system and the adaptive immune response. In this report, we examined the antileukemia activity of MBD2 in a murine model of acute lymphoid leukemia (ALL) L1210. L1210 cells were engineered to secrete biologically functional MBD2. MBD2-modified L1210 (L1210-MBD2) showed significantly reduced leukemogenecity, resulting in a 80% rate of complete leukemia rejection. Inoculation of mice with L1210-MBD2 induced enhanced CTL and natural killer (NK) activity and augmented interleukin-12 and IFN-gamma production. All the recovered mice from the inoculation showed a protective immunity to the following challenge with parental L1210 cells and generate leukemia-specific memory CTL. Vaccines with irradiated L1210-MBD2 cells could cure 50% leukemia-bearing mice. Depletion of CD8+ T cells but not CD4+ T cells completely abrogated the antileukemia activity of MBD2. Interestingly, NK cells were also required for the MBD2-mediated antileukemia response, although ALL generally display a high degree of resistance to NK-mediated lysis. Our results suggest that MBD2 can activate both innate and adaptive immunity to generate potent antileukemia response, and MBD2 immunotherapy warrants further evaluation as a potential treatment for ALL.     

Single-cell cloning of human, donor-derived antileukemia T-cell lines for in vitro separation of graft-versus-leukemia effect from graft-versus-host reaction

In previous studies, we showed the possibility of expanding in vitro polyclonal CTL lines directed against patient leukemia cells using effector cells derived from both HLA-matched and HLA-mismatched hematopoietic stem cell donors. Some CTL lines, especially those derived from an HLA-disparate donor, displayed residual alloreactivity against patient nonmalignant cells. In this study, we evaluated the possibility of separating in vitro CTLs with selective graft-versus-leukemia (GVL) activity from those potentially involved in the development of graft-versus-host disease (GVHD) through single T-cell cloning of antileukemia polyclonal CTL lines. We showed that CTLs that were expanded from a single T-cell clone (TCC), able to selectively kill leukemia blasts and devoid of alloreactivity towards nonmalignant cells, can be obtained from antileukemia alloreactive polyclonal CTL lines. TCCs expressed a wide repertoire of different T-cell receptor (TCR)-Vbeta families, mainly produced IFNgamma and interleukin 2, irrespective of CD8 or CD4 phenotype, and could be extensively expanded in vitro without losing their peculiar functional features. The feasibility of our approach for in vitro separation of GVL from GVH reaction opens perspectives for using TCCs, which are selectively reactive towards leukemia blasts, for antileukemia adoptive immune therapy approaches after hematopoietic stem cell transplantation, in particular from HLA-mismatched donors.     

Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2

Adoptive immunotherapy with tumor-specific T cells has emerged as a valid approach for prevention or treatment of diseases, such as melanoma and EBV-associated lymphoma. As interleukin (IL) 15 promotes survival of CD8(+) memory CTLs, we hypothesized that it could be used to enhance antitumor immunity in vivo through the maintenance of adoptively transferred memory CTL. To test this, we treated mice bearing P1A(+) tumors with adoptively transferred T cells possessing a transgenic Valpha8(+) T-cell receptor specific for the P1A tumor antigen (called P1CTL). Mice were then randomized to receive daily low-dose IL-15 (0.5 microg/day) or PBS. Mice receiving the transgenic P1CTL and IL-15 experienced a significantly delayed tumor relapse or complete tumor regression (P < 0.002 compared with PBS), with a striking persistence of the CD8(+) Valpha8(+) P1CTL compared with mice receiving the CD8(+) Valpha8(+) P1CTL and PBS vehicle (26.3 versus 5.1% P < 10(-5)). Animals exhibiting complete tumor regression had a significant population of CD8(+) Valpha8(+) P1CTL (46%) that persisted with IL-15 treatment until 140 days after adoptive transfer and successfully defended them against tumor rechallenge without IL-15. Low-dose IL-2 afforded no protection over vehicle and resulted in lower percentages of T cells with an activated memory phenotype, lower Bcl-2 expression, and lower ex vivo antitumor cytotoxicity compared with mice treated with IL-15. Collectively, the data support the notion that exogenous low-dose IL-15 therapy can enhance and even reverse the limited efficacy of adoptively transferred tumor-specific T-cell therapy and may do so in a fashion that is superior and distinct from exogenous IL-2 therapy.     

5-Fluorouracil and Interleukin-2 Immunochemotherapy Enhances Immunogenicity of Non-Small Cell Lung Cancer A549 Cells through Upregulation of NKG2D Ligands

Background: The aim of this study was to investigate the anti-cancer effects and mechanisms ofimmunochemotherapy of 5-fluorouracil (5-FU) and interleukin-2 (IL-2) on non-small cell lung cancer (NSCLC)A549 cells. Materials and Methods: In order to detect whether 5-FU+IL-2 could effectively inhibit tumorgrowth in vivo, we established an A549-bearing nude mouse model. The cytotoxicity of natural killer (NK) cellswas evaluated using a standard chromium release assay. To evaluate the relevance of NK cells in 5-FU+IL-2-mediated tumor inhibitory effects, we depleted NK cells in A549-bearing mice by injecting anti-asialo-GM-1antibodies. Effects of 5-FU+IL-2 on the expression and promoter activity of NKG2D ligands (MICA/MICB) inA549 cells in vitro were also assessed. Results: In A549-bearing nude mice, combination therapy significantlyinhibited tumor growth in comparison with monotherapy with 5-FU or IL-2 and enhanced the recognitionand lysis of tumor cells by NK cells. Further study of mechanisms showed that NK cells played a vital role inthe anticancer immune response of 5-FU+IL-2 immunochemotherapy. In addition, the combination therapysynergistically stimulated the expression and promoter activity of MICA/MICB. Conclusions: 5-FU and IL-2immunochemotherapy significantly inhibited tumor growth and activated NK cytotoxicity in vivo, and theseeffects were partly impaired after depleting NK cells in tumor-bearing mice. Combination treatment of 5-FUand IL-2 upregulated the expression and the promoter activity of MICA/MICB in A549 cells, which enhancedthe recognition of A549 cells by NK cells. All of the data indicated that immunochemotherapy of 5-FU and IL-2may provide a new treatment option for patients with lung cancer     

Interleukin-12 augments cytolytic activity of peripheral blood mononuclear cells against autologous lung cancer cells in combination with IL-2

The majority of patients with advanced lung cancer die within a few years. Accordingly, new therapeutic modalities need to be developed. Interleukin (IL)-12 was previously known as natural killer (NK) cell stimulatory factor or cytotoxic lymphocyte maturation factor. By virtue of its effects on T cells and NK cells, IL-12 seems to be one of the key cytokines that regulates cell-mediated anti tumor immune responses. Recently, there has been a substantial interest in the potential applications of IL-12 in the treatment of lung cancer. However, there have been no reports about the effect of IL-12 on peripheral blood mononuclear cells (PBMCs) obtained from lung cancer patients in an autologous setting. In this study, we examined the cytotoxicity of PBMC activated by IL-2, IL-12 or both against K562 or autologous lung cancer cells. In contrast to the effect of IL-2 on NK activity, IL-12 alone augmented NK activity against K562 cells, but not against autologous lung cancer cells. IL-12 augmented the IL-2 mediated cytotoxicity of PBMC against both K562 and autologous lung cancer cells. In the absence of IL-2, IL-12 alone cannot induce an autologous anti-tumor effect in vivo. In summary, our results clearly demonstrated that IL-12 can augment the cytolytic activity of PBMC against K562 and autologous lung cancer cells when combined with IL-2, although, IL-12 alone was unable to induce a marked increase in the cytotoxicity against autologous lung cancer cells. These results suggest that an administration of IL-12 in combination with IL-2 may be a useful therapeutic option for solid tumors.     

Synergistic antitumor effects of interleukin 2 and the monoclonal Lym-1 against human Burkitt lymphoma cells in vitro and in vivo

Interleukin 2 (IL-2) regulates immune responses by inducing proliferation and differentiation of T-cells into cytotoxic cells, inducing lymphokine activated killer activity and enhancing antibody dependent cellular cytotoxicity (ADCC). Lym-1, a monoclonal antibody, recognizes a membrane antigen present on the surface of B-lymphoma cells and can be used for ADCC. We therefore used Raji (human Burkitt lymphoma) cells to study the efficacy of combination therapy with IL-2, lymphokine activated killer activity, and Lym-1. In vitro ADCC assays using Lym-1 showed that preincubation of peripheral blood lymphocytes with IL-2 had a synergistic antitumor effect. The maximum synergism was achieved when peripheral blood lymphocytes were incubated with IL-2 for 3 days as compared to 1 or 2 days, with the optimal concentration of IL-2 being 1000 units/ml. This effect was specific for Lym-1 as demonstrated by experiments using an irrelevant (antimelanoma) monoclonal antibody or an irrelevant target cell (A375). The ADCC was blocked by an anti-Fe receptor antibody (3G8). In vivo experiments performed by growing Raji tumors in nude mice also demonstrated the increase in ADCC and the synergism between IL-2 and Lym-1 in terms of decreased tumor size and growth. The mechanism of this synergy is probably from activation of cells mediating ADCC. This raises the possibility that treatment of patients with low doses of IL-2 in combination with Lym-1 may enhance immune responses and thereby antitumor activity.     

Role of interleukin 2 and interferon gamma in induction of activated natural killer cells by the streptococcal preparation OK432

The natural killer (NK) cell mediated cytotoxicity to syngeneic tumor cells can be augmented by in vivo priming and subsequent in vitro challenge with the streptococcal preparation OK432. Supernatants of coculture of spleen cells with OK432 contained Interleukin 2 (IL-2) and Interferon (IFN), mainly IFN-gamma. As the anti-mouse IFN-gamma monoclonal antibody but not anti-mouse IFN-alpha antibody inhibited the induction of activated NK cells with OK432, the IFN-gamma participated in this response. The incubated spleen cells with IL-2 receptors increased with OK432 treatment by flow cytometry, and the NK cell and IFN activities of supernatants were also abrogated by the treatment with anti-mouse IL-2 receptor monoclonal antibody to block the interaction between IL-2 and these receptors of effector cells. By panning method, it was clarified that the incubated spleen cells with IL-2 receptors were responsible for the production of IFN-r. These results suggest that IL-2 plays a major role to induce the activated NK cells from murine spleen cells primed in vivo and subsequently challenged in vitro with OK432, by the production of IFN-gamma.