IL-2 expansion of T and NK cells from growth factor-mobilized peripheral blood stem cell products: monocyte inhibition.

The expansion of T and natural killer (NK) cells in growth factor-mobilized peripheral blood stem cell (PSC) products with interleukin-2 (IL-2) requires a reduction in monocyte frequency. Monocytes are enriched with stem cells during apheresis and, in this series of growth factor-mobilized PSC products from breast cancer patients, represented 36 +/- 6% of the cells in the product. Immunophenotyping studies revealed that monocytes inhibited the proliferation of NK cells (CD56+ and CD3- CD8+ CD56+ cells) and T cells (CD3+, CD4+, and CD8+ cells) during IL-2 co-culture for 7, 14, or 21 days. A reduction in monocytes resulted in 61-fold expansion of CD3- CD8+ CD56+ cells compared with a 3.7-fold increase of CD3+ cells by day 21. In addition, following IL-2 co-culture, cells from PSC products with a reduced frequency of monocytes had a significantly increased T cell mitogenic response and NK cell activity in PSC products compared with intact products. We suggest that monocytes inhibit the IL-2-dependent proliferation and augmented function of NK and T cells from growth factor-mobilized PSC products.

Comparative evaluation of multiple lymphoid and recombinant human interleukin-2 preparations.

Six lymphoid human interleukin-2s (nIL-2s) [four from peripheral blood mononuclear cells (PBMC) and two from JURKAT cells] and six recombinant IL-2s (rIL-2s) were obtained for comparative evaluation. The main issues addressed were possible differences among the preparations in potency in T cell growth assays and other functional assays, and the possible presence of other cytokine activities or contaminants. Each preparation was assigned a standardized IL-2 activity in reference units (RU) by comparing its T cell growth promoting activity against the Biological Response Modifiers Program IL-2 (JURKAT) reference reagent. Relative to the IL-2 unitage indicated by the suppliers, the RU varied from 110-fold less to 38.5-fold more for the various preparations. Two nIL-2s and two rIL-2s contained significant levels of endotoxin. One nIL-2 contained low levels of both alpha and gamma interferon (IFN), and one nIL-2 had a high level of gamma IFN. All other IL-2s were negative for IFN activity. All IL-2 preparations significantly augmented human natural killer (NK) activity, although the amount of RU required varied from 0.1 to 50 RU. Four nIL-2s and three rIL-2s induced human PBMC to produce gamma IFN, whereas two nIL-2s and one rIL2 did not. All nIL-2s had substantial amounts of B cell growth factor activity, whereas none of the rIL-2s consistently displayed this activity. All IL-2s stimulated the tritiated thymidine [3H]TdR incorporation of human PBMC in the absence of other stimuli, in addition to augmenting the response to mitogen or alloantigens. Some nILs and IL-2s had effects on human monocytes such as inhibiting migration, inducing cytotoxic or growth inhibitory activity against tumor cells, and causing changes in cell surface markers. The IL-2s were also tested for activity in vitro and in vivo in mice. Although there was a 12-fold variation in activity among the preparations, all but one of the IL-2s showed augmentation of the mixed lymphocyte reaction activity and all IL-2s tested stimulated macrophage cytotoxicity in vitro. All IL-2s tested enhanced the mixed lymphocyte-allogeneic tumor cell reaction resulting in greater production of cytotoxic T cells. However, significant quantitative differences in potency were evident among the various IL-2 preparations, especially the nIL-2s. Only very high doses of IL-2 (intraperitoneal injection of 100,000 RU/animal) induced in vivo augmentation of splenic or peritoneal NK cells, although all IL-2s tested increased NK activity against tumor target cells in vitro with substantially lower doses (10-100 RU/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Immunomodulatory effects in mice of polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose.

In this report, we describe the immunomodulatory characteristics of poly(I,C)-LC, a synthetic, double-stranded nucleic acid polymer, polyinosinic-polycytidylic acid, that is complexed with poly-L-lysine and solubilized by the addition of carboxymethylcellulose. We consistently observed, both in vitro and in vivo, stimulation of macrophage cytotoxicity and augmentation of natural killer-cell activity by poly(I,C)-LC. This immunomodulator also increased the allogeneic mixed-lymphocyte response, without any blastogenic effect on responder cells cultured in the absence of allogeneic stimulator cells. Further, the addition of poly(I,C)-LC to an allogeneic mixed-lymphocyte tumor reaction did not stimulate the development of cytotoxic effector T-cells. Poly(I,C)-LC did, however, have adjuvant activity when admixed with irradiated tumor cells in the immunization of syngeneic mice. Unlike classic adjuvants, poly(I,C)-LC also enhanced the development of specific cytotoxic T-lymphocytes when it was injected either i.v. or i.p. in conjunction with a vaccine delivered at an intradermal site. The results indicate that poly(I,C)-LC has considerable potential as an immunotherapeutic agent, with the ability not only to induce macrophage and NK cell activation but also to stimulate specific cytotoxic T-lymphocytes.

Immunotherapeutic potential in murine tumor models of polyinosinic-polycytidylic acid and poly-L-lysine solubilized by carboxymethylcellulose.

The systemic administration of multiple, nontoxic doses of polyinosinic-polycytidylic acid and poly-L-lysine solubilized by carboxymethylcellulose [poly(I,C)-LC] eradicated established experimental and spontaneous pulmonary metastases. Optimal immunotherapy was schedule dependent, requiring three to five injections of poly(I,C)-LC per week for a minimum of 4 weeks; in addition, therapeutic efficiency was partially dosage independent. Immunotherapy by poly(I,C)-LC was found to be limited by tumor burden, although when combined with chemotherapy as a debulking regimen it resulted in increased survival with protocols in which poly(I,C)-LC alone was insufficient. These data suggest that the systemic administration of poly(I,C)-LC may provide a successful adjuvant therapeutic modality against cancer metastasis.

Lymphokines, monoclonal antibodies, and other biological response modifiers in the treatment of cancer.

Biologicals and biological response modifiers (BRMs) represent a new class of agents for cancer therapy. Historically, there have been many attempts to stimulate the immune response with nonspecific immunomodulators in the form of bacterial extracts, viruses, and chemicals. Although these approaches have occasionally proven useful under defined conditions in experimental models, their extension to the clinic has been largely unsuccessful. Recent advances in molecular biology and hybridoma technology have made available genetically engineered lymphokines and cytokines, as well as monoclonal antibodies, as highly purified biologicals for cancer treatment. These agents may act directly on tumor cells and/or may act on the patient's own biological responses to induce an antitumor response. Selective defects in T-cell function have recently been identified in cancer patients and in patients with acquired immunodeficiency syndrome (AIDS). Simultaneously, the availability of gamma interferon (gamma-IF) and interleukin-2 (IL-2) may allow for the selective correction of these T-cell deficits, leading to restoration of the patient's immune responses and perhaps correction of the clinical syndromes. Preliminary data suggest that gamma-IF and IL-2 have in vitro activity on these T-cell defects, and the preliminary evidence that these agents have activity in vivo will be reviewed. Extensive trials are being conducted at the National Cancer Institute with monoclonal antibodies as anticancer agents. Animal model experiments have demonstrated considerable antitumor activity of immunoconjugates using monoclonal antibodies tied to toxins. Preliminary clinical results suggest that T-101 in leukemia and lymphoma and 9.2.27 in malignant melanoma may prove useful as specific reagents in the treatment of these disorders. While the antitumor effects with these antibodies have not been dramatic, our preliminary data in approximately 30 patients with leukemia, lymphoma, and melanoma clearly demonstrate the ability of intravenous monoclonal antibody to locate and specifically label tumor cells bearing the target antigens. It has been possible to localize antibody on the tumor cells in melanoma deposits that are barely visible in the skin. These data and radioimaging data suggest a future role for immunoconjugates as anticancer agents.

The NCI preclinical screen of biological response modifiers.

The development of successful approaches to immunotherapy is dependent upon proper consideration of the pathobiology of metastasis and the establishment of a better scientific understanding of the principles of biological response modification. The studies we have undertaken are aimed at the rational development of a preclinical data base to help provide this understanding. Ultimately, these studies will be correlated with clinical trials to assess the predictive value of the preclinical screen. We anticipate that the testing of biological response modifiers (BRMs) in a controlled but evolving system will help eliminate arbitrary decisions on the preparations and use of a given BRM and ultimately will contribute to the development of novel approaches for the treatment of disseminated cancer. Immunotherapy alone or in combination with other therapeutic modalities is an area of rapid evolution with tremendous potential as an adjuvant therapy of disseminated cancer.