Anti-myeloma activity of endogenous and adoptively transferred activated natural killer cells in experimental multiple myeloma model.

OBJECTIVE: Despite advances in autologous stem cell transplantation and chemotherapy, multiple myeloma (MM) remains an incurable disease. Due to the role of natural killer (NK) cells in host resistance against several tumors, it is of interest to explore the anti-MM activity of NK cells. For this reason, we aimed to determine if NK cells provide anti-MM activity following interleukin-2 (IL-2) administration, and if ex vivo activated and intravenously administered NK cells prolong survival in MM-bearing C57BL/KaLwRij mice. METHODS: The anti-MM effect of IL-2 was tested by intraperitoneal injection into the 5T33MM-inoculated mice. Subsequently, in vivo effector cell depletions were performed by administration of anti-NK1.1 or anti-CD8 monoclonal antibodies. Finally, magnetically separated and activated NK cells from splenocytes of C57BL/KaLwRij mice were adoptively transferred to tumor-bearing mice in conjunction with IL-2 treatment. RESULTS: IL-2 administration into MM-bearing mice significantly prolonged their survival. This effect was diminished by in vivo depletion of NK cells. Adoptive transfer of activated NK cells showed a significant in vivo anti-MM effect that was dependent on cell dose. Biodistribution of the marked adoptively transferred NK cells correlated with MM cells' homing sites. CONCLUSION: These data suggest that activated NK cells have a promising potential in adoptive immunotherapy for MM.

Efficient gene transfer into primary human natural killer cells by retroviral transduction.

OBJECTIVE: To optimize retroviral gene transfer into primary human natural killer (NK) cells. MATERIALS AND METHODS: NK cells from healthy donors were expanded ex vivo for a period of 21 days. Retroviral transductions were carried out by replacing culture media with retrovirus-containing supernatant during 2-hour incubations on days 3, 4, 5, 6, 10, 15, or 20. In some experiments, NK cells were transduced on 2 consecutive days (days 5 and 6). Green fluorescent protein served as a marker for detection of transduced cells. RESULTS: NK cells showed a median of 27.2% transduction efficiency after a single transduction round (transduction on day 5) and a median of 47.1% transduction efficiency after two rounds of transduction (transduction on days 5 and 6), 24 hours after exposure to retrovirus-containing supernatants. On day 21 after initial culture, 51.9% of NK cells were transduced after a single transduction round (transduction on day 5) and 75.4% after two rounds of transduction (transduction on days 5 and 6). Gene transfer did not change the function or phenotype of NK cells as determined by phenotypical analysis, nor did the proliferative ability or cytotoxic function change. CONCLUSION: The results show that NK cells can successfully be transduced with retroviral vectors, without any detectable changes in phenotype or function. This may open up new possibilities in the studies of NK cell biology and the development of NK cells for immunotherapy regimens.

Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells.

OBJECTIVE: Anti-tumor effects mediated by adoptively transferred natural killer (NK) cells are dependent on the presence of interleukin-2 (IL-2). IL-2 is considered to be a survival factor for NK cells and an enhancer of their cytotoxic potential. However, systemic administration of IL-2 is frequently impeded by undesirable side effects, such as high toxicity and nonlocalized administration. Genetic modification of NK cells expressing IL-2 in a localized and controlled manner could be a powerful tool for overcoming these obstacles. METHODS: Consequently, we have cloned the IL-2 gene using PCR and designed constructs that target IL-2 to specific subcellular compartments. The IL-2-dependent NK-92 cell line was used to verify the functionality of the subcellularly targeted IL-2 constructs. RESULTS: IL-2 targeted specifically to the endoplasmic reticulum (ER) was sufficient to support growth of NK-92 cells. In such cell lines, IL-2 was verified to be localized to the ER. IL-2 was not detected in the supernatant and growth of non-IL-2-modified NK-92 cells was not supported during coculturing experiments. IL-2-transduced NK-92 cell lines showed comparable functional activity and cytotoxicity to parental NK-92 cells. CONCLUSION: We demonstrate the ability of ER-retained IL-2 to provide autocrine growth stimulation to NK-92 cells, without secretion of the cytokine to the extracellular compartment. Therapy with IL-2 gene-modified autoactivating NK cells may avoid side effects imposed by exogenously administered IL-2.

Visualization of 5T33 myeloma cells in the C57BL/KaLwRij mouse: establishment of a new syngeneic murine model of multiple myeloma.

OBJECTIVE: Lack of good models for in vivo detection of multiple myeloma (MM) cells hampers our understanding of the disease. Our objective was to establish a murine model for MM, allowing sensitive and labor-free tracing and quantification of MM cells in an immunocompetent host. METHODS: 5T33MM cells were retrovirally transduced, expressing enhanced green fluorescent protein (eGFP) and/or herpes simplex virus thymidine kinase (HSV-tk) as a control. Flow cytometric eGFP detection accuracy and sensitivity were assessed. Functional characteristics of transduced cells, including growth rate and production of IgG2b paraprotein and interleukin-6, were compared to those of nontransduced cells in vitro. For induction of MM, C57BL/KaLwRij mice were injected intravenously with transduced and nontransduced cells. Survival kinetics and distribution of eGFP cells in tissues were evaluated. RESULTS: Flow cytometric eGFP detection was accurate at 1:1000 transduced/nontransduced cell ratio. Transduced and nontransduced 5T33MM cells exhibited similar growth rates, producing comparable IgG2b and interleukin-6 levels. Intravenous injection of both nontransduced and eGFP-transduced MM cells to C57BL/KaLwRij mice resulted in paraplegia. At the time of paraplegia, eGFP-transduced MM cells were detected substantially in the bone marrow, spleen, and liver, less in lymph nodes, but not in the thymus. The bone marrow of paraplegic mice contained higher eGFP-transduced MM cells compared to that of nonparaplegic animals. CONCLUSIONS: In the established eGFP-5T33 MM model, MM cells are easily traced in an immunocompetent host. This model simplifies the analysis of homing pattern studies, the evaluation of therapeutic effects of various treatment approaches and contributes towards better understanding of MM.