Generation of cytokine-induced killer cells using exogenous interleukin-2, -7 or -12

Abstract

Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells.

Activation-Induced Expression of CD56 by T Cells Is Associated With a Reprogramming of Cytolytic Activity and Cytokine Secretion Profile In Vitro

“…In the present study, we confirm previous reports that human CD56 ϩ T cells can be expanded from fresh PBMCs by TCR-mediated activation, particularly when grown in the presence of exogenous IL-2 [4,11,[13][14][15][16][17]. We found that a single CD3/TCR stimulation of PBMCs in the absence of added growth factors resulted in threefold expansions of CD56 ϩ T cells but no increase in total cell numbers.…”

“…T cells with NKRs, including CD56 ϩ T cells, are often present in cultures of stimulated T cells [4,11,[13][14][15][16][17], and they frequently express memory phenotypes [2,36,37]. They can be activated by TCR ligation and by antigen-nonspecific signals through NKRs and cytokine receptors [4,11,25,[33][34][35] and display potent MHC-unrestricted cytotoxicity against a range of tumor cells in vitro and in vivo [3,11,12,18,19,25].…”

“…They are capable of potent perforin/granzyme-mediated killing of a variety of tumor target cells in vitro and in vivo [3,11,12]. This MHC-unrestricted cytotoxicity does not require prior antigenic exposure but appears to involve the ligation of stimulatory NKRs and/or the TCR, and exposure to cytokines of diverse cellular origins, including interferon-␥ (IFN-␥), interleukin-1 (IL-1) and IL-2 [11], IFN-␣, IL-2, IL-12 and/or IL-18 [13], IL-2 and IL-12 [13][14][15][16][17], and IL-15 [4]. The cytotoxic activities of CD56 ϩ T cells against autologous and allogeneic leukemic cells are reported to be superior to those of NK cells and lymphokine activated killer cells [11,18,19], and phase I trials for leukemia by using autologous CD56…”

Adenovirus-mediated LIGHT gene modification in murine B-cell lymphoma elicits a potent antitumor effect

“…The NK or CTL activity was determined by a standard lactate dehydrogenase (LDH) release assay (Promega, Madison, MI, USA). 21 The amount of LDH-released was detected by absorbance at 490 nm using an ELISA reader. Calculations were carried out according to the following formula: % of specific lysis51003(experimental-effectorspontaneous-target spontaneous)/(target maximum-target spontaneous).…”

Lymphotactin cotransfection enhances the therapeutic efficacy of dendritic cells genetically modified with melanoma antigen gp100

“…The NK activity and CTL activity were determined by lactate dehydrogenase (LDH) release assay with CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI, USA). 23 The target cells (YAC-1 cells, B16 cells or EL-4 transfected with or without Adgp100) were washed three times with RPMI 1640 medium containing 5% FCS to remove adherent LDH derived from lysed cells. The cell suspension was diluted with RPMI 1640 medium containing 5% FCS to give final concentration of 1 × 10 5 cells/ml.…”