Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

Abstract Background aims. Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results. Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.     

The role of IL-4 in proliferation and differentiation of human natural killer cells. Study of an IL-4-dependent versus an IL-2-dependent natural killer cell clone

The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.     

The effects of IL-4 on human natural killer cells. A potent regulator of IL-2 activation and proliferation

NK cells are directly activated by rIL-2 and subsequently undergo rIL-2-dependent proliferation in vitro. Herein, we report that rIL-4 is a potent regulator of human NK cells. Although rIL-4 had no effect on the cytotoxic activity of resting NK cells, it was capable of inhibiting in a concentration-dependent manner the rIL-2-induced cytolytic activation of NK cells against NK cell-resistant tumor cell targets. rIL-4 acted directly on NK cells and did not require accessory cells. rIL-4-induced inhibition of NK cell activation was specific for rIL-2 in that activation of NK cell cytolysis by IFN-alpha was not affected. These results represent the first direct evidence that rIL-2 and IFN-alpha activate NK cells by different pathways. rIL-4 also effectively blocked the rIL-2-dependent proliferation of NK cells. The results presented in this study clearly demonstrate that rIL-4 is a potent regulator of IL-2-dependent mechanisms of NK cell activation and proliferation and thus may play an important physiologic role in vivo.     

Human T cells constitutively express IL-15 that promotes ex vivo T cell homeostatic proliferation through autocrine/juxtacrine loops

Homeostatic proliferation of T cells in vivo is responsible for the maintainance of the T cell pool, and IL-15 is a pivotal cytokine implicated in this process. Known cell sources providing physiologically active IL-15 are monocytes/macrophages, dendritic cells, and stromal cells. T lymphocyte expression of functionally active IL-15 and its possible role in T cell biology have not been investigated. In this study, we demonstrate that human T cells constitutively express IL-15 that acts through autocrine/juxtacrine loops to promote ex vivo homeostatic T cell proliferation.     

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.     

Anti-tumor efficacy of interleukin-2-activated killer cells in human neuroblastoma ex vivo

We studied the ex vivo sensitivity of continuously cultured neuroblastoma cells from 3 different patients towards interleukin-2-induced cell-mediated cytotoxicity. A mean (+/- SD) target cell lysis (4 h 51Cr release) of 49 +/- 11, 46 +/- 8, and 32 +/- 11% in SMS-SAN, LA-N-1, and SK-N-BE2 cell lines, respectively, was achieved when neuroblastoma cells were co-cultured at an effector-to-target (E:T) ratio of 50:1 with peripheral blood mononuclear cells (PBMC) that had been preincubated for 4 days in the presence of recombinant interleukin-2 (rIL-2; 100 U/ml). Under identical conditions, 93 +/- 9% of Daudi cells (a standard target for rIL-2-activated killer cells) were lysed. Preincubation of rIL-2-induced PBMC cultures in the presence of irradiated neuroblastoma targets (LA-N-1, SK-N-BE2) resulted in a significant cytolytic augmentation. At E:T ratios of 50:1 and 10:1, day-4 rIL-2/LA-N-1-stimulated PBMC produced 69 +/- 7 and 41 +/- 11% lysis of LA-N-1 cells, as compared to 46 +/- 8 and 22 +/- (mean +/- SD) 7% lysis by untargeted PBMC that were preincubated with rIL-2 (100 U/ml) in the absence of LA-N-1 target cells (p less than 0.05). Co-incubation of rIL-2-induced PBMC preparations with irradiated LA-N-1 and SK-N-BE2 cells, respectively, did not significantly enhance the cytolytic activity against other neuroblastoma targets and the standard Daudi cell line (p greater than or equal to 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-18 acts synergistically with IL-15 in stimulating natural killer cell proliferation

Mature natural killer (NK) cells are able to vigorously proliferate in response to infectious stimuli such as viral infections. The factors driving NK cell proliferation under these circumstances are only beginning to be characterized. NK cells constitutively express interleukin-18 receptor alpha and are stimulated by IL-18 to produce IFNgamma. Although IL-18 alone is not sufficient to drive NK cell proliferation, we demonstrate that IL-18 is able to act synergistically with IL-15 in stimulating in vitro NK cell proliferation. Furthermore using a NK cell line, we show that this effect occurs through direct stimulation of NK cells by IL-18 rather than through a secondary signal generated by an intermediary cell type. This raises the possibility that IL-18 may act synergistically with IL-15 in driving pathogen-induced NK cell proliferation in addition to its contribution in enhancing IL-12 stimulation of NK cell IFNgamma production.     

IFN-beta 2/IL-6 augments the activity of human natural killer cells

MHC nonrestricted cytotoxic cells play an important role in the killing of tumor cells in vitro and potentially in vivo. The activity of these cells is regulated by several cytokines such as IL-2 and IFN. In the present study we provide first evidence that IL-6 significantly augments the cytotoxic activity of human NK cells. IL-6 is produced by many different cells and is also known as IFN-beta 2, B cell stimulatory factor 2, hybridoma growth factor, hepatocyte-stimulating factor, and 26 kDa protein. IL-6 stimulates the activity of human CD3- NK cells but not that of CD3+ non-MHC-restricted cytotoxic T lymphocytes. As is the case with IL-2, the IL-6-mediated augmented cytotoxicity was a result of a more efficient lysis, but was not caused by an increased effector to target cell binding. Moreover, the effect of IL-6 on NK cell activity was blocked by a mAb directed against IL-2, and IL-6 itself was found to be a potent inducer of IL-2 production in cultured human PBMC. Thus it may be concluded that IL-6 enhances the cytotoxic activity of NK cells via IL-2. This newly recognized property of IL-6, which is produced by almost any cell, may be of importance in host defense against microbes and malignancies and therefore could contribute to improve the adoptive immunotherapy by using lymphokine-activated killer cells.     

Lymphokine-activated killer cells are rejected in vivo by activated natural killer cells

A 4-h in vivo cytotoxicity assay was used to study the fate of implanted IL-2-generated, lymphokine-activated killer (LAK) cells in mice undergoing an activated NK cell response. 125Iododeoxyuridine-labeled LAK cells were rejected from selected organs of C57BL/6 mice infected with lymphocytic choriomeningitis virus or treated with IL-2 or the IFN inducer poly I:C. This rejection was abrogated by the selective depletion of NK cells with antibodies to asialo-GM1 and NK1.1 Ag. Similar results were noted when LAK cells were generated from the spleens of B and T cell-deficient severe combined immunodeficiency mice and when LAK cells were implanted into severe combined immunodeficiency mice. These data indicate that NK cells activated by virus infections or by IL-2 infusions directly or indirectly eliminate implanted LAK cells. Because LAK cells are used in the treatment of certain human cancers, the strategy of accompanying this therapy with IL-2 infusions should be reassessed in light of these results.     

The proinflammatory cytokines IL-2, IL-15 and IL-21 modulate the repertoire of mature human natural killer cell receptors

Natural killer (NK) cells play a crucial role in the immune response to micro-organisms and tumours. Recent evidence suggests that NK cells also regulate the adaptive T-cell response and that it might be possible to exploit this ability to eliminate autoreactive T cells in autoimmune disease and alloreactive T cells in transplantation. Mature NK cells consist of a highly diverse population of cells that expresses different receptors to facilitate recognition of diseased cells and possibly pathogens themselves. Ex vivo culture of NK cells with cytokines such as IL-2 and IL-15 is an approach that permits significant expansion of the NK cell subpopulations, which are likely to have potent antitumour, antiviral, or immunomodulatory effects in autoimmunity. Our data indicate that the addition of IL-21 has a synergistic effect by increasing the numbers of NK cells on a large scale. IL-2 and IL-15 may induce the expression of killer cell immunoglobulin-like receptors (KIRs) in KIR-negative populations, the c-lectin receptor NKG2D and the natural cytotoxic receptor NKp44. The addition of IL-21 to IL-15 or IL-2 can modify the pattern of the KIR receptors and inhibit NKp44 expression by reducing the expression of the adaptor DAP-12. IL-21 also preserved the production of interferon-γ and enhanced the cytotoxic properties of NK cells. Our findings indicate that the proinflammatory cytokines IL-2, IL-15 and IL-21 can modify the peripheral repertoire of NK cells. These properties may be used to endow subpopulations of NK cells with specific phenotypes, which may be used in ex vivo cellular immunotherapy strategies.     

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

Ultraviolet radiation inhibits human natural killer activity and lymphocyte proliferation

Exposure to ultraviolet radiation (UVR) has been implicated in the predisposition to certain neoplasms and leads to viral reactivation. Natural killer (NK) activity may play a role in immunosurveillance and response to certain viral infections. We have evaluated the sensitivity to UVR of human NK activity, a nonproliferative function, and the proliferative response to the mitogen phytohemagglutinin (PHA). In vitro exposure to UVR resulted in a dose-dependent inhibition of NK activity and response to PHA. The wavelength dependence for UVR inhibition of NK activity and of the PHA response of lymphocytes were virtually superimposable at wavelengths at or above 300 nm, but NK activity was less sensitive to UVR at 260 and 280 nm. Maximal sensitivity for both functions was found at 260 nm, consistent with a nucleic acid chromophore mediating UVR inhibition of both activities. The DNA-directed drugs mitomycin C, acridine orange, and adriamycin at concentrations that inhibit proliferation are poor inhibitors of NK activity. These results suggest that UVR inhibition of NK activity as well as proliferation may be mediated by a nucleic acid chromophore. NK activity, however, is less sensitive to direct damage of DNA by alkylation, distortion, or oxidation. At 300 nm, the amount of radiation required to inhibit NK activity and proliferation is within the range penetrating to the dermis and capillaries during environmental exposure to sunlight.     

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Abstract

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.     

IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.     

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.