Population dynamics of human activated natural killer cells in culture (original) (raw)
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Journal of Immunological Methods, 1985
We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and punfied large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2 The results indicated that PBL had a 30% increase m cumulatwe net cell growth and had as high or higher cytotoxlc activity after growth in SF medium than in medium containing FBS Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than m SF medium However, the c)totoxlc activity of cells grown in SF medium persisted dunng the imtlal 3 weeks of culture Purified LGL that were maintained and were subcultured at cell densities of 106 cells or greater per mllhhter of either SF or FBS-contalning medium had equivalent levels of cytotoxlclty over a 44-day period in either medium compared with cells subcultured at a density of 5 × 105 cells per malllhter of medium NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxlc activity was blocked by 10% FBS We conclude that the SF medmm supplemented with IL-2 can be used as an alternative to FBS-contmnlng medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF Key words yerum-free culture medium-human mononuclear cells-natural killer cells
Japanese Journal of Cancer Research, 2002
An anchorage-dependent Wilms tumor cell line HFWT was found to stimulate selective and remarkable expansion of human natural killer (NK) cells from human peripheral blood mononuclear cells (PBMC). After PBMC of healthy donors were cultured on irradiated HFWT cells for 10-21 days, the lymphocytes expanded 58-to 401-fold. This NK cell expansion required direct contact of PBMC with live, but not fixed, HFWT cells. The PBMC from an end-stage brain tumor patient also expanded 156-fold, whereas those cultured with irradiated NK-sensitive K562 grew only 30.5-fold. CD16 + CD56 + NK cells accounted for more than 70% of the population expanded on HFWT cells. No essential difference in expression of NK receptors was observed in the expanded NK cells on HFWT and K562 and without feeder cells. The expanded NK cells killed not only fresh HFWT cells but, unexpectedly, also MHC class I-expressing autologous brain tumor cells at an effector/target ratio of 4 for 24 h. These results will contribute to the development of a large-scale preparation method for human NK cells, which will aid studies of NK cell biology and possible treatment of brain tumors.
Blood, 1989
Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16 cells and were devoid of CD3 T cells. CD1 5 monocytes. and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete. a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony-inhibiting activity (CIA) in the culture supernatants. Such CIA was
Journal of Experimental Medicine, 1989
Human B lymphoblastoid cell lines facilitate the growth in vitro of human NK cells and of T cell clones (1-4), and together with a source of IL-2, have been successfully used to maintain both NK and T cell clones in culture (1, 2). We have shown that irradiated B lymphoblastoid cell lines induce proliferation of purified human NK cells only in synergy with IL-2 (3). They also facilitate continued proliferation and enhance the cloning efficiency of purified human NK cells in limiting dilution assays in the presence of IL-2 without increasing the proportion (>507o) of NK cells entering the cell cycle in response to IL-2 (4). During culture of total PBMC with irradiated B cell lines, NK cells become activated, as shown by increased cytotoxic activity, by proliferation, and by expression of surface activation antigens such as class II HLA antigens, transferrin receptors, and IL-2 receptors (5, 6). In these cultures, a preferential proliferation of CD16+ CD56(NKH-1)+ CD3 -NK cells is observed (6) : in 10-d cultures, NK cell number is increased 25-fold, whereas T cell number is increased only 3-fold . Elimination of CD4 + cells or the presence of an anti-IL-2 antiserum completely prevents NK cell proliferation (6), suggesting that this probably depends on the production of IL-2 by CD4 + T cells upon allogeneic stimulation . However, the B cell lines also contribute directly to the proliferation of NK cells because in the absence of B cell lines neither high doses of IL-2 alone nor stimulation by allogeneic PBMC induce preferential proliferation of NK cells (6) .
Human natural killer cells enhance a mixed leukocyte reaction
Journal of Leukocyte Biology
Natural killer cells (NK) have been reported to down-regulate the Initiation of T cell responses In animal models. in the current study, highly purified CD16 + human NK cells were obtained by cell sorting and their effect on the stimulation of allogeneic T cells (MLR) determined. NK cells did not directly stimulate T cell proliferation. However, when added to a population of loosely adherent mononuclear cells (LAM), NK enhanced the ability of these accessory cells to stimulate T proliferation. This effect was not reproduced by the addition of sorted CD5 + T cells, sorted CD16 -cells, or control lymphocytes to the MLR. The effect of NK on the MLR was not restricted by class II antigens and was similar to the effect of adding IL-i to MLR cultures. These results demonstrate that human NK cells are capable of enhancing a I cell response.
Generation of a cloned NK cell line derived from the "null cell" fraction of human peripheral blood
The Journal of Immunology
The present studies were designed to determine the conditions for generation of human NK clones. First the "null cell" (NC) fraction of human peripheral blood mononuclear cells, which contains the NK effectors, was purified using negative selection with anti-T3, anti-T8, anti-B1, and anti-Mo2 monoclonal antibodies. Subsequently, NC were cloned by limiting dilution using a combination of phytohemagglutinin (PHA) and lymphocyte-conditioned medium (LCM) as an initial stimulus. Colonies could be easily obtained with this procedure, but the maintenance of long-term growth of the cultures represented a major problem. A cloned cell line termed JT1 was generated and has been proliferating continuously in culture for more than 6 mo. The phenotype of JT1 cells was analyzed several times with a series of monoclonal antibodies. These cells did not express surface markers related to thymic (T3-, T4-, T8-, T11-), B cells (B1-, J5-), or myelomonocytic (Mo1-, Mo2-, MY7-) differentiation. ...
Biology of Blood and Marrow Transplantation, 2004
shown that natural killer (NK) cells from PBPC collections are less expandable in vitro than those obtained during steady-state hematopoiesis. We show here that the extent of this proliferation deficit is related to the number of circulating CD34 ؉ cells in vivo at the time of PBPC apheresis. Likewise, addition of autologous CD34 ؉ cells to unseparated PBL reduced the expansion of the NK-cell subset by 22.2% ؎ 6.0% (n ؍ 10; P <.005). In contrast, when using purified NK cells, their proliferation remained unimpaired by autologous CD34 ؉ cells. Supernatants from CD34 ؉ cells cultured with autologous PBLs had an inhibitory effect on proliferation of purified NK cells (n ؍ 16; P ؍ .03), indicating that an interaction between CD34 ؉ cells and lymphocytes is essential for the suppressive effect on NK cells. To investigate the role of T cells in this interaction, intracellular cytokines were determined in T cells cultured for 7 days with or without autologous CD34 ؉ cells. When cultured with CD34 ؉ cells, the frequency of IL-2-producing CD4 ؉ and CD8 ؉ T cells was reduced by 19% and 24%, respectively, compared with T cells cultured alone (n ؍ 7; P ؍ .016). Interferon-␥-producing T cells were slightly reduced (P ؍ not statistically significant [ns]). Finally, the influence of T cells and NK cells on the recovery of myeloid colony-forming cells (CFU-GMs) from purified CD34 ؉ cells was examined. In the presence of T cells, 16% ؎ 6% of the input CFU-GM recovered after 7 days, compared with 5% ؎ 4% in the presence of NK cells (n ؍ 5; P ؍ ns). Our findings point to an inhibition of NK-cell proliferation mediated by an interaction of CD34 ؉ cells and T cells occurring during PBPC mobilization with G-CSF.
Human natural killer cell development in a xenogeneic culture system
British Journal of Haematology, 2002
In vivo and in vitro xenogeneic models have shown the ability of a non-human environment in supporting human haemopoiesis. In the present study, we evaluated the effect of fetal sheep thymic stroma in the in vitro development of natural killer (NK) cells from human haemopoietic progenitors. CD34 + HLA-DR + (CD34 + DR + )Linand CD34 + DR -Linbone marrow (BM) progenitors were cultured for 3 weeks with or without interleukin 2 (IL-2), in fetal sheep thymic stroma contact and transwell cultures. Both progenitors gave rise to NK cells, defined as CD45 + CD56 + cells, in the presence or absence of IL-2; however, the percentage of NK cells originated in cultures with IL-2 was significantly higher. Direct contact with stroma seemed to be required for the most immature progenitors, CD34 + DR -Lin -, to differentiate along the NK cell lineage. Functional assays revealed that only cells grown in the presence of IL-2 were cytolytic against K562 targets and, curiously, NK cells derived from CD34 + DR -Linprogenitors were more cytotoxic that NK cells derived from CD34 + DR + Linprogenitors. These studies suggest that the ability of fetal sheep thymic stroma in promoting the generation of human NK cells from haemopoietic progenitors may have relevance in terms of NK cell ontogeny and induction of tolerance in transplantation.
Growth of Murine Natural-Killer-Cells from Bone-Marrow Invitro - Role of TNF-Alpha and Ifn-Gamma
International Journal of Immunopharmacology, 1991
It has been previously shown that natural killer (NK) cell growth can be induced by interleukin-2 (IL-2) in bone marrow (BM) cultures and that other cytokines (CKs), including IL-la, act synergistically with IL-2. However, as the effect of IL-2 and IL-la could be due to direct stimulation of NK progenitor cell growth, as well as to the induction of other factors, we analysed the role of the endogenous production of CKs in BM cultures. Results show that mRNAs specific for tumour necrosis factor-a (TNFa) and interferongamma (IFNy) are detectable within hours in BM cultures supplemented with IL-2 and IL-la, and that the amount is higher when both IL-2 and IL-la are present. Antibodies directed against TNFa and IFNy abrogate the NK cell development, indicating that these CKs play an essential role. The antibodies, however, had no effect on mature NK cells. Furthermore, pretreatment of BM cells with TNFa or IFNy before culturing with IL-2, enhances IL-2 responsiveness and NK cell growth. These results suggest that induction of cytokines production may be important for growth of NK cells from BM precursors and that the synergistic effect of IL-I~ could be due, at least in part, to increased TNFa and IFNy production. Interleukin-2 and IFNs have been shown to stimulate the in vivo and in vitro growth of NK cells in both man and animals (