RESUMO
The ability to perform biological studies on Natural Killer (NK) cells requires effective methods for their isolation from hematopoietic cells of other lineages. NK cells are a discrete lymphocyte subset distinguishable from B- and T-lymphocytes on the basis of both physical and phenotypic characteristics that can be exploited for then purification. Techniques based on differential cell buoyancy (centrifugation on discontinuous density gradients, such as Percoll [1]) have been used to enrich NK cells from mixed lymphocyte populations, but do not allow purification of these cells to homogeneity. The mononuclear cell suspensions obtained, although enriched in NK cells, also contain variable proportions of other cell types (notably monocytes and/or activated T- and B-lymphocytes) (2) and subsets of NK cells of higher density are lost in these preparations.
RESUMO
Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL-2, NKSF/IL-12 enhances NK cell-mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)-alpha, IFN-beta, IFN-gamma, IL-2 or tumor necrosis factor (TNF)-alpha and, unlike the induction of IFN-gamma production by peripheral blood lymphocytes, it does not require HLA class II-positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor-derived target cells, NKSF/IL-12 is also a potent stimulator of cytotoxicity against virus-infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus-1. NK cell-mediated antibody-dependent cytotoxicity or anti-CD16 antibody-redirected lysis is not significantly enhanced by NKSF/IL-12. However, the ability of resting peripheral blood T cells to mediate anti-CD3 antibody-redirected lysis is enhanced by 18-h incubation with NKSF/IL-12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Interleucinas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Animais , Células CHO , Células Cultivadas , Cricetinae , Antígenos HLA-DR/análise , Humanos , Interleucina-12 , Células Matadoras Naturais/imunologia , Receptores de IgG/fisiologia , Células Tumorais Cultivadas , Vírus/imunologiaRESUMO
In this report, we show that in vitro stimulation of human peripheral blood mononuclear cells (PBMC) with B lymphoblastoid cell lines results in preferential proliferation of cells with the phenotypic, genotypic and functional characteristics of natural killer (NK) cells. This culture system offers a useful method for obtaining large numbers of pure NK cells on which biochemical, molecular and functional studies can be performed. Using this culture system, and average 25-fold increase in NK cell number is obtained, whereas the number of T cells is increased only 3-fold. At early times, activation of both T and NK cells occurs, as detected by the presence of activation antigens on both cell types, but actual proliferation of NK cells starts on day 6 of culture. Elimination of CD3(+)/CD5(+) T cells from the cultured cells gives homogeneous preparations of large numbers of CD16(+)/NKH-1(+) cells that have the morphology of large granular lymphocytes, are powerful effectors of both spontaneous and antibody-dependent cell-mediated cytotoxicity, and rapidly proliferate in the presence of recombinant interleukin 2 (rIL-2). As fresh NK cells, NK cell-enriched preparations from 10-day cultures of Daudi-stimulated PBMC do not show rearrangement of the gene for the beta-chain of the T cell antigen receptor; the 1.3-kb functional transcript of the beta-chain gene was not expressed in NK cells, but the 1.0-kb truncated transcript was present in all preparations. Our data indicate that proliferation of both T and NK cells is dependent upon IL-2 production in the culture, because an anti-IL-2 antiserum completely suppresses proliferation. Because T cells, and in particular CD4(+) T cells, are required for preferential proliferation of NK cells, the NK cell stimulation induced by the B cell line is probably in part indirect, and due to induction of IL-2 production by allogeneic stimulation of CD4(+) cells. However, the B cell lines also need to interact directly with NK cells because neither allogeneic PBMC nor high doses of rIL-2 are sufficient to induce preferential proliferation of NK cells.