Comparative study of splenic natural killer cells and prothymocytes

An increase in the concentration of the acute-phase reactant, serum amyloid A (SAA), following endotoxin treatment, is a consequence of the action of lipopolysaccharide (LPS) on macrophages to produce a monokine, the SAA inducer, which in turn, triggers SAA synthesis by hepatocytes. We have found that murine SAA inducer is closely related, if not identical, to murine lymphocyte activating factor (LAF), otherwise known as Interleukin 1 (IL 1). Furthermore, both rabbit endogenous pryrogen (EP), which is believed to be identical to LAF (IL 1), and human LAF (IL 1), induced elevated SAA concentrations in C3H/HeJ mice. Antiserum previously shown to block both pyrogenic and thymocyte proliferating activities of the species of rabbit EP exhibiting an isoelectric point of pH 7.3 (EP 7), also blocked the SAA inducing activity of EP7. Phenylglyoxal treatment of highly purified murine LAF (IL 1) abrogated both thymocyte proliferating activity and the SAA inducing activity. These studies support and extend previous reports suggesting that within 2 hr of an inflammatory stimulus, macrophages produce a monokine that acts systemically to alter body temperature, activate T cells, and induce hepatic protein synthesis of acute-phase reactants.     

Radiosensitivity of human natural killer cells: binding and cytotoxic activities of natural killer cell subsets

The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.     

Cytotoxic action of natural killer cells on human tumor cells

Natural killer cell cytotoxicity was studied in a 18-hour 51Cr-release assay in the cultures of human tumor target cells: K562 leukemia and lung adenocarcinoma (LAC) cells. The mean cytotoxic value was similar for K562 and LAC cells: 36.13 +/- 3.23% and 40.78 +/- 3.43%, respectively, although significant individual variability was recorded. The similar cytolytic action of blood mononuclear cells (MNC) on the two tumor lines was observed in 30% of normal donors. MNC from 30% donors produced more pronounced lytic action on K562 cells while MNC from other 30% donors lysed mainly LAC cells. In the competitive inhibition test cold K562 cells more effectively than cold LAC cells suppressed the MNC-induced lysis of both K562 and LAC cells.     

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.     

Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

CD3 negative "small agranular lymphocytes" are natural killer cells

We describe here that CD3-, CD16+ and/or CD56+ small lymphocytes, in a highly reproducible fashion, mediate a significant level of K562 killing that is, on a "per cell" basis, comparable to the cytolytic activity of CD3- LGL. The CD3- small lymphocytes appeared to have no granules based on light and electron microscopy and lack of right-angle scatter on the FACS; we thus refer to them as small "agranular" lymphocytes (SAL). The lytic activity against K562 is inhibited by treatment with either L-leucine methyl ester or EGTA, which are reported to effect granule-dependent killing. We suggest that the SAL have lytic molecules in their cytoplasm (which are sensitive to these treatments) but that these molecules are not organized into discrete granules as found in LGL. The CD3- SAL are phenotypically very similar to LGL and both SAL and LGL mediated equal and reproducible antibody-dependent cell-mediated cytotoxicity. These observations force redefinition of the concept of NK cells to include both CD3- LGL and CD3- SAL.     

Adhesion and activation molecules expressed by human natural killer cells

Our study concerns the expression and the regulation of adhesion and activation receptors on human NK cells. In particular we provide evidence on: a) the expression on fresh human NK cells of VLA-4, VLA-5 and VLA-6, extracellular matrix (ECM) receptors of integrin family capable of mediating their adhesion to FN and LM; b) the role of PKC on the regulation of CD16, a differentiation antigen associated with FcR type III expressed by all NK cells, which mediate ADCC activity and trigger lymphokine production.     

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells

We observed that lysophosphatidylglycerol (LPG) stimulates chemotactic migration in human natural killer (NK) cells. The LPG-induced chemotactic migration of NK cells was completely inhibited by pertussis toxin (PTX). LPG also stimulated the extracellular signal-regulated kinase (ERK) and Akt activities in NK cells. LPG-stimulated ERK activity was inhibited by PTX, indicating the involvement of PTX-sensitive G-proteins. The preincubation of NK cells with an ERK inhibitor (PD98059) or phosphoinositide-3-kinase (PI3K) inhibitors (wortmannin and LY294002) completely inhibited LPG-induced chemotactic migration, suggesting the essential role of ERK and PI3K in the process. Moreover, LPG-induced chemotactic migration in NK cell was inhibited by Ki16425, an LPA_1/3 receptor-selective antagonist, suggesting the involvement of the Ki16425-sensitive G-protein coupled receptor (GPCR) in the process. Taken together, the results indicate that LPG stimulates chemotactic migration in NK cells through GPCR, suggesting a new function of LPG as a modulator of NK cell functioning.     

Participatory role of natural killer and natural killer T cells in atherosclerosis: lessons learned from in vivo mouse studies

Atherosclerosis is a multifactor, highly complex disease with numerous aetiologies that work synergistically to promote lesion development. One of the emerging components that drive the development of both early- and late-stage atherosclerotic lesions is the participation of both the innate and acquired immune systems. In both humans and animal models of atherosclerosis, the most prominent cells that infiltrate evolving lesions are macrophages and T lymphocytes. The functional loss of either of these cell types reduces the extent of atherosclerosis in mice that were rendered susceptible to the disease by deficiency of either apolipoprotein E or the LDL (low density lipoprotein) receptor. In addition to these major immune cell participants, a number of less prominent leukocyte populations that can modulate the atherogenic process are also involved. This review will focus on the participatory role of two "less prominent" immune components, namely natural killer (NK) cells and natural killer T (NKT) cells. Although this review will highlight the fact that both NK and NKT cells are not sufficient for causing the disease, the roles played by both these cells types are becoming increasingly important in understanding the complexity of this disease process.     

Immunologic and clinical aspects of natural killer cells in human leukemia

We have studied peripheral-blood, splenic and bone marrow natural killer (NK) activity in patients with leukemia. These studies demonstrated that leukemic patients displayed defective NK activity in all of these tissues. However, NK defect could be corrected by culture of effector cells with interleukin-2 (IL-2). The phenotypic analysis of IL-2 cultures showed clearly the heterogeneity of lymphocyte subsets. The characterization studies demonstrated that CD56+, CD3- NK cells manifested most potent lysis of leukemia, CD56+, CD3+ T cells mediated some, but low, antileukemia activity and CD56-, CD3+ T lymphocytes were devoid of cytotoxicity.     

Divergence in activation by poly I:C of human natural killer and killer cells

Summary Interferons consistently enhance spontaneous cellular cytotoxicity (SCC) mediated by natural killer (NK) cells. More controversial is the ability of interferons to enhance antibody-dependent cellular cytotoxicity (ADCC) mediated by killer (K) cells. Since NK and K cells appear to represent overlapping subpopulations of lymphocytes, the present study was undertaken to examine in greater detail the relationship between NK and K cell functional modulation by the potent interferon inducer, poly I:C. Utilizing peripheral mononuclear cells from a panel of 21 healthy individuals, treatment in vitro with poly I:C resulted in modulation of both SCC and ADCC. SCC was significantly enhanced in 52 of a series of 55 trials (95%), whereas ADCC was significantly enhanced in parallel in only 18 of the trials (33%). Cells which mediated enhanced ADCC were plastic-nonadherent, which is characteristic of K cells. SCC was consistently enhanced in all but two of the 14 individuals who were tested two or more times. By contrast, the ability of poly I:C to enhance ADCC varied between trials in 11 of these individuals. In the other three, ADCC enhancement never occurred. No correlation existed between SCC and ADCC augmentation despite use of the same target cell to assess the two lytic activities in parallel. Poly I:C exclusively enhanced SCC in 36 trials (65%) and exclusively enhanced ADCC in two trials (4%). Discordance between SCC and ADCC enhancement also occurred in three of eight trials (38%) in which lymphocytes were treated directly with interferon a. Results in long-term (18-h) ⁵¹Cr-release assays indicated that poly I:C accelerated the kinetics of ADCC without affecting the proportion of target cells lysed by K cells. By contrast, an increased proportion of target cells was killed by poly I:C-stimulated NK cells. These results suggest that the controversy concerning relative interferon effects upon NK and K cells derives from differences both quantitative and qualitative in nature. K cell activity is enhanced but at a relatively low frequency. Enhancement of NK cell activity is selective in the sense that it occurs independently of and with greater frequency than enhancement of K cell activity. Distinct biological mechanisms may, therefore, be involved in regulation and expression of NK and K cell activation by interferons.     

Recognition of vaccinia virus-infected cells by human natural killer cells depends on natural cytotoxicity receptors

Natural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.     

Evidence for a beta-adrenoceptor-mediated regulation of human natural killer cells

Pretreatment of lymphocytes (16 hr, 37 degrees C) with adrenaline at final concentrations of 10(-7) to 10(-9) M, followed by removal of the drug, increased natural killer (NK) cell activity vs K562 leukemic cells in a 4-hr 51Cr-release assay. The most efficient concentration of adrenaline was 10(-8) M; mean increase of NK activity over base-line activity for all donors examined was 30%. However, the individual response to adrenaline pretreatment was variable; in some donors, the effect was equal to maximal interferon (IFN) stimulation. Effects of adrenaline pretreatment were consistently reduced to base-line activity by co-incubation with the nonselective beta-adrenoceptor antagonist propranolol at 100-fold higher concentrations. The enhancing effect of adrenaline (10(-8) M) pretreatment was also observed after 1-hr pretreatment; this effect was prevented by simultaneous incubation with propranolol but was not affected by dex-propranolol. Direct addition of adrenaline to lymphocyte/target cell mixtures was inhibitory at 10(-6) M adrenaline concentration. The inhibitory effect of adrenaline in this assay was again completely prevented by propranolol and unaffected by dex-propranolol. The observed stimulatory effect of adrenaline pretreatment could not be ascribed to IFN production. Data presented indicate a dual effect of adrenaline on NK cell activity and suggest both a positive and a negative beta-adrenoceptor-mediated regulation of human NK cells.     

Characterization of an antigen expressed by human natural killer cells

A monoclonal antibody, anti-N901, was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human CML cells. This antibody was reactive with a subpopulation of peripheral blood LGL, including the natural killer cells. Monocytes, granulocytes, B cells, T cells (T3+ cells), erythrocytes, and platelets were nonreactive. The N901-positive cells in the peripheral blood were heterogeneous with respect to expression of other cell surface antigens. The majority of N901+ cells co-expressed T11, Mo1, and HNK-1, whereas a smaller percentage expressed T8. Ia, T3, T4, Mo2, or B1 antigens were very uncommon on N901+ cells. The heterogeneity of the N901+ LGL was further investigated by examining the expression of N901 antigen on a series of cloned normal human NK cell lines. N901 antigen was expressed by each of the NK cell lines tested, and by a minority of cloned T cell lines without NK activity. Anti-N901 does not block NK activity and can be used to rapidly purify functional NK cells for further study.     

Population dynamics of human activated natural killer cells in culture

The growth kinetics and population dynamics of recombinent interleukin-2 (rlL-2) stimulated human natural killer (NK) cell-enriched populations were studied in vitro. The NK-enriched populations was obtained from normal peripheral blood mononuclear cells (PBMNC) by immunomagnetic bead depletion of CD3(+) and CD5(+) T cells. The growth kinetics of NK cells, T cells, monocytes, and total cells are shown. In the absence of PBMNC accessory cells, the NK-enriched population showed limited expansion. In the presence of PBMNC accessory cells, the NK-enriched population expanded threefold more than in the absence of accessory cells due to increased NK cell growth rate and increased duration of exponential growth. Using a Transwell system, which separates two cell population by a polycarbonate membrane, the accessory cells were shown to act on the NK-enriched population via a diffusible factor. Accessory cell conditioned media was able to replace the accessory cell population to stimulate NK cell expansion. A monocyte-enriched population prepared by sheep red blood cell rosetting of T cells was extensively phenotyped and compared with the NK-enriched populations. Although the final cultured cells were phenotypically homogeneous for CD56(+)/CD3(-) NK cells, the initial NK precusor populations appear to be different. Namely, the NK cell precursors in the monocyte-enriched population were predominantly CD56(+)/CD2(-). Kinetic equations were formulated for this culture system and the effects of major culture variables are investigated.