In vitro generation of human activated lymphocyte killer cells. II. N-acetyl-D-galactosamine inhibits a distinct subpopulation of human activated lymphocyte killer cells generated in mixed lymphocyte culture

A range of monosaccharides was tested for its ability to inhibit the generation of cytotoxic cells during mixed lymphocyte culture. The most discriminatory effect was produced by N-acetyl-D-galactosamine (NADG). The presence of this sugar at the initiation of the coculture significantly inhibited in a dose-dependent manner the induction of a subset of nonspecific activated lymphocyte (ALK) cells preferentially able to lyse the K562 target cell (natural killer, NK-like cells) but had no effect on the generation of either specific cytotoxic T lymphocytes or another separate subset of ALK cells mediating lysis of an NK-insensitive melanoma cell line. The addition of conditioned medium containing interleukin 2 and interferon (IFN) at the start of culture reversed the inhibitory effect of the sugar. Under conditions of limiting dilution, the frequency of NK-like precursors ranged from 1/50 to 1/1200 with different mononuclear cells (MNC) and in all cases the presence of NADG from Day 0 of culture selectively decreased the frequency of these precursors. At the concentrations used NADG had no effect on NK-like cell cytolysis once generated. The addition of recombinant gamma-IFN did not abrogate the inhibitory effect of NADG and in MLC of some individuals decreased the frequencies of ALK cell precursors. These data provide further evidence for the heterogeneity of ALK cells and indicate that what is usually referred to as NK-like cell activity in in vitro culture is mediated by a subpopulation of MNC which are activated and induced to differentiate along a pathway independent of that of other ALK subsets.     

In vitro generation of natural killer cells from SJL/J bone marrow precursors

The development of natural killer (NK) cells from undifferentiated bone marrow (BM) precursors of low-NK-reactive SJL/J mice was studied. Results indicate that BM cells of untreated mice are not able to generate NK effector cells in cultures supplemented with recombinant interleukin-2 (IL-2). On the other hand in the presence of IL-2, NK cells are generated in cultures of BM from mice pretreated with 5-fluorouracil (5-FU, 150 mg/kg iv 4 days before harvesting), a treatment which has been shown to eliminate more differentiated but spare less differentiated BM precursors. The 5-FU resistant BM progenitor is asialoGM1-, Thy.1+, Lyt.1- and Lyt.2-. The cells generated by culturing with IL-2 are asialoGM1+, Thy.1+, Lyt.5+, Lyt.1-, Lyt.2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2 receptor (IL-2/r) antibodies. These studies demonstrate that it is possible to generate NK effectors from SJL/J BM cells by in vitro culturing with IL-2.     

High frequency of precursors of anomalous killer cells in human peripheral blood: evidence for T-cell regulation

The frequency of precursors (P) of the anomalous killer (AK) cells able to kill a melanoma target cell line without prior sensitization was determined by limiting dilution analysis. The frequencies obtained from the peripheral blood mononuclear cells of six healthy individuals ranged from 1/250 to 1/750, which was considerably higher than those of alloreactive cytolytic T-lymphocyte (CTL) precursors induced in the same cultures (range 1/900 to 1/7500). The presence of phytohemagglutinin (PHA) inhibited the appearance of both CTL and AK in bulk cocultures, and in limiting dilution analysis the presence of the lectin resulted in multiphasic cell dose-response curves rather than linear single hit responses for both types of precursor cells. The results suggest that AK-P are under the same type of regulation as are CTL-P.     

In vitro generation of tumour-specific lymphocyte reactivity to colonic carcinoma cells

Summary Purified tumour cells and normal mucosa cells from fresh human colorectal cancer resection specimens, and T-cell-enriched autologous peripheral blood lymphocytes, were mixed in short-term (6 day) mixed lymphocytetumour cell (MLTC) microcultures. Lymphocyte stimulation was measured by ³H-thymidine uptake, and a stimulation index (SI=[lymphocytes vs tumour cells (cpm)–tumour cells (cpm)]/[lymphocytes (cpm)])>3 was regarded as significant. Significant lymphocyte reactivity was found in 10/15 patients with colon carcinoma. However, 1 patient with autologous tumour reactivity, also showed significant stimulation against autologous normal mucosa cells, suggesting tumour-associated reactivity. Maximum stimulation occurred most frequently at a lymphocyte:tumour cell ratio of 2:1 and with nylon wool-passaged lymphocytes.Project was supported by a grant from the Cancer Research CampaignSupported by New South Wales Cancer Council Fellowship     

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c⁺ CD11b⁺ DCs derived from murine CD34-positive precursors. DCs derived from CD34⁺ cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34⁺ cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.     

Tac antigen-positive T cells activated in autologous mixed lymphocyte reaction regulate the generation of killer T cells against hapten-modified autologous cells

T cells that proliferate in the autologous mixed lymphocyte reaction (auto-MLR) have been shown to acquire some suppressor or regulatory activities. In the present study, we examined the suppressive effects of T cells activated in the auto-MLR on the induction of hapten-specific cytotoxic T cells. NRFT (depletion of ARFT from UT) were used as the responder cells of TNP-MLR. After primary and secondary TNP-MLR, the cells were harvested and tested for their cytotoxic activities against TNP-modified autologous cells by 51Cr-release assay. When UT cells cultured for 1 wk in auto-MLR were added to primary TNP-MLR at the beginning of culture, the cytotoxic activity tested at the end of the culture was suppressed from 15.6% +/- 2.7 to 5.8% +/- 1.1 (percent cytotoxicity, mean +/- SE). However, these auto-MLR-activated UT cells had little suppressive activity against cytotoxic T cells when they were added to the final assay of TNP-CTR. Suppressive activities of these cells on the generation of cytotoxic T cells during secondary TNP-MLR were also tested. The addition of auto-MLR-activated UT cells to the secondary TNP-MLR at the beginning of the culture reduced the cytotoxic activities of NRFT from 23.8% +/- 2.3 to 9.7% +/- 1.7 after secondary TNP-MLR. Allo-activated T cells, PHA blasts, and fresh autologous T cells were used as the controls, but none of the cells had suppressive effects on the generation of CTL. Characteristics of these suppressor cells were examined. Auto-MLR-activated cells from ARFT fractions exhibit very powerful suppressor activity. Treatment of the auto-MLR-activated T cells with mitomycin C eliminated their suppressive effects on the generation of CTL; 21.2% +/- 6.3 of UT cells became anti-Tac positive after 1 wk of auto-MLR. Treatment of auto-MLR-activated UT cells with anti-Tac antibody plus complement eliminated their suppressive activities on the induction of CTL. Thus, T cells stimulated in auto-MLR were shown to have suppressive effects on the induction of cytotoxic T cells against TNP-modified autologous cells. These cells were mitomycin C sensitive. Because anti-Tac antibody is reactive to activated T cells, activation of T cells during auto-MLR was thought to be necessary for the acquisition of the suppressive activity.     

In vitro lymphocyte cytotoxicity. I. Evidence of multiple cytotoxic molecules secreted by mitogen activated human lymphoid cells in vitro.

Multiple families of cytotoxic molecules [Lymphotoxin (LT)] have been identified in phytohemagglutinin (PHA-P) activated human lymphocyte supernatants and lymphocyte homogenates, using gel filtration chromatography on Sephadex G-150. These macromolecules have molecular weights of 80–90,000, 50,000, and 10–15,000 daltons and have been termed LT₂, LT₂ and LT₃, respectively. They are secreted by cells from a variety of lympboid tissues, i.e., tonsil, adenoid, and peripheral blood. The kinetics of appearance of the cytotoxins indicate that all three are present within 16 hr after lymphocyte activation. However, while LT₁ and LT₂ persist in these cultures through day 5, LT₃ is not detectable after day 3. These molecules can also be detected when either PHA or concanavalin A are employed as the stimulating agent. Moreover, the relative amounts of LT₁, LT₂ and LT₃ activity in a given supernatant vary dramatically from culture to culture. Extracellular levels of LT accumulate and peak by 4 to 5 days in culture, however, intracellular levels of LT reach a maximum on day 3 and decrease to very low levels on day 5. Mitogen-stimulated lymphocytes at 3 days contain intracellular levels of LT which are several logs higher than that detectable in unstimulated cells. This observation suggests that both the biosynthesis and secretion of lymphotoxin is governed by a regulatory control process(es).     

Functional studies on the precursors of human lymphokine-activated killer cells

Human peripheral blood lymphocytes cultured for 4 days in the interleukin 2 (IL-2)-containing cell-free supernatant of the MLA144 cell line (MLA144CM) are cytolytic to NK-susceptible and NK-resistant tumor target cells. This lymphokine-activated killer (LAK) activity is dependent on IL-2 as development of LAK activity is inhibited in the presence of a monoclonal antibody (MoAb) reacting with the IL-2 receptor (anti-Tac). Addition of cyclosporin A (CyA) to mixed lymphocyte cultures inhibits the development of allospecific cytotoxic activity and inhibits the development of IL-2 responsiveness. However, development of LAK activity is unaffected by the inclusion of CyA in the cultures, showing that the LAK precursor can be functionally distinguished from the allospecific cytotoxic precursor cell. Development of LAK activity does not require mature NK cells as shown by the generation of LAK activity from NK inactive human thymocytes and lymph node cells. In addition, depletion of NK activity from human PBL does not impair the development of LAK activity.     

In vitro generation of human cells with cancer stem cell properties

Cancer stem cells (CSCs) have been implicated in the maintenance and progression of several types of cancer. The origin and cellular properties of human CSCs are poorly characterized. Here we show that CSC-like cells can be generated in vitro by oncogenic reprogramming of human somatic cells during neoplastic transformation. We find that in vitro transformation confers stem-cell properties to primary differentiated fibroblasts, including the ability to self-renew and to differentiate along multiple lineages. Tumours induced by transformed fibroblasts are hierarchically organized, and the cells that act as CSCs to initiate and maintain tumour growth are marked by the stage-specific embryonic antigen SSEA-1. Heterogeneous lineages of cancer cells in the bulk of the tumour arise through differentiation of SSEA-1(+) fibroblasts, and differentiation is associated with loss of tumorigenic potential. These findings establish an experimental system to characterize cellular and molecular properties of human CSCs and demonstrate that somatic cells have the potential to de-differentiate and acquire properties of CSCs.     

In vitro differentiation of transplantable neural precursors from human embryonic stem cells.

The remarkable developmental potential and replicative capacity of human embryonic stem (ES) cells promise an almost unlimited supply of specific cell types for transplantation therapies. Here we describe the in vitro differentiation, enrichment, and transplantation of neural precursor cells from human ES cells. Upon aggregation to embryoid bodies, differentiating ES cells formed large numbers of neural tube-like structures in the presence of fibroblast growth factor 2 (FGF-2). Neural precursors within these formations were isolated by selective enzymatic digestion and further purified on the basis of differential adhesion. Following withdrawal of FGF-2, they differentiated into neurons, astrocytes, and oligodendrocytes. After transplantation into the neonatal mouse brain, human ES cell-derived neural precursors were incorporated into a variety of brain regions, where they differentiated into both neurons and astrocytes. No teratoma formation was observed in the transplant recipients. These results depict human ES cells as a source of transplantable neural precursors for possible nervous system repair.     

Expression of human T lymphocyte antigens by killer cells.

K cells, the effectors of antibody-dependent cell-mediated cytotoxicity, were found to express human T but not B lymphocyte antigens detected by rabbit anti-HTLA and anti-HBLA. Pretreatment of effector cells with anti-HTLA+C inhibited ADCC by specifically lysing K cells: no inhibition of ADCC by anti-HTLA occurred when deltaC was substituted for C. By contrast, pretreatment of effector cells with anti-HBLA nonspecifically inhibited ADCC, probably for forming antigen-antibody complexes with HBLA+ cells in effector suspensions: a) treatment with anti-HBLA deltaC was more inhibitory of ADCC than treatment with anti-HBLA+C, and b) the inhibitory effect of anti-HBLA on ADCC was either eliminated or markedly reduced if effector suspensions were first passed through a nylon fiber column, a procedure that removed most HBLA+ cells without affecting K cell activity. HTLA antigens expressed by K cells and NK cells are the same as HTLA antigens expressed by thymocytes since thymocytes completely absorb the anti-K cell and NK cell reactivity of anti-HTLA.     

In vitro tumour cell growth inhibition: a comparative study between allosensitized cytotoxic T lymphocytes and lymphokine activated killer cells

There is general agreement that several distinct subpopulations of lymphocytes, including major histocompatibility complex (MHC)-restricted T lymphocytes and non-restricted natural killer, or lymphokine-activated killer (LAK), cells are active in lysing neoplastic cells. In this study experiments were designed to compare the inhibitory effects of LAK cells and allosensitized cytotoxic T lymphocytes (CTL) on in vitro growth of an Epstein–Barr virus-transformed B-cell line (BSM) and of a HTLV-I producer T-cell line (MT-2). It was found that allosensitized CTL are more efficient at inducing BSM, or MT-2, cell growth inhibition than LAK cells. These results are consistent with the hypothesis that MHC-restricted T effector cells could mediate higher tumour suppressive effects than non-MHC restricted LAK cells.     

The DNA methylation profile of activated human natural killer cells

Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states.     

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.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anti-CD3 + IL-2-stimulated murine killer cells. In vitro generation and in vivo antitumor activity

The growth, phenotype, in vitro cytolytic characteristics, and in vivo antitumor activity of murine splenocytes stimulated with anti-murine CD3 mAb in combination with IL-2 as compared with IL-2 alone was investigated. When cultured for 12 days with anti-CD3 mAb + IL-2, murine splenocytes increased 100- to 4000-fold in number compared with only 6- to 20-fold for cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2 activated cultures developed high lymphokine-activated killer activity against NK-resistant targets including the P815 mastocytoma cell line and fresh MCA 106 sarcoma. Peak cytotoxicity on a per cell basis developed by day 8 after anti-CD3 mAb + IL-2 activation. A large proportion of the total cytolytic activity of long term anti-CD3 mAb + IL-2-stimulated cultures was related to the presence of anti-CD3 in the assay, indicating enhancement of cytotoxicity by activated CD3+ T cells. Phenotypic analysis indicated that anti-CD3 mAb + IL-2-stimulated cultures contained heterogeneous populations of T cells with increased percentages of both CD4+ and CD8+ phenotypes compared with cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2-stimulated cells were tested for their in vivo antitumor activity by using C57BL/6 mice bearing MCA 106 sarcoma pulmonary metastases. IL-2-activated murine killer cells were given in combination with in vivo IL-2 and indomethacin, the latter of which was shown to potentiate the antitumor effect of IL-2. When given on day 5 after tumor inoculation, cell doses as low as 5 x 10(6) anti-CD3 mAb + IL-2-stimulated cells per mouse significantly reduced the number of pulmonary metastases (p less than 0.005). Thus, activation with the combination of anti-CD3 mAb + IL-2 produces rapidly expanding cultures of cytolytic cells with demonstrated in vivo antitumor efficacy.     

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

Summary The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10–200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of TNF suggesting interferon-independent activation. In further experiments after i.p. injection of TNF peritoneal exudate cells (PECs) only killed YAC-1 targets in a 4-h assay. There was no additional killing in an 18-h assay towards neither YAC-1 cells or P815 cells, suggesting that macrophages were not involved. Furthermore TNF was also active in vitro by activating NK cells in isolated human peripheral blood cells. However in the PECs stimulated in vitro no significant induction of cytotoxic capacities by TNF was measured. Our data suggest that the action of TNF is not restricted to the lysis of tumor cells but can also induce immunological properties in the host defense against virus infections and neoplasms.     

In vitro regulation of human lymphocyte proliferation by selenium

A chemoprotective role for dietary selenium in malignancy has been well documented in numerous epidemiological and experimental studies. The precise mechanisms of this relationship are not understood, but may be related to observations that selenium can inhibit the proliferation of various normal and neoplastic cells, both in vivo and in vitro. In this study, we present evidence that selenium at physiologic concentrations can effectively inhibit the overall proliferation of human lymphocyte populations in response to various immune stimuli in vitro, including mixed lymphocyte response and response to soluble antigen (tetanus toxoid). This inhibition was reversible, indicating that selenium was not toxic to the lymphocytes at these concentrations. Preliminary data from our laboratory indicate that the antiproliferative effects of selenium may be specific for certain lymphocyte subsets. Similar modulation of immune responses in vivo could enhance various humoral and cellular immune mechanisms. Together with published evidence that selenium can inhibit tumor cell proliferation, these data may help to explain the decreased incidence of cancer associated with elevated selenium intake.     

Epinephrine-induced mobilization of natural killer (NK) cells and NK-like T cells in HIV-infected patients

HIV infection is known to cause changes in phenotype and function of natural killer (NK) cells. The aim of this study was to characterize the NK cells mobilized from peripheral reservoirs in human immunodeficiency virus (HIV)-infected patients and controls. Seventeen HIV-infected patients and eight age- and sex-matched controls received a 1-h epinephrine infusion. Epinephrine induced mobilization of high numbers of NK-like T cells with no difference between HIV-infected patients and controls. Interestingly, all subjects mobilized NK cells containing increased proportions of perforin, in particular the CD3(-)CD16(+)CD56(+) NK cell subset. The HIV-infected patients mobilized CD3(-)CD16(-)CD56(+) and CD3(-)CD16(+)CD56(+) NK cells to a lesser extent than did controls. In contrast, the HIV-infected patients mobilized relatively more CD3(-)CD16(+)CD56(-) NK cells independent of antiretroviral treatment. It is suggested that these cells represent an immature NK cell subpopulation possibly resulting from an impaired cytokine tissue environment in HIV-infected patients.