K562 Target Cells Research Articles

Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition

Granzymes are key components of the immune response that play important roles in eliminating host cells infected by intracellular pathogens. Several granzymes are potent inducers of cell death. However, whether granzymes use additional mechanisms to exert their antipathogen activity remains elusive. Here, we show that in adenovirus-infected cells in which granzyme B (gzmB) and downstream apoptosis pathways are inhibited, granzyme H (gzmH), an orphan granzyme without known function, directly cleaves the adenovirus DNA-binding protein (DBP), a viral component absolutely required for viral DNA replication. We directly addressed the functional consequences of the cleavage of the DBP by gzmH through the generation of a virus that encodes a gzmH-resistant DBP. This virus demonstrated that gzmH directly induces an important decay in viral DNA replication. Interestingly, gzmH also cleaves the adenovirus 100K assembly protein, a major inhibitor of gzmB, and relieves gzmB inhibition. These results provide the first evidence that granzymes can mediate antiviral activity through direct cleavage of viral substrates, and further suggest that different granzymes have synergistic functions to outflank viral defenses that block host antiviral activities.

B7-1 and 4-1BB ligand expression on a myeloma cell line makes it possible to expand autologous tumor-specific cytotoxic T cells in vitro

The aim of this study was to confer an antigen-presenting cell (APC) ability on multiple myeloma cell lines (HMCLs) using B7-1 and/or 4-1BBL gene transfer. HMCLs were retrovirally transduced with B7-1 and/or 4-1BBL cDNAs. Allogeneic or autologous T cells were stimulated by coculture with B7-1- and/or 4-1BBL-transduced HMCLs in the presence of interleukin-2. T cell clones were obtained by limiting dilution. T-cell activation was assessed by interferon-gamma Elispot assays and cytotoxicity by (51)Cr release assays. Neither primary multiple myeloma cells (MMCs) nor HMCLs expressed B7-1 or 4-1BBL, and these molecules could not be induced by CD40 triggering. HMCLs failed to stimulate allogeneic or autologous T cells. Transduction of HMCLs with B7-1 and/or 4-1BBL retroviruses induced a high expression of B7-1 and 4-1BBL molecules and a strong T-cell activation ability. Long-term cultured CD8(+) T-cell lines could be obtained by stimulation with the autologous B7-1/4-1BBL XG-19 HMCL. These cytotoxic T lymphocytes (CTL) efficiently killed the autologous parental XG-19 HMCL as well as autologous primary MMCs and allogeneic HMCLs. They did not kill autologous CD34 cells and autologous EBV cell line or natural killer target K562 cells. Cloned CTL could recognize allogeneic HMCLs, demonstrating that a shared anti-MMC repertoire was expanded. Transduction with B7-1 and 4-1BBL retroviruses turned HMCLs into efficient APCs. It permitted the long-term expansion of autologous anti-tumor CTL with a shared anti-MMC repertoire, for one HMCL. These data suggest developing an immunotherapy using modified tumor cells in patients with multiple myeloma.

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell–target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25–50% higher, and the amount of lytic protein released was 55–65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

Validation of Flow Cytometric Measurements of NK Cytotoxicity Against Chromium Release Assay for Use in Clinical Monitoring of Immune Function.

Chromium (51Cr) release assays have long been a standard technique for measuring the cytolytic activity of various immune effector cells. Although relatively easy to perform, these assays suffer from problems with sensitivity and require special radiation precautions limiting their routine use in clinical laboratories. Several flow cytometric techniques measuring cytotoxicity have been introduced and used as a measure of immune response. These assays are more rapid than the 51Cr based assay and have the added advantage of freeing the laboratory from the precautions required when working with radioactivity. Such testing has however largely been restricted to the research setting. In an attempt to extend these benefits to a clinical laboratory, we attempted to validate two commercially available flow cytometry kits (CyToxiLux Plus® & GranToxiLux®, OncoImmunin, Gathersburg, MD) measuring either caspase or granzyme B activity inside K562 target cells induced by interaction with NK cells. These assays were then compared with a standard NK cytotoxicity assay using 51Cr release technique. With K562 as a target, we parallel tested three CD56+CD3-KIR- NK clones; both the 51Cr and caspase based cytotoxicity values were highly positive. With WH autologous EBV cell line as target, we parallel tested five NK clones and five normal control donors as effectors. As expected, all cytotoxicity values for both assays were <10%. The caspase and granzyme assays were then compared and the degree of separation in the flow cytometric plots of the granzyme based assay were found to be marginally superior facilitating analysis of the data. The granzyme B assay therefore was chosen for investigation in the clinical samples. The granzyme and 51Cr assays were then compared both in 49 normal control donors and in 10 patients. There was a high level of concordance between the two assays. Comparing all results using linear correlation yielded a squared correlation (R2) value of 0.6878. However, given the wide range of NK activity in normal control donors the degree of positivity may not be clinically significant. Therefore, the data was examined in the context of a positive 51Cr response. Using a 2×2 table to exam the level of agreement, the kappa statistic was 0.63, indicating good agreement between the assays. When progressively fewer effector cells were added to a fixed number of target cells, the % cytotoxicity decreased from 39% to 0% with the flow cytometric assay and from 26% to 1% with the 51Cr assay. In the normal control donors the positive values ranged from 17% to 65% cytotoxicity using the flow assay compared with the previous range of 15% to 72% established with 51Cr. Patients having cytotoxicity values below 17% were deemed as having an unreactive or abnormal low result. One of the patients with few CD56+CD3- NK cells had consistently low cytotoxicity each time tested. For the patients who have either undergone allogeneic stem cell transplantation or have a known immunodeficiency, further clinical follow-up will serve to establish the clinical relevance of these measurements. These preliminary data suggest that the flow cytometric technique can be used in the clinical setting for immune monitoring.

Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells.

NO inhibits cytotoxic T lymphocyte killing of target cells, although the precise mechanism is unknown. We hypothesized that NO decreases exocytosis of cytotoxic granules from activated lymphocytes. We now show that NO inhibits lymphokine-activated killer cell killing of K562 target cells. Exogenous and endogenous NO decreases the release of granzyme B, granzyme A, and perforin: all contents of cytotoxic granules. NO inhibits the signal transduction cascade initiated by cross-linking of the T cell receptor that leads to granule exocytosis. In particular, we found that NO decreases the expression of Ras, a critical signaling component within the exocytic pathway. Ectopic expression of Ras prevents NO inhibition of exocytosis. Our data suggest that Ras mediates NO inhibition of lymphocyte cytotoxicity and emphasize that alterations in the cellular redox state may regulate the exocytic signaling pathway.

Effector Activity of Decidual CD8+ T Lymphocytes in Early Human Pregnancy1

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.

The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity

Toll-like receptor (TLR) ligands are potent inducers of the innate immune system, of which NK and NKT cells play an important role. We examined the direct activation of highly purified human NK and/or NKT cells with known TLR ligands. NK/NKT cells were positive for all known TLR mRNA (TLR1–10). Ligands for TLR2–5 induced production of significant amounts of IFN-γ by purified NK cells. However, a TLR9 ligand failed to induce significant levels of the cytokine. NK cells were depleted from PBMCs to confirm that they were the main source of IFN-γ following treatment with TLR ligands, which resulted in a significant decrease in cytokines. The direct effects of TLR ligands on NK cytotoxicity were determined using 51Cr-labeled K562 target cells. Ligands for TLR2–5 were potent inducers of NK cell cytotoxicity, a TLR9 ligand was not. Our results suggest that TLR ligands can directly stimulate and enhance NK cell cytokine production and induce cytotoxic activities.

Human Congenital Infection With Trypanosoma cruzi Induces Phenotypic and Functional Modifications of Cord Blood NK Cells

We studied the phenotype and activity of cord blood natural killer (NK) cells in newborns congenitally infected with Trypanosoma cruzi. We found that the proportion of CD56(bright) NK cells was significantly decreased in cord blood from these newborns, suggesting they may have been recruited to secondary lymphoid organs. The remaining CD56(bright) NK cells exhibited a defective ability in the production of interferon (IFN)-gamma following in vitro activation with interleukin (IL)-12 + IL-2 or IL-12 + IL-15 cytokines, as compared with NK cells from uninfected newborns. In addition, cord blood NK cells from congenitally infected newborns stimulated with cytokines have a decreased release of granzyme B (GrB) when incubated with K562 target cells. This defect in cytotoxic effector function is associated with a reduced surface expression of activating NK receptors (NKp30, NKp46, and NKG2D) on CD56(dim) NK cells compared with uninfected newborns. These alterations of fetal NK cells from congenitally infected newborns may reflect a down-regulation of the NK cell response after an initial peak of activation and could also be the result of T. cruzi modulating the immune response.

Expression of Glycosylphosphatidylinositol-Anchored CD59 on Target Cells Enhances Human NK Cell-Mediated Cytotoxicity

NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs previously shown to lack blocking capacity for C-mediated lysis. We investigated the consequences for alternative membrane attachment modalities, namely bis-myristoylated-peptidyl (BiMP) and GPI anchoring, on CD59-negative U937 cells. Expression of GPI-anchored CD59 either via transfection or incorporation rendered U937 targets more susceptible to NK cytotoxicity, whereas incorporation of CD59 via a BiMP anchor to similar levels did not alter susceptibility to NK cytotoxicity. Localization of both BiMP- and GPI-anchored CD59 proteins was shown to be within the lipid raft microdomain. A role for the GPI anchor and independence from glycosylation status was confirmed by expression of transmembrane-anchored CD59 or unglycosylated CD59 and by testing in NK cytotoxicity assays. To investigate mechanisms, we compared the signaling capacity of the various forms of expressed and incorporated CD59 following Ab cross-linking in calcium flux assays. GPI-anchored CD59, with or without glycosylation, mediated activation events, whereas CD59 forms lacking the GPI anchor did not. The data show that the increased susceptibility of target cells expressing CD59 to NK cytotoxicity requires GPI anchor-mediating signaling events, likely mediated by interactions between GPI-anchored CD59 on targets and NK receptors.

Tumour-expressed tissue factor inhibits cellular cytotoxicity.

The association between tissue factor (TF) expression and increased rate of tumour metastasis is well established. In this study, we have examined the hypothesis that the expression of TF by disseminated tumour cells confers protection against immune recognition and cytotoxicity. A hybrid EGFP-TF protein was expressed in HT29 colon carcinoma and K562 lymphoblast cell lines. To assess the cytotoxic activity against tumour cells over-expressing TF, a novel method was used, based on the direct measurement of fluorescently labelled HT29 or K562 target cells. Upon challenge with peripheral blood mononuclear cells (PBMC), tumour cells expressing TF partially evaded cellular cytotoxicity (Delta=15-40% reduction in cytotoxicity). Moreover, the influence of TF was not primarily dependent on its procoagulant function, although the inclusion of 20% (v/v) plasma did lower the rate of cytotoxicity against untransfected cells. However, expression of a truncated form of TF, devoid of the cytoplasmic domain, did not mediate any degree of inhibition of cytotoxicity, suggesting that the protective function of TF is principally due to this domain. We conclude that TF can promote immune evasion in tumour cells expressing this protein leading to increased survival and therefore metastatic rate in such cells.

Natural killer cell activity and lymphocyte activation: Investigating the effects of a selection of essential oils and components in vitro

A selection of essential oils and components were tested in vitro for potential immunomodulating effects on natural killer cell activity (NKCA) and lymphocyte activation through CD69 expression. Matricaria recutita , Boswellia carteri , Pelargonium graveolens , Lavandula angustifolia , Citrus limon , Melaleuca alternifolia , Melaleuca viridiflora , Santalum spicatum , Cedrus atlantica , and Thymus vulgaris ct. linalool essential oils were solubilised with ethanol and methylated β-cyclodextrin 1:5:25 (w/v) and pre-incubated at 10 μg/mL and 2 μg/mL ( M. alternifolia and C. atlantica : 2 and 1 μg/mL) with human peripheral blood mononuclear cells (PBMC) for 2 h. They were then incubated for 2 h with K562 target cells before determination of NKCA. The first 6 of these oils were similarly solubilised, pre-incubated for 2 h with aliquots of whole blood, then incubated with PHA for 4 h before measurement of CD69 antigen expression. Reference components α-pinene, S -(−)-limonene, linalool, geraniol, thymol, 1,8-cineole, linalyl acetate, eugenol, (+)-terpinen-4-ol, and trans -caryophyllene were solubilised with ethanol 1:5 and similarly tested. Dose-dependent stimulation of NKCA was found for trans -caryophyllene and linalyl acetate. No other effects of essential oils or components were found in these assays. Methylated β-cyclodextrin proved to be a less than ideal agent for enhancing solubility of essential oils, due to substantial effects on assay outcomes.

A study of CD33 (SIGLEC-3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing

The expression of CD33, a restricted leukocyte antigen considered specific for myeloid lineage, has been studied extensively on lymphoid cells. We demonstrated that wide subsets of mitogen- or alloantigen-activated human T and natural killer (NK) cells express CD33 at protein and nucleic acid levels. CD33+ and CD33- T and NK cell populations showed identical surface expression of activation markers such as CD25, CD28, CD38, CD45RO, or CD95. Myeloid and lymphoid CD33 cDNA were identical. However, lymphoid CD33 protein had lower molecular weight, suggesting cell type-specific, post-translational modifications. Additionally, reverse transcriptase-polymerase chain reaction and Northern blot analysis showed an unknown CD33 isoform (CD33m) expressed on all CD33+ cell lines or T cell clones tested. CD33m was identical to CD33 (CD33M) in the signal peptide, the immunoglobulin (Ig) domain C2, the transmembrane, and the cytoplasmic regions but lacked the extracellular ligand-binding variable Ig-like domain encoded by the second exon. CD33m mRNA was mostly detected on NKL and myeloid cell lines but poorly expressed on B cell lines and T lymphocytes. The CD33m extracellular portion was successfully expressed as a soluble fusion protein on transfected human cells, suggesting a functional role on cell membranes. Cross-linking of CD33 diminished the cytotoxic activity of NKL cells against K562 and P815 target cells, working as an inhibitory receptor on NK cells. These data demonstrate that CD33 expression is not restricted to the myeloid lineage and could exist as two different splicing variants, which could play an important role in the regulation of human lymphoid and myeloid cells.

Generation of in vitro B-CLL specific HLA class I restricted CTL responses using autologous dendritic cells pulsed with necrotictumor lysate

New approaches in the treatment of chronic B lymphocytic leukemia (B-CLL) have led to improved clinical response rates. In this setting there is a need to evaluate novel therapeutic approaches that aim to eradicate minimal residual B-CLL cells following an initial favorable response. The use of tumor lysate-pulsed dendritic cells (DC) represents a potentially important development in the field of cancer vaccination. B-CLL is ideally suited for DC-based vaccination since tumor cells are readily available (peripheral blood) and both known (tumor idiotype) and unknown antigens can be exploited to stimulate immune responses. In the current study we have evaluated the ability to stimulate in vitro autologous immune reactivity against target B-CLL cells using autologous DCs pulsed with B-CLL tumor lysate. Enhanced specific T cell IFN-γ expression was detected in 9 of 14 patients evaluated. These responses were specific with increased levels of IFN-γ mRNA measurable in T-cells stimulated with NC-DCs and not unpulsed DCs or DCs pulsed with normal B cell lysate. CTLs demonstrating increased levels of IFN-γ mRNA also lysed autologous B-CLL targets cells in an MHC class 1-restricted manner by 51chromium release assay. Priming target leukemic cells with CD40 ligand and IL-4 enhanced CTL killing. The effector CTL displayed negligible toxicity against NK susceptible target cells K-562 and spared CD19+CD5− normal B cells in cytotoxicity assays. The specificity of the CTL response was confirmed by blocking HLA class I molecules and cold target inhibition assays.

Human CD56bright and CD56dim Natural Killer Cell Subsets Respond Differentially to Direct Stimulation with Mycobacterium bovis Bacillus Calmette‐Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.

A differential assay of NK-cell-mediated cytotoxicity in K562 cells revealing three sequential membrane impairment steps using three-color flow-cytometry

Annexin V and propidium iodide (PI) staining is a general technique for detecting apoptosis by flow-cytometry (FCM). The release of 2′,7′-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein (BCECF), a non-lipophilic membrane-impermeable labeling dye, from the cytoplasm of target cells is an indicator of increased membrane permeability. This study aimed to devise a three-color FCM technique involving the BCECF-release parameter in addition to conventional Annexin V and PI staining for the analysis of target K562 cells undergoing cytotoxic/apoptotic processes mediated by natural killer (NK) cells. The results demonstrated the following step-wise process of membrane impairment: (1) initiation of Annexin V staining accompanied by increasing forward scatter (FSC) before BCECF-release, indicating membrane impairment without permeabilization by necrosis; (2) BCECF-release with decreasing FSC before PI influx; and (3) PI staining with the lowest FSC state. Therefore, the early stage of cytotoxicity/apoptosis conventionally defined by the flow-cytometric criteria of Annexin V staining before PI staining could be sub-divided into two stages before and after BCECF-release. Annexin-V staining in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was also initiated without BCECF-release. Although the underlying mechanism of the transition process from stage 1 to stage 2 is still unknown, this FCM technique should be a useful tool for differential assays of target cells regarding the sequential processes of NK-induced cytotoxicity.

Comparative study of NK cell-mediated cytotoxicity using radioactive and flow cytometric cytotoxicity assays

Cell-mediated cytotoxicity is a major effector pathway of the immune system. Thus far, radioactive assays have been widely used, but have significant disadvantages. Meanwhile, flow cytometric assays have been established but have not all been assessed simultaneously relative to the radioactive assays. Here, we have evaluated flow cytometric enumeration of surviving target cells, annexin-V binding and detection of activated caspase-3 and caspase-6 in direct comparison to the 51chromium ( 51Cr) release assay, and the JAM test. For assay evaluation NKL effector cells Fas-resistant K562 and Fas-sensitive Jurkat target cells were studied. Percent specific lysis measured for each E : T ratio was fitted to a sigmoid dose response curve. Both the flow cytometric and radioactive cytotoxicity assays showed equivalent background lysis (1–13%) but differed considerably with respect to maximum cytotoxicity (11–82% in K562 and 49–75% in Jurkat cells). Half maximum lysis ranged from 4 : 1 to 28 : 1 E : T ratios in K562 cells and from 1 : 3 to 24 : 1 in Jurkat cells, respectively. Flow cytometric enumeration of surviving target cells was the only assay which permitted detection of cytotoxicity at considerable lower E : T ratios (in K562 cells 1 : 4 to 2 : 1 and in Jurkat cells 1 : 4 to 1 : 1) than the conventional assays. Prolonged incubation over 24 h did not improve the sensitivity for flow cytometric enumeration of surviving target cells or the JAM test. The observed differences in the lysis of target cells are likely to reflect different sensitivity of cell death-associated changes which are measured by each assay. Thus, the particular choice of a cytotoxicity assay must be carefully adapted to the experimental situation under study.

A novel telomerase-based approach to detect natural cell-mediated cytotoxic activity against tumor cells in vitro

This study was designed to develop a novel technical approach based on tumor-associated telomerase activity to detect cytotoxic activity of effector cells of the natural immune system against neoplastic cells. Human K562, DAUDI or Raji leukemia cells were co-cultured with NK or LAK effector cells at 37 °C for 4 h. Target cell killing was evaluated by 51Cr-release assay (CRA) or reduction of telomerase activity (R-TRAP CTX) of the target after exposure to effector cells. NK and LAK effector cells tested against K562 target cells at effector/target ratio of 50:1 showed cytotoxicity of 65% and 78%, respectively, with CRA and 51% and 74%, respectively, with R-TRAP CTX. Incorrect results were obtained with CRA when target cells were admixed with normal fibroblasts, whereas R-TRAP CTX was not influenced by the presence of normal cells. Control experiments performed with telomerase-negative cells showed that telomerase activity of effector cells was not altered during the cytolytic reaction. Moreover, supernatants obtained from effector–target cell co-cultures did not influence telomerase activity of targets. This novel R-TRAP CTX method to assay anti-tumor natural and possibly antigen-dependent cell-mediated cytotoxicity appears to provide sensible advantages over classical CRA or gamma-interferon release by effector cells in presence of target cells (ELISPOT), since (a) it furnishes reliable data on effector cell killing against neoplastic cells, even when malignant cells are admixed with normal cells, as frequently occurs in tumor biopsies, not manageable with CRA; (b) it provides an actual measure of target cell killing, not furnished by ELISPOT technique.

Reply to ‘NK cell subsets and CD107a mobilization assay’ by Zimmer et al

In the extensive comment by Zimmer et al on our article three points of criticism are expressed: (1) a simultaneous publication dealing with the same topic has not been cited, (2) the reported results are not specific for CD56 NK-cells because CD3 NKT-cells were not excluded from the analysis and (3) the degranulation of CD56CD16 in response to tumor targets does not fit to previous studies reporting cytotoxic effects after co-incubation with CD56CD16 NK cells. First, although we have not cited the online version of the most recent paper of Alter et al, we have cited the earlier work by this group on the assessment of NK-cell activity by CD107a surface expression in the introduction of our article. Second, the analysis of CD3 CD56 NK-T cells (after staining with anti-CD3 FITC mAb, anti-CD56 PE and antiCD107a PE-Cy5) revealed that ca 2% of these cells degranulated in response to K562 cells. Since the finding that a part of the CD56CD16 cells are NK-T cells, which to some extent mediate natural cytotoxicity towards tumor targets, is not new we have not discussed it explicitly in our paper. Nevertheless (and most importantly), our observation that neither CD56 nor CD56/CD16 NK cells degranulated in response to tumor targets is independent from the presence or absence of CD56CD3 cells. Third, the fact that the surface expression of CD107a in response to tumor targets is restricted to the CD56/CD16 subpopulation has been reproducibly and unambiguously shown under different experimental conditions. This finding and the close correlation of degranulation and cytotoxicity can indeed not easily be linked to the results of previous studies showing that CD56/CD16 NK cells are able to kill K562 target cells, as indicated in chromium release cytotoxicity assays. We apologize that we have not further discussed this incongruity by ourselves and so far cannot provide a convincing explanation for these discrepant but clear results. However, there are many examples for scientific progress through results that are somehow discrepant to previous and well-established assumptions. We hope that our reply has helped to clarify methodological aspects and that our findings may have stimulated the discussion in this field.

Erratum

American Journal of Reproductive ImmunologyVolume 54, Issue 2 p. 124-124 Free Access Erratum First published: 05 August 2005 https://doi.org/10.1111/j.1600-0897.2005.00303.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Am J Reprod Immunol 2005; 53:13–20. Loss of surface CD200 on stored allogeneic leukocytes may impair anti-abortive effect in vivo. Clark DA, Chaouat G. In the above paper1, Figure 5 was published with incorrect IVIG concentrations on the x-axis. The amended figure is printed below. Figure 5Open in figure viewerPowerPoint Anti-CD200 abrogates suppressive effect of human intravenous immunoglobulin G (IVIG) in natural killer (NK) cytotoxicity assay. Fresh peripheral blood lymphocyte were tested at a ratio of 50:1 with 10451Cr-labeled K562 target cells in 200 μL medium with 10% fetal bovine serum. In some sets of wells, Bayer IVIG was added at different doses at the final concentration indicated at the beginning of the assay. Six replicate wells were utilized for each test groups. Percent suppression of NK lytic activity (LU20) was calculated as described in Materials and methods, and is expressed as mean + 1 S.E.M. In the untreated control for IVIG suppression, 51Cr release at 18 hr in this experiment was 69.1% (LU20 = 5.26). The effect of addition of 2 μg monoclonal anti-human CD200 antibody was tested in control and IVIG-supplemented wells (for flow cytometry, 1 μg/106 cells is normally used, and 2 μg was used in the experiments to ensure binding/blocking of binding of CD200 to CD200R). *P < 0.05 abrogation of suppression of IVIG by anti-CD200. Reference 1 Clark DA, Chaouat G. Loss of surface CD200 on stored allogeneic leukocytes may impair anti-abortive effect in vivo. Am J Reprod Immunol 2005; 53: 13– 20. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume54, Issue2August 2005Pages 124-124 FiguresReferencesRelatedInformation

Activated CD8+ T-lymphocytes in obstructive sleep apnoea

T-lymphocytes are implicated in the development of atherosclerosis. The aim of this study was to assess whether the CD8+ T-lymphocytes of obstructive sleep apnoea (OSA) patients undergo phenotypic and functional changes that may exaggerate atherogenic sequelae in OSA. A total of 36 OSA patients, 17 controls and 15 single-night-treated OSA patients were studied. Phenotype and cytotoxicity against K562 target cells were analysed by flow cytometry. Cytotoxicity against human umbilical vein endothelial cells (HUVECs) was assessed by 51Cr release assay. The cytotoxicity of the CD8+ T-lymphocytes of OSA patients against K562 and HUVECs was significantly greater than controls. This increased cytotoxicity directly depended on the presence of perforin and natural killer receptors (CD56, CD16), which were significantly increased in OSA CD8+ T-lymphocytes. Also the percentage of the CD56bright subset, which mediates initial interactions with vascular endothelium, significantly increased in OSA. Nasal continuous positive airway pressure treatment significantly decreased CD8+ T-cell cytotoxicity and CD56 expression, and was positively correlated with natural killer inhibitory NKB1 receptor expression either after a single-night treatment or after a prolonged treatment. In conclusion, the CD8+ T-lymphocytes of obstructive sleep apnoea patients undergo phenotypic and functional changes, rendering them cytotoxic to target cells via increased CD56+/perforin+ expression, which can be ameliorated by nasal continuous positive airway pressure treatment. These results are compatible with the current authors' hypothesis of atherogenic sequelae in obstructive sleep apnoea.