Peripheral blood lymphocytes resistant to Epstein-Barr virus immortalization manifest high natural killer (NK) type activity against NK-resistant target cells

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. We found recently that PBL from two EBV-seropositive healthy adults were exceptionally resistant to immortalization by EBV. In contrast to PBL from other EBV-seropositive donors sensitive to immortalization by EBV (S-PBL), the "resistant" PBL (R-PBL) respond to EBV infection with an early interleukin-2 (IL-2) synthesis and high interferon gamma (IFN gamma) production. In order to determine whether these differences in cytokine responses between R-PBL and S-PBL could be associated with a detectable difference in lymphocyte cytotoxicity, we compared the natural killer (NK) activity of R-PBL and S-PBL effectors by using both NK-sensitive (i.e. K562) and NK-resistant (i.e. Raji) targets. We found that, while effectors from EBV-infected R-PBL and S-PBL cultures exhibited comparable NK activity against the K562 targets, they differed remarkably in their cytolytic activity against Raji cells. At days 3 and 5 of culture, effectors from EBV-infected R-PBL showed a significantly higher lytic activity against Raji targets, whereas S-PBL did not. Culture of EBV-infected R-PBL and S-PBL effectors in the presence of recombinant IL-2 (rIL-2) for 5 days resulted in increases of their lytic activity against Raji cells, whereas pretreatment of these effectors with recombinant IFN gamma (rIFN gamma) was found to increase only R-PBL cytotoxicity. These results suggest that the resistance of R-PBL to EBV immortalization could be associated with a lymphokine-mediated early cellular cytotoxic response of the NK/LAK (lymphokine-activated killer cell) type against EBV-infected cells.     

Activated Natural Killer Cells Suppress Myelopoiesis in Mice with Severe Combined Immunodeficiency

The in vivo effect of natural killer (NK) cell activation on aulologous myelopoiesis was studied in an environment deficient of functional Tand B cells. Administration of 3.6-bis[2-(Dimethylamino)-ethoxy]-9H-xanthen-9-one dihydrochloride) Tilorone) or recombinant interleukin-2 (rIL-2) to mice with severe combined immunodeficiency (C. B. -I7 scid/scid) resulted in an increase in YAC-1 lysis by their splenocytes as well as bone marrow cells. Recombinant IL-2 furthermore led to a fivefold increase in the cellularity of the spleen. When assayed against human NK/lymphokine-activated killer (LAK) target, K562 cell line. the IL-2-activated mouse cells exhibited no cytotoxicity across the species barrier. Both agents induced a profound suppression of myelopoietic progenitor cells as measured in a 7-day granulocyte-macrophage colony forming cell (GM-CFC) assay. We conclude that the presence of neither functional T nor B cells is necessary for NK cells to mediate inhibition cf myelopoiesis in the autologous host.     

Biological response modifier enhances the activity of natural killer cell against human cytomegalovirus-infected cells

Peripheral blood lymphocytes (PBL) from two human cytomegalovirus (CMV)-seronegative donors and eight CMV-seropositive donors were cultured for 3 days with or without the biological response modifier OK-432 and examined for lysis of K562 cells and CMV-infected MRC-5 cells. OK-432-stimulated PBL exhibited significantly greater natural killer (NK) activity than did unstimulated PBL. There was no difference in activity of NK cells in PBL prepared from CMV-seronegative and -seropositive donors. Antibody-complement depletion studies suggested that OK-432-stimulated NK activity was associated with Leu-7-positive cells. The ability of OK-432 to sustain the NK activity in PBL was decreased when the CD4-positive population of lymphocytes was eliminated by antibody-complement depletion prior to OK-432 stimulation. The ability of OK-432 to sustain the NK activity of PBL was also significantly decreased in the presence of monoclonal antibody against recombinant human interleukin-2. The results suggest that the activity of human NK cells against K562 and CMV-infected MRC-5 target cells can be sustained in vitro by OK-432-stimulated T-helper cells and that the effect of the T-helper cells is mediated, at least in part, by interleukin-2.     

Defective generation of killer cells against spontaneously Epstein-Barr virus (EBV)-transformed autologous B cells in a fatal EBV infection.

Killer cell activities were analysed in a 16-month-old boy with a sporadic form of fatal Epstein-Barr virus (EBV) infection, and compared with those in three patients with acute infectious mononucleosis (IM). We used spontaneously EBV-transformed autologous lymphoblastoid B cell lines (LCL) as target cells, because the results obtained with such targets can be expected to reflect most accurately the killer-versus-target reaction in vivo. The patient's fresh peripheral blood mononuclear cells (PBMC) had relatively high natural killer (NK) cell activity against K562 cells (128% of the control value), but they did not kill his autologous LCL. The patient's PBMC, unlike PBMC of acute IM, showed no cytotoxicity against Raji cells and autologous LCL after 5 days' culture in the presence of recombinant interleukin 2 (rIL-2), indicating defective generation of lymphokine-activated killer (LAK) cells. The patient's PBMC, unlike PBMC of acute IM, also could not induce cytotoxicity against autologous LCL when cocultured with mitomycin C-treated respective autologous LCL for 7 days. The addition of rIL-2 to the culture significantly restored their ability to generate cytotoxic T lymphocytes (CTL) against his LCL: the percent cytotoxicity value rose from 3.0% to 37.7%. With respect to this, the endogenous IL-2 production by the patient's PBMC was deficient. These results suggest that the defective EBV-selective CTL generation was due to deficient IL-2 production. The failure of the killer cells to eliminate EBV-infected cells seems to have been responsible for the patient's unusual course after primary EBV infection.     

Association of decreased T-cell-mediated natural cytotoxicity and interferon production in Down's syndrome

Total peripheral blood lymphocytes (PBL) and isolated subpopulations from children with Down's Syndrome (DS) and age-matched healthy controls were investigated for their (1) natural killer (NK) and antibody-dependent cellular cytotoxic activities, (2) interleukin 2 (IL-2)-induced augmentation of NK activity, (3) lectin-dependent cellular cytotoxicity (LDCC), (4) ability of serum- and culture-derived soluble suppressor factor(s) to inhibit NK activity of normal lymphocytes, and (5) capacity to produce interferon (IFN) against tumor targets in vitro. T lymphocytes from DS patients demonstrated significantly decreased NK activity against K562 target cells compared to controls. DS lymphocytes also demonstrated a significant reduction in LDCC activity and IL-2-induced enhancement of NK activity. Furthermore, the ability of DS lymphocytes to produce IFN in vitro against K562 target cells was also significantly lower than that for normal PBL. Although sera from DS patients showed a significantly greater inhibitory effect on the NK activity of allogeneic normal PBL than normal sera, culture supernates from DS lymphocytes demonstrated suppressive effects comparable to culture supernates from normal PBL. These studies suggest an association between the decreased NK activity of T-cell subpopulations and lower IFN production by PBL from patients with DS.     

Bone marrow and peripheral blood natural killer cell activity in lymphomas. Its response to IL-2.

Natural killer (NK) cytotoxic activity was simultaneously investigated in bone marrow mononuclear cells (BMMC) and peripheral blood lymphocytes (PBL) from nine Hodgkin's disease (HD) and 15 non-Hodgkin lymphoma (NHL) untreated patients. Twenty-five PBL samples and seven bone marrow specimens from healthy individuals were also included as control group (C). NK cell activity was evaluated in basal condition and post-stimulation with human recombinant IL-2 (rIL-2). Data were expressed in K values (number of BMMC or PBL needed to lyse 50% of the target cells). In basal condition, both HD and NHL patients showed a NK cell activity comparable to the C group, both in BMMC (HD, K = 2.48 +/- 1.3; NHL, K = 3.8 +/- 2.0; C, K = 3.2 +/- 0.7) and PBL (HD, K = 2.0 +/- 1.0; NHL, K = 2.3 +/- 1.0; C, K = 2.2 +/- 0.2). Stimulation with rIL-2 induced a significant and comparable enhancement of the NK activity in PBL from HD, NHL and C while the response to rIL-2 of the BMMC in most of the HD and NHL patients was significantly greater than the C group. Responder cells were characterized by negative selection with specific MoAb plus complement as a CD3-, CD16+, CD56+ cytotoxic cell and further confirmed by flow cytometry. We postulate that IL-2 activation of bone marrow NK cell precursors, in addition to enhancing the activity of circulating NK, may be of value for the therapeutic rationale of IL-2 in patients with lymphoma.     

Natural cell-mediated cytotoxicity of peripheral blood lymphocytes against target cells transfected with epidermodysplasia verruciformis-specific human papillomavirus type 8 L1 DNA sequences

The aim of the present study was to characterise natural cell-mediated cytotoxicity against COS-7 cells transfected with potentially oncogenic HPV-8 L1 DNA sequences cloned in sense and antisense orientation and to evaluate their lysis by peripheral blood lymphocytes (PBL) from patients with epidermodysplasia verruciformis (EV), a rare disease associated with life-long infection by specific HPV types. COS-7 cells were transfected with HPV-8 Hinc II restriction fragment (nucleotide positions 5434-7654) cloned in sense (COS-L1S) and antisense (COS-L1A) orientation into pCB6 expression vector. Cytotoxic activity of isolated PBL against COS cell lines as well as K562 erythroleukaemic cells was evaluated by 51Cr-release assay. We found that lymphocytes responsible for natural lysis of COS and K562 cells are CD3-negative CD56-positive natural killer (NK) cells. Analysis of NK cell cytotoxic activity against different COS cell lines has revealed that lymphocytes from healthy subjects killed COS-L1S cells significantly more efficiently than wild COS-7 and COS-L1A cells. Significantly more efficient lysis of COS-L1S cells was also observed in EV patients. Thus, expression of HPV L1 renders target cells more susceptible to NK-mediated cytotoxicity that may enable more effective elimination of transformed cells.     

Defective NK cell activity following thermal injury.

Peripheral blood mononuclear lymphocytes (PBL) from thermal injury patients were examined for their ability to mediate natural killer (NK) cell activity against K562 tumour cells and against herpes simplex virus type 1 (HSV-1) infected Raji tumour cells. Using fluorescein isothiocyanate-conjugated monoclonal antibodies, the number of T3, T4, T8, Leu11, and Leu7 positive cells in PBL obtained from patients and normal controls was determined. Thermal injury patients had decreased levels of T3+ cells and a T4:T8 ratio which was significantly lower than that found in normal control individuals. Although patients had normal percentages of Leu7+ and Leu11+ cells, they had depressed NK cell activity against both K562 tumour cells and HSV-1 infected Raji cells. NK cell activity against K562 tumour cells was severely depressed during the first 20 days after injury. This defective NK cell activity did not appear to be due to a defect in PBL binding to the K562 tumour cells. In patients, the level of NK cell activity against HSV-1 infected cells did not correlate with the level of NK cell activity against K562 tumour cells. This finding further supports previous reports showing that NK cells which kill K562 tumour cells are different from the NK cell population which kills HSV-1 infected cells. Pretreatment of PBL obtained from patients with IL-2 or IFN-alpha, in some cases greatly enhanced NK cell killing of K562 tumour cells. However, IL-2 or IFN-alpha did not enhance NK cell activity in patients who had severely depressed levels of NK cell activity. Interestingly, in some patients, differential responsiveness to IL-2 and IFN-alpha was observed. In some patients, NK cell activity was enhanced by IL-2 but not by IFN-alpha. These results, while only suggestive, may indicate that different populations of NK cells respond preferentially to IL-2 and that IFN-alpha and/or IL-2 enhance NK cell activity in PBL obtained from some, but not all, thermal injury patients. Finally, this study clearly shows that thermal injury patients have defective NK cell activity not only against K562 tumour cells but also against virus-infected cells.     

Human T lymphocyte clones with killer or natural killer activity

We describe a reliable method for obtaining a significantly higher frequency of human cloned T lymphocytes with killer and/or NK-like activity. Human peripheral blood mononuclear cells were treated with recombinant interferon-γ (rIFN-γ) and recombinant interleukin-2 (rIL-2) in a culture medium containing autologous serum and were then cloned by single cell micromanipulation. The cloned T lymphocyte populations were tested simultaneously for their ability to proliferate in response to exogeneous IL-2, to exhibit lectin-dependent cytolysis and to kill the tumor cell line K562. Results indicate that the cloning technique allowed each isolated T lymphocyte to undergo cell expansion. Furthermore when T cells were pretreated with rIFN-γ and rIL-2, 88% of the T cell clones were capable of mediating cytotoxicity in the presence of PHA. Moreover one third of the clones which exhibited lectin-dependent lysis were able to kill K562 target cells.     

Morphological, phenotypic and functional characteristics of a pure population of CD56+ CD16- CD3- large granular lymphocytes generated from human duodenal mucosa.

Interleukin-2 (IL-2)-dependent large granular lymphocytes (LGL) with a distinctive surface phenotype were generated from histologically normal duodenal biopsy tissues. Immunoperoxidase staining of the mucosa with an anti-CD56 monoclonal antibody revealed LGL localized in the lamina propria rather than in the epithelium. Light and electron microscopy demonstrated azurophilic and electron-dense cytoplasmic granules. Flow cytometry analysis revealed that these cells express CD45, CD56, CD2, CD7, CD11a, CD18, CD69 and the intermediate affinity (p70) IL-2 receptor (IL-2R) but not CD57, CD16, CD3, CD4, CD5, CD8, CD45RA, CD25, or the high affinity p55 IL-2R. The LGL proliferated when cultured in the presence of human rIL-2 but not in the presence of human rIL-4. Functional studies demonstrated that the LGL had strong cytotoxicity against natural killer (NK) target cells, K562, but not NK-resistant targets such as Colo 205, Melanoma and Epstein-Barr virus (EBV)-transformed B-cell lines. The LGL expressed genes for IL-5, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha) and the corresponding cytokines were detected in culture supernatant. These results provide evidence for an important role of gut mucosal LGL in the induction and regulation of inflammation and immunity in the gut.     

NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways

INTRODUCTION: Ewing sarcoma (EWS) is a malignant bone-associated sarcoma, with poor prognosis in case of metastasis or relapse. To explore the feasibility of natural killer (NK) cell mediated immunotherapy and to identify molecular mechanisms involved, the susceptibility of EWS to NK cells was investigated. METHODS AND RESULTS: All EWS cell lines tested (n=7) were lysed by purified allogeneic NK cells from healthy donors, and the efficacy of lysis was increased by activating NK cells with interleukin-15 (IL-15). FACS analysis and immunohistochemistry revealed that EWS cell lines as well as primary tumor cells expressed ligands for the activating NK cell receptors NKG2D and DNAM-1. NK cell cytotoxicity to EWS cells critically depended on the combination of NKG2D and DNAM-1 signaling, since blocking either of these receptors abrogated lysis by resting NK cells. Cytokine-activated NK cells more efficiently recognized EWS cells, since only combined, but not single blockade of NKG2D and DNAM-1 by antibodies inhibited lysis of EWS cells. Induction or blockade of HLA class I on EWS cells did not significantly influence lysis. This suggests that predominantly activating, rather than inhibitory signals on EWS cells determined susceptibility to NK cell cytotoxicity. NK cell cytotoxicity to EWS cells and K562 was reduced in EWS patients at diagnosis (n=11) compared to age matched controls, despite normal NK cell numbers and increased expression of NKG2D. The impaired function of these NK cells was restored after activation with IL-15 in vitro. CONCLUSION: These results demonstrate that EWS cells are potentially susceptible to NK cell cytotoxicity due to the expression of activating NK cell receptor ligands. The use of cytokine-activated NK cells rather than resting NK cells in immunotherapy may be instrumental to optimize NK cell reactivity to EWS.     

Activation of human NK cells and monocytes with cisplatin in vitro

Human natural killer (NK) cells and monocytes treated in vitro concomitantly with cisplatin and rIFN-γ enhanced lysis of K562 cells. Lysis was dependent upon the duration of treatment. Cisplatin and rIFN-γ treated monocytes were equally cytotoxic to NK sensitive (K562) and NK resistant (Daudi & Raji) cell lines whereas NK cells were not rendered cytotoxic against NK resistant tumor cells. NK- and monocyte-mediated cytotoxicity against K562 cells was further enhanced when the effector cells were primed with rIFN-γ and were subsequently treated with cisplatin.     

Enhancement of natural killer cell activity in vitro against human tumor cells by some plants from Jordan

The effect of different concentrations (0, 0.01, 0.1, and 0.5 mg/ml) of plant aqueous extracts on the anti-tumor activity of natural killer (NK) cells isolated from human blood was examined. Plant extracts induced significant enhancement of (26.6-67.7%) of NK cell activity against K562 tumor cells. This increase in NK cell cytotoxicity was found to be due to the enhancement of NK cell production of interferon-gamma (87-337%), and on tumor necrosis factor-alpha (60-200%). Furthermore, the release of both granzyme A and N-acetyl-beta-D-glucosaminidase was increased significantly when compared with controls. Activation of granzyme A and N-acetyl-beta-D-glucosaminidase was clearly observed ranging from 24.2-106.4% to 26.8-110.7%, respectively. Lastly, in the absence of IL-2, plant extracts caused a significant increase in NK-cell-induced cytotoxicity (256%) against K562 tumor cells, and in the presence of IL-2 stimulated cells plant extracts caused an increase in NK cell-cytotoxicity (112%).     

Non-major histocompatibility complex-restricted killer cells in human cord blood: generation and cytotoxic activity in recombinant interleukin-2-supplemented cultures.

Interleukin-2 (IL-2)-induced generation of non-major histocompatibility complex (MHC)-restricted killer cells among human cord blood lymphocytes (CBL) was investigated. After 1 week in culture with recombinant (r)IL-2 and human serum (HuSer), the cytotoxicity of CBL against K562 and COLO cells greatly exceeded the cytotoxicity of cultured adult peripheral blood lymphocytes. Culturing of CBL with rIL-2 and HuSer led to preferable generation of CD56+ cells. After 1 month in culture, the number and frequency of CD56+ cells had increased by more than 50 and nine times, respectively. The generation of CD56+ cells in CBL cultures may at least partially be explained by their comparatively strong expression of the IL-2 receptor (IL-2R) beta-chain (p75).     

Surface markers of human natural killer cells as analyzed in a modified single cell cytotoxicity assay on poly-L-lysine coated cover slips

A modified single cell cytotoxicity assay using poly-L-lysine coated cover slips (PLL-SCCA) was employed to study the frequency and surface marker profile of human peripheral blood lymphocytes (PBL) with NK reactivity against K 562 target cells. When compared with the previously described agarose single cell cytotoxicity assay (A-SCCA) identical results were obtained. For 13 donors tested 18.1 +/- 4.4% of the PBL formed conjugates with K 562 and 2.7 +/- 1.6% displayed NK reactivity. In contrast to the A-SCCA, the PLL-modified assay permits direct identification of both conjugate forming (TBC) and cytolytic PBL (NK) by means of surface markers. Indirect immunofluorescence studies with monoclonal anti-PBL antibodies revealed that neither the plating procedures nor the incubation conditions employed affected the expression of the antigens recognized by these reagents. This method of directly identifying NK cells showed that OKM1+ cells were enriched among the NK cells as compared to PBL and TBC (55% vs. 23% and 43%, respectively). In contrast, the OKT3+ or Leu1+ fraction of the NK cells was reduced as compared to PBL and TBC. However, using this method of identification at the effector cell level, a substantial proportion of the NK cells were OKT3+ or Leu1+ (57% or 58% respectively, 7 donors). Approximately 25% of the NK cells were Leu2a+ and 30% were Leu3a+, respectively. However, the size of the Leu3a+ fraction varied considerably with individual donors and the size of this fraction appeared to be inversely related to that of the donors NK pool.     

Activation of swine peripheral blood lymphocytes with human recombinant interleukin-2.

These studies examined the effect of the lymphokine interleukin-2 (IL-2) on porcine peripheral blood lymphocytes (PBL). Cultures of porcine (Yorkshire) PBL in human recombinant (r) IL-2 had a time-dependent increase in activation of killer cells. Previous reports of porcine killer cells derived from PBL indicated maximal lysis 18 hr after killer and target cells were mixed, with very little lytic activity at 4 hr. After culturing for 2 days in rIL-2, the cells acquire enhanced lytic capabilities resulting in substantial target lysis in the 4-hr assay. Enhanced lytic activity in the 18 hr assay occurs as early as 30 min after exposure to rIL-2, with a more significant increase after 2 days' exposure. The IL-2-treated cells have an increase in the incorporation of tritiated thymidine and an increase in percentage of granular cells. Tumour targets, such as Daudi, which are resistant to lysis, and L929, Mdt4 and P815, which are moderately susceptible to lysis by untreated swine PBL, had a substantial increase in lysis in IL-2-treated swine PBL. These results indicate human rIL-2 induces functional and morphological changes in swine peripheral blood lymphocytes.     

Interleukin 2 enhances natural killing of varicella-zoster virus-infected targets.

Preincubation of peripheral blood non-adherent mononuclear cells with purified or recombinant interleukin 2 (IL-2) significantly enhanced natural killer (NK) activity against uninfected and varicella-zoster virus (VZV)-infected targets, while antibody-dependent cellular cytotoxicity (ADCC) against VZV-infected targets was not increased. Preincubation of effector cells with IL-2 had no effect on conjugate formation, but lysis of both targets was increased in single cell assays. IL-2-enhanced NK against VZV-infected targets was independent of gamma-interferon (gamma-IFN) production.     

Cytolytic mechanisms involved in non-MHC-restricted cytotoxicity in Chediak-Higashi syndrome.

To determine the mechanisms responsible for the impaired lymphocyte-mediated cytotoxicity in Chediak-Higashi syndrome (CHS), we investigated the killing ability of peripheral blood lymphocytes (PBL) from three patients with CHS using several kinds of target cells that were sensitive to perforin, Fas ligand (FasL), and/or tumour necrosis factor-alpha (TNF-alpha). Freshly isolated CHS PBL did not kill K562 target cells, killing of which by normal PBL was perforin-dependent, as demonstrated by complete inhibition by concanamycin A (CMA), an inhibitor of perforin-based cytotoxicity. In contrast, the CHS PBL exhibited substantial cytotoxicity against Jurkat cells, which was only partially inhibited by CMA treatment but not by the addition of neutralizing anti-FasL or anti-TNF-alpha antibodies. IL-2-activated CHS PBL exhibited substantial levels of cytotoxicity against K562 and Jurkat cells, the levels being 74% and 83% of the respective normal control values, respectively. CMA treatment showed that while the cytotoxicity of IL-2-activated CHS PBL against K562 was largely dependent on perforin, that against Jurkat was largely not. IL-2-activated CHS PBL expressed FasL mRNA, and killed Fas transfectants. These findings indicate that CHS PBL have an ability to kill some target cells via a perforin-mediated pathway, especially when they are activated by IL-2. It was also demonstrated that CHS PBL can exert cytotoxicity against certain target cells by utilizing FasL and an undefined effector molecule other than perforin, FasL, or TNF-alpha.     

Ontogeny of natural killer cell activity and antibody dependent cell mediated cytotoxicity in pigs

Cells from pigs of various ages were collected from peripheral blood, mesenteric lymph node, spleen and thymus and their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity was determined. ADCC against chicken red blood cell (CRBC) was present in cells from peripheral blood, lymph node and spleen, but was absent in thymic cells. There were no age-related differences in ADCC to CRBC and cells from fetal pigs had similar activities to cells from adult pigs. Maximal cytotoxicity against CRBC was found in the polymorphonuclear leukocyte (PMN) cell fraction. In contrast to the good response against CRBC, PMN cells were not lytic in the ADCC assay when PI3 virus-infected cells were used as target cells. Peripheral blood lymphocytes (PBL) had low but significant lytic activity against PI3 virus-infected cells in the presence of high concentrations of specific antiserum. NK cell activity against K562 target cells was readily detected in PBL of pigs older than 2 weeks but was not observed with cells from spleen, lymph node or thymus from pigs of any age. PBL of pigs younger than 2 weeks of age had low but detectable NK activity: however, fetal pigs had no NK activity against K562 target cells. In contrast, when PI3 virus infected Vero cells were used as target cells, NK cells were detected in spleen and PBL, but not lymph nodes or thymus, of pigs greater than one day of age. Similar to the absence of activity to K562, none of the lymphoid cells from fetal pigs had NK activity against PI3 virus-infected Vero cells. The present results suggest that the effector cells that mediate ADCC are distinct from those that mediate NK activity in that cells mediating ADCC develop earlier and are found in different organs than the NK cells. Additionally, the cells that mediate NK activity against viral infected cells may be different from those that mediate NK activity for K562 target cells; however, regardless of the target, NK cells are not present before birth in the pig.     

Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay

In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 results to increase CD69 expression on NK and NKT but not on T lymphocytes. Only a part of peripheral blood NK cells became CD69 positive after incubation with excess of K562 cells. CD69+ NK cell frequencies did not increase after elevation of K562/NK ratio or incubation period that confirmed existence of subset of NK cells able to response to K562. CD69 elevation on NK significantly correlated with NK cytotoxicity (r=0.726). CD69 increases were similar when whole blood or isolated PBMC was used in assay. We also found different capacity to activation in NK subsets that express CD62L at various densities. The results demonstrated that K562 induced CD69 expression displays NK lymphocyte functional condition that associated with cytotoxic function.