Separation methods of T cells, natural killer, and dendritic cells from peripheral blood of cancer patients using interleukin-2 and functional analysis of natural killer cells after separation

We aimed to induce three different immune cell subsets from a single blood sample from cancer patients to target different biological characters of cancer cells. In the presence of 6000 IU/ml IL-2, natural killer (NK) cells adhere to plastic. By using this ability, we could separate dendritic cells, T cells, and NK cells from peripheral blood mononuclear cells. The cultured NK cells demonstrated higher nonspecific cytotoxicity against tumor cell lines than did the T cells. Furthermore, adherent NK cells demonstrated higher cytotoxicity than nonadherent NK cells, although there was no difference between adherent and nonadherent NK cells in natural cytotoxicity receptors (NKp30, NKp44, NKp46) and NKG2D expression. With these results, we confirmed that we could induce dendritic cell, T cell, and higher cytotoxic NK cells from a single blood draw, and this methodology facilitates to the use of these cells for clinical grade conditions.     

Natural killer cells become tolerogenic after interaction with apoptotic cells

NK cells are effectors in innate immunity and also participate in immunoregulation through the release of TGF‐β1 and lysis of activated/autoreactive T cells. Apoptotic cells (AC) have been shown to induce tolerogenic properties in innate immune cells, including macrophages and dendritic cells, but not NK cells. In this study, we demonstrated that after interaction with AC, NK cells released TGF‐β1, which in turn suppressed the production of IFN‐γ by NK cells upon IL‐12 and IgG activation. We further identified phosphatidylserine as a potential target on AC for the NK cells, as phosphatidylserine could stimulate NK cells to release TGF‐β1, which in turn suppressed CD4⁺ T‐cell proliferation and activation. Moreover, AC‐treated NK cells displayed cytotoxicity against autologous‐activated CD4⁺ T cells by upregulating NKp46. This lysis occurred in part through the NKp46‐vimentin pathway, as activated CD4⁺ T cells expressed vimentin on the cell surface and blocking of vimentin or NKp46, but not other NK‐cell receptors, significantly suppressed the NK‐cell cytotoxicity. We report here a novel interaction between NK cells and AC, resulting in the tolerogenic properties of NK cells required for immune contraction.     

Interaction of Mycobacterium tuberculosis Cell Wall Components with the Human Natural Killer Cell Receptors NKp44 and Toll‐Like Receptor 2

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll‐like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl‐arabinogalactan‐peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN‐γ production at levels comparable to M. bovis Bacillus Calmette–Guérin‐stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG‐exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN‐γ production were inhibited by anti‐TLR2 MAb, but not by anti‐NKp44 MAb. Differently, anti‐NKp44 MAb partially inhibited CD69 expression on NK cells pre‐activated with IL‐2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR‐2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN‐γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.     

Target-Effector Interaction in the Natural Killer Cell System

These results show that two subpopulations of target-binding cells (TBC) can be detected in the lymphoid organs of normal, nonimmunized mice. One cell type is not adherent to nylon wool columns and binds selectively to a large number of tumour cell targets which are susceptible to lysis by the natural killer (NK) cell. The rise and fall in the frequency of these nylon nonadherent TBC, with age, closely parallels the NK cell activity in these mice. Nylon nonadherent TBC were specific since they could be inhibited by subcellular sonicates of sensitive targets but not insensitive targets. The presence of these TBC in nude mice and their ability to pass through nylon wool columns is compatible with the suggestion that, like the NK cell, they may not be mature T cells, macrophages or B cells and hence represent a distinct but not yet defined subpopulation of lymphocytes. The genes controlling the frequency of TBC are inherited in a dominant fashion and are linked to the H-2 region. The strong correlation between the frequency of TBC in a population and the level of lysis provides strong indirect evidence that the TBC may represent, or be closely related to, the NK cell. In contrast, the second cell type(s), a nylon-adherent population, was not subject to any detectable genetic control and bound to targets nonspecifically. Furthermore, these nylon-adherent TBC differed from nylon-nonadherent TBC in their lack of correlation with lysis, age variations and organ distribution. We believe these observations provide the basis for the eventual understanding of the target structures and receptors involved in the NK cell system.     

The Use of Salmonella Schottmulleri for Mapping and Separation of Human Lymphocyte Subpopulations1

Human lymphocyte subpopulations (B cells, B₁, B₂, T₁, T₂, T₃, and T₄ cells; our denomination) have been previously identified and isolated by bacterial adherence and functional differences between them have been demonstrated. Here we examined the binding properties of Salmonella schottmulleri to human lymphocytes in peripheral blood smears and found that it binds to more lymphocyte subpopulations, namely B, T₁, T₂ and T₃ cells, than any bacteria previously tested. Thus, using only four bacteria: Salmonella schottmulleri, Brucella melitensis, Arizona hinshawii and Bacillus globigii we identified in blood smears B cells, two B and four T cell subpopulations. When we used gelatin-coupled monolayers of Sal. schottmulleri to isolate lymphocyte subpopulations, we showed that the nonadherent (T₄) cells could be efficiently separated from the adherent cells. Furthermore, we tested the isolated subpopulations for natural killing (NK) activity and for antibody-dependent cell-mediated cytotoxicity (ADCC). Using both NK and ADCC assays, we observed a significantly higher cytotoxic activity in the nonadherent cell population than in the unseparated or adherent cell populations. Also the nonadherent cells contained most of the lymphocytes that have receptors for the Fc portion of IgG and those cells described as large granular lymphocytes. We concluded that Sal. schottmulleri is a valuable new reagent for the identification and separation of human lymphocyte subpopulations.     

Changes of NK Cells in Preeclampsia

The regulation of uterine and circulating peripheral blood natural killer (NK) cells has been associated with reproductive immunology such as recurrent pregnancy losses, implantation failures, or preeclampsia. Preeclampsia is a hypertensive disorder of pregnancy characterized by increased blood pressure accompanied by proteinuria and is a major cause of maternal and fetal mortality. Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. The relation of NCRs to reproduction is not fully characterized yet. The different profile of NCRs expression may suggest presence of abnormal regulation of NK cell in women with reproductive failures. Pregnant women with preeclampsia carry immunological abnormalities of NCRs on peripheral blood NK cells during pregnancy. The lower expression of NKp46⁺ NK cells in women with preeclampsia may account for the higher production of NK1 cytokine that is known as NK1 shift in pregnant women with preeclampsia. Evaluation of NKp46 on peripheral blood NK cells may be applicable to find the onset of preeclampsia. In this review, various expressions of NK cell surface markers including NCRs on NK cells, NK cell cytotoxicity, and production of cytokines and angiogenic factors by NK cells were reviewed in relation to preeclampsia.     

Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

不明原因自然流产与蜕膜NK细胞表面活化性受体表达的相关性研究

探讨不明原因自然流产患者蜕膜NK细胞杀伤活性与其细胞表面活化性受体NKp46、NKp44、NKp30和NKG2D表达的相关性。选取21例早孕不明原因自然流产患者为病例组,25例正常早孕人流妇女为对照组,收集两组的蜕膜组织,Ficoll密度梯度离心分离淋巴细胞,MACS磁珠分选CD3-CD56+NK细胞。以K562细胞为靶细胞,用细胞染色及流式细胞技术检测两组蜕膜NK细胞杀伤活性,用流式细胞技术检测两组蜕膜CD56brightCD16-NK和CD56dimCD16+NK细胞上活化性受体NKp46、NKp44、NKp30和NKG2D的表达,并与NK细胞杀伤活性进行相关性分析。结果:(1)早孕蜕膜NK细胞具有杀伤活性;(2)病例组蜕膜NK细胞的杀伤活性较正常对照组显著增强(P=0.014);(3)病例组蜕膜CD56brightCD16-NK细胞中NKp44的表达比正常对照组显著升高(P=0.021);病例组蜕膜CD56dimCD16+NK细胞中NKp46和NKp44的表达比正常对照组显著升高(分别P=0.026,P=0.041);其余活化性受体的表达两组未见明显差异;(4)蜕膜NK细胞杀伤功能与蜕膜CD56brightCD16-NK细胞中NKp44的表达呈显著正相关(r=0.677,P<0.05),和蜕膜CD56dimCD16+NK细胞中NKp46的表达呈显著正相关(r=0.634,P<0.05)。蜕膜NK细胞活化性受体NKp46和NKp44表达增加,从而使蜕膜NK细胞的杀伤功能增强可能在不明原因自然流产的发病中起重要作用。     

Characterization of Autoantibodies to Natural Killer Cells in HIV-Infected Patients

Natural killer (NK) cells in HIV-infected patients have a reduced ability to generate non-MHC restricted cytotoxicity to a variety of target cells. The authors investigated antibodies to NK cells in HIV-infected patients and evaluated effects of these antibodies to NK cell numbers and function. Antibodies to NK cells were determined in 160 HIV-infected patients and 35 healthy controls. Flow cytometric whole blood methods were developed to detect antibodies to NK cells. Antibodies to asialo-GM1 were detected by TLC immunostaining. The presence of antibodies to NK cells was demonstrated in plasma of about one-third (54/160) of HIV-infected patients but rarely in controls (2/35). Autoantibodies bound to NK cells in vivo and were detected by a strong increase of surface immunoglobulin (Ig) on NK cells of HIV-infected patients. Anti-NK cell antibodies were warmreactive antibodies rather of IgG than of IgM phenotype. The prevalence of specific antibodies to asialo-GM1 was low (12.5%). Numbers of circulating NK cells did not differ significantly between antibody positive (99.5/μl) and antibody negative (141/μl) patients ( P  = 0.3). However, pre-incubation of healthy donors' NK cells with autoantibody positive plasma significantly inhibited cytotoxicity to K562 leukaemic cells ( P  = 0.002). Autoantibodies to NK cells in HIV-infected patients are present in the plasma of one-third of HIV-infected patients and are bound to NK cells in vivo . There is evidence that these autoantibodies can induce NK cell defects similar to those seen in vivo     

Enriched natural killing and antibody-dependent cellular cytotoxicity in lymphocyte populations adherent to tumor cell monolayers

We have previously shown that human peripheral blood lymphocytes that do not adhere to natural killer (NK)-sensitive target cell monolayers are at least partially depleted of NK activity. To demonstrate directly that NK effector cells adhere to the monolayers, we have now recovered the adherent populations by treatment with ethylenediaminetetraacetic acid. When tested immediately after isolation, these adherent populations show NK activity that is intermediate between that of the nonadherent and control cells. When antibody-dependent cellular cytotoxicity is tested concomitantly, the adherent fractions are consistently found to be enriched relative to control, unfractionated lymphocytes. In further studies, control populations and fractions nonadherent and adherent to HSB monolayers are incubated overnight at 37 degrees C and then tested for NK activity. We find that the adherent fractions are selectively enhanced in activity, causing the NK activity of the adherent fractions to exceed that of control populations. The possible involvement of interferon in the augmentation of NK activity is considered.     

NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human NK cells. Correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells.

NKp46 is a novel triggering receptor expressed by all human NK cells that is involved in natural cytotoxicity. In this study we show that the surface density of NKp46 may vary in different NK cells and that a precise correlation exists between the NKp46 phenotype of NK clones and their natural cytotoxicity against HLA-class I-unprotected allogeneic or xenogeneic cells. Thus, NKp46bright clones efficiently lysed human and murine tumor cells while NKp46dull clones were poorly cytolytic against both types of target cells. We also show that the NKp46 phenotype of NK clones correlates with their ability to lyse HLA-class I-unprotected autologous cells. Finally, NKp46 was found to be deeply involved in the natural cytotoxicity mediated by freshly derived NK cells. This was indicated both by the inhibition of cytolysis after monoclonal antibody-mediated masking of NKp46 and by the correlation existing between the natural cytotoxicity of fresh NK cells derived from different donors and their NKp46 phenotype. In conclusion, these studies strongly support the concept that NKp46 plays a central role in the physiological triggering of NK cells and, as a consequence (in concert with killer inhibitory receptors), in the NK-mediated clearance of abnormal cells expressing inadequate amounts of HLA-class I molecules.     

Siglec‐7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell‐Mediated Activities

Sialic acid‐binding immunoglobulin‐like lectin‐7 (Siglec‐7) is an inhibitory receptor expressed on natural killer (NK) cells. In this study, we investigated the relationship between Siglec‐7 expression and NK cell functions. Siglec‐7 was highly expressed on NK cells and was preferentially expressed by mature NK cells from peripheral blood of healthy adults. Siglec‐7⁺ NK cells displayed higher levels of activating receptors CD38, CD16, DNAM1, NKp30 and NKp46, but lower levels of inhibitory receptors such as NKG2A and CD158b, compared with Siglec‐7^– NK cells. Functional tests showed that Siglec‐7⁺ NK cells displayed more CD107a degranulation and IFN‐γ production than Siglec‐7^– NK cells. Siglec‐7 inhibited NK cell functions when interacting with specific antibodies. These data suggest that Siglec‐7 defines a highly functional NK cell subset and suppresses NK cell‐mediated functions when cross‐linked with specific antibodies.     

Phenotypic and Functional Heterogeneity of Natural Killer Cells from Umbilical Cord Blood Mononuclear Cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-γ and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56^hiCD16^? NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-γ expression by CD56^hiCD16^? and CD56^loCD16^? subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.     

Immunomodulatory Activity of A Novel Nucleoside, 7-thia-8-oxoguanosine: I. Activation of Natural Killer Cells in Mice

We reported recently that a novel immunomodulator, 7-thia-8-oxoguanosine (7T80G)² inhibited formation of pulmonary melanoma metastases (1), prevented against viral infection in mice (2) and potentiated the efficacy of a weakly immunogenic leukemia vaccine (3). Since certain tumor metastases and virus infected cells are targets to natural killer cells (NK cells), we now investigated whether 7T80G is capable of activating NK cells in mice using NK cell sensitive YAC-1 and B16 and NK cell insensitive P815 targets. CBA/CaJ spleen cells incubated in vitro with 7T80G at concentrations ranging from 0.005 to 0.5 mM responded with increased NK cell activity (32-62 %) compared to controls (4-8%) to YAC-1 targets. Similar levels of augmentation in NK cell activity were observed when 40-168 mg/kg of 7T80G was administered in vivo. In addition to the spleen, 7T80G activated NK cells in the bone marrow (BM), the lungs, the liver, and in peritoneal exudate cells (PE). Although 7T80G elicited activation of NK cells was observed as early as three hours after treatment, the maximal activity was observed after 24 h in the spleen; 12 h in the BM; 48 h in the lungs, and 72 h in PE. Administration of the drug by s.c, i.v., and i.p. routes all induced activition of NK cells in spleen, BM and PE. 7T80G was found to activate NK cells in seven inbred and an outbred mouse strain, suggesting that the induced cytotoxicity against allogeneic and syngeneic tumor cells is not strain specific as well as independent of MHC restriction. C3H/He, CBA/CaJ and BDF/1 displayed higher levels of increased NK cell activity, whereas AKR mice were low responders. Low concentrations of IL-2 (0.25-5 U/ml) that induce little or no NK cell activity, when used in combination with 7T80G, elicited significant enhancement of NK cell cytotoxicity. In contrast, IFN and 7T80G showed no such synergism.     

Expansion of 2B4+ natural killer (NK) cells and decrease in NKp46+ NK cells in response to influenza

Several studies have highlighted the importance of murine natural killer (NK) cells in the control of influenza virus infection, notably through the natural cytotoxicity receptor NKp46. However, little is known about the involvement of NK cells in human influenza infection. Here, we show that upon in vitro exposure to influenza, NKp46 expression on NK cells decreases, whereas expression of 2B4, an activating receptor that can enhance natural cytotoxicity in synergy with NKp46, is up-regulated. Consistent with these observations, NKp46(dull) and 2B4(bright) NK cells had a higher functional activity in response to influenza than NK cells expressing high levels of NKp46 or low levels of 2B4, respectively. Importantly, we assessed whether the expression of these receptors was also modified in vivo in response to influenza antigens and showed that an increase in 2B4-expressing NK cells and a decrease in NKp46(+) NK cells occurred following intramuscular influenza vaccination. Altogether, our results further suggest that NKp46 may play an important role in the innate immune response to human influenza and reveal that exposure to influenza antigens is associated with a previously unrecognized increase in 2B4 expression that can impact NK cell activity against the virus.     

ORIGINAL ARTICLE: Suppression of Natural Killer Cell Cytotoxicity in Postpartum Women

Citation Groer M, El‐Badri N, Djeu J, Harrington M, Van Eepoel J. Suppression of natural killer cell cytotoxicity in postpartum women. Am J Reprod Immunol 2010; 63: 209–213 Problem Natural Killer (NK) cell numbers and cytotoxicity are suppressed during pregnancy. Little is known about postpartum NK number and function. Method of study Postpartum women (n = 39) were studied at one week and then monthly over the first six postpartum months. The standard natural killer cell cytotoxicity assay (NKCA) was performed. This is a Cr51 release assay from K562 cells cultured with peripheral blood mononuclear cells (PBMCs). Results Data indicate suppression of NK cytotoxicity in postpartum women. Cytotoxicity at each effector:target (E:T) ratio showed a drop from 1 week postpartum, reaching a nadir at around 2 months, and a trend towards recovery of cytotoxicity from 3 to 6 months. Lytic units (LUs) from pre‐incubated cells from postpartum women were lower than age‐matched, non‐pregnant, non‐postpartum controls through the fifth postpartum month. Conclusion These data suggest that the postpartum period, like pregnancy, is characterized by decreased NK cytotoxicity activity. This suppressed NK cytotoxic effect may result as a response to interaction with tolerized fetal microchimeric cells accumulated during pregnancy in maternal blood and tissues.     

Evolutionary Conservation of a Human Function-Associated Molecule on Murine Natural Killer Cells: Expression and Function

Using a novel anti-natural killer (NK) cell monoclonal antibody (MoAb), we have recently identified an evolutionary conserved function-associated molecule (FAM) present on fish, rat and human NK cells. This molecule is involved in NK cell function as anti-FAM MoAbs inhibit cytotoxicity, stimulate lymphokine secretion and inhibit conjugate formation between effector cells and target cells. We now have examined murine NK cells for the presence of this structure. It was observed by two-colour flow cytometric analysis that the anti-FAM MoAb 5C6 specifically bound to a subpopulation of nylon wool non-adherent splenic lymphocytes (19–20%). The expression of the FAM molecule was restricted to NK cells that expressed the NK1.1 antigen. Neither T cells, B cells, nor macrophages reacted with the anti-FAM MoAb. Analysis of FAM expression in various lymphoid tissues revealed that splenocytes expressed the greatest numbers of MoAb(+) cells. Generation of lymphokine-activated killer (LAK) cells and adherent tymphokine-activated killer (ALAK) cells resulted in higher levels of FAM expression. The anti-FAM MoAb 5C6 also detected the presence of FAM on fresh SCID NK cells. It was demonstrated that the anti-FAM MoAb 5C6 inhibited the lysis of target cells by endogenous NK cells, activated NK cells, 5d LAK cells, ALAK cells and SCID NK cells. Moreover, conjugate assays demonstrated involvement of this molecule in recognition between NK cells and target cells.