Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perforin and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7-AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of 51Cr release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly.     

转Wee1基因靶细胞抗CTL介导的细胞毒效应

目的 :研究Wee1转基因细胞抗CTL介导的细胞毒效应及抗穿孔素抗体对抗细胞毒效应的影响。方法 :体外混合NIT及淋巴细胞培养获得CTL ,并测定淋巴细胞增殖指数 (MTT法 ) ;采用脂质体将重组体Wee1Hu GFP导入哺乳动物细胞NIT ;以转染重组体前后的NIT为靶细胞 ,检测CTL介导的胞毒效应 (LDH法 ) ,同时测定抗穿孔素抗体对此效应的影响。结果 :混合细胞培养可诱导CTL的增殖 ;转染Wee1基因及加入抗穿孔素抗体NIT组发生的细胞溶解的数量最少。结论 :转Wee1基因靶细胞在一定程度上可抵抗CTL介导的细胞毒作用 ,穿孔素抗体可协同增强此抗胞毒作用。     

Monoclonal antibodies detecting discrete epitopes of human perforin

Perforin is a cytolytic protein of natural killer (NK) cells and cytotoxic T cells (CTL). Purified perforin has been shown to cause cell lysis and to form stable pores in the target cell membrane, but its relevance to cytolysis in vivo is not clear. The gene for human perforin has been cloned, but monoclonal antibodies (mabs) have not been available. In order to study further its role in cytotoxicity, we have generated mabs to different regions of human perforin. Four mabs were produced from mice immunized with hybrid proteins comprising E. coli TrpE protein at the N-terminus and different regions of human perforin at the C-terminus. These proteins were made using the pATH expression plasmids into which fragments of perforin cDNA were subcloned. Monoclonal antibody PA1 was made from a mouse immunized with a hybrid protein containing the C-terminal 240 amino acids (AA) of perforin, PE1 - the N-terminal 118 AA, and PB1 and PB2 - the central 199 AA. The three plasmid constructs contained non-overlapping cDNA segments which covered the entire sequence of perforin. All mabs reacted with the immunizing hybrid protein, but not with the other hybrid proteins, indicating that at least three epitopes are recognized by this set of mabs. All mabs immunoprecipitated a molecule of about 68 kd from lysates of metabolically-labelled cytolytic large granular lymphocytic leukemia cells, but not from control lysates of non-cytolytic promyelocytic U937 cells. These mabs should be of use in determining structure-function relationships for perforin.     

Study of diverse mechanisms of cell-mediated cytotoxicity in gene-targeted mice using flow cytometric cytotoxicity assay

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms-(1) release of lytic granules (containing perforin and granzymes), and (2) Fas ligand (FasL)/Fas or TNF-initiated apoptosis. We have examined mechanisms of target cell lysis by effector T cells from gene-targeted and mutant mice using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH67 dye. Cell death was estimated by 7-AAD inclusion and annexin V-PE binding. A direct correlation has been found between the percentage of dead target cells in FC Assay and the results of 111In release cytotoxicity assay when effector T cells from either Pfp -/- (perforin knockout) or gld (non-functional Fas Ligand) mice were used. As shown by the 4 h FC assay, the granule-mediated mechanism was utilized by T cells from gld mice. In contrast, T cells from Pfp -/- mice used death receptor-mediated lysis. Therefore, cytotoxic cells from gene-targeted and mutant mice can serve as valuable tools for studying different mechanisms of cell-mediated cytotoxicity, and the FC assay could be applied irrespective of which cytotoxic effector pathway is involved.     

Transfection of human CD59 complementary DNA into rat cells confers resistance to human complement.

We have examined the role of the human CD59 antigen in inhibiting complement-mediated lysis by transfer and expression of a CD59 cDNA in rat cells. A cDNA encoding CD59 was subcloned into the expression vector pSFSVneo and stably transfected into the rat T cell line NB2-6TG. Indirect immunofluorescence staining using the anti-CD59 monoclonal antibody YTH53.1 demonstrated the presence of human CD59 antigen on transfected cells and its attachment to the cell surface by a rat glycolipid anchor. Transfected cells were found to contain a single 3.3-kb species of CD59 mRNA by Northern blot hybridization. Immunoblotting revealed that this encoded a protein band of the same size as that observed in human erythrocytes. To determine the biological effect of expression of human CD59 in rat cells, an assay was devised which measured the relative lysis of transfected cells compared to untransfected cells in the presence of human complement and a lytic monoclonal antibody. It was observed that CD59-transfected rat cells are less susceptible to lysis by human complement and that this effect was blocked by a F(ab')2 fragment of YTH53.1. These experiments provide a direct demonstration that CD59 can function as an homologous complement restriction factor for nucleated cells.     

Involvement of protein kinase C-delta in CD28-triggered cytotoxicity mediated by a human leukaemic cell line YT.

Ligation of CD28 molecules expressed on the surface of human leukaemic natural killer-like YT cells triggers intracellular signals leading to cytolysis of target cells expressing CD80 or CD86 molecules. Known intracellular events include tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C (PKC). In this study, we report that PKC-delta isoenzyme activity is required for CD28-triggered cytotoxicity mediated by YT cells and we also demonstrate that one of the primary targets of bryostatin 1, a modulator of PKC activity, is PKC-delta. Treatment of YT cells with bryostatin 1 caused degradation of PKC-delta, but not other PKC isoenzymes, and completely blocked the cytolytic activity of YT cells. In addition, PKC-delta-specific antibody introduced into YT cells by electroporation inhibited partially the YT cell-mediated cytotoxicity of B-lymphoblastoid cell line JY. This effect was specific, since addition of anti-PKC-delta antibody-blocking peptide in combination with anti-PKC-delta antibody to YT cells for electroporation, neutralized the effect of this antibody. These results demonstrate that YT cell cytolytic activity is dependent on PKC-delta, which is selectively down-regulated by bryostatin 1.     

Analysis of Perforin Expression in Human Peripheral Blood Lymphocytes, CD 56+ Natural Killer Cell Subsets and Its Induction by Interleukin-2

In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin postive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25-30% of this population represents the CD56^bright+ subset, while 70-75% represents the CD56^dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97-99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin postive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2.     

Analysis of Perforin Expression in Human Peripheral Blood Lymphocytes,CD 56+ Natural Killer Cell Subsets and Its Induction by Interleukin-2

In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin postive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25–30% of this population represents the CD56^bright+ subset, while 70–75% represents the CD56^dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97–99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin postive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2.     

A study of complement fixation by rheumatoid factor using a haemolytic assay system.

Complement fixation by rheumatoid factor (RF) in sera from rheumatoid arthritis patients has been investigated by means of a haemolytic assay system employing sheep erythrocytes (SRBC) coated with reduced and alkylated rabbit IgG anti-SRBC antibody. Haemolysis was almost invariably detected with RF-positive sera whereas haemolysis was not observed with RF-negative sera; It was evident from a marked increase in haemolysis, obtained following isolation of IgM-RF of high purity from several RF-positive sera, that the full in vitro complement-fixing ability and hence lytic potential of RF in serum is masked. Parallel observations were also made with synovial fluids. The inhibition of haemolysis by RF in sera and synovial fluids commonly involved a reduction in degree of lysis at high concentration of test material as well as a more generalized inhibition in overall percentage lysis. Complete replication of typical RF-positive serum and synovial fluid patterns of lysis on dilution was achieved by addition of heat-aggregated human IgG to isolated IgM-RF preparations and demonstrated that the two inhibitory effects are separable in terms of complement depletion and reduction of free rheumatoid factor antibody activity, respectively.     

Comparison of fluorochrome-labeled and 51Cr-labeled targets for natural killer cytotoxicity assay

An alternative method for measuring in vitro cellular cytotoxicity has been developed utilizing the carboxyfluorescein derivative 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) as the target cell label. Target cells labeled with the fluorescent dye are incubated with effector cells, if killing of targets occurs, BCEDF is released analogous to 51Cr release. Measurement of specific lysis in this assay is based on the direct measurement of dye retained by the remaining viable target cells using the Pandex FCA. In paired experiments we have compared the fluorochrome assay to the standard 51Cr release assay in measuring porcine natural killer cytotoxicity. The target labeling time with BCECF is 30 min as opposed to 1 h with 51Cr; and there is no significant dye reincorporation after release. The optimal target number per incubation well for the BCECF assay is 5 X 10(3) cells. In both the BCECF and 51Cr release assays, maximum percent specific lysis is reached after 3-4 h incubation. By 2 h incubation, the BCECF assay reaches the maximum seen with 51Cr and in a 4 h assay the maximum NK activity measured with BCECF labeled targets is always higher than that measured with 51Cr-labeled targets. In paired experiments, we have shown the reproducibility of the BCECF assay and that the BCECF assay measures NK enhancement by NK enhancing monoclonal antibody and inhibition by NK inhibiting monoclonal antibody as good as the 51Cr release assay, if not better. In conclusion, the BCECF assay is a reliable and reproducible measure of in vitro cellular cytotoxicity, eliminates the use of radioisotopes and is cost efficient.     

Staphylococcal enterotoxin B induces potent cytotoxic activity by intraepithelial lymphocytes

In food poisoning, Staphylococcus aureus secretes staphylococcal enterotoxin B (SEB), a superantigen that causes intense T-cell proliferation and cytotoxicity. The effects of SEB on lytic activity by human intestinal intraepithelial lymphocytes (IEL) were investigated. Jejunal IEL, from morbidly obese individuals undergoing gastric bypass operations, were tested for SEB-induced cytotoxicity against C1R B-lymphoblastoid cells, HT-29 adenocarcinoma cells, or CD1d-transfected cells using the 51Cr-release assay. Fas and Fas ligand expression were detected by immunofluorescence and flow cytometry and soluble ligand by enzyme-linked immunosorbent assay (ELISA). In the presence of SEB, IEL became potently cytotoxic against C1R cells and interferon-gamma (IFN-gamma)-precultured HT-29 cells, causing 55+/-10% and 31+/-6% lysis, respectively, greater than that by phytohaemagglutinin (PHA)-, interleukin-2 (IL-2)-, or anti-T-cell receptor (TCR)-activated IEL. SEB-stimulated peripheral blood (PB) CD8+ T cells lysed similar numbers of C1R cells but fewer HT-29 cells (53+/-13% and 8+/-5%, respectively). IEL killing of C1R cells involved interaction of major histocompatibility complex (MHC) class II with TCR, CD2 with CD58, and CD11a with CD54, and was perforin mediated. SEB-induced IEL lysis of HT-29 cells, in contrast, was caused by an unknown target cell structure, not MHC class II or CD1d, and resulted from a combination of perforin and Fas-mediated events. The potent cytotoxic activities of IEL promoted by SEB utilize two different mechanisms, depending on the surface receptors expressed by the target cells.     

Two Distinct Mechanisms of Cytotoxity Mediated by Herpes Simplex Virus-Specific CD4+Human Cytotoxic T Cell Clones

The present study was undertaken to elucidate the mechanisms responsible for the cytotoxicity of herpes simplex virus (HSV)-specific CDA4⁺human cytotoxic T lymphocyte (CTL) clones, focusing on perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-β). Two HSV-specific CDA4⁺CTL clones, which expressed both perforin and membrane-bound LT, exerted HSV-specific cytotoxicity and cytotoxicity against LT-sensitive L929 cells. These CDA4⁺CTL clones lysed HSV-infected cells directly in an HLA class II-restricted manner and did not exhibit "bystander killing." The culture supernatants of these clones stimulated with HSV antigen showed no cytotoxicity against HSV-infected cells or L929 cells, suggesting that adhesion to target cells is essentail to their antigen-specific and antigen-nonspecific cytotoxicities. The cytotoxicities of these clones against HSV-infected autologous cells were inhibited by an anti-CD3 monoclonal antibody but not by an anti-LT antibody. Conversely, their cytotoxicities against L929 cells appeared to be partially inhibited by the anti-LT antibody but not by the anti-CD3 monoclonal antibody. Furthermore, target cell DNA fragmentation induced by these CDA4⁺CTL clones was apparently observed in L929 cells but only faintly detected in HSV-infected autologous cells. L929 cell DNA fragmentation was also inhibited by adding the anti-LT antibody to CDA4⁺CTL cultures. these data suggest that some CDA4⁺CTL possess at least two cytolytic mediators, i.e., perforin and membrane-bound LT simultaneously, and can exert both antigen-specific and antigen-nonspecific cytotoxicity via two distinct mechanisms, necrosis and apoptosis.     

The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis.

The splicing isoform of HLA-G that is expressed in xenogeneic cells, and its effect on NK-mediated direct cytotoxicity was examined, using stable Chinese hamster ovary (CHO) cell or swine endothelial cell (SEC) transfectants. cDNAs of HLA-G (G1 and G3) and human beta2-microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA-G1 was easily expressed on SEC, and co-transfection with human beta2-microglobulin led to an enhanced level of HLA-G1 expression, as evidenced by flow cytometry. The expressed HLA-G1 significantly suppressed NK-mediated SEC cell lysis, which is an in vitro delayed-type rejection model of a xenograft. On the other hand, the swine leucocyte antigen (SLA) class I molecules could be up-regulated as the result of the transfection of human beta2-microglobulin, but did not down-regulate human NK-mediated SEC lysis. The HLA-G3 was not expressed on CHO and SEC in contrast to HLA-G1, as the result of the transfection. The gene introduction of HLA-G3 in SEC showed no protective effect from human NK cells. However, indirect evidence demonstrated that HLA-G3 transfection resulted in HLA-E expression, but not itself, when transfected to the human cell line, 721.221, thus providing some insight into its natural function in human cells. The present findings suggest that the expression of HLA-G1 on the cell surface could serve as a new approach to overcoming NK-mediated immunity to xenografts.     

Human antibody-dependent cell-mediated cytotoxicity against target cells infected with respiratory syncytial virus.

A chromium release assay was established to study human antibody-dependent cell-mediated cytotoxicity (ADCC) of HEp 2 cells infected with respiratory syncytial (RS) virus. Human peripheral blood lymphocytes in the presence of specific antibody to RS virus caused in vitro lysis of RS virus infected target cells. ADCC was detected in sera of infants with RS virus infections and in specimens of colostrum. The ability of serum or colostrum to mediate the cytotoxic reaction appeared to be related to the level of specific IgG, or IgA antibody to RS virus, as detected by membrane fluorescence. Separation of effector cells by their glass adherence properties showed that the ability to produce cytotoxicity resided in non-adherent effector cells.     

Stable expression of EBV-gp350 on the surface of NC37 cells confers natural killer (NK)-cell susceptibility or resistance, depending on the assay used to assess NK-mediated function

NC37 cells containing the Epstein-Barr virus (EBV) genome do not express the viral glycoprotein-350 (gp350) on the cell surface. Despite being a cancer cell line, NC37 cells show resistance to natural killer (NK) cell cytotoxicity by the standard chromium (⁵¹Cr) release assay (CRA). EBV-gp350 has been identified as a ligand for antibody dependent cell-mediated cytotoxicity (ADCC). The stable expression of gp350 on the NC37 cell surface membrane could make this cell line a suitable target for measuring ADCC antibody. The pcDNA3.1-gp350 was transfected into the stably expressing enhanced green fluorescent protein (EGFP)-NC37 cell line. The transfected cells were then selected for expression of gp350 on the cell surface using immunomagnetic bead-based sorting. The gp350-EGFP-NC37 cell line was then re-examined for resistance to NK cytotoxicity, and compared with the standard K562 and EGFP-K562 cell lines using the CRA and a flow cytometric method, respectively. Surprisingly, the gp350-EGFP-NC37 cells, like the parental NC37 cell line, showed comparable resistance to NK cell-mediated cytotoxic activity by the CRA, while demonstrating susceptibility to NK cell cytotoxicity comparable to EGFP expressing K562 cells by the flow cytometric method. The susceptibility of gp350-EGFP-NC37 cells to NK cell cytotoxic activity is dependent on the type of assay.     

Antibody-dependent cell-mediated cytotoxicity (ADCC) toward human O+ red cells coated with anti-D antibody: comparison between lymphocyte and monocyte ADCC activity

We investigated the antibody dependent cell-mediated cytotoxicity (ADCC) of lymphocytes and monocytes toward human O+ red cells coated with anti-D antibody using a 51Cr release assay. Lysis of sensitized red cells by lymphocytes occurred rapidly, but monocyte-mediated lysis occurred slowly. This difference might be due to postphagocytic 51Cr release by monocytes. ADCC of lymphocytes increased in proportion to the effector cell number, but large amounts of antibodies were required. In contrast, ADCC of monocytes was independent of the effector/target ratio and very small amounts of antibodies could produce red cell lysis. Large amounts of fluid phase IgG were required to inhibit the lymphocyte ADCC, whereas the monocyte ADCC was markedly inhibited by small amounts of IgG. Monocyte-mediated lysis was completely inhibited by the addition of 10% human AB serum, but lymphocyte-mediated lysis was only slightly inhibited. Purified IgG1 and IgG3 were much more inhibitory to the lysis by both effectors than IgG2 and IgG4 (IgG2 greater than IgG4). Erythrophagocytosis also was inhibited by IgG1 and IgG3. These studies demonstrate that lymphocytes as well as monocytes can cause the lysis of antibody sensitized red cells, and IgG1 and IgG3 subclasses are more important than IgG2 and IgG4 in causing lysis of anti-D coated red cells.     

Expression of the CD80 and CD86 molecules enhances cytotoxicity by human natural killer cells

In contrast to the inhibitory pathway of NK cell regulation, much less is known about stimulatory or activation signals in NK cells. Both CD80 and CD86 function as costimulatory molecules in T-cell cytotoxicity. Several previous reports, most of them in the murine system, have indirectly or directly indicated the possible role of B7 molecules (CD80 and CD86) triggering NK cell-mediated cytotoxicity in vitro. Nevertheless, only little is known about the role of these molecules on human target cells. Therefore, anti-CD80 and anti-CD86 mAbs were used in blocking experiments and both were shown to inhibit lysis by human NK cells. The degree of inhibition observed was variable. 64% of these NK clones were strongly inhibited by both anti-CD80 and anti-CD86 (Type 1). A small number (19%) were only moderately inhibited by both of these antibodies (Type 2), and 17% of these NK clones were inhibited strongly by anti-CD86 but weakly or not at all by anti-CD80 (Type 3). To further examine the importance of these proteins, B7.1 (CD80) and B7.2 (CD86) genes were transfected into the mouse mastocytoma P815 cell line that could not be killed by the human NK cells. These transfectant cell lines were then tested in cytotoxicity assays using a number of human NK lines. Expression of the CD80 and CD86 molecules resulted in enhanced lysis of P815 by most of the NK lines tested. Thus, both CD80 and CD86 molecules are involved in triggering of human NK cells.     

The use of passive haemolysis in the quantitative estimation of anti-protein antibodies

The conditions for the establishment of a method for the quantitative assay of antiprotein antibodies by the use of passive haemolysis were studied by using an anti-BSA—BSA system as a model.

When haemolytic assaying of antibody was carried out under standard conditions with a fixed concentration of optimally sensitized red cells, in the presence of excess complement (10 C′H₅₀) reproducible titrations could be performed to detect 0.12 μg. N Ab with an error of ±10 per cent.

Factors affecting the specific lysis of optimally sensitized cells were studied and some parameters fixed. The amounts of coated erythrocytes and complement and the length of time of reaction were seen to influence the final degree of lysis given by a fixed amount of antibody.

The conditions of optimal sensitization of erythrocytes with protein antigen by the use of bis-diazotized benzidine (BDB) are indicated. It was found that, when conjugation was carried out at 0° with proper amounts of a standardized cell suspension, BDB and antigen, the resulting coated cells were highly sensitive to immune haemolysis and highly resistant to `spontaneous' lysis.

     

血管内皮细胞(ECV304)表达的HLA-G1对NK细胞杀伤活性的抑制作用

目的:证实HLA-G1分子能够抑制NK细胞对同种血管内皮细胞系的杀伤作用。方法:采用脂质体介导的DNA转染技术,以构建的真核表达质粒pcDNA3-HLA-G1转染人脐静脉内皮细胞系ECV304,再用免疫荧光法检测表达的HLA-G1分子。并用MTT比色法检测HLA-G1对NK细胞杀伤活性的影响。结果:ECV304细胞上可稳定表达HLA-G1。NK细胞对空质粒pcDNA3转染的ECV304细胞的杀伤率为(50.6±18.1)%;而对pcDNA3-HLA-G1转染的ECV304细胞的杀伤率为(29.7±11.4)%,两者差异具有显著意义(P<0.01)。结论:HLA-G1分子可明显抑制NK细胞对同种血管内皮细胞的杀伤效应。