~(51)Cr释放法检测杀伤性T细胞的细胞毒作用和评价

本文报道了国产铬酸钠(Na_2~(51)CrO_4)标记P815细胞的条件,和用~(51)Cr释放法检测杀伤性T细胞(CTL)的细胞毒作用。~(51)Cr标记P815细胞的适宜条件是:2×10~5个P815细胞加入100μCi~(51)Cr,37℃恒温下温育2小时。效应细胞与靶细胞作用的条件是:效靶比100:1,作用时间4.5小时。C57小鼠脾脏CTL的特异杀伤作用在P815细胞致敏后第7天开始出现,第11天达高峰,以后开始下降。环磷酰胺50mg/kg可明显抑制小鼠CTL的细胞毒作用。     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. I. Phagocytic and non-phagocytic effector cell activity against erythrocyte and tumour target cells in a 51Cr release cytotoxicity assay and [125I]IUdR growth inhibition assay.

Both phagocytic and non-phagocytic effector cells were able to kill rabbit antibody-coated chicken erythrocytes (CRBC) while only non-phagocytic effector cells were active against alloantibody-coated SL2 lymphoma. In addition to the variation in susceptibility of erythrocyte and tumour target cells to various effector cell populations, it was found that different tumour cells can vary markedly in their ability to be killed by non-immune spleen cells in the presence of antibody. It is postulated that both the type of antibody and certain characteristics of the cell membrane are important in determining whether target cells are susceptible to antibody-dependent cell-mediated cytotoxicity detected by the 51Cr release assay. It was also demonstrated that alloantibody-coated P-815-Y mastocytoma, which showed very little evidence of cytotoxicity in the 51Cr release assay, was markedly inhibited in its ability to incorporate [125I]IUdR after incubation with antiserum and non-immune spleen cells. This growth inhibition in the absence of cytotoxicity, or cytostasis, is discussed in relation to the potential mechanisms of target cell damage, and in the light of recent observations (Plata, Gomard, LeClerc and Levy, 1974; Newlands and Roitt, 1975) that cytotoxicity and growth inhibition assays detect different effector cell populations in tumour-bearing animals.     

The decay of memory of cell-mediated cytotoxicity after primary sensitization in vivo against a tumor allograft.

The length of time mice were able to show a secondary cell-mediated response after a tumor allograft was investigated. C57B1/6 mice were inoculated ip with P-815 mastocytoma cells and, at selected time intervals up to 12 months after immunization, spleen cells were placed in JLC in an attempt to generate secondary CTL activity. It was found, as measured by the 51Cr-release assay, that all mice tested had significant secondary CTL activity 3 months after allograft. The ability to generate a secondary response then decreased with considerable individual variation up to 6 months post-immunization. No significant difference in CTL activity was found between immunized and age-matched control mice 12 months after tumor allograft.     

One-step simple assay to determine antigen-specific cytotoxic activities by single-color flow cytometry

Assays for cytotoxicity of CTLs in vivo using a fluorescent-based dye, 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE), have been established and widely used. On the basis of this experience, we applied it to in vitro assay system and established a simpe, highly sensitive flow cytometric assay for CTL activity. In our assay, specific activities of CTLs could be detected by a reduction in sensitive target cell numbers on single-color histogram plot analysis. By using this assay, we could determine the changes in cytotoxic activity by single amino acid substitution within an epitope peptide. Adherent cells were also used as target cells in this assay by treatment with excess EDTA and trypsin reagents after incubation with effector CTLs. Furthermore, when fluorescent calibration beads were used as a control, we could determine the cytotoxicity of CTLs against tumor cells. The results obtained from our assay were almost consistent with those from the conventional ( 51)Cr-release assay.Because our assay uses only a stable non-radioactive reagent, CFSE, this assay is safe, inexpensive and extremely easy. These results indicated that this new assay (FACS-CTL assay) would be sufficiently acceptable alternative to classical (51)Cr-release assay.     

Phorbol myristate acetate-induced modulation of antibody-dependent cellular cytotoxicity by human polymorphonuclear leukocytes.

The effect of phorbol myristate acetate (PMA) on the antibody-dependent cellular cytotoxicity (ADCC) of human polymorphonuclear leukocytes (PMNs) against human erythroleukemic K562 cells was studied by the use of a 3-h 51Cr-release assay. Pretreatment of PMNs with PMA (10 ng/ml) for 60 min resulted in inhibition of subsequent ADCC. This inhibition was dependent on doses of PMA. The effect of pretreatment of PMNs with PMA on O2- generation of the cells was also studied. The ability of the cells to generate O2- was not suppressed, and the expression of Fc receptors on the cell membrane was well preserved. In contrast, the addition of PMA to the ongoing ADCC (5 to 30 min after the start of the ADCC assay) enhanced the activity of the cells for ADCC. This augmentation was abolished by catalase, whereas ADCC itself was not affected by the agent. These results imply divalent effects of PMA on the ADCC of PMNs. The suppression of ADCC activity of PMNs by pretreatment with PMA is not due to inhibition of the reactive oxygen burst of the cells. The augmentation of ongoing ADCC by the addition of PMA is due to secretion of hydrogen peroxide from the cells induced by PMA, and this augmentation occurs only when the interaction between effector and target cells exists through Fc receptor.     

The Decay of Memory of Cell-Mediated Cytotoxicity after Primary Sensitization in Vivo Against a Tumor Allograft

The length of time mice were able to show a secondary cell-mediated response after a tumor allograft was investigated. C57B1/6 mice were inoculated ip with P-815 mastocytoma cells and, at selected time intervals up to 12 months after immunization, spleen cells were placed in MLC in an attempt to generate secondary CTL activity. It. was found, as measured by the ⁵¹Cr-release assay, that all mice tested had significant secondary CTL activity 3 months after allograft. The ability to generate a secondary response then decreased with considerable individual variation up to 6 months post-immunization. No significant difference in CTL activity was found between immunized and age-matched control mice 12 months after tumor allograft.     

Increased cell-mediated lysis of chicken erythrocytes following the addition of metabolic inhibitors.

The effect of a variety of metabolic inhibitors on the cell-mediated lysis of chicken erythrocytes by immune spleen cells was investigated using the Cr-release assay. The addition of cycloheximide, puromycin, emetine, pactamycin, actinomycin D or EDTA during the early stages of the reaction (0-2 h) produced partial to complete inhibition of the cytotoxic reaction, while the addition of these compounds at later time periods (2-4 h) resulted in the progressive loss of inhibitory effects. Later additions (4-6 h) of all compounds, except EDTA, resulted in a significant increase in target cell lysis. The ability of these compounds to induce increased cytotoxicity required complete inhibition of protein synthesis and the presence of reactive effector cells. It did not appear to be due to an increase in the rate of 51Cr release from previously damaged target cells, or inhibition of a target-cell repair mechanism dependent on protein synthesis. At least a portion of the increased reactivity was due to effector cell-target cell adhesions which formed after the addition of the inhibitor. The data suggests that the addition of metabolic inhibitors during the later stages of the reaction induced an increase in the efficiency or number of cytotoxic attacks.     

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.     

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.     

Distinct Patterns of Virus-Specific T Cell-Mediated Cytolysis of Transformed versus Primary Target Cells

Ideas about the mechanism(s) by which cytotoxic T lymphocytes (CTL) lyse appropriate target cells are still controversial. We studied the action of established murine CTL clones as well as of freshly prepared primarily induced CTL against two types of target cells. Transformed tumour cell lines (MC57G and L929) and untransformed cells such as peritoneal exudate cells (PEC) or fibroblasts were examined as target cells by scanning and transmission electron microscopy and with the 51Cr-release assay. We found independent of which CTL used, that in transformed cells the cell membrane seemed to be the first target of CTL attack, whereas in untransformed cells the first noticeable events appeared to take place in the nucleus of the target cells; the membrane of attacked untransformed PEC or fibroblasts was found to be intact at a time when the cellular organelles already were disintegrated. The morphological observations were paralleled by differences in the kinetics of 51Cr-release; untransformed target cells released their label only after a 2 h long period, whereas transformed cells released 51Cr considerably earlier.     

In vitro induction of tumor-specific immunity. VI: analysis of specificity of immune response by cellular competitive inhibition: limitations and advantages of the technique.

The cellular competitive inhibition 51Cr-release assay makes two distinct contributions to the in vitro study of cell-mediated immunity. It allows target cells which are not amenable to isotopic labelling to be investigated for their antigenic specificity, and it provides a means, complementary to the direct cytotoxicity assay, of estimating qualitative and quantitative differences in antigen expression on intact normal and neoplastic cells. Various parameters of a micro-51Cr-release inhibition assay have been studied, and it was found that the assay conditions markedly influenced both the sensitivity and specificity. It is concluded that optimal assay conditions for specificity include: 1) moderate levels of lysis on the linear part of the CL/T titration curve, 2) avoidance of prolonged assay times, and 3) low ratios of blocker to target cells. When tumor cells with large cell volumes are used as competitive inhibitor (blocker) cells, non-specific blocking will occur; limits have been defined for this particular micro-inhibition assay which, in general, exclude these effects.     

The cytotoxic T lymphocyte response in reticuloendotheliosis virus-infected chickens is mediated by alpha beta and not by gamma delta T cells.

We induced a virus-specific cytotoxic T lymphocyte (CTL) response in B2 chickens by i.v. inoculation with 100 TCID50 of the reticuloendotheliosis virus (REV). Chickens were sacrificed 7 days after the infection and cytotoxic activity of the spleen cells against various target cells was assayed in a 4 h 51Cr-release assay at an effector to target ratio of 100:1. In addition, T cell receptor (TCR) alpha beta and TCR gamma delta cells were negatively selected from the REV-immune spleen cells and used as effector cells against REV-infected B2 target cells. (On average 40% of spleen T cells express TCR gamma delta in the chicken.) By inhibition of the cytotoxic activity of the immune spleen cells against REV-infected syngeneic target cells with monoclonal antibodies specific for chicken CD3 and CD8 molecules, the effector cells could be identified as CD8+ T cells. The cytotoxic activity was MHC-restricted, as only syngeneic but not allogeneic REV-infected target cells were lysed by REV-immune spleen cells, and virus-specific, as no cytotoxic activity could be found using uninfected syngeneic target cells. When assaying the activity of the negatively selected, > 98% pure alpha beta and gamma delta T cells, it was found that alpha beta T cells exerted virus-specific CTL activity ranging from 26 to 62% specific 51Cr-release, while gamma delta T cells showed only 2-4% 51Cr-release. These data indicate that REV-specific CTL response is mediated by alpha beta T cells and that gamma delta T cells are not involved in virus-specific CTL activity in the spleen of REV-infected chickens.     

The fluorolysis assay,a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard 51Cr-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few (> or =6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers (> or =25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies.     

Cytolysis of actinomycin D-treated target cells by cell-free supernatants from human monocytes

The lytic mechanism of human peripheral blood monocytes was studied by using as targets actinomycin D-treated WEHI-164, an NK-insensitive murine fibrosarcoma cell line. Monocytes, but not lymphocytes, lysed WEHI-164 target cells pre-treated with actinomycin D within 6 h in 51Cr-release assays. Because cytolysis could not be inhibited competitively by unlabeled WEHI/D target cells, contact-independent mechanisms of cytolysis were investigated. Cell-free supernatants collected from monocytes cultured for 4-6 h at 37 degrees C lysed target cells as effectively as effector cell preparations of monocytes. Supernatants from lymphocytes cultured in parallel were not cytolytic. Cytolytic activity was not detected in supernatants from preparations of monocytes that were held on ice. However, monocytes produced cytolytic activity whether they were isolated by adherence or remained unseparated in suspensions of mononuclear cells. The cytolytic activity in cell-free supernatants (CFS) from monocytes was unaffected by incubation with protease inhibitors. CFS activity was destroyed by heat. Storage of CFS at 37 degrees, 22 degrees, 4 degrees, or -20 degrees C for 24 h decreased cytolytic activity; however, loss of cytotoxicity was minimized by storage at 4 degrees C. The cytolytic substance detected in 4-h CFS from monocytes appeared to be a protein(s) based on the sensitivity of the cytolytic activity to proteases. Cytolytic activity of CFS eluted from Sephacryl 200 in a single peak with an apparent molecular weight between 25,000 and 45,000 daltons.     

Modulation of natural killing by cyclo- and lipo-oxygenase inhibitors.

The effect on NK activity of two oxygenase inhibitors, indomethacin which is cyclo-oxygenase-specific at 10(-6) M and BW755C which blocks both cyclo- and lipo-oxygenase, has been analysed. The NK activity of human peripheral blood mononuclear cells (PBMC) has been studied in a 18-hr 51Cr-release assay. PBMC were incubated with the drugs for different periods of time before and during NK assay in order to analyse the correlations between the time of incubation and the modulation of NK activity. The results show that the inhibition of the cyclo-oxygenase alone, by indomethacin, enhances the NK activity if the drug is added both during and prior to the assay. In the latter case the enhancement is time dependent, reaching a maximum level after 18 hr of incubation. The simultaneous inhibition of lipo- and cyclo-oxygenase by BW755C has a rather more complex effect. The NK activity is similar to that of untreated cells after 6 hr incubation with BW755C, but is inhibited or greatly enhanced after 4 and 18 hr of incubation, respectively. These effects seem largely independent of the presence of monocytes because they can be observed with non-adherent PBMC (NA-PBMC).     

Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing

We have developed a highly sensitive biophotonic luciferase assay as an alternative to (51)Cr-release for assessment of cell-mediated cytotoxicity. The luciferin/ATP-dependent luminescent signal of target cells stably or transiently transfected with a firefly luciferase reporter gene (fLuc:Zeo) linearly correlates with viable target cell number. Upon incubation of fLuc:Zeo(+) target cells with CD8(+) CTLs, a rapid decrease in bioluminescence was detected that correlated with antigen-specific target cell lysis. The levels of specific lysis measured by (51)Cr-release assays correlated with the attenuation in biophotonic target cell signal, thus validating this approach as a sensitive and accurate method for the measurement of cytolysis. We show that this luminescent-based cytolytic assay (LCA) is amenable for high-throughput screening of effector cell cytolytic activity, allows for the rate of cytolysis to be measured in a single micro-plate, and permits the multiplexing of cytolytic killing with other lymphocyte functional assays such as cytokine release. Importantly, this method accurately measures the cytolytic killing of target cells that are either stably or transiently transfected with a fLuc reporter gene, and thus is ideal for monitoring cytolysis of both primary autologous and immortalized target cell lines. The versatility of the non-radioactive, high-throughput, biophotonic cytolytic assay should make this method an attractive alternative to chromium-release for quantifying effector cell cytolytic activity.     

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 degrees C for 4 h. Target cell killing was evaluated by 51Cr-release assay (CRA) or reduction of telomerase activity (R-TRAPCTX) 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-TRAPCTX. Incorrect results were obtained with CRA when target cells were admixed with normal fibroblasts, whereas R-TRAPCTX 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-TRAPCTX 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.     

The Role of Viral Glycoproteins in Mumps Virus-Mediated Antibody-Dependent Cellular Cytotoxity in Vitro

Treatment of human peripheral blood lymphocytes with live or UV-inactivated mumps virions enhances antibody-mediated cellular cytotoxicity (ADCC), reflected by increased target cell lysis in a 51Cr-release assay or an increased number of plaque-forming cells on monolayers of bovine erythrocytes (Eb) in the presence of anti-Eb antibodies. Virus treatment of the Eb targets causes a similar enhancement. The role of viral glycoproteins in ADCC enhancement was investigated by using a panel of monoclonal antibodies raised in mice against mumps virions. Most of the lymphocytes bound mumps virions, as ascertained by indirect immunofluorescence. A high proportion of virus-treated lymphocytes also formed rosettes with Eb. Anti-HN antibodies inhibited rosetting to various degrees. Although antibodies with high haemagglutination inhibition titres were most efficient inhibitors, antibodies without this serological activity were also inhibitory. Anti-F antibodies were only weakly inhibitory, and anti-NP antibodies had no effect. Anti-HN antibodies also abrogated target cell lysis in the 51Cr-release assay and effector cell recruitment in the ADCC plaque assay by inhibiting virus-mediated Eb-lymphocyte interactions both at the target cell and at the effector cell level. Anti-F or anti-NP antibodies were only weakly or not at all inhibitory. The results suggest that virus-mediated enhancement of ADCC is caused by the HN glycoprotein, primarily (although perhaps not exclusively) by its improvement of the effector cell-target cell contacts necessary for the efficient execution of target cell lysis.     

Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells.

A tetrazolium compound, XTT, bioreducible to a water-soluble formazan was used to develop a simplified cellular cytotoxicity assay. Most (13/15 melanoma and 2/3 colon carcinoma cell lines tested metabolized XTT greater than 50 times more efficiently than the lymphoid effector cells, and thus the test could be performed without separation of the effector from the target cells. The XTT assay (XTT-A) was compared to the standard 51chromium-release assay (51CrA) in terms of sensitivity as well as intra- and interassay variability using low effector to target cell (E:T) ratios and both short and long incubation periods. The correlation coefficient (r) for percent specific lysis (%SL: 35.0 +/- 15.0 versus 30.2 +/- 15.8) or lytic units (LU20/10(7) effector cells: 405 +/- 208 versus 357 +/- 227) between XTT-A and 51CrA was 0.86 for 4 h XTT-A and 51CrA (n = 37). Due to a poor performance of the 51CrA after 24 h incubation of effector and target cells, the correlation coefficient for 24 h assays was reduced to 0.79 (n = 44,%SL = 63.3 +/- 23.9 versus 55.5 +/- 26.6, and LU = 1267 +/- 982 versus 1017 +/- 691). Inter- and intra-assay variability of XTT-A were significantly lower than those for 51CrA. The total background values for XTT-A and 51CrA were similar in 4 h cytotoxicity assay and lower for XTT-A in assays with 24 h incubation. The sensitivity, in terms of discrimination between effector cells with different lytic capacity and targets with different susceptibility, was identical. The XTT-A was simpler, cheaper, and safer to perform than the 51CrA. Furthermore, the XTT-A was suitable for long-term assays and allowed experiments without requiring trypsinization of tumor cells grown in 96-well plates prior to testing.