A Comparative Study of the JAM Test and 51Cr‐Release Assay to Assess the Cytotoxicity of Dendritic Cells on Hematopoietic Tumor Cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti‐tumor cytotoxic ability. Nevertheless, the mechanism of anti‐tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H‐TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr‐labeled tumor cells; 3) and to induce DNA fragmentation on ³H‐TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H‐TdR. However no cytolysis was verified by ⁵¹Cr‐release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr‐release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4‐h and the 10‐h ⁵¹Cr‐release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation An easy and low cost alternative to radioactive thymidine incorporation

The classical in vitro assay for the determination of cell mediated immune responses is the lymphocyte transformation test (LTT) in which cell proliferation is measured by incorporation of radioactive labeled thymidine (³H-TdR). The LTT assay using ³H-TdR is less suited for modestly equipped laboratories as it is costly, laborious and involves the need to handle radioactive isotopes and specialized equipment.

Here we describe an improved alternative LTT method which is capable of detecting specific cellular immune reactions (CMI) against (mycobacterial) antigens in vitro. This assay, the bromodeoxyuridine-ELISA LTT test, is simple, less expensive, reproducible and is as sensitive as the ³H-TdR test. The specific advantages of the test are a simple denaturation step and the fact that no radioactive isotopes are needed. The test is specifically suited for research laboratories in tropical countries which study CMI in those human infectious diseases where this arm of the immune response plays a pivotal role in the generation of immunity, e.g., in tubercolosis, leprosy and leishmaniasis.     

Simultaneous Analysis of In Vivo CD8+ T Cell Cytotoxicity Against Multiple Epitopes using Multicolor Flow Cytometry

CD8+ T cells play a critical role in host defense against infections and tumors. Analysis of cytotoxic function of antigen-specific CD8+ T cells in animal models would be important in optimizing vaccine design against infections and tumors. In vivo cytotoxicity assays using fluorescent cellular dyes have been used as a popular alternative to traditionally used in vitro ⁵¹Cr-release assays. With the identification of multiple epitopes in various pathogen models, methods to simultaneously analyze cytotoxicity of CD8+ T cells to multiple epitopes in vivo would assist studies which aim to generate protective CD8+ T cell immunity to multiple epitopes. In this study, we evaluate the use of multiple fluorescent cellular dyes for the in vivo cytotoxicity assay. The use of 3 dyes allowed us to analyze the cytotoxicity of antigen-specific CD8+ T cell populations to multiple epitopes generated by virus infections, as well as their functional avidity, in vivo. Our studies extend the use of in vivo cytotoxicity assays to allow direct comparisons of cytotoxicity to various epitopes in the same animal and may also be applicable to assessment of in vitro cytotoxicity of human CD8+ T cells specific for multiple viral or tumor antigens in clinical settings.     

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.     

Tumor transfected with CCL21 enhanced reactivity and apoptosis resistance of human monocyte-derived dendritic cells

Chemokine CCL21 can effectively attract CCR7⁺ dendritic cells (DCs), however its role in this event is poorly understood. In this report, we investigated the effect of exogenous CCL21 expressed in breast cancer MCF-7 on human monocyte-derived DCs. CCL21-transfected MCF-7 stimulation prompted DC functions: migration, antigen-uptake and presentation. The stimulated DCs facilitated Th1 type cytokines production, perforin-forming CD8⁺ T cell transformation and final T cell-associated clearance of MCF-7. Moreover, the MCF-7-resourced CCL21 protected DCs from apoptosis significantly, involving up-regulations of Bcl-2 expression and NF-κB activity, and reduction of caspase-3. This study provides evidence that tumor-derived CCL21 increases the presentation and apoptosis resistance of DCs, suggesting such a mechanism may be useful for the improvement of tumor cell immunogenicity and anti-tumor response.     

Bacteria-Immune System Interactions. VIII. Modulation of Human Cytotoxic Reactions by Crude Lipoteichoic Acid Extracts of Bacillus Globigii

Crude lipoteichoic acid (LTA)¹ extracts were prepared from Bacillus globigii (Bg-LTA). The addition of Bg-LTA to mixed lymphocyte reactions (MLR) produced a dose dependent inhibition of specific cytotoxic reactions and of tritiated thymidine (³H-TdR) incorporation. Much less Bg-LTA was needed to inhibit the initiation (addition at day 0) than the effector phase (addition at day 6) of the cytotoxic reactions. None of the LTA quantities used had a toxic effect on the lymphocytes. LTA pretreated stimulator cells also had a modulatory effect on the cytotoxic reactions. Stimulators pretreated with very low or very high LTA concentrations had no effect; however, pretreatment with intermediate concentrations induced a drastic inhibition of cytotoxicity. LTA pretreated stimulator cells did not significantly affect ³H-TdR Incorporation. Suppression of the cytotoxic reaction could also be obtained by LTA-pretreatment of the effector cells.     

不同大鼠胶质瘤抗原致敏的DC体外诱导CTL活性的研究

目的: 比较大鼠骨髓来源的树突状细胞(dendritic cells, DC)体外经由大鼠C6胶质瘤细胞由不同方式制备的不同抗原致敏后,对特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)的诱导作用。方法: 自大鼠骨髓分离DC前体细胞,经重组大鼠粒细胞巨噬细胞集落刺激因子(rrGM-CSF)+白细胞介素4(rrIL-4)诱导培养、扩增;由C6胶质瘤细胞经由反复冻融、煮沸灭活及超声破碎细胞抽提其总蛋白的方法制备各种不同抗原致敏DC,致敏的DC与T淋巴细胞进行共培养诱导CTL;以ELISA法检测CTL诱导过程中淋巴细胞趋化因子(lymphocyte chemoattractant factor)及细胞因子IFN-γ分泌水平:以 -TdR掺入法检测DC诱导T细胞增殖及其特异性CTL杀伤活性。 结果: 体外应用煮沸灭活瘤细胞制备的肿瘤抗原致敏DC,能诱导更强的刺激T细胞增殖的能力、并且可以诱导杀伤活性更强的CTL。结论: 应用煮沸灭活的瘤细胞制备瘤抗原负载DC获得瘤苗可获得更强的抗肿瘤保护作用。     

Influence of Ara-C on Effector Activity of Cytotoxic T-Lymphocytes in Vitro?

Mouse T-lymphocytes carrying the H-2?^d haplotype were sensitized in vitro? against allogeneic spleen cells homozygous for the H-2?^b haplotype. Cytolytic T-lymphocytes (CTL) were tested in a 4 hr cytotoxicity assay against ⁵¹Cr-labeled H-2?^b RBL-5 lymphoma cells. The cytotoxic activity of CTL was markedly increased by preincubation with Ara-C (1 through 1000 μg/ml, 1 hr at 37°C). Limited but significant increase of cytotoxicity occurred when the drug was added to the effector-target mixture, or when target cells were preincubated with Ara-C using the same conditions adopted for the in vitro ?treatment of CTL. It is suggested that the present findings would contribute to elucidate the mechanism underlying anti-tumor synergistic effects between host's responses and treatment with antineoplastic agents.     

Application of Non-Radioactive Europium(EU3+) Release Assay to a Measurement of Human Natural Killer Activity of Healthy and Patient Populations

Europium (Eu³⁺) release assay is a non-radioactive method for a measurement of cytotoxicity of lymphocytes and has several advantages compared with a conventional ⁵¹Cr release assay. However, the Eu³⁺ release assay has not been applied to a natural killer (NK) activity measurement of a large number of the human population mainly due to a lack of comparability with the ⁵¹Cr release assay. With some modifications of the procedures and careful manipulation of cells, constant and reproducible results were obtained by the Eu³⁺ release assay. NK activity of several individuals was measured by the Eu³⁺ release assay and was compared with data obtained by ⁵¹Cr release assay performed simultaneously. The obtained values by the two methods were almost identical. We applied the Eu³⁺ method to measure NK activity of a large number of individuals, including 68 apparently healthy donors and 36 autoimmune and 21 cancer patients. Some of these diseases are known to show abnormal NK activity. The obtained cytotoxicities were mostly consistent with the previously reported data obtained by the ⁵¹Cr release assay. These results indicated that the Eu³⁺ release assay could be used as an alternative method for a measurement of human NK activity of mass population including patients.     

Inhibition of tumor growth by peptide specific cytotoxic T lymphocytes in a three-dimensional collagen matrix

Tumor associated, MHC I restricted antigenic peptides have been identified in both human and mouse tumors. Cytotoxic T lymphocytes (CTL) which recognize these tumor associated antigenic peptides are potential anti-cancer effectors. The anti-tumor activity of CTL is usually measured in vitro by the ⁵¹Cr release assay and in mice by tumor growth inhibition which is the most direct assessment of anti-tumor effect. In clinical studies, an in vivo tumor growth inhibition assay is not an option and an in vitro assay which corroborates with in vivo tumor growth is needed to assess the long-term outcome of CTL activity. Here, a three-dimensional (3-D) collagen gel assay was developed to measure in vitro the inhibition of mouse mammary tumor growth by anti-tumor CTL. BALB/c mouse CTL were induced with peptide E474 SFAVATTAL which was expressed by mouse mammary tumor cells D2F2. To measure D2F2 tumor growth inhibition in vitro, a mixture of tumor cells and anti-E474 CTL in a 1 μl cell bolus was embedded in the collagen gel. Complete eradication of tumor growth was observed at E:T ratio of or greater than 1:1. rIL-2 supplementation was necessary to achieve long-term tumor growth inhibition. Even spontaneous D2 tumor explant could be grown in the collagen gel and addition of anti-E474 to this culture reduced tumor growth. This assay system provides a realistic and sensitive alternative to the in vivo tumor growth inhibition assay and allows easy adaptation to test additional therapeutic reagents.     

Screening of high cytotoxic tumor killer cells using a sensitive adherent target detachment assay

Screening of high cytotoxic tumor killer cells is of great importance in adoptive immunotherapy. Here, we describe a more sensitive assay, as compared to traditional 51Cr- or Calcein-release assay, to measure cytolytic activity of killer cells. This adherent target detachment (ATD) assay is carried out in microwells of Terasaki tissue culture trays using adherent tumor cells as targets. Target tumor cells are seeded at the concentration of 300-400 cells/well and incubated overnight to allow for the adhesion of cells to the plastic surface of the wells. Effector cells were added at various effector: target (E:T) ratios, and incubated for 24 h. During incubation, dead target cells became nonadherent and together with the added effector cells were removed by washing. The remaining viable adherent target cells were stained with acrydine orange contained in the quencher and optically counted by microcomputer. A notable dose-dependent killing on target tumor cells was reproducibly obtained by tested effector cells. Cytotoxic activities of most effector cells were significantly higher in the 24-h ATD assay than those of concordant 4-h target cell lysis test. Chloroquine (chq) inhibition test in the 24-h ATD assay showed negative or weak inhibition on cytotoxicity against adherent targets. Linear regression analysis also manifested the lack of a close correlation between 4- and 24-h target cell lysis tests, indicating these two assays measured different killing activities of the same effector cells. Because of its high sensitivity and reproducibility, this semiautomatic 24-h assay with computerized fluorescence measurement system could serve as a sensitive screening assay to select high cytotoxic tumor killer cells in adoptive immunotherapy.     

Cell mediated lympholysis; a modified technique using indium-oxine-labelled targets

The isotope ⁵¹Cr generally used in the cell mediated lympholysis (CML) assay suffers from the disadvantage of low specific activity, poor incorporation and high spontaneous release, limiting the CML assay to 4–6 h. We have labelled PHA derived human lymphoblasts with the isotope ¹¹¹indium (using ¹¹¹indium-oxine) and evaluated these cells as targets in CML. The level of ¹¹¹In-oxine incorporation decreased rapidly in the presence of serum; in the absence of serum approximately 85% of the available isotope in the supernatant was incorporated into the blasts. Under the labelling conditions used, spontaneous release was 1.6–2%/h on average allowing an effector phase of 18 h. About 5–8% of the released isotope was reutilized by the effector cells during an 18 h incubation period. Extending the CML assay from 6 to 18 h greatly increased the cytotoxicity. At an effector to target ratio of 25:1, the average per cent specific release increased from 15 to 50%. The use of ¹¹¹In-oxine labelled targets in the CML therefore increases the sensitivity of the test and allows fewer effector and target cells to be used as compared with ⁵¹Cr techniques.     

Complement Dependent Cytotoxicity of Sensory Ganglion Neurons Mediated by the gp120 Glycoprotein of HIV-1

Patients infected with HIV-1 frequently have a sensory neuropathy, but the cause is unknown. We found that the gp120 glycoprotein of HIV-1 bound to the surface of cultured rat dorsal root ganglia (DRG) neurons, and activated the complement cascade to lyse the neuronal cells. Cytotoxicity was measured by a ⁵¹Cr-release assay, and deposits of the C5b-9 complement complex were detected on the affected cells. As controls, gp120 or complement alone, or rgp120 plus deactivated complement, did not damage the neuronal cells, and fibroblasts were unaffected. The gp120 glycoprotein can thereby damage DRG neurons by complement dependent mechanisms. This interaction may contribute to the development of the sensory neuropathy in AIDS.     

Lactoferrin-inducible monocyte cytotoxicity for K562 cells and decay of natural killer lymphocyte cytotoxicity.

Monocyte-enriched and lymphocyte-enriched fractions of peripheral blood from three healthy volunteers were obtained by percoll density gradient centrifugation. The cytotoxic activity of each fraction against 51Cr-labelled K562 cells was quantified in a 2-h assay using freshly isolated cells of each fraction and cells of each fraction which had been incubated with and without lactoferrin in complete medium for 18 h before performing the assay. We have thereby shown that cytotoxicity was not demonstrable in the lymphocyte fraction (containing 7.3 +/- 2% large granular lymphocytes) after 18 h in medium, whereas the cytotoxicity of the monocyte fraction (containing 3 +/- 0.4% large granular lymphocytes) was still significantly increased (P less than or equal to 0.01) and that lactoferrin had no effect on lymphocyte fraction cytotoxicity while producing an 11-fold increase in the cytotoxicity of the monocyte fraction. It is therefore possible to perform a relatively simple test of monocyte cytotoxicity using lactoferrin as a stimulant in a 2-h 51Cr-labelled K562 assay system by allowing 18 h to elapse for lymphocyte natural killer cytotoxicity to decay.     

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

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

FL-CTL assay: fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells

Cytotoxic T lymphocytes (CTLs) are crucial effectors against intracellular pathogens and cancer. Accurate and efficient assessment of CTL activity is important for basic and clinical studies. Widely used CTL assays, including the chromium release, JAM test and ELISPOT, involve either radioisotopes or lengthy procedures. Here, we developed a new fluorolysometric CTL assay based on cell-mediated cytolysis of fluorescent protein (GFP or DsRed) expressing cells quantified by one of the fluoro-based methods: flow cytometry, fluorescence microplate reader, or fluorescence microscopy. With flexible detection methods and lentiviral vector transduced stable lines of either GFP⁺ or DsRed⁺ cells as targets for antigen presentation and equal number of the other as internal reference for consistency and accuracy, this assay is easy to perform and to scale-up for simultaneous multi-sample analyses. Using two different antigen systems, we demonstrated that this assay is very sensitive to determine primary CTL activity of both in vitro and in vivo primed antigen-specific T cells. Thus, this FL-CTL assay is highly sensitive, reliable, reproducible, economical, convenience and supports broad applications compared to conventional CTL assays.     

冷冻预处理温度对胎兔许旺细胞活性的影响

背景：许旺细胞作为周围神经损伤修复的重要桥接材料,其活性对临床修复周围神经损伤成功与否具有重要影响。 目的：观察不同维持温度冷冻预处理超深低温保存对胎兔许旺细胞活性的影响。 方法：设计胎兔许旺细胞冷冻维持温度为-30~-70 ℃,每间隔-5 ℃为一组,以10%二甲基亚砜为低温保护剂,对胎兔许旺细胞进行上述温度冷冻预处理,液氮中保存48 h,快速复温后继续培养48 h,透射电镜观察细胞超微结构改变,MTT法和³H-TdR掺入法检测细胞活性。 结果与结论：透射电镜下见-45 ℃处理的细胞超微结构良好,其余各温度组细胞均出现细胞器肿胀、坏死等轻重不同的冷冻损伤表现。MTT法和³H-TdR检测结果均显示-45 ℃冷冻预处理对许旺细胞的生长抑制率最低,分别为(3.83±0.56)%、(4.41±0.71)%,和其余各温度组之间相比,差异有显著性意义(P < 0.05)。提示-45 ℃是胎兔许旺细胞冷冻预处理的最佳维持温度值,对细胞活性影响最小,可较好保持其生物学活性。     

Concomitant presence of anti-tumor effector cells and suppressor cells in the spleen of tumor-bearing mice : the nature of suppressor cells

In order to investigate the immunological responsiveness of tumor-bearing hosts to tumor cells, splenic suppressor cells from Ehrlich tumor-bearing mice that inhibited anti-tumor effector cell activity were characterized. In vitro cell-mediated cytoxicity and cytostasis assays were performed to test for the existence of anti-tumor immunity. suppressive activity assayed by cell mixture experiments became apparent with decline of anti-tumor immunity and progressive tumor growth. The cells mediating the suppression were found to be nylon wool column adherent T cells and inhibited T cell dependent cytotoxicity rather than non-T cell dependent cytostasis. In vivo cell transfer experiments demonstrated that intravenous injection of suppressor cells to a host already inoculated with tumor cells mixed with antitumor effector cells resulted in significant enhancement of tumor growth. This inhibition of in vivo neutralization assay be suppressor cells was found in not only allogeneic but also syngeneic tumor system. Splenectomy at the time of tumor resection endowed the host with stronger resistance against subsequent reinoculated tumor than sham-splenectomy did, reflected by prolonged survival times. These results suggest that splenectomy combined with surgical removal of the tumor is a useful treatment of clinical malignancies.     

Potent Inhibition of Interleukin 1β-Mediated Human Melanoma (A375.6) Lysis by Corticosteroids, Staurosporine, and Tilorone

The mechanism of human interleukin (IL)-1 β-mediated cytolysis was studied in a human melanoma cell line, A375.6. Purified recombinant human IL-1β produced 50% cytocidal activity at 50 pg/ml. A variety of compounds were tested for their ability to interfere with A375.6 lysis. Compounds were added simultaneously with IL-1β (100 pg/ml), and tumor cytolysis was measured after 72 hr of culture by release of ¹²⁵I from DNA of A375.6 cells labeled with [¹²⁵I]-dUrd. A variety of antiinflammatory/immunosuppressive agents (including auranofin, chloroquine, cyclosporin A, d-penicillamine) and several cyclo-oxygenase/lipoxygenase inhibitors (AA-861, BW755c, and indomethacin) lacked protective activity. Similarly, phospholipase inhibitors (mepacrine and 4-bromophenacyl bromide), putrescine, inhibitors of lysosomal activity (chloroquine and NH4CI), calcium channel blockers (nifedipine and verapamil), calmodulin inhibitors (W-7 and calmidazolium), and inhibitors of ADP ribosylation (nicotinamide and 3-aminobenzamide) were inactive. In contrast, corticosteroids (dexamethasone, hydrocortisone, and paramethasone acetate), tilorone, and protein kinase C inhibitors (1-[5-isoquinolinyl-sulfonyl]-2-methylpiperazine and stauro-sporine) significantly inhibited IL-1 β-mediated A375.6 cytolysis. These compounds also interfered with tumor necrosis factor-mediated lysis of A375.6, suggesting common mechanisms of tumor cytotoxicity by these monokines. This model may be useful for delineating intracellular biochemical events integral to IL-1 action.