Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species

Piriou, Laurence; Chilmonczyk, Stéfan; Genetet, N.; Albina, Emmanuel

doi:10.1002/1097-0320(20001201)41:4<289::aid-cyto7>3.0.co;2-5

Cited by 89 publications

(52 citation statements)

References 30 publications

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“…The permanent expression of CSFV antigens was confirmed by labeling with an anti-CSFV monoclonal antibody (PPL 282C10A, kindly provided by Merial, France) and subsequent analysis by flow cytometry. CTL activity was measured by flow cytometry as described by Piriou et al [18]. Briefly, this assay is based on a dual fluorescent staining of target cells.…”

Section: Studies Of Csf-specific Cytotoxic T Lymphocytesmentioning

confidence: 99%

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

et al. 2003

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-A better understanding of cell-mediated immune responses to classical swine fever virus (CSFV) is essential for the future development of improved vaccines. We analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination. Viral infection induced high T cell responses with high primary and secondary CTL activity correlated with high IFN-g production, whereas vaccination with a live vaccine followed by infection mainly induced neutralizing antibody but low cell-mediated responses. Moreover, high IgG1 response was associated with high IFN-g response following infection whereas a weak IFN-g response was related to a good IgG2 response but a low IgG1 production. These data could reflect Th1/Th2-like balance of immune responses depending upon immunization protocols, which has not yet been described in the pig. T-cell responses to CSFV were evidenced by CSFV-specific CD25 upregulation on CD4 -CD8 + , but not on CD4 + CD8 -cells, which further illustrated the importance of CTL responses after infection. Our results indicated that generation of cell-mediated immune responses was much higher following intranasal/oral CSFV infection than after intramuscular vaccination, which implies that the capacity of new CSFV vaccines to induce higher T-cell responses should be considered. immunity / cytotoxicity / CSFV / d/d histocompatible pig

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Section: Studies Of Csf-specific Cytotoxic T Lymphocytesmentioning

confidence: 99%

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

et al. 2003

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“…values for cytolytic activity and simultaneously the evaluation of other functional and effector functions in samples. The present work expands on previously published flow-cytometrybased cytotoxicity assays (3,5,6,8À14) in two significant ways by demonstrating the methodological approach and potential to quantify cytolytic activity and the significant potential and advantage for multiplexing the FBCA assay with other platforms to obtain more comprehensive and integrated datasets.…”

Section: Discussionmentioning

confidence: 61%

Development and application of a multiplexable flow cytometry‐based assay to quantify cell‐mediated cytolysis

et al. 2010

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Although target cell cytolysis has been widely employed to describe effector function of cells, cytolysis assays as commonly employed do not generate quantitative data. In this report we describe the development and application of a statistically supported flow cytometry-based assay to quantify cell-mediated cytolysis. The assay depends on the use of the fluorescent dye CFSE to distinguish target from effector cells, the DNA intercalating dye 7AAD to distinguish dead from live cell events, and on the establishment of a cytolysis curve that allows for the derivation of statistically robust data. We demonstrate that the cytolysis curve is well described by a four parameter logistic regression model provided that (i) the range of effector to target (E:T) ratios studied allows for full description of the logistic curve, and (ii) an adequate number of data points are collected to estimate the model parameters. We show that the assay is highly reproducible and accurate, and comparable in sensitivity with the standard 51 Cr assay. We report on the potential for this assay to generate quantitative data on the cytolytic activity of both CD8 T and NK cells; describe a relationship between the efficiency of effector cell degranulation and target cell cytolysis throughout a range of E:T ratios, and demonstrate the potential to multiplex with other platforms to obtain broader datasets for the effector phenotype of cells. Appropriate use of this assay will enhance the ability to derive quantitative and integrated correlative datasets from basic, translational, and clinical studies. ' The most commonly employed assay to measure target cell lysis is the 51 Cr release assay (1). Although the 51 Cr release assay has enjoyed wide use and has provided useful data in a large number of clinical and basic research studies, the assay suffers from certain intrinsic limitations. Specifically, (i) the use of a radioactive element ( 51 Cr) with attendant issues of licensing, isotope half-life, storage, disposal, and compliance, (ii) the requirement for metabolic labeling, since metabolically slow cells can label very poorly, and cells at different metabolic states may be differentially resistant to lysis, and (iii) despite the fact that cytolysis is reported in the context of effector:target cell ratios (E:T), the read-out from 51 Cr-based assays is not based on a per-cell measurement but rather on bulk release of radioactivity into the culture medium.

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“…The median duration of infection in the patients at inclusion in the study was 11 years (range, [7][8][9][10][11][12][13][14][15][16][17][18][19]. The median viral load was 3,100 copies/ml (range, 333) and the median CD4 T-cell count was 639 cells/ll (range, 469-1,126).…”

Section: Materials and Methods Study Subjectsmentioning

confidence: 99%

“…Several flow cytometric assays for the assessment of NK-cell cytolysis (11)(12)(13)(14)(15)(16)(17)(18)(19) and a handful assays for the assessment of CD8 1 T-cell mediated cytotoxicity (18,(20)(21)(22)(23) have been described. In these assays different approaches to discriminate target cells from effector cells have been used, including scatter characteristics (24,25), monoclonal antibodies (15,20), and membrane-binding dyes (14,16,18,19,21). In our experience, the major problem with membrane-binding dyes is their spectral overlap with the vital fluorochromes used for the detection of cell death.…”

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confidence: 99%

A novel assay for assessment of HIV‐specific cytotoxicity by multiparameter flow cytometry

et al. 2005

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BackgroundAssessment of CD8+ T‐cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV‐specific cytotoxicity by multiparameter flow cytometry.MethodsTarget cells, pulsed with peptide pools (Gag or Nef), were stained with 5‐ (and –6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSEhiPI−) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The 51Cr‐release assay and IFN‐γ ELISpot were performed by standard procedures.ResultsA comparison with the Cr‐release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01).ConclusionThis new assay provides both specific and sensitive results when employed for the detection of HIV‐specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV‐infected subjects, especially if such responses are present at low levels. © 2005 Wiley‐Liss, Inc.

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Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species

Cited by 89 publications

References 30 publications

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

Development and application of a multiplexable flow cytometry‐based assay to quantify cell‐mediated cytolysis

A novel assay for assessment of HIV‐specific cytotoxicity by multiparameter flow cytometry

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