Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen‐Specific Cell‐Mediated Cytotoxicity

Baalen, Carel A. van; Kwa, David; Verschuren, Esther J.; Reedijk, Mariska L.; Boon, Adrianus C. M.; Mutsert, Gerrie de; Rimmelzwaan, Guus F.; Osterhaus, Albert D. M. E.; Gruters, Rob A.

doi:10.1086/444546

Cited by 25 publications

(34 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The open reading frames (ORF) of the M1 protein of influenza viruses A/Netherlands/018/1994 (H3N2) and A/Vietnam/1194/2004 (H5N1) without their stop codons were cloned in frame with the ORF of enhanced green fluorescent protein (eGFP) into the pEGFP-N1 plasmid (Becton Dickinson) as described previously (46). Next, the ORF of the NP 383-391 epitope (SRYWAIRTR), including 50 N-and C-terminal amino acids (all derived from A/Puerto Rico/8/1934), was cloned in frame between the ORFs of the M1 and eGFP protein (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The fluorescent antigen-transfected target cell-CTL (FATT-CTL) assay was used for the detection of lytic activity of the M1 58 -66 -and NP 383-391 -specific CD8 ϩ T cell clones as described previously (46). Briefly, the cell line Nucleofector kit V (Lonza), program T16, was used to transfect 5 ϫ Kinetics of CD8 ؉ T cell activation in the FATT-CTL assay.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Differential Recognition of Influenza A Viruses by M1_58–66Epitope-Specific CD8⁺T Cells Is Determined by Extraepitopic Amino Acid Residues

Sandt

Kreijtz

Geelhoed-Mieras

et al. 2016

J Virol

View full text Add to dashboard Cite

Natural influenza A virus infections elicit both virus-specific antibody and CD4؉ and CD8 ؉ T cell responses. Influenza A virusspecific CD8؉ cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M1 58 -66 ) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M1 58 -66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8 ؉ T cell recognition of the M1 58 -66 epitope. These data indicate that human influenza A viruses can impair recognition by M1 58 -66 -specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8؉ cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M1 58 -66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M1 58 -66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Differential Recognition of Influenza A Viruses by M1_58–66Epitope-Specific CD8⁺T Cells Is Determined by Extraepitopic Amino Acid Residues

Sandt

Kreijtz

Geelhoed-Mieras

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…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: 62%

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

et al. 2010

View full text Add to dashboard Cite

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.

show abstract

“…The virus suppression assay as described here has several advantages over the more traditional methods to study cellular immunity: it directly assesses antiviral activity rather than parameters that are associated with the antiviral capacity, like IFN-g production or cytolytic activity. Compared to the fluorescent antigen-transfected target cell-CTL described by Van Baalen et al [21] this assay is closer to the natural situation, since it is based on infection rather than transfection of APC. Consequently, viral antigens are processed and presented as they would be during natural measles in vivo, and antiviral capacity is measured against the entire virus instead of single proteins or peptides.…”

Section: Infectionmentioning

confidence: 99%

“…Recently, a functional assay to assess virus-specific T-lymphocyte-mediated killing was developed based on transfection of autologous target cells with a viral gene linked to GFP. This assay was referred to as the fluorescent antigen-transfected target cell-CTL assay [21]. We adapted this set-up to a more dynamic system, in which autologous target cells were infected with a recombinant MV strain expressing enhanced GFP (EGFP) [22] and co-cultured with MV-specific TCC.…”

mentioning

confidence: 99%

Specific CD8⁺ T‐lymphocytes control dissemination of measles virus

et al. 2010

View full text Add to dashboard Cite

Measles continues to be an important cause of childhood mortality in developing countries. Measles virus (MV) is lymphotropic and infects high percentages of B-and T-lymphocytes in lymphoid tissues. Cellular immunity is considered crucial for viral clearance; however, MV-specific T-lymphocytes generated during primary infection also constitute a potential target for MV infection. We therefore aimed to identify T-lymphocyte subsets that can clear MV infection without becoming infected. To this end, we infected human EBV transformed B-lymphoblastic cell lines (B-LCL) with a recombinant MV strain expressing enhanced GFP, and co-cultured these with non-infected B-LCL resulting in rapid viral spread. MV-specific CD8 1 T-cell clones efficiently suppressed MV dissemination in autologous and HLA-matched, but not in HLA-mismatched B-LCL. In contrast, CD41 T-cell clones could not control MV dissemination but became a target for MV infection themselves. Furthermore, PBMC collected 6-9 months after acute measles and stimulated with autologous MV-infected B-LCL also efficiently suppressed MV dissemination; this was mediated by the fraction containing CD8 1 T-lymphocytes. In conclusion, we have developed a powerful tool to study cellular immunity against measles, and demonstrate that control of MV dissemination is mediated by virus-specific CD8 1 rather than by CD4 1 T-lymphocytes.Key words: Clearance . Measles virus . Suppression . T-lymphocytes IntroductionDespite significant progress of global control programs, measles continues to be an important cause of morbidity and mortality in developing countries. Measles virus (MV) is a highly contagious agent that is transmitted by aerosols or direct contact with contaminated respiratory secretions. Measles starts with a prodromal phase of fever, cough and coryza, followed by a generalized maculopapular skin rash and conjunctivitis [1]. This phase is associated with a transient but profound immune suppression that can persist for months after convalescence, resulting in an increased susceptibility to opportunistic infections.Paradoxically, measles also induces strong MV-specific humoral and cellular immune responses, resulting in lifelong immunity [2]. Virus neutralizing serum antibodies are considered the main correlate of protection against MV infection [3]. However, viral clearance is predominantly mediated by cellular immune responses. This is illustrated by the fact that hypogammaglobulinemic children recover normally from MV infection [4,5], whereas children with deficits in cellular immunity develop severe disease and display prolonged viral shedding [4,6,7]. This could be partly due to the spread of MV via cell-cell fusion, thus potentially avoiding neutralizing antibodies in the bloodstream. Ex vivo experiments have shown that CD81 MV-specific Currently, most studies on antiviral cellular immunity focus on quantification of virus-specific T cells rather than on the functional capacity of these cells. These assays either assess lymphoproliferation (e.g. CFSE dilution assay...

show abstract

Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen‐Specific Cell‐Mediated Cytotoxicity

Cited by 25 publications

References 32 publications

Differential Recognition of Influenza A Viruses by M1_58–66Epitope-Specific CD8⁺T Cells Is Determined by Extraepitopic Amino Acid Residues

Differential Recognition of Influenza A Viruses by M1_58–66Epitope-Specific CD8⁺T Cells Is Determined by Extraepitopic Amino Acid Residues

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

Specific CD8⁺ T‐lymphocytes control dissemination of measles virus

Contact Info

Product

Resources

About

Fluorescent Antigen–Transfected Target Cell Cytotoxic T Lymphocyte Assay for Ex Vivo Detection of Antigen‐Specific Cell‐Mediated Cytotoxicity

Cited by 25 publications

References 32 publications

Differential Recognition of Influenza A Viruses by M158–66Epitope-Specific CD8+T Cells Is Determined by Extraepitopic Amino Acid Residues

Differential Recognition of Influenza A Viruses by M158–66Epitope-Specific CD8+T Cells Is Determined by Extraepitopic Amino Acid Residues

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

Specific CD8+ T‐lymphocytes control dissemination of measles virus

Contact Info

Product

Resources

About

Differential Recognition of Influenza A Viruses by M1_58–66Epitope-Specific CD8⁺T Cells Is Determined by Extraepitopic Amino Acid Residues

Differential Recognition of Influenza A Viruses by M1_58–66Epitope-Specific CD8⁺T Cells Is Determined by Extraepitopic Amino Acid Residues

Specific CD8⁺ T‐lymphocytes control dissemination of measles virus