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Measuring Cellular Immunity to Influenza: Methods of Detection, Applications and Challenges.

Coughlan L, Lambe T - Vaccines (Basel) (2015)

Bottom Line: Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza.However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome.Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX1 7DQ, UK. lynda.coughlan@ndm.ox.ac.uk.

ABSTRACT
Influenza A virus is a respiratory pathogen which causes both seasonal epidemics and occasional pandemics; infection continues to be a significant cause of mortality worldwide. Current influenza vaccines principally stimulate humoral immune responses that are largely directed towards the variant surface antigens of influenza. Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza. Vaccination approaches targeting conserved viral antigens have the potential to provide broadly cross-reactive, heterosubtypic immunity to diverse influenza viruses. However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome. Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs. T-cell directed immunity has long been known to play a role in ameliorating the severity and/or duration of influenza infection, but the precise phenotype, magnitude and longevity of the requisite protective response is unclear. In order to progress the development of universal influenza vaccines, it is critical to standardize assays across sites to facilitate direct comparisons between clinical trials.

No MeSH data available.


Related in: MedlinePlus

Tetramers increase avidity for epitope specific TCR interactions. (A) Schematic showing the recognition of peptide loaded biotinylated MHC I by Ag-specific TCR. Individual molecules have weak binding and cannot be stained; (B) Tetramerization of MHC I subunits is achieved by addition of fluorescently labelled streptavidin which interacts with biotin. Tetramerization increases avidity, TCR specificity and allows detection of Ag-specific T-cells by flow cytometry. Adapted from Klenerman and colleagues [84].
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vaccines-03-00293-f002: Tetramers increase avidity for epitope specific TCR interactions. (A) Schematic showing the recognition of peptide loaded biotinylated MHC I by Ag-specific TCR. Individual molecules have weak binding and cannot be stained; (B) Tetramerization of MHC I subunits is achieved by addition of fluorescently labelled streptavidin which interacts with biotin. Tetramerization increases avidity, TCR specificity and allows detection of Ag-specific T-cells by flow cytometry. Adapted from Klenerman and colleagues [84].

Mentions: Tetramers (pentamers, hexamers or dextramers are also available) are synthetic MHC complexes made up of four or more identical versions of HLA molecules held together by non-covalent interactions (Figure 2). Each subunit is biotinylated, and tetramerization is facilitated by conjugation to a fluorescently-labelled streptavidin molecule [84]. These homo-tetrameric complexes are subsequently loaded with an antigen-specific peptide, allowing them to specifically identify and label CD8+ T-cells which express T-cell receptors (TCRs) specific for that peptide-MHC complex. The tetrameric nature of these complexes increases the affinity of the interaction between MHC I-TCR due to their ability to bind up to three separate TCRs simultaneously. Steric constraints can affect the ability to engage all four peptide complexes [85]. Specialised tubes, such as BD TruCOUNTTM tubes, can facilitate the accurate enumeration of absolute leucocyte counts and following analysis using flow cytometry, antigen-specific responses can be quantified as a percentage of total CD8+ T-cells. Although, it is important to note that not all tetramer-positive cells represent functional antigen-specific T-cells. Tetramer assays are specific and sensitive and can be combined with other functional assays thus facilitating the enrichment or isolation of rare antigen-specific T-cell populations. Indeed, cloning of low frequency antigen-specific T-cells has been demonstrated in the past using HLA-A2.1-restricted CTL clones recognising the influenza matrix protein peptide 58–66 [86,87]. In addition, tetramer assays have been shown to have minimal intra-assay variation, better precision and linearity than ICS or ELISPOTs performed using frozen PBMCs from the same donors [88].


Measuring Cellular Immunity to Influenza: Methods of Detection, Applications and Challenges.

Coughlan L, Lambe T - Vaccines (Basel) (2015)

Tetramers increase avidity for epitope specific TCR interactions. (A) Schematic showing the recognition of peptide loaded biotinylated MHC I by Ag-specific TCR. Individual molecules have weak binding and cannot be stained; (B) Tetramerization of MHC I subunits is achieved by addition of fluorescently labelled streptavidin which interacts with biotin. Tetramerization increases avidity, TCR specificity and allows detection of Ag-specific T-cells by flow cytometry. Adapted from Klenerman and colleagues [84].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494351&req=5

vaccines-03-00293-f002: Tetramers increase avidity for epitope specific TCR interactions. (A) Schematic showing the recognition of peptide loaded biotinylated MHC I by Ag-specific TCR. Individual molecules have weak binding and cannot be stained; (B) Tetramerization of MHC I subunits is achieved by addition of fluorescently labelled streptavidin which interacts with biotin. Tetramerization increases avidity, TCR specificity and allows detection of Ag-specific T-cells by flow cytometry. Adapted from Klenerman and colleagues [84].
Mentions: Tetramers (pentamers, hexamers or dextramers are also available) are synthetic MHC complexes made up of four or more identical versions of HLA molecules held together by non-covalent interactions (Figure 2). Each subunit is biotinylated, and tetramerization is facilitated by conjugation to a fluorescently-labelled streptavidin molecule [84]. These homo-tetrameric complexes are subsequently loaded with an antigen-specific peptide, allowing them to specifically identify and label CD8+ T-cells which express T-cell receptors (TCRs) specific for that peptide-MHC complex. The tetrameric nature of these complexes increases the affinity of the interaction between MHC I-TCR due to their ability to bind up to three separate TCRs simultaneously. Steric constraints can affect the ability to engage all four peptide complexes [85]. Specialised tubes, such as BD TruCOUNTTM tubes, can facilitate the accurate enumeration of absolute leucocyte counts and following analysis using flow cytometry, antigen-specific responses can be quantified as a percentage of total CD8+ T-cells. Although, it is important to note that not all tetramer-positive cells represent functional antigen-specific T-cells. Tetramer assays are specific and sensitive and can be combined with other functional assays thus facilitating the enrichment or isolation of rare antigen-specific T-cell populations. Indeed, cloning of low frequency antigen-specific T-cells has been demonstrated in the past using HLA-A2.1-restricted CTL clones recognising the influenza matrix protein peptide 58–66 [86,87]. In addition, tetramer assays have been shown to have minimal intra-assay variation, better precision and linearity than ICS or ELISPOTs performed using frozen PBMCs from the same donors [88].

Bottom Line: Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza.However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome.Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX1 7DQ, UK. lynda.coughlan@ndm.ox.ac.uk.

ABSTRACT
Influenza A virus is a respiratory pathogen which causes both seasonal epidemics and occasional pandemics; infection continues to be a significant cause of mortality worldwide. Current influenza vaccines principally stimulate humoral immune responses that are largely directed towards the variant surface antigens of influenza. Vaccination can result in an effective, albeit strain-specific antibody response and there is a need for vaccines that can provide superior, long-lasting immunity to influenza. Vaccination approaches targeting conserved viral antigens have the potential to provide broadly cross-reactive, heterosubtypic immunity to diverse influenza viruses. However, the field lacks consensus on the correlates of protection for cellular immunity in reducing severe influenza infection, transmission or disease outcome. Furthermore, unlike serological methods such as the standardized haemagglutination inhibition assay, there remains a large degree of variation in both the types of assays and method of reporting cellular outputs. T-cell directed immunity has long been known to play a role in ameliorating the severity and/or duration of influenza infection, but the precise phenotype, magnitude and longevity of the requisite protective response is unclear. In order to progress the development of universal influenza vaccines, it is critical to standardize assays across sites to facilitate direct comparisons between clinical trials.

No MeSH data available.


Related in: MedlinePlus