Beyond 51Cr release: New methods for assessing HIV-1-specific CD8+ T cell responses in peripheral blood and mucosal tissues

Abstract

Much scientific effort has been directed towards elucidating the complexities of cell-mediated immune responses to HIV-1(reviewed in [1,2]). These studies have attempted to explain the immune system's ultimate failure to contain viral replication, leading to development of AIDS disease, and to identify immune responses that will be useful in developing immunomodulatory therapies and novel vaccine strategies. Although many of the complex interactions involved in AIDS pathogenesis remain unsolved, great progress has been made in characterizing the kinetics, specificity and functional dynamics of HIV-1-specific T cell responses. These investigations have come at a time when advances in virology, cellular immunology and molecular biology have converged to provide a variety of methodological approaches not available at the onset of the AIDS pandemic. Application of these tools to other infectious diseases and immunopathological conditions will provide a fertile area of research for future years. This review focuses on recent developments in the assessment of HIV-1-specific T cell responses in peripheral blood and tissues, with a particular emphasis on flow cytometry-based approaches.

Fig. 1

Effector functions of CD8⁺ T cells. As described in the text, CD8⁺ T cells perform MHC class I restricted lysis of target cells. Cytotoxicity may be mediated by exocytosis of cytotoxic granules containing perforin and granzymes (shown in figure), or by Fas/Fas ligand or TNF/TNFR interactions (not shown). In addition, CD8⁺ T cells secrete soluble factors including cytokines (IL-2, IFN-γ, TNF-α and others) and chemokines (MIP-1α, -β, RANTES and others). Chemokines may be a component of cytotoxic granules.

Fig. 2

Methods for assessing CD8⁺ T cells. (a)–(c): Cytotoxicity assays. (a) Chromium release assay. Radiolabelled target cells (T) are mixed with effector cells (E) at a predefined ratio (E : T ratio). When lysed, target cells release ⁵¹Cr, which is detected by scintillation counting. (b) The FATAL assay. Target cells are labelled with a membrane dye, PKH-26, and a cytoplasmic stain, CFSE. After incubation with effector cells, the percent specific lysis is determined by monitoring loss of fluorescent target cells by flow cytometry. (c) The FCC assay. Target cells are loaded with a fluorescent caspase substrate. Induction of apoptosis leads to caspase activation and cleavage of the fluorescent substrate, and the resultant signal is measured by flow cytometry. (d) Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS). CFC detects cytokine that has accumulated within antigen-stimulated cells following treatment with an inhibitor of vesicular protein transport (i.e. Brefeldin A or Monensin). To detect intracellular cytokine, the cells are permeabilized and incubated with fluorescently labelled monoclonal antibodies. (e) The ELISPOT assay. ELISPOT detects cytokine released by antigen-stimulated T cells in a culture plate previously coated with specific antibody. Cells may be stimulated with recombinant poxviruses, peptides, proteins or viral lysates. After overnight incubation, cells are removed. Antibody-bound cytokine is detected by an enzyme-linked secondary antibody. Antigen-specific cells are enumerated by counting cytokine ‘footprints’. (f) Surface staining with MHC class I complexes. Two configurations are used commonly to link MHC class I monomers. Four monomers may be linked to biotin and joined via streptavidin, which has four biotin binding sites (top). Alternatively, two monomers may be linked via mouse IgG1 (bottom). Tetrameric complexes are directly labelled with fluorochromes; dimer-Ig complexes are detected with fluorochrome-labelled anti-mouse antibodies.

Fig. 2

Methods for assessing CD8⁺ T cells. (a)–(c): Cytotoxicity assays. (a) Chromium release assay. Radiolabelled target cells (T) are mixed with effector cells (E) at a predefined ratio (E : T ratio). When lysed, target cells release ⁵¹Cr, which is detected by scintillation counting. (b) The FATAL assay. Target cells are labelled with a membrane dye, PKH-26, and a cytoplasmic stain, CFSE. After incubation with effector cells, the percent specific lysis is determined by monitoring loss of fluorescent target cells by flow cytometry. (c) The FCC assay. Target cells are loaded with a fluorescent caspase substrate. Induction of apoptosis leads to caspase activation and cleavage of the fluorescent substrate, and the resultant signal is measured by flow cytometry. (d) Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS). CFC detects cytokine that has accumulated within antigen-stimulated cells following treatment with an inhibitor of vesicular protein transport (i.e. Brefeldin A or Monensin). To detect intracellular cytokine, the cells are permeabilized and incubated with fluorescently labelled monoclonal antibodies. (e) The ELISPOT assay. ELISPOT detects cytokine released by antigen-stimulated T cells in a culture plate previously coated with specific antibody. Cells may be stimulated with recombinant poxviruses, peptides, proteins or viral lysates. After overnight incubation, cells are removed. Antibody-bound cytokine is detected by an enzyme-linked secondary antibody. Antigen-specific cells are enumerated by counting cytokine ‘footprints’. (f) Surface staining with MHC class I complexes. Two configurations are used commonly to link MHC class I monomers. Four monomers may be linked to biotin and joined via streptavidin, which has four biotin binding sites (top). Alternatively, two monomers may be linked via mouse IgG1 (bottom). Tetrameric complexes are directly labelled with fluorochromes; dimer-Ig complexes are detected with fluorochrome-labelled anti-mouse antibodies.

Fig. 2

Methods for assessing CD8⁺ T cells. (a)–(c): Cytotoxicity assays. (a) Chromium release assay. Radiolabelled target cells (T) are mixed with effector cells (E) at a predefined ratio (E : T ratio). When lysed, target cells release ⁵¹Cr, which is detected by scintillation counting. (b) The FATAL assay. Target cells are labelled with a membrane dye, PKH-26, and a cytoplasmic stain, CFSE. After incubation with effector cells, the percent specific lysis is determined by monitoring loss of fluorescent target cells by flow cytometry. (c) The FCC assay. Target cells are loaded with a fluorescent caspase substrate. Induction of apoptosis leads to caspase activation and cleavage of the fluorescent substrate, and the resultant signal is measured by flow cytometry. (d) Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS). CFC detects cytokine that has accumulated within antigen-stimulated cells following treatment with an inhibitor of vesicular protein transport (i.e. Brefeldin A or Monensin). To detect intracellular cytokine, the cells are permeabilized and incubated with fluorescently labelled monoclonal antibodies. (e) The ELISPOT assay. ELISPOT detects cytokine released by antigen-stimulated T cells in a culture plate previously coated with specific antibody. Cells may be stimulated with recombinant poxviruses, peptides, proteins or viral lysates. After overnight incubation, cells are removed. Antibody-bound cytokine is detected by an enzyme-linked secondary antibody. Antigen-specific cells are enumerated by counting cytokine ‘footprints’. (f) Surface staining with MHC class I complexes. Two configurations are used commonly to link MHC class I monomers. Four monomers may be linked to biotin and joined via streptavidin, which has four biotin binding sites (top). Alternatively, two monomers may be linked via mouse IgG1 (bottom). Tetrameric complexes are directly labelled with fluorochromes; dimer-Ig complexes are detected with fluorochrome-labelled anti-mouse antibodies.

Fig. 3

Excitation and emission spectra of fluorescent dyes used for multi-color flow cytometry. Shown are the excitation and emission spectra for dyes used in multi-colour flow cytometry. Also depicted are commonly used bandpass filters through which the emitted light is passed prior to collection on photomultiplier tubes. Lasers are listed on the far right. Currently up to 11 dyes can be used simultaneously. Abbreviations used are: CasB, cascade blue; CasY, cascade yellow; FITC, fluorescein isothiocyanate; PE, phycoerythrin; TR, Texas red; Cy, cychrome; APC, allophycocyanin. *Due to the spectral overlap of Texas red-PE (TR-PE), the use of this dye in combination with TR, excited by a dye laser tuned to give a 595-nm line, is problematic. Use of other lasers exciting in the red (for example 632 nm HeNe, also 647 nm Krypton and 625 nm diode lasers) in combination with a 488-nm Krypton laser do allow use of TR-PE in combination with all other dyes. However, TR is not excited by these lasers. —, excitation; emission; , bandpass filter.

Fig. 4

TCR clonotype analysis. (a) As described by Douek et al. [76], antigen-specific cells were stimulated with peptide. Cytokine-producing cells were stained using a ‘cytokine capture’ method in which released IFN-γ is captured at the cell surface by a bispecific reagent recognizing CD45 and IFN-γ. The cells were then stained with a phycoerythrin (PE)-labelled detection antibody and viably sorted on the basis of PE fluorescence. B. RNA extracted from viable sorted cells was subjected to RT-PCR and sequencing of the TCRB locus. (c) Fluorescent probes were designed to identify unique VDJ rearrangements. Individual clones were tracked in longitudinal samples using clonotype-specific quantitative real-time PCR (qPCR).