.2
 Detection of Antigen-spe

.2
 Detection of Antigen-specific T Cells
It is indisputable that improvement of vaccination strategies often requires stronger in vivo induction of antigen-specific effector and memory T cells or, for therapeutic vaccination, modulation of already existing antigen-specific T cell populations. In- deed, many vaccination protocols and reagents have been developed over the past decades with the specific goal of improving T cell immunogenicity. However, im- provement of T cell-based vaccination is dependent on accurate monitoring of the success of Tcell vaccination and correlation of vaccine-induced Tcell status with the quality of protection. The presence and frequencies of antigen-specific Tcells in vivo can be detected by ‘function-dependent’ and ‘function-independent’ methods. Until recently, the limit- ing dilution assay (LDA) was the only technique available for determination of anti- gen-specific Tcell frequencies [4]. LDA is based on sequential dilution andin vitro ex- pansion of cell samples in 96-well plates, followed by functional assays (such as pro- liferation or cytotoxicity assays) to ascertain the number of wells containing antigen- specific cell lines or clones. Recent studies using more advanced methods for ex vivo T cell analysis demonstrated that the numbers obtained by LDA assays often sub- stantially underestimated the true in vivo T cell frequencies [5–7]. The development of assays that require only very brief in vitro restimulation, such as the enzyme- linked immunospot assay (ELISPOT) [8] and intracellular cytokine staining [5], con- siderably improved determination of T cell frequencies (Figures 5.1 and 5.2). In these assay systems, T cells responding specifically to antigen are detected through their ability to rapidly produce effector cytokines such as IFN , TNF , IL-2, or IL-4. Cytokine capture is achieved by using an affinity matrix,which can be established ex- tracellularly (e.g., by using nitrocellulose, ELISPOT) or on the cell surface (a so- called cytokine capture assay, CCA [9]). Another method, intracellular cytokine stain- ing (ICS), is based on accumulation of cytokines within the cell by blocking the se- cretion apparatus (e.g., in the presence of brefeldin A or monensin) and subsequent intracellular staining for the cytokine of interest (Figure 5.1). All these assays have proven to be very sensitive, allowing ex vivo detection of antigen-specific Tcell popu- lations with frequencies as low as 0.02%. One advantage of the ELISPOT assay is that relatively little cell material is needed in comparison to flow cytometry-based de- tection systems (CCA, ICS). However, flow cytometry allows simultaneous staining of different cytokines and surface antigens, providing a more precise phenotypic analysis of the responding cell populations. Multiparameter ELISPOTassays have re- cently been developed to compensate for some of the limitations of this method [10]. The CCA keeps cells alive, and its combination with cell separation techniques such as FACS or sorting with paramagnetic beads allows purification of antigen-specific Tcells for further analysis of the cell population. All these assays detect antigen-spe- cific Tcells based on an effector function – rapid production of cytokines in response to in vitro restimulation with antigen. Only T cells capable of responding with the readout effector function under the chosen in vitro restimulation conditions are de- tected. Because antigen-specific T cell populations under in vivo conditions seem t

Kết quả (Việt) 1:

.2 phát hiện tế bào kháng nguyên cụ thể TIt is indisputable that improvement of vaccination strategies often requires stronger in vivo induction of antigen-specific effector and memory T cells or, for therapeutic vaccination, modulation of already existing antigen-specific T cell populations. In- deed, many vaccination protocols and reagents have been developed over the past decades with the specific goal of improving T cell immunogenicity. However, im- provement of T cell-based vaccination is dependent on accurate monitoring of the success of Tcell vaccination and correlation of vaccine-induced Tcell status with the quality of protection. The presence and frequencies of antigen-specific Tcells in vivo can be detected by ‘function-dependent’ and ‘function-independent’ methods. Until recently, the limit- ing dilution assay (LDA) was the only technique available for determination of anti- gen-specific Tcell frequencies [4]. LDA is based on sequential dilution andin vitro ex- pansion of cell samples in 96-well plates, followed by functional assays (such as pro- liferation or cytotoxicity assays) to ascertain the number of wells containing antigen- specific cell lines or clones. Recent studies using more advanced methods for ex vivo T cell analysis demonstrated that the numbers obtained by LDA assays often sub- stantially underestimated the true in vivo T cell frequencies [5–7]. The development of assays that require only very brief in vitro restimulation, such as the enzyme- linked immunospot assay (ELISPOT) [8] and intracellular cytokine staining [5], con- siderably improved determination of T cell frequencies (Figures 5.1 and 5.2). In these assay systems, T cells responding specifically to antigen are detected through their ability to rapidly produce effector cytokines such as IFN , TNF , IL-2, or IL-4. Cytokine capture is achieved by using an affinity matrix,which can be established ex- tracellularly (e.g., by using nitrocellulose, ELISPOT) or on the cell surface (a so- called cytokine capture assay, CCA [9]). Another method, intracellular cytokine stain- ing (ICS), is based on accumulation of cytokines within the cell by blocking the se- cretion apparatus (e.g., in the presence of brefeldin A or monensin) and subsequent intracellular staining for the cytokine of interest (Figure 5.1). All these assays have proven to be very sensitive, allowing ex vivo detection of antigen-specific Tcell popu- lations with frequencies as low as 0.02%. One advantage of the ELISPOT assay is that relatively little cell material is needed in comparison to flow cytometry-based de- tection systems (CCA, ICS). However, flow cytometry allows simultaneous staining of different cytokines and surface antigens, providing a more precise phenotypic analysis of the responding cell populations. Multiparameter ELISPOTassays have re- cently been developed to compensate for some of the limitations of this method [10]. The CCA keeps cells alive, and its combination with cell separation techniques such as FACS or sorting with paramagnetic beads allows purification of antigen-specific Tcells for further analysis of the cell population. All these assays detect antigen-spe- cific Tcells based on an effector function – rapid production of cytokines in response to in vitro restimulation with antigen. Only T cells capable of responding with the readout effector function under the chosen in vitro restimulation conditions are de- tected. Because antigen-specific T cell populations under in vivo conditions seem t