Tetramers-ppt
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This document discusses the use of peptide-MHC (pMHC) tetramers to identify antigen-specific T cells. It begins by explaining how pMHC monomers are biotinylated and tetramerized with streptavidin to form stable complexes that can bind multiple T cell receptors. These fluorescent pMHC tetramers allow detection of antigen-specific T cells by flow cytometry. Later sections discuss how pMHC tetramers can be used to map T cell epitopes, monitor vaccine responses and disease-related T cell responses, and even track tumor-specific T cells in vivo. Challenges in staining low-affinity T cells are also addressed, such as using a secondary antibody to amplify fluorescence intensity from
Tetramers-ppt
- 2. Introduction 2 Figure 1. Model for the interaction of an MHC-peptide complexon one cell with a T cell receptor on another cell.
- 3. TCRs recognize and bind to complexes composed of MHC molecules and specific peptides expressed on the surface of antigen-presenting cells. antigen-specific T cells could be detected using soluble MHC/ peptide complexes, monomeric MHC/peptide complexes proved to be impractical due to their instability and low affinity to the TCR. To overcome this difficulty, MHC/ peptide monomers are biotinylated and tetramerized with streptavidin to maintain stable binding to multiple TCR, enabling MHC/peptide Tetramers to be used as detection tools. MHC Tetramers are labeled with fluorescent molecules including phycoerythrin (PE) or allophycocyanin (APC) and thus allow detection of antigenspecific T cells by flow cytometry or fluorescence microscopy. 3
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- 5. 5 Fluorochrome-conjugated peptide–MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct exvivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell- surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II–restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs.
- 6. 6 to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome conjugated Abs to amplify fluorescence intensity. an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with phycoerythrin (PE) , allophycocyanin (APC), or FITC-based reagents.
- 7. 7 FIGURE 2. Schematic representation of the test and control conditions used in this study. Alongside a standard pMHC multimer (tetramer or dextramer) staining protocol (test 1), the binding of a mouse anti-fluorochrome unconjugated 1° Ab to the pMHC multimer associated fluorochrome followed by a goat anti-mouse conjugated 2° Ab (test 2).
- 8. Recombinant a and b Chains of MHC Class II Tetramers with Affinity-Tagged Peptides Targeting CD4+ T cells through their unique antigen- specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. 8
- 9. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses. 9
- 10. As an approach to the preparation of peptide-HLA class II monomers, the antigenic peptides were synthetically extended with a C-terminal H6 sequence, which subsequently allowed functional, monomeric peptide-HLA complexes to be concentrated and purified by IMAC chromatography. This raises the questionwhether HLA class II in general will accept this kind of extension. 10
- 11. A reasonable explanation of this apparent discrepancy is that the a and b chains were kept together by the leucine zipper in a soluble, but inactive, peptide-free form, and that this complex was bound to the Ni2+-IDA-matrix through the weak b chain interaction. In contrast, for the molecules found in the later eluting peak 2 , the presence of a and b chains in a 1:1 stoichiometry, and of L243 staining. demonstrated the presence of properly folded monomer. 11
- 12. Applications of tetramers 1. T cell epitope identification 2. Monitoring vaccine studies 3. Monitoring effect of Immunotherapy 4. Monitoring T-cell responses in infectious disease, cancer, autoimmunity and other malignancies 5. Detection of antigen-specific T-cell responses in immunological studies 12
- 13. Epitope mapping Epitope identification using a tetramer guided mapping strategy. Large peptide libraries, separated into sets of pooled sequences, are loaded into the binding groove of recombinant class II molecules and assembled into tetramers. Direct flow cytometry analysis of T cell populations identifies peptides containing actual epitopes representative of the natural immune response . 13
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- 15. MHC tetramers assay for analysis of virus- specific T cells MHC tetramer production (A) Soluble MHC I heavy chain tagged at the carboxyl terminus with BirA Substrate Peptide (BSP) is expressed in E. coli. (B) The heavy chain is refolded with β2-microglobulin and the specific peptide. This refolded MHC monomer is biotinylated with the BirA enzyme at BSP site. (C) A tetramer is assembled from four MHC–biotin complexes through biotin-streptavidin interaction. The streptavidin is conjugated to a fluorochrome that allows it to be detected on the flow cytometer. (D) Tetramer when added to the lymphocytes binds to specific T-cell receptors on CD8 T cells, and with the anti-CD8 antibody the antigen-specific T cell population can be identified. 15
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- 17. Tetramer profiling of low-avidity T cells in autoimmunity class II pMHC tetramers containing autoantigenic specificities Have been used to study T cell responses in a variety of diseases, including type 1 diabetes (T1D), celiac disease, pemphigus vulgaris, rheumatoid arthritis, multiple sclerosis, and uveitis. tetramer analyses in the infectious pathogen and allergen studies cited above, in which most of the responding T cells have a high-avidity binding to appropriate pMHC tetramers, tetramer binding to peripheral T cells in the context of autoimmunity displays a much wider spectrum of relative strength of interaction. 17
- 18. In vivo tracking of tumor-specific T cells The intensity of tetramer staining can also be correlated with TCR affinity, which may be critical when vaccination strategies to induce high-affinity tumor-reactive T cells are evaluated. Furthermore, following sorting of T cells, TCR usage or cDNA microarray analysis of specific T cells can be performed Recently, the addition of confocal laser scanning microscopy to tetramer analysis has enabled precise anatomic localization of antigen-specific T cells in tissues. 18
- 19. Reference 1-cellular and molecular immunology, 6th ed, 2007 2-MHC Class II Tetramers. Gerald T. Nepom. doi:10.4049/jimmunol.1102398. J Immunol 2012; 188: 3-Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs. The Journal of Immunology, 2015, 194: 463–474. 4-MHC Class II Tetramers Made from Isolated Recombinant a and b Chains Refolded with Affinity-Tagged Peptides. Francesco Dieli, University of Palermo, Italy. June 3, 2013; Accepted July 22, 2013; Published September 2, 2013 5-MHC tetramers assay for analysis of virus-specific T cells.Pervaiz A. Dar and Sabrin H. Beituni 6- Class II major histocompatibility complex tetramer staining: progress, problems, and prospects. Sabrina S. Vollers and Lawrence J. Stern. Received 5 December 2007; revised 10, 1 December 2007; accepted 13 December 2007. 7- In vivo tracking of tumor-specific T cells. Cassian Yee, Stanley R Riddell and Philip D Greenberg 19