The greater pMHC valency and scaffold flexibility of dextramers compared to tetramers could also increase the cell surface interaction avidity, potentially leading to higher molecular occupancy. of human leucocyte antigen (HLA)-A2 monomer made up of epitopes from your BMLF-1259C267 protein of EpsteinCBarr computer virus (EBV) (GLCTLVAML) (a), MART-126C35 (ELAGIGILTV; heteroclitic amino acid shown in strong) (b) and preproinsulin (PPI)15C24 (ALWGPDPAAA) (c). Dextramers and tetramers made up of the PPI epitope were used to stain peripheral blood mononuclear cells (PBMC) directly from HLA-A2+ patients with type 1 diabetes (T1Ds) (c), whereas all other PBMC was sourced from HLA-A2+ non-TID donors (a,b). The percentage of dextramer or tetramer-positive cells of CD8+ T cells and the mean fluorescence intensity Baloxavir marboxil (MFI) of this population is displayed in strong. The gates were set on: 1, lymphocytes; 2, single cells; 3, amine-CD3+CD14CCD19C cells. The subsequent gate was set Baloxavir marboxil on CD8 dextramer and tetramer and events displayed using large dots. cei0177-0047-SD2.eps (1.8M) GUID:?96A71C9E-8CCC-4271-868F-B3D986DC2646 Fig. S3. High-resolution version of Fig. 1a. cei0177-0047-SD3.pdf (950K) GUID:?5BDB1882-A641-4977-B220-52348955CF35 Abstract Fluorochrome-conjugated peptideCmajor histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidinCbiotin-based tetramers, can be manufactured readily in the laboratory. Unfortunately, you will find large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from answer and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter transporting greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCRCpMHC affinity is usually low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art circulation cytometry platforms and will probably find particular power in the fields of autoimmunity and malignancy immunology. by using a spectrum of fluorochrome-conjugated antibodies specific for other T cell markers. More recent improvements have expanded Baloxavir marboxil possibilities by using heavy atom-conjugated pMHC multimers and antibodies in combination with mass spectrometry 3. The success of pMHC multimers is demonstrated by their use in thousands of published studies and the commercialization of several different pMHC multimerization platforms 1. The original such platform, an avidinCbiotin-based tetramer 4, is still the most common format in use, as it can be readily manufactured in laboratories equipped with basic protein expression and purification facilities. However, several other multimerization strategies are now available to investigators, including pMHC pentamers and octamers 1. Dextran-based pMHC multimers (dextramers) are a more recent addition to this toolbox 5. We have determined previously that the binding affinity threshold for pMHC class I (pMHC-I) tetramers is significantly higher than that required for T cell activation 6. As a result, pMHC-I tetramers can often fail to stain antigen-specific T cells where the interaction between pMHC and T cell receptor (TCR) is weaker than KD = 80 M. Such weak TCRCpMHC affinities are not usually characteristic of CD8+ T cells specific for foreign, pathogen-derived antigens, and pMHC-I tetramers have excelled when used to characterize virus-specific cytotoxic T lymphocyte (CTL) populations. In contrast, the use of pMHC-I tetramers can be more problematic when the reagents are used to identify T cells specific for self-derived peptides (anti-tumour and autoimmune T cells). Self-reactive T cells are known to generally bear weaker binding TCRs 7. This is thought to be the result of thymic editing that culls T cells bearing higher-affinity self-reactive TCRs 7. Thus, at present, pMHC-I tetramers cannot be Rabbit Polyclonal to NCAPG2 used to detect all antigen-specific CD8+ T cells 1,6. There are further issues with the use of pMHC class II (pMHC-II) tetramers for the detection of T helper cells 1,8. First, antigen-specific CD4+ T cell populations tend to be considerably smaller in number than the larger anti-viral CD8+ T cell populations. Secondly, MHC-II-restricted TCRs bind with.