The choice of antigen format impacts upon the frequency of respon

The choice of antigen format impacts upon the frequency of responding T cells. An islet extract comprises the full spectrum of islet antigens, whereas at the other extreme synthetic peptides comprise one, sometimes two, epitopes [30,31]. Hence, one would expect responses to islet lysates to be detected more readily because a larger pool of potentially responsive T cells is present in the blood. However, tissue extracts are susceptible to protease digestion Romidepsin cost and other modifications that may alter the immunogenicity of the tissue. Furthermore, the composition of tissue extracts cannot be defined in the same ways as peptides or recombinant

proteins. Recombinant protein preparations can vary in quality and purity, and these changes can impact upon T cell responses [32]. Synthetic peptides have also Selleckchem BTK inhibitor been reported to give misleading results. Attempts to detect CD8+ T cell responses to proinsulin-derived peptides lead to CD4+ T cell responses against a minor (<5%) peptide contaminant [33]. Responses to other peptide contaminants

have been described in attempts to detect T cell responses to other autoimmune diseases [34]. Given the low frequency of antigen-specific T cells, assays designed to measure islet autoantigen-specific T cell function are particularly susceptible to the technical problems outlined above. The solution is to use the appropriate controls to demonstrate the islet antigen specificity

of the T cell responses being measured, and thorough testing with samples from individuals with and without T1D, ifenprodil to demonstrate disease specificity. Broadly, current assays for measuring islet antigen-specific T cell responses measure cytokine production, T cell proliferation or the frequency of epitope-specific T cells using HLA-peptide multimers with or without in vitro expansion. Examples of each type of assay, their strengths and weaknesses, are discussed below. While we have highlighted published assays with which the authors have direct experience, it should be noted that there are many variations on each assay format. Furthermore, description of an assay here does not imply that it is, in some way, endorsed by the Immunology of Diabetes Society (IDS). At this point ‘head-to-head’ comparisons of the different assays are beginning to be published, but it is not clear [35] which assay, if any, is the ‘best’ assay. Indeed, the most appropriate assay may differ depending upon the aim of the analysis. For example, the best assays for detecting islet antigen-specific T cell responses in the blood of people at risk of T1D may not be the most appropriate assay for monitoring changes in epitope-specific T cell function following antigen-based therapy. Clearly, much work is required before there is sufficient evidence to promote one assay above any other. Background.

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