In this way, T cell assays may provide immune surrogate marker(s) of clinical efficacy and provide evidence that the treatment had impacted upon the subject’s immune system. This would confirm that the route and dose chosen was sufficient to stimulate changes in immune function. Importantly, if the trial did not identify an effective therapy, knowledge of changes
in T cell function, or the failure to induce them, would guide the development of future therapeutic approaches. see more The ideal T cell assay would require a small amount of blood (<5 ml), be technically very simple, have very low intra- and inter-assay variability, be specific for the appropriate islet antigens, work equally well with fresh and cryopreserved peripheral blood mononuclear cells (PBMCs) and give a quantitative measure of islet antigen-specific effector and regulatory T cell responses. Although this ideal may not become a reality, this list highlights the technical challenges to be overcome if an informative assay is
to be developed. None the less, an assay that achieved some, if not all, the criteria listed above would still be very useful. What has prevented the development of T cell assays for islet antigen-specific Talazoparib mw T cell responses? The major problem is that the frequency of islet antigen-specific T cells is very low in the blood. The frequency of proinsulin76–90-specific CD4+ T cells has been estimated to be ∼1 in 300 000 . The frequency of flu matrix 58–66-specific CD8+ T cells has been estimated to be ∼1 in 200 cells , and the frequency of self-reactive proINS- (proINS34–42, proINS101–109) or GAD65 (GAD65536–545, GAD65114–123)-specific CD8+ T cells has been assessed on ∼1 in 1000 cells and ∼1 in 2500 cells, respectively [23–25] (and James and Durinovic-Belló, unpublished observation). In almost all cases, peripheral venous blood is the only tissue available for routine analysis in humans. Another hurdle is that autoreactive T cells are
not only rare but are also of low functional avidity, making it more difficult to detect them. This feature stems from the fact that most high-avidity autoreactive T cells are deleted in the thymus, so that the repertoire of T cells reaching find more periphery becomes skewed towards lower-avidity T cell receptors. The third challenge is to determine which antigens are the targets of the pathogenic autoimmune response and hence the most appropriate for stimulating T cell responses in vitro. Several formats of antigen have been used. Brooks-Worrell et al.  have used protein extracts from human islets, separated by electrophoresis and transferred to nitrocellulose, to measure T cell responses. The use of islet protein extracts avoids the need to choose a single protein or epitope.