Combinatorial detection of autoreactive CD8+ T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop

Aims/hypothesis: Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8+ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting. Methods: The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers. Results: The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8+ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8+ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors. Conclusions/interpretation Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting

Plasmid-Encoded Proinsulin Preserves C-Peptide While Specifically Reducing Proinsulin-Specific CD8+ T Cells in Type 1 Diabetes

In type 1 diabetes (T1D) an intense inflammatory response destroys β cells in the pancreas, where insulin is produced and released. A therapy for T1D that reduces the specific autoimmune response in this disease while leaving the remainder of the immune system intact has long been sought. Proinsulin is a major target of adaptive immunity in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve β cell function in T1D patients through reduction of insulin-specific T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide served as an exploratory measure of efficacy and safety. Islet-specific CD8+ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides containing pancreatic or unrelated antigens. No serious adverse events related to BHT-3021 occurred. C-peptide levels improved relative to placebo at all doses, most notably at 1 mg at 15 weeks (+19.5% BHT-3021 versus −8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8+ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8+ T cells reactive to proinsulin while preserving C-peptide over the course of dosing

Immunological Balance Is Associated with Clinical Outcome after Autologous Hematopoietic Stem Cell Transplantation in Type 1 Diabetes

Transplantation and autoimmunit

Immune Monitoring of Islet and Pancreas Transplant Recipients

Transplantation and autoimmunit

CD8 T cell autoreactivity to preproinsulin epitopes with very low human leucocyte antigen class I binding affinity

Transplantation and autoimmunit

Present status of the an GR DNA Bank in Brazil

Ex situ conservation include the cryopreservation of genetic material: semen, oocytes, embryos, somatic cells and DNA (FAO, 1998). Although, it is not possible to regenerate whole animals from isolated DNA, it has been proved that DNA banking was useful in population genetic studies and epidemiological investigations. The Animal Genetics Laboratory (AGL) at Embrapa Genetic Resources and Biotechnology - Cenargen, Brasília - DF, Brazil, works with genetic characterization of animal populations that belong to the Brazilian conservation program. At the same time, a DNA Bank is being set up, which already has samples from several animals species. The DNA was extracted from cryopreserved leukocyte pellets or buffy coats. After extraction it was quantified and checked for its integrity and quality. Each specimen extracted was divided in at least two samples. One of them was used for characterization studies (-20°C) while the other was stored at -80°C at the DNA bank. All animals that were collected received a code number and were catalogued in a data bank where all the available information was registered. The refinement of molecular biology techniques increase the usefulness of banked material that will provide a ready reservoir of valuable scientific information. At this time, the AGL DNA bank has specimens of several breeds of six domestic species. Most of these breeds are in risk of extinction and are involved in the Conservation Program of Embrapa/Cenargen. DNA banking is proving to be useful for characterization of domestic animal populations that are in Brazilian conservation program. In near future, it might be the method of choice when many representatives of a breed are to be stored