Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell–Associated Epitopes Using Combinatorial MHC Multimers

Abstract

OBJECTIVEType 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8+ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8+ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive.RESEARCH DESIGN AND METHODSHere, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10–18, prepro-insulin (PPI)15–24, islet antigen (IA)-2797–805, GAD65114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)265–273, and prepro islet amyloid polypeptide (ppIAPP)5–13–specific CD8+ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients.RESULTSUsing this kit, islet autoreactive CD8+ T-cells recognizing insulin B10–18, IA-2797–805, and IGRP265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome.CONCLUSIONSA kit was developed that allows simultaneous detection of CD8+ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

Clinical Potential of Antigen-Specific Therapies in Type 1 Diabetes

“…We recently demonstrated that the presence of insulin autoantibodies prior to treatment with oral insulin/anti-CD3 combination therapy predicts efficacy in the NOD mouse [37]. To adequately apply potential immune biomarkers such as regulatory cytokine responses to islet antigens [38][39][40] or autoreactive T cell frequencies [41], in future trials, we need to better understand how these immune markers vary per individual during the natural course of T1D development and progression.…”

The Enlarging List of Phenotypic Characteristics That Might Allow the Clinical Identification of Families at Risk for Type 1 Diabetes

“…Unfortunately, monitoring multiple epitope-specific CD8 T cell populations poses many technical problems. Recently, monitoring of CD8 T cells reactive to beta-cell-derived antigens has been performed using the combinatorial quantum dot technique, which has been validated using peripheral blood cells from recent-onset type 1 diabetic patients, their siblings, and control subjects (Velthuis et al, 2010). Moreover, during the progression of autoimmune diabetes, memory autoreactive regulatory CD8 T cells can be expanded that could effectively suppress the expansion of dominant and subdominant effectors (Khadra et al, 2010).…”

Beta cells under attack: toward a better understanding of type 1 diabetes immunopathology

“…The recent development of combinatorial major histocompatibility complex (MHC) class I multimers to simultaneously detect multiple islet-specific CD8 T cells in small sample volumes should add to the established array of protocols [20]. Using conventional HLA class I tetramers, Velthuis and colleagues were able to detect isletreactive specificities in an inflamed pancreas allograft of a T1D patient, be it in much higher frequencies than in the blood [21].…”

“…Dr. Serreze has recently pioneered the design of humanized NOD mice carrying human class I constructs. These animals thus present epitopes restricted to human HLA molecules and develop a repertoire of diabetogenic, islet-infiltrating CD8 T cells that shows remarkable correspondence with specificities found in human peripheral blood [20,49]. This kind of approach may aid in bridging the gap between mice and men and suggests that animal models will continue to play a crucial role in T1D research for many years to come.…”