Phenotypic characterization of HLA-DQ8-restricted insulin-specific CD4+ T cells in longterm-autoantibody positive children

Scientific question: The appearance of multiple islet-autoantibodies in children denotes a point of limited return in the immunepathogenesis of Type 1 Diabetes. However, once islet autoimmunity is established the time frame to overt diabetes is variable ranging from several months to more than two decades. We ask whether such longterm non-progressors will help to identify states of ongoing immune activation vs. regulation when compared to children with recent development of autoantibodies or fast diabetes cases.

Methods: HLA-DQ8-restricted CD4⁺ T-cells purified from peripheral blood are identified by two newly developed HLA-DQ8 insulin-specific tetramer reagents (Tet) based on human insulin mimetopes and combined with intracellular Foxp3-staining and multicolor-flowcytometry permitting the identification of insulin-specific Treg (Tet⁺CD4⁺CD127^lowCD25^highFoxp3⁺) alongside T follicular helper cells (Tet⁺CD4⁺CXCR5⁺CCR7^lowPD1^high). Tetramer⁺ cells will be cloned from single cells for further phenotypic characterization. Percentages of respective T cell populations are presented as averages ± SEM with numbers as indicated.

Results and conclusions: Preliminary experiments using peripheral blood from children with multiple islet-autoantibody positivity for > 5 years without metabolic disease (n = 6) showed the presence of HLA-DQ8-restricted naïve CD45RA⁺ and memory CD45RO⁺-insulin-specific CD4⁺ T-cells. Further characterization of these insulin-specific CD4⁺T-cells revealed the presence of Foxp3⁺Treg cells (Tet⁺CD4⁺CD127^lowCD25^highFoxp3⁺ T cells: average = 7.2 ± 2.2%). In contrast, these insulin-specific Foxp3⁺Treg cells were absent in peripheral blood from children with recent activation of islet-autoantibodies (n = 3). These pilot data provide first evidence that regulatory mechanisms may be operating in these non-progressors and we now aim to delineate whether such a tolerance phenotype is associated with slow progression.