TSHR-specific lymphocytes in Ad-TSHR-immunized native and HLA-DR3-transgenic mice and in Graves´ disease patient blood using MHCII tetramers.

BACKGROUND Antigen-specific lymphocytes are increasingly investigated in autoimmune diseases and immune therapies. We sought to identify thyroid stimulating hormone receptor (TSHR)-specific lymphocytes in mouse models of Graves´ disease including Graves´ patient-specific immunotype HLA-DR3, and in frozen and thawed Graves´ patient blood samples. METHODS AND RESULTS Splenic lymphocytes of adenovirus (Ad)-TSHR-immunized BALB/c mice were stimulated with TSHR-specific peptides C, D, or J. Furthermore, CD154 expressing cells were enriched, expanded in vitro, and analyzed for binding of peptide-MHCII multimers ("tetramers", immunotype H2-IAd). Only peptides C and J were able to elicit increased expression/secretion of CD154 and interferon (IFN)- and tetramers which were loaded with peptide C resulted in antigen-specific signals in splenic lymphocytes from Ad-TSHR-immunized mice. Accordingly, TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 specifically bound to human HLA-DR3-(allele B1*03:01)-transgenic Bl/6 mouse splenic T lymphocytes. In addition, we fine-tuned a protocol to reliably measure thawed human peripheral blood mononuclear cells (PBMCs), which resulted in very acceptable recovery after freezing and thawing with regard to vitality and B and T cell subpopulation markers including regulatory T cells (CD3, CD4, CD25, FoxP3, CD25high, CD127low). TSHR-specific HLA-DR3-MHC class II tetramers loaded with peptide p10 identified antigen-specific T cells in HLA-DR3-positive Graves´ patients´ thawed PBMCs. Moreover, stimulation-dependent release of interleukin (IL)-1s, IL-6, tumor necrosis factor (TNF)-alpha from thawed PBMCs occurred at the expected levels. CONCLUSIONS Novel MHC II tetramers identified TSHR-specific T lymphocytes in Ad-TSHR-immunized hyperthyroid BALB/c or HLA-DR3-transgenic mice, and in thawed human PBMCs from Graves´ patients. These assays may contribute significantly to measure both, disease severity and effects of novel immune therapies in future animal studies and clinical investigations of Graves´ disease.