The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide-specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T cell epitope GAD-555-567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555–567 complexes in APC. I561M single mutation at the central position (P5) of the GAD-555-567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555-567 specific, DR4-restricted T cell response in vitro and in vivo in HLA-DR4 transgenic mice.

Our findings constitute a proof of concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools, which enable studies related to antigen presentation, as well as novel therapeutic agents, that may be used to modulate autoimmune disorders such as T1D.

Interpretation
We have demonstrated that G3H8 can be used to detect and visualize GAD presentation by APC. The TCR-like fine specificity and requirement for both GAD-555–567 peptide and the HLA-DR4 molecule for G3H8 recognition were verified. Moreover, we have identified the P5 position in the center of the peptide to be essential for G3H8 binding of the DR4/GAD complex. This position was found to play a critical role in T cell recognition [24], and thus the overlap in P5 dependency between the TCR and our G3H8 TCRL antibody implies that our antibody has fine specificity and TCR-like characteristics. Once the fine specificity of G3H8 was determined, we used it to detect naturally processed GAD epitopes. G3H8 stained specific MHC class II
complexes derived from processing of the whole GAD antigen by GAD-transfected Priess cells. The lack of extracellular staining by G3H8 suggests that most of the DR4/GAD complexes in these cells were located inside the cells, whereas the number of complexes presented on the cell surface was below the detection threshold of G3H8. Our failure to stain nPOD pancreata is likely due to this same issue, and in addition we had background issues that were never resolved. We concluded that use of this novel reagent needs a substantial amount of the epitope on the APC for detection (only seen when peptide was added to cells or after an immunization in mice).