In this study we have shown the ability to select, from a large n

In this study we have shown the ability to select, from a large non-immune repertoire of human Fab fragments, a panel of recombinant Abs with TCRL specificity directed to auto-reactive T-cell epitopes in the form of self-peptide presented by MHC-II. Abs directed to MHC-II–peptide complexes have been generated before, using epitope-specific immunization as the initial step for further conventional hybridoma technology or construction of a phage display library 35–39. We report here, for the first time, the generation of MHC-II–peptide TCRL Fabs from a naïve human Ab library.

Moreover, due to the Natural Product Library large size of our phage display library, we were able to isolate several different Fabs directed to each targeted MHC-II peptide complex. Based R428 datasheet on our successful

experience in the generation of MHC I–peptide TCRLs and the current data, we believe that the described method can be duplicated for a relatively rapid generation of TCRL Fabs directed to other MHC-II–peptide complexes. We isolated five different TCRL Fab clones directed to the minimal two-domain DR2–MOG-35-55 (RTL1000) complex. Characterization of these Fabs indicated a requirement for both DR2 and MOG-35-55 peptide for recognition. The Fabs could further discern conformational differences in the P42S variant of DR2-bound MOG-35-55 peptide present in RTL342m, demonstrating individual variation in binding to specific contact residues within the DR2–MOG-35-55 complex. Moreover, cross-recognition of RTL342m by the 2E4 this website Fab allowed neutralization of RTL treatment of mMOG-35-55-induced EAE, illustrating the functional activity of this highly characterized Fab in vivo. These Abs therefore mimic the fine specificity of TCRs with the advantages of high-affinity and stable characteristics of the recombinant Fab fragment. Our TCRLs exhibited high structural sensitivity while firmly distinguishing two- versus

four-domain MHC-II–peptide complexes. None of the anti-RTL1000 TCRL Fabs were able to recognize four-domain DR2–MOG-35-55 presented by APC or in a recombinant form. Similarly, two panels of TCRL Fabs directed to two- or four-domain DR4–GAD-555-567 complexes clearly distinguished these two conformational MHC-II peptide determinants. While our previous biophysical and biochemical data suggest a similar secondary structure content for the RTL constructs and the peptide-binding domains of native MHC, our novel TCRL Fabs have identified distinct conformational differences between MHC-II–peptide and RTL–peptide complexes. This novel finding suggests that autoreactive four- versus two-domain MHC-II TCR ligands have distinct conformational shapes that can be distinguished by human Fab molecules and that apparently confer opposing immunological functions (peptide-specific T-cell activation versus tolerance).

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