Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation

Recombinant TCR ligands (RTLs) consisting of covalently linked α ₁, and β₁, domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP_72-89) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP_72-89 peptide-specific CD4 ⁺ T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP _72-89-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP _72-89 peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3ζ p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3ε treatment produced stronger activation of these cellular events with additional activation of NF-κB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-κB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4⁺ T cells.