N-terminal functional region of the invariant chain efficiently targets the binding of a CTL epitope to MHC class I molecules during cross-presentation

Abstract

ABSTRACT. Cross-presentation (CP) is important for priming T cell responses to many viral, bacterial, and tumor antigens. Here, we designed two Ii mutants, based on evidence that the invariant chain (Ii, also named CD74) binds newly synthesized MHC class I molecules with the class II-associated invariant chain peptide (CLIP) region of Ii, which occupies the peptide-binding groove. Specifically, we designed (1) Ii-O257, which is a CLIP-substituted Ii chimer, in which OVA257-264 (SIINFEKL) was substituted for CLIP, and (2) Ii-, also named CT257, which is a C-terminal truncated form of Ii-O257 that contains the N-terminal flanking region of Ii. We immunized C57BL/6 mice with these recombinant proteins. Real-time PCR detected that mice immunized with either Ii-O257 or Ii-CT257 recombinant proteins exhibited increased IFN-γ mRNA expression (approximately 11-fold and 13-fold, respectively) and increased IL-2 mRNA expression (approximately 9-fold and 11-fold, respectively), compared to mice immunized with the OVA257-264 peptide. In vivo cytokine analysis showed that recombinant Ii proteins were highly efficient at activating T cells. Confocal microscopy and co-immunoprecipitation showed that the 2 Ii-OVA257-264 chimers are associated intracellularly with H-2K b molecules. Thus, Ii-CT257 (amino acids 1-89) binds stably to MHC class I with high affinity, indicating that it is a minimal functional fragment of the Ii immune vector. In conclusion, the N-terminal functional region of the Ii fusion protein containing CTL epitopes might prove to be useful for developing peptide or DNA vaccines that use CP as the main mechanism for CD8 + T cell stimulation.

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

“…While fusion of full-length Ii to endogenous antigen results in inconsistent activation of CD4+ T cells, most likely due to the presence of class II associated Ii peptide (CLIP) on the Cterminus, fusion of Ii derivatives accounting for various lengths of N-terminal sequence have been shown to effectively direct antigen to lysosome (161,162). Exogenously administered fusion proteins that have been tagged with an Ii portion thought to assist with MHC class II loading (LRMK, Ii-Key) have also been explored, resulting in potentiation of both CD4+ and CD8+ T cell immune response (163)(164)(165)(166)(167)(168). Finally, since APCs are the only cell type capable of processing antigen via the MHC class II pathway, the targeting of APC uptake is also a viable means of targeting CD4+ T cell activation.…”