Boosting immune response with the invariant chain segments via association with non-peptide binding region of major histocompatibility complex class II molecules

Abstract

BackgroundBased on binding of invariant chain (Ii) to major histocompatibility complex (MHC) class II molecules to form complexes, Ii-segment hybrids, Ii-key structure linking an epitope, or Ii class II-associated invariant chain peptide (CLIP) replaced with an epitope were used to increase immune response. It is currently unknown whether the Ii-segment cytosolic and transmembrane domains bind to the MHC non-peptide binding region (PBR) and consequently influence immune response. To investigate the potential role of Ii-segments in the immune response via MHC II/peptide complexes, a few hybrids containing Ii-segments and a multiepitope (F306) from Newcastle disease virus fusion protein (F) were constructed, and their binding effects on MHC II molecules and specific antibody production were compared using confocal microscopy, immunoprecipitation, western blotting and animal experiments.ResultsOne of the Ii-segment/F306 hybrids, containing ND (Asn–Asp) outside the F306 in the Ii-key structure (Ii-key/F306/ND), neither co-localized with MHC II molecules on plasma membrane nor bound to MHC II molecules to form complexes. However, stimulation of mice with the structure produced 4-fold higher antibody titers compared with F306 alone. The two other Ii-segment/F306 hybrids, in which the transmembrane and cytosolic domains of Ii were linked to this structure (Cyt/TM/Ii-key/F306/ND), partially co-localized on plasma membrane with MHC class II molecules and weakly bound MHC II molecules to form complexes. They induced mice to produce approximately 9-fold higher antibody titers compared with F306 alone. Furthermore, an Ii/F306 hybrid (F306 substituting CLIP) co-localized well with MHC II molecules on the membrane to form complexes, although it increased antibody titer about 3-fold relative to F306 alone.ConclusionsThese results suggest that Ii-segments improve specific immune response by binding to the non-PBR on MHC class II molecules and enabling membrane co-localization with MHC II molecules, resulting in the formation of relatively stable MHC II/peptide complexes on the plasma membrane, and signal transduction.

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

“…Likewise, Ii-CT257 was also coprecipitated with H-2Kb molecules. This observation was consistent with our previous observations that the N-terminal region of Ii containing F306 epitopes could bind to MHC class II molecules (Chen et al, 2012). These results indicated that, in addition to the CLIP region, the N-terminal flanking region of CLIP and DN residues might also play an important role in interacting with MHC class I molecules.…”

“…In addition, the residues Asn-Asp (DN), which lay just outside the C-terminal of CLIP (Figure 1a), might promote the association of Th epitopes with the peptide-binding site, as shown in our previous study (Chen et al, 2012).…”

“…The TM of Ii also helps form αβ/ Ii trimers (Ashman and Miller, 1999). In addition, the Ii-key segment of the Ii protein acts as an allosteric site on MHC class II molecules to facilitate the charging of antigenic peptides (Kallinteris et al, 2006), while DN facilitates the effective and stable binding of peptides to MHC class II molecules (Chen et al, 2012). Consequently, we speculate that the TM domain might also bind to sites in MHC class I molecules outside the peptide-binding groove, and that Ii-key and DN segments facilitate OVA257-264 epitope loading onto MHC class I molecules.…”

“…cDNA coding for murine Ii p31 in our previous study (Chen et al, 2012) was used as a cassette vector in which the CLIP-encoding sequence may be replaced with immunodominant OVA peptide SIINFEKL, OVA257-264. The sequence of the double-stranded oligonucleotides was as follows: 5'-AGTATAATCAACTTTGAAAAACTG-3', 5'-CAGTTTTCAAAGTTATT ATACT-3'.…”

“…The murine p31 Ii cDNA clones were previously described (Chen et al, 2012). We constructed a CLIP-replaced Ii hybrid, in which the CLIP region had been substituted with an OVA257-264 epitope.…”