TLR9 activation increases TAP-independent vesicular MHC class I processing in vivo.

Area of Science:

• Immunology
• Cellular Biology
• Molecular Biology

Background:

• Dendritic cells (DCs) present soluble protein antigens on MHC class I molecules via pathways that can be dependent or independent of TAP peptide transporters.
• The TAP-independent endolysosomal vesicular pathway (el-VP) for cross-presentation has been observed in vitro, but its in vivo activity and modulation remain less understood.
• CpG oligodeoxynucleotides are known immunostimulants that engage Toll-like receptor 9 (TLR9).

Purpose of the Study:

• To investigate whether immunostimulatory CpG oligodeoxynucleotides can enhance the activity of the TAP-independent endolysosomal vesicular pathway (el-VP) in vivo.
• To elucidate the role of cathepsin S (CatS) and TLR9 in CpG-mediated enhancement of antigen presentation via the el-VP.

Main Methods:

• Utilized CFSE-labeled OT-1 T cells transferred into TAP1-deficient (TAP1-/-) mice to assess in vivo T-cell responses to ovalbumin (OVA).
• Administered soluble OVA with or without CpG oligodeoxynucleotides and analyzed OT-1 T-cell proliferation as a readout for el-VP activity.
• Employed confocal microscopy to visualize the co-localization of MHC class I-peptide complexes (H-2K(b)/SIINFEKL) with endolysosomal markers (LAMP1) and cellular compartments (ER, cis-Golgi).

Main Results:

• Soluble OVA alone induced a poor OT-1 T-cell response in TAP1-/- mice, which was significantly enhanced by co-administration of CpG.
• CpG-mediated enhancement of the T-cell response required both endolysosomal cathepsin S (CatS) and Toll-like receptor 9 (TLR9).
• Confocal microscopy revealed increased co-localization of H-2K(b)/SIINFEKL complexes with LAMP1 in DCs upon CpG treatment, with no evidence of classical MHC-I processing in the ER/cis-Golgi of TAP1-/- mice.

Conclusions:

• In vivo activation of TLR9 by CpG significantly increases the efficiency of the TAP-independent endolysosomal vesicular pathway (el-VP) for antigen cross-presentation.
• This CpG-induced enhancement of the el-VP likely contributes to the potent adjuvant activity of CpG compounds.
• The findings highlight a critical role for TLR9 and CatS in facilitating cross-presentation via an endolysosomal route, independent of TAP transporters.