Human immune receptors as potential targets for antigen delivery

Abstract

Targeting antigen to recycling receptors present on immune cells may represent a strategy for increasing antigen uptake. The C-type lectin receptors CD206, CD209 and MGL, as well as MHC-II were identified as potential targets for antigen delivery. To elucidate the use of these receptors as antigen delivery vehicles, the endocytic trafficking patterns were examined to determine each receptor's ability to deliver antigen to appropriate endocytic compartments for processing and presentation. Advanced confocal microscopy techniques were used to quantitatively assess the trafficking of these receptors to early, late and recycling endosomes as identified by co-localization with protein markers EEAI, LAMPI, Rab5, Rab7 and Rabi 1. This thesis also investigated the potential use of a non-toxic superantigen variant, SMEZ2.Ml to target antigen through its high affinity binding to MHC-II. Characterization of SMEZ2.M 1 revealed it formed stable complexes with MHC-II that were re-distributed to lipid rafts. SMEZ2.Ml binding to MHC-II also resulted in a loss of CD4+ T cell receptor (TCR) recognition of allogenic MHC-II: peptide complexes at high concentrations. Analysis of hepatitis B core antigen (HBcAg) trafficking following conjugation to SMEZ2.M 1 revealed that SMEZ2.M 1 facilitated the internalization of HBcAg in human dendritic cells (DCs) and targeted HBcAg to MHC-1 and MHC-II positive endosomes. Investigation of the use of LPS and Poly (l:C) as maturation stimulants for human dendritic cells revealed concentration dependent effects on the up-regulation of maturation markers, secretion of the cytokine IL-12p70 and enhanced phagocytosis. Analysis of the effects of receptor binding by anti-CD206, CD209, MGL and MHC-11 antibodies found modest changes in the expression of dendrite cell maturation markers CD86, MHC-1 and MHC-11. Receptor-mediated endocytosis of HBcAg via CD209, MGL and MHC-11, revealed that these receptors were capable of delivering HBcAg to compartments involved in crosspresentation. Comparative analysis of receptor-mediated endocytosis and phagocytosis of HBcAg revealed that phagocytosis was more effective at inducing CDS+ T cell activation. Furthermore, LPS enhanced cross-presentation in cells that phagocytosed antigen, but down-regulated cross-presentation in cells that delivered HBcAg by receptor-mediated endocytosis. This result suggested that receptor-mediated signaling, when combined with TLR3 or TLR4 triggering, may negatively impact the cross-presentation pathway.