Conformational Variation of Surface Class II MHC Proteins during Myeloid Dendritic Cell Differentiation Accompanies Structural Changes in Lysosomal MIIC

Abstract

Dendritic cells (DC), uniquely among APC, express an open/empty conformation of MHC class II (MHC-II) proteins (correctly folded molecules lacking bound peptides). Generation and trafficking of empty HLA-DR during DC differentiation are investigated here. HLA-DR did not fold as an empty molecule in the endoplasmic reticulum/trans-Golgi network, did not derived from MHC/Ii complexes trafficking to the cell surface, but was generated after invariant chain degradation within lysosomal-like MHC-II rich compartments (MIIC). In pre-DC, generated from monocytes cultured in the presence of GM-CSF, Lamp-1+MHC-II+ compartments are predominantly electron dense and, in these cells, empty MHC-II molecules accounts for as much as 20% of total surface HLA-DR. In immature DC, generated in presence of GM-CSF and IL-4, empty HLA-DR reside in multilamellar MIIC, but are scarcely observed at the cell surface. Thus, the morphology/composition of lysosomal MIIC at different DC maturational stages appear important for surface egression or intracellular retention of empty HLA-DR. Ag loading can be achieved for the fraction of empty HLA-DR present in the “peptide-receptive” form. Finally, in vivo, APC-expressing surface empty HLA-DR were found in T cell areas of secondary lymphoid organs.

GMCSF in the absence of other cytokines sustains human dendritic cell precursors with T cell regulatory activity and capacity to differentiate into functional dendritic cells

“…1) and in DC areas of human tonsil and other lymphoid tissue [54], we suggest that GMCSF-DC may be useful both as a source of precursors to generate functional DC, and also as an in vitro model to investigate tolerogenic function as a developmental feature of DC and to understand the role of IL-4 in priming of immature DC for maturation.…”

Cholesterol lowering drug may influence cellular immune response by altering MHC II function

“…It may be recalled that binding of conformation-specifi c antibody is a reliable tool to study conformational change of MHC II (22)(23)(24)(25)(26). Thus, it is likely that in the absence of cholesterol, conformation of the peptide binding groove may have changed with decreased binding of immunogenic peptide to the peptide binding groove of MHC II protein.…”

“…The conformational changes of MHC II during biosynthesis, folding, and in the MHC class II-containing compartment were detected by monoclonal antibody (mAb ) binding (22)(23)(24)(25). The subtle conformational changes of MHC II upon binding of peptide were detected by mAb binding ( 26 ).…”

Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying)

“…Comparing distributions of gold particles between the same compartments in different cells or conditions is relatively straightforward, because it relies on direct comparisons of raw gold counts found on those compartments (Mayhew et al 2002Mayhew and Desoye 2004;Potolicchio et al 2005;Santambrogio et al 2005). Comparing compartments within a given cell is more demanding because it requires some estimates of compartment sizes in order to calculate their labelling intensities.…”