HLA-DM Constrains Epitope Selection in the Human CD4 T Cell Response to Vaccinia Virus by Favoring the Presentation of Peptides with Longer HLA-DM–Mediated Half-Lives

Abstract

HLA-DM (DM) is a non-classical major histocompatibility complex II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during antigen presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of DM in epitope selection is still unclear, especially in human response to infectious disease. In this study, we addressed this question in the context of the human CD4 T cell response to vaccinia virus. We measured the IC50, intrinsic dissociation half-life, and DM-mediated dissociation half-life for a large set of peptides derived from the major core protein A10L and other known vaccinia epitopes bound to HLA-DR1, and compared these properties to the presence and magnitude of peptide-specific CD4+ T cell responses. We found that MHC II-peptide complex kinetic stability in the presence of DM distinguishes T cell epitopes from non-recognized peptides in A10L peptides and also in a set of predicted tight binders from the entire vaccinia genome. Taken together, these analyses demonstrate that DM-mediated dissociation half-life is a strong and independent factor governing peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability in the presence of DM.

Susceptibility to HLA-DM Protein Is Determined by a Dynamic Conformation of Major Histocompatibility Complex Class II Molecule Bound with Peptide

“…Fluorescence Polarization Assay-Fluorescence polarization assay was used to measure the IC 50 value of each peptide, using Alexa488-HA(306 -318) as probe peptide as described previously (13,38).…”

“…Because the immunogenicity of epitopes after infection and vaccination is strongly linked to their relative DM susceptibility (8,13,58,59), understanding these determinants is crucial to follow immune responses, improve vaccines, and understand the etiology of autoimmune disease. Previous models for predicting DM susceptibility have variously implicated particular conserved hydrogen bonds near pocket 1 (17,18,20,21), spontaneous dissociation of the peptide N terminus (22,60), conformational lability of the 3 10 helical region adjacent to pocket 1 (23), an SDS-sensitive flexible conformation determined by pocket 1 occupancy (28,45), and an "compare-exchange-pushoff" mechanism (25).…”

“…Peptide Dissociation Assay and DM Susceptibility Calculation-Peptide dissociation kinetics was measured by europium time-resolved fluorescence as described previously (13). DM susceptibility, the specific rate enhancement defined as the slope of the off-rate versus DM concentration curve, was calculated as (k off, DM Ϫ k off, in )/[DM], where k off, in is the intrinsic off-rate; k off, DM is the DM-mediated off-rate; and [DM] is concentration of DM.…”

The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity

“…A fluorescence polarization (FP) assay was used to measure the IC 50 of each peptide as described (61,62). Soluble recombinant MHC class II molecules DR1 (HLA-DRA*01:01;DRB1*01: 01) and DM were expressed in Drosophila S2 cells and purified as described previously (63,64).…”

“…DR1 (100 nM) was incubated with probe peptide Alexa Fluor 488-HA-(306 -318) (25 nM 50 is the 50% inhibition concentration of the test peptide. To measure DM susceptibility, 0.25 M DM was included in the binding competition assay, and DM susceptibility was reflected by the ratio of IC 50 measured in the presence and absence of DM after incubation at 37°C for 24 h, as described elsewhere (61,65).…”