Strategy for Identifying Dendritic Cell-Processed CD4+ T Cell Epitopes from the HIV Gag p24 Protein

Leonia Bozzacco,Nawei Zhang,David Fenyö,Victoria Küttner,Ralph M Steinman,Haiteng Deng,Beatrix M Ueberheide,Brian T Chait,Christine Trumpfheller,Henry A Zebroski,Haiqiang Yu,Jörn Dengjel,Svetlana Mojsov,Silke Appel

doi:10.1371/journal.pone.0041897

Leonia Bozzacco, Nawei Zhang + Show 12 more

Open Access

https://doi.org/10.1371/journal.pone.0041897

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Abstract

Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4+ T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.

Highlights

Cellular immune responses against any foreign non-self antigens depend upon recognition of peptides presented on the surface of antigen presenting cells (APCs), such as dendritic cells (DCs) [1]
EpiSifter for Rapid Detection of Specific major histocompability complexes (MHC) II Peptides To detect, identify and measure exogenous MHC II-bound peptides presented by DCs among the abundant and complex mixture of endogenous peptides, Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized DCs are pulsed in vitro with synthetic peptides or with a protein [1,7,16,17]
DC MHC II-complexes are enriched by immunoprecipitation, whereupon bound peptides are eluted and purified for Mass Spectrometry (MS) analysis (Figure 1A) [7]

Summary

Introduction

Cellular immune responses against any foreign non-self antigens depend upon recognition of peptides presented on the surface of antigen presenting cells (APCs), such as dendritic cells (DCs) [1]. Assarsson et al [6] identified 54 novel epitopes from more than 4000 computer-defined peptides with HLA binding motifs, including 38 that were CD8+ T cell specific It is not known whether these are immunodominant or subdominant within any given CD8+ T cell response. If a potential match is found, this is tested by targeted tandem mass spectrometry To evaluate this strategy we chose HIV gag p24 peptide sequences because the gag protein plays an important role in inducing protective T cell immunity to HIV [11] and is a component of several experimental vaccines [3,12,13,14]. We demonstrate that presentation of MHC II-associated HIV gag peptides by mature DCs takes place intracellularly

Results

Discussion

Materials and Methods