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Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

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.

SUBMITTER: Bozzacco L

PROVIDER: S-EPMC3408443 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

Bozzacco Leonia L Yu Haiqiang H Dengjel Jörn J Trumpfheller Christine C Zebroski Henry A HA Zhang Nawei N Küttner Victoria V Ueberheide Beatrix M BM Deng Haiteng H Chait Brian T BT Steinman Ralph M RM Mojsov Svetlana S Fenyö David D

PloS one 20120730 7

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 ...[more]

PMID: 22860026

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Project description:Dendritic cell (DC) therapy is a promising technology for the treatment of HIV infected individuals. HIV-1 Gag- and Nef RNA-loaded DC have previously been shown to induce immune responses ex vivo following coculture with autologous lymphocytes. However, polyfunctionality and memory responses following coculture have not been evaluated. In addition, little is known regarding whether specific HIV-1 proteome components, such as highly conserved regions of the HIV-1, could enhance clinical responses following DC therapy.To determine the breadth of the immune responses to antigen loaded DC, we analyzed polyfunctional T cell response ex vivo to Gag RNA loaded DC. Blood samples were used to generate monocyte derived DC, which were then matured and cocultured with autologous lymphocytes. We found that cytokine-matured DC loaded with Gag RNA was able to induce Gag-specific IFN-? and IL-2 responses after a 12-day coculture. We characterized these responses by polyfunctional intracellular cytokine staining and evaluation of T cell memory phenotypes. Central memory CD8+ T cells were induced ex vivo after DC coculture from each of 3 patients, and the effector memory pool was increased by DC coculture from 2 patients. We also observed a decrease in the terminal effector and intermediate CD8+ T cell pool and an increase in the naïve/other population. There was a reduction in terminal effector and intermediate CD4+ T cells, and a corresponding increase in naïve/other CD4+ T cells. Finally, we evaluated conserved regions of Gag as a novel DC therapy immunogen and found that a conserved element (CE) p24 Gag antigen elicited IFN-? and IL-2 responses comparable to those induced by a full-length Gag antigen.We showed that RNA-loaded DC therapy induced a polyfunctional T cell response ex vivo, supporting the use of such DC-therapy for HIV infection. However, the central and effector memory phenotypes of T cells did not appear to be enhanced during coculture with Gag RNA-loaded DC. Furthermore, comparable antigen-specific responses were induced in HIV infected individuals using full-length Gag or only conserved elements of the Gag p24 protein. This indicates that immune responses can be focused onto the conserved elements of Gag in the absence of other Gag components.

Dataset Information

Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

Publications

Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

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