Lipopeptide presentation pathway in dendritic cells

doi:10.1016/S0165-2478(01)00271-1

Immunology Letters

Volume 79, Issues 1–2, 1 November 2001, Pages 97-100

https://doi.org/10.1016/S0165-2478(01)00271-1 Get rights and content

Abstract

Lipopeptides are currently being evaluated as candidate vaccines in human volunteers. They elicit cytotoxic responses from CD8⁺ T lymphocytes, whereas peptides without a lipidic moiety usually do not. The exact processing and presentation pathways leading to association with MHC class I molecules has not yet been defined. This is of particular interest in dendritic cells, which are required for primary T cell stimulation. We have tracked lipopeptides derived from an HLA-A2.1-restricted HIV-1 Reverse Transcriptase epitope, by N-terminal addition of an N-epsilon-palmitoyl-lysine. Entry of the lipopeptides into human monocyte-derived dendritic cells (MDC) was mediated by endocytosis, as assessed by colocalization using analogs labelled with rhodamine, and by confocal microscopy. This internalization in DC induced functional stimulation of CD8⁺ T lymphocytes specific for the epitopes, quantified by Interferon-γ ELISPOT assays. The peptide alone was not visualized inside the DC and was only presented through direct surface association to HLA-A*0201. Therefore, lipopeptides provide a model system to define precisely the cross-presentation pathways that lead exogenous proteins to associate with class I MHC molecules within dendritic cells. Using this approach, cross-presentation pathways can be better defined and vaccine lipopeptides can be further optimized for MHC class I association in human dendritic cells.

Introduction

The mechanisms of antigen presentation to the immune system need to be studied primarily in the professional antigen presenting cells, dendritic cells (DC). Convergent evidence shows that these cells are the only antigen presenting cells able to initiate primary T cell responses against antigens. Consequently they have a central role in the induction of protective CD4, CD8 and antibody responses during HIV infection [1]. The mechanisms of CD8⁺ T cell response induction by DC are very important to study for vaccine research against HIV. Indeed, CD8⁺ T lymphocytes are major protective effectors against HIV as well as against other viruses. In HIV infection, their fundamental protective role, in association with CD4⁺ T lymphocytes and antibody responses, has been shown [2], [3], [4]. No vaccine protocol has yet been found to consistently generate protection against SIV_mac or SHIV infection, except live attenuated vaccines. Since these could revert into infectious virus, alternative non-replicative vaccines are currently being sought [3].

CD8⁺ T lymphocytes respond to peptides associated with class I major histocompatibility complex molecules (MHC I). Antigenic proteins are degraded as peptides which associate with MHC I. Classically, this degradation is made by the proteasome in the cytoplasm, therefore antigenic proteins need to be synthesized within the presenting cells. This is an apparent paradox with the necessity of DC for primary immunization. How can tissue-specific antigens, which we are tolerized to, and antigens from pathogens which do not infect DC directly, induce class I-restricted immune responses? HIV itself is only infrequently found in DC from patients [5]. In fact, cross-presentation mechanisms allow soluble antigen proteins that are not synthesized in DC to be presented in association with MHC I, although they are endocytosed and classically directed towards association with class II MHC molecules [6]. Recent data point to DC as the main agents of cross-presentation [7], [8]. Cross-presentation pathways are currently under study for application to vaccinology.

Among the non-replicative vaccines, lipopeptides induce levels of CD8⁺ T cell responses in vivo that are compatible with protection, whereas peptides without a lipid moiety usually do not [9], [10], [11], [12]. However, their presentation pathway has not been elucidated. In this study, the presentation pathway of lipopeptides derived from an HLA-A2.1-restricted HIV-1 Reverse Transcriptase (RT) epitope was studied in human dendritic cells using confocal microscopy and functional T lymphocyte recognition assays.

Section snippets

Dendritic cells

Peripheral blood monocytes were isolated by continuous flow centrifugation leukapheresis and counterflow centrifugation [13]. Immature monocyte-derived DC were obtained after culture with recombinant GM-CSF (Schering-Plough, Dardilly, France) and IL-4 (Peprotech, Le Perray en Yvelines, France) for 7–8 days as described [14].

Peptides and lipopeptides

Labeled and non-labeled peptides and lipopeptides derived from the HLA-A2.1-restricted HIV-1 RT 476-484 epitope (ILKEPVHGV) and from the longer sequence 461–484 were

Entry of the lipopeptides into human dendritic cells

To follow entry into human monocyte derived-DC, rhodamine-labelled analogs of the RT 476-484-peptide or -lipopeptide were used. As shown in Fig. 1, the lipid moiety induced entry of the lipopeptide into DC, whereas the peptide alone was not visualized in the cells. Entry was not dependent on the expression of HLA-A2.1, since it also occured with A2.1 negative DC (Fig. 1). Entry was energy dependent, since it was inhibited at 4 °C or by treatment of the cells with azide and deoxyglucose [14].

Discussion

The lipid moiety of the short lipopeptide derived from the HIV-1 RT 476-484 epitope induced its internalization by endocytosis into human DC, and the epitope was recognized in association with HLA-A2.1 presumably through an intracellular cross-presentation pathway. A more precise study of this pathway was hampered by the direct surface binding of this short lipopeptide: competition with high concentrations of M58-66 peptide was necessary to eliminate the contribution of this binding to T-cell

Acknowledgements

This work was supported by grants from the Fondation de la Recherche Médicale (FRM), the Agence Nationale de Recherches sur le SIDA (ANRS), the Pasteur Institute, Ensemble Contre le SIDA and the Etablissement de Transfusion Sanguine de Strasbourg. We are grateful to David Ojcius for helpful discussion.

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Lipopeptide presentation pathway in dendritic cells

Abstract

Introduction

Section snippets

Dendritic cells

Peptides and lipopeptides

Entry of the lipopeptides into human dendritic cells

Discussion

Acknowledgements

Immunol. Today

Curr. Opin. Immunol.

Immunol. Today

Immunol. Today

Curr. Opin. Immunol.

Res. Immunol.

Mol. Cell Biol.

Ann. Rev. Immunol.

Science

J. Virol.

Nature

J. Immunol.

J. Clin. Invest.

J. Virol.