Peptide-loaded dendritic cells prime and activate MHC ...

Abstract

Undifferentiated and differentiated dendritic cells (uDC and dDC, respectively), derived from the bone marrow, were studied in vitro and in vivo. Ovalbumin (OVA) and two OVA-derived peptides binding to H-2Kb and I-Ab, respectively, were used. Two IL-2 secreting T cell hybridomas specific for the OVA-derived epitopes were used in the in vitro read-out. The ability to cross-present the H-2Kb binding OVA257–264-peptide (SIINFEKL) was restricted to dDC, which express CD11c+, CD86+, and MHC-II+. In vitro, the antigenicity of SIINFEKL-loaded DC declined at a slower rate than that of OVA-pulsed DC. Moreover, SIINFEKL-loaded DC were up to 50 times more efficient than DC-pulsed with OVA-protein for generation of an H-2Kb-restricted response. Immunization of mice with SIINFEKL-loaded DC resulted in a much stronger H-2Kb-restricted response than immunization with OVA-pulsed DC. These data might have important implications for the choice of antigen source in the design of DC-based vaccines.

Keywords

Dendritic cells

;

Cross-presentation

;

Ovalbumin

;

Immunization

;

Interleukin 2

;

Cytotoxic T lymphocyte

;

ELISPOT

Introduction

In contrast to other antigen presenting cells (APC), dendritic cells (DC)1 are highly efficient in taking up exogenous antigen and to present them on MHC-class I molecules; a phenomenon known as cross-presentation [1], [2], [3], [4]. This pathway is accessible to various antigens, including soluble protein, cell-associated antigens (apoptotic or necrotic), and heat shock proteins [3], [5], [6], [7], [8], [9]. Cross-presentation has been shown to play a significant role in the priming of cytotoxic T lymphocyte (CTL) against various antigens, including minor histocompatibility antigens, tumor-associated antigens, and viral antigens, in a process referred to as cross-priming (reviewed in [2], [10]).

DC are candidate target cells for manipulation of the immune system, particularly for the generation of specific CTL-mediated immunotherapy against tumors [11]. Thus, it has been shown that both murine and human DC pulsed with tumor-associated peptides or proteins are able to promote prophylactic and therapeutic anti-tumor responses [12], [13], [14], [15]. Immature DC are capable of taking up antigenic material from the environment by pinocytosis and phagocytosis. However, only mature DC are capable of cross-presenting the antigenic peptide and thereby activate naive CTL precursors [8], [16].

In the present work, we have studied in vitro conditions for the generation of bone marrow derived OVA cross-presenting DC. Our data clearly show that the class I-restricted OVA-derived epitope H-2Kb/SIINFEKL is far more efficiently presented when DC are loaded directly with the SIINFEKL-peptide than when the peptide is cross-presented by H-2Kb molecules after OVA protein-pulsing. These data might be of importance for the design of DC-based vaccines.

Section snippets

Mice

Five weeks old female C57Bl/6 (H-2b) mice were purchased from Bomholtgaard (Ry, Denmark). All mice were kept in a controlled microbial environment at The Department of Experimental Medicine at The Panum Institute (Copenhagen, Denmark) and used at the age of 8–10 weeks.

Generation and culture of DC from bone marrow

Bone marrow-derived DC were generated as described previously [17] with modifications. Briefly, bone marrow was flushed from femurs and tibia and cells were depleted of erythrocytes with lysis buffer. The cells were washed and

Phenotype of the DC-populations and specificity of the two T-cell hybridomas

Bone marrow-derived DC were harvested after 8 days of culture. Differentiation of DC was induced by the addition of LPS in the culture for the final 24 h (see Section 2). The phenotype of the DC cell cultures were analyzed by a two-color flow cytometry (Fig. 1). Only 6% of the cells in the uDC cultures coexpressed the DC markers CD11c and CD86, whereas these markers were coexpressed on 63% of the cells in the dDC cultures. MHC-class II was expressed on 20% and 64% of the CD11c positive cells in

Discussion

The results of the present study show that only differentiated CD11c+ DC are capable of cross-presenting the H-2Kb-restricted OVA-derived SIINFEKL epitope. In contrast, both undifferentiated and differentiated OVA pulsed DC process and express the MHC-class II/OVA323–339-epitope with comparable efficiency, indicating that the lysosomal compartment necessary for MHC-class II processing functions independently of the stage of DC maturation. These data are in accordance with previous studies

Acknowledgments

This work is part of the IHWG, and is supported by the Danish MRC (grant 22-01-0272), the 5th Framework programme of the European Commission (grant QLRT-1999-00173), the NIH (grant A149213-02), and also supported by various grants from the Danish Medical research Council, the THOR program, the Danish Cancer Society, Fam. Hede Nielsen’s, Dir. Ib Henriksen’s, Dir. E. Danielsen and Wife’s, Enid Intgemand’s, and Grosser M. Brogaard and Wife’s Foundations.

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