Dendritic cells transfected with Her2 antigen-encoding RNA replicons cross-prime CD8 T cells and protect mice against tumor challenge
Introduction
Tumor associated antigens (TAAs) are primarily expressed by tumor cells and frequently involved in tumorigenesis [1]. Immunotherapy as an alternative type of cancer treatment aims at stimulating the immune system to selectively react against neoplastic cells by breaking the tolerance to TAA [2]. A particularly promising approach takes advantage of the unique properties of dendritic cells (DCs) to induce and regulate immune responses. Subsequent to antigen acquisition, DCs mature and migrate to secondary lymphoid organs where they present immunogenic peptides in the context of major histocompatibility complex (MHC) molecules to T cells and deliver critical co-stimulatory signals [3]. Following the first clinical study that employed autologous, peptide-pulsed DCs for vaccination of patients with B cell lymphoma [4] multiple trials were conducted, which showed DC vaccination to be safe, well tolerated and immunogenic [5].
The immunogenicity of DC-presented foreign antigens depends on the activation/maturation status of the cells, on the efficiency of antigen processing, and on a sustained presentation of high numbers of immunogenic peptides on the MHC [6], [7]. Thus, a critical determinant of the immunogenicity of DC-based vaccines is the “loading” of vaccine DCs with antigen. For example, this is achieved by pulsing DCs with recombinant tumor proteins or peptides, or by transfecting DCs with antigen-encoding nucleic acids [5]. Another important determinant of the immunogenicity of DC-based vaccines is the transfer of cellular material from vaccine DCs to endogenous antigen presenting cells (APCs) [6]. The latter comprise macrophages and endogenous DCs [8], which internalize proteins, cellular fragments and apoptotic cells [9] and reprocess antigens in the endosomal compartment or cytosol for presentation on their own MHC molecules [8], [10]. Vaccination with antigen-expressing DCs may induce rapid, effective and maintained T cell responses against viral or tumor antigens that are otherwise not accessible to endogenous APCs. This immunological pathway is termed “cross-priming” and may be particularly useful in vaccination approaches [11], [12].
In a recent study, we used the cross-priming pathway to induce a protective T cell response against hepatitis C virus [13]. For this purpose, vaccine DCs were transfected with self-replicating viral RNAs (“RNA replicons”) of bovine viral diarrhea virus (BVDV), in which the genetic units coding for the virus structural proteins were replaced by a heterologous open reading frame coding for an antigen of choice (Fig. 1A). Such “bi-cistronic” BVDV replicons turned out to be advantageous for vaccination because they express high amounts of the antigen in the cytoplasm of the transfected cells and do not form infectious virus particles. An additional advantage is the cytopathogenicity of BVDV replicons that express the viral NS3 protein resulting in apoptosis 24–48 h after transfection [14], [15]. This time-delayed apoptosis of the replicon-transfected vaccine DCs was shown to be crucial for efficient cross-priming of an antigen-specific T cell response [13].
The aim of the current study was to employ the DC/BVDV replicon system to vaccinate against neoplastic cells that over-express Her2 as a model TAA. Her2 (ErbB2; neu) is overexpressed by breast tumors, gastric carcinomas, lung tumors and ovarian cancer [16]. In an attempt to enhance the vaccination process, the DC/replicon system was used to co-deliver the cytokine IL-12, which plays a key role in the activation of T cells. Our results demonstrate that the DC/BVDV replicon system can be used as a valuable tool in vaccination approaches targeting tumor cells as well as a delivery system of immuno-stimulatory molecules. Most interestingly, induction of T cell responses via cross-priming was shown to be sufficient to induce a potent, preventive anti-tumor response in the absence of direct priming of T cells, and in the absence of antibodies.
Section snippets
Cell lines
The dendritic cell line DC2.4 (haplotype: H-2b) was kindly provided by Dr. K.L. Rock (Dana Farber Cancer Institute, Boston, MA) [17] and cultured in RPMI 1640 containing 10% heat-inactivated bovine calf serum (FBS), 2 mM l-glutamine, 100 μM nonessential amino acids, 100 U/ml penicillin/streptomycin (all from Cellgro, Manassas, VA) (complete medium) and 50 μM 2-mercaptoethanol (Gibco BRL, Grand Island, NY). Sublines of highly transfectable cells were established by limiting dilution cloning. The
Generation of cytokine and antigen expressing RNA replicons
The initial task of this study was the construction of cDNAs that allowed the transcription of bi-cistronic BVDV replicon RNAs encoding IL-12 and immunogenic domains of Her2. While the procedures for the generation of the cDNA platforms were the same as described by Racanelli et al. [13] (see Section 2 for details), the heterologous ORFs were constructed in such a way that a fusion protein comprising (N- to C-terminus) the pestiviral autoprotease NPRO and the heterologous protein was generated.
Discussion
Multiple phase I and II clinical trials proved DC-based immunotherapies to be safer and less toxic than conventional cancer therapies. However, to date, the response to DC-based cancer immunotherapies is frequently unsatisfying as exemplified by the complete failure of a phase III trial in melanoma patients [23]. Accordingly, various parameters such as the DC maturation status, the route of administration and, most prominently, the mode of antigen-delivery require further improvement [24]. Many
Acknowledgements
We thank Dr. E.M. Jaffee for the NT-2 cell line, Dr. K.L. Rock for the DC2.4 cell line and Dr. N. Tautz for the anti BVDV NS3 antibody. This study was supported by the Martin-Luther-University Halle-Wittenberg and the intramural research program of NIDDK, NIH.
References (52)
- et al.
Dendritic cells transfected with cytopathic self-replicating RNA induce crosspriming of CD8+ T cells and antiviral immunity
Immunity
(2004) - et al.
A phase I/II study of recombinant human interleukin-12 in patients with chronic hepatitis B
J Hepatol
(2000) - et al.
Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG
Ann Oncol
(2006) - et al.
Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis
Immunity
(2006) - et al.
Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes
Vaccine
(2004) - et al.
Ability to differentiate between cp and ncp BVDV by microarrays: towards an application in clinical veterinary medicine?
Vet Immunol Immunopathol
(2005) - et al.
TLR signalling regulated antigen presentation in dendritic cells
Curr Opin Immunol
(2010) - et al.
Type I interferon as a stimulus for cross-priming
Cytokine Growth Factor Rev
(2008) - et al.
Adoptive cell therapy for the treatment of patients with metastatic melanoma
Curr Opin Immunol
(2009) - et al.
CD4+ T-cell induction of Fas-mediated apoptosis in Burkitt's lymphoma B cells
Blood
(1996)
Human T cell responses against melanoma
Annu Rev Immunol
Vaccines for tumour prevention
Nat Rev Cancer
Antigen presentation and T cell stimulation by dendritic cells
Annu Rev Immunol
Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells
Nat Med
Current approaches in dendritic cell generation and future implications for cancer immunotherapy
Cancer Immunol Immunother
Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting
Nat Rev Immunol
Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines
Nat Rev Cancer
Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens
Immunol Rev
Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes
J Exp Med
Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation
Nat Immunol
Cross-priming as a predominant mechanism for inducing CD8(+) T cell responses in gene gun DNA immunization
J Immunol
Cross-priming of cytotoxic T cells dictates antigen requisites for modified vaccinia virus Ankara vector vaccines
J Virol
Characterization of an autonomous subgenomic pestivirus RNA replicon
J Virol
Establishment and characterization of cytopathogenic and noncytopathogenic pestivirus replicons
J Virol
Novel anticancer targets: revisiting ERBB2 and discovering ERBB3
Nat Rev Cancer
Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules
J Immunol
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These authors contributed equally.