Autologous dendritic cells loaded with apoptotic tumor cells induce T cell-mediated immune responses against breast cancer in vitro

Abstract

Dendritic cell (DCs) based immunotherapy has received increased interest in the treatment of specific malignancies including breast cancer. In this in vitro study, T cell responses, which are induced by monocyte-derived DCs pulsed with apoptotic breast tumor cells (ApTC), were analyzed in terms of proliferation, specific cytotoxicity, and cytokine release. Nylon wool-enriched T lymphocytes from five patients with breast cancer stimulated with monocyte-derived DCs pulsed with apoptotic tumor cells in vitro and their proliferation response were analyzed by [³H] thymidine uptake and specific cytotoxic activity of tumor antigen-primed T cells after three rounds of weekly stimulation by flow cytometry. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) cytokine release assay was carried out 24 h after the last stimulation. The supernatant from primed T cells was collected and analyzed using commercially available ELISA kits. T cell proliferation assays revealed that DCs pulsed with apoptotic tumor cell could stimulate an autologous T cell proliferation response with stimulation indices of 5-21. The T cell-mediated cytotoxicity assay demonstrated that tumor antigen-primed T cells could kill significantly more autologous tumor cells than normal cells (P < 0.05). These cells had variable amounts of cytotoxic activity against K562 cells. Primed T cells released both IFN-γ and IL-4 in response to re-stimulation by antigen-pulsed DCs, but were dominated by IFN-γ production in two out of five patients and IL-4 production in three out of five patients. In conclusion, our results suggested that DCs pulsed with apoptotic breast tumor cells could elicit effective specific antitumor T cell responses in vitro. Therefore, vaccination with DCs pulsed with apoptotic tumor cells may be considered as a novel strategy for immunotherapy of patients with breast cancer refractory to standard modalities.

Introduction

Breast cancer is one of the most common cancers among females. The incidence of this type of cancer varies widely around the world with North America and European countries having the highest rates and Asian and African countries having the lowest. Current incidence rates predict that one in eight women in the United States will develop breast cancer during their lifetime. Breast cancer is the second leading cause of cancer death in American women following lung cancer [1].

The modern era of breast cancer treatment has developed with great rapidity due to the efforts of an extremely broad spectrum of basic and clinical scientists whose efforts have redefined our standards for appropriate therapeutic strategies. Immunotherapy is a therapeutic strategy that manipulates the host’s immune responses against tumor cells. This type of therapy marks a new area of cancer therapies that are directed passively or actively against tumor cells [2].

Dendritic cells (DCs) are the most potent antigen presenting cells for naive T cell activation [3]. DCs originate from the bone marrow and reside in a resting or immature state in non-lymphoid tissues in which they efficiently capture and process antigens. Upon stimulation with bacterial products, inflammatory cytokines, or CD40 ligation, DCs undergo a maturation process that results in enhanced antigen presenting capacity and expression of MHC, upregulation of co-stimulatory molecules, and migration into secondary lymphoid organs where they prime naive T cells [4], [5]. The presence of DCs at the tumor site and regional lymph nodes suggested that these cells have a crucial role in the antitumor immune response [6], [7]. Because of their unique capacity to stimulate resting T cells, DCs are the most promising option for immunization protocols, especially since they can potently induce antitumor immunity in patients with malignant disease [8], [9].

The rationale for this approach is based on the observation that DCs can be pulsed with tumor antigen and subsequently administered as a cellular vaccine to induce a specific antitumor response [10]. Methods that allow for large scale in vitro generation of DCs from peripheral blood monocytes using granulocyte–macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) have recently developed and facilitated induction of immune responses in vitro as well as in clinical vaccination trials [11].

It is generally accepted that tumors growing in vivo naturally provide antigens to APCs either by shedding from the surface of viable cells or by fragmentation of dead tumor cells. Previous studies have shown that multiple tumor antigens do exist and can be used to induce autologous tumor-specific T cell responses in vitro. Thus, they present an alternative strategy for effective vaccination due to the use of unfractionated tumor-derived antigens such as tumor cell lysates [12], peptides eluted from tumor cell membrane [13], apoptotic tumor cells [14], and fusion of tumor and dendritic cells [15]. Indeed, feeding DCs with apoptotic tumor cells provides a full array of antigenic peptides that rapidly gain access to both MHC class I (cross-presentation) and class II pathways, leading to a diversified immune response involving cytotoxic T lymphocytes as well as CD4+ T cells [16], [17], [18]. This method does not require the identification of tumor-associated antigens. Despite protein alterations during apoptosis induction, cross-presentation of apoptotic bodies (apobodies) allows for the presentation of the MHC-peptide density as efficiently as peptide loading for priming, naïve CTLs [19].

In the present study, we have evaluated whether DCs pulsed with apoptotic tumor cells derived from patients with breast cancer are able to elicit T cell responses in terms of proliferation, cytotoxicity, and cytokine release against autologous tumor cells. Our aim was to obtain initial preclinical evidence for the potential efficacy of DC therapy. Importantly, a major objective of our study was to establish an experimental model that would allow us to evaluate and subsequently optimize the immunostimulatory capacity of DCs under autologous conditions. We hope that such an approach may therefore hold potential for treatment with active or adoptive immunotherapy for patients with breast cancer who have residual or resistant disease after standard surgical and cytotoxic treatments.