Abstract
Dendritic cells (DCs) are the main antigen presenting cells and play a pivotal role in the stimulation of T-cell immune responses. DCs cultured in the presence of a single tumor antigen can elicit an immune response against tumor cells expressing that antigen. However, simultaneous use of several tumor antigens may be advantageous since polyclonal activation of T cells against different tumor antigens may be a better approach to eradicate tumor cells. In this sense, fusions of dendritic and tumor cells (FCs) show a broad spectrum of tumor antigens, both known and unidentified, to be presented by class I and II MHC. Although prophylactic vaccines were successful in murine models, the results in the therapeutic setting have been unsatisfactory. We hypothesised that enhancing costimulation of FCs would help to break tumor tolerance once the tumor is established. To this purpose, we transduced FCs with a recombinant adenovirus encoding CD40L (AdvCD40L or AdvGFP as control) and we studied the therapeutic antitumoral effect of the administration of FC-CD40L in a murine model of myeloma. DCs obtained from day 7-bone marrow cultures of Balb/c mice were fused with tumor cells, a syngeneic murine myeloma cell line (4TOO). FCs hybrids were generated with PEG and selected after culturing in HAT medium plus GM-CSF for 7 days. FC were quantified by determining the percentage of cells that coexpress specific DC (CD11c) and tumor markers (CD138). Mean fusion efficiency was 30% (20–40%) and FCs expressed moderate levels of CD80, CD83, CD86, CD54, CD40 and MHC II and did not express CD40L. FC-CD40L showed a significant increase of expression of costimulatory molecules (CD80, CD86, CD54, and MHC II) compared to FC-GFP (p=0.011). Moreover, in a syngeneic mixed lymphocyte reaction, FC-CD40L induced a two-fold higher T-cell proliferation than FC-GFP or FC alone. In addition, FC-CD40L had improved migration to lymphoid tissues, preferentially to spleen, in comparison with FC-GFP (2.8% versus 1.6%). The antitumor effect of FC-CD40L was analyzed in vivo. Mice (n=10 per group) were injected i.v. with 2.5×105 tumor cells and treated with irradiated FC, FC-GFP or FC-CD40L (1×106 cells each) on days 2, 6 and 10 after tumor challenge. 40% of mice treated with FC-CD40L had long-term survival (>120 days). In contrast, all of mice treated with FC or FC-GFP died between days 25 and 35 (p=0.012). In parallel, treatment with mixed cells (not fused DC+ tumor cells), mix transduced with AdvGFP, or mix transduced with AdvCD40L did not provide any significant antitumor effect. We conclude that FCs transduced with AdvCD40L better stimulate in vitro and in vivo immune responses than FC alone and may provide a new strategy for treating patients with multiple myeloma or lymphoma.
Targeting Tumor Antigens to Secreted Membrane Vesicles In vivo Induces Efficient Antitumor Immune Responses
