Abstract
BackgroundOur previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer.MethodsIn the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.ResultsCCL21 protein production from transduced DC was detected in supernatants (24–72 hours, 3.5–6.7 ng/4–5 × 106 cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro.ConclusionViable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.
Highlights
Our previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability
DC were obtained from cryopreserved MNC by culture of adherent monocytes with laboratory grade GM-CSF and IL-4, as described in Materials and Methods
We report that 1) human DC can be efficiently transduced ex vivo with clinical grade adenoviral vector expressing the CCL21 gene, 2) transduction of DC with AdCCL21 at room temperature (RT) improved cell viability over that observed after transduction at 37°C, 3) CCL21-DC demonstrate an immature DC phenotype with the ability to phagocytose and present antigens, 4) keyhole limpet hemocyanin (KLH) inhibits the ability of CCL21-DC to induce T cell proliferation and cytokine production, 5) CCL21-DC maintain the ability to be activated and produce IL-12 and transduction does not impair DC generation of IP-10/CXCL10 and MIG/CXCL9, and 6) CCL21DC secrete CCL21 protein within 24 h of transduction and demonstrate biological activity with induction of chemotaxis
Summary
Our previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. Intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer. DC produce high levels of cytokines and chemokines, attracting antigen-specific T cells in vivo. These properties, combined with the efficient capture of antigens by immature DC, allow them to efficiently present antigenic peptides and costimulate antigen-specific naïve T cells [8]. DC transfer has been demonstrated to be a safe approach in clinical studies [13,14,15,16]
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