siRNA silencing of PD-1 ligands on dendritic cell vaccines boosts the expansion of minor histocompatibility antigen-specific CD8+ T cells in NOD/SCID/IL2Rg(null) mice

Abstract

Allogeneic stem cell transplantation (allo-SCT) can be a curative therapy for patients suffering from hematological malignancies. The therapeutic efficacy is based on donor-derived CD8+ T cells that recognize minor histocompatibility antigens (MiHAs) expressed by patient’s tumor cells. However, these responses are not always sufficient, and persistence and recurrence of the malignant disease are often observed. Therefore, application of additive therapy targeting hematopoietic-restricted MiHAs is essential. Adoptive transfer of MiHA-specific CD8+ T cells in combination with dendritic cell (DC) vaccination could be a promising strategy. Though effects of DC vaccination in anti-cancer therapy have been demonstrated, improvement in DC vaccination therapy is needed, as clinical responses are limited. In this study, we investigated the potency of program death ligand (PD-L) 1 and 2 silenced DC vaccines for ex vivo priming and in vivo boosting of MiHA-specific CD8+ T cell responses. Co-culturing CD8+ T cells with MiHA-loaded DCs resulted in priming and expansion of functional MiHA-specific CD8+ T cells from the naive repertoire, which was augmented upon silencing of PD-L1 and PD-L2. Furthermore, DC vaccination supported and expanded adoptively transferred antigen-specific CD8+ T cells in vivo. Importantly, the use of PD-L silenced DCs improved boosting and further expansion of ex vivo primed MiHA-specific CD8+ T cells in immunodeficient mice. In conclusion, adoptive transfer of ex vivo primed MiHA-specific CD8+ T cells in combination with PD-L silenced DC vaccination, targeting MiHAs restricted to the hematopoietic system, is an interesting approach to boost GVT immunity in allo-SCT patients and thereby prevent relapse.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1668-6) contains supplementary material, which is available to authorized users.

Hematopoietic stem cell-derived myeloid and plasmacytoid DC-based vaccines are highly potent inducers of tumor-reactive T cell and NK cell responsesex vivo

“…pDCs were stimulated with 3.2 µg/mL CpG-A (ODN 2216) or 5 µg/mL R848 (Resiquimod; both from Enzo Life Sciences, catalog# ALX-746–005 and ALX-420–038, respectively) in medium supplemented with IL-3 (10 ng/mL, Immunotools, catalog# 11340035), while BDCA1 + mDCs were stimulated with a combination of 5 µg/mL R848 and 20 µg/mL Poly I:C (Polyinosinic:polycytidylic acid, Sigma-Aldrich, catalog# P0913), referred to as RPI:C, in the presence of GM-CSF (800 IU/mL) and IL-4 (500 IU/mL). MoDCs were matured with RPI:C or conventional cytokine mixture (cytomix) containing 5 ng/mL IL-1β, 15 ng/mL IL-6, 20 ng/mL TNF-α and 1 µg/mL PGE2, as described previously 51 . Cell culture supernatants were analyzed with ELISA for IFN-α (pan-specific, MabTech, catalog# 3425–1H-20), IL-12p70 (eBioscience, catalog# 14–8129 (standard), 16–8126 (coating Ab) and 13–7129 (detection Ab)) and TNF-α (Sanquin, catalog# M9323) levels, according to manufacturer's instructions.…”

Dendritic cell vaccines: A review of recent developments and their potential pediatric application

“…Further, infusion of these Ag-specific CTL simultaneously with vaccination of PD-L1 silenced DC induced an increased and sustained antigen-specific CTL response. 59 These modifications to PD-1/ PD-L1 expression on DC did not result in any systemic toxicity.…”

“…60 A study by van der Waart, et al investigated this further using an in vivo murine AML model. 59 In this study DC derived from PBMC were treated with short interfering RNA (siRNA) against PD-L1 and or PD-L2 and were then shown to increase in vitro proliferation of antigen specific CD8…”

“…This concern has led some groups to investigate ways of achieving a more focused silencing of PD-1/PD-L1. 59,60 In one study, DC from healthy volunteers were infected with a lentiviral vector encoding short hairpin RNA (shRNA) for PD-L1 which resulted in abrogation of PD-L1 production. They found no change in the standard DC phenotype except loss of expression of surface PD-L1, but cells treated in this way had increased ability to stimulate T-cell proliferation, secretion of IL-12 and interferon-g, and in vitro tumor cell killing.…”

Minor histocompatibility Ags: identification strategies, clinical results and translational perspectives

“…Moreover, knockdown of the PD ligands on mHag-loaded DCs resulted in enhanced induction of mHag-specific T cells. [96][97][98] These results have already led to the initiation of two current trials in which mHag-loaded moDCs silenced for PD-L1/2 with short hairpin RNAs are being used. Also, the induction of cell-adhesion-mediated immune resistance, in parallel to the well-known phenomenon of cell-adhesionmediated drug resistance, may have an important role in tumor escape from immunotherapy.…”