siRNA knockdown of PD-L1 and PD-L2 in monocyte-derived dendritic cells only modestly improves proliferative responses to Gag by CD8(+) T cells from HIV-1-infected individuals

Abstract

Introduction: Due to their capacity to elicit and regulate immunity, dendritic cells (DCs) are important targets to improve vaccination. Knowing that programmed death-1 (PD-1) high virus-specific T cells become functionally exhausted during chronic exposure to human immunodeficiency virus-1 (HIV-1), the development of a therapeutic DC-based HIV-1 vaccine might include strategies that downregulate PD-L1 and PD-L2 counter-receptors.

Methods: After showing that monocyte-derived DCs rapidly upregulated PD-L1 and PD-L2 expression upon maturation with a variety of stimuli, e.g., Toll-like receptor ligands and cytokines, we determined that PD-L1 and PD-L2 expression could be knocked down by electroporation of a single small interfering RNA (siRNA) sequence twice at the monocyte and immature stages of DC development. This knockdown approached completion and was specific and lasting for several days.

Results: We then added the PD-L1 and PD-L2 silenced monocyte-derived DCs to peripheral blood mononuclear cells from HIV-1-infected individuals along with pools of 15-mer HIV-1 Gag p24 peptides. However, in cultures from six patients, there was only a modest enhancing effect of PD-L1 and PD-L2 silencing on CD8(+) T cell proliferative responses to the DCs.

Discussion: These findings suggest that, in monocyte-derived DCs, additional strategies than PD-L1 or PD-L2 blockade will be needed to improve the function of PD-1 high T cells.

Figure 1

Expression of PD-L1 (upper panel) and PD-L2 (lower panel) on monocyte-derived DCs is up regulated upon maturation with cytokines or agonists for TLR. iDCs were generated from healthy donors by culturing CD14⁺ cells in RPMI 2% human serum in which GM-CSF and IL-4 were added on day 0, 2 and 4. On day 5, iDCs were matured for 24, 48, 72 and 96 hour with either pro-inflammatory cytokines (arrows), TLR ligands (LPS, Poly I:C, ssRNA, Imiquimod, Zymosan, Flagellin), IFN-α2b or IFN-γ. The cell surface expression of PD-L1 and PD-L2 was assessed by FACS (gating on living cells). PD-L1 (clone MIH1) and PD-L2 (clone MIH18) antibodies were used for staining. Isotype control was done with iDCs at 72 hour. The data are representative of three independent experiments on three different donors.

Figure 2

PD-L1 and PD-L2 siRNA knockdown in monocyte-derived DCs is efficient, specific and long lasting. A. Identification of PD-L1 and PD-L2 siRNA duplexes. 4 million iDCs from a healthy donor were electroporated with 1 nmol of PD-L1 or PD-L2 siRNA and maturation was induced right after electroporation with IL-1β, IL-6, TNF-α and PGE₂. FACS analysis of the surface expression of the protein was performed at 48 hour. B. Electroporation of cells twice improved significantly PD-L1 knockdown. Cells of a healthy donor were electroporated either at 5 days of monocyte culture in GM-CSF and IL-4 (1X), or twice in monocyte at day 0 and immature DC at day 5 (2X) with 1 nmol of PD-L1 siRNA or scrambled siRNA. FACS analysis of the surface expression of the protein was performed at 48 hour. C. PD-L1 knockdown is specific. Surface expression of CD83, CD80, CD86 and HLA-DR on PD-L1 silenced DCs were analyzed by flow cytometry 48 hour after electroporation. D. PD-L1 knockdown is long lasting. As in C, but PD-L1 surface expression was assessed 24, 48, 72 and 96 hour after electroporation and induction of maturation. The data in A, B, C and D are representative of three different donors.

Figure 3

PD-L1 and PD-L2 siRNA silenced DCs show a modest increase in their capacity to expand Gag specific CD8⁺ T cells in vitro. A. Example of CD8⁺ T cell proliferative responses after Gag and Ova (control) stimulation of PBMCs with either scrambled siRNA treated DCs (−), PD-L1 silenced DCs, scrambled siRNA treated DCs in presence of PD-L1 blocking antibody, PD-L2 silenced DCs and scrambled siRNA treated DCs in presence of PD-L2 blocking antibody (DC:PBMC ratio is 1:100). CFSE dilution of peptide specific T cells was evaluated after 6-7 days of stimulation using flow cytometry. One of 6 patients is shown. B. Summary data of CD8⁺ T cell proliferative responses to Gag and Ova in six different patients. Each condition is performed in triplicate. Statistical comparisons were made using Wilcoxon match pairs test.