Effects of herpes simplex virus amplicon transduction on murine dendritic cells

Abstract

The herpes simplex virus (HSV)-based amplicon is a versatile vaccine platform that has been preclinically vetted as a gene-based immunotherapeutic for cancer, HIV, and neurodegenerative disorders. Although it is well known that injection of dendritic cells (DCs) transduced ex vivo with helper virus-free HSV amplicon vectors expressing disease-relevant antigens induces antigen-specific immune responses, the cellular receptor(s) by which the amplicon virion gains entry into DCs, as well as the effects that viral vector transduction impinges on the physiological status of these cells, is less understood. Herein, we examine the effects of amplicon transduction on mouse bone marrow-derived DCs. We demonstrate that HSV-1 cellular receptors HveC and HveA are expressed on the cell surface of murine DCs, and that HSV amplicons transduce DCs at high efficiency (>90%) with minimal effects on cell viability. Transduction of dendritic cells with amplicons induces a transient DC maturation phenotype as represented by self-limited upregulation of MHCII and CD11c markers. Mature DCs are less sensitive to HSV amplicon transduction than immature DCs regarding DC-related surface marker maintenance. From this and our previous work, we conclude that HSV amplicons transduce DCs efficiently, but impart differential and transient physiological effects on mature and immature DC pools, which will facilitate fine-tuning of this vaccination platform and further exploit its potential in immunotherapy.

FIG. 1.

Expression levels of nectin-1 and HveA on bone marrow-derived dendritic cells (DCs). Bone marrow (BM) samples were isolated from wild-type B6 mice and cultured as described in Materials and Methods. Cells were harvested and analyzed by flow cytometric analysis on day 9, using anti-nectin-1 antibody (CK6) or anti-HveA antibody (R140) combined with phycoerythrin (PE)-conjugated CD11c and allophycocyanin (APC)-conjugated MHCII antibodies. Propidium iodide (PI) was added to cells before flow cytometric analysis. (A) Dot-plot analysis of total cells. The R1 gate represents live cells that were PI negative. FSC, forward scatter. (B) Quadrant analysis of live cells. Live cells (R1 gated) were further classified into four different distinct cell populations (designated as R2 to R5) according to their surface expression of CD11c and MHCII markers. Expression levels of HveC or nectin-1 (C) or HveA (D) were analyzed by overlaid histogram analysis. Expression levels of HveC in live CD11c⁺MHCII⁺ double-positive cells (R1 and R3 gated) and in the CD11c⁺MHCII⁻ population (R1 and R5 gated) are shown by solid green and open pink curves, respectively. The expression level of HveA is shown in blue (right). The black curve represented staining of the isotype control.

FIG. 2.

HSV amplicon transduces BM-derived DCs efficiently. Day 9 BM-derived DCs were transduced with HSVeGFP at a multiplicity of infection (MOI) of 0 (A), 0.5 (B), or 1.0 (C). Cells were subsequently resuspended in R7 medium in the presence of rGM-CSF for 10 hr to allow expression of eGFP. The histogram analysis portrays the percentage of eGFP⁺ cells in the total cell population.

FIG. 3.

HSV amplicons are able to transduce all subsets of BM-derived cells. Day 9 BM-derived DCs were transduced with HSVeGFP at an MOI of 0.5 for 2 hr and cultured in R7 medium supplemented with rGM-CSF to allow expression of eGFP. Cells were then washed extensively, blocked, and stained with CD11c–PE/MHCII–APC. PI was added to the cells before flow cytometric analysis. BM-derived DCs were classified into four groups, R2 (A), R3 (B), R4 (C), and R5 (D), according to their surface expression of CD11c and MHCII as defined in Fig. 1. Solid green curves represent the expression level of eGFP in the corresponding untransduced cell population (background), and pink curves represent the expression level of eGFP on HSVeGFP-transduced cells.

FIG. 4.

mDCs (+LPS) and iDCs (–LPS) respond differently to HSV amplicon transduction. BM-derived DCs were generated as stated in the caption to Fig. 1. On day 8, half the cells were treated with LPS for 16 hr and used as mDCs, and half the cells were left untreated and used as iDCs. Before HSV amplicon transduction, the phenotypes of iDCs and mDCs were examined by flow cytometry. (A and B) Distribution of BM-derived cell populations with LPS (A) or without LPS (B) before HSV amplicon transduction. Quadrant analysis was gated on live cells (PI negative). On day 9, cells were subject to amplicon transduction at various MOIs (0, 0.5, 1.0, and 5.0), followed by phenotype characterization by flow cytometric analysis. (C) Dynamic changes in R3 and R5 populations (CD11c⁺MHCII⁺ and CD11c⁺MHCII⁻, respectively, as defined in Fig. 1) after amplicon transduction (MOI, 0 to 5). Solid columns and open columns represent the percentage of R3 and R5 cells within total live cells, respectively. Error bars indicate the standard deviation and an asterisk (*) indicates p < 0.05 as determined by paired Student t test. (D) Percentage of dead cells (PI positive) in LPS-treated and untreated BM-derived cells after HSV amplicon transduction. Error bars indicate the standard deviation.

FIG. 5.

Dynamic changes in DC surface markers occur after HSV amplicon transduction. Day 9 BM-derived cells were harvested and subjected to HSVPrPuc amplicon transduction at an MOI of 2. Excessive amplicon particles were removed by thorough washing and amplicon-transduced cells were resuspended in medium in the presence of rGM-CSF. Aliquots of these cells were collected at 2-hr intervals after transduction and their phenotypes were analyzed by flow cytometric analysis. Quadrant analysis was gated on live cells (PI negative). Expression of CD11c and MHCII on BM-derived cells was monitored in untransduced control cultures (A, G, and M) and 0 hr (B, H, and N), 2 hr (C, I, and O), 4 hr (D, J, and P), 6 hr (E, K, and Q), and 8 hr (F, L, and R) after HSV amplicon transduction. Numbers shown in quadrants of (A−F) represent the percentage of R3 (CD11c⁺MHCII⁺) in the gated live cell pool. Mean fluorescence intensity (MFI) profiles were also analyzed for MHCII⁺ cells (G−L) and CD11c⁺ cells (M−R). (S) Dynamic changes in MHCI, MHCII, CD11c, and CD80 markers on the BM-derived CD11c⁺MHCII⁺ cell subset (R3) were measured 0–8 hr after HSV amplicon transduction. The expression levels of each marker at various time points were first assessed by histogram analysis and compared with those profiles found on nontransduced cells. All flow cytometric analyses were performed independently three times and representative plots from a single experimental run are shown.

FIG. 5.

Dynamic changes in DC surface markers occur after HSV amplicon transduction. Day 9 BM-derived cells were harvested and subjected to HSVPrPuc amplicon transduction at an MOI of 2. Excessive amplicon particles were removed by thorough washing and amplicon-transduced cells were resuspended in medium in the presence of rGM-CSF. Aliquots of these cells were collected at 2-hr intervals after transduction and their phenotypes were analyzed by flow cytometric analysis. Quadrant analysis was gated on live cells (PI negative). Expression of CD11c and MHCII on BM-derived cells was monitored in untransduced control cultures (A, G, and M) and 0 hr (B, H, and N), 2 hr (C, I, and O), 4 hr (D, J, and P), 6 hr (E, K, and Q), and 8 hr (F, L, and R) after HSV amplicon transduction. Numbers shown in quadrants of (A−F) represent the percentage of R3 (CD11c⁺MHCII⁺) in the gated live cell pool. Mean fluorescence intensity (MFI) profiles were also analyzed for MHCII⁺ cells (G−L) and CD11c⁺ cells (M−R). (S) Dynamic changes in MHCI, MHCII, CD11c, and CD80 markers on the BM-derived CD11c⁺MHCII⁺ cell subset (R3) were measured 0–8 hr after HSV amplicon transduction. The expression levels of each marker at various time points were first assessed by histogram analysis and compared with those profiles found on nontransduced cells. All flow cytometric analyses were performed independently three times and representative plots from a single experimental run are shown.