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Interplay between modified vaccinia virus Ankara and dendritic cells: phenotypic and functional maturation of bystander dendritic cells - PubMed

Interplay between modified vaccinia virus Ankara and dendritic cells: phenotypic and functional maturation of bystander dendritic cells

María F Pascutti et al. J Virol. 2011 Jun.

Abstract

Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus strain, currently under evaluation as a vaccine vector in various clinical settings. It has been reported that human dendritic cells (DCs) mature after infection with MVA, but reports on the functionality of DCs have so far been controversial. In this work, we studied the phenotype and functionality of MVA-infected DCs. As previously reported, we found that human monocyte-derived DCs upregulated CD86 and HLA-DR in response to MVA infection. Moreover, infected DCs produced a broad array of chemokines and cytokines and were able to activate and induce gamma interferon (IFN-γ) production both in CD4(+) and in CD8(+) allogeneic T cells and in specific autologous peripheral blood lymphocytes (PBLs). Analysis of DC maturation following infection with a recombinant green fluorescent protein (GFP)-expressing MVA revealed that upregulation of CD86 expression was mainly observed in GFP(neg) (bystander) cells. While GFP(pos) (infected) DCs produced tumor necrosis factor alpha (TNF-α), they were unable to produce CXCL10 and were less efficient at inducing IFN-γ production in CEF-specific autologous PBLs. Maturation of bystander DCs could be achieved by incubation with supernatant from infected cultures or with apoptotic infected cells. Type I IFNs were partially responsible for the induction of CXCL10 on bystander DCs. Our findings demonstrate for the first time that, in MVA-infected DC cultures, the leading role with respect to functionality and maturation characteristics is achieved by the bystander DCs.

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Figures

Fig. 1.
Fig. 1.

MVA induces phenotypic maturation of immature DCs. DCs were either infected with MVA at different MOIs (0.1 and 1), incubated with 100 ng/ml LPS, or mock-infected and then cultured for 24 h. Cell surface expression of CD83, CD86, HLA-A, -B, and -C, and HLA-DR was analyzed by flow cytometry. (A) A representative histogram is shown for each condition and marker. (B) Results are depicted for 4 different donors as the relative mean fluorescence intensity (rMFI), calculated as [(MFI for sample − MFI for isotype)/(MFI for mock infection − MFI for isotype)]. The average rMFI value is shown for each condition and marker. Statistical differences between groups are also shown for each marker (**, P < 0.01).

Fig. 2.
Fig. 2.

MVA infection of immature DCs induces a characteristic pattern of cytokines and chemokines. Supernatant from MVA-infected (MOI = 3) or mock-infected DCs was collected at 24 h p.i., centrifuged to pellet cells, and frozen at −80°C until further use. Then, samples from 5 different donors were thawed, and an array of mediators was assayed by Luminex technology. Results are presented for chemokines (A), proinflammatory mediators (B), and T-cell polarizing cytokines (C) as the concentration of mediator found on each supernatant in ng/ml (left y axis for IL-6, TNF-a, and IL-16 [A and B] and left y axis for IL-12p40 [C]) or pg/ml (right y axis for IL-1b [B] and right y axis for IL-12p70, IFN-a2, and IL-10 [C]), as appropriate. Statistical differences between levels for MVA-infected and mock-infected DCs are shown (*, P < 0.05, and **, P < 0.01).

Fig. 3.
Fig. 3.

MVA-infected DCs are functionally mature and efficiently activate CD4+ and CD8+ T cells. (A) DCs were infected with MVA at an MOI of 1 and then washed and left to mature. As controls, DCs were either matured in the presence of 100 ng/ml LPS or mock treated. After 24 h, DCs were harvested, washed, irradiated, and placed in coculture with 2.105 allogeneic PBLs at different DC/PBL ratios. Proliferation was assessed by [3H]thymidine incorporation on day 2 of coculture. Results are shown in cpm (mean ± standard deviation [SD]) for triplicate wells of a representative experiment. (B) Cytokines in supernatants obtained after 5 days of allogeneic coculture were measured by Luminex technology. Results are presented as the concentration of mediator found on each supernatant in ng/ml (left y axis for IFN-γ) or pg/ml (right y axis for IL-17, IL-4, IL-5 and IL-13) (n = 5). Statistical difference from the level for mock-infected DCs is shown (*, P < 0.05, and **, P < 0.01). (C) Staining for activation markers was performed after 5 days of allogeneic coculture. Cells were washed, stained simultaneously with anti-CD3, anti-CD4, anti-CD8, anti-CD38, and anti-CD69 antibodies, and analyzed in a 6-color BD FACSCanto flow cytometer. Graphs show the expression of CD38 and CD69 observed on gated CD3+ CD8+ and CD3+ CD4+ cells after culture in complete medium or with mock-treated (DC mock) or MVA-infected (DC MVA) DCs, and the percentages are specified for each quadrant. (D) IFN-γ expression was analyzed by flow cytometry after 3 days of allogeneic coculture. Brefeldin A was added 12 h before the end of the culture, and then cells were harvested and stained with antibodies against human CD3, CD4, CD8, and IFN-γ. Results are shown as the percentage of IFN-γ+ cells in gated CD3+ CD4+ or CD3+ CD8+ cells. (E) DCs were infected at an MOI of 1, stimulated with 100 ng/ml LPS, or mock treated for 24 h. Then, they were washed, loaded with CEF peptides, and placed in culture with syngeneic PBLs, at different ratios, on ELISPOT plates coated with an antibody against human IFN-γ. The average number of CEF-specific IFN-γ spot-forming cells (SFC)/106 PBLs (± SD; n = 3) is depicted for each condition. In panels A, C, D, and E, results for a representative experiment of 3 are shown.

Fig. 4.
Fig. 4.

Differential phenotype and functionality of infected and bystander DCs. (A) DCs were infected with MVA-GFP at different MOIs, and GFP expression was analyzed by flow cytometry at 24 h p.i. Data are expressed as percentages of GFPpos cells for each donor. (B) Expression of maturation markers CD86 and HLA-DR was analyzed for GFPpos (infected cells) or GFPneg (bystander cells) in MVA-infected DC cultures (MOI = 2) or in mock-treated DCs. Matched isotype controls are also included, and a representative set of histograms is presented for each marker. (C) The rMFIs (calculated as described for Fig. 1B) of CD86 and HLA-DR are depicted as a function of MOI for GFPneg (bystander; upper panels) and GFPpos (infected; lower panels) cells present in the same MVA-infected culture. Each line represents data from one donor. (D) Production of TNF-α and IP-10 in DCs that were mock-treated, matured with 100 ng/ml LPS, or infected with MVA-GFP at an MOI of 2 was analyzed by flow cytometry at 20 h p.i. Brefeldin A was added during the last 12 h of culture. Results are presented as percentages of cytokine-positive cells in each quadrant from one representative experiment. (E) DCs were infected at increasing MOI, stimulated with 100 ng/ml LPS, or mock treated for 24 h. Then, they were washed, loaded with CEF peptides, and placed in culture with syngeneic PBLs at a 1:25 DC/PBL ratio on ELISPOT plates coated with an antibody against human IFN-γ. The average number of CEF-specific IFN-γ spot-forming cells (SFC)/106 PBLs (± SD; n = 3) is depicted for each condition. In panels B, D, and E, results for a representative experiment of 3 are shown.

Fig. 5.
Fig. 5.

Activation of bystander DCs can be mimicked by stimulation with supernatant from infected cultures or infected apoptotic cells. DCs were infected at an MOI of 5, washed, and left in culture. After 24 h, supernatants (SN) were harvested and filtered with a 0.22-μm syringe filter while infected cells (DC MVA) were washed repeatedly to eliminate remaining virus. (A to D) To study bystander maturation, both preparations were added in graded doses to autologous DCs. CD86 (for panel A, SN, and for panel C, DC MVA) and HLA-DR (for panel B, SN, and for panel D, DC MVA) expression was analyzed by flow cytometry after another 24 h. Results are shown as rMFIs for each marker, donor, and condition. Lines represent the mean rMFIs for each condition. Statistical difference from the level for mock-infected DCs is shown (*, P < 0.05). (E) DCs were incubated with SN at a 1:2 dilution of DC MVA at a 1:1 ratio or complete medium (DC mock) for 24 h, and then they were washed and added to allogeneic PBLs at a 1:20 ratio. PBLs incubated in complete medium were included as negative controls. IFN-γ production was assessed by flow cytometry after 3 days of coculture. Brefeldin A was added for the last 12 h, and then cells were harvested and stained with antibodies against human CD3, CD4, CD8, and IFN-γ. Results are shown as the percentage of IFN-γ+ cells in gated CD3+ CD4+ or CD3+ CD8+ cells. Results for a representative experiment of 3 are shown.

Fig. 6.
Fig. 6.

Type I IFNs contribute to IP-10 production by bystander DCs but do not affect their phenotypic maturation. (A and B) Supernatants (SN) from MVA-infected DCs were prepared as described for Fig. 5. Autologous DCs were pretreated with an antibody against human IFNAR (50 μg/ml) or isotype control antibody for 15 min at 37°C, and then SN was added, leaving the antibody at a final concentration of 10 μg/ml. Incubation with complete medium served as a negative control (mock). (A) IP-10 production was analyzed by flow cytometry after 20 h. (B) Expression of CD86 and HLA-DR was analyzed after 24 h. Results are shown as rMFIs (mean ± SD; n = 3) for each condition and marker. **, statistically different from SN (P < 0.01). (C to E) DCs were infected with MVA-GFP at an MOI of 1 in the presence of anti-IFNAR antibody (DC MVA + αIFNAR), isotype control (DC MVA), or complete medium (DC mock). (C) TNF-α and IP-10 production was analyzed as described for panel A. (D) CD86 expression was analyzed by flow cytometry after 24 h in the bulk culture (total) or in the GFPpos and GFPneg populations. Results are shown as rMFIs (mean ± SD; n = 3) for each treatment. (E) MVA-infected DCs in the presence or absence of anti-IFNAR were incubated for 24 h, washed, loaded with CEF peptides, and placed in culture with syngeneic PBLs at a 1:25 DC/PBL ratio on ELISPOT plates coated with an antibody against human IFN-γ. LPS-matured and mock-treated DCs were included as positive and negative controls, respectively. The average number of CEF-specific IFN-γ-spot forming cells (SFC)/106 PBLs (± SD; n = 3) is depicted for each condition. In all cases, results for a representative experiment of 3 are shown.

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