Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II

Abstract

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-gamma, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC.

Figure 1

Vaccinia virus (VACV) infection of primary antigen-presenting cells (APCs) inhibits the amount of interleukin (IL)-2 produced by cognate CD4⁺ T cells and the amount of nitric oxide (NO) produced by the APCs. Freshly isolated peritoneal exudate cells (PECs) (a), splenocytes (b) or dendritic cells (c) were isolated from a rat, infected with VACV Western Reserve [multiplicity of infection (MOI) = 2] for 5 hr, pulsed for 30 min with 50 nm guinea pig myelin basic protein (GPMBP), and then incubated with the cognate CD4⁺ RsL.11 T-cell line. Uninfected PECs served as a control. At 15 to 48 hpi, supernatants (50 μl) were collected and assayed for either IL-2 using the IL-2-dependent cell line CTLL (a–c) or for NO using Greiss reagent (d). For the CTLL bioassay, medium only was used to define the background control level and known IL-2-containing supernatants were used as a positive control. Proliferation was read as absorbance at 492 nm. IL-2 production was determined as the mean optical density (OD) value of the experimental group minus the background control level. *P < 0·05.

Figure 2

Vaccinia virus (VACV) effects on antigen presentation are not attributable to a decreased capacity of the RsL.11 T cell to produce interleukin (IL)-2 or the CTLL to respond to IL-2. (a) RsL.11 T cells were infected with WR [multiplicity of infection (MOI) = 4] for 4 hr and then incubated with decreasing numbers of antigen (Ag)-pulsed peritoneal exudate cells (PECs). After 24–48 hr, supernatants (50 μl) were collected and assayed for IL-2 production using the CTLL IL-2 bioassay. (b) RsL.11 T lymphocytes were uninfected or infected for 3–4 hr (dark bars) and then stimulated with PECs/50 nm GPMBP, 25 μg/ml Con A, or 100 nm PMA/2 μm ionomycin. Supernatants (50 μl) were collected at 20 and 40 hr and assayed for IL-2. (c) RsL.11 T cells were infected with VACV for 10 hr, and growth metabolism was measured by reduction of 10 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt/phenazine methosulphate (MTS/PMS); absorbance at 492 nm. (d) CTLL cells were infected with VACV (MOI = 5) for 4 hr and then placed in media with varying amounts of an IL-2-containing supernatant. Proliferation was measured by adding 10 μl of MTS/PMS.

Figure 3

Increased antigen concentration augments the antigen presentation capacity of infected antigen-presenting cells (APCs). Peritoneal exudate cells (PECs) were infected with vaccinia virus (VACV) [multiplicity of infection (MOI) = 2] for 4 hr and then pulsed with a titration of guinea pig myelin basic protein (GPMBP; 50–250 nm) before being co-cultured with antigen-specific T cells. Supernatants (50 μl) were collected at 24 to 48 hours post infection (hpi) and assayed for interleukin (IL)-2 using the CTLL IL-2 bioassay.

Figure 4

Vaccinia virus (VACV)-infected mouse B cells are impaired in their ability to stimulate antigen-specific CD4⁺ T cells to produce interleukin (IL)-2. (a) 25 000 1153 B cells were infected for 6 hr, and then pulsed with the HEL74-88 antigen for 1 hr, followed by the addition of 25 000 B04 T cells. After a 24- to 48-hr incubation, supernatants (50 μl) were collected and assayed for IL-2 using the CTLL IL-2 bioassay. (b) 25 000 B04 T cells were infected for 24 hr. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt/phenazine methosulphate (MTS/PMS) was then added to measure metabolism. *P < 0.01.

Figure 5

Vaccinia virus (VACV) decreases the amount of nitric oxide (NO) that is produced by stimulated macrophages. Peritoneal exudate cells (PECs) (a) or RAW 264.7 macrophages (b) were infected at a multiplicity of infection (MOI) of 1 for 4 hr, and were then stimulated to produce NO by the addition of lipopolysaccharide (LPS), interferon (IFN)-γ, or a combination of both. Supernatants (50 μl) were collected at 24–48 hr and then assayed for NO production by the addition of Greiss reagent. Absorbance was read at 540 nm. *P < 0·01.

Figure 6

Vaccinia virus (VACV) globally reduces cytokine synthesis by antigen-presenting cells (APCs) and responding T cells. Peritoneal exudate cells (PECs) were harvested from Lewis rats, infected with VACV Western Reserve [multiplicity of infection (MOI) = 2] for 5 hr, pulsed with 50 nm guinea pig myelin basic protein (GPMBP), and then co-cultured with the cognate CD4⁺ RsL.11 T-cell line. Supernatants (50 μl) were collected at 24 hours post infection (hpi) and assayed for the production of cytokines using the LincoPlex 24 rat cytokine/chemokine Luminex bead immunoassay kit. Interleukin (IL)-2 and interferon (IFN)-α values were divided by 20 and 100, respectively (hatch marks) to fit to scale. *P < 0·05.

Figure 7

Vaccinia virus (VACV) decreases major histocompatibility complex (MHC) class II expression. Peritoneal exudate cells (PECs) or 1153 cells were infected with VACV for 5 hr, washed and incubated with no primary antibody [mean fluorescence intensity (MFI) for 1153: uninfected cells, 8·4 ± 0·59; VACV-infected cells, 5·4 ± 0·44; PECs uninfected 2·35 ± 0·06; VACV-infected 2·38 ± 0·48], anti-class II MHC (MFI for uninfected cells, 145 ± 13·4; VACV-infected cells, 76 ± 2·8), or anti-CD45 and then fluorescein isothiocyanate (FITC)-conjugated secondary antibody and analysed by flow cytometry.

Figure 9

Vaccinia virus (VACV) induces apoptosis in macrophages. Peritoneal exudate cells (PECs) (a) or RAW 264.7 macrophages (b) were infected with VACV for 4 hr [MOI = 2]. 1153 B cells (c) were infected at an MOI = 5 for 6 hr. Apoptosis was assessed by staining cells with annexinV-fluorescein isothiocyanate (FITC) and propidium iodide (PI) and analysed by flow cytometry. Variation was not higher than 2% between duplicate samples.

Figure 8

Vaccinia virus (VACV) inhibits the metabolism of macrophages. Peritoneal exudate cells (PECs) (a) were infected with VACV [multiplicity of infection (MOI) = 2], and 1153 B cells (b) at an MOI of 5 and metabolism was measured by the reduction of 10 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt/phenazine methosulphate (MTS/PMS). Standard deviation error bars are shown on all figures but are too small to be visible on some graphs. *P < 0·05.

Figure 10

Vaccinia virus (VACV) does not replicate to high titres in peritoneal exudate cells (PECs). BS-C-1 cells or PECs were infected with VACV [multiplicity of infection (MOI) = 10]. Cells were collected at designated times post-infection. Cell-associated virus was titrated on BS-C-1 monolayers. PFU, plaque-forming units.

Figure 11

Vaccinia virus (VACV) decreases peptide–major histocompatibility complex (MHC) complexes. Spleens were harvested from B10.A-H2^i5 H2-T18^a/(5R)SgSnJ mice and uninfected or VACV-infected with purified virus for 3 hr [multiplicity of infection (MOI) = 10] and then incubated with biotin-conjugated Y-Ae and streptavidin-R-phycoerythrin. C57Bl/6 mice and were used as a negative control and showed no staining above background.