Murine cytomegalovirus inhibits interferon gamma-induced antigen presentation to CD4 T cells by macrophages via regulation of expression of major histocompatibility complex class II-associated genes

Abstract

CD4 T cells and interferon gamma (IFN-gamma) are required for clearance of murine cytomegalovirus (MCMV) infection from the salivary gland in a process taking weeks to months. To explain the inefficiency of salivary gland clearance we hypothesized that MCMV interferes with IFN-gamma induced antigen presentation to CD4 T cells. MCMV infection inhibited IFN-gamma-induced presentation of major histocompatibility complex (MHC) class II associated peptide antigen by differentiated bone marrow macrophages (BMMphis) to a T cell hybridoma via impairment of MHC class II cell surface expression. This effect was independent of IFN-alpha/beta induction by MCMV infection, and required direct infection of the BMMphis with live virus. Inhibition of MHC class II cell surface expression was associated with a six- to eightfold reduction in IFN-gamma induced IAb mRNA levels, and comparable decreases in IFN-gamma induced expression of invariant chain (Ii), H-2Ma, and H-2Mb mRNAs. Steady state levels of several constitutive host mRNAs, including beta-actin, cyclophilin, and CD45 were not significantly decreased by MCMV infection, ruling out a general effect of MCMV infection on mRNA levels. MCMV effects were specific to certain MHC genes since IFN-gamma-induced transporter associated with antigen presentation (TAP)2 mRNA levels were minimally altered in infected cells. Analysis of early upstream events in the IFN-gamma signaling pathway revealed that MCMV did not affect activation and nuclear translocation of STAT1alpha, and had minor effects on the early induction of IRF-1 mRNA and protein. We conclude that MCMV infection interferes with IFN-gamma-mediated induction of specific MHC genes and the Ii at a stage subsequent to STAT1alpha activation and nuclear translocation. This impairs antigen presentation to CD4 T cells, and may contribute to the capacity of MCMV to spread and persist within the infected host.

Figure 1

MCMV infection impairs IFN-γ–enhanced peptide presentation by MHC class II. 129 mouse BMMφs were mock infected or infected with MCMV at an MOI of 5.0 for 1 h, and stimulated with IFN-γ (100 IU/ml) or medium alone. After 24 h BMMφs were assayed for ability to present peptide antigen, and analyzed for MHC class II expression by flow cytometry. (A) Mφs were plated at 10³, 5 × 10³, 10⁴, and 5 × 10⁴ cells per well, and incubated with the T cell hybridoma B11 (β-galactosidase 429-441, IA^b-restricted) in the presence of β-galactosidase peptide (429–441) at a concentration of 10 μM. Supernatants were harvested and assayed for IL-2 content using [³H]thymidine incorporation by CTLL2 cells. Mock + IFN-γ and MCMV + IFN-γ BMMφs were mixed 1:1 and assayed as a control for potential toxic effects of MCMV-infected cells. Similar data was obtained at a peptide concentration of 1.0 μM. Data is from one of two similar experiments. All groups were plated in triplicate and are shown as mean cpm ± SEM. (B) Cell surface expression of the MHC class II molecule IA^b on BMMφs infected with MCMV and stimulated with IFN-γ as above was measured by flow cytometry.

Figure 1

MCMV infection impairs IFN-γ–enhanced peptide presentation by MHC class II. 129 mouse BMMφs were mock infected or infected with MCMV at an MOI of 5.0 for 1 h, and stimulated with IFN-γ (100 IU/ml) or medium alone. After 24 h BMMφs were assayed for ability to present peptide antigen, and analyzed for MHC class II expression by flow cytometry. (A) Mφs were plated at 10³, 5 × 10³, 10⁴, and 5 × 10⁴ cells per well, and incubated with the T cell hybridoma B11 (β-galactosidase 429-441, IA^b-restricted) in the presence of β-galactosidase peptide (429–441) at a concentration of 10 μM. Supernatants were harvested and assayed for IL-2 content using [³H]thymidine incorporation by CTLL2 cells. Mock + IFN-γ and MCMV + IFN-γ BMMφs were mixed 1:1 and assayed as a control for potential toxic effects of MCMV-infected cells. Similar data was obtained at a peptide concentration of 1.0 μM. Data is from one of two similar experiments. All groups were plated in triplicate and are shown as mean cpm ± SEM. (B) Cell surface expression of the MHC class II molecule IA^b on BMMφs infected with MCMV and stimulated with IFN-γ as above was measured by flow cytometry.

Figure 2

Infectious MCMV impairs IFN-γ–induced MHC class II expression on Mφs in a dose-dependent manner. IFN-α/βR^−/− BMMφs were mock infected or infected with live or UV-inactivated MCMV at an MOI of 1.5, 3.0, or 6.0 for 1 h before addition of 100 IU/ml IFN-γ. Mφs were harvested 48 h after infection and assayed for MHC class II expression (IA^b) by flow cytometry. Shown is one of three experiments, each of which yielded similar results.

Figure 3

MCMV-infected BMMφs express low levels of MHC class II. IFN-α/βR^−/− BMMφs were either mock infected or infected with recombinant MCMV expressing β-galactosidase (RM427) at an MOI of 0.6. 1 h after infection, cells were treated with IFN-γ (100 IU/ml) or medium alone for 48 h. Cells were harvested, stained for MHC class II (IA^b) expression, and separated into MHC class II high and low populations by fluorescent activated cell sorting. The numbers of cells expressing β-galactosidase in total and sorted cell populations was determined by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (x-gal) staining. (A) Mock-infected or infected cells (top panels), and IA^b high and low populations from infected cultures (bottom panels) were stained for IA^b. The dotted line represents staining on unstimulated, mock-infected cells. The solid line depicts staining of IFN-γ– treated groups. (B) x-gal staining of mock- or MCMV-infected, and MHC class II high and low, populations is shown from one of three similar experiments.

Figure 3

MCMV-infected BMMφs express low levels of MHC class II. IFN-α/βR^−/− BMMφs were either mock infected or infected with recombinant MCMV expressing β-galactosidase (RM427) at an MOI of 0.6. 1 h after infection, cells were treated with IFN-γ (100 IU/ml) or medium alone for 48 h. Cells were harvested, stained for MHC class II (IA^b) expression, and separated into MHC class II high and low populations by fluorescent activated cell sorting. The numbers of cells expressing β-galactosidase in total and sorted cell populations was determined by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (x-gal) staining. (A) Mock-infected or infected cells (top panels), and IA^b high and low populations from infected cultures (bottom panels) were stained for IA^b. The dotted line represents staining on unstimulated, mock-infected cells. The solid line depicts staining of IFN-γ– treated groups. (B) x-gal staining of mock- or MCMV-infected, and MHC class II high and low, populations is shown from one of three similar experiments.

Figure 4

Supernatants from MCMV-infected Mφs inhibit IFN-γ induction of MHC class II on normal but not IFN-α/βR^−/− BMMφs. Wild-type 129 BMMφs were either mock infected or infected with MCMV at an MOI of 5.0. 48 h after infection, supernatants were removed, ultracentrifuged to remove free virus, and placed on naive cultures of either 129 or IFN-α/βR^−/− BMMφs. At the time of supernatant transfer, additional cultures were infected with MCMV (MOI = 5.0) as positive controls. After 1 h of incubation with the supernatants or MCMV, cells were treated with IFN-γ (100 IU/ml), incubated for 48 h, and harvested. MHC class II (IA^b) expression was determined by flow cytometry. Shown is data from one of two experiments yielding similar results. (US, unstained).

Figure 5

MCMV infection reduces IFN-γ–induced MHC class II and Ii mRNA levels. IFN-α/βR^−/− BMMφs were either mock infected or infected with MCMV or UV-inactivated MCMV at an MOI of 5.0. 1 h after infection, cells were treated with medium alone or with IFN-γ (100 IU/ml). Total cellular RNA was harvested at either 24 or 48 h after infection and analyzed by Northern blot hybridization for the IA^b beta chain or Ii. β-actin hybridization is also shown. Similar results were obtained in three separate experiments.

Figure 8

MCMV infection inhibits H-2Ma, H-2Mb, and TAP1 mRNA expression, but not IFN-γ–induced TAP2 mRNA expression. IFN-α/ βR^−/− BMMφ were either mock infected or infected with MCMV or UV-inactivated MCMV at an MOI of 5.0 in the presence or absence of IFN-γ (100 IU/ml) for 24 h. Total cellular RNA was harvested and analyzed by Northern blot. Blots were probed using radiolabeled probes for murine TAP1, TAP2, H-2Ma, H-2Mb, cyclophilin, and 28S ribosomal RNA. Shown is one of three experiments which yielded similar results.

Figure 6

STAT1α activation and nuclear translocation after IFN-γ stimulation is normal in MCMV-infected Mφs. IFN-α/ βR^−/− BMMφs were infected with MCMV at an MOI of 5.0 or mock infected for 1 or 24 h. Harvested cells were incubated with IFN-γ for 10 min and nuclear extracts were prepared. Extracts were assayed for STAT1α activation by EMSA against a ³²P-labeled IFN-γ activation sequence containing oligonucleotide derived from the murine FcγR1 promoter. The presence of STAT1α in the complex was determined by antibody super-shift using STAT1α-specific antiserum. Shown are STAT1α activation by IFN-γ at 1 h (100 IU/ml IFN-γ) or 24 h (5 IU/ml IFN-γ) after infection. Similar results were obtained in four separate experiments.

Figure 7

IRF-1 mRNA and DNA binding activity is inducible by IFN-γ in MCMV-infected Mφs. (A) IFN-α/βR^−/− were either mock infected or infected with MCMV at an MOI of 5.0 for 3 h. Cultures were treated with IFN-γ for the final 2 h of infection and assayed for IRF-1 mRNA levels by Northern blot hybridization. Shown is one of two experiments yielding similar results. (B) IFN-α/βR^−/− BMMφs were infected with MCMV at an MOI of 5.0 or mock infected for 5 h. Cells were treated with IFN-γ for the final 4 h of infection and nuclear extracts were prepared. IRF-1 DNA binding activity was assessed by incubating extract with a ³²P-labeled IRF-E oligonucleotide probe, which was then analyzed by EMSA. The presence of IRF-1 within the complex was confirmed by incubation with antiserum against murine IRF-1 or STAT1α. Three separate experiments yielded similar results.

Figure 7

IRF-1 mRNA and DNA binding activity is inducible by IFN-γ in MCMV-infected Mφs. (A) IFN-α/βR^−/− were either mock infected or infected with MCMV at an MOI of 5.0 for 3 h. Cultures were treated with IFN-γ for the final 2 h of infection and assayed for IRF-1 mRNA levels by Northern blot hybridization. Shown is one of two experiments yielding similar results. (B) IFN-α/βR^−/− BMMφs were infected with MCMV at an MOI of 5.0 or mock infected for 5 h. Cells were treated with IFN-γ for the final 4 h of infection and nuclear extracts were prepared. IRF-1 DNA binding activity was assessed by incubating extract with a ³²P-labeled IRF-E oligonucleotide probe, which was then analyzed by EMSA. The presence of IRF-1 within the complex was confirmed by incubation with antiserum against murine IRF-1 or STAT1α. Three separate experiments yielded similar results.