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Comparative Study
. 2001 Jun;75(11):5174-81.
doi: 10.1128/JVI.75.11.5174-5181.2001.

Reduced expression of HLA class II molecules and Iinterleukin-10- and transforming growth factor beta1-independent suppression of T-cell proliferation in human cytomegalovirus-infected macrophage cultures

J Odeberg  1 , C Söderberg-Nauclér
Affiliations
Comparative Study

Reduced expression of HLA class II molecules and Iinterleukin-10- and transforming growth factor beta1-independent suppression of T-cell proliferation in human cytomegalovirus-infected macrophage cultures

J Odeberg et al. J Virol. 2001 Jun.

Abstract

After a primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in myeloid lineage cells, and the virus has developed several mechanisms to avoid immune recognition and destruction of infected cells. In this study, we show that HCMV utilizes two different strategies to reduce the constitutive expression of HLA-DR, -DP, and -DQ on infected macrophages and that infected macrophages are unable to stimulate a specific CD4+ T-cell response. Downregulation of the HLA class II molecules was observed in 90% of the donor samples and occurred in two phases: at an early (1 day postinfection [dpi]) time point postinfection and at a late (4 dpi) time point postinfection. The early inhibition of HLA class II expression and antigen presentation was not dependent on active virus replication, since UV-inactivated virus induced downregulation of HLA-DR and inhibition of T-cell proliferation at 1 dpi. In contrast, the late effect required virus replication and was dependent on the expression of the HCMV unique short (US) genes US1 to -9 or US11 in 77% of the samples. HCMV-treated macrophages were completely devoid of T-cell stimulation capacity at 1 and 4 dpi. However, while downregulation of HLA class II expression was rather mild, a 66 to 90% reduction in proliferative T-cell response was observed. This discrepancy was due to undefined soluble factors produced in HCMV-infected cell cultures, which did not include interleukin-10 and transforming growth factor beta1. These results suggest that HCMV reduces expression of HLA class II molecules on HCMV-infected macrophages and inhibits T-cell proliferation by different distinct pathways.

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Figures

FIG. 1
FIG. 1
Expression of HCMV antigens, CD14, and HLA-DR, -DQ, and -DP in HCMV-infected macrophages. (A) HCMV-infected macrophages were fixed at 7 dpi and stained for the IE and glycoprotein B (gB) antigens. The figure shows the mean percentage (± standard error) of HCMV-infected macrophages at 7 dpi (n = 6). (B) Flow cytometric analysis was performed with uninfected (gray line) and HCMV-infected (black line) macrophages by using antibodies directed against HLA-DR, -DQ, and -DP and CD14. The figure shows a representative example of an analysis of HCMV's effect on the expression of HLA class II molecules and CD14 on macrophages from one donor at 4 dpi.
FIG. 2
FIG. 2
Importance of the HCMV US1 to -9 and US11 genes for downregulation of HLA class II molecules at 4 dpi, but not at 1 dpi. Macrophages were infected with HCMV AD169 or the HCMV AD169 mutant strain RV670, which lacks the genes US1 to -9 and US11 (n = 17). At 1 dpi, HCMV infection or HCMV RV670 infection resulted in decreased expression of HLA-DR, -DQ, or -DP in 88 and 80% of the samples, respectively. A decreased expression of HLA class II molecules was observed in 72% of the donors at 4 dpi. In 77% of these samples, RV670 did not affect the expression of HLA class II molecules.
FIG. 3
FIG. 3
Importance of viral replication for downregulation of HLA class II surface expression at 1 dpi. Flow cytometric analysis of HLA-DR expression was performed at 1 dpi on uninfected and HCMV-, UV-HCMV-, and HCMV-IVIG-infected macrophages. The reduced expression of HLA-DR on HCMV AD169-infected macrophages (A) was similar to the reduced expression induced by UV-irradiated HCMV (B). However, an effect on the HLA-DR expression was not detected on macrophages infected with IVIG-neutralized HCMV (C).
FIG. 4
FIG. 4
Decreased CD4+ T-cell proliferation in HCMV-infected macrophage cultures. Uninfected (gray) and HCMV-infected (black) macrophages were tested at 1 and 4 dpi for their ability to present tetanus peptides to specific CD4+ T-cell clones. Panels A and B show representative examples of the experiments. (A) The proliferative T-cell response was reduced by 66 to 90% at 1 dpi and by 76 to 89% at 4 dpi in HCMV-infected macrophage cultures. (B) Macrophages were infected with UV-treated HCMV (white) and tested for their ability to present tetanus toxoid peptides to CD4+ T-cell clones. The proliferative T-cell response was reduced by 72 to 86% by UV-treated HCMV at 1 dpi. Thus, UV treatment of HCMV also results in inhibition of CD4+ T-cell proliferation.
FIG. 5
FIG. 5
Soluble factor or factors produced by HCMV-infected macrophages suppress T-cell proliferation. Supernatants from uninfected or HCMV-infected macrophage cultures were incubated with PBMCs and PHA. T-cell proliferation was measured (A), and expression of the activation markers CD69 and CD45RO was analyzed by flow cytometry (B) at 3 days poststimulation. Panel A shows inhibition of PHA-induced T-cell proliferation by 97% in the presence of supernatants from HCMV-infected macrophage cultures (gray), compared to that in uninfected macrophages (hatched bar). In panel B; CD4 + T cells express the activation markers CD69 and CD45RO after PHA stimulation in the presence of supernatants from uninfected or HCMV-infected macrophage cultures; unstimulated PBMCs (a and d), PHA-stimulated PBMCs in the presence of supernatants from uninfected macrophage cultures (b and e), and PHA-stimulated PBMCs in the presence of supernatants from HCMV-infected macrophage cultures (c and f).
FIG. 6
FIG. 6
IL-10 (A) and TGF-β1 (B) production is not affected by HCMV infection in macrophages. The production of IL-10 or TGF-β1 was measured in supernatants from uninfected (hatched bars) or HCMV (gray bars)- and UV-HCMV (white bars)-infected macrophages at 4 and 7 dpi. A significant difference in the production of IL-10 or TGF-β1 was not demonstrated in supernatants obtained from uninfected or HCMV- and UV-HCMV-infected macrophages.
FIG. 7
FIG. 7
Blocking of the IL-10R or the TGF-βIIR does not affect T-cell proliferation. IL-10R and TGF-βIIR on PBMCs were blocked with polyclonal antibodies and then stimulated with PHA in the presence of supernatants from uninfected or HCMV-infected macrophages. Antibody blocking of the IL-10R (A) or the TGF-βIIR (B) did not enhance T-cell proliferation in the presence of supernatants from HCMV-infected macrophage cultures.
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