Viral replication-independent blockade of dendritic cell maturation and interleukin-12 production by human herpesvirus 6

Abstract

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive CD4(+)-T-lymphotropic betaherpesvirus that causes severe human thymocyte depletion in heterochimeric SCID-hu thy/liv mice and has been implicated as a potential cofactor in the progression of AIDS. However, the mechanisms of HHV-6-mediated immunosuppression have not yet been fully elucidated. We investigated the phenotypic and functional alterations induced by HHV-6 on peripheral blood-derived human dendritic cells (DC). The infection of DC with HHV-6 A or B was nonproductive, as revealed by calibrated real-time PCR measuring the accumulation of viral genome equivalents over time. Nevertheless, preexposure to HHV-6 markedly impaired the maturation of DC driven by gamma interferon and lipopolysaccharide, as shown by the reduced surface expression of major histocompatibility complex class I molecules, HLA-DR, CD40, and CD80. Moreover, HHV-6, but not the closely related betaherpesvirus HHV-7, dramatically suppressed the secretion of interleukin-12 (IL-12) p70 by DC, while the production of other cytokines that influence DC maturation, i.e., IL-10 and tumor necrosis factor alpha, was not significantly modified. Likewise, the secretion of the CC chemokines macrophage inflammatory protein 1beta and RANTES was unaltered. Functionally, a pretreatment with HHV-6 impaired the ability of DC to stimulate allogeneic T-cell proliferation. Altogether, these data identify interference with the functional maturation of DC as a potential mechanism of HHV-6-mediated immunosuppression.

FIG. 1.

Lack of productive HHV-6 infection in DC. DC or PHA-activated CBMC were infected with purified HHV-6 A virions at an approximate MOI of 1. After 1, 3, and 6 days, the cells were harvested and analyzed for viral DNA content by use of a quantitative calibrated real-time PCR assay. Human β-actin DNA was measured in parallel to normalize the cell numbers. The data shown are from two representative DC donors and one CBMC donor and are expressed as HHV-6 genome equivalents per cell.

FIG. 2.

Inhibition of IFN-γ- and LPS-induced DC maturation by HHV-6. DC were preexposed to purified HHV-6 A virions and then cultured for 40 h in the absence (A) or presence (B) of IFN-γ and LPS. The cells were then harvested and analyzed for surface expression of CD40, MHC classes I and II, and CD80 by fluorocytometry. Profiles of HHV-6-treated DC are shown as black lines, while those of uninfected controls are shown as gray lines. The number on each line denotes the mean fluorescence intensity value. Representative data from one of three donors tested are shown.

FIG. 3.

Inhibition of IL-12 p70 production from primary human DC by HHV-6. Human peripheral blood-derived DC were preexposed to purified HHV-6 virions and then stimulated with IFN-γ plus LPS or left unstimulated. Control DC were not preexposed to HHV-6. Culture supernatants were analyzed for IL-12 p70 levels by ELISA 24 h after the addition of LPS. (A) Mean results (± SD) for DC from four donors that were pretreated with HHV-6 subgroup A (strain GS). (B) Mean results (± SE) for DC from two donors that were pretreated with HHV-6 subgroup B (strain PL-1).

FIG. 4.

Failure of HHV-6 to modulate IL-10, TNF-α, MIP-1β, and RANTES production from activated DC. Culture supernatants of DC that had been preexposed to purified HHV-6 A virions and then either left unstimulated or stimulated with IFN-γ and LPS were tested for IL-10 (A), TNF-α (B), MIP-1β (C), and RANTES (D) levels by ELISAs. Mean results (± SD) for five donors are shown in panels A to C, and mean results for three donors are presented in panel D.

FIG. 5.

Failure of HHV-7 to inhibit IL-12 p70 production from primary human DC. Human peripheral blood-derived DC were preexposed to purified HHV-7 virions at an approximate MOI of 1 and then stimulated with IFN-γ plus LPS or left unstimulated. Control DC were not preexposed to HHV-7. Culture supernatants were analyzed for IL-12 p70 levels by ELISA 24 h after the addition of LPS. Mean results (± SE) for DC from two donors are shown.

FIG. 6.

Reduced proliferation of T cells by allogeneic DC pretreated with HHV-6. Peripheral blood-derived DC were treated with purified UV-inactivated HHV-6 virions overnight and then matured with IFN-γ and LPS or left immature; mock-treated DC were exposed to uninfected culture medium under the same conditions. A total of 10⁵ allogeneic T cells were incubated with various numbers of either immature (A) or mature (B) DC. Five days later, the cells were incubated with radiolabeled thymidine and then were harvested and analyzed. Representative data (means ± SD for triplicate wells) for one of three donors tested are shown.