Pathogenic effects of human herpesvirus 6 in human lymphoid tissue ex vivo

Abstract

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of human immunodeficiency virus disease. However, the lack of suitable experimental models has hampered the elucidation of the mechanisms of HHV-6-mediated immune suppression. Here, we used ex vivo lymphoid tissue to investigate the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both HHV-6 subgroups (A and B) were able to productively infect human tonsil tissue fragments in the absence of exogenous stimulation. The majority of viral antigen-expressing cells were CD4(+) T lymphocytes expressing a nonnaive phenotype, while CD8(+) T cells were efficiently infected only with HHV-6A. Accordingly, HHV-6A infection resulted in the depletion of both CD4(+) and CD8(+) T cells, whereas in HHV-6B-infected tissue CD4(+) T cells were predominantly depleted. The expression of different cellular antigens was dramatically altered in HHV-6-infected tissues: whereas CD4 was upregulated, both CD46, which serves as a cellular receptor for HHV-6, and CD3 were downmodulated. However, CD3 downmodulation was restricted to infected cells, while the loss of CD46 expression was generalized. Moreover, HHV-6 infection markedly enhanced the production of the CC chemokine RANTES, whereas other cytokines and chemokines were only marginally affected. These results provide the first evidence, in a physiologically relevant study model, that HHV-6 can severely affect the physiology of secondary lymphoid organs through direct infection of T lymphocytes and modulation of key membrane receptors and chemokines.

FIG. 1.

Replication of HHV-6 in human lymphoid tissue ex vivo. (A) Kinetics of HHV-6A and -6B replication. Culture medium was changed every three days, and HHV-6 DNA was quantified in these medium samples. Typical replication kinetics are presented. The insets represent the average kinetic of replication of HHV-6A (n = 5) and HHV-6B (n = 5). Results are shown as mean ± SEM (error bars). To pool the data from different experiments, the amount of HHV-6 DNA for each experiment was normalized as percent of the maximum production of HHV-6 over the 15-day period. (B) Expression of HHV-6 antigen by lymphocytes as detected by flow cytometry. A representative experiment (out of nine) is shown. Contour plots (at 2% probability, gated on lymphocytes) represent CD2 versus HHV-6 pp41/38 for control and HHV-6A- and HHV-6B-infected tissues.

FIG. 2.

Phenotype of HHV-6-infected cells in human lymphoid tissue ex vivo. (A) Mean proportion of CD4⁺ and CD8⁺ T cells productively infected with HHV-6. At day 12 or 13 postinfection, the cells were stained with anti-CD2 TriColor, anti-CD4-APC or anti-CD8-APC, as well as with anti-HHV-6 nuclear phosphoprotein pp41 antibody coupled with Alexa 488. The results represent the mean ± SEM (error bars) from five experiments with HHV-6A and three experiments with HHV-6B, each performed with 54 tissue blocks derived from an individual tonsil donor. (B) Mean proportion of naive and memory cells productively infected with HHV-6. At day 12 or 13 postinfection, the cells were stained with anti-CD2 PE, anti CD62L TriColor and anti-CD45 RA APC and with HHV-6 nuclear protein pp41 antibody coupled with Alexa 488. Average data obtained from tissues from 54 blocks of tissues from five donors for HHV-6A and from three donors for HHV-6B are presented. Results are shown as mean ± SEM (error bars).

FIG. 3.

Depletion of CD4⁺ and CD8⁺ T cells by HHV-6 in human lymphoid tissue ex vivo. At day 12 or 13 postinfection, the cells were stained with anti-CD3 fluorescein isothiocyanate, anti-CD4 PE, and anti-CD8 TriColor. To enumerate the cells, we used TrueCount tubes containing a known number of fluorescent beads. The numbers of cells in infected tissue blocks were expressed as percent of that in matched uninfected tissues. The results represent the mean ± SEM (error bars) from seven experiments with HHV-6A and 6 experiments with HHV-6B, each performed with 54 tissue blocks derived from an individual tonsil donor.

FIG. 4.

Downmodulation of cell surface antigens by HHV-6 in human lymphoid tissue ex vivo. At day 12 or 13 postinfection, the cells were stained with anti-CD46 PE, CD2 TriColor, CD3 APC and anti-HHV-6 nuclear protein pp41/38 antibody coupled with Alexa 488. (A) The left panel shows downmodulation of CD46, expressed as the percent median fluorescence intensity (MFI) in control tissue. The right panel shows typical contour plots at 2% probability representing CD46 versus HHV-6 pp41/38 for a HHV-6A infected tissue gated on CD2⁺ lymphocytes. (B) The left panel shows downmodulation of CD3 expressed as percent MFI in control tissue. The right panel shows typical contour plots at 2% probability representing CD3 versus HHV-6 pp41/38 for an HHV-6A-infected tissue gated on CD2⁺ lymphocytes. (C) Upregulation of CD4 is expressed as a percent MFI in control tissue. The right panel shows typical contour plots at 2% probability representing CD3 versus HHV-6 pp41/38 for a HHV-6A infected tissue gated on CD2⁺ lymphocytes. In all panels, the results represent the mean ± SEM (error bars) from five experiments with HHV-6A and three with HHV-6B, each performed with 54 tissue blocks derived from an individual tonsil donor.

FIG. 5.

Modulation of cytokine and chemokine secretion by HHV-6 in human lymphoid tissue ex vivo. Production of cytokines and chemokines accumulated in tissues infected with HHV-6 over 13 days of infection. The results represent the mean ± SEM (error bars) from 8 to 11 experiments with HHV-6A and 3 to 6 experiments with HHV-6B, each performed with 54 tissue blocks derived from an individual tonsil donor. The production of chemokines in infected tissues is expressed as percent of the production in matched uninfected tissues.

FIG. 6.

Correlation between RANTES production and HHV-6 replication in ex vivo-infected tissues. Log regression analysis of data obtained from tissues of five donors infected ex vivo with HHV-6A (A) or HHV-6B (B) over a 12-day period is shown. The regression analysis was performed by plotting the concentration of RANTES and the number of HHV-6 genome equivalents per microliter of culture supernatant (log-transformed).