Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent

Abstract

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4(+) and CD8(+) T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14(+) monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-gamma) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-gamma was crucial for reactivation of latent HCMV, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-gamma in the reactivation of HCMV.

FIG. 1

Expression of HCMV antigens IE and gB and virus growth in Allo-MDM and ConA-MDM. Parallel cultures of Allo-MDM and ConA-MDM were established as described in Materials and Methods, and the cultures (n = 4 with duplicates) were infected with HCMV. Cells were fixed at different time points after infection, and the expression of HCMV proteins was detected by double-label immunocytochemistry. HCMV IE proteins were observed earlier in Allo-MDM than in ConA-MDM. In addition, a sixfold increase in the number of HCMV-positive cells was detected in Allo-MDM compared to ConA-MDM (A). HCMV infection of fibroblasts, Allo-MDM, or PBMC from HLA-identical twins was performed at an MOI of 1 and with ConA-MDM at an MOI of 10 (B). Infectious virus was detected at 3 days postinfection in HF as well as Allo-MDM, whereas similar levels of infectious virus were detected in ConA-MDM first at 7 days postinfection. Virus was not produced in macrophage cultures, which were established by mixing PBMC from two HLA-identical twins. Similar amounts of HCMV were produced in the supernatants of infected Allo-MDM as well as HF cells, but not ConA-MDM. Bars represent the standard deviation of the mean.

FIG. 2

Allogeneically induced differentiation of HCMV-permissive Allo-MDM is dependent on the presence of CD4⁺ and CD8⁺ T lymphocytes and HLA class I and II molecules. Allo-MDM cultures were established by negative selection of either CD4⁺ or CD8⁺ T lymphocytes from the respective donors before allogeneic stimulation (n = 6 with duplicates). The expression of the HCMV-encoded IE antigen in Allo-MDM was determined by immunofluorescence staining at day 7 postinfection. Depletion of CD4⁺ cells inhibited IE expression by 55 to 60%, while depletion of CD8⁺ cells reduced viral expression by 60 to 80% (A). Depletion of CD4⁺ and CD8⁺ T cells also inhibited the production of virus in Allo-MDM cultures by 1,000-fold and 10,000-fold, respectively (B). Blocking of HLA class I or HLA class II molecules with neutralizing antibody prior to allogeneic stimulation of PBMC also inhibited HCMV IE expression (C) and viral production (D), which correlated with the CD4⁺ and CD8⁺ depletion experiments.

FIG. 3

Allo-MDM and ConA-MDM display differences in kinetics and production of cytokines. To compare cytokine production in Allo-MDM and ConA-MDM cultures, PBMC from six histoincompatible donors were stimulated by either allogeneically matching different pairs within the group or stimulating cells from individuals mitogenically. Following adherence of activated PBMC, nonadherent cells were removed from the cultures at 24 h poststimulation, and supernatants were collected from the cultures at the indicated intervals. Parallel cultures of Allo-MDM (red), ConA-MDM (blue), and unstimulated PBMC (green) were established as described in Materials and Methods. Cytokine-specific ELISA analysis was used to determine cytokine expression kinetics in cell-free culture supernatants collected at 6, 12, 24, 36, and 48 h and at 3, 5, and 8 days postisolation. Cytokines analyzed include IFN-γ (A), TNF-α (B), IL-2 (C), IL-3 (D), GM-CSF (E), G-CSF (F), M-CSF (G), IL-10 (H), IL-13 (I), IL-7 (J), TGF-β (K), IL-1β (L), and IL-6 (M).

FIG. 3

Allo-MDM and ConA-MDM display differences in kinetics and production of cytokines. To compare cytokine production in Allo-MDM and ConA-MDM cultures, PBMC from six histoincompatible donors were stimulated by either allogeneically matching different pairs within the group or stimulating cells from individuals mitogenically. Following adherence of activated PBMC, nonadherent cells were removed from the cultures at 24 h poststimulation, and supernatants were collected from the cultures at the indicated intervals. Parallel cultures of Allo-MDM (red), ConA-MDM (blue), and unstimulated PBMC (green) were established as described in Materials and Methods. Cytokine-specific ELISA analysis was used to determine cytokine expression kinetics in cell-free culture supernatants collected at 6, 12, 24, 36, and 48 h and at 3, 5, and 8 days postisolation. Cytokines analyzed include IFN-γ (A), TNF-α (B), IL-2 (C), IL-3 (D), GM-CSF (E), G-CSF (F), M-CSF (G), IL-10 (H), IL-13 (I), IL-7 (J), TGF-β (K), IL-1β (L), and IL-6 (M).

FIG. 4

Allogeneically induced differentiation of HCMV-permissive MDM is dependent on the presence of IL-2 and IFN-γ. HCMV infection was inhibited in Allo-MDM that were established by neutralization of IL-2 and IFN-γ, whereas an effect was not observed by neutralization of IL-1, TNF-α, or TGF-β before establishment of the respective Allo-MDM cultures (n = 6 with duplicates). (A) Percent HCMV IE-expressing cells in the Allo-MDM cultures. (B) Production of cell-associated HCMV. All viral titer assays were performed on Allo-MDM collected by scraping at 14 days postinfection, and the expression of IE was determined in Allo-MDM fixed at 7 days postinfection.

FIG. 5

Reactivation of latent HCMV in Allo-MDM is dependent on production of IFN-γ. Allo-MDM were established by mixing PBMC from two healthy blood donors (n = 3 with duplicates), and reactivation of HCMV was detected by expression of the HCMV proteins IE and gB. (A) Confocal microscopy analysis of a representative sample of an Allo-MDM culture which reactivated HCMV. The cells expressed both HCMV IE (red) and gB (green). Magnification, ×630. (B) Reactivation of HCMV was inhibited in Allo-MDM, which were established in the presence of neutralizing antibodies to IFN-γ. In contrast, reactivation of virus was not affected by the addition of neutralizing antibodies directed against IL-1, IL-2, TNF-α, TGF-β, or GM-CSF at the time of establishment of the Allo-MDM cultures. (B) Percent HCMV IE-expressing cells in the Allo-MDM cultures at 52 days poststimulation.

FIG. 6

Reactivation of HCMV in monocytes stimulated with Allo-MDM-conditioned medium. Monocytes from single donors were isolated by adherence to plastic dishes for 2 h, after which nonadherent cells were removed by extensive washing. Supernatants from the enriched monocyte cultures were replaced at 1, 3, 5, and 7 days postisolation with Allo-MDM-conditioned medium obtained from allogeneic Allo-MDM cultures at parallel time points. (A) Allo-MDM generated from allogeneic stimulation of PBMC. Enriched monocyte cultures treated with canditioned medium are shown at 7 days poststimulation (B) in comparison to untreated adhered monocytes at the same time (C). HCMV reactivation in conditioned-medium-stimulated monocytes is demonstrated in panel D by immunofluorescence at 16 days poststimulation, with HCMV gB in green and IE in red. A graphical representation of reactivation in conditioned-medium-treated CD14⁺ monocytes derived from HCMV-seropositive donors is shown in panel E. Reactivation was determined by positive fluorescent staining for the HMCV proteins IE and gB.

FIG. 7

Model for cellular and cytokine factors necessary for generation of ConA-MDM and Allo-MDM. This model describes the cellular and cytokine components involved in the generation of ConA-MDM and Allo-MDM. While CD8 T-cell contact and IFN-γ and TNF-α are critical in the formation of ConA-MDM, both CD8 and CD4 T-cell contact and IFN-γ and IL-2 are important in the generation of Allo-MDM, which can reactivate and replicate latent HCMV.