Figure 3.
Figure 3. HCMV infection of immature moDCs is cytopathic with lytic transmission of virus, while mature moDCs survive and do not productively transmit virus despite HCMV replication. (A) Inverse phase micrographs showing preinfection immature moDCs (left; original magnification, × 10) compared with enlarged, HCMV-infected immature moDCs (right; original magnification, × 10), further identified in panel B as the IE1-positive population (gated, thick solid line histogram, all large FSC) on the forward scatter histogram (gated IE1-negative cells plotted along the thin line histogram, all low FSC; and total population not gated for IE1 expression and showing a bimodal FSC distribution in the filled histogram). (C) Forward scatter histograms from uninfected cultures (filled histograms) and day-3 HCMV-infected cultures (thick lines, unfilled histograms) of immature moDCs (left) and mature moDCs (right) at similar rates of infection (40% IE1-positive immature moDCs vs 30% IE1-positive mature moDCs). (D) Viable cell counts were determined by trypan blue exclusion. The ratio of viable cells from infected cultures to viable cells from uninfected cultures × 100 was calculated to provide a percent relative viability. Each column represents the mean of 3 to 6 experiments ± SD; 100% viability was indexed according to the absolute number of viable cells in uninfected cultures on the assessment day indicated along the x-axis. Increased viability in the infected mature moDC cultures did not reflect expansion, as these moDCs were nondividing, but rather reflected enhanced viability relative to the uninfected cultures where there was some normal, unavoidable cell death in the absence of cytokines or other stimulus. Decreased viability of the infected immature moDCs reflected the lytic nature of the infection. Error bars indicate SD. (E) Expression of the immediate early-1 antigen IE1 (FITC) and early-late antigen UL44 (Texas-Red) (upper panels, all × 25 original magnification), and expression of the late antigen gpB (FITC) with PI (red fluorescence) (lower panels, both × 63 original magnification) with different patterns between immature and mature moDCs (see text). (F) HCMV titers from cell-free supernatants assessed by a rapid culture assay on MRC5 (1 experiment representative of 3). (G-H) HCMV cell-to-cell spread: 2500 EC-HCMV-infected moDCs were cultured on permissive fibroblast (MRC5) monolayers. Over 6 days, HCMV foci of infected fibroblasts were stained by anti-IE1-FITC and counted (nd indicates not done on day 2). Immature and mature moDCs were infected in parallel at different MOIs to achieve similar rates of infection (G) or in parallel at the same MOI, which resulted in different rates of infection (H) (1 experiment representative of 3 shown for each). (I) Confocal micrographs of fibroblast layers after coculture with day +5 HCMV-infected immature or mature moDCs. Infected cells were detected by green staining with anti-IE1-FITC, and anti-HLA-DR-PE identified moDCs as red cells.

HCMV infection of immature moDCs is cytopathic with lytic transmission of virus, while mature moDCs survive and do not productively transmit virus despite HCMV replication. (A) Inverse phase micrographs showing preinfection immature moDCs (left; original magnification, × 10) compared with enlarged, HCMV-infected immature moDCs (right; original magnification, × 10), further identified in panel B as the IE1-positive population (gated, thick solid line histogram, all large FSC) on the forward scatter histogram (gated IE1-negative cells plotted along the thin line histogram, all low FSC; and total population not gated for IE1 expression and showing a bimodal FSC distribution in the filled histogram). (C) Forward scatter histograms from uninfected cultures (filled histograms) and day-3 HCMV-infected cultures (thick lines, unfilled histograms) of immature moDCs (left) and mature moDCs (right) at similar rates of infection (40% IE1-positive immature moDCs vs 30% IE1-positive mature moDCs). (D) Viable cell counts were determined by trypan blue exclusion. The ratio of viable cells from infected cultures to viable cells from uninfected cultures × 100 was calculated to provide a percent relative viability. Each column represents the mean of 3 to 6 experiments ± SD; 100% viability was indexed according to the absolute number of viable cells in uninfected cultures on the assessment day indicated along the x-axis. Increased viability in the infected mature moDC cultures did not reflect expansion, as these moDCs were nondividing, but rather reflected enhanced viability relative to the uninfected cultures where there was some normal, unavoidable cell death in the absence of cytokines or other stimulus. Decreased viability of the infected immature moDCs reflected the lytic nature of the infection. Error bars indicate SD. (E) Expression of the immediate early-1 antigen IE1 (FITC) and early-late antigen UL44 (Texas-Red) (upper panels, all × 25 original magnification), and expression of the late antigen gpB (FITC) with PI (red fluorescence) (lower panels, both × 63 original magnification) with different patterns between immature and mature moDCs (see text). (F) HCMV titers from cell-free supernatants assessed by a rapid culture assay on MRC5 (1 experiment representative of 3). (G-H) HCMV cell-to-cell spread: 2500 EC-HCMV-infected moDCs were cultured on permissive fibroblast (MRC5) monolayers. Over 6 days, HCMV foci of infected fibroblasts were stained by anti-IE1-FITC and counted (nd indicates not done on day 2). Immature and mature moDCs were infected in parallel at different MOIs to achieve similar rates of infection (G) or in parallel at the same MOI, which resulted in different rates of infection (H) (1 experiment representative of 3 shown for each). (I) Confocal micrographs of fibroblast layers after coculture with day +5 HCMV-infected immature or mature moDCs. Infected cells were detected by green staining with anti-IE1-FITC, and anti-HLA-DR-PE identified moDCs as red cells.

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