Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells

Abstract

Measles causes a profound immune suppression which is responsible for the high morbidity and mortality induced by secondary infections. Dendritic cells (DC) are professional antigen-presenting cells required for initiation of primary immune responses. To determine whether infection of DC by measles virus (MV) may play a role in virus-induced suppression of cell-mediated immunity, we examined the ability of CD1a+ DC derived from cord blood CD34+ progenitors and Langerhans cells isolated from human epidermis to support MV replication. Here we show that both cultured CD1a+ DC and epidermal Langerhans cells can be infected in vitro by both vaccine and wild type strains of MV. DC infection with MV resulted within 24-48 h in cell-cell fusion, cell surface expression of hemagglutinin, and virus budding associated with production of infectious virus. MV infection of DC completely abrogated the ability of the cells to stimulate the proliferation of naive allogeneic CD4+ T cell as early as day 2 of mixed leukocyte reaction (MLR) (i.e., on day 4 of DC infection). Mannose receptor-mediated endocytosis and viability studies indicated that the loss of DC stimulatory function could not be attributed to the death or apoptosis of DC. This total loss of DC stimulatory function required viral replication in the DC since ultraviolet (UV)-inactivated MV or UV-treated supernatant from MV-infected DC did not alter the allostimulatory capacity of DC. As few as 10 MV- infected DC could block the stimulatory function of 10(4) uninfected DC. More importantly, MV-infected DC, in which production of infectious virus was blocked by UV treatment or paraformaldehyde fixation, actively suppressed allogeneic MLR upon transfer to uninfected DC-T-cultures. Thus, the mechanisms which contribute to the loss of the allostimulatory function of DC include both virus release and active suppression mediated by MV-infected DC, independent of virus production. These data suggest that carriage of MV by DC may facilitate virus spreading to secondary lymphoid organs and that MV replication in DC may play a central role in the general immune suppression observed during measles.

Figure 1

MV infection induces syncytia in cultured DC and skin LC. DC derived from day 9 cultures of cord blood CD34⁺ progenitors were either mock-infected (a and d  ) or infected with either MV-Hallé (b and e) or MV–LYS-1 (c and f  ) at an MOI of 0.05. DC syncytia were observed on day 2 of infection by phase-contrast microscopy analysis (final magnification, ×400) (a–c) and by May-Grunwald Giemsa staining (final magnification ×400 (d  ); ×1,000 (e and f   )). LC-enriched epidermal cell suspensions either uninfected (g) or infected with the Edmonston strain at an MOI of 0.05 (h) were cultured for 3 d in the presence of 100 ng/ml of rGM-CSF. On day 3 of infection, the cells were processed for staining with a polyclonal rabbit antiserum directed against S100 and counterstained with hematoxylin. No staining was detected with control normal rabbit serum (not shown). (g) Specific staining of LC for S100 in uninfected LC-enriched suspension; (h) S100 expression by LC syncytia in MV-infected LC-enriched suspension. ×400. The results are representative of five experiments.

Figure 2

FACS^® analysis of HA expression by CD1a⁺ DC. DC were either mock-infected (a) or infected with MV-Hallé (b) or MV–LYS-1 (c). On day 2 of infection, cell surface HA expression by single MV-infected DC was analyzed by indirect immunofluorescence using the MV-HA specific antibody, mAb 55, and an FITC-conjugated F(ab′)2 fragment of goat anti–mouse IgG (shaded histogram). A mouse IgG2b was used as isotype control (white histogram). In double immunofluorescence labeling, HA⁺ cells were electronically gated and the percentages of HA⁺ cells expressing CD1a or CD14 in both Hallé-infected (d) and LYS-1–infected (e) DC were determined using PE-conjugated anti-CD1a or anti-CD14 mAbs. The dotted lines represent the position of the PE-conjugated control IgG. The data are from a representative experiment out of 10.

Figure 3

Electron microscopy analysis of MV replication in DC. Transmission electron microscopic analysis performed on day 2 after infection with LYS-1, shows the complete replication cycle of MV in DC. (a) Syncytia containing ⩾10 DC nuclei and cytoplasmic structures typical of the viral nucleocapsid (arrow) (×7,600); (b) A higher magnification of the viral nucleocapsid close to a nucleus (×28,000); (c) Viral glycoproteins identified at sites of membrane rufflings (×34,000); (d) Virus budding leading to the release of intact virions (×46,000); (e) Fragments of DC syncytia resulting from cell clasmatosis, and containing viral nucleocapsids in the cytoplasm (×28,000); and (f) Apoptosis of DC syncytia with condensed chromatin in the nuclei (×8,600).

Figure 4

Productivity of MV infection of DC. Day 9 DC were infected with either MV-Hallé (•) or MV-LYS-1 (○) at an MOI of 0.05 and seeded at 2 × 10⁵ cells/well. The supernatants were harvested at the indicated time of culture and titrated for the presence of infectious virions in a standard plaque assay using the permissive cell line B95a. The results are expressed as PFU/ml.

Figure 5

MV infection of DC abrogates their ability to stimulate naive CD4⁺ T cell proliferation allogeneic MLR. Naive CD45 RA⁺ CD4⁺ T cells (2 × 10⁴ cells) were cultured for 6 d in triplicate wells with either (A) various numbers or (B) a fixed number (10⁴) of either mock- infected (□) or day 2-Hallé- infected (▪) or LYS-1–infected (▨ ) allogeneic DC. T cell proliferation was measured either on day 6 (A) or at various times (B) of culture by [³H]thymidine uptake during the last 16 h of culture. (C) 2 × 10⁴ naive T cells were cultured for 6 d with 10⁴ DC which had been either infected with MV or pulsed with UV-irradiated MV. Thymidine incorporation was determined using a β counter. The results, expressed as mean cpm ± SD, are from a representative experiment out of three to five.

Figure 6

Flow cytometry analysis of dextran-FITC internalization and Annexin V FITC and PI double staining of DC after MV infection. DC were either mock-infected (DC/0) or infected with MV (DC/Hallé; DC-LYS-1) at an MOI of 0.05. (A) Dextran-FITC internalization by mock-infected DC (a), DC infected with MV Hallé (b) and DC infected with MV LYS-1 (c) on day 2 after infection. The cells (2 × 10⁵) were incubated for 15 min in the presence of dextran-FITC either at 37°C (white histogram) or at 0°C for control (shaded histogram). Dextran-FITC uptake by viable cells was analyzed by flow cytometry, after exclusion of dead cells using PI. The numbers in parenthesis represent the percentage of cells which have internalized dextran-FITC. (B and C) On days 2, 3, and 4 after infection, cell viability was determined by flow cytometry analysis of double staining with Annexin V–FITC and PI. (B) Dot plot representation of Annexin V–FITC and PI double stainings. The lower left quadrants of each panel show the viable DC (Annexin V⁻ PI ⁻ ). The upper right quadrants contain the nonviable necrotic DC (Annexin V⁺ PI ⁺). The lower right quadrants represent the apoptotic DC (Annexin V⁺ PI ⁻). (C) Histogram representation of the percentages of intact viable, apoptotic, and necrotic DC among total DC present at various time of culture of either uninfected (□), Hallé-infected (•), or LYS-1–infected (○) DC.

Figure 7

Inhibitory effect of MV-infected DC and of DC supernatants in allogeneic MLR. (A and B) Supernatants from either mock-infected (□), day 2 Hallé- infected (▪), or LYS-1–infected (▨ ) DC were either untreated (A) or UV-irradiated (B) and added to cocultures of various numbers of uninfected DC and 2 × 10⁴ naive allogeneic CD4⁺ T cells. (C) Various numbers of either uninfected (□), Hallé-infected (♦), or LYS-1–infected (▪) DC were cocultivated for 6 d with 10⁴ uninfected DC and 2 × 10⁴ allogeneic CD4⁺ T cells. T cell proliferation was analyzed on day 6 of culture by thymidine uptake over the last 16 h of culture. The results are expressed as mean cpm ± SD of triplicate wells.

Figure 8

UV-treated or PF-fixed MV-infected DC can inhibit T cell proliferation in allogeneic DC–T cell MLR. Uninfected (□) or day 2 LYS-1–infected (○) DC were either UV-treated (A) or PF-fixed (B) before addition in graded numbers to cultures containing 10⁴ DC and 2 × 10⁴ CD45RA⁺CD4⁺T cells. T cell proliferation was analyzed on day 6 of culture by [³H]thymidine uptake for the last 16 h of culture. The results, expressed as mean cpm ± SD of triplicate wells, are representative of one experiment out of three.