Interplay between virus-specific effector response and Foxp3 regulatory T cells in measles virus immunopathogenesis.

Measles is a highly contagious childhood disease associated with an immunological paradox: although a strong virus-specific immune response results in virus clearance and the establishment of a life-long immunity, measles infection is followed by an acute and profound immunosuppression leading to an increased susceptibility to secondary infections and high infant mortality. In certain cases, measles is followed by fatal neurological complications. To elucidate measles immunopathology, we have analyzed the immune response to measles virus in mice transgenic for the measles virus receptor, human CD150. These animals are highly susceptible to intranasal infection with wild-type measles strains. Similarly to what has been observed in children with measles, infection of suckling transgenic mice leads to a robust activation of both T and B lymphocytes, generation of virus-specific cytotoxic T cells and antibody responses. Interestingly, Foxp3(+)CD25(+)CD4(+) regulatory T cells are highly enriched following infection, both in the periphery and in the brain, where the virus intensively replicates. Although specific anti-viral responses develop in spite of increased frequency of regulatory T cells, the capability of T lymphocytes to respond to virus-unrelated antigens was strongly suppressed. Infected adult CD150 transgenic mice crossed in an interferon receptor type I-deficient background develop generalized immunosuppression with an increased frequency of CD4(+)CD25(+)Foxp3(+) T cells and strong reduction of the hypersensitivity response. These results show that measles virus affects regulatory T-cell homeostasis and suggest that an interplay between virus-specific effector responses and regulatory T cells plays an important role in measles immunopathogenesis. A better understanding of the balance between measles-induced effector and regulatory T cells, both in the periphery and in the brain, may be of critical importance in the design of novel approaches for the prevention and treatment of measles pathology

Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response▿ †

Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-β) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-β response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-α/β production, an amplified IFN-β response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-β gene deficiencies were trans complemented to induce IFN-β production. Production of IFN-β and IFN-α was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-α/β. The amplification of IFN-β production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-α/β production in infected cells, and this indicates that MGC contribute to the antiviral immune response

T lymphocyte infiltration at the sites of MV-brain infection.

<p>Brain sections from suckling CD150-transgenic (A–E) and nontransgenic littermate mice (F) infected with MV were analyzed by immunohistofluorescence for MV nucleoprotein (N) localization (A–F, in red) and the presence of CD4⁺ (A, in green), CD8⁺ (B, in green) and Foxp3⁺ T cells (C–F, in green). Cell nuclei were counterstained with DAPI (in blue). Infiltrating T CD4⁺ and CD8⁺ lymphocytes were detected in brains from CD150 transgenic mice (A and B, respectively) at the sites of MV infection identified by a N-specific labelling (red dots) but not in their nontransgenic littermates (not shown). Images are shown at 40× original magnification and are representative of three to five mice per group.</p

Characterization of Treg function following MV infection.

<p>(A) Analysis of suppressor activity of Tregs isolated from CD150 mice, inoculated with MV (open symbol) or with medium (full symbol), in cocultures with CD4⁺CD25⁻ effector T cells from either uninfected CD150 mice (left panel), or infected CD150 mice (right panel) in the presence of irradiated CD4⁺ T cell–depleted splenic APCs and Con A (3 to 5 pooled mice per group). Proliferation of Tregs from either control or CD150 infected mice in response to Con A was ∼300–600 cpm. The results are shown as the mean percentage of proliferation inhibition in triplicate cultures±SD. Results are representative of three different experiments. (B) Splenocytes isolated from either uninfected (left panel) or MV-infected (right panel) CD150 mice and their nontransgenic littermates (wild type) were stimulated with either irradiated Balb/c or C57Bl/6 splenocytes in MLR in triplicate cultures, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004948#s4" target="_blank">Methods</a>. Proliferation is expressed as mean proliferation index±SD and is representative from two independent experiments (* P<0,01, Student t-test).</p

MV infection induces a strong activation of the immune response in CD150 transgenic mice.

<p>CD150 transgenic mice and nontransgenic littermates (control) were inoculated i.n. with MV or medium (uninfected). Spleens were harvested 13 dpi and cells were analyzed by flow cytometry. (A) Expression of MV hemagglutinin (H) antigen on the surface of spleen cells from uninfected (grey line) and MV-infected (black line) CD150 transgenic mice, gated on CD19⁺ (B cells) or CD3⁺ (T cells) cells. Numbers are percentages of cells expressing MV H antigen in infected conditions. (B) Staining for CD4⁺ and CD8⁺ T cells and (C) CD19⁺ B cells, among analyzed splenocytes. Results are representative of 8 different experiments, each involving 3–6 mice. Differences between CD150 transgenic infected mice and the other groups were statistically significant (p<0.05, Student t-test).</p

MV-induced immunosuppression in adult transgenic mice.

<p>(A) Transgenic CD150×IFNα/βR KO mice were infected i.n. with MV at different ages and monitored for survival by Kaplan-Meier analysis. (B, C) Groups of 5 mice, 6 to 7 week-old, were infected i.n. with MV or left untreated. Splenocytes were harvested at 11 dpi and stained for CD4 and CD25 followed by anti-Foxp3 intracellular staining and analyzed by flow cytometry. Results are presented: (B) as the percentage of CD25⁺Foxp3⁺ cells within CD4⁺ compartment, for each analyzed animal and (C) as a percentage of CD25⁺ cells within the CD4⁺Foxp3⁺ population. Horizontal bars correspond to mean values. (D) Groups of 10 mice, 6 to 7 weeks old, were infected i.n. with MV or left untreated. Seven days later, mice were sensitized with DNFB 0.5% on the ventral skin or left unsensibilized (unsens). All mice were challenged 5 days later with DNFB 0.1% on the left ear. Results are presented as the individual ear swelling of two independent experiments and horizontal bars correspond to mean values. (** p<0.01, * p<0.05, Mann-Whitney test).</p

MV infection increases the proportion of CD4⁺CD25⁺Foxp3⁺ Tregs.

<p>(A) Splenocytes from CD150 or nontransgenic mice (control), inoculated i.n. with either MV or medium (uninfected), were harvested 13 dpi and stained for CD4 and CD25 followed by anti-Foxp3 intracellular staining and analyzed by flow cytometry. (B, C) CD150×Foxp3-GFP transgenic mice and Foxp3-GFP littermates (control) were inoculated i.n. with MV. Brains were harvested 8 dpi and analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004948#s4" target="_blank">Methods</a>. (B) Proportion of infiltrating CD4⁺ and CD8⁺ T lymphocytes in the brain (two left panels); expression of the CD44 activation marker on CD4⁺ T lymphocytes (right panel, CD150 transgenic in red and nontransgenic control in blue). (C) Tregs detected by the co-expression of Foxp3 and CD25 or ICOS. Results are representative of 4 independent experiments, each involving 4–7 mice per group. Differences between infected and noninfecetd mice were statistically significant (p<0.05, Student t-test).</p

MV infection induces a specific antibody and cytotoxic response in CD150 transgenic mice.

<p>(A, B) Production of anti-MV nucleoprotein (N) antibodies (IgG) was measured in serum of individual mice by ELISA and the number of tested animals is indicated in parenthesis. Titers are expressed as relative units. (A) Mice were immunized with low titre of MV (200 PFU) and serum was collected on days 13 and 28, or (B) mice were immunized with higher dose of MV (10³ PFU) and serum was collected on day 13 post infection. (C) To analyze cellular anti-viral response, splenocytes were harvested and restimulated with target cells expressing MV N gene (P815-N) for one week (3 to 8 pooled mice per group). Cytotoxic activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004948#s4" target="_blank">Methods</a>; results are expressed as the mean percentage of N-specific cytotoxic activity from duplicate cultures (+/−SD) and the data are from one representative experiment out of three. Cytotoxic activity of lymphocytes obtained from MV-infected CD150 mice was significantly higher compared to the other groups, (p<0.05, Mann-Whitney U test).</p