Inclusion of CD80 in HSV Targets the Recombinant Virus to PD-L1 on DCs and Allows Productive Infection and Robust Immune Responses

CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC

Iron administration reduces airway hyperreactivity and eosinophilia in a mouse model of allergic asthma

The prevalence of allergic diseases has increased dramatically during the last four decades and is paralleled by a striking increase in iron intake by infants in affluent societies. Several studies have suggested a link between increased iron intake and the marked increase in prevalence of allergic diseases. We hypothesized that the increased iron intake by infants offers an explanation for the increased prevalence of allergic disease in industrialized societies during the past four decades. A well-established mouse model of ovalbumin (OVA)-driven allergic asthma was used to test the effects of differences in iron intake and systemic iron levels on the manifestations of allergic asthma. Surprisingly, iron supplementation resulted in a significant decrease in airway eosinophilia, while systemic iron injections lead to a significant suppression of both allergen-induced airway eosinophilia and hyperreactivity compared to placebo. In contrast, mice fed on an iron-deprived diet did not show any difference in developing experimentally induced allergic asthma when compared to those fed on an iron-sufficient control diet. In contrast to our hypothesis, airway manifestations of allergic asthma are suppressed by both increased levels of iron intake and systemic iron administrations in the mouse model

Contribution of regulatory T cells to alleviation of experimental allergic asthma after specific immunotherapy

Background Allergen-specific immunotherapy (SIT) has been used since 1911, yet its mechanism of action remains to be elucidated. There is evidence indicating that CD4+FOXP3+ regulatory T cells (Treg cells) are induced during SIT in allergic patients. However, the contribution of these cells to SIT has not been evaluated in vivo. Objective To evaluate the in vivo contribution of (i) CD4+ CD25+ T cells during SIT and of (ii) SIT-generated inducible FOXP3+ Treg cells during allergen exposure to SIT-mediated suppression of asthmatic manifestations. Methods We used a mouse model of SIT based on the classical OVA-driven experimental asthma. Treg cells were quantified by flow cytometry 24 and 96h post SIT treatment. We depleted CD4+CD25+ T cells prior to SIT, and CD4+FOXP3+ T cells prior to allergen challenges to study their contribution to the suppression of allergic manifestations by SIT treatment. Results Our data show that depletion of CD4+CD25+ T cells at the time of SIT treatment reverses the suppression of airway hyperresponsiveness (AHR), but not of airway eosinophilia and specific IgE levels in serum. Interestingly, the number of CD4+CD25+FOXP3+ T cells is transiently increased after SIT in the spleen and blood, suggesting the generation of inducible and presumably allergen-specific Treg cells during treatment. Depletion of CD4+FOXP3+ Treg cells after SIT treatment partially reverses the SIT-induced suppression of airway eosinophilia, but not of AHR and serum levels of specific IgE. Conclusion and clinical relevance We conclude that SIT-mediated tolerance induction towards AHR requires CD4+CD25+ T cells at the time of allergen injections. In addition, SIT generates CD4+CD25+FOXP3+ T cells that contribute to the suppression of airway eosinophilia upon allergen challenges. Therefore, enhancing Treg cell number or their activity during and after SIT could be of clinical relevance to improve the therapeutic effects of SIT

Expression of CD80 by HSV-CD80 in infected DCs.

<p>A<u>) Detection of HSV-1 gC in infected DCs</u>. Subconfluent monolayers of DCs isolated from C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c and anti-HSV-1 gC antibodies and FACS analysis was performed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>). Experiments were repeated twice; <u>B) FACS analyses of infected DCs for expression of HSV-1 gC and CD80</u>. Monolayers of DCs were treated as above. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-HSV-1 gC, and anti-CD80 antibodies and FACS analysis was performed. CD11c⁺ cells were gated for expression of HSV-1 gC and CD80. Experiments were repeated twice; <u>C) Immunostaining of HSV-CD80 infected DCs</u>. DCs were infected with 1 or 10 PFU/cell of HSV-CD80 or parental virus. At 12 and 24 hr PI, cells were stained with anti-HSV-1 gC antibody and subjected to IHC as descried in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>; and <u>D) Quantification of photomicrographs from C</u>. Different areas of 3 slides/virus/pfu/time point from IHC described above were imaged and the number of HSV-1 gC⁺ cells was counted. Each point represents the mean ± SEM of HSV-1 gC⁺ DCs from 12 images.</p

Costimulation of type-2 innate lymphoid cells by GITR promotes effector function and ameliorates type 2 diabetes

Type-2 innate lymphoid cells (ILC2s) are an immune population secreting Th2 cytokines playing a role in the regulation of adipose metabolic homeostasis. Here the authors show that engagement of GITR, a member of the TNF superfamily, in activated ILC2s is protective against insulin resistance in both a preventive and a therapeutic manner in the context of obesity

Subcutaneous immunotherapy suppresses Th2 inflammation and induces neutralizing antibodies, but sublingual immunotherapy suppresses airway hyperresponsiveness in grass pollen mouse models for allergic asthma

BackgroundBoth subcutaneous and sublingual allergen immunotherapy (SCIT and SLIT) have been shown to effectively suppress allergic manifestations upon allergen exposure, providing long-term relief from symptoms in allergic disorders including allergic asthma. Clinical studies directly comparing SCIT and SLIT report a different kinetics and magnitude of immunological changes induced during treatment. Comparative studies into the mechanisms underlying immune suppression in SCIT and SLIT are lacking. ObjectiveWe aimed to establish an experimental model for grass pollen (GP) SCIT and SLIT that would allow a head-to-head comparison of the two treatments. MethodsBALB/c mice were sensitized with GP extract, followed by SCIT and SLIT treatments with various GP dosages. Subsequently, we challenged mice with GP and measured airway responsiveness (AHR), GP-specific immunoglobulins, ear swelling tests (EST), eosinophilic inflammation in bronchoalveolar lavage fluid (BALF), and T cell cytokine release after restimulation of lung cells (IL-5, IL-10, and IL-13). ResultsWe find that SLIT treatment was able to suppress allergen-induced AHR, while allergic inflammation was not effectively suppressed even at the highest GP dose in this model. In contrast, SCIT treatment induced higher levels of GP-specific IgG1, while SLIT was superior in inducing a GP-specific IgG2a response, which was associated with increased Th1 activity in lung tissue after SLIT, but not SCIT treatment. Interestingly, SCIT was able to suppress Th2-type cytokine production in lung cell suspensions, while SLIT failed to do so. Conclusions and clinical relevanceIn conclusion, GP-SCIT suppresses Th2 inflammation and induced neutralizing antibodies, while GP-SLIT suppresses the clinically relevant lung function parameters in an asthma mouse model, indicating that the two application routes depend on partially divergent mechanisms of tolerance induction. Interestingly, these data mirror observations in clinical studies, underscoring the translational value of these mouse models

PD-L1 and PD-L2 affect the cytokines produced by lung iNKT cells after IAV infection.

<p>iNKT cells were positively selected from influenza A infected lungs on day 5 from PD-L1^−/−, PD-L2^−/− and wild type BALB/c mice and different cytokines were analysed by quantitative RT-PCR normalized to β-actin levels. Data presented as mean ± SEM. * P>0.05, ** P>0.01 and *** P>0.001 as calculated by a one-way ANOVA with Tukey post hoc test and is representative of five independent experiments with n of 5 per group.</p

Colocalization of PD-L1 on DCs to CD80 expressed by HSV-CD80 and its effect on virus replication.

<p><u>A) FACS analyses of infected WT DCs</u>. Subconfluent monolayers of DCs isolated from WT C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-HSV-1 gC, and anti-PD-L1 antibodies and three-color FACS analysis was performed. CD11c⁺ gated cells were analyzed for expression of HSV-1 gC and PD-L1. Experiments were repeated twice; <u>B) FACS analyses of DCs from knockout mice.</u> DCs from C57BL/6 WT, C57BL/6-PD-L1^-/- and C57BL/6-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-CD80, and anti-PD-L1 antibodies and three-color FACS analysis was performed. CD11c⁺ gated cells were analyzed for expression of CD80 and PD-L1. Experiments were repeated twice; <u>C) Immunostaining of DCs from knockout mice</u>. DCs from WT BALB/c, BALB/c-PD-L1^-/- and BALB/c-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV-CD80 or parental virus. At 24 hr PI, cells were reacted with anti-HSV-1 gC, anti-CD80, and anti-PD-L1 antibodies. DAPI is shown as a nuclear counter-stain; <u>D) Replication of HSV-CD80 in DCs isolated from knockout mice</u>. Subconfluent monolayers of DCs isolated from WT BALB/c, BALB/c-PD-L1^-/- and BALB/c-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV- CD80 or parental virus as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. Virus yield was determined at the indicated times by standard plaque assays. Each point represents the mean ± SEM (n = 6) from two separate experiments; and <u>E) Replication of HSV-CD80 in WT C57BL/6 DCs in presence of blocking antibody</u>. Subconfluent monolayers of DCs were incubated with 10F.2H11 antibody, irrelevant antibody, or no antibody and infected with 1 PFU/cell of HSV-CD80 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. Virus yield was determined at the indicated times by standard plaque assays. Each point represents the mean ± SEM (n = 6).</p

Replication of HSV-CD80 recombinant virus in BM-derived DCs.

<p>Subconfluent monolayers of DCs isolated from C57BL/6, 129SVE, and BALB/c mice were infected with 10 PFU/cell of HSV-CD80 or parental virus as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. In some experiments DCs from C57BL/6 mice were infected with 1 PFU/cell of each virus, while DCs from 129SVE were infected with 10 PFU/cell of wt HSV-1 strain McKrae. Virus yield was determined at the indicated times PI by standard plaque assays. Panels: A) DCs from C57BL/6 mice were infected at 1 PFU/cell of HSV-CD80 or parental virus; B) DCs from C57BL/6 mice were infected at 10 PFU/cell of HSV-CD80 or parental virus; C) DCs from 129SVE mice were infected at 10 PFU/cell of HSV-CD80, parental, and wt McKrae viruses; and D) DCs from BALB/c mice were infected at 10 PFU/cell of HSV-CD80 or parental virus. Each point represents the mean ± SEM (n = 6) from two separate experiments.</p