Caterina Vizzardelli

Background: Allergen‐specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE‐mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT‐induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy.Methods: Nonobese diabetic severe combined immunodeficient γc^−/− (NSG) mice received intraperitoneally allergen‐reactive PBMC from birch pollen‐allergic patients together with birch pollen extract and human IL‐4. Engraftment was assessed by flow cytometry. Airway hyperresponsiveness (AHR) and bronchial inflammation were analyzed after intranasal challenges with allergen or PBS. Sera collected from patients before and during AIT with birch pollen were added to the allergen prior to intranasal challenge. The IgE‐blocking activity of post‐AIT sera was assessed in vitro.Results: Human cells were detected in cell suspensions of murine lungs and spleens indicating successful humanization. Humanized mice displayed a more pronounced AHR and bronchial inflammation when challenged with allergen compared to negative controls. Post‐AIT sera exerted IgE‐blocking activity. In contrast to pre‐AIT sera, the presence of heterologous and autologous post‐AIT sera significantly reduced the allergic airway inflammation and matched their IgE‐blocking activity determined in vitro.Conclusion: Our data demonstrate that post‐AIT sera with IgE‐blocking activity ameliorate allergic airway inflammation in a human/mouse chimeric model of respiratory allergy independently of AIT‐induced cellular changes.

The lysosome-associated membrane protein (LAMP) family includes the dendritic cell endocytic receptors DC-LAMP and CD68, as well as LAMP-1 and LAMP-2. In this study we identify LAMP-1 (CD107a) and LAMP-2 (CD107b) on the surface of human monocyte-derived dendritic cells (MoDC) and show only LAMP-2 is internalized after ligation by specific Abs, including H4B4, and traffics rapidly but transiently to the MHC class II loading compartment, as does Ag conjugated to H4B4. However, pulsing MoDC with conjugates of primary (keyhole limpet hemocyanin; KLH) and recall (Bet v 1) Ags (H4B4*KLH and H4B4*Bet v 1) induced significantly less CD4 cell proliferation than pulsing with native Ag or Ag conjugated to control mAb (ISO*KLH and ISO*Bet v 1). In H4B4*KLH-pulsed MoDC, the duration of KLH residence in MHC class II loading compartments was significantly reduced, as were surface HLA-DR and DR-bound KLH-derived peptides. Paradoxically, MoDC pulsed with H4B4*KLH, but not the other KLH preparations, induced robust proliferation of CD4 cells separated from them by a transwell membrane, indicating factors in the supernatant were responsible. Furthermore, extracellular vesicles from supernatants of H4B4*KLH-pulsed MoDC contained significantly more HLA-DR and KLH than those purified from control MoDC, and KLH was concentrated specifically in exosomes that were a uniquely effective source of Ag in standard T cell proliferation assays. In summary, we identify LAMP-2 as an endocytic receptor on human MoDC that routes cargo into unusual Ag processing pathways, which reduces surface expression of Ag-derived peptides while selectively enriching Ag within immunogenic exosomes. This novel pathway has implications for the initiation of immune responses both locally and at distant sites.

Lipocalin 2 (LCN2), which is highly expressed by dendritic cells (DCs) when treated with dexamethasone (Dex) and lipopolysaccharide (LPS), plays a key role in the defence against bacteria and is also involved in the autocrine apoptosis of T-cells. However, the function of LCN2 when secreted by DCs is unknown: this is a critical gap in our understanding of the regulation of innate and adaptive immune systems. Tolerance, stimulation and suppression are functions of DCs that facilitate the fine-tuning of the immune responses and which are possibly influenced by LCN2 secretion. We therefore examined the role of LCN2 in DC/T-cell interaction. WT or Lcn2^−/− bone marrow-derived DCs were stimulated with LPS or LPS+IFN-γ with and without Dex and subsequently co-cultured with T-cells from ovalbumin-specific TCR transgenic (OT-I and OT-II) mice. We found that CD8⁺ T-cell apoptosis was highly reduced when Lcn2^−/− DCs were compared with WT. An in vivo CTL assay, using LPS-treated DCs, showed diminished killing ability in mice that had received Lcn2^−/− DCs compared with WT DCs. As a consequence, we analysed T-cell proliferation and found that LCN2 participates in T-cell-priming in a dose-dependent manner and promotes a T_H1 microenvironment. DC-secreted LCN2, whose function has previously been unknown, may in fact have an important role in regulating the balance between T_H1 and T_H2. Our results yield insights into DC-secreted LCN2 activity, which could play a pivotal role in cellular immune therapy and in regulating immune responses.

The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs’ initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.

While lipopolysaccharides (LPS) induce dendritic cell (DC) maturation and migration to lymph nodes, glucocorticoids such as dexamethazone (Dex) have a profound suppressive effect on immune response. The mechanisms that might control this suppressive effect of Dex have been extensively investigated in lymphocytes as possible targets. Much less is known on the effects of Dex on DC, although they are recognized to regulate immunity. To get insights into possible combined effects of Dex and LPS on DC functions, we have undertaken a genome‐wide analysis of differentially expressed genes of DC treated with Dex alone, LPS alone, or both, using high‐density oligonucleotide microarrays. Hierarchical clustering and principal component analysis (PCA) agreed in identifying 24 h as the time point that best discriminated the three treatments. Among the counteracting effects we have observed an inhibition of Dex on the LPS‐induced up‐regulation of the chemokine receptor CCR7. In vivo, Dex treatment blocked the LPS‐induced migration of DC, which lost their ability to reach the draining lymph nodes. In addition, we observed a synergistic effect of Dex and LPS on the expression of the secreted lipocalin 24p3, which has been reported to induce apoptosis in T cells and thus may be related to immune suppression.

Recognition of pathogen‐associated molecular patterns (PAMP) influences the response of dendritic cells (DC) and therefore development of innate and adaptive immunity. Different forms of Leishmania mexicana have distinct effects on DC, with promastigotes and amastigotes being activating and apparently neutral, respectively. We investigated whether stage‐specific differences in surface composition might account for these distinct effects. Amastigotes and promastigotes lacking the lpg1 gene needed for lipophosphoglycan (LPG) biosynthesis could not activate DC in vitro. Genome‐wide transcriptional profiling of DC infected with wild‐type or mutant promastigotes or wild‐type amastigotes revealed that wild‐type promastigotes induce an inflammatory signature that is lacking in DC exposed to the other parasite forms. The proinflammatory response pattern was partly recovered by reconstitution of lpg1 expression in lpg1^–/– parasites, and exposure to purified LPG increased the expression of MHC class II and CD86 on DC. Infection with wild‐type but not lpg1^–/– promastigotes increased the number of activated DC in draining lymph nodes, and this was correlated with lower early parasite burdens in wild‐type‐infected animals. These in vivo and in vitro results suggest an LPG‐dependent activation of DC that contributes to host defense and agree with the notion that the parasites evolved under immune pressure to down‐regulate PAMP expression in mammalian hosts.