Macrophage progenitors are an effective and long-lasting therapy of hereditary pulmonary alveolar proteinosis.

CMV can infect dendritic cells (DCs), and direct Ag presentation could, therefore, lead to the priming of CMV-specific CD8⁺ T cells. However, CMV-encoded immune evasins severely impair Ag presentation in the MHC class I pathway; thus, it is widely assumed that cross-presentation drives the priming of antiviral T cells. We assessed the contribution of direct versus cross priming in mouse CMV (MCMV) infection using recombinant viruses. DCs infected with an MCMV strain encoding the gB₄₉₈ epitope from HSV-1 were unable to stimulate in vitro naive gB₄₉₈-specific CD8⁺ T cells from TCR transgenic mice. Infection of C57BL/6 mice with this recombinant virus led, however, to the generation of abundant numbers of gB₄₉₈-specific T cells in vivo. Of the DC subsets isolated from infected mice, only CD8α⁺ DCs were able to stimulate naive T cells, suggesting that this DC subset cross-presents MCMV-encoded Ag in vivo. Upon infection of mice with MCMV mutants encoding Ag that can either be well or hardly cross-presented, mainly CD8⁺ T cells specific for cross-presented epitopes were generated. Moreover, even in the absence of immune evasion genes interfering with MHC class I–mediated Ag presentation, priming of T cells to Ag that can only be presented directly was not observed. We conclude that the host uses mainly DCs capable of cross-presentation to induce the CMV-specific CD8⁺ T cell response during primary, acute infection and discuss the implications for the development of a CMV vaccine.

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediatorRbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S‐[2,3‐bispalmitoyiloxy‐(2R)‐propyl]‐R‐cysteinyl‐amido‐monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide‐2, is a potent adjuvant for cross‐priming against cellular antigens. Systemic administration of BPP‐induced maturation of CD8α⁺ DC and CD8α⁻ DC in the spleen and resulted in enhanced cross‐presentation of intravenously co‐administered antigen in mice. In addition, administration of BPP and cell‐associated OVA generated an effective CTL response against OVA in vivo in a CD4⁺ T helper cell‐dependent manner, but independent of IFN‐α. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut‐down of antigen presentation by splenic DC subsets, although cross‐priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross‐priming.

Different models of experimental allergic asthma have shown that the TLR7/8 agonist resiquimod (R848) is a potential inhibitor of type 2 helper cell–driven inflammatory responses. However, the mechanisms mediating its therapeutic effects are not fully understood. Using a model of experimental allergic asthma, we show that induction of IL-27 by R848 is critical for the observed ameliorative effects. R848 significantly inhibited all hallmarks of experimental allergic asthma, including airway hyperreactivity, eosinophilic airway inflammation, mucus hypersecretion, and Ag-specific Ig production. Whereas R848 significantly reduced IL-5, IL-13, and IL-17, it induced IFN-γ and IL-27. Neutralization of IL-27 completely reversed the therapeutic effect of R848 in the experimental asthma model, demonstrating dependence of R848-mediated suppression on IL-27. In vitro, R848 induced production of IL-27 by murine alveolar macrophages and dendritic cells and enhanced expression of programmed death–ligand 1, whose expression on monocytes and dendritic cells has been shown to regulate peripheral tolerance in both murine and human studies. Moreover, in vitro IL-27 enhanced secretion of IFN-γ whereas it inhibited IL-5 and IL-13, demonstrating its direct effect on attenuating Th2 responses. Taken together, our study proves that R848-mediated suppression of experimental asthma is dependent on IL-27. These data provide evidence of a central role of IL-27 for the control of Th2-mediated allergic diseases.