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MPYS/STING (stimulator of IFN genes) senses cyclic dinucleotides (CDNs), generates type I IFNs, and plays a critical role in infection, inflammation, and cancer. In this study, analyzing genotype and haplotype data from the 1000 Genomes Project, we found that the R71H-G230A-R293Q (HAQ) MPYS allele frequency increased 57-fold in East Asians compared with sub-Saharan Africans. Meanwhile, the G230A-R293Q (AQ) allele frequency decreased by 98% in East Asians compared with sub-Saharan Africans. We propose that the HAQ and AQ alleles underwent a natural selection during the out-of-Africa migration. We used mouse models of HAQ and AQ to investigate the underlying mechanism. We found that the mice carrying the AQ allele, which disappeared in East Asians, had normal CDN–type I IFN responses. Adult AQ mice, however, had less fat mass than did HAQ or wild-type mice on a chow diet. AQ epididymal adipose tissue had increased regulatory T cells and M2 macrophages with protein expression associated with enhanced fatty acid oxidation. Conditional knockout mice and adoptive cell transfer indicate a macrophage and regulatory T cell–intrinsic role of MPYS in fatty acid metabolism. Mechanistically, AQ/IFNAR1−/− mice had a similar lean phenotype as for the AQ mice. MPYS intrinsic tryptophan fluorescence revealed that the R71H change increased MPYS hydrophilicity. Lastly, we found that the second transmembrane (TM) and the TM2–TM3 linker region of MPYS interact with activated fatty acid, fatty acyl–CoA. In summary, studying the evolution of the human MPYS gene revealed an MPYS function in modulating fatty acid metabolism that may be critical during the out-of-Africa migration.

Induction of lung mucosal immune responses is highly desirable for vaccines against respiratory infections. We recently showed that monocyte-derived dendritic cells (moDCs) are responsible for lung IgA induction. However, the dendritic cell subset inducing lung memory TH cells is unknown. In this study, using conditional knockout mice and adoptive cell transfer, we found that moDCs are essential for lung mucosal responses but are dispensable for systemic vaccine responses. Next, we showed that mucosal adjuvant cyclic di-GMP differentiated lung moDCs into Bcl6+ mature moDCs promoting lung memory TH cells, but they are dispensable for lung IgA production. Mechanistically, soluble TNF mediates the induction of lung Bcl6+ moDCs. Our study reveals the functional heterogeneity of lung moDCs during vaccination and paves the way for an moDC-targeting vaccine strategy to enhance immune responses on lung mucosa.

The lung is a naturally tolerogenic organ. Lung regulatory T cells (T-regs) control lung mucosal tolerance. Here, we identified a lung IFNAR1hiTNFR2+ conventional DC2 (iR2D2) population that induces T-regs in the lung at steady state. Using conditional knockout mice, adoptive cell transfer, receptor blocking antibodies, and TNFR2 agonist, we showed that iR2D2 is a lung microenvironment-adapted dendritic cell population whose residence depends on the constitutive TNFR2 signaling. IFNβ-IFNAR1 signaling in iR2D2 is necessary and sufficient for T-regs induction in the lung. The Epcam+CD45- epithelial cells are the sole lung IFNβ producer at the steady state. Surprisingly, iR2D2 is plastic. In a house dust mite model of asthma, iR2D2 generates lung TH2 responses. Last, healthy human lungs have a phenotypically similar tolerogenic iR2D2 population, which became pathogenic in lung disease patients. Our findings elucidate lung epithelial cells IFNβ-iR2D2-T-regs axis in controlling lung mucosal tolerance and provide new strategies for therapeutic interventions.

Induction of lung T-cell responses, including memory CD4+THand TFHcells, are highly desirable for vaccines against respiratory infections. We recently showed that the non-migratory monocytes-derived DCs (moDCs) induced lung TFHcells. However, the DCs subset inducing lung CD4+memory THcells is unknown. Here, using conditional knockout mice and adoptive cell transfer, we first established that moDCs are essential for lung mucosal, but are dispensable for systemic, vaccine responses. Next, we showed that intranasal administration of adjuvant cyclic di-GMP differentiated lung moDCs into Bcl6+and Bcl6-moDCs promoting lung memory THcells and lung TFHcells, respectively. Mechanistically, soluble TNF from lung TNFR2+cDC2 subpopulation mediates the induction of lung Bcl6+moDCs. Last, we designed fusion proteins targeting soluble or transmembrane TNF to lung moDCs and generated Bcl6+, Bcl6-lung moDCs respectively. Together, our study revealed lung mature moDCs heterogeneity and showed a moDCs-targeting strategy to enhance lung mucosal vaccine responses.

Cyclic dinucleotides (CDNs) are promising vaccine adjuvants inducing balanced, potent humoral, and cellular immune responses. How aging influences CDN efficacy is unclear. We examined the vaccine efficacy of 3′,5′-cyclic diguanylic acid (cyclic di-GMP, CDG), the founding member of CDNs, in 1-year-old (middle-aged) and 2-year-old (aged) C57BL/6J mice. We found that 1- and 2-year-old C57BL/6J mice are defective in CDG-induced memory T helper (Th)1 and Th17 responses and high-affinity serum immunoglobulin (Ig)G, mucosal IgA production. Next, we generated two novel tumor necrosis factor (TNF) fusion proteins that target soluble TNF (solTNF) and transmembrane TNF (tmTNF) to monocyte-derived dendritic cells (moDCs) to enhance CDG vaccine efficacy in 1- and 2-year-old mice. The moDC-targeting TNF fusion proteins restored CDG-induced memory Th1, Th17, and high-affinity IgG, IgA responses in the 1- and 2-year-old mice. Together, the data suggested that aging negatively impacts CDG vaccine adjuvanticity. MoDC-targeting TNF fusion proteins enhanced CDG adjuvanticity in the aging mice.

Cyclic dinucleotides (CDNs), including cyclic di-GMP (CDG), are promising vaccine adjuvants in preclinical/clinical trials. The in vivo mechanisms of CDNs are not clear. Here we investigated the roles of lung DC subsets in promoting CDG mucosal adjuvant responses in vivo. Using genetically modified mice and adoptive cell transfer, we identified lung conventional DC 2 (cDC2) as the central player in CDG mucosal responses. We further identified two functionally distinct lung cDC2 subpopulations: TNFR2+pRelB+ and TNFR2−pRelB− cDC2. The TNFR2+ cDC2 were mature and migratory upon intranasal CDG administration while the TNFR2− cDC2 were activated but not mature. Adoptive cell transfer showed that TNFR2− cDC2 mediate the antibody responses of CDG, while the TNFR2+ cDC2 generate Th1/17 responses. Mechanistically, immature TNFR2− cDC2 activate monocyte-derived DCs (moDCs), which do not take up intranasally administered CDG. moDCs promote CDG-induced generation of T follicular helper- and germinal center B cells in the lungs. Our data revealed a previously undescribed in vivo mode of DCs action, whereby an immature lung TNFR2− cDC2 subpopulation directs the non-migratory moDCs to generate CDG mucosal responses in the lung.

MHC class II regulates B cell activation, proliferation, and differentiation during cognate B cell-T cell interaction. This is, in part, due to the MHC class II signaling in B cells. Activation of MHC Class II in human B cells or "primed" murine B cells leads to tyrosine phosphorylation, calcium mobilization, AKT, ERK, JNK activation. In addition, crosslinking MHC class II with monoclonal Abs kill malignant human B cells. Several humanized anti-HLA-DR/MHC class II monoclonal Abs entered clinical trials for lymphoma/leukemia and MHC class II-expressing melanomas. Mechanistically, MHC class II is associated with a wealth of transmembrane proteins including the B cell-specific signaling proteins CD79a/b, CD19 and a group of four-transmembrane proteins including tetraspanins and the apoptotic protein MPYS/STING. Furthermore, MHC class II signals are compartmentalized in the tetraspanin-enriched microdomains. In this review, we discuss our current understanding of MHC class II signaling in B cells focusing on its physiological significance and the therapeutic potential.

Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. IMPORTANCE Like most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of glycosphingolipids in the HCV life cycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.