Macrophage P2Y6 Receptor Signaling Selectively Activates NFATC2 and Suppresses Allergic Lung Inflammation.

Innate immune responses to innocuous Ags can either prevent or facilitate adaptive type 2 allergic inflammation, but the mechanisms are incompletely understood. We now demonstrate that macrophage UDP-specific type 6 purinergic (P2Y6) receptors selectively activate NFATC2, a member of the NFAT family, to drive an innate IL-12/IFN-γ axis that prevents type 2 allergic inflammation. UDP priming potentiated IL-12p40 production in bone marrow-derived macrophages (BMMs) stimulated by the house dust mite Dermatophagoides farinae (Df) in a P2Y6-dependent manner. Inhibitions of phospholipase C, calcium increase, and calcineurin eliminated UDP-potentiated Df-induced IL-12p40 production. UDP specifically induced nuclear translocation of NFATC2, but not NFATC1 and NFATC3, in BMMs in a P2Y6-dependent manner. UDP-potentiated IL-12p40 production by BMMs and Df-induced IL-12p40 gene expression by alveolar macrophages were abrogated in cells from Nfatc2 knockout mice. Pulmonary transplantation of wild-type but not Nfatc2 knockout macrophages increased Df-induced IL-12 production and IFN-γ expression in P2ry6 fl/fl/Cre/+ recipient mice. Finally, Nfatc2 knockout mice showed significantly increased indices of type 2 immunopathology in response to Df challenge, similar to P2ry6 fl/fl/Cre/+ mice. Thus, macrophage P2Y6 receptor signaling selectively utilizes NFATC2 to potentiate an innate IL-12/IFN-γ axis, a potential mechanism that protects against inappropriate type 2 immune responses.

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity-suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants.

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.

Dissecting strategies to tune the therapeutic potential of SARS-CoV-2-specific monoclonal antibody CR3022.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross-SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection.

Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production.

Antibodies are key immune effectors that confer protection against pathogenic threats. The nature and longevity of the antibody response to SARS-CoV-2 infection are not well defined. We charted longitudinal antibody responses to SARS-CoV-2 in 92 subjects after symptomatic COVID-19. Antibody responses to SARS-CoV-2 are unimodally distributed over a broad range, with symptom severity correlating directly with virus-specific antibody magnitude. Seventy-six subjects followed longitudinally to ∼100 days demonstrated marked heterogeneity in antibody duration dynamics. Virus-specific IgG decayed substantially in most individuals, whereas a distinct subset had stable or increasing antibody levels in the same time frame despite similar initial antibody magnitudes. These individuals with increasing responses recovered rapidly from symptomatic COVID-19 disease, harbored increased somatic mutations in virus-specific memory B cell antibody genes, and had persistent higher frequencies of previously activated CD4+ T cells. These findings illuminate an efficient immune phenotype that connects symptom clearance speed to differential antibody durability dynamics.

P2Y6 signaling in alveolar macrophages prevents leukotriene-dependent type 2 allergic lung inflammation.

Antagonists of the type 1 cysteinyl leukotriene receptor (CysLT1R) are widely used to treat asthma and allergic rhinitis, with variable response rates. Alveolar macrophages express UDP-specific P2Y6 receptors that can be blocked by off-target effects of CysLT1R antagonists. Sensitizing intranasal doses of an extract from the house dust mite Dermatophagoides farinae (Df) sharply increased the levels of UDP detected in bronchoalveolar lavage fluid of mice. Conditional deletion of P2Y6 receptors before sensitization exacerbated eosinophilic lung inflammation and type 2 cytokine production in response to subsequent Df challenge. P2Y6 receptor signaling was necessary for dectin-2-dependent production of protective IL-12p40 and Th1 chemokines by alveolar macrophages, leading to activation of NK cells to generate IFN-γ. Administration of CysLT1R antagonists during sensitization blocked UDP-elicited potentiation of IL-12p40 production by macrophages in vitro, suppressed the Df-induced production of IL-12p40 and IFN-γ in vivo, and suppressed type 2 inflammation only in P2Y6-deficient mice. Thus, P2Y6 receptor signaling drives an innate macrophage/IL-12/NK cell/IFN-γ axis that prevents inappropriate allergic type 2 immune responses on respiratory allergen exposure and counteracts the Th2 priming effect of CysLT1R signaling at sensitization. Targeting P2Y6 signaling might prove to be a potential additional treatment strategy for allergy.