Resident CD8 1 DCs perform several functions, including cross-presenting antigen and rapidly engulfing the Gram-positive intracellular pathogen Listeria monocytogenes. Little is known about how these functions of CD8 1 DCs are modulated. Here, we show that granulocyte-macrophage CSF (GM-CSF), a cytokine that exists at low levels at steady state but is elevated during infection and inflammation, enhances cross-presentation and rapid uptake of L. monocytogenes by resident CD8 1 DCs. This previously unrecognized functional enhancement of CD8 1 DCs by GM-CSF was independent of promoting DC survival in vitro. Enhancement of these functions by GM-CSF was also marked by CD103 expression on CD8 1 DCs that was strongly regulated by GM-CSF. Our findings not only identify GM-CSF as a key molecule regulating CD8 1 DC function, but also as a factor responsible for functional heterogeneity of CD8 1 DCs that is at least substantially demarcated by CD103 expression.Keywords: CD8 1 DC . CD103 . Cross-presentation . Granulocyte-macrophage colony-stimulating factor . Listeria monocytogenes Supporting Information available online IntroductionDCs found in the spleen can be grossly separated into CD8 1 DCs and CD8 À DCs. The latter includes CD8 À CD4 1 and CD8 À CD4 À conventional DCs and CD45RA 1 plasmacytoid DCs. Different DC subsets have different functions. Notably, the resident CD8 1 DCs show superior capacity for ingesting cell-associated antigens [1], for cross-presenting antigens (i.e. presenting exogenous antigens to CD8 1 T cells) [2][3][4], for the rapid uptake of certain bacterial pathogens [5] and for producing . It has been previously reported that a small proportion of CD8 1 DCs express the integrin CD103 and these CD103-expressing CD8 1 DCs are enriched in cells engulfing cellular antigens and cross-priming CD8 1 T cells [7]. However, it is unclear what regulates the above à Co-senior authors. Here, we investigated whether GM-CSF might influence the function of CD8 1 DCs and CD103 expression. Functionally, DCs from GM-CSF transgenic (GMtg) mice had enhanced crosspresentation, whereas in the absence of GM-CSF signaling, crosspresentation was reduced. Rapid uptake of Listeria monocytogenes by CD8 1 DCs was also reduced in the absence of GM-CSF. Phenotypically, we showed that bacterial infection preferentially increased CD103 expression by CD8 1 DCs and this increase was dependent on GM-CSF. Together, our results identify a critical role for GM-CSF in preferentially regulating expression of the integrin CD103 by CD8 1 DCs and in regulating certain functions of CD8 1 DCs. ResultsGM-CSF enhances cross-presentation by CD8 1 DCs GM-CSF is a cytokine that exists at very low levels at steady state but is greatly induced during infection and inflammation [13]. To understand the role of GM-CSF in regulating the functions of CD8 1 DCs, we chose mouse models with GM-CSF overexpression. We first compared the cross-presentation by CD8 1 DCs from GMtg mice and control littermates. When soluble OVA was used as the antigen in vitro, CD...

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.

Infection by SARS-CoV-2 is initiated by binding of viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. While antibodies that block this interaction are in emergency use as early COVID-19 therapies, precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that all potently block ACE2 binding, yet exhibit divergent neutralization efficacy against live virus. Strikingly, these neutralizing antibodies can either inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in COVID-19 patients. Multiple cryogenic electron microscopy structures of Spike-antibody complexes reveal distinct binding modes that not only block ACE2 binding, but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion, with profound implications in COVID-19 pathology and immunity.

SARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31, a potent SARS-CoV-2 neutralizing IgG1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL-6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFNγ-driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite FcγR binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. Therapeutic efficacy was confirmed in SARS-CoV-2-infected hamsters, where SC31 again significantly reduced viral load, decreased lung lesions and inhibited progression to severe disease manifestations. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development.