Although extracellular DNA is known to form immune complexes (ICs) with autoantibodies in systemic lupus erythematosus (SLE), the mechanisms leading to the release of DNA from cells remain poorly characterized. Here, we show that the pore-forming protein, gasdermin D (GSDMD), is required for nuclear DNA and mitochondrial DNA (mtDNA) release from neutrophils and lytic cell death following ex vivo stimulation with serum from patients with SLE and IFN-γ. Mechanistically, the activation of FcγR downregulated Serpinb1 following ex vivo stimulation with serum from patients with SLE, leading to spontaneous activation of both caspase-1/caspase-11 and cleavage of GSDMD into GSDMD-N. Furthermore, mtDNA oxidization promoted GSDMD-N oligomerization and cell death. In addition, GSDMD, but not peptidyl arginine deiminase 4 is necessary for extracellular mtDNA release from low-density granulocytes from SLE patients or healthy human neutrophils following incubation with ICs. Using the pristane-induced lupus model, we show that disease severity is significantly reduced in mice with neutrophil-specific Gsdmd deficiency or following treatment with the GSDMD inhibitor, disulfiram. Altogether, our study highlights an important role for oxidized mtDNA in inducing GSDMD oligomerization and pore formation. These findings also suggest that GSDMD might represent a possible therapeutic target in SLE.

Excessive self-reactive and inadequate affinity-matured antigen-specific antibody responses have been reported to coexist in lupus, with elusive cellular and molecular mechanisms. Here, we report that the antigen-specific germinal center (GC) response―a process critical for antibody affinity maturation―is compromised in murine lupus models. Importantly, this defect can be triggered by excessive autoimmunity-relevant CD11c ⁺ Tbet ⁺ age-associated B cells (ABCs). In B cell-intrinsic Ship-deficient (ShipΔB) lupus mice, excessive CD11c ⁺ Tbet ⁺ ABCs induce deregulated follicular T-helper (T _FH ) cell differentiation through their potent antigen-presenting function and consequently compromise affinity-based GC selection. Excessive CD11c ⁺ Tbet ⁺ ABCs and deregulated T _FH cell are also present in other lupus models and patients. Further, over-activated Toll-like receptor signaling in Ship-deficient B cells is critical for CD11c ⁺ Tbet ⁺ ABC differentiation, and blocking CD11c ⁺ Tbet ⁺ ABC differentiation in ShipΔB mice by ablating MyD88 normalizes T _FH cell differentiation and rescues antigen-specific GC responses, as well as prevents autoantibody production. Our study suggests that excessive CD11c ⁺ Tbet ⁺ ABCs not only contribute significantly to autoantibody production but also compromise antigen-specific GC B-cell responses and antibody-affinity maturation, providing a cellular link between the coexisting autoantibodies and inadequate affinity-matured antigen-specific antibodies in lupus models and a potential target for treating lupus.

Microfluidics system enables high-throughput functional screening of antibodies for cancer immunotherapy.

Human immunoglobulin G (IgG) agonistic antibodies targeting costimulatory immunoreceptors represent promising cancer immunotherapies yet to be developed. Whether, and how, human IgG hinge and Fc impact on their agonistic functions have been disputed. Here, we show that different natural human IgGs confer divergent agonistic anti-CD40 immunostimulatory and antitumour activities in FcγR-humanized mice, including inactive IgG3 and superior IgG2. This divergence is primarily due to their CH1-hinges despite all human IgGs requiring Fc-FcγR binding for optimal agonistic activities. Unexpectedly, biophysical flexibility of these CH1-hinges inversely correlates with, and can modulate, their agonistic potency. Furthermore, IgG Fcs optimized for selective FcγR binding synergize with and still require IgG hinge, selected for rigidity, to confer improved anti-CD40 immunostimulatory and antitumour activities. These findings highlight the importance of both hinge rigidity and selective FcγR binding in antibody agonistic function, and the need for newer strategies to modulate antibody agonism for improved clinical application.

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform.