Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of Ripk1 results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8–driven apoptosis. IFNs are thought to contribute to the lethality of Ripk1-deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8–mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either Zbp1 or core IFN signaling components prolong viability of Ripk1−/− mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.

Maturation of antibody responses entails somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell differentiation, and generation of memory B cells, and it is thought to require T cell help. We showed that B cell Toll-like receptor 4 (TLR4)–B cell receptor (BCR) (receptor for antigen) coengagement by 4-hydroxy-3-nitrophenyl acetyl (NP)–lipopolysaccharide (LPS) ( Escherichia coli lipid A polysaccharide O-antigen) or TLR5-BCR coengagement by Salmonella flagellin induces mature antibody responses to NP and flagellin in Tcr β ^−/− Tcr δ ^−/− and NSG/B mice. TLR-BCR coengagement required linkage of TLR and BCR ligands, “linked coengagement.” This induced B cell CSR/SHM, germinal center–like differentiation, clonal expansion, intraconal diversification, plasma cell differentiation, and an anamnestic antibody response. In Tcr β ^−/− Tcr δ ^−/− mice, linked coengagement of TLR4-BCR by LPS or TLR5-BCR by flagellin induced protective antibodies against E. coli or Salmonella Typhimurium. Our findings unveiled a critical role of B cell TLRs in inducing neutralizing antibody responses, including those to microbial pathogens, without T cell help.

The maintenance of regulatory T (T _reg ) cells critically prevents autoimmunity. Pre–B cell leukemia transcription factor 1 ( Pbx1 ) variants are associated with lupus susceptibility, particularly through the expression of a dominant negative isoform Pbx1-d in CD4 ⁺ T cells. Pbx1-d overexpression impaired T _reg cell homeostasis and promoted inflammatory CD4 ⁺ T cells. Here, we showed a high expression of Pbx1 in human and murine T _reg cells, which is decreased in lupus patients and mice. Pbx1 deficiency or Pbx1-d overexpression reduced the number, stability, and suppressive activity of T _reg cells, which increased murine responses to immunization and autoimmune induction. Mechanistically, Pbx1 deficiency altered the expression of genes implicated in cell cycle and apoptosis in T _reg cells. Intriguingly, Rtkn2 , a Rho-GTPase previously associated with T _reg homeostasis, was directly transactivated by Pbx1. Our results suggest that the maintenance of T _reg cell homeostasis and stability by Pbx1 through cell cycle progression prevent the expansion of inflammatory T cells that otherwise exacerbates lupus progression in the hosts.

Necroptosis is an antiviral form of programmed cell death modulated by proteins that interact via RIP Homotypic Interaction Motifs (RHIMs). The result of the signaling pathways depends on which RHIM-containing proteins are involved: although both host and viral proteins contain RHIMs, virally encoded RHIM proteins, such as murine cytomegalovirus (MCMV)-encoded viral inhibitor of RIP activation (vIRA) serve to prevent cell death. Although every RHIM contains the same core four-amino-acid pattern, there are variations in individual sequences that we hypothesized would determine the differential outcomes in necroptotic signaling. As such, we replaced the RHIM in vIRA with the RHIMs from other proteins involved in the signaling cascade (RIPK1, RIPK3, ZBP1, ICP6) to assess the effect on necroptosis during MCMV infection. Although these RHIM-swap vIRA constructs remained able to bind to RIPK3, in the context of MCMV infection, they lost the ability to prevent necroptosis. These results are consistent with other studies that demonstrate that RHIM-containing proteins form amyloid fibrils unique to the proteins interfacing. Our results provide biological context for the growing model that the outcome of RHIM-based signaling is influenced by the specific amyloid fibril structures that are driven by the unique amino-acid sequences of each RHIM present.