Systemic Lupus Erythematosus Pathogenesis: Interferon and Beyond.

Autoreactive B cells and interferons are central players in systemic lupus erythematosus (SLE) pathogenesis. The partial success of drugs targeting these pathways, however, supports heterogeneity in upstream mechanisms contributing to disease pathogenesis. In this review, we focus on recent insights from genetic and immune monitoring studies of patients that are refining our understanding of these basic mechanisms. Among them, novel mutations in genes affecting intrinsic B cell activation or clearance of interferogenic nucleic acids have been described. Mitochondria have emerged as relevant inducers and/or amplifiers of SLE pathogenesis through a variety of mechanisms that include disruption of organelle integrity or compartmentalization, defective metabolism, and failure of quality control measures. These result in extra- or intracellular release of interferogenic nucleic acids as well as in innate and/or adaptive immune cell activation. A variety of classic and novel SLE autoantibody specificities have been found to recapitulate genetic alterations associated with monogenic lupus or to trigger interferogenic amplification loops. Finally, atypical B cells and novel extrafollicular T helper cell subsets have been proposed to contribute to the generation of SLE autoantibodies. Overall, these novel insights provide opportunities to deepen the immunophenotypic surveillance of patients and open the door to patient stratification and personalized, rational approaches to therapy.

TFH cells in bystander and cognate interactions with B cells.

Follicular T-helper (TFH ) cells play a crucial role in three aspects of the germinal center (GC) response. They promote GC formation, arbitrate competition among GC B cells to determine the outcome of affinity maturation, and regulate GC output of memory and plasma cells to shape the long-lived humoral immune memory. Of fundamental importance are dynamic physical interactions between TFH and B cells, which are the main platform for TFH cells to deliver "help" factors to B cells and also for reciprocal signaling from B cells to maintain the helper state of TFH cells. Recent work has significantly expanded our understanding of how T-B interactions are spatiotemporally regulated and molecularly orchestrated to fulfill those TFH functions. In this review, we elaborate two modes of T-B interactions, the antigen-specific or cognate mode in which TFH cells engage individual antigen-presenting B cells and the antigen nonspecific bystander mode in which TFH cells are engaged with the ensemble of follicular B cells. We discuss findings that indicate how short-lived cognate T-B contacts coupled with an intercellular positive feedback drive affinity-based selection and how bystander interactions between T and B cells regulate follicular T-cell recruitment and maintenance of an appropriate helper state. We argue that this combination of bystander and cognate interactions with B cells constantly shapes the internal state of TFH cells and provides the platform to execute their helper functions.

T-B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction.

The germinal centre (GC) reaction supports affinity-based B-cell competition and generates high-affinity bone-marrow plasma cells (BMPCs). How follicular T-helper (TFH) cells regulate GC selection is not clear. Using competitive mixed chimaera, we show here that, beyond the role in promoting TFH development, ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) is important for individual B cells to competitively participate in the GC reaction and to develop into BMPCs. Using intravital imaging aided by a calcium reporter, we further show that ICOSL promotes an 'entangled' mode of TFH-B-cell interactions, characterized by brief but extensive surface engagement, productive T-cell calcium spikes, and B-cell acquisition of CD40 signals. Reiterated entanglement promotes outer-zone co-localization of outcompeting GC B cells together with TFH cells, affording the former increased access to T-cell help. ICOSL on GC B cells is upregulated by CD40 signals. Such an intercellular positive feedback between contact-dependent help and ICOSL-controlled entanglement promotes positive selection and BMPC development, as evidenced by observations that higher-affinity B-cell receptor variants are enriched in the ICOSL(high) fraction, that numerically disadvantaged ICOSL-deficient GC B cells or BMPCs exhibit strong affinity compensation in competitive chimaera, and that when GC competition proceeds without ICOSL, selection of high-affinity variants in otherwise normal GC reactions is impaired. By demonstrating entanglement as the basic form of GC TFH-B-cell interactions, identifying ICOSL as a molecular linkage between T-B interactional dynamics and positive selection for high-affinity BMPC formation, our study reveals a pathway by which TFH cells control the quality of long-lived humoral immunity.

B cell-reactive triad of B cells, follicular helper and regulatory T cells at homeostasis.

Autoreactive B cells are silenced through receptor editing, clonal deletion and anergy induction. Additional autoreactive B cells are ignorant because of physical segregation from their cognate autoantigen. Unexpectedly, we find that follicular B cell-derived autoantigen, including cell surface molecules such as FcγRIIB, is a class of homeostatic autoantigen that can induce spontaneous germinal centers (GCs) and B cell-reactive autoantibodies in non-autoimmune animals with intact T and B cell repertoires. These B cell-reactive B cells form GCs in a manner dependent on spontaneous follicular helper T (TFH) cells, which preferentially recognize B cell-derived autoantigen, and in a manner constrained by spontaneous follicular regulatory T (TFR) cells, which also carry specificities for B cell-derived autoantigen. B cell-reactive GC cells are continuously generated and, following immunization or infection, become intermixed with foreign antigen-induced GCs. Production of plasma cells and antibodies derived from B cell-reactive GC cells are markedly enhanced by viral infection, potentially increasing the chance for autoimmunity. Consequently, immune homeostasis in healthy animals not only involves classical tolerance of silencing and ignoring autoreactive B cells but also entails a reactive equilibrium attained by a spontaneous B cell-reactive triad of B cells, TFH cells and TFR cells.

An unconventional mechanism of IL-1ß secretion that requires Type I IFN in lupus monocytes.

Systemic Lupus Erythematosus (SLE) is characterized by autoreactive B cell activation, upregulation of Type I Interferon (IFN) and widespread inflammation. Mitochondrial nucleic acids (NAs) are increasingly recognized as triggers of IFN 1 . Thus, defective removal of mitochondria from mature red blood cells (Mito + RBCs), a feature of SLE, contributes to IFN production by myeloid cells 2 . Here we identify blood monocytes (Mo) that have internalized RBCs and co-express IFN-stimulated genes (ISGs) and interleukin-1ß (IL-1ß) in SLE patients with active disease. We show that ISG expression requires the interaction between Mito + RBC-derived mitochondrial DNA (mtDNA) and cGAS, while IL-1ß production entails Mito + RBC-derived mitochondrial RNA (mtRNA) triggering of RIG-I-like receptors (RLRs). This leads to the cytosolic release of Mo-derived mtDNA that activates the NLRP3 inflammasome. Importantly, IL-1ß release depends on the IFN-inducible myxovirus resistant protein 1 (MxA), which enables the translocation of this cytokine into a trans-Golgi network (TGN)-mediated unconventional secretory pathway. Our study highlights a novel and synergistic pathway involving IFN and the NLRP3 inflammasome in SLE.

A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines.

BACKGROUND: Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. RESULTS: We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV) to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116) and HDAC2 KO cells from the colorectal cancer cell line (DLD1). CONCLUSIONS: Our method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.

Breaching self-tolerance by targeting the gatekeeper.

Loss-of-function mutations in DNaseL13, the enzyme that restricts the amount of microparticle-associated DNA, cause SLE in humans and mice. In this issue of JEM, Hartl et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20201138) uncover a reduction in plasma DNASE1L3 enzymatic activity due to the presence of autoantibodies in patients with nonfamilial SLE.

Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids.

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).

Transmembrane domain-mediated Lck association underlies bystander and costimulatory ICOS signaling.

The B7-family inducible costimulator (ICOS) activates phosphoinositide-3 kinase (PI3K) and augments calcium mobilization triggered by the T-cell receptor (TCR). We surprisingly found that the entire cytoplasmic domain of ICOS is dispensable for its costimulation of calcium mobilization. This costimulatory function relies on the unique transmembrane domain (TMD) of ICOS, which promotes association with the tyrosine kinase Lck. TMD-enabled Lck association is also required for p85 recruitment to ICOS and subsequent PI3K activation, and Lck underlies both the bystander and costimulatory signaling activity of ICOS. TMD-replaced ICOS, even with an intact cytoplasmic domain, fails to support TFH development or GC formation in vivo. When transplanted onto a chimeric antigen receptor (CAR), the ICOS TMD enhances interactions between T cells and antigen-presenting target cells. Therefore, by revealing an unexpected function of the ICOS TMD, our study offers a new perspective for the understanding and potential application of costimulation biology.