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IL-23 promotes autoimmune disease, including Th17 CD4 T cell development and autoantibody production. In this study, we show that a deficiency of the p19 component of IL-23 in the autoimmune BXD2 (BXD2-p19−/−) mouse leads to a shift of the follicular T helper cell program from follicular T helper (Tfh)–IL-17 to Tfh–IFN-γ. Although the germinal center (GC) size and the number of GC B cells remained the same, BXD2-p19−/− mice exhibited a lower class-switch recombination (CSR) in the GC B cells, leading to lower serum levels of IgG2b. Single-cell transcriptomics analysis of GC B cells revealed that whereas Ifngr1, Il21r, and Il4r genes exhibited a synchronized expression pattern with Cxcr5 and plasma cell program genes, Il17ra exhibited a synchronized expression pattern with Cxcr4 and GC program genes. Downregulation of Ighg2b in BXD2-p19−/− GC B cells was associated with decreased expression of CSR-related novel base excision repair genes that were otherwise predominantly expressed by Il17ra+ GC B cells in BXD2 mice. Together, these results suggest that although IL-23 is dispensable for GC formation, it is essential to promote a population of Tfh–IL-17 cells. IL-23 acts indirectly on Il17ra+ GC B cells to facilitate CSR-related base excision repair genes during the dark zone phase of GC B cell development.

To determine the extent to which the gut microbiome influences systemic autoimmunity in a mouse model of lupus.We generated germ-free (GF) lupus-prone BXD2 mice, which under normal conditions develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. GF status was confirmed by gut bacterial culture. The autoimmune phenotypes of 6- and 12-month-old gnotobiotic GF BXD2 mice and specific pathogen-free (SPF) BXD2 mice were compared. Serum levels of autoantibodies were measured by enzyme-linked immunosorbent assay. Histologic sections of the mouse kidney and joints were evaluated. Flow cytometry was used to analyze GCs and age-associated B cells. CD4+ T cells were analyzed for PD-1+ICOS+ activated T cells, T follicular regulatory (Tfr) cells (Foxp3+CD25+ PD-1+CXCR5+), and PD-1+ICOS+ T cells expressing interleukin-17A (IL-17A) or interferon-γ (IFNγ) after stimulation with phorbol myristate acetate (PMA)/ionomycin.In 6-month-old mice, GF status did not affect splenomegaly, GC B cells, age-associated B cells, or serum autoantibody levels, except for IgG antihistone. GF BXD2 mice exhibited a significantly higher percentage of Tfr cells compared to their SPF counterparts (P0.05). At 12 months of age, however, GF BXD2 mice had significantly diminished IgG autoantibody levels and a lower percentage of GC B cells and age-associated B cells (P0.05). Following stimulation with PMA/ionomycin, PD-1+ICOS+ CD4+ T cells expressed significantly lower IL-17A, but not IFNγ, levels in GF BXD2 mice compared to SPF BXD2 mice (P0.01). SPF BXD2 mice and GF BXD2 mice developed equivalent renal and joint disease with no significant differences in severity.Our results suggest a model in which genetics plays a dominant role in determining the initial development of autoimmunity. In contrast, gut microbiomes may regulate the persistence of certain aspects of systemic autoimmunity.

Protein kinase CK2 (also known as Casein Kinase 2) is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α′) and two regulatory CK2β subunits. CK2 is overexpressed and overactive in B cell acute lymphoblastic leukemia and diffuse large B cell lymphomas, leading to inappropriate activation of the NF-κB, JAK/STAT, and PI3K/AKT/mTOR signaling pathways and tumor growth. However, whether CK2 regulates normal B cell development and differentiation is not known. We generated mice lacking CK2α specifically in B cells (using CD19-driven Cre recombinase). These mice exhibited cell-intrinsic expansion of marginal zone B cells at the expense of transitional B cells, without changes in follicular B cells. Transitional B cells required CK2α to maintain adequate BCR signaling. In the absence of CK2α, reduced BCR signaling and elevated Notch2 signaling activation increased marginal zone B cell differentiation. Our results identify a previously unrecognized function for CK2α in B cell development and differentiation.

The transitional stage of B cell development is a formative stage in the spleen where autoreactive specificities are censored as B cells gain immune competence, but the intrinsic and extrinsic factors regulating survival of transitional stage 1 (T1) B cells are unknown. We report that B cell expression of IFN-β is required for optimal survival and TLR7 responses of transitional B cells in the spleen and was overexpressed in T1 B cells from BXD2 lupus-prone mice. Single-cell gene expression analysis of B6 Ifnb+/+ versus B6 Ifnb–⁄– T1 B cells revealed heterogeneous expression of Ifnb in wild-type B cells and distinct gene expression patterns associated with endogenous IFN-β. Single-cell analysis of BXD2 T1 B cells revealed that Ifnb is expressed in early T1 B cell development with subsequent upregulation of Tlr7 and Ifna1. Together, these data suggest that T1 B cell expression of IFN-β plays a key role in regulating responsiveness to external factors.

The onset and severity of autoimmunity can be attributed to both genetic predisposition and environmental triggers including intestinal microbiota dysbiosis. To determine if development of spontaneous autoimmunity is dependent on gut microbiota, we generated germ-free (GF) BXD2 lupus mice which otherwise develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. The microbiome and immune phenotypes in specific pathogen free (SPF) and GF BXD2 and normal B6 strains of mice were analyzed. The GF status was confirmed by gut bacterial culture. Surprisingly, the GF status did not affect disease onset and severity in 6-mo-old mice. There were comparable serum levels of IgG or IgM autoantibodies against lupus autoantigens (DNA, histone, and RNP) in SPF versus GF BXD2 mice. Flow cytometry analysis showed that the frequency and absolute numbers of GC B cells, CD4+ T cells, follicular T-helper (Tfh) cells in the spleen were also indistinguishable between SPF and GF BXD2 mice although GF BXD2 mice exhibited a higher number of CD19+ B cells and Tregs, compared to the SPF counterpart. Histologic examination of kidney and lung also showed similar disease scores between SPF and GF BXD2 mice. At older age (12-mo-old), however, there were diminished GC B cells and serum levels of autoantibodies in GF BXD2 mice compared to SPF mice. The results suggest a model in which genetic factors play a dominant role in determining the onset and susceptibility of autoimmunity and that endogenous self-antigens are the primary driver of autoreactive T and B cells leading to spontaneous GCs in BXD2 mice. In contrast, environmental factors such as gut microbiomes may play a modifying role in regulating the persistence of systemic autoimmunity.

The role of IL-23 in promoting pathogenic autoantibody development in autoimmune disease is unclear. The purposes of this study are to use the IL-23p19 knockout in the BXD2, a lupus mouse strain, to determine: (i) if IL-23 is required for germinal center (GC) and pathogenic autoantibody development; and (ii) if IL-23 acts through follicular T-helper cells (Tfh) lineage switching to induce autoantibody formation. Sera autoantibodies were measured by ELISA. GC development and the expression of AID were determined by confocal imaging. IL-17 and IFN-γ producing Tfh cells were detected by intracellular staining. The expression of GC program genes and T cell program genes was measured by qRT-PCR. Exogenous IL-23 was administered to B6 mice using an IL-23 expressing adenovirus (AdIL-23). There was significantly increased IgM but decreased IgG autoantibodies in BXD2-p19−/− mice compared to BXD2 mice. The expression of AID was decreased in BXD2-p19−/− mice. Surprisingly, the size and number of GC were increased in BXD2-p19−/− mice. There was also similar percentage of PD1+CXCR5+Tfh cells in BXD2 and BXD2-p19−/− mice. However, the percentage of IL-17A expressing Tfh cells was suppressed but the IFN-γ producing Tfh was increased in BXD2-p19−/− mice. Consistent with this, AdIL-23 administration suppressed the expression of Tbet, Ifng, Bcl6, Il21, Gata3, and Il4 but enhanced the expression Il17a, Il17f in Tfh cells. Our data suggest Tfh-IFN-γ and Tfh-IL-17 each plays an important role to determine the development of autoantibody producing B cells. While IL-23-mediated Tfh-IL-17 does not promote the size of autoreactive GCs, it regulates the induction of AID and thereby plays an important role to determine autoantibody affinity maturation.

Targeting IL-23 to treat autoimmune disease and chronic inflammation is under development based on its proinflammatory function and pro-Th17 cell effects. IL-23 was previously shown to be important for promoting pathogenic autoantibody production in B6-Faslpr/lpr mice. In contrast, the lack of IL-23 did not influence germinal center (GC) formation and IgG anti-CII autoantibodies in type II collagen immunized DBA/1 mice. We determined if IL-23 regulated development of spontaneous GCs and autoantibody production in lupus-prone BXD2 mice by generating IL-23 deficient BXD2-p19−/− mice. There was significantly decreased total IgG, but increased total IgM and IgM anti-DNA and RF autoantibodies in BXD2-p19−/− mice compared to BXD2 mice. Flow cytometry and confocal imaging analyses showed that both the size and number of GL-7+Fas+ GC B cells were increased, yet there was a decreased development of GC dark zone (DZ) (CD86−CXCR4+) B cells in BXD2-p19−/− mice. Interestingly, the expression of activation-induced cytidine deaminase (AICDA or Aicda) was significantly decreased in GC B cells in BXD2-p19−/− mice. However, the expression of GC program genes (Bach2, Pax5) was increased except Bcl6. In contrast, the expression of key plasma cell program genes Prdm1 in GC B cells was diminished in the absence of IL-23. Our data suggest a novel pathway for IL-23 to promote IgG autoantibody production by upregulation of AICDA in the GC DZ. Importantly, such pathway is independent of the canonical GC program genes that have been implicated for the induction of AICDA. These results suggest a potential novel action of anti-IL-23 therapies to inhibit adaptive immune responses in addition to its known inhibitory role of innate response in autoimmune diseases.

We previously showed that B-cell endogenous expression of interferon beta (IFNβ) is associated with the development of autoreactive B cells in BXD2 autoimmune mice. As among all type I IFNs, IFNβ exhibits the highest affinity to IFNAR1 and IFNAR2 and IFNβ also can stimulate IFNαs, there is a pressing need for a better understanding of the source and responses of IFNβ in systemic lupus erythematosus (SLE). Flow cytometry and super-resolution imaging analysis showed increased expression of IFNβ in B cells obtained from PBMCs of SLE patients compared to healthy controls. The importance of SLE B-cell endogenous IFNβ in regulating TLR7-mediated responses was identified when CL264 (a TLR7 ligand) plus anti-Ig-induced CD69 and survival of purified B cells were significantly and equivalently suppressed by an anti-IFNβ or an anti-IFNAR1 blocking antibody. We next carried out single cell qRT-PCR analysis to determine if B-cell endogenous IFNβ acts in an autocrine or paracrine manner to modulate IFN stimulated genes (ISGs). Hierarchical analysis revealed three prominent and consistent clusters, consisting of an IFNB+ cluster, an ISG+ cluster, and an IFNA+ cluster in naïve B cells isolated from 3 SLE patients. Cells within the IFNB+ cluster expressed higher levels of IFNB, IFNA1, IFNA8, and TLR7. Cells within the ISG+ cluster expressed higher levels of IFNAR1, IFNAR2, BAFFR, MX1, RIG1 and TLR9. Cells within the IFNA+ cluster expressed higher levels of IFNA4, IFNA10, IFNA14, IFNA16, IFNA17, and TLR3. Together, the results reveal (1) B cells as an important source of IFNβ in modulating TLR7 responses in SLE; (2) a well-orchestrated program in modulating IFNβ donor versus responder B cells; and (3) some patients may benefit from specific targeting of IFNβ.