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Activated naive (aNAV) B cells have been shown to be the precursor of the CD11c+T-bet+ IgD−CD27− double-negative (DN)2 or atypical memory (aMEM) B cells in systemic lupus erythematosus (SLE). To determine factors that maintain resting naive (rNAV) B cells, the transcriptomic program in naive (IGHD+IGHM+) B cells in human healthy control subjects (HC) and subjects with SLE was analyzed by single-cell RNA-sequencing analysis. In HC, naive B cells expressed IL-4 pathway genes, whereas in SLE, naive B cells expressed type I IFN-stimulated genes (ISGs). In HC, aNAV B cells exhibited upregulation of the gene signature of germinal center and classical memory (cMEM) B cells. In contrast, in SLE, aNAV B cells expressed signature genes of aMEM. In vitro exposure of SLE B cells to IL-4 promoted B cell development into CD27+CD38+ plasmablasts/plasma and IgD−CD27+ cMEM B cells. The same treatment blocked the development of CD11c+Tbet+ aNAV and DN2 B cells and preserved DN B cells as CD11c−Tbet− DN1 B cells. Lower expression of IL-4R and increased intracellular IFN-β in naive B cells was correlated with the accumulation of CD21−IgD− B cells and the development of anti-Smith and anti-DNA autoantibodies in patients with SLE (n = 47). Our results show that IL-4R and type I IFN signaling in naive B cells induce the development of distinct lineages of cMEM versus aMEM B cells, respectively. Furthermore, diminished IL-4R signaling shifted activated B cell development from the DN1 to the DN2 trajectory in patients with SLE. Therapies that enhance IL-4R signaling may be beneficial for ISGhi SLE patients.

Here, we report a case of atopic dermatitis (AD) in a patient who received biweekly doses of dupilumab, an antibody against the IL-4 receptor α chain (IL-4Rα). Single cell RNA-sequencing showed that naïve B cells expressed the highest levels of IL4R compared to other B cell subpopulations. Compared to controls, the dupilumab-treated patient exhibited diminished percentages of IL4R+IGHD+ naïve B cells and down-regulation of IL4R, FCER2 (CD23), and IGHD. Dupilumab treatment resulted in upregulation of genes associated with apoptosis and inhibition of B cell receptor signaling and down-regulation of class-switch and memory B cell development genes. The dupilumab-treated patient exhibited a rapid decline in COVID-19 anti-spike and anti-receptor binding domain antibodies between 4 and 8 and 11 months post COVID-19 vaccination. Our data suggest that intact and persistent IL-4 signaling is necessary for maintaining robust survival and development of naïve B cells, and maintaining a long term vaccine response.

Objective: To determine if circulating autoantibodies (autoAbs) and T-helper cell cytokines correlate with a different class of lupus nephritis (LN), including class III/IV, compared to class V and other manifestations of systemic lupus erythematosus (SLE). Methods: All patients (N=62) met the SLE classification criteria of the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) or Systemic Lupus International Collaborating Clinics (SLICC) classification criteria. Demographic, clinical data, and serologic manifestations included anti-DNA, anti-Smith (Anti-Sm), C3, and C4 were included. Plasma levels of interferon gamma (IFNɣ), interleukin 17 (IL-17), interleukin 10 (IL-10) isotype-specific (IgG) anti-DNA, anti-Ro/SSA (SSA), and anti-Sm were measured using enzyme-linked immunosorbent assay (ELISA).Results: The most common manifestations by history were arthritis in 61% (N=38), photosensitivity rash in 53% (N=33), LN in 44% (N=27), and oral/nasal ulcer in 31% (N=19) SLE patients. AutoAbs (anti-DNA, anti-SSA, and anti-Sm), IFNɣ, and IL-17, but not IL-10, were significantly elevated in SLE patients compared to healthy controls. There were elevated plasma levels of anti-DNA (p = 0.0101) and anti-Sm (p = 0.0499) in patients with a history of LN compared to patients without LN. In contrast, plasma levels of anti-Sm were decreased in patients who had a history of acute mucocutaneous manifestations, including photosensitivity rash and/or malar rash (p = 0.0152). Among the three cytokines that were analyzed, IL-10 was significantly elevated in patients with a history of LN compared to patients without LN (p = 0.0216). IL-17 was positively correlated with anti-SSA (p = 0.0130) and was significantly higher in patients with discoid rash (p = 0.0238) and history of class V LN (p = 0.0055). IFNɣ was positively correlated with anti-DNA (p = 0.0355) and anti-SSA (p = 0.0402). Conclusion: This cross-sectional study supports the role of different T-helper cell cytokines that may be associated with the development of different autoAbs in influencing the diversity of SLE clinical manifestations. The results suggests that elevated IFNɣ and IL-17 are more generalized features in SLE patients. In contrast, higher levels of IL-10 were observed in patients with a history of LN. This provide insights into the pathogenic mechanisms of LN that can help guide future diagnosis and therapies.

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.

Background In systemic lupus erythematosus (SLE), detection of interferon-β (IFNβ) in B cells was found to be most prominent in patients with high anti-Smith (Sm) and renal disease, but a mechanistic connection was not clear. The objective of the present study is to determine the association of IFNβ in peripheral blood naïve B cells with the histopathological features of lupus nephritis (LN). Methods The percentage of IFNβ+ cells in IgD+CD27− naïve CD19+ B cells (B cell IFNβ) among peripheral blood mononuclear cells (PBMCs) from 80 SLE patients were analyzed using flow cytometry. Serological and clinical data were collected. The correlations of B cell IFNβ with LN classification and with histopathological findings (light, electron, and immunofluorescence [IF] microscopic analyses for deposition of IgM, IgG, IgA, C1q, and C3) were determined in 23 available biopsy specimens. Results B cell IFNβ is positively associated with anti-Sm (p = 0.001), anti-DNA (p = 0.013), and LN (p < 0.001) but was negatively associated with oral/nasal ulcer (p = 0.003) and photosensitivity (p = 0.045). B cell IFNβ positively correlated with immune complex (IC) deposit in the glomerular basement membrane (GBM) (p = 0.002) but not in the mesangial (p = 0.107) or tubular region (p = 0.313). Patients with high B cell IFNβ had statistically increased development of the proliferative LN (Classes III, IV and/or V), compared to patients with low B cell IFNβ (p p = 0.023), chronic glomerular lesions indicated by segmental sclerosis (p = 0.033), and a membranous pattern of renal damage indicated by spike/holes (p = 0.015). Conclusion B cell IFNβ correlates with history of severe LN, glomerular basement membrane (GBM) IC deposition, and anatomical features of both active and chronic glomerular lesions.

In systemic lupus erythematosus (SLE), type I IFNs promote induction of type I IFN–stimulated genes (ISG) and can drive B cells to produce autoantibodies. Little is known about the expression of distinct type I IFNs in lupus, particularly high-affinity IFN-β. Single-cell analyses of transitional B cells isolated from SLE patients revealed distinct B cell subpopulations, including type I IFN producers, IFN responders, and mixed IFN producer/responder clusters. Anti-Ig plus TLR3 stimulation of SLE B cells induced release of bioactive type I IFNs that could stimulate HEK-Blue cells. Increased levels of IFN-β were detected in circulating B cells from SLE patients compared with controls and were significantly higher in African American patients with renal disease and in patients with autoantibodies. Together, the results identify type I IFN–producing and –responding subpopulations within the SLE B cell compartment and suggest that some patients may benefit from specific targeting of IFN-β.

Fucosylation is a biological process broadly observed in vertebrates, invertebrates, plants, bacteria, and fungi. Fucose moieties on cell-surface glycans are increasingly recognized as critical to many cell-cell interaction and signaling processes. One of the characteristic roles of fucose is its regulation of selectin-dependent leukocyte adhesion that has been well studied over the last two decades. Recent studies of fucose in immune cell development and function regulation have significantly expanded the contemporary understanding of fucosylation. From cellular adhesion to immune regulation, herein we discuss the use of gene knockout studies, competitive inhibitors of fucose-containing glycan, and metabolic inhibitors of fucose biosynthesis to probe fucosylated glycan biosynthesis and signaling and its functional consequences. Promising clinical and preclinical applications in sickle cell disease, rheumatoid arthritis, tumor inhibition, metastasis prevention, antibody-dependent cell-mediated cytotoxicity, chemoresistance reversal, and in improving chemotherapy-related side effects and recovery are reviewed.

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 current concepts for development of autoreactive B cells in SLE (systemic lupus erythematosus) focus on extrinsic stimuli and factors that provoke B cells into tolerance loss. Traditionally, major tolerance loss pathways are thought to be regulated by factors outside the B cell including autoantigen engagement of the B-cell receptor (BCR) with simultaneous type I interferon (IFN) produced by dendritic cells, especially plasmacytoid dendritic cells (pDCs). Later, in autoreactive follicles, B-cells encounter T-follicular helper cells (Tfh) that produce interleukin (IL)-21, IL-4 and pathogenic cytokines, IL-17 and IFN gamma (IFNɣ). This review discusses these mechanisms and also highlights recent advances pointing to the peripheral transitional B-cell stage as a major juncture where transient autocrine IFNβ expression by developing B-cells imprints a heightened susceptibility to external factors favoring differentiation into autoantibody-producing plasmablasts. Recent studies highlight transitional B-cell heterogeneity as a determinant of intrinsic resistance or susceptibility to tolerance loss through the shaping of B-cell responsiveness to cytokines and other environment factors.

Background Dysregulated responses to type I interferons (IFNs) is a hallmark of autoreactive B-cell development in SLE. This study investigated the source of IFN, the major type I IFN responsive B cells, and the disparities associated with B-cell IFN production and type I IFN responses. Methods IFN expression in B, CD4 T and plasmacytoid dendritic cells (pDCs) in PBMCs were analyzed by flow cytometry. Single cell gene expression analysis was carried out using the Fluidigm/BioMark system for targeted expression of low abundance genes, and the 10x Chromium platform for unbiased transcriptome and BCR V(D)J analysis of approximately 2,000 B cells per subject. Autoantigen epitope targets were analyzed using a 4287 high-throughput PEPperPrint Autoimmune Epitope Microarray and a conventional ELISA analysis. Results IFN was analyzed in B cells, CD4 T cells and pDCs in PBMCs of SLE patients and healthy controls (HCs). Endogenous IFN was significantly increased in transitional (Tr), mature naive, and memory B cells of SLE patients compared to HCs. Endogenous IFN in B cells was equivalent to that in pDCs. B-cell endogenous IFN was highly correlated with renal disease, anti-dsDNA, anti-Sm and anti-SSA. Strikingly, the highest correlation of IFN with clinical manifestations was observed in African-American (AA) patients with IgG autoAbs against snRNP323-339, U1snRNP-C97-113. At the single cell transcriptome levels, Tr B cells could be divided into type I IFN expressing (IFN+) or type I IFN stimulated gene (ISG+) subpopulations. TLR7 and TLR3 were mainly expressed by IFN +cells whereas TLR9 was mainly expressed by ISG +B cells. Unbiased single cells analysis of B cells indicated highly expressed ISG gene set in IGHM+, IGHD+, and IGHG +B cells in AA patients with autoantibodies and renal disease. Further, ISG highly expressing SLE B cells exhibited unique heavy- and light-chain repertoires including expression of the autoreactive IGHV4-34 gene, targeted with the 9 G4 anti-idiotype antibody that recognizes DNA- and RBP-autoreactive B cells. Conclusions (i) B cells are an important source of type I IFNs in modulating TLR and BCR responses in SLE; (ii) there are well-orchestrated distinct programs in type I IFN expression and response genes in subsets of B cells, (iii) distinct pathways of autoreactive B cell survival and activation are effected by combined signaling through BCR, TLR, and IFNAR with resultant distinct BCR heavy- and light-chain repertoire. Funding Source(s): R01-AI-071110, R01 AI134023, LRA Distinguished Innovator Award and Novel Research Award, VA Merit Review Award I01B × 004049, Immunology T32 Training Grant 2T32AI007051-39, the LFA Finzi Summer Fellowship.

Lupus nephritis (LN) is histologically evident yet early diagnosis can be challenging since some patients do not exhibit overt clinical manifestations until advanced stages. The objective of the present study is to determine if B-cell expression of interferon beta (IFNβ) can be a unique systemic lupus erythematosus (SLE) clinical prognostic marker and if specific histopathologic features of LN can be identified in patients with elevated B-cell expression of IFNβ. A total of 80 patients who met the ACR/EULAR classification criteria of SLE were recruited. LN was identified in 41% (N=33). Multivariate regression analysis indicated a significant positive correlation between naïve B-cell IFNβ with race (African American > European American; P=0.006) and LN class (P

Abnormal expansion of type I interferon (IFN) stimulated B cells and T-bet+CD11c+IgD−CD27− DN2 atypical memory B cells are both hallmarks of SLE. To determine how they are interrelated, we have carried out single cell transcriptomics analysis using B cells derived from 3 SLE patients (anti-DNA+ and anti-Smith+) and 3 healthy controls (HCs)(all African Americans). We have identified that elevation of IL4R in pre-switched IGHM+/IGHD+ B cells is the opposing signature of type I IFN stimulated genes (ISGs) and DN2 B cells (TBX21, ITGAX, FCRL3, FCRL5, and ZEB2). In HCs, IGHM+/IGHD+ B cells expressed elevated IL4R. Further, activated naïve B cells (IGHM+IGHD+IGHG+) exhibited upregulation of the germinal center-oriented classical memory B-cell signature genes, CD27, TNFRSF13B (the gene encoding TACI), and GPR183 (the gene encoding EBI2). Flow cytometry analysis confirmed that down-regulation IL-4R and upregulation of intracellular IFN-β in naïve IgD+CD27− B cells correlated with the accumulation of atypical CD21−IgD−CD27−B cells and a reduction in the percentage of classical IgD−CD27+memoryB cells in SLE patients (n=47). In vitro exposure of SLE B cells to IL-4 prior to treatment with anti-Ig+IL-21+IFN-gamma+TLR7+BAFF stimulation significantly enhanced B-cell development into CD27+CD38+ plasmablasts/plasma cells as well as IgD−CD27+memoryB cells but it significantly blocked the development of DN2 B cells. Our results show that upregulation of type I IFN and down-regulation of IL-4R signaling in naïve B cells lead to the accumulation of atypical memory B cells in SLE. Type I IFN and IL-4R signaling in naïve B cells induce the development of distinct lineages of atypical versus classical memory B cells, respectively.

IL-17 receptor A (IL-17RA) expression has been identified in B cells and macrophages. There was low expression of IL-17RA on T cells. Surprisingly, we previously showed that IL-17RA signaling through NF-κB p65/p50 has a complex effect on both B and T-cell localization and germinal center (GC) architecture, making it difficult to dissect the specific effects of IL-17RA on B cells. To isolate the effects of the absence of IL-17RA specifically on B cells, we have generated a B-cell specific IL-17RA knockout (Il17rafloxed/floxed x Cd19.Cre) BXD2 mice. Flow cytometry confirmed the loss of IL-17RA expression in only CD19+ B cells but not CD3+ T cells or CD11b+ macrophages. There was a 2-fold decreased spontaneous GC B cell population in Il17rafloxed/floxed x Cd19.Cre BXD2, compared to Il17rawt/floxed x Cd19.Cre BXD2 mice. Interestingly, the abnormal activation and expansion of T-follicular helper cell (Tfh) phenotype in BXD2-Il17ra−/− mice was abrogated in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. In the BXD2-Il17ra−/− mice, B cells were anergic to TLR7 stimulation and exhibited a strong stimulus-specific transcription repressor p50/p50 homodimer and a lower NF-κB phospho-p65. To demonstrate that this anergy is due specifically to IL-17RA on B cells but not other cells, we have identified that there was a lower TLR7-induced CD69 in vitro in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. Taken together, our results suggest that IL-17RA signaling directly regulates B cell autoimmune phenotype in the BXD2 mice through an enhancement of B-cell responses stimulated by the NF-κB signaling pathway. A deficiency of such signal enforces B cell anergy and prevents autoreactive GC formation.

We have recently identified that down-regulation of interleukin-4 receptor (IL-4R) is a novel hallmark of B-cell dysregulation in SLE patients. Single cell transcriptomics analysis revealed that elevation of IL4R in pre-switched B cells is the opposing signature of increased type I interferon stimulated genes (ISGs) and double negative B cells (TBX21, ITGAX, FCRL3, FCRL5, and ZEB2). Flow cytometry analysis further confirmed that down-regulation IL-4R and upregulation of intracellular IFN-β correlated with the loss of CD21+IgD+CD27− resting naïve (rNVA) B cells and accumulation of CD21−IgD+CD27− activated naïve (aNAV) and double negative CD21−IgD+CD27− B cells in SLE patients (n=47). Interestingly, exposure of SLE B cells to IL-4 prior to treatment with low-dose IL-2 (0.2 ng/mL) blocks the anti-Ig, IL-21, IFN-gamma, TLR7 and BAFF-stimulated development of T-bet+CD11c+IgD−CD27− DN2 B cells and CD27+CD38+ plasmablasts (PBs) and preserves B cells in the rNAV state. Such effect was diminished in the absence of IL-4 or when low-dose of IL-2 was substituted with high-dose of IL-2 (20 ng/mL). Stimulation of B cells with IL-4 promoted the induction of CD25 (IL-2RA) on B cells. Together, our results suggest a mechanism of action underlying the synergism between IL-4 and low-dose IL-2. Specifically, the IL-4R signaling pathways upregulate CD25 on B cells, thereby promoting formation of the high affinity IL-2R complexes required for low-dose IL-2 signaling that induces a regulatory signal that restores SLE B cells to the rNAV stage. Our results suggest that the process of development of autoreactive B cells in SLE can be directly measured in a clinical lab, and can be directly abrogated using cytokine therapy available for clinical use.

The most specific predictor for systemic lupus erythematosus (SLE) and lupus nephritis (LN) is the presence of anti-Smith (Sm) autoAb. Mechanisms leading to selection bias for anti-Sm development is unclear. We found that anti-Sm levels in SLE correlated with increased endogenous IFN-β and interferon-stimulated genes (ISGs) in combination with low (IL-4R) network genes. Using flow cytometry analysis of 47 SLE patients, we found that anti-Sm+ patients exhibited an increased ratio of IFN-β+ activated naive (aNAV) B cells and a decrease in IL-4R+ resting naive (rNAV) B cells. Using a combined 10X single-cell transcriptomics and BCR usage analysis, anti-Sm+LN+ISGhiIL4Rlopatients (Sm+) were compared with anti-Sm−LN− ISGloIL4Rhi patients (Sm−). BCR heavy chain analysis showed that while B cells from Sm+ patients exhibit greater usage of IGHV1-2, IGHV4-34, and IGHV4-39 genes, Sm− SLE patients exhibit greater usage of IGHV3-30, IGHV3-33, and IGHV4-31 genes. Preferential Ig light chain usage for ISGhi B cells was IGLV1-51, IGLV3-21, IGLV6-57, IGKV1-5, IGKV3-15 and IGKV 6-21. Dominant heavy-light chain pair usages for the Sm+ patients were IGHV4/IGKV1 and IGHV4/IGKV3. IGHV4-34, which is intrinsically autoreactive and the target of the 9G4 anti-idiotype Ab, was found to be expressed by the ISGhiIL4Rlo B cells derived from the same Sm+ patients. The results indicated that increased levels of ISGs and decreased levels of IL-4R pathway genes are the two dominant signatures and the underlying mechanisms leading to selection and activation of anti-Sm+ B cells. Verification that the B-cell IFN-β and IL-4R phenotypes are associated with race (AA vs. EA), repertoire (anti-Sm+) and disease (LN+) would lay the basis for targeted rational therapies.

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.

Although multiple and overlapping mechanisms are ultimately responsible for the immunopathology observed in patients with systemic lupus erythematosus, autoreactive Abs secreted by autoreactive plasma cells (PCs) are considered to play a critical role in disease progression and immunopathology. Given that PCs derive from the germinal centers (GC), long-term dysregulated GC reactions are often associated with the development of spontaneous autoantibody responses and immunopathology in systemic lupus erythematosus patients. In this review, we summarize the emerging evidence concerning the roles of T follicular helper cells in regulating pathogenic GC and autoreactive PC responses in lupus.

Background Increased type I interferon (IFN) has been shown to affect survival and activation of B cells in SLE. This study investigated novel mechanisms of endogenous production and autocrine activity of IFNβ in SLE B cells at the single-cell level. Methods IFNβ in B cells from SLE patients was analyzed using t-SNE platform based high dimensional flow cytometry. Intracellular IFNβ expression was visualized and analyzed by super-resolution confocal imaging and ImageStream analysis. Single cell gene expression analysis was carried out using the Fluidigm/BioMark system for targeted expression of low abundance genes, and the 10x Chromium platform for unbiased transcriptome analysis of up to 4,000 B cells per subject. Functional production of type I IFNs by B cells was analyzed using a human type I IFNs SEAP reporter HEK293 cell line. Results High dimension flow cytometry analysis identified intracellular IFNβ expression in pDCs, B cells, and CD4 T cells. B-cell endogenous IFNβ was required for optimal in vitro BCR and TLR7-induced activation and survival of B cells. Using a Fluidigm targeted-gene approach, B cells could be divided into type I IFN expressing (IFN+) or type I IFN stimulated gene (ISG+) subpopulations, suggesting B cells not only respond to type I IFNs but also express type I IFNs including IFNB and different IFNA genes. TLR7 and TLR3 were mainly expressed by IFN +cells whereas TLR9 was mainly expressed by ISG +B cells. The production of functional IFNβ and IFNα protein by single B cells from SLE subjects and was verified using a novel alkaline phosphatase live staining of HEK-blue reporter cells. There was enhanced IFNAR signaling by reporter cells in direct contact with SLE B cells which was blocked by anti-IFNβ and anti-IFNα. Interesting, anti-Ig crosslinking was required for optimal B-cell endogenous type I IFNs to stimulate responder cells. Unbiased single cells transcriptome analysis of SLE B cells using the 5’ 10X Chromium platform and Loupe V(D)J Browser indicated that gene clusters in type I IFN expressing or responding SLE B cells exhibited unique heavy- and light-chain gene expression repertoires. Conclusion (i) B cells are an important source of type I IFNs in modulating TLR and BCR responses in SLE; (ii) well-orchestrated and distinct programs in type I expression and responses genes in subsets of B cells, and (iii) distinct pathways of B cell survival and activation based on combined signaling through BCR, TLR, and IFNAR with a distinct BCR heavy- and light-chain repertoire. Acknowledgements This work was supported by grants from R01-AI-071110, R01 AI134023, Lupus Research Alliance Distinguished Innovator Award, I01B × 004049, and 1I01B × 000600 to J.D.M, 2T32AI007051–39 Immunology T32 Training Grant and the LFA Finzi Summer Fellowship to J.A.H, and the LRA Novel Research Award to H.-C.H.

Background Dysregulated responses to type I interferons (IFNs) is a hallmark of autoreactive B cell development in SLE patients. High sera levels of type I IFN protein were recently shown to occur in the absence of increased circulating pDCs and in the absence of increased pDC IFNs, suggesting the likelihood of other important sources of type I IFN that may act on B cells. The present study determined the cellular source of IFNβ and its association with disease activities in SLE. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from 31 SLE patients meeting ACR 1997 revised criteria for SLE and 9 healthy controls. Intracellular IFNβ was determined using flow cytometry with FITC-anti-IFNβ mAb. Comprehensive clinical data was recorded for each SLE subject and the clinical data and laboratory analysis of B-cell intracellular IFNβ expression were collected in a double-blind fashion. Results IFNβ was detected in various cell types including CD4 T cells, B cells and pDCs in PBMCs of SLE patients. Endogenous IFNβ in B cells was significantly higher than endogenous IFNβ in CD4 T cells and were equivalent to that seen in pDCs. Within B cells, there was a significant increase in endogenous IFNβ in all B cell subpopulations of SLE patients compared to healthy controls. The most significant increase was found in the CD27+IgD– memory subpopulation. B-cell endogenous IFNβ was not a result of B-cell uptake of exogenous IFNβ as coculture of SLE B cells with HEK293 reporter cells resulted in induction of interferon stimulatory genes as determined by the secreted alkaline phosphatase assay. This was further blocked by an anti-IFNβ neutralization antibody. Interestingly B-cell endogenous IFNβ was highly correlated with clinical disease including renal disease and autoantibodies including anti-dsDNA, anti-Sm and anti-SSA. Strikingly, the highest correlation of IFNβ with clinical manifestations was observed in African-American patients. B-cell IFNβ expression was significantly correlated with CD19loCD38hiCD27+ plasma cell formation. Conclusion Intracellular IFNβ production by B cells is a novel and important B-cell intrinsic factor that may be essential for B-cell development into autoantibody producing B cells. The present work suggests a need for future human lupus studies into type I IFN dysregulation that pioneer beyond the view of pDC produced IFNα. These results also provided a mechanistic basis for development of more effective therapies to target the high-affinity IFNβ or the enhanceosome components that promote its induction in a subgroup of lupus patients. Acknowledgements This work was supported by grants from R01-AI-071110, R01 AI134023, Lupus Research Alliance Distinguished Innovator Award, I01B × 004049, and 1I01B × 000600 to J.D.M, 2T32AI007051–39 Immunology T32 Training Grant and the LFA Finzi Summer Fellowship to J.A.H, and the LRA Novel Research Award to H.-C.H.

Type I interferons (IFNs) have a prominent role in many aspects of normal innate and adaptive immunity and autoimmunity. However, cell-type specific information about type I IFN expression and autocrine/paracrine signaling is sparse and mostly focused on non-lymphocyte and non-immune cell populations. A major function of B cells is cytokine production, but surprisingly, type I IFN production by B cells in systemic lupus erythematosus (SLE) has not been thoroughly investigated. This is due, in part, to the established view that plasmacytoid dendritic cells (pDCs) are the primary source of pathogenic type I IFN in lupus. Recent studies, however, have provided evidence to challenge this paradigm. Here, we discuss data supporting a new concept that the production of type I IFN, especially IFNβ, by early stage transitional B cells may be an important source of type I IFN to support autoreactive B cell development in lupus. These findings, if confirmed, may provide a new paradigm in designing and developing more effective therapies for preventing the formation of autoreactive B cells.

Periodontitis is a bacteria-induced inflammatory disease mainly affecting periodontal tissues, leading to periodontal inflammation, bone breakdown, and loss of the tooth. The main obstacle for treating periodontitis effectively is the difficulty in finding a target that can inhibit bone loss and inflammation simultaneously. Recent studies showed that cathepsin K (CTSK) might have functions in the immune system besides its role in osteoclasts. Thus, targeting CTSK would have a potential therapeutic effect in both the bone system and the immune system during the progression of periodontitis.In the current study, a small molecular inhibitor (odanacatib [ODN]) is explored to inhibit the function of CTSK in a bacteria-induced periodontitis mouse model.The application of ODN decreased the number of osteoclasts, macrophages, and T cells, as well as the expression of Toll-like receptors (TLRs) in the periodontitis lesion area. Furthermore, lack of CTSK inhibited the expression of TLR4, TLR5, and TLR9 and their downstream cytokine signaling in the gingival epithelial cells in periodontitis lesions, demonstrating that the innate immune response was inhibited in periodontitis.The present results show that inhibition of CTSK can prevent bone loss and the immune response during the progression of periodontitis, indicating that CTSK is a promising target for treating inflammatory diseases such as periodontitis by affecting both osteoclasts and the immune system.

The thymus is a complex organ that performs its critical function through the action of several key groups of cells that are located in spatially distinct areas of the thymus: cortical thymic epithelial cells, medullary thymic epithelial cells, and medullary dendritic cells. Negative selection occurs in the thymus when dendritic cells (DCs) presenting self-antigens interact with CD4+CD8+ double-positive thymocytes that express a self-reactive T-cell receptor. Expression of major histocompatibility and costimulatory molecules on the DCs are considered the major factors determining the fate of thymocytes. Accumulating evidence suggests that additional factors including cell-to-cell interactions between thymocytes and permanent thymic resident cells as well as the presence of local cytokines and soluble mediators that act on thymocytes through the JAK-STAT pathway, or through Nur77 or RORγt signaling pathways, can also play an important role to refine the selection process. This chapter will describe key cytokines and nuclear hormone receptors that act together at different stages of thymocyte development to mediate thymocyte survival and apoptosis. The connection of IL-23 in influencing T-cell diversity and regulation in the periphery through a central mechanism is also described.

Objective Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular helper T (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently showed that interleukin-21 (IL-21) promoted Tfh cell differentiation in autoimmune BXD2 mice that develop spontaneous GCs. This study was undertaken to determine the modulatory effects of IL-21 on Tfr cells and the Tfr cell to Tfh cell balance in BXD2 mice. Methods The percentage and phenotype of Tfr cells were determined in BXD2 and BXD2-IL21−/− mice. The effects of IL-21 on Tfr cells and the Tfr cell:Tfh cell ratio were evaluated. Sorted Tfr cells from BXD2-IL21−/− mice were cocultured with Tfh cells and B cells, or transferred into BXD2 mice to determine their function. Results The percentages and numbers of GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-IL21−/− mice than in wild-type BXD2 mice. Administration of AdIL-21 to BXD2-IL21−/− mice decreased the percentages and numbers of Tfr cells and the Tfr cell:Tfh cell ratio but increased the number of GC B cells in the spleen. Recombinant murine IL-21 suppressed FoxP3 and significantly reduced Tgfb1, Il2, and Gitr but enhanced Il21, Il6, Pd1, Cxcr5, and Icos expression in Tfr cells. IL-21 also counteracted Tfr cell–mediated inhibition of antibody secretion in the Tfh cell–B cell coculture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased the numbers of autoantibody-producing B cells. Conclusion Our findings indicate that high levels of IL-21 selectively enhance Tfh cell differentiation but inhibit Tfr cell commitment and the suppressive function of Tfr cells on Tfh cells and B cells, suggesting that IL-21 skews the balance from Tfr cells to Tfh cells to promote autoreactive GC reactions in BXD2 mice.

Objective Fucosylation catalyzed by fucosyltransferases (FUTs) is an important post-translational modification involved in a variety of biological processes. The purpose of the current study is to determine the roles of fucosylation in rheumatoid arthritis (RA) and the efficacy of reestablishing the immune homeostasis by using 2-Deoxy-D-galactose (2-D-gal), a fucosylation inhibitor.

The development of pathogenic autoantibody producing B cells is normally limited at the early transitional (Tr) stage and a later T-dependent developmental checkpoint. The precise molecular mechanisms underlying defective autoreactive B cell deletion/anergy at each checkpoint have been difficult to define. We utilized a combined single-cell B cell transcriptome and high dimensional flow cytometry analysis approach for a parallel understanding of B-cell developmental checkpoint defects at multiple stages in SLE. We found that while high levels of B-cell endogenous interferon-β (IFNβ) is an important risk factor for autoantibody positive African American (AA) SLE patients, low expression of IL-4R was a risk factor of both AA and European-Americans (EA) patients for development of pathogenic autoantibodies. The molecular signature of autoreactive B cells that escape deletion at the Tr stage include type I IFN stimulated genes (ISGs), including IRF7, ISG15, ISG20, MX1, and STAT1. The molecular signature of autoreactive B cells that escape the second T-dependent checkpoint, include low expression of IL4R and the transcriptional repressor BACH2, and high levels of TBX21, ITGAX, FCRL5 and IFI30. Interestingly, in vitro culture stimulation confirmed that the presence of IL-4 signaling significantly suppressed the development of pathogenic CD21−CD27−IgD−CD11c+T-bet+ double negative 2 (DN2) B cells in SLE. These results suggest that B-cell checkpoint defects can be used to stratify SLE patients according to high type I IFN or low IL-4R and their response signatures. These immune phenotype and transcriptome signatures offer the potential to predict therapy responses to type I IFN blockade or other therapies.

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.

While research on human SLE and mouse models of lupus have focused on mechanisms of B cell tolerance loss, these efforts have been complicated by multiple networks of immune regulatory genes, thereby impeding current understanding of the development of autoantibody producing B cells in SLE. We applied an unsupervised single cell RNA-seq (scRNA-seq) approach to determine the network and pathways-associated with the development of plasmablasts (PBs) in SLE. PBs were isolated as CD19+CD21−IgD−CD27hiCD38hi cells from PBMCs of SLE patients (n=3). A high-throughput scRNA-seq was carried out using a droplet-based 10x Chromium approach (~ 2,000 cells per sample). We identified two populations of PBs based on the expression of MKI67 encoding for Ki67, a nuclear protein associated with cellular proliferation. Representative genes elevated in MKI67+ PBs included cell cycle related genes as well as TNFR2. Gene ontology analysis further suggest that TNFR2+ B cells expressed increased levels of genes that are associated with cell cycle G1/S transition, DNA synthesis and elongation, and replication. Flow cytometry analysis identified TNFR2+ B cells as a population of antibody secreting cells expressing higher levels of CD86, CD11c, and CD38 but lower levels of CD20. As both CD86 and CD11c poise B cells to receive T-cell help, our studies suggest that T-cell stimulation enables development of cycling PBs in SLE. The strong association of TNFR2 with cell cycle genes in PBs suggest that blockade of TNFR2 may be a novel strategy to inhibit PB proliferation and differentiation.

Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) mediate both regulation and inflammation in multiple sclerosis, neuromyelitis optica, and in experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanism for these Janus-like activities of type I and II IFNs in neuroinflammation remains unclear. Although endogenous type I IFN signaling provides a protective response in neuroinflammation, we find that when IFN-γ signaling is ablated, type I IFNs drive inflammation, resulting in exacerbated EAE. IFN-γ has a disease stage–specific opposing function in EAE. Treatment of mice with IFN-γ during the initiation phase of EAE leads to enhanced severity of disease. In contrast, IFN-γ treatment during the effector phase attenuated disease. This immunosuppressive activity of IFN-γ required functional type I IFN signaling. In IFN-α/β receptor–deficient mice, IFN-γ treatment during effector phase of EAE exacerbated disease. Using an adoptive transfer EAE model, we found that T cell–intrinsic type I and II IFN signals are simultaneously required to establish chronic EAE by encephalitogenic Th1 cells. However, in Th17 cells loss of either IFN signals leads to the development of a severe chronic disease. The data imply that type I and II IFN signals have independent but nonredundant roles in restraining encephalitogenic Th17 cells in vivo. Collectively, our data show that type I and II IFNs function in an integrated manner to regulate pathogenesis in EAE.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5+ICOS+ TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra−/− mice, the majority of CXCR5+ TFH cells from BXD2-Il17ra−/− mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra−/−, disrupted TFH–B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16−/− mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA+ TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Marginal zone macrophages (MZMs) act as a barrier to entry of circulating apoptotic debris into the follicles of secondary lymphoid organs. In autoimmune BXD2 mice, there is a progressive reduction in the function and numbers of MZMs. Absence of MZMs results in retention of apoptotic cell (AC) debris within the marginal zone (MZ) and increased loading of AC Ags on MZ B cells and MZ-precursor (MZ-P) B cells. The MZ-P B cells are capable of translocating the AC Ags to the follicular zone and stimulating T cells. Both MZMs and MZ-P B cells from BXD2 mice express low levels of tolerogenic signals and high levels of inflammatory signals. Thus, the current study suggests a multifaceted mechanism in which MZMs maintain tolerance to apoptotic autoantigens and suppress their translocation to follicles. Lack of clearance of apoptotic debris by MZMs drives follicular Ag–transportation by MZ-P B cells to stimulate an autoimmune response.

Given the protective roles of 25-hydroxyvitamin D (25(OH)D or vitamin D) in musculoskeletal health and the potential beneficial effects of vitamin D supplementation in reducing the risk of various chronic diseases, intensive repletion of vitamin D has been widely advocated. Of note, CD8 T cells have the highest levels of the vitamin D receptor compared with other major immune cells. The effects of vitamin D on CD8 T cells during aging, however, remain unclear. This study determined the relationship between vitamin D levels and CD8 T cell status in 34 healthy female subjects (all >60 years old). The CD8 T-cell phenotype was defined by the surface expression of CD28 and CD95. The low-25(OH)D serum groups (≤30 ng/ml) had higher percentages of CD28+CD95–CD8+ (na?ve) T cells and lower percentages of CD28+CD95+CD8+ (effector) T cells. By contrast, subjects with high levels of 25(OH)D had very low percentages of na?ve CD8 T cells but very high percentages of effector CD8 T cells. There was a significant inverse correlation between 25(OH)D levels and the frequency of na?ve CD8 T cells. The results show that higher levels of vitamin D are correlated with decreased frequencies of na?ve CD8 T cells during early aging, suggesting that higher levels of 25(OH)D accelerate CD8 T cell senescence. These results warrant further evaluation of the effects of vitamin D supplementation in immune aging.

Macrophages play a central role in the pathogenesis of rheumatoid arthritis (RA). There is an imbalance of inflammatory and antiinflammatory macrophages in RA synovium. Although the polarization and heterogeneity of macrophages in RA have not been fully uncovered, the identity of macrophages in RA can potentially be defined by their products, including the co-stimulatory molecules, scavenger receptors, different cytokines/chemokines and receptors, and transcription factors. In the last decade, efforts to understand the polarization, apoptosis regulation, and novel signaling pathways in macrophages, as well as how distinct activated macrophages influence disease progression, have led to strategies that target macrophages with varied specificity and selectivity. Major targets that are related to macrophage development and apoptosis include TNF-α, IL-1, IL-6, GM-CSF, M-CSF, death receptor 5 (DR5), Fas, and others, as listed in Table 1. Combined data from clinical, preclinical, and animal studies of inhibitors of these targets have provided valuable insights into their roles in the disease progression and, subsequently, have led to the evolving therapeutic paradigms in RA. In this review, we propose that reestablishment of macrophage equilibrium by inhibiting the development of, and/or eliminating, the proinflammatory macrophages will be an effective therapeutic approach for RA and other autoimmune diseases.

Objective To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. Methods We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN–transgenic mice). Real-time quantitative reverse transcriptase–polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6–8-month-old mice. Results Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage–related genes in GC B cells of BXD2-Aicda-DN–transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN–transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM– and anti-CD40 plus anti-Ig–induced B cell proliferative responses were decreased in BXD2-Aicda-DN–transgenic mice. Conclusion Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

Numerous studies have been performed in vitro and in various animal models to modulate the interaction of dendritic cells (DC) and T cells by Fas (CD95/Apo-1) signalling to delete activated T cells via induction of activation-induced cell death (AICD). Previously, we could demonstrate that Fas ligand (FasL/CD95L)-expressing 'killer-antigen-presenting cells' can be generated from human monocyte-derived mature DC (mDC) using adenoviral gene transfer. To evaluate whether these FasL-expressing mDC (mDC-FasL) could eliminate alloreactive primary human T cells in vitro, co-culture experiments were performed. Proliferation of human T cells was markedly reduced in primary co-cultures with allogeneic mDC-FasL, whereas a strong proliferative T-cell response could be observed in co-cultures with enhanced green fluorescent protein-transduced mDC. Inhibition of T-cell proliferation was related to the transduction efficiency, and the numbers of mDC-FasL present in co-cultures. In addition, proliferation of pre-activated alloreactive CD4(+) and CD8(+) T cells could be almost completely inhibited in secondary co-cultures using mDC-FasL as stimulatory cells, which was the result of induction of apoptosis in the majority of preactivated T cells. The specific deletion of alloreactive T cells by mDC-FasL was confirmed by an unaffected proliferative response of surviving T cells towards allogeneic 'third-party' peripheral blood mononuclear cells in a third stimulation, or upon unspecific stimulation with anti-CD3/CD28 beads. The results of this study demonstrate that allospecifically activated T cells are efficiently eliminated by mDC-FasL, supporting further investigations to apply FasL-expressing 'killer-DC' as a novel strategy for the treatment of allograft rejection.

New MRI techniques have been developed to assess not only the static anatomy of synovial hyperplasia, bone changes and cartilage degradation in patients with rheumatoid arthritis (RA), but also the activity of the physiological events that cause these changes. This enables an estimation of the rate of change in the synovium, bone and cartilage as a result of disease activity or in response to therapy. Typical MRI signs of RA in the pre-erosive phase include synovitis, bone marrow edema and subchondral cyst formation. Synovitis can be assessed by T2-weighted imaging, dynamic contrast-enhanced MRI or diffusion tensor imaging. Bone marrow edema can be detected on fluid-sensitive sequences such as short-tau inversion recovery or T2-weighted fast-spin echo sequences. Detection of small bone erosions in the early erosive phase using T1-weighted MRI has sensitivity comparable to CT. Numerous MRI techniques have been developed for quantitative assessment of potentially pathologic changes in cartilage composition that occur before frank morphologic changes. In this Review, we summarize the advances and new directions in the field of MRI, with an emphasis on their current state of development and application in RA.

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.

The role of immune responses in tumor development is a central issue for tumor biology and immunology. IL-17 is an important cytokine for inflammatory and autoimmune diseases. Although IL-17–producing cells are detected in cancer patients and tumor-bearing mice, the role of IL-17 in tumor development is controversial, and mechanisms remain to be fully elucidated. In the current study, we found that the development of tumors was inhibited in IL-17R–deficient mice. A defect in IFN-γR increased tumor growth, whereas tumor growth was inhibited in mice that were deficient in both IL-17R and IFN-γR compared with wild-type animals. Further experiments showed that neutralization of IL-17 by Abs inhibited tumor growth in wild-type mice, whereas systemic administration of IL-17 promoted tumor growth. The IL-17R deficiency increased CD8 T cell infiltration, whereas it reduced the infiltration of myeloid-derived suppressor cells (MDSCs) in tumors. In contrast, administration of IL-17 inhibited CD8 T cell infiltration and increased MDSCs in tumors. Further analysis indicated that IL-17 was required for the development and tumor-promoting activity of MDSCs in tumor-bearing mice. These data demonstrate that IL-17–mediated responses promote tumor development through the induction of tumor-promoting microenvironments at tumor sites. IL-17–mediated regulation of MDSCs is a primary mechanism for its tumor-promoting effects. The study provides novel insights into the role of IL-17 in tumor development and has major implications for targeting IL-17 in treatment of tumors.