An Aged/Autoimmune B-cell Program Defines the Early Transformation of Extranodal Lymphomas.

A third of patients with diffuse large B-cell lymphoma (DLBCL) present with extranodal dissemination, which is associated with inferior clinical outcomes. MYD88L265P is a hallmark extranodal DLBCL mutation that supports lymphoma proliferation. Yet extranodal lymphomagenesis and the role of MYD88L265P in transformation remain mostly unknown. Here, we show that B cells expressing Myd88L252P (MYD88L265P murine equivalent) activate, proliferate, and differentiate with minimal T-cell costimulation. Additionally, Myd88L252P skewed B cells toward memory fate. Unexpectedly, the transcriptional and phenotypic profiles of B cells expressing Myd88L252P, or other extranodal lymphoma founder mutations, resembled those of CD11c+T-BET+ aged/autoimmune memory B cells (AiBC). AiBC-like cells progressively accumulated in animals prone to develop lymphomas, and ablation of T-BET, the AiBC master regulator, stripped mouse and human mutant B cells of their competitive fitness. By identifying a phenotypically defined prospective lymphoma precursor population and its dependencies, our findings pave the way for the early detection of premalignant states and targeted prophylactic interventions in high-risk patients. SIGNIFICANCE: Extranodal lymphomas feature a very poor prognosis. The identification of phenotypically distinguishable prospective precursor cells represents a milestone in the pursuit of earlier diagnosis, patient stratification, and prophylactic interventions. Conceptually, we found that extranodal lymphomas and autoimmune disorders harness overlapping pathogenic trajectories, suggesting these B-cell disorders develop and evolve within a spectrum. See related commentary by Leveille et al. (Blood Cancer Discov 2023;4:8-11). This article is highlighted in the In This Issue feature, p. 1.

Adaptive immune responses in patients requiring revision after total knee arthroplasty.

Dissatisfaction occurs in nearly 20% of patients after total knee arthroplasty (TKA); however, there remains only limited understanding of the biologic mechanisms that may contribute to suboptimal postoperative outcomes requiring revision surgery. Expansion of effector T and B cells, could promote an abnormal healing response via local or peripheral immune system mechanisms and contribute to inferior outcomes necessitating revision TKA. In this pilot study, we hypothesized that patients suffering from complications of arthrofibrosis or instability may exhibit differences in adaptive immune function. Patients (n = 31) undergoing revision TKA for an indication of arthrofibrosis or instability were prospectively enrolled. Whole blood and synovial fluid (SF) from the operative knee were collected at time of surgery. Peripheral blood mononuclear cells were isolated and analyzed by flow cytometry. Serum and SF were assessed for immunoglobulin levels by Luminex and antiphospholipid antibodies by enzyme-linked immunoassay. No significant differences were observed in peripheral blood T/B cell populations or serum immunoglobulins levels between groups. SF analysis demonstrated significant differences between the two groups, with higher levels of immunoglobulin G1 (IgG1) (p = 0.0184), IgG3 (p = 0.0084) and antiphosphatidyl serine IgG (p = 0.034) in arthrofibrosis relative to instability patients. Increased levels of both IgG subclasses and antiphospholipid antibodies in the SF suggest that intra-articular T-B cell interactions, potentially triggered by exposure to apoptotic components generated during post-op healing, could be functioning as a source of immune complexes that fuel fibrous tissue growth in arthrofibrotic patients.

Serotonin-1A receptor, a psychiatric disease risk factor, influences offspring immunity via sex-dependent genetic nurture.

Serotonin-1A receptor (5HT1AR) is highly expressed in corticolimbic regions and its deficit is associated with anxiety and depression. A similar reduction in 5HT1AR heterozygous knockout (Het) mice results in anxiety-like and increased stress-reactivity phenotypes. Here we describe immunological abnormalities in Het females, characterized by an activated state of innate and adaptive immune cells. Het males showed only limited immune dysregulation. Similar immune abnormalities were present in the genetically WT female (F1) but not male offspring of Het mothers, indicating sex-specific immune system abnormalities that are dependent on the mother's 5HT1AR deficit, known as maternal genetic effect or "genetic nurture". Expression profiling of the maternal-fetal interface revealed reduced immune cell invasion to decidua and accelerated trophoblast migration. These data suggest that 5HT1AR deficit, by altering the maternal immune system and midgestational in utero environment, leads to sex-biased outcomes, predominantly immune dysregulation in the female and anxiety-like behavior in the male offspring.

Interleukin-13 Receptor α1-Mediated Signaling Regulates Age-Associated/Autoimmune B Cell Expansion and Lupus Pathogenesis.

OBJECTIVE: Age-associated/autoimmune B cells (ABCs) are an emerging B cell subset with aberrant expansion in systemic lupus erythematosus. ABC generation and differentiation exhibit marked sexual dimorphism, and Toll-like receptor 7 (TLR-7) engagement is a key contributor to these sex differences. ABC generation is also controlled by interleukin-21 (IL-21) and its interplay with interferon-γ and IL-4. This study was undertaken to investigate whether IL-13 receptor α1 (IL-13Rα1), an X-linked receptor that transmits IL-4/IL-13 signals, regulates ABCs and lupus pathogenesis. METHODS: Mice lacking DEF-6 and switch-associated protein 70 (double-knockout [DKO]), which preferentially develop lupus in females, were crossed with IL-13Rα1-knockout mice. IL-13Rα1-knockout male mice were also crossed with Y chromosome autoimmune accelerator (Yaa) DKO mice, which overexpress TLR-7 and develop severe disease. ABCs were assessed using flow cytometry and RNA-Seq. Lupus pathogenesis was evaluated using serologic and histologic analyses. RESULTS: ABCs expressed higher levels of IL-13Rα1 than follicular B cells. The absence of IL-13Rα1 in either DKO female mice or Yaa DKO male mice decreased the accumulation of ABCs, the differentiation of ABCs into plasmablasts, and autoantibody production. Lack of IL-13Rα1 also prolonged survival and delayed the development of tissue inflammation. IL-13Rα1 deficiency diminished in vitro generation of ABCs, an effect that, surprisingly, could be observed in response to IL-21 alone. RNA-Seq revealed that ABCs lacking IL-13Rα1 down-regulated some histologic characteristics of B cells but up-regulated myeloid markers and proinflammatory mediators. CONCLUSION: Our findings indicate a novel role for IL-13Rα1 in controlling ABC generation and differentiation, suggesting that IL-13Rα1 contributes to these effects by regulating a subset of IL-21-mediated signaling events. These results also suggest that X-linked genes besides TLR7 participate in the regulation of ABCs in lupus.

Molecular mechanisms controlling age-associated B cells in autoimmunity.

Age-associated B cells (ABCs) have emerged as critical components of immune responses. Their inappropriate expansion and differentiation have increasingly been linked to the pathogenesis of autoimmune disorders, aging-associated diseases, and infections. ABCs exhibit a distinctive phenotype and, in addition to classical B cell markers, often express the transcription factor T-bet and myeloid markers like CD11c; hence, these cells are also commonly known as CD11c+ T-bet+ B cells. Formation of ABCs is promoted by distinctive combinations of innate and adaptive signals. In addition to producing antibodies, these cells display antigen-presenting and proinflammatory capabilities. It is becoming increasingly appreciated that the ABC compartment exhibits a high degree of heterogeneity, plasticity, and sex-specific regulation and that ABCs can differentiate into effector progeny via several routes particularly in autoimmune settings. In this review, we will discuss the initial insights that have been obtained on the molecular machinery that controls ABCs and we will highlight some of the unique aspects of this control system that may enable ABCs to fulfill their distinctive role in immune responses. Given the expanding array of autoimmune disorders and pathophysiological settings in which ABCs are being implicated, a deeper understanding of this machinery could have important and broad therapeutic implications for the successful, albeit daunting, task of targeting these cells.

Altered function and differentiation of age-associated B cells contribute to the female bias in lupus mice.

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c- effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis.

Selective dysregulation of ROCK2 activity promotes aberrant transcriptional networks in ABC diffuse large B-cell lymphoma.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive subtype of lymphoma usually associated with inferior outcomes. ABC-DLBCL exhibits plasmablastic features and is characterized by aberrancies in the molecular networks controlled by IRF4. The signaling pathways that are dysregulated in ABC-DLBCL are, however, not fully understood. ROCK2 is a serine-threonine kinase whose role in lymphomagenesis is unknown. Here we show that ROCK2 activity is constitutively dysregulated in ABC-DLBCL but not in GCB-DLBCL and BL. We furthermore show that ROCK2 phosphorylates IRF4 and that the ROCK2-mediated phosphorylation of IRF4 modulates its ability to regulate a subset of target genes. In addition to its effects on IRF4, ROCK2 also controls the expression of MYC in ABC-DLBCL by regulating MYC protein levels. ROCK inhibition furthermore selectively decreases the proliferation and survival of ABC-DLBCL in vitro and inhibits ABC-DLBCL growth in xenograft models. Thus, dysregulated ROCK2 activity contributes to the aberrant molecular program of ABC-DLBCL via its dual ability to modulate both IRF4- and MYC-controlled gene networks and ROCK inhibition could represent an attractive therapeutic target for the treatment of ABC-DLBCL.

Serine-threonine kinase ROCK2 regulates germinal center B cell positioning and cholesterol biosynthesis.

Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. RHO-associated coiled-coil-containing protein kinase 2 (ROCK2), a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA GTPases. Although RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we found that ROCK2 was activated in response to key T cell signals like CD40 and IL-21 and that it regulated GC formation and maintenance. RNA-Seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing) and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated interferon regulatory factor 8 (IRF8), a crucial mediator of GC responses, and promoted its interaction with sterol regulatory element-binding transcription factor 2 (SREBP2) at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.

Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

Regulation of age-associated B cells by IRF5 in systemic autoimmunity.

Age-associated B cells (ABCs) are a subset of B cells dependent on the transcription factor T-bet that accumulate prematurely in autoimmune settings. The pathways that regulate ABCs in autoimmunity are largely unknown. SWAP-70 and DEF6 (also known as IBP or SLAT) are the only two members of the SWEF family, a unique family of Rho GTPase-regulatory proteins that control both cytoskeletal dynamics and the activity of the transcription factor IRF4. Notably, DEF6 is a newly identified human risk variant for systemic lupus erythematosus. Here we found that the lupus syndrome that developed in SWEF-deficient mice was accompanied by the accumulation of ABCs that produced autoantibodies after stimulation. ABCs from SWEF-deficient mice exhibited a distinctive transcriptome and a unique chromatin landscape characterized by enrichment for motifs bound by transcription factors of the IRF and AP-1 families and the transcription factor T-bet. Enhanced ABC formation in SWEF-deficient mice was controlled by the cytokine IL-21 and IRF5, whose variants are strongly associated with lupus. The lack of SWEF proteins led to dysregulated activity of IRF5 in response to stimulation with IL-21. These studies thus elucidate a previously unknown signaling pathway that controls ABCs in autoimmunity.

Identification of Three Rheumatoid Arthritis Disease Subtypes by Machine Learning Integration of Synovial Histologic Features and RNA Sequencing Data.

OBJECTIVE: In this study, we sought to refine histologic scoring of rheumatoid arthritis (RA) synovial tissue by training with gene expression data and machine learning. METHODS: Twenty histologic features were assessed in 129 synovial tissue samples (n = 123 RA patients and n = 6 osteoarthritis [OA] patients). Consensus clustering was performed on gene expression data from a subset of 45 synovial samples. Support vector machine learning was used to predict gene expression subtypes, using histologic data as the input. Corresponding clinical data were compared across subtypes. RESULTS: Consensus clustering of gene expression data revealed 3 distinct synovial subtypes, including a high inflammatory subtype characterized by extensive infiltration of leukocytes, a low inflammatory subtype characterized by enrichment in pathways including transforming growth factor ß, glycoproteins, and neuronal genes, and a mixed subtype. Machine learning applied to histologic features, with gene expression subtypes serving as labels, generated an algorithm for the scoring of histologic features. Patients with the high inflammatory synovial subtype exhibited higher levels of markers of systemic inflammation and autoantibodies. C-reactive protein (CRP) levels were significantly correlated with the severity of pain in the high inflammatory subgroup but not in the others. CONCLUSION: Gene expression analysis of RA and OA synovial tissue revealed 3 distinct synovial subtypes. These labels were used to generate a histologic scoring algorithm in which the histologic scores were found to be associated with parameters of systemic inflammation, including the erythrocyte sedimentation rate, CRP level, and autoantibody levels. Comparison of gene expression patterns to clinical features revealed a potentially clinically important distinction: mechanisms of pain may differ in patients with different synovial subtypes.

Regulation of systemic autoimmunity and CD11c+ Tbet+ B cells by SWEF proteins.

Recent studies have revealed the existence of a T-bet dependent subset of B cells, which expresses unique phenotypic and functional characteristics including high levels of CD11c and CD11b. In the murine system this B cell subset has been termed Age/autoimmune-associated B cells (ABCs) since it expands with age in non-autoimmune mice and it prematurely accumulates in autoimmune-prone strains. The molecular mechanisms that promote the expansion and function of ABCs are largely unknown. This review will focus on the SWEF proteins, a small family of Rho GEFs comprised of SWAP-70 and its homolog DEF6, a newly identified risk variant for human SLE. We will first provide an overview of the SWEF proteins and then discuss the complex array of biological processes that they control and the autoimmune phenotypes that spontaneously develop in their absence, highlighting the emerging involvement of these proteins in regulating ABCs. A better understanding of the pathways controlled by the SWEF proteins could help provide new insights into the mechanisms responsible for the expansion of ABCs in autoimmunity and potentially guide the design of novel therapeutic approaches.

The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity.

Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (TH) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (TFH) cells, is critical as aberrant TFH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1-4E-BP-eIF4E axis, secondary to aberrant assembly of a raptor-p62-TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant TFH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity.Excessive expansion of the T follicular helper (TFH) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of TFH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

Targeting the RhoA-ROCK pathway to reverse T-cell dysfunction in SLE.

OBJECTIVES: Deregulated production of interleukin (IL)-17 and IL-21 contributes to the pathogenesis of autoimmune disorders such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Production of IL-17 and IL-21 can be regulated by ROCK2, one of the two Rho kinases. Increased ROCK activation was previously observed in an SLE cohort. Here, we evaluated ROCK activity in a new SLE cohort, and an RA cohort, and assessed the ability of distinct inhibitors of the ROCK pathway to suppress production of IL-17 and IL-21 by SLE T cells or human Th17 cells. METHODS: ROCK activity in peripheral blood mononuclear cells (PBMCs) from 29 patients with SLE, 31 patients with RA and 28 healthy controls was determined by ELISA. SLE T cells or in vitro-differentiated Th17 cells were treated with Y27632 (a pan-ROCK inhibitor), KD025 (a selective ROCK2 inhibitor) or simvastatin (which inhibits RhoA, a major ROCK activator). ROCK activity and IL-17 and IL-21 production were assessed. The transcriptional profile altered by ROCK inhibitors was evaluated by NanoString technology. RESULTS: ROCK activity levels were significantly higher in patients with SLE and RA than healthy controls. Th17 cells exhibited high ROCK activity that was inhibited by Y27632, KD025 or simvastatin; each also decreased IL-17 and IL-21 production by purified SLE T cells or Th17 cells. Immune profiling revealed both overlapping and distinct effects of the different ROCK inhibitors. CONCLUSIONS: ROCK activity is elevated in PBMCs from patients with SLE and RA. Production of IL-17 and IL-21 by SLE T cells or Th17 cells can furthermore be inhibited by targeting the RhoA-ROCK pathway via both non-selective and selective approaches.

Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis.

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

The RhoA-ROCK pathway in the regulation of T and B cell responses.

Effective immune responses require the precise regulation of dynamic interactions between hematopoietic and non-hematopoietic cells. The Rho subfamily of GTPases, which includes RhoA, is rapidly activated downstream of a diverse array of biochemical and biomechanical signals, and is emerging as an important mediator of this cross-talk. Key downstream effectors of RhoA are the Rho kinases, or ROCKs. The ROCKs are two serine-threonine kinases that can act as global coordinators of a tissue's response to stress and injury because of their ability to regulate a wide range of biological processes. Although the RhoA-ROCK pathway has been extensively investigated in the non-hematopoietic compartment, its role in the immune system is just now becoming appreciated. In this commentary, we provide a brief overview of recent findings that highlight the contribution of this pathway to lymphocyte development and activation, and the impact that dysregulation in the activation of RhoA and/or the ROCKs may exert on a growing list of autoimmune and lymphoproliferative disorders.

Cigarette smoke inhibits ROCK2 activation in T cells and modulates IL-22 production.

Gene-environment interactions are known to play a key role in the development of rheumatoid arthritis (RA). Exposure to cigarette smoke (CS) is one of the strongest environmental risk factors associated with RA and has been shown to mediate a range of complex immunomodulatory effects from decreased T and B cell activation to depressed phagocytic function. The effects of CS on the function of TH17 cells, one of the key TH effector subsets implicated in RA pathogenesis, are not fully understood. IRF4 is one of the crucial transcription factors involved in TH-17 differentiation and is absolutely required for the production of IL-17 and IL-21 but, interestingly, inhibits the synthesis of IL-22. The production of IL-17 and IL-21 by IRF4 can be augmented by its phosphorylation by the serine-threonine kinase ROCK2. Given that CS has been reported to increase ROCK activity in endothelial cells, here we investigated the effects of CS on the ROCK2-IRF4 axis in T cells. Surprisingly, we found that CS leads to decreased ROCK2 activation and IRF4 phosphorylation in T cells. This effect was associated with increased IL-22 production. Using a GEF pull-down assay we furthermore identify ARHGEF1 as a key upstream regulator of ROCK2 whose activity in T cells is inhibited by CS. Thus CS can inhibit the ROCK2-IRF4 axis and modulate T cell production of IL-22.

Regulation of Effector Treg Cells in Murine Lupus.

OBJECTIVE: Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF-4) is an essential controller of effector Treg cells and regulates their ability to express interleukin-10 (IL-10). In non-Treg cells, IRF-4 activity is modulated by interactions with DEF-6 and its homolog switch-associated protein 70 (SWAP-70). Although mice lacking both DEF-6 and SWAP-70 (double-knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF-6 and SWAP-70 to restrain IRF-4 activity. METHODS: Treg cells were evaluated by fluorescence-activated cell sorting. The B lymphocyte-induced maturation protein 1 (BLIMP-1)/IL-10 axis was assessed by crossing DKO mice with BLIMP-1-YFP-10BiT dual-reporter mice. Deletion of IRF-4 in Treg cells from DKO mice was achieved by generating FoxP3(Cre) IRF-4(fl/fl) DKO mice. RESULTS: The concomitant absence of DEF-6 and SWAP-70 led to increased numbers of Treg cells, which acquired an effector phenotype in a cell-intrinsic manner. In addition, Treg cells from DKO mice exhibited enhanced expression of the BLIMP-1/IL-10 axis. Notably, DKO effector Treg cells survived and expanded as disease progressed. The accumulation of Treg cells from DKO mice was associated with the up-regulation of genes controlling autophagy. IRF-4 was required for the expansion and function of effector Treg cells from DKO mice. CONCLUSION: This study revealed the existence of mechanisms that, by acting on IRF-4, can fine-tune the function and survival of effector Treg cells in lupus. These findings suggest that the existence of a powerful effector Treg cell compartment that successfully survives in an unfavorable inflammatory environment could limit disease development.

Rho Kinases in Autoimmune Diseases.

The Rho kinases, or ROCKs, are a family of serine-threonine kinases that serve as key downstream effectors for Rho GTPases. The ROCKs are increasingly recognized as critical coordinators of a tissue response to injury due to their ability to modulate a wide range of biological processes. Dysregulated ROCK activity has been implicated in several human pathophysiological conditions ranging from cardiovascular and renal disorders to fibrotic diseases. In recent years, an important role for the ROCKs in the regulation of immune responses is also being uncovered. We provide an overview of the role of the ROCKs in immune cells and discuss studies that highlight the emerging involvement of this family of kinases in the pathogenesis of autoimmune diseases. Given the potential promise of the ROCKs as therapeutic targets, we also outline the approaches that could be employed to inhibit the ROCKs in autoimmune disorders.