Myeloablation Followed by Hematopoietic Stem Cell Transplantation and Long-Term Normalization of Systemic Sclerosis Molecular Signatures.

OBJECTIVE: In the randomized Scleroderma: Cyclophosphamide or Transplantation (SCOT) trial, myeloablation, followed by hematopoietic stem cell transplantation (HSCT), led to the normalization of systemic sclerosis (SSc) peripheral blood cell (PBC) gene expression signature at the 26-month visit. Herein, we examined long-term molecular changes ensuing 54 months after randomization for individuals receiving an HSCT or 12 months of intravenous cyclophosphamide (CYC). METHODS: Global PBC transcript studies were performed in study participants at pretreatment baseline and at 38 months and 54 months after randomization, as well as in healthy controls using Illumina HT-12 arrays. RESULTS: Thirty (HSCT = 19 and CYC = 11) participants had 38-month samples available, and 26 (HSCT = 16 and CYC = 11) had 54-month samples available. In the paired comparison to baseline, a significant down-regulation of interferon modules and an up-regulation of cytotoxic/natural killer module were observed at the 38-month and 54-month visits in the HSCT arm, indicating a long-term normalization of baseline SSc gene expression signature. No differentially expressed modules were detected in the CYC arm. In comparison to samples from healthy controls, 38-month visit samples in the HSCT arm showed an up-regulation of B cell and plasmablast modules and a down-regulation of myeloid and inflammation modules. Importantly, 54-month HSCT samples did not show any differentially expressed modules compared to healthy control samples, suggesting completion of immune reconstitution. Participants in the CYC arm continued to show an SSc transcript signature in comparison to controls at both time points. CONCLUSION: Paralleling the observed clinical benefit, HSCT leads to durable long-term normalization of the molecular signature in SSc, with completion of immune resetting to 54 months after HSCT.

Minimal Clinically Important Change of Movement Pain in Musculoskeletal Pain Conditions.

Movement pain, which is distinct from resting pain, is frequently reported by individuals with musculoskeletal pain. There is growing interest in measuring movement pain as a primary outcome in clinical trials, but no minimally clinically important change (MCIC) has been established, limiting interpretations. We analyzed data from 315 participants who participated in previous clinical trials (65 with chronic Achilles tendinopathy; 250 with fibromyalgia) to establish an MCIC for movement pain. A composite movement pain score was defined as the average pain (Numeric Rating Scale: 0-10) during 2 clinically relevant activities. The change in movement pain was calculated as the change in movement pain from pre-intervention to post-intervention. A Global Scale (GS: 1-7) was completed after the intervention on perceived change in health status. Participants were dichotomized into non-responders (GS ≥4) and responders (GS <3). Receiver operating characteristic curves were calculated to determine threshold values and corresponding sensitivity and specificity. We used the Euclidean method to determine the optimal threshold point of the Receiver operating characteristic curve to determine the MCIC. The MCIC for raw change in movement pain was 1.1 (95% confidence interval [CI]: .9-1.6) with a sensitivity of .83 (95% CI: .75-.92) and specificity of .79 (95% CI: .72-.86). For percent change in movement pain the MCIC was 27% (95% CI: 10-44%) with a sensitivity of .79 (95% CI: .70-.88) and a specificity of .82 (95% CI: .72-.90). Establishing an MCIC for movement pain will improve interpretations in clinical practice and research. PERSPECTIVE: A minimal clinically important change (MCIC) of 1.1- points (95% CI: .9-1.6) for movement pain discriminates between responders and non-responders to rehabilitation. This MCIC provides context for interpreting the meaningfulness of improvement in pain specific to movement tasks.

Optimism and Sleep in Aging Women: Bidirectional Relationships.

OBJECTIVE: Sleep quality and duration are important for biological restoration and promotion of psychological well-being. Optimism may facilitate or result from sufficient sleep, but questions remain as to directionality. The present study tested how optimism is associated with levels of and variability in sleep quantity and quality in a longitudinal burst design. METHODS: Midlife and older women ( N = 199) reported their sleep quantity and quality in online diaries for a 7-day period, every 3 months for 2 years. Optimism was measured at baseline and end-of-study. Multilevel models tested the effects of optimism on sleep. Linear regression models tested the effect of sleep on optimism. RESULTS: Baseline optimism was associated with higher sleep quality ( γ = 2.13 [1.16 to 3.11], p < .0001) and lower intraindividual variability (IIV; night-to-night and wave-to-wave) in sleep quantity (night-to-night: γ = -0.07 [-0.13 to -0.005], p = .03; wave-to-wave: b = -0.07 [-0.12 to -0.02], p = .003). In turn, higher average sleep quality (but not quantity) was associated with higher optimism at end-of-study ( b = 0.02 [0.007 to 0.03], p = .002). Variability in sleep was unrelated to optimism. CONCLUSIONS: Optimism may play an important role in maintaining sleep quality and consistency in sleep quantity, perhaps by buffering stress. Similarly, sleep quality may play an important role in maintaining optimism. The cycle whereby optimism and sleep enhance one another could improve physical health and psychological well-being among aging adults.

Implications of the accuracy of diagnostic algorithms for systemic lupus on our understanding of racial disparities in pregnancy outcomes.

OBJECTIVE: Disparities in pregnancy outcomes among women with SLE remain understudied, with few available racially diverse datasets. We sought to identify disparities between Black and White women in pregnancy outcomes within academic institutions in the United States. METHODS: Using the Common Data Model electronic medical record (EMR)-based datasets within the Carolinas Collaborative, we identified women with pregnancy delivery data (2014-2019) and ≥1 SLE International Classification of Diseases 9 or 10 code (ICD9/10) code. From this dataset, we identified four cohorts of SLE pregnancies, three based on EMR-based algorithms and one confirmed with chart review. We compared the pregnancy outcomes identified in each of these cohorts for Black and White women. RESULTS: Of 172 pregnancies in women with ≥1 SLE ICD9/10 code, 49% had confirmed SLE. Adverse pregnancy outcomes occurred in 40% of pregnancies in women with ≥1 ICD9/10 SLE code and 52% of pregnancies with confirmed SLE. SLE was frequently over-diagnosed in women who were White, resulting in 40-75% lower rates of adverse pregnancy outcomes in EMR-derived vs confirmed SLE cohorts. Over-diagnosis was less common for Black women with pregnancy outcomes 12-20% lower in EMR-derived vs confirmed SLE cohorts. Black women had higher rates of adverse pregnancy outcomes than White women in the EMR-derived, but not the confirmed cohorts. CONCLUSION: EMR-derived cohorts of pregnancies in women who are Black, but not White, provided accurate estimations of pregnancy outcomes. The data from the confirmed SLE pregnancies suggest that all women with SLE, regardless of race, referred to academic centres remain at very high risk for adverse pregnancy outcome.

Unmet need in rheumatology: reports from the Advances in Targeted Therapies meeting, 2023.

The Advances in Targeted Therapies meets annually, convening experts in the field of rheumatology to both provide scientific updates and identify existing scientific gaps within the field. To review the major unmet scientific needs in rheumatology. The 23rd annual Advances in Targeted Therapies meeting convened with more than 100 international basic scientists and clinical researchers in rheumatology, immunology, infectious diseases, epidemiology, molecular biology and other specialties relating to all aspects of immune-mediated inflammatory diseases. We held breakout sessions in five rheumatological disease-specific groups including: rheumatoid arthritis (RA), psoriatic arthritis (PsA), axial spondyloarthritis (axSpa), systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and vasculitis, and osteoarthritis (OA). In each group, experts were asked to identify and prioritise current unmet needs in clinical and translational research. An overarching theme across all disease states is the continued need for clinical trial design innovation with regard to therapeutics, endpoint and disease endotypes. Within RA, unmet needs comprise molecular classification of disease pathogenesis and activity, pre-/early RA strategies, more refined pain profiling and innovative trials designs to deliver on precision medicine. Continued scientific questions within PsA include evaluating the genetic, immunophenotypic, clinical signatures that predict development of PsA in patients with psoriasis, and the evaluation of combination therapies for difficult-to-treat disease. For axSpA, there continues to be the need to understand the role of interleukin-23 (IL-23) in pathogenesis and the genetic relationship of the IL-23-receptor polymorphism with other related systemic inflammatory diseases (eg, inflammatory bowel disease). A major unmet need in the OA field remains the need to develop the ability to reliably phenotype and stratify patients for inclusion in clinical trials. SLE experts identified a number of unmet needs within clinical trial design including the need for allowing endpoints that reflect pharmacodynamic/functional outcomes (eg, inhibition of type I interferon pathway activation; changes in urine biomarkers). Lastly, within SSc and vasculitis, there is a lack of biomarkers that predict response or disease progression, and that allow patients to be stratified for therapies. There remains a strong need to innovate clinical trial design, to identify systemic and tissue-level biomarkers that predict progression or response to therapy, endotype disease, and to continue developing therapies and therapeutic strategies for those with treatment-refractory disease. This document, based on expert consensus, should provide a roadmap for prioritising scientific endeavour in the field of rheumatology.

Influence of Transcutaneous Electrical Nerve Stimulation (TENS) on Pressure Pain Thresholds and Conditioned Pain Modulation in a Randomized Controlled Trial in Women With Fibromyalgia.

Transcutaneous electrical nerve stimulation (TENS) effectively reduces pain in fibromyalgia (FM). The purpose of this study was to examine the influence of TENS use on pressure pain thresholds (PPT) and conditioned pain modulation (CPM) in individuals with FM using data from the Fibromyalgia Activity Study with TENS trial (NCT01888640). Individuals with FM were randomly assigned to receive active TENS, placebo TENS, or no TENS for 4 weeks. A total of 238 females satisfied the per-protocol analysis among the active TENS (n = 76), placebo TENS (n = 68), and no TENS (n = 94) groups. Following 4 weeks of group allocation, the active TENS group continued for an additional 4 weeks of active TENS totaling 8 weeks (n = 66), the placebo and no TENS groups transitioned to receive 4 weeks of active TENS (delayed TENS, n = 161). Assessment of resting pain, movement-evoked pain (MEP), PPT, and CPM occurred prior to and following active, placebo, or no TENS. There were no significant changes in PPT or CPM among the active TENS, placebo TENS, or no TENS groups after 4 weeks. Individuals who reported clinically relevant improvements in MEP (≥30% decrease) demonstrated increases in PPT (P < .001), but not CPM, when compared to MEP non-responders. There were no significant correlations among the change in PPT or CPM compared to MEP and resting pain following active TENS use (active TENS + delayed TENS). PPT and CPM may provide insight to underlying mechanisms contributing to pain; however, these measures may not relate to self-reported pain symptoms. PERSPECTIVE: Pressure pain threshold increased in individuals with clinically relevant improvement (≥30%) in MEP, indicating the clinical relevance of PPT for understanding mechanisms contributing to pain. CPM was not a reliable indicator of treatment response in MEP responders.

Albumin-binding RNAi Conjugate for Carrier Free Treatment of Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin 'hitchhiking' for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary: Lipophilic siRNA conjugates optimized for albumin binding and "hitchhiking" can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.

Neurophysiological and psychosocial mechanisms of fibromyalgia: A comprehensive review and call for an integrative model.

Research into the neurobiological and psychosocial mechanisms involved in fibromyalgia has progressed remarkably in recent years. Despite this, current accounts of fibromyalgia fail to capture the complex, dynamic, and mutual crosstalk between neurophysiological and psychosocial domains. We conducted a comprehensive review of the existing literature in order to: a) synthesize current knowledge on fibromyalgia; b) explore and highlight multi-level links and pathways between different systems; and c) build bridges connecting disparate perspectives. An extensive panel of international experts in neurophysiological and psychosocial aspects of fibromyalgia discussed the collected evidence and progressively refined and conceptualized its interpretation. This work constitutes an essential step towards the development of a model capable of integrating the main factors implicated in fibromyalgia into a single, unified construct which appears indispensable to foster the understanding, assessment, and intervention for fibromyalgia.

Low Gut Microbial Diversity Augments Estrogen-Driven Pulmonary Fibrosis in Female-Predominant Interstitial Lung Disease.

Although profibrotic cytokines, such as IL-17A and TGF-ß1, have been implicated in the pathogenesis of interstitial lung disease (ILD), the interactions between gut dysbiosis, gonadotrophic hormones and molecular mediators of profibrotic cytokine expression, such as the phosphorylation of STAT3, have not been defined. Here, through chromatin immunoprecipitation sequencing (ChIP-seq) analysis of primary human CD4+ T cells, we show that regions within the STAT3 locus are significantly enriched for binding by the transcription factor estrogen receptor alpha (ERa). Using the murine model of bleomycin-induced pulmonary fibrosis, we found significantly increased regulatory T cells compared to Th17 cells in the female lung. The genetic absence of ESR1 or ovariectomy in mice significantly increased pSTAT3 and IL-17A expression in pulmonary CD4+ T cells, which was reduced after the repletion of female hormones. Remarkably, there was no significant reduction in lung fibrosis under either condition, suggesting that factors outside of ovarian hormones also contribute. An assessment of lung fibrosis among menstruating females in different rearing environments revealed that environments favoring gut dysbiosis augment fibrosis. Furthermore, hormone repletion following ovariectomy further augmented lung fibrosis, suggesting pathologic interactions between gonadal hormones and gut microbiota in relation to lung fibrosis severity. An analysis of female sarcoidosis patients revealed a significant reduction in pSTAT3 and IL-17A levels and a concomitant increase in TGF-ß1 levels in CD4+ T cells compared to male sarcoidosis patients. These studies reveal that estrogen is profibrotic in females and that gut dysbiosis in menstruating females augments lung fibrosis severity, supporting a critical interaction between gonadal hormones and gut flora in lung fibrosis pathogenesis.

Low Gut Microbial Diversity Augments Estrogen-driven Pulmonary Fibrosis in Female-Predominant Interstitial Lung Disease.

Although profibrotic cytokines such as IL-17A and TGF-ß1 have been implicated in interstitial lung disease (ILD) pathogenesis, interactions between gut dysbiosis, gonadotrophic hormones and molecular mediators of profibrotic cytokine expression, such as phosphorylation of STAT3, have not been defined. Here we show by chromatin immunoprecipitation sequencing (ChIP-seq) analysis of primary human CD4+ T cells that regions within the STAT3 locus are significantly enriched for binding by the transcription factor estrogen receptor alpha (ERa). Using the murine model of bleomycin-induced pulmonary fibrosis, we found significantly increased regulatory T cells compared to Th17 cells in the female lung. Genetic absence of ESR1 or ovariectomy in mice significantly increased pSTAT3 and IL-17A expression in pulmonary CD4+ T cells, which was reduced after repletion of female hormones. Remarkably, there was no significant reduction in lung fibrosis under either condition, suggesting that factors outside of ovarian hormones also contribute. Assessment of lung fibrosis among menstruating females in different rearing environments revealed that environments favoring gut dysbiosis augment fibrosis. Furthermore, hormone repletion following ovariectomy further augmented lung fibrosis, suggesting pathologic interactions between gonadal hormones and gut microbiota on lung fibrosis severity. Analysis in female sarcoidosis patients revealed a significant reduction in pSTAT3 and IL-17A levels and a concomitant increase in TGF-ß1 levels in CD4+ T cells, compared to male sarcoidosis patients. These studies reveal that estrogen is profibrotic in females and that gut dysbiosis in menstruating females augments lung fibrosis severity, supporting a critical interaction between gonadal hormones and gut flora in lung fibrosis pathogenesis.

Characterising the autoantibody repertoire in systemic sclerosis following myeloablative haematopoietic stem cell transplantation.

OBJECTIVES: Results from the SCOT (Scleroderma: Cyclophosphamide Or Transplantation) clinical trial demonstrated significant benefits of haematopoietic stem cell transplant (HSCT) versus cyclophosphamide (CTX) in patients with systemic sclerosis. The objective of this study was to test the hypothesis that transplantation stabilises the autoantibody repertoire in patients with favourable clinical outcomes. METHODS: We used a bead-based array containing 221 protein antigens to profile serum IgG autoantibodies in participants of the SCOT trial. RESULTS: Comparison of autoantibody profiles at month 26 (n=23 HSCT; n=22 CTX) revealed antibodies against two viral antigens and six self-proteins (SSB/La, CX3CL1, glycyl-tRNA synthetase (EJ), parietal cell antigen, bactericidal permeability-increasing protein and epidermal growth factor receptor (EGFR)) that were significantly different between treatment groups. Linear mixed model analysis identified temporal increases in antibody levels for hepatitis B surface antigen, CCL3 and EGFR in HSCT-treated patients. Eight of 32 HSCT-treated participants and one of 31 CTX-treated participants had temporally varying serum antibody profiles for one or more of 14 antigens. Baseline autoantibody levels against 20 unique antigens, including 9 secreted proteins (interleukins, IL-18, IL-22, IL-23 and IL-27), interferon-α2A, stem cell factor, transforming growth factor-ß, macrophage colony-stimulating factor and macrophage migration inhibitory factor were significantly higher in patients who survived event-free to month 54. CONCLUSIONS: Our results suggest that HSCT favourably alters the autoantibody repertoire, which remains virtually unchanged in CTX-treated patients. Although antibodies recognising secreted proteins are generally thought to be pathogenic, our results suggest a subset could potentially modulate HSCT in scleroderma.

Clinical and Molecular Findings After Autologous Stem Cell Transplantation or Cyclophosphamide for Scleroderma: Handling Missing Longitudinal Data.

OBJECTIVE: Among individuals with systemic sclerosis (SSc) randomized to cyclophosphamide (CYC) (n = 34) or hematopoietic stem cell transplantation (HSCT) (n = 33), we examined longitudinal trends of clinical, pulmonary function, and quality of life measures while accounting for the influence of early failures on treatment comparisons. METHODS: Assuming that data were missing at random, mixed-effects regression models were used to estimate longitudinal trends for clinical measures when comparing treatment groups. Results were compared to observed means and to longitudinal trends estimated from shared parameter models, assuming that data were missing not at random. Longitudinal trends for SSc intrinsic molecular subsets defined by baseline gene expression signatures (normal-like, inflammatory, and fibroproliferative signatures) were also studied. RESULTS: Available observed means for pulmonary function tests appeared to improve over time in both arms. However, after accounting for participant loss, forced vital capacity in HSCT recipients increased by 0.77 percentage points/year but worsened by -3.70/year for CYC (P = 0.004). Similar results were found for diffusing capacity for carbon monoxide and quality of life indicators. Results for both analytic models were consistent. HSCT recipients in the inflammatory (n = 20) and fibroproliferative (n = 20) subsets had superior long-term trends compared to CYC for pulmonary and quality of life measures. HSCT was also superior for modified Rodnan skin thickness scores in the fibroproliferative subset. For the normal-like subset (n = 22), superiority of HSCT was less apparent. CONCLUSION: Longitudinal trends estimated from 2 statistical models affirm the efficacy of HSCT over CYC in severe SSc. Failure to account for early loss of participants may distort estimated clinical trends over the long term.

Hybrid Decoders for Marked Point Process Observations and External Influences.

OBJECTIVE: Internal physiological processes govern multiple state variables within the human body. Estimating these from point process-type bioelectric and biochemical observations is a challenge. Here we seek to estimate cortisol-related energy production and sympathetic arousal based on point process and continuous-valued data while permitting an external influence to affect the state estimates. METHODS: Traditional point process state-space methods, such as those used for estimating the aforementioned quantities from cortisol and skin conductance measurements respectively, suffer from the inability to permit the state estimates to also fit to an external influence (e.g. labels) or be guided by it. Here we modify an existing recurrent neural network (RNN) approach for state-space estimation through a weighted cost-function to enable a hybrid estimator that has this capability. RESULTS: Results on cortisol data based on a hypothetical sleep-wake influence term show how energy production can be estimated by permitting the estimates to fit to the external influence as much as desired. We further show how overfitting may be reduced by using circadian rhythm-based influence terms. Results on skin conductance data also indicate how the method can be used to estimate sympathetic arousal in an experiment containing stressors and relaxation, and permit an external influence as well. CONCLUSION: The RNN-based hybrid method is thus able to recover internal physiological states from point process and continuous-valued observations while permitting an external influence to guide the estimates. SIGNIFICANCE: The hybrid estimator could be embedded within wearable monitors that can be tailored based on domain expertise or individual feedback.

Myeloablative autologous haematopoietic stem cell transplantation resets the B cell repertoire to a more naïve state in patients with systemic sclerosis.

OBJECTIVES: Myeloablative autologous haematopoietic stem cell transplant (HSCT) was recently demonstrated to provide significant benefit over cyclophosphamide (CYC) in the treatment of diffuse cutaneous systemic sclerosis (dcSSc) in the Scleroderma: Cyclophosphamide or Transplantation (SCOT) trial. As dysregulation of the B cell compartment has previously been described in dcSSc, we sought to gain insight into the effects of myeloablative autologous HSCT as compared with CYC. METHODS: We sequenced the peripheral blood immunoglobulin heavy chain (IGH) repertoires in patients with dcSSc enrolled in the SCOT trial. RESULTS: Myeloablative autologous HSCT was associated with a sustained increase in IgM isotype antibodies bearing a low mutation rate. Clonal expression was reduced in IGH repertoires following myeloablative autologous HSCT. Additionally, we identified a underusage of immunoglobulin heavy chain V gene 5-51 in patients with dcSSc, and usage normalised following myeloablative autologous HSCT but not CYC treatment. CONCLUSIONS: Together, these findings suggest that myeloablative autologous HSCT resets the IGH repertoire to a more naïve state characterised by IgM-expressing B cells, providing a possible mechanism for the elimination of pathogenic B cells that may contribute to the benefit of HSCT over CYC in the treatment of dcSSc.

Emotion regulation and the salience network: a hypothetical integrative model of fibromyalgia.

Fibromyalgia is characterized by widespread pain, fatigue, sleep disturbances and other symptoms, and has a substantial socioeconomic impact. Current biomedical and psychosocial treatments are unsatisfactory for many patients, and treatment progress has been hindered by the lack of a clear understanding of the pathogenesis of fibromyalgia. We present here a model of fibromyalgia that integrates current psychosocial and neurophysiological observations. We propose that an imbalance in emotion regulation, reflected by an overactive 'threat' system and underactive 'soothing' system, might keep the 'salience network' (also known as the midcingulo-insular network) in continuous alert mode, and this hyperactivation, in conjunction with other mechanisms, contributes to fibromyalgia. This proposed integrative model, which we term the Fibromyalgia: Imbalance of Threat and Soothing Systems (FITSS) model, should be viewed as a working hypothesis with limited supporting evidence available. We hope, however, that this model will shed new light on existing psychosocial and biological observations, and inspire future research to address the many gaps in our knowledge about fibromyalgia, ultimately stimulating the development of novel therapeutic interventions.

Multiparameter analysis of human B lymphocytes identifies heterogeneous CD19+ CD21lo subsets.

Autoreactive B cell subsets have been described in a variety of settings, using multiple classification schemes and cell surface markers also found on healthy cells. CD19+ CD21lo B cells have been identified as an autoreactive-prone subset of B cells, although the downregulation of CD21 has been observed on a variety of B cell subsets in health and disease. This variation has led to confusion regarding the meaning and applicability of the loss or reduction of CD21 in peripheral B cells. To better understand the relationships between commonly used B cell markers and their associated characteristics, we analyzed human B cells from healthy participants using multiparameter flow cytometry and the visualization algorithm, tSNE. This approach revealed significant phenotypic overlap amongst five previously described autoimmune-prone B cell subsets, including CD19+ CD10- CD27- CD21lo B cells. Interestingly, 12 different subpopulations of CD19+ CD21lo B cells were identified, some of which mapped to previously described autoreactive populations, while others were consistent with healthy B cells. This suggests that CD21 is downregulated in a variety of circumstances involving B cell activation, all of which are present in low numbers even in healthy individuals. These findings describe the utility of unbiased multiparameter analysis using a relatively limited panel of flow cytometry markers to analyze autoreactive-prone and normal activated B cells.