Interstitial lung disease (ILD) represents a significant cause of morbidity and mortality in systemic sclerosis (SSc). The purpose of this study was to examine recirculating lymphocytes from SSc patients for potential biomarkers of interstitial lung disease (ILD). Peripheral blood mononuclear cells (PBMCs) were isolated from patients with SSc and healthy controls enrolled in the Vanderbilt University Myositis and Scleroderma Treatment Initiative Center cohort between 9/2017–6/2019. Clinical phenotyping was performed by chart abstraction. Immunophenotyping was performed using both mass cytometry and fluorescence cytometry combined with t-distributed stochastic neighbor embedding analysis and traditional biaxial gating. This study included 34 patients with SSc-ILD, 14 patients without SSc-ILD, and 25 healthy controls. CD21lo/neg cells are significantly increased in SSc-ILD but not in SSc without ILD (15.4 ± 13.3% vs. 5.8 ± 0.9%, p = 0.002) or healthy controls (5.0 ± 0.5%, p < 0.0001). While CD21lo/neg B cells can be identified from a single biaxial gate, tSNE analysis reveals that the biaxial gate is comprised of multiple distinct subsets, all of which are increased in SSc-ILD. CD21lo/neg cells in both healthy controls and SSc-ILD are predominantly tBET positive and do not have intracellular CD21. Immunohistochemistry staining demonstrated that CD21lo/neg B cells diffusely infiltrate the lung parenchyma of an SSc-ILD patient. Additional work is needed to validate this biomarker in larger cohorts and longitudinal studies and to understand the role of these cells in SSc-ILD.
The idiopathic inflammatory myopathies (IIM) are a rare clinically heterogeneous group of conditions affecting the skin, muscle, joint, and lung in various combinations. While myositis specific autoantibodies are well described, we postulate that broader immune endotypes exist in IIM spanning B cell, T cell, and monocyte compartments. This study aims to identify immune endotypes through detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) in IIM patients compared to healthy controls. We collected PBMCs from 17 patients with a clinical diagnosis of inflammatory myositis and characterized the B, T, and myeloid cell subsets using mass cytometry by time of flight (CyTOF). Data were analyzed using a combination of the dimensionality reduction algorithm t-distributed stochastic neighbor embedding (t-SNE), cluster identification, characterization, and regression (CITRUS), and marker enrichment modeling (MEM); supervised biaxial gating validated populations identified by these methods to be differentially abundant between groups. Using these approaches, we identified shared immunologic features across all IIM patients, despite different clinical features, as well as two distinct immune endotypes. All IIM patients had decreased surface expression of RP105/CD180 on B cells and a reduction in circulating CD3+CXCR3+ subsets relative to healthy controls. One IIM endotype featured CXCR4 upregulation across all cellular compartments. The second endotype was hallmarked by an increased frequency of CD19+CD21loCD11c+ and CD3+CD4+PD1+ subsets. The experimental and analytical methods we describe here are broadly applicable to studying other immune-mediated diseases (e.g., autoimmunity, immunodeficiency) or protective immune responses (e.g., infection, vaccination).
Objectives: The idiopathic inflammatory myopathies (IIM) are a clinically heterogeneous group of conditions affecting the skin, muscle, joint, and lung in various combinations. This study aims to investigate the immunologic heterogeneity through detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) in IIM patients and healthy controls. Methods: We collected PBMCs from 17 patients with a clinical diagnosis of inflammatory myositis in the inpatient or outpatient setting and performed immunophenotyping using mass cytometry by time of flight (CyTOF) to simultaneously characterize B, T, and myeloid cell subsets. Data were analyzed using a combination of supervised biaxial gating and unsupervised clustering algorithms including t-distributed stochastic neighbor embedding (tSNE), cluster identification, characterization, and regression (CITRUS), and marker enrichment modeling (MEM). Results: We identified two distinct immune signatures amongst IIM patients. In one signature, increased CD19+CXCR4hiCCR7hi cells correlated with increased CD3+CXCR4hiCD38hi (r=0.62, p=0.009) and CD14+CD16-CXCR4+CD38+HLADR- (r=0.61, p=0.01) populations. In the second signature, increased CD19+CD21loCD11c+ cells correlated with an increased CD3+CD4+PD1+ (r=0.60, p=0.01) population. Other shared immunologic features amongst IIM patients compared to healthy controls included decreased surface expression of RP105/CD180 on B cells (median mass intensity 39.9 ± 16.0 v. 60.9 ± 20.1, p=0.002). In the T cell compartment, all circulating CD3+CXCR3+ subsets (2.7 ± 2.4 v. 9.6 ± 8.1% of all PBMCs, p=0.0004) were reduced. Conclusion: Based on circulating B cell phenotype, we identified two distinct immunologic signatures in IIM patients. Future work is needed to determine the significance of these immune signatures for clinical manifestations and treatment responses.
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.
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