Standardization of flow cytometry and cell sorting to enable a transcriptomic analysis in a multi-site sarcoidosis study.

The contribution and regulation of various CD4+ T cell lineages that occur with remitting vs progressive courses in sarcoidosis are poorly understood. We developed a multiparameter flow cytometry panel to sort these CD4+ T cell lineages followed by measurement of their functional potential using RNA-sequencing analysis at six-month intervals across multiple study sites. To obtain good quality RNA for sequencing, we relied on chemokine receptor expression to identify and sort lineages. To minimize gene expression changes induced by perturbations of T cells and avoid protein denaturation caused by freeze/thaw cycles, we optimized our protocols using freshly isolated samples at each study site. To accomplish this study, we had to overcome significant standardization challenges across multiple sites. Here, we detail standardization considerations for cell processing, flow staining, data acquisition, sorting parameters, and RNA quality control analysis that were performed as part of the NIH-sponsored, multi-center study, BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints (BRITE). After several rounds of iterative optimization, we identified the following aspects as critical for successful standardization: 1) alignment of PMT voltages across sites using CS&T/rainbow bead technology; 2) a single template created in the cytometer program that was used by all sites to gate cell populations during data acquisition and cell sorting; 3) use of standardized lyophilized flow cytometry staining cocktails to reduce technical error during processing; 4) development and implementation of a standardized Manual of Procedures. After standardization of cell sorting, we were able to determine the minimum number of sorted cells necessary for next generation sequencing through analysis of RNA quality and quantity from sorted T cell populations. Overall, we found that implementing a multi-parameter cell sorting with RNA-seq analysis clinical study across multiple study sites requires iteratively tested standardized procedures to ensure comparable and high-quality results.

T-bet Expression in Peripheral Th17.0 Cells Is Associated With Pulmonary Function Changes in Sarcoidosis.

Background: Interferon-gamma (IFN-γ) is a key mediator of sarcoidosis-related granulomatous inflammation. Previous findings of IFN-γ-producing Th17 cells in bronchoalveolar lavage fluid from sarcoidosis patients invokes the transition of Th17.0 cells to Th17.1 cells in the disease's pathogenesis. Since the T-bet transcription factor is crucial for this transition, the goal of this study was to determine if T-bet expression in Th17.0 cells reflects the extent of granulomatous inflammation in sarcoidosis patients as assessed by clinical outcomes. Methods: Using a case-control study design, we identified two groups of sarcoidosis subjects (total N = 43) with pulmonary function tests (PFTs) that either (1) changed (increased or decreased) longitudinally or (2) were stable. We used flow cytometry to measure the transcription factors T-bet and RORγt in Th1, Th17.0, and Th17.1 cell subsets defined by CCR6, CCR4 and CXCR3 in blood samples. We compared the percentages of T-bet+ cells in RORγt+Th17.0 cells (defined as CCR6+CCR4+CXCR3-) based on subjects' PFT group. We also assessed the relationship between the direction of change in PFTs with the changes in %T-bet+ frequencies using mixed effects modeling. Results: We found that T-bet expression in subjects' RORγt+Th17.0 cells varied based on clinical outcome. The T-bet+ percentage of RORγt+Th17.0 cells was higher in the cases (subject group with PFT changes) as compared to controls (stable group) (27 vs. 16%, p = 0.0040). In comparisons before and after subjects' PFT changes, the T-bet+ frequency of RORγt+Th17.0 cells increased or decreased in the opposite direction of the PFT change. The percentage of these T-bet+ cells was also higher in those with greater numbers of involved organs. Serum levels of interferon-γ-induced chemokines, CXCL9, CXCL10, and CXCL11, and whole blood gene expression of IFN-γ-related genes including GBP1, TAP1, and JAK2 were independently positively associated with the T-bet+ frequencies of RORγt+Th17.0 cells. Conclusions: These data suggest that expression of T-bet in Th17.0 cells could reflect the extent of granulomatous inflammation in sarcoidosis patients because they represent a transition state leading to the Th17.1 cell phenotype. These findings indicate that Th17 plasticity may be part of the disease paradigm.

Molecular profiling in sarcoidosis.

PURPOSE OF REVIEW: Sarcoidosis is a systemic disease characterized by granulomatous inflammation of unknown cause. There is extensive heterogeneity between patients with respect to the number and types of organs involved, disease course, and response to therapy. Recent research in the field has leveraged 'omics' techniques such as transcriptomics to identify important 'molecular profiles' in the disease. These tools may help in identifying clinically useful biomarkers and targets for therapy. RECENT FINDINGS: Several studies have used gene expression profiling of predesignated lists or the entire genome to find genes and markers that differentiate sarcoidosis from healthy controls, but only a few have compared sarcoidosis patients based on disease phenotypes and organ involvement. The common gene pathways that have been repeatedly identified include those related to the interferon response, T-cell receptor signaling, and the major histocompatibility complex. SUMMARY: While the molecular profiling studies to date offer the ability to compare sarcoidosis and health as well as across tissues, further longitudinal studies that include sarcoidosis patients with varying outcomes with respect to organ involvement and response to treatment are needed to identify clinically important phenotypes in the disease that can then be differentiated based on molecular features.

CXCL9 and CXCL10 are differentially associated with systemic organ involvement and pulmonary disease severity in sarcoidosis.

BACKGROUND: Sarcoidosis is a granulomatous inflammatory disease with limited blood markers to predict outcomes. The interferon-gamma (IFN-γ)-inducible chemotactic cytokines (chemokines), CXCL9 and CXCL10, are both increased in sarcoidosis patients, yet they possess important molecular differences. Our study determined if serum chemokines correlated with different aspects of disease severity. METHODS: We measured CXCL9 and CXCL10 serum levels at initial study visits and longitudinally in sarcoidosis subjects using ELISA. We examined these chemokines' relationships with pulmonary and organ involvement outcomes, their gene expression, peripheral blood immune cell populations, and immunosuppression use. RESULTS: Higher CXCL10 levels negatively correlated with FVC, TLC, and DLCO at subjects' initial visit and when measured repeatedly over two years. CXCL10 also positively correlated with longitudinal respiratory symptom severity. Additionally, for every log10(CXCL10) increase, the risk of longitudinal pulmonary function decline increased 8.8 times over the 5-year study period (95% CI 1.6-50, p = 0.014, log10(CXCL0) range 0.84-2.7). In contrast, CXCL9 levels positively correlated with systemic organ involvement at initial study visit (1.5 additional organs involved for every log10(CXCL9) increase, 95% CI 1.1-2.0, p = 0.022, log10(CXCL9) range 1.3-3.3). CXCL10, not CXCL9, positively correlated with its own blood gene expression and monocyte level. Immunosuppressive treatment was associated with lower levels of both chemokines. CONCLUSIONS: In sarcoidosis subjects, serum CXCL9 levels correlated with systemic organ involvement and CXCL10 levels strongly correlated with respiratory outcomes, which may ultimately prove helpful in clinical management. These differing associations may be due to differences in cellular regulation and tissue origin.

Serum CXCL11 correlates with pulmonary outcomes and disease burden in sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic granulomatous disease of unknown etiology that affects the lungs in 90% of patients, but has a wide range of disease manifestations and outcomes including chronic and progressive courses. Noninvasive biomarkers are needed to assess these outcomes and guide decisions for long term monitoring and treatment. Interferon-gamma (IFN-γ)-inducible chemotactic cytokines (chemokines), CXCL9, CXCL10 and CXCL11, show promise in this regard because they have been implicated in the pathogenesis of and reflect the burden of granulomatous inflammation. CXCL11 has been reported to have unique functional properties in modulating adaptive immunity in model systems so our goal was to examine serum levels of CXCL11 in relation to clinical outcomes in a heterogeneous cohort of sarcoidosis subjects. METHODS: CXCL19, CXCL10, and CXCL11 serum levels were measured in sarcoidosis and healthy subjects using ELISA assay. We determined relationships between CXCL11 and standard clinical inflammatory markers, expression of IFN-γ-related genes in whole blood, organ involvement, dyspnea scores, and measures of pulmonary function. RESULTS: In a cross-sectional analysis of 104 sarcoidosis subjects, serum CXCL11 was significantly elevated compared to 49 healthy controls (p < 0.001). CXCL11 was positively correlated with CXCL9 and CXCL10 (p < 0.001), sedimentation rate (p < 0.01), and mean expression of three IFN-γ-related genes in whole blood (GBP1, STAT1, and STAT2) (p < 0.001). CXCL11 was inversely correlated with FVC %predicted (%pred) and FEV1 %pred and higher levels were associated with higher patient-reported dyspnea scores. We found positive correlations between CXCL11 and number of organs involved. Using survival analyses, we found that CXCL11 levels were predictive of future pulmonary function test (PFT) decline (log rank <0.001 and HR of log10(CXCL11) = 5.1, 95% CI 1.2-21, p = 0.026). CONCLUSIONS: The pattern of expression of serum CXCL11 in sarcoidosis patients suggests that this blood measure could be helpful in identifying patients that need longer-term monitoring for progressive thoracic and extra-thoracic sarcoidosis.

Clinical and Biological Insights from the University of California San Francisco Prospective and Longitudinal Cohort.

INTRODUCTION: Sarcoidosis is a systemic inflammatory disease characterized by non-necrotizing granulomas in involved organs, most commonly the lung. Description of patient characteristics in the Western United States is limited. Furthermore, blood-based measures that relate to clinical sarcoidosis phenotypes are lacking. We present an analysis of a prospective, longitudinal sarcoidosis cohort at a Northern Californian academic medical center. METHODS: We enrolled 126 sarcoidosis subjects and 64 healthy controls and recorded baseline demographic and clinical characteristics. We used regression models to identify factors independently associated with pulmonary physiology. We tested whether blood transcript levels at study entry could relate to longitudinal changes in pulmonary physiology. RESULTS: White, non-Hispanics composed ~70% of subjects. Hispanics and Blacks had a diagnostic biopsy at an age ~7 years younger than whites. Obstructive, but not restrictive, physiology characterized Scadding Stage IV patients. Subjects reporting use of immunosuppression had worse FEV1%p, FVC%p, and DLCO%p compared to subjects never treated, regardless of Scadding stage. We defined sarcoidosis disease activity by a drop in pulmonary function over 36 months and found that subjects meeting this definition had significant repression of blood gene transcripts related to T cell receptor signaling pathways, referred to as the "TCR factor." CONCLUSION: Obstructive pulmonary physiology defined Stage IV patients which were mostly white, non-Hispanics. Genes comprising the composite gene expression score, TCR factor, may represent a blood-derived measure of T-cell activity and an indirect measure of active sarcoidosis inflammation. Validation of this measure could translate into individualized treatment for sarcoidosis patients.