Profiling cellular heterogeneity in asthma with single cell multiparameter CyTOF

Stewart, Edgar I.; Wang, Xiaomei; Chupp, Geoffrey; Montgomery, Ruth R.

doi:10.1002/jlb.5ma0720-770rr

Cited by 13 publications

(16 citation statements)

References 54 publications

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“…Although our analysis was limited to assessment of eosinophilic activity, there might be other aspect of cellular inflammation that are often not taken into consideration leading to inaccurate characterization as a non-T2 endotype. These include lymphocyte [ 20 ] and mast cell numbers [ 21 ] in sputum that can be identified particularly with more advanced microscopy, flow cytometry, and mass cytometry. They could also be markers of corticosteroid responsiveness and it remains to be seen how often these endotypes may occur in the absence of eosinophilia.…”

Section: Discussionmentioning

confidence: 99%

Underestimation of airway luminal eosinophilia by quantitative sputum cytometry

Kjarsgaard

Adatia

Bhalla

et al. 2021

Allergy Asthma Clin Immunol

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Rationale On Wright-stained sputum cytospins, eosinophil differential of ≥ 1.2% is considered abnormal, and ≥ 2.3% identifies an eosinophilic endotype. We hypothesized that failure to consider free eosinophil granules (FEG), and the re-emergence (unmasking) of eosinophilia due to various reasons underestimate the prevalence of the eosinophilic endotype. Methods This is a retrospective analysis of our Institutional Review Board-approved clinical sputum database. Of the 24,176 examinations of sputa from patients with various airway diseases, 17,693 were viable cell counts from 9570 patients (6604 on a single occasion, 2967 from multiple occasions). The prevalence of intact eosinophil % at 1.2 and 2.3% thresholds was first examined. Then, additional evidence of eosinophilia was assessed by semi-quantitative enumeration of FEGs. In those patients whose sputa were examined on multiple occasions (at the time of an exacerbation or after corticosteroid dose was reduced), re-emergence (unmasking) of eosinophilia was assessed . Results Using the threshold of eosinophilia ≥ 1.2%, 6289/17693 (35.6%) of sputa were classified as eosinophilic. This increased to 7850/17693 (44.4%) when the presence of FEGs was considered. Using the threshold of eosinophilia ≥ 2.3%, 4647/17693 (26.3%) of sputa were classified as eosinophilic. This increased to 5435/17693 (30.7%) when the presence of FEG were considered. Extrapolating from the prevalence of re-emergence observed in the 2967 patients who had sputa examined on multiple occasions to the whole sample, we estimated that eosinophilia at 1.2% threshold would be observed in at least 60% of the samples, and a clinically relevant eosinophilia at 2.3% threshold would be observed in at least 48.5% of the samples. Conclusions Using a large sputum cytometry clinical database (17,693 viable cell counts), we demonstrate that a single time point intact cell count underestimates the prevalence of eosinophilia in a variety of airway diseases. The prevalence of eosinophilia increases from 35.6 to 60% (40% underestimation) at the 1.2% threshold, and from 26.3 to 48.5% (45% underestimation) at the 2.3% clinically relevant threshold, when free granules and a second examination are considered. This has important implications to identify the eosinophilic and Th2 high endotype both for clinical trials of anti-eosinophil therapies, and to select patients who may respond well to glucocorticosteroids and anti-IL5 therapies.

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Section: Discussionmentioning

confidence: 99%

Underestimation of airway luminal eosinophilia by quantitative sputum cytometry

Kjarsgaard

Adatia

Bhalla

et al. 2021

Allergy Asthma Clin Immunol

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“…Although our analysis was limited to assessment of eosinophilic activity, there might be other aspect of cellular in ammation that are often not taken into consideration leading to inaccurate characterization as a non-T2 endotype. These include lymphocyte (20) and mast cell numbers (21) in sputum that can be identi ed particularly with more advanced microscopy, ow cytometry, and mass cytometry. They could also be markers of steroid responsiveness and it remains to be seen how often these endotypes may occur in the absence of eosinophilia.…”

Section: Discussionmentioning

confidence: 99%

Underestimation of Luminal Eosinophilia by Quantitative Sputum Cytometry

Kjarsgaard

Adatia

Bhalla

et al. 2021

Preprint

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Rationale: On Wright-stained sputum cytospins, eosinophil differential of >1.2% is considered abnormal and ≥3% clinically relevant. We hypothesized that failure to consider free eosinophil granules (FEG), and the re-emergence of eosinophilia (at ≥1.2 and ≥2.3%) underestimate the prevalence of the eosinophilic phenotype.Methods: This is a retrospective analysis of our Institutional Review Board-approved clinical sputum database. Of the 24,176 examinations, 17,693 had viable cell counts from 9570 patients (6604 on one occasion, 2967 with two or more) with various airway diseases. In all samples, FEGs were semi-quantified to estimate the prevalence of eosinophilia at <1.2 and <2.3%. In those patients with sputum examined on more than one occasion, subsequent results identified re-emergence of eosinophilia at ³1.2 and ³2.3%.Results: Of those with intact cell counts (n=15,278), 8562 (56.0%) and 9690 (63.4%) would have been classified as clinically non-relevant eosinophilia of <1.2% and <2.3% respectively. Among those with two or more intact cell counts, 1142 (38.5%) and 1083 (36.5%) of samples had re-emergence of eosinophilia (at 1.2 and 2.3%), either when a previous neutrophilia resolved (n= 218/19.1%, n=312/28.8%) or due to a reduction in the dose of corticosteroids (n=634/21.4%, n=666/22.4%) with no significant differences between groups (p=0.018). There was a significant difference between the proportions of eosinophilic versus non-eosinophilic at 1.2 and 2.3%, using the presence of FEG and the non-intact cell count (p<0.001).Conclusions: A total of 3180 (32.8%) samples identified as non-eosinophilic, had clinically relevant airway eosinophilia that would not have been identified if the phenotypic classification was limited to ≥2.3% intact eosinophils on one occasion. This underestimation is likely to be significantly more if other means of airway eosinophilic activity were included and if mast cell activity and lymphocyte numbers, (not routinely quantified by sputum cytometry), could be contributing to the under-appreciation of airway T2 inflammatory processes.

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“…Maximum sample loading rate was 300 events per second. Then, the data files were standardized and analyzed using the bead-based normalizer 15 and cell analysis bank The clustering of PhenoGraph map and the dimensionality reduction were completed using R software package cytofkit and the default k value = 30. 16,17 According to the median of each phenological group marker expression, two meta clusters were determined by hierarchical clustering method.…”

Section: Mass Cytometrymentioning

confidence: 99%

A cellular census of human peripheral immune cells identifies novel cell states in lung diseases

Song

Yan

et al. 2021

Clinical & Translational Med

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Increasing evidence supports a central role of the immune system in lung diseases. Understanding how immunological alterations between lung diseases provide opportunities for immunotherapy. Exhausted T cells play a key role of immune suppression in lung cancer and chronic obstructive pulmonary disease was proved in our previous study. The present study aims to furthermore define molecular landscapes and heterogeneity of systemic immune cell target proteomic and transcriptomic profiles and interactions between circulating immune cells and lung residential cells in various lung diseases. We firstly measured target proteomic profiles of circulating immune cells from healthy volunteers and patients with stable pneumonia, stable asthma, acute asthma, acute exacerbation of chronic obstructive pulmonary disease, chronic obstructive pulmonary disease and lung cancer, using single‐cell analysis by cytometry by time‐of‐flight with 42 antibodies. The nine immune cells landscape was mapped among those respiratory system diseases, including CD4 + T cells, CD8 + T cells, dendritic cells, B cells, eosinophil, γδT cells, monocytes, neutrophil and natural killer cells. The double‐negative T cells and exhausted CD4 + central memory T cells subset were identified in patients with acute pneumonia. This T subset expressed higher levels of T‐cell immunoglobulin and mucin domain‐containing protein 3 (Tim3) and T‐cell immunoreceptor with Ig and ITIM domains (TIGIT) in patients with acute pneumonia and stable pneumonia. Biological processes and pathways of immune cells including immune response activation, regulation of cell cycle and pathways in cancer in peripheral blood immune cells were defined by bulk RNA sequencing (RNA‐seq). The heterogeneity among immune cells including CD4 + , CD8 + T cells and NK T cells by single immune cell RNA‐seq with significant difference was found by single‐cell sequencing. The effect of interstitial telocytes on the immune cell types and immune function was finally studied and the expressions of CD8a and chemokine C–C motif receptor 7 (CCR7) were increased significantly in co‐cultured groups. Our data indicate that proteomic and transcriptomic profiles and heterogeneity of circulating immune cells provides new insights for understanding new molecular mechanisms of immune cell function, interaction and modulation as a source to identify and develop biomarkers and targets for lung diseases.

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Profiling cellular heterogeneity in asthma with single cell multiparameter CyTOF

Cited by 13 publications

References 54 publications

Underestimation of airway luminal eosinophilia by quantitative sputum cytometry

Underestimation of airway luminal eosinophilia by quantitative sputum cytometry

Underestimation of Luminal Eosinophilia by Quantitative Sputum Cytometry

A cellular census of human peripheral immune cells identifies novel cell states in lung diseases

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