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Randomized Controlled Trial
. 2024 Aug;154(2):297-307.e13.
doi: 10.1016/j.jaci.2024.01.031. Epub 2024 Mar 12.

Activated sputum eosinophils associated with exacerbations in children on mepolizumab

Gabriella E Wilson  1 , James Knight  2 , Qing Liu  1 , Ashish Shelar  2 , Emma Stewart  3 , Xiaomei Wang  1 , Xiting Yan  1 , Joshua Sanders  4 , Cynthia Visness  4 , Michelle Gill  5 , Rebecca Gruchalla  6 , Andrew H Liu  7 , Meyer Kattan  8 , Gurjit K Khurana Hershey  9 , Alkis Togias  10 , Patrice M Becker  10 , Matthew C Altman  11 , William W Busse  12 , Daniel J Jackson  13 , Ruth R Montgomery  1 , Geoffrey L Chupp  14
Affiliations
Randomized Controlled Trial

Activated sputum eosinophils associated with exacerbations in children on mepolizumab

Gabriella E Wilson et al. J Allergy Clin Immunol. 2024 Aug.

Abstract

Background: MUPPITS-2 was a randomized, placebo-controlled clinical trial that demonstrated mepolizumab (anti-IL-5) reduced exacerbations and blood and airway eosinophils in urban children with severe eosinophilic asthma. Despite this reduction in eosinophilia, exacerbation risk persisted in certain patients treated with mepolizumab. This raises the possibility that subpopulations of airway eosinophils exist that contribute to breakthrough exacerbations.

Objective: We aimed to determine the effect of mepolizumab on airway eosinophils in childhood asthma.

Methods: Sputum samples were obtained from 53 MUPPITS-2 participants. Airway eosinophils were characterized using mass cytometry and grouped into subpopulations using unsupervised clustering analyses of 38 surface and intracellular markers. Differences in frequency and immunophenotype of sputum eosinophil subpopulations were assessed based on treatment arm and frequency of exacerbations.

Results: Median sputum eosinophils were significantly lower among participants treated with mepolizumab compared with placebo (58% lower, 0.35% difference [95% CI 0.01, 0.74], P = .04). Clustering analysis identified 3 subpopulations of sputum eosinophils with varied expression of CD62L. CD62Lint and CD62Lhi eosinophils exhibited significantly elevated activation marker and eosinophil peroxidase expression, respectively. In mepolizumab-treated participants, CD62Lint and CD62Lhi eosinophils were more abundant in participants who experienced exacerbations than in those who did not (100% higher for CD62Lint, 0.04% difference [95% CI 0.0, 0.13], P = .04; 93% higher for CD62Lhi, 0.21% difference [95% CI 0.0, 0.77], P = .04).

Conclusions: Children with eosinophilic asthma treated with mepolizumab had significantly lower sputum eosinophils. However, CD62Lint and CD62Lhi eosinophils were significantly elevated in children on mepolizumab who had exacerbations, suggesting that eosinophil subpopulations exist that contribute to exacerbations despite anti-IL-5 treatment.

Keywords: Interleukin-5; asthma; mass cytometry; sputum.

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Conflict of interest statement

Declaration of Interests:

MG, MK, and DJJ report grants from the US National Institutes of Health (NIH)-NIAID during the study. CV reports Institutional Cooperative Agreement funding support from the US National Institutes of Health (NIH)-NIAID during the study. RRM reports grants from the US National Institutes of Health (NIH) during the study. GW and XY report grants from the US National Institutes of Health (NIH) outside of the submitted work. MG reports personal fees from the Society for Pediatric Research outside of the submitted work, and Leadership roles within the Society for Pediatric Research and Pediatric Academic Societies. GLC reports investigator-initiated research grant, consulting fees from GSK, AZ, Sanofi, Regeneron, Amgen, Genentech, Novartis, RAPT, and Teva, as well as speaking honoraria from GSK, AZ, Genentech, Regeneron, Sanofi, Amgen, and RAPT outside of the submitted work. MCA reports personal fees from Sanofi-Regeneron outside of the submitted work. WWB reports personal fees from Elsevier, Sanofi, Regeneron, GlaxoSmithKline, Novartis, AstraZeneca, and Genentech outside of the submitted work. DJJ reports funding support from GlaxoSmithKline during the current study, funding support from the NIH-US National Heart, Lung, and Blood Institute (NHLBI) outside of the submitted work, and personal fees from AstraZeneca, Avillion, GlaxoSmithKline, Regeneron, Sanofi, Genentech, OM Pharma, Areteia, and Pfizer outside of the submitted work. JK, QL, AS, ES, XW, JS, RG, AHL, GKKH, AT, PMB, MCA have nothing to disclose outside of the submitted work.

Figures

Figure 1:
Figure 1:. Relative abundances of sputum leukocytes according to treatment arm.
A, Manually gated leukocytes are shown as a proportion of total CD45+ cells in mepolizumab and placebo treated participants. B, manually gated eosinophils are shown as a proportion of the total CD45+ cells in mepolizumab and placebo treated participants. Individual leukocyte proportions per sample and median and interquartile range for each cell type are shown.
Figure 2:
Figure 2:. Eosinophil abundance according to exacerbation status after 12 months of treatment.
A, Eosinophil abundance shown as a proportion of total CD45+ cells in placebo participants separated by the number of exacerbations during the trial period. “PlacNonEx” indicates participants with 0 exacerbations during treatment and “PlacEx” indicates participants with ≥1 exacerbation during treatment. B, Eosinophil abundance shown as a proportion of total CD45+ cells in mepolizumab participants separated by the number of exacerbations during the trial period. “MepoNonEx” indicates participants with 0 exacerbations during treatment and “MepoEx” indicates participants with ≥1 exacerbation during treatment. Individual eosinophil proportions per sample and median and interquartile range for each group are shown.
Figure 3:
Figure 3:. Clustering analysis of manually gated eosinophils.
A, Median scaled expression of eosinophil functional markers separates eosinophils into 8 Clusters. Inter-cluster similarity is shown on the dendrogram on the left of the heatmap. B, UMAP representation of eosinophil subtypes after dendrogram-based cluster merging results in 3 eosinophil subtypes labeled CD62Llo, CD62Lint, and CD62Lhi. Clustering was performed on sputum eosinophils from both mepolizumab and placebo participants, and markers used for clustering are shown in Table S4.
Figure 4:
Figure 4:. Functional marker expression across eosinophil subtypes.
A, Median expression of eosinophil activation and mobilization markers across eosinophil subtypes in all cells. B, Median expression of T1/T3 inflammatory mediators across eosinophil subtypes in all cells. C, Median expression of T2 inflammatory mediators across eosinophil subtypes in all cells. Individual median expression values per sample and median and interquartile range for each marker are shown.
Figure 5:
Figure 5:. Eosinophil subpopulation proportions separated by treatment group and number of exacerbations.
A, CD62Llo, CD62Lint, and CD62Lhi eosinophil abundances are shown as a proportion of total CD45+ cells for each placebo participant. Participants are separated by number of exacerbations; “PlacNonEx” indicates participants with 0 exacerbations and “PlacEx” indicates participants with ≥1 exacerbation during the trial period. B, CD62Llo, CD62Lint, and CD62Lhi eosinophil abundances are shown as a proportion of total CD45+ cells for each mepolizumab participant. Participants are separated by number of exacerbations; “MepoNonEx” indicates participants with 0 exacerbations and “MepoEx” indicates participants with ≥1 exacerbation during the trial period. Individual eosinophil subtype proportions per sample and median and interquartile range for each group are shown.
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