D1.131 - Unconventional Group 2 Innate Lymphoid Cells Expressing C-Kit and IL-17A are Increased in Sputum from Severe Asthma with Mixed Granulocytic Airway Inflammation

Conventional group 2 innate lymphoid cells (cILC2) expressing T2 cytokines are increased in prednisone-dependent severe asthma (SA) with sputum eosinophilia (>3%) indicating a role in the persistence of airway inflammation. Detection of C-kit+IL17A+cILC2 in SA with mixed granulocytic inflammation indicates a potential for trans-differentiation of cILC2 to an intermediate ILC2/3 phenotype instructed by changing microenvironmental cues (IL-1β+IL18) which may determine the predominant airway inflammatory profile. Recently, unconventional ILC2 (UncILC2) that lack CD127 (IL7Rα) have been described and shown to arise following stimulation of cILC2 with alarmin cytokines. This study enumerated UncILC2 in SA.

Consenting subjects with SA [pre-bronchodilator FEV₁ <80% predicted; FEV₁ reversibility ≥12% and ≥200 mL, or a historical methacholine PC20 ≤8 mg/mL] receiving high-dose ICS/LABA (>880 μg/day) and OCS (7.5-40 mg daily prednisone or equivalent) were recruited when stable and categorized by differential sputum cell counts as eosinophilic (≥3%), neutrophilic (≥61% and ≥15×10⁶ /ml TCC) or mixed granulocytic (both) airway inflammation. Sputum cells were stained, acquired on BD LSRFortessa^TM and analysed by FlowJo V10 or R studio (Seurat package) to phenotype and enumerate ILCs.

In stable SA, total UncILC2 (Lin-CD45+CD127-CRTH2+) and the proportion of cells expressing intracellular IL-5/IL-13 were significantly greater than cILC2 (Lin-CD45+CD127+CRTH2+) (35.2±15.6 vs 5.9±1.9 x10⁶ cells/ml; 3.56±1.0 vs 1.05±1.7 %Lin- cells, respectively, P<0.05).  Levels of IL-17A+UncILC2 were significantly greater in SA with mixed granulocytic airway inflammation (P<0.05), were associated with c-Kit expression, higher OCS therapy (≥ 7.5 mg/day) and were inversely correlated with % predicted FEV1 (R²= 0.374, P<0.01). FACS sort-purified cILC2 cultured with IL-1β+IL-18 compared to IL-2 for 7 days, showed a significant loss of CD127 expression, in vitro (21±11 % vs 47±16 Lin– cells, respectively P<0.05).

Unconventional ILC2s expressing IL-17A and c-kit are markedly elevated in sputum from subjects with stable SA and mixed granulocytic airway inflammation. These cells are associated with increased disease severity and lung dysfunction, highlighting a potential role in driving the severity of corticosteroid-insensitive asthma. This underscores the importance of broadening cell profiling to fully understand the role of innate lymphoid cells in SA