Clustering analysis of asthma patients based on lymphocytes subpopulations: CD4+CD26-/lo T cells with a TEM/TEMRA phenotype blur the innate‑adaptive frontier in allergic asthma

Asthma is a chronic inflammatory disease which encompasses a heterogeneous group of patients. Despite sharing respiratory symptoms produced by airway inflammation and remodelling, the underlying pathophysiological mechanism varies among individuals. Here, we aimed to explore the association between effector/memory lymphocyte subsets defined based on CD26 levels (CD26^−/lo, CD26^int and CD26^hi) and specific asthma phenotypes. To do so, we have measured CD26 by flow cytometry in conventional CD4⁺ T cells, CD4⁻ lymphocytes, γδ-T cells, NK, and NKT cells from healthy donors (N = 30) and asthma patients (N = 119) with different phenotypes/severity. Cluster analysis including flow cytometry and clinical data resulted in 4 groups of donors, two with opposite types of inflammation (eosinophilic vs. neutrophilic). Additionally, we have confirmed the expansion of CD4⁺CD26^−/lo T cells (T_EM/T_EMRA) in asthma (<italic>p</italic> = 0.009), especially in atopic patients compared to HC (<italic>p</italic> = 0.016). Furthermore, their characterisation by LC-MS/MS (N = 4) revealed increased levels (log₂ fold-change > 1) of proteins related to different innate cells (e.g., MPO, RNASE2) and extracellular matrix (e.g., MMP9) compared with total CD4⁺ T cells. Finally, immunofluorescence analysis validated these findings, alongside with an enrichment in cells with "flower-like" nuclei. Therefore, our study demonstrates an association between asthma phenotypes and different memory populations, and especially a potential role of a highly differentiated effector-memory Th cell population (CD4⁺CD26^−/lo T cells) to sustain the disease and cause relapses.