Exploring CD26^-/lo subpopulations of lymphocytes in asthma phenotype and severity: A novel CD4⁺ T cell subset expressing archetypical granulocyte proteins.

Abstract

Asthma pathology may induce changes in naïve/memory lymphocyte proportions assessable through the evaluation of surface CD26 (dipeptidyl peptidase 4/DPP4) levels. Our aim was to investigate the association of asthma phenotype/severity with the relative frequency of CD26^-/lo, CD26^int and CD26^hi subsets within different lymphocyte populations. The proportion of CD26^-/lo, CD26^int and CD26^hi subsets within CD4⁺ effector T cells (T_eff), total CD4^- lymphocytes, γδ-T cells, NK cells and NKT cells was measured in peripheral blood samples from healthy (N = 30) and asthma (N = 119) donors with different phenotypes/severities by flow cytometry. We performed K-means clustering analysis and further characterised the CD4⁺CD26^-/lo T_eff cell subset by LC-MS/MS and immunofluorescence. Cluster analysis including clinical and flow cytometry data resulted in four groups, two of them with opposite inflammatory profiles (neutrophilic vs. eosinophilic). Neutrophilic asthma presented reduced CD4^-CD26^hi cells, which negatively correlated with systemic inflammation. Eosinophilic asthma displayed a general expansion of CD26^-/lo subsets. Specifically, CD4⁺CD26^-/lo T_eff expansion was confirmed in asthma, especially in atopic patients. Proteomic characterisation of this subset with a T_EM/T_EMRA phenotype revealed upregulated levels of innate (e.g. MPO and RNASE2) and cytoskeleton/extracellular matrix (e.g. MMP9 and ACTN1) proteins. Immunofluorescence assays confirmed the presence of atypical proteins for CD4⁺ T cells, and an enrichment in 'flower-like' nuclei and MMP9/RNASE2 levels in CD4⁺CD26^-/lo T_eff compared to CD4⁺ T lymphocytes. There is an association between CD26 levels in different lymphocyte subsets and asthma phenotype/severity. CD4⁺CD26^-/loT_EMRA cells expressing innate proteins specific to eosinophils/neutrophils could be determinant in sustaining long-term inflammation in adult allergic asthma.