Background: 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.Methods: 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.Results: 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.Conclusion: 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.

Several studies have described the proteomic profile of different immune cell types, but only a few have also analysed the content of their delivered small extracellular vesicles (sEVs). The aim of the present study was to compare the protein signature of sEVs delivered from granulocytes (i.e., neutrophils and eosinophils) and CD4⁺ T cells (i.e., TH1, TH2, and TH17) to identify potential biomarkers of the inflammatory profile in chronic inflammatory diseases. Qualitative (DDA) and quantitative (DIA-SWATH) analyses of in vitro-produced sEVs revealed proteome variations depending on the cell source. The main differences were found between granulocyte- and TH cell-derived sEVs, with a higher abundance of antimicrobial proteins (e.g., LCN2, LTF, MPO) in granulocyte-derived sEVs and an enrichment of ribosomal proteins (RPL and RPS proteins) in TH-derived sEVs. Additionally, we found differentially abundant proteins between neutrophil and eosinophil sEVs (e.g., ILF2, LTF, LCN2) and between sEVs from different TH subsets (e.g., ISG15, ITGA4, ITGB2, or NAMPT). A “proof-of-concept” assay was also performed, with TH2 biomarkers ITGA4 and ITGB2 displaying a differential abundance in sEVs from T2^high and T2^low asthma patients. Thus, our findings highlight the potential use of these sEVs as a source of biomarkers for diseases where the different immune cell subsets studied participate, particularly chronic inflammatory pathologies such as asthma or chronic obstructive pulmonary disease (COPD).

This study aims to develop a protocol for respiratory disease-associated biomarker discovery by combining urine proteome studies with urinary exosome components analysis (i.e., miRNAs). To achieve this, urine was DTT treated to decrease uromodulin, then concentrated and ultracentrifuged. Proteomic analyses of exosome-free urine were performed using LC-MS/MS. Simultaneously, miRNA expression from urine exosomes was measured using either RTqPCR (pre-amplification) or nCounter Nanostring (non-amplication) analyses. We detected 548 different proteins in exosome-free urine samples (N = 5) with high confidence (FDR < 1%), many of them being expressed in different non-renal tissues. Specifically, lung-related proteins were overrepresented (Fold enrichment = 1.31; FDR = 0.0335) compared to whole human proteome, and 10–15% were already described as protein biomarkers for several pulmonary diseases. Urine proteins identified belong to several functional categories important in respiratory pathology. We could confirm the expression of miRNAs previously connected to respiratory diseases (i.e., miR-16-5p, miR-21-5p, miR-146a-5p, and miR-215-5p) in urine exosomes by RTqPCR. Finally, we detected 333 miRNAs using Nanostring, 15 of them up-regulated in T2^high asthma (N = 4) compared to T2^low asthma (N = 4) and healthy subjects (N = 4). Therefore, this protocol combining the urinary proteome (exosome free) with the study of urinary exosome components (i.e., miRNAs) holds great potential for molecular biomarker discovery of non-renal and particularly respiratory pathologies.