Objectives: To examine functional features of CD4+CD8+ double-positive T-cells in patients with granulomatosis with polyangiitis (GPA) using phenotypic and transcriptomic analysis.
Methods: Staining of cellular surface marker was performed using freshly collected whole blood. For intracellular cytokine staining freshly collected whole blood was stimulated with phorbol myristate acetate and ionomycin. Multicolor flow cytometric analysis was performed on a FACSCanto II cytometer using FACSDiva software. Lymphocytes were gated on CD3, CD4, and CD8 staining. FACS-sorted CD4+CD8+ double-positive T-cells of GPA-patients and HC (n=3 each) were subjected to transcriptional profiling using an Affymetrix Human Genome 2.0 microarray. Differently expressed genes were analysed using biological databases.
Results: Frequency of CD4+CD8+ double-positive T-cells was increased within the total CD3+ T-cell population in GPA, but no difference was detected between patients with active disease and remission. Percentages of interferon γ (Th1-type), interleukin 17 and interleukin 22 (Th17-type) producing CD4+CD8+ double-positive T-cells exceeded the percentage of interleukin 4 (Th2-type) producing cells. There were no significant differences in the percentages of the respective cytokine-positive CD4+CD8+ double-positive T-cells between GPA and HC. Up-regulated genes of CD4+CD8+ double-positive T-cells in GPA were enriched within Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to nuclear factor kappa-lightchain-enhancer of activated B cells signalling, toll-like receptor signalling, nucleotide-binding oligomerisation domain-like receptor signalling as well as major histocompatibility complex class-II antigen presentation.
Conclusions: Employing a combined phenotypic and transcriptomic approach we disclosed a Th1/Th17 phenotype as well as innate and adaptive functions of CD4+CD8+ double-positive T-cells in GPA.