gms | German Medical Science

Background: An increased frequency of CD4CD8 double positive (DP) T-cells showing a memory phenotype has been observed in the peripheral blood in several autoimmune diseases, viral infections and cancer. However, many questions regarding the factors driving DP T-cell differentiation have remained unanswered. Therefore we analyzed the proliferative response to different cytokines, the antigen specificity and the transcriptome signature of DP T-cells in granulomatosis with polyangiitis (GPA) and healthy controls.

Methods: PBMC from 20 GPA-patients and healthy controls were stained for phenotypic markers (CD3, CD4, CD8, CCR4, CCR6, CCR7, CD45RA, CD24, CD28, TdT, CFSE) and intracellular cytokines (interleukin (IL)-4, IL-17, IL-22, interferon (IFN)-γ). Cells were subsequently analyzed by flow cytometry. Antigen-specificity was determined by staining of DP T-cells with HLA-A*0201-restricted peptide / MHC class I dextramers directed against Epstein Barr virus (EBV), cytomegalovirus (CMV), influenza A virus (IFV), coronavirus (COV), respiratory syncytial virus (RSV) and proteinase 3 (PR3) after stimulation with peptides for 14 days. For microarray analysis RNA of sorted CD4 single positive (SP), CD8 SP and DP T-cells was extracted and subsequent transcriptome analysis was performed.

Results: The frequency of DP T-cells was significantly increased in GPA-patients compared to healthy controls. DP T-cells displayed features of memory T-cells and expressed the co-stimulatory molecule CD28, chemokine receptors CCR4 and CCR6 and the natural-killer-cell receptor NKG2D, but lacked thymic markers suggestive of recent thymic emigration. DP T-cells produced predominantly Th1 (IFN-γ) - and Th17 (IL-17, IL-22) cytokines and proliferated in response to common γ-chain cytokines and anti-CD3 stimulation. Virus- and proteinase 3 (PR3) -specific cells were detected within the DP T-cell population in GPA-patients and healthy controls following peptide stimulation for 14 days and corresponding peptide/ MHC class I dextramer staining. Transcriptome analysis revealed a relationship of DP T-cells to CD8 SP T-cells in GPA patients, whereas a CD4 SP T-cell related signature was found in healthy controls.

Conclusion: DP memory T-cells are expanded in peripheral blood of patients with GPA. Viral infections, autoreactivity and common γ-chain cytokines appear to be major driving forces of DP T-cell generation. Unlike previously suggested, DP T-cells are not terminally differentiated, but can proliferate in response to different stimuli.