METHODS: Circulating CD8+ T cells were analysed for differentiation status (CD45RO, CCR7), markers of activation (CD69 and CD25) and proliferation (Ki-67) in 14 newly diagnosed GCA patients and 18 healthy controls by flow cytometry. Proliferative capacity of CD8+ T cells upon anti-CD3 and anti-CD3/28 in vitro stimulation was assessed. Single-cell RNA sequencing of peripheral blood mononuclear cells of patients and controls (n = 3 each) was performed for mechanistic insight. Immunohistochemistry was used to detect CD3, CD8, Ki-67, TNF-α and IFN-γ in GCA-affected tissues.
RESULTS: GCA patients had decreased numbers of circulating effector memory CD8+ T cells but the percentage of Ki-67-expressing effector memory CD8+ T cells was increased. Circulating CD8+ T cells from GCA patients demonstrated reduced T cell receptor activation thresholds and displayed a gene expression profile that is concurrent with increased proliferation. CD8+ T cells were detected in GCA temporal arteries and aorta. These vascular CD8+ T cells expressed IFN-γ but not Ki-67.
CONCLUSION: In GCA, circulating effector memory CD8+ T cells demonstrate a proliferation-prone phenotype. The presence of CD8+ T cells in inflamed arteries seems to reflect recruitment of circulating cells rather than local expansion. CD8+ T cells in inflamed tissues produce IFN-γ, which is an important mediator of local inflammatory responses in GCA.
METHODS: This study belongs to a part of an ongoing Singapore/Malaysia cross-sectional genetics and epidemiological study (SMCSGES). We assessed the associations of FOXO1 transcript expression levels in peripheral blood mononuclear cells (PBMC) with AR phenotype, total nasal symptom score (TNSS), and SNP genotype in a sub-cohort of n = 658 individuals from the SMCSGES population. Associations of FOXO1 SNPs with AR were assessed in a cohort of n = 5,072 individuals from the SMCSGES population. In vitro promoter luciferase assay was used to evaluate the effect of AR-associated SNPs on FOXO1 promoter activity.
RESULTS: FOXO1 transcript expression in PBMC was significantly associated with the risk of AR (p < 0.05) and TNSS among AR patients (p < 0.0001). We identified a significant association between tag-SNPs rs9549246 and FOXO1 transcript expression in PBMC from the SMCSGES sub-cohort and the multiethnic eQTLGen consortium (false discovery rate-adjusted p < 0.05). The minor allele "A" of tag-SNP rs9549246 was significantly associated with a higher risk of AR (p = 0.04422, odds ratio = 1.21, 95% confidence interval = 1.01-1.45) in the SMCSGES genotyping cohort (n = 5,072). In vitro luciferase assay showed the minor allele "A" of rs35594717 (tagged by rs9549246) was significantly associated with a higher FOXO1 promoter activity (p < 0.05).
CONCLUSION: FOXO1 transcript expression in PBMC has a strong association with the risk and symptom severity of AR. Genetic variants tagged by rs9549246 were shown to affect the expression of FOXO1 and contribute to the development of AR in the SMCSGES population.
MATERIALS AND METHODS: The PBMC (n = 7) were cultured either in RPMI-1640 containing L-glutamine and 50 units/ml Penicillin-Streptomycin (BM) or in BM with either AuHS or FBS. Viability, proliferation and differentiation of PBMC were evaluated. Paracrine factors present in the secretomes (n = 6) were analysed using ProcartaPlex Human Cytokine panel (17 plex). Ingenuity Pathway Analysis (IPA) was performed to predict activation or inhibition of biological functions related to tissue regeneration.
RESULTS: The viability of PBMC that were cultured with FBS supplement was significantly reduced at 96 h compared to those at 0 and 24 h (P