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Clonal hematopoiesis (CH) is common among older people and is associated with an increased risk of atherosclerosis, inflammation, and shorter overall survival. Age and inflammation are major risk factors for ischemic stroke, yet the association of CH with risk of secondary vascular events and death is unknown. We investigated CH in peripheral blood DNA from 581 patients with first-ever ischemic stroke from the Prospective Cohort With Incident Stroke–Berlin study using error-corrected targeted sequencing. The primary composite end point (CEP) consisted of recurrent stroke, myocardial infarction, and all-cause mortality. A total of 348 somatic mutations with a variant allele frequency ≥1% were identified in 236 of 581 patients (41%). CH was associated with large-artery atherosclerosis stroke (P = .01) and white matter lesion (P < .001). CH-positive patients showed increased levels of proinflammatory cytokines, such as interleukin-6 (IL-6), interferon gamma, high-sensitivity C-reactive protein, and vascular cell adhesion molecule 1. CH-positive patients had a higher risk for the primary CEP (hazard ratio [HR], 1.55; 95% confidence interval [CI], 1.04-2.31; P = .03), which was more pronounced in patients with larger clones. CH clone size remained an independent risk factor (HR, 1.30; 95% CI, 1.04-1.62; P = .022) in multivariable Cox regression. Although our data show that, in particular, larger and TET2- or PPM1D-mutated clones are associated with increased risk of recurrent vascular events and death, this risk is partially mitigated by a common germline variant of the IL-6 receptor (IL-6R p.D358A). The CH mutation profile is accompanied by a proinflammatory profile, opening new avenues for preventive precision medicine approaches to resolve the self-perpetuating cycle of inflammation and clonal expansion.

Background Age-related dysfunction in immune cells (immunosenescence), such as T cell dysfunction, may contribute to the development of rheumatoid arthritis (RA). In aged people, senescent T cells tend to produce low amounts of pro-inflammatory cytokines leading to low-grade inflammation. However, cellular metabolism modulates effector functions such as cytokine production and proliferation in T cells by providing energy and building blocks. Metabolically, naive and memory CD4+ T cells are relatively quiescent immune cells. Currently, the metabolic phenotype of naive and memory CD4+ T cells and how metabolism affects functions of naive and memory CD4+ T cells in aged people are not well understood. Objectives Therefore, we analysed the differences in the metabolic phenotype of peripheral naive and memory CD4+ T cells in young and aged healthy donors to explore fundamental processes of immune-aging in the pathogenesis of RA. Methods Naive and memory CD4+ T cells were isolated from PBMCs of young donors (18-35 years) and old donors (>50 years) by using MACSTM technology. Purity of isolated cell fractions was assessed by flow cytometry. Ex vivo naive and memory CD4+ T cells were analysed by SeahorseTM Technology to determine proton efflux rate (PER) as a measure of glycolysis (glycPER) and oxygen consumption rate (OCR) as a measure of mitochondrial respiration (mitoOCR). Cytokine expression and secretion was measured by flow cytometry and multiplex assay. Finally, TCR-stimulated memory CD4+ T cell proliferation was determined using CSFE and Ki-67 after 3 days and 4 days by flow cytometry. Results We included 9 young (20-32 years, 25.0±3.4 years) and 9 aged gender-matched donors (52-67 years, 57.8±5.7 years) for PER and OCR measurement. Memory CD4+ T cells demonstrated higher basal glycolysis, compensatory glycolysis as well as basal OCR and spare respiratory capacity than naive CD4+ T cells. Memory CD4+ T cells from young donors had higher basal glycolysis, and compensatory glycolysis than aged donors, but lower ratio of basal mitoOCR/glycPER. Although we did not observe differences in intercellular cytokine expression measured by flow cytometry after 5h stimulation of memory CD4+ T cells, we determined a significant higher amount of secreted IL-6, IL-9, IP-10, monocyte chemotactic and activating factor in the supernatant of memory CD4+ T cells from aged donors as compared to those from young donors. Cell division index, proliferation index, percentage of divided cell, and Ki-67 expression after 3 and 4 days of stimulation showed no statistical differences between both groups. Conclusion Here, we demonstrate a higher basal glycolysis, basal OCR, mitochondrial and glycolytic capacity of human ex vivo memory CD4+ T cells as compared to naive T cells. A decrease of basal glycolysis, compensatory glycolysis in memory CD4+ T cells of aged people which results in an enhanced cytokine expression can be assumed to culminate in T cell dysfunction leading to the development of RA during aging. Acknowledgement The work of Yuling Chen was funded by the Chinese Scholarship Council (201606230248). The work of Timo Gaber was funded by the Deutsche Forschungsgemeinschaft (353142848) Disclosure of Interests Yuling Chen: None declared, Pelle Lowe: None declared, Hao Wu: None declared, Frank Buttgereit: None declared, Timo Gaber Grant/research support from: Pfizer