Furthermore, the myocardial gene expression signature found in T-cell-transferred NSG-DR1 mice overlapped with an conserved age-related gene set described in several tissues of senescent mice, rats and humans ( Figure 5F ) (36). donor subjects, the CD4+ T cell compartment was primarily composed of na?ve cells defined as CCR7+CD45RO-. However, when transplanted into young lymphocyte-deficient mice, CD4+ T cells underwent homeostatic expansion, upregulated expression of PD-1 receptor and strongly shifted towards effector/memory (CCR7- CD45RO+) and terminally-differentiated phenotypes (CCR7-CD45RO-), as typically seen in elderly. Differentiated CD4+ T cells also infiltrated the myocardium of recipient mice at comparable levels to what is observed during physiological aging. In addition, young mice harboring an expanded CD4+ T cell compartment showed increased numbers of infiltrating monocytes, macrophages and dendritic cells in the heart. Bulk mRNA sequencing analyses further confirmed that expanding T-cells promote myocardial inflammaging, marked by a distinct age-related transcriptomic signature. Altogether, these data indicate that exaggerated CD4+ T-cell expansion and differentiation, a hallmark of the aging immune system, is sufficient to promote myocardial alterations MA-0204 compatible with inflammaging in juvenile healthy mice. procedures were approved by the local authorities (analysis. FACS analysis of freshly isolated cells (heart and spleen) was performed. The samples intended for RNA analysis were stored in RNAlater (Qiagen, Hilden, Germany) for 24 h and then stored at -80C. Samples intended for histological analysis were embedded in Tissue-TeK optimum cutting temperature medium (Sakura Finetek, Alphen ann den Rijn, The Netherlands) and then stored at -80C. Heart DNA extraction was performed from heart slices by trimming the Tissue-TeK content and performing tissue digestion and DNA purification using a GeneJET genomic DNA purification kit (Thermo Scientific, Vilnius, Lithuania) according to the manufacturers instructions. Stimulation Assay 1 million PBMC (pretransfer), NSG-DR1 recipients splenocytes or digested heart tissues were resuspended in complete RPMI media containing 10% FCS, 1% L-Glutamine, 1% Sodium pyruvate, 1% non-essential amino acids, 1%Pen/Strep, 1 M 2-ME (Gibco C Grovemont Cir, USA) and seeded in U-bottom 96-well plate. Cells were stimulated for 3 h with a T-cell stimulation cocktail containing Phorbol 12-Myristat 13-Acetat (81 M) und Ionomycin (1.34 M) supplemented with protein transport inhibitors Brefeldin A (10.6 M) and Monensin (2M) (eBioscience C San Diego, USA). Following incubation time, cells were washed and used in intracellular flow cytometry analysis. Flow Cytometry Immunophenotyping of spleen and digested heart samples obtained from control and CD4+ T cell transfer mice and human PBMCs was performed. The heart samples were enzymatically digested in type MA-0204 II collagenase (1,000 IU/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA) for 30 min at 37C and then ground against a 70-m mesh (Miltenyi Biotec, Bergisch Gladbach, Germany) in 0.5% BSS/BSA. The lymphoid organs were ground against a 30-m cell strainer, and the splenocyte preparations underwent erythrocyte lysis. All samples were stained with zombie aqua fixable viability dye (BioLegend, San Diego, USA) for 15 min at room temperature and protected from light. Afterwards, samples were washed and resuspended in FACS buffer, and surface staining was performed in the presence of FC-blocking antibody (anti-CD16/CD32, clone 2.4G2, BD Pharmingen) for host NSG-DR1 cell Rabbit Polyclonal to EPHA3 panels. The following antibodies conjugated with different fluorophores were used: Human: anti-CD45RO (clone UCHL1), anti-CD4 (OKT4), anti-CCR7 (clone G043H7), anti-CD25 (clone M-A251)anti-CD127 (clone A010D5) obtained from BioLegend (San Diego, USA), and anti- PD-1 (clone J105), anti-FoxP3 (clone 236A/E7), anti-TNF (clone Mab11), anti-IFN- (clone 45.B3), anti-IL-17a (clone eBio64DEC17) and anti-IL-13 (clone 85BRD) obtained from eBioscience (San Diego, USA). Mouse: anti-CD45 (clone 30-F11), anti-CD11b (clone M1/70), anti-Ly6G (clone 1A8), anti-CD11c (clone N418), anti-CD62L (MEL-14), anti-CD3e (145-2C11), anti-CD44 (IM7), and anti-CD4 (RM4-5) conjugated with different fluorophores were obtained from BioLegend (San Diego, USA). Flow cytometry measurements were performed on an Attune-NxT (Thermo Scientific, Darmstadt, Germany). Data analysis was performed using FlowJo software (FlowJo LLC Ashland, OR, USA). Compensation MA-0204 for spectral overlap was conducted based on single staining controls, and flow cytometry gates were set based on unstained controls and fluorescence minus one controls. Gene Expression Analysis RNA was extracted from mouse myocardial samples (apical region) using the tissue.