RNA extraction kit was from Qiagen (Qiagen Inc, CA, USA). DAPI(#4083) and antibodies against Nrf2 (#12721), Akt (#9272), HO-1 (#43966), CAT (#14097), GSK-3β (#12456), NQO1 (#62262), P16 (#80772), P21 (#2947), p-Akt (#4060), p-GSK-3β (#5558), Fyn (#4023), Histon H3 (#4499) and β-actin (#4970) were obtained from Cell signing technology corporation (Massachusetts, USA). CD4+ T Cell Isolation Kit II were obtained from Miltenyi (Bergisch Gladbach, Germany). SA-β-Gal staining kit were obtained from Beyotime (Shanghai, China). LY294002 (L9908) and ML385 (SML1833) were obtained from Sigma-Aldrich (St. Louis., MO, USA).
2.2. Animal models
Male C57BL/6 mice (8 weeks) were obtained from Binzhou Medical University (Yantai, China). Mice were housed in a standard environment with a regular light/dark cycle and free access to water and chow diet. The project was approved by the Ethics Committee of Binzhou Medical University, Yantai, China.
The mice were randomly divided into four groups (n = 10 animals per group): (1) control group, treated with saline (20 mL kg-1 day-1) as a vehicle for 7 weeks; (2) D-gal group, treated with D-gal (200 mg kg-1 day-1) for 7 weeks; and (3) mice were treated with D-gal (200 mg kg-1 day-1) for 7 weeks, followed by 1×106 hPMSCs week-1 or (4) 150 μL PBS kg-1 week-1 intravenously on the first day of weeks 5-7 (hPMSC group and PBS group, respectively). hPMSCs (1×106 hPMSCs precipitate in 150 μL PBS per mice) or an equal volume of PBS (150 μL) were administered intravenously over a period of 2 min via the tail vein. D-gal and saline were administered intraperitoneally. Detailed animal experimental designs are presented in Fig. 1A. CD4+ T cells were isolated from single-cell suspensions of splenocytes using a magnetic cell separator as described previously .
2.3. Isolation of hPMSCs
hPMSCs were isolated from human term placentas of donors as described previously . Isolated hPMSCs were identified by detection of cell morphology using microscopy and cell surface antigens CD34, CD105, CD90, CD19, CD73, CD14 and HLA-DR using flow cytometry (FCM). The FCM results indicated that more than 95% of isolated hPMSCs expressed CD73, CD90, and CD105 but not CD14, CD19, CD34, or HLA-DR (Fig. S1D). These results are in accordance with the well-established markers of hPMSCs. The hPMSCs displayed typical fibroblastic morphology (Fig. S1A). The hPMSCs were cultured in adipogenic and osteogenic induction medium to differentiate into adipocytes and osteoblasts, respectively. Osteoblasts were verified by Alizarin Red staining of intracellular calcium deposits (Fig. S1B). Oil Red O staining of fat globules was performed in adipocyte induction medium to verify the presence of adipocytes (Fig. S1C).
2.4. Naive CD4+ T cell isolation and Co-culture with hPMSCs
Human naive CD4+ T (CD4CD45RA) cells were prepared using a naive CD4+ T cell isolation kit II. CD4+ T cells were pretreated for 1 h with the Akt inhibitor LY294002 (30 μM) or Nrf2 inhibitor ML385 (10 μM). In direct co-cultures, hPMSCs were added at a 1:10 ratio to CD4+ T cells (4×106) in direct contact and cultured at 37°C in 5% CO2 for 72 h in the absence or presence of anti-CD3/CD28 Dynabeads (1 μg/ml) and IL-2 (2.5 ng/ml) as a mitogenic stimulus. In transwell cultures, hPMSCs (4×105) were seeded in the upper chamber whereas CD4+ T cells (4×106) were seeded in the lower chamber.
2.5. Intracellular ROS detection
The intracellular production of ROS was assessed by 2',7'-dichlorofluorescein diacetate (H2DCF-DA) (Sigma, St Louis, MO, USA) . CD4+ T cells that were not labeled with H2DCF-DA probe used as the negative control group. Briefly, collected CD4+ T cells were washed with PBS. Then, added H2DCF-DA (10 μM) and incubated at 37℃ for 30 min. The levels of intracellular ROS in CD4+ T cells were analyzed by FCM after washed with PBS.
2.6. Antioxidant Biomarkers detection
The activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in CD4+ T cells were measured by colorimetric analysis. GSH-Px activity was detected using the DTNB method, CAT activity was measured using the ammonium molybdate method  and SOD activity was measured using the xanthine-oxidase method .
2.7. SA-β-Gal staining
SA-β-Gal activity in senescent T cells were tested as described previously . Briefly, T cells were fixed with 3% formaldehyde ather washed in PBS, and followed to incubate overnight at 37°C with SA-β-Gal staining solution. After washing with PBS, senescent cells were observed under microscopy (Leica, Germany).
2.8. Western Blot Analysis
Western blot analyses were performed as previously described . In short, total proteins were electro-transferred to PVDF membranes after separated by SDS-PAGE. Then, membranes were incubated with indicated primary antibody overnight at 4°C after blocked in 5% BSA dissolved in TBST for 2h at room temperature, followed by incubation with appropriate secondary antibody 2 h at room temperature. ECL plus detection reagents (Beyotime, Shanghai, China) was used for visualized protein bands. The Image J gel analysis software was used for densitometric analysis.
2.9. RNA extraction and quantitative real-time PCR
The levels of mRNA was measured using quantitative real-time PCR assay. Relative abundance of genes was calculated using 2-∆∆CT formula, and β-actin as internal control. Primers attached in the additional file 1.
2.10. Immunofluorescence assay
CD4+ T cells were fixed in 4% paraformaldehyde for 10 min after washed with cold PBS. Then, cells were permeabilized for 15 min using 1% Triton X-100. After washed with PBS, cells were incubation with primary antibody overnight. After washed with PBS, cells were incubation in fluorescence-tagged secondary antibody for 1 h. Nuclei were counter-stained with DAPI. Laser scanning confocal microscope (FV3000, Olympus Corporation, Japan) was used for fluorescence images capture.
2.11. Statistical analysis
Data represent as mean ± SEM. Statistical significance is determined by unpaired two-tailed Student’s t-test (or nonparametric test), one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Significance was defined as P < 0.05.