Anti cd3 cd28 beads

CD62L^lowCD45RO⁺CD45RA⁻CD25⁻CD8⁻CD4⁺ T cell subsets: CCR6⁺CXCR3⁻, CCR6⁺CXCR3⁺, and CCR6⁻CXCR3⁺ were FACS sorted for functional studies according to the gating strategy depicted in Figure 2A. Transcriptomic studies examined sorted CCR6⁺CXCR3⁻CD62L^lowCD45RO⁺CD45RA⁻CD25⁻CD8⁻CD4⁺ T cells treated in the absence or presence of IL12. Cell isolation was performed using FACS Aria II cell sorter and data were analyzed using FACS Diva 6 (BD Biosciences).
The three purified CD4⁺ T cell subsets were stimulated with anti-CD3/CD28 beads (Miltenyi Biotec) and cultured with or without IL12 (20 ng/ml, R&D system) or IL23 (10 ng/ml, R&D system) for 6 days. Cultures were performed in RPMI 1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin; 20,000–50,000 cells per well. For intracytoplasmic staining, PMA-ionomycin was added for 6 h in cell cultures and Brefeldin A for the last 3 h, cells were then fixed and stained with CD3 monoclonal antibody followed by intracytoplasmic staining for IL17 and IFNγ.

Cells were isolated from peripheral blood donated by anonymous healthy volunteers via the National Blood Service with informed consent according to our GMP protocols.13 (link), 14 (link), 15 (link) PBMCs were separated by Lymphoprep density gradient centrifugation (PAA Laboratories). CD4⁺ T cells were enriched using RosetteSep (STEMCELL Technologies), followed by selection and separation of CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ Teffs using CD25 microbeads (Miltenyi Biotec). PBMCs were also depleted of CD25⁺ cells by using CD25 microbeads (Miltenyi) and are referred to hereafter as CD25⁻ PBMCs. Aliquots of CD25⁻ PBMCs, Tregs, and Teffs were cryopreserved in 5% (v/v) human serum (BioSera) containing 10% (v/v) DMSO. Tregs were activated with anti-CD3/CD28 beads (1:1 bead:cell ratio, Miltenyi) and cultured in X-Vivo15 medium (Lonza) supplemented with 5% (v/v) human AB serum (BioSera; Treg medium) and 100 nM rapamycin (LC-Laboratories). 1,000 U/mL recombinant human IL-2 (Proleukin-Novartis) was added 72 h after cell isolation. After 10 days, Tregs were activated with anti-CD3/CD28 beads (1:1 bead:cell ratio) for further expansion, which was repeated every 10 days for expansion and longevity. X-Vivo15 medium supplemented with human IL-2 and rapamycin (see above) was used to feed the cells every twodays. Tregs were cultured at 37°C in the presence of 5% (v/v) CO₂ in a humidified incubator.

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples of SLE patients and healthy controls by Ficoll density gradient centrifugation (STEMCELL Technologies, Vancouver, CA). Human CD4⁺ or naïve CD4⁺ T cells were purified from fresh PBMCs by using the EasySep Human CD4⁺ or Naïve CD4⁺ T Cell Isolation kits (STEMCELL Technologies), stimulated with anti-CD3/CD28 beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, CD3/CD28 beads-stimulated naïve CD4⁺ T cells were treated with tofacitinib, STAT3 inhibitor (NSC74859), AKT inhibitor (GSK690693), or p38 MAPK inhibitor (SB203580) for 24 h. NSC74859, GSK690693, and SB203580 were purchased from ApexBio (Houston, TX, USA). Tofacitinib was added at the determined concentrations (0.1 µM, 1 µM, and 10 µM) for 24 h. In some experiments, CD4⁺ T cells were stimulated with anti-CD3/CD28 beads in the presence of recombinant cytokines, such as TGF-β (5 ng/ml; PeproTech, Rocky Hill, NJ) and IL-6 (100 ng/ml; PeproTech).
Spleens were isolated, and the erythrocytes in splenocyte suspensions were lysed with red cell lysate. Mouse CD4⁺ T cells were purified from splenocytes by using the EasySep Mouse CD4⁺ T Cell Isolation kit (STEMCELL Technologies) and processed for real-time PCR or Western blotting analysis.

All T cell activation assays to determine changes in metabolic flux were performed as previously described [5 (link)]. Briefly, 2×10⁵ purified T cells were seeded per well in a poly-D-lysine (100 μg/ml) coated Seahorse 96 well plate and centrifuged for 5 minutes at 300xg. Cells were incubated for 1 hour in a non-CO₂, 37°C incubator prior to analysis. After 4 baseline measurements, T cells were activated by injection of anti-CD3/CD28 beads (Miltenyi Biotec) at a ratio of 8:1 beads:T cell followed by injection of oligomycin (2μM) and then 2-deoxyglucose (2-DG; 50 mM). The extracellular acidification rate (ECAR) was measured using a Seahorse XFe96 Bioanalyzer (Agilent) with readings collected approximately every 6.5 minutes.

To assess the efficiency of Breg-mediated conversion of Tregs, freshly purified CD4⁺CD25⁻ T cells were cultured in a 24-well plate alone or with Bregs at a ratio of 1:1 at 37 °C with 5% CO₂ in X-VIVO™15 medium (Lonza) supplemented with 5% FBS and recombinant Interleukin-2 (100 units/ml; R&D Systems) and were stimulated with anti-CD3/CD28 beads (Miltenyi Biotec). After 5 days, the cells were analyzed for the expression of CD4, CD25 and Foxp3 on a Canto II flow cytometer (BD Biosciences).