Anti cd28 antibody

Single-cell suspensions of splenocytes were harvested as described above. According to the manufacturer’s instructions, CD4⁺ T cells were purified with an EasySep Mouse CD4⁺ T cell enrichment kit (STEMCELL Technologies, Canada), and the purity was confirmed to be greater than 90% confirmed by FACS (Zhang et al., 2014 (link)). The purified naïve CD4⁺ T cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (HyClone) at a density of 1 × 10⁵ in a 5% CO₂ humidified incubator at 37°C. In addition, CD4⁺ T cells were induced by incubation with an anti-CD3 antibody (2.5 μg/ml, Invitrogen), an anti-CD28 antibody (5 μg/ml, Invitrogen), IL-2 (20 U/ml, Miltenyi Biotec) and TGF-β (2 ng/ml, Miltenyi Biotec) in the presence or absence of 10 nM RvD1 for 5 days (Chiurchiu et al., 2016 (link)). Cultures were supplemented with RvD1 every other day. After 5 days, cells were collected for FACS and real-time PCR analyses. In some cases, the purified naïve CD4⁺ T cells were preincubated with anti-GPR32 neutralizing antibodies (2 μg/ml, GeneTex) and/or anti-ALX/FPR2 neutralizing antibodies (2 μg/ml, Genovac) for 30 min before the incubation with RvD1 or vehicle and then stimulated with anti-CD3/CD28, IL-2, and TGF-β.

Naïve T cells were isolated using an EasySep™ Human Naïve CD4+ T Cell Isolation Kit (Miltenyi Biotech, Auburn, CA, USA). T cell purity was assessed through a flow cytometry analysis of CD4 markers and were typically >95% pure. For direct T cell stimulation and proliferation, a 96-well u-bottom plate was coated with 10 µg/mL anti-CD3 antibody (Invitrogen, Thermofisher Scientific West Columbia, SC, USA; Cat#: 16-0037-85) and incubated at 37 °C with 5% CO2 for 2 h. The plate was then rinsed twice with PBS, and cells in the presence or absence of 10⁸/mL MoDC exo subtypes were added to the plate at 200,000 cells per well in complete RPMI 1640 medium (10% fetal bovine serum (FBS), 1% Pen Strep, 1x nonessential amino acids, 0.1% b-mercaptoetanol) containing 10 µg/mL anti-CD28 antibody (Invitrogen, Thermofisher Scientific West Columbia, SC, USA; Cat#:16-0289-85). Cells were incubated at 37 °C with 5% CO2 for 72 h, and T cell polarization/differentiation were assessed using flow cytometry. Regulatory T cells (Tregs) were identified by gating on double-positive cells for FOXP3 and CD25 in CD4⁺ cell gate populations, while T helper 17 induction was identified by measuring IL17A⁺ cells in CD4⁺ cell populations. Inhibitory molecules’ CTLA4 and PD1 expressions were measured by gating on CTLA4⁺ cells and PD1⁺ cells in CD4⁺ cell populations, respectively.

An allogeneic mixed lymphocyte reaction (MLR) assay was conducted using DCs from an 8-week-old female C57BL/6 mouse and T cells from an 8-week-old female BALB/c mouse. First, DCs were incubated with 100 ng/mL LPS for 2 h and then treated with AYC-EVs (20 μg/mL) for 24 h. These cells were then used in the MLR assay. Spleen cells were isolated from the BALB/c mouse and labeled with CD4 or CD8 microbeads (Miltenyi Biotec) at 4 °C for 15 min. After incubation, CD4- or CD8-labeled cells were isolated using MACS LS separation columns, according to the manufacturer’s protocol. Isolated T cells were stained using the CellTrace CFSE Cell proliferation kit reagent (2 μM, Invitrogen) for 10 min in a 37 °C water bath. Next, 96-well plates were coated with anti-CD3e (1 μg/mL; Invitrogen) antibody for 2 h at 37 °C, and the cells (DCs; 2 × 10⁵ cells per well and CFSE-labeled T cells; 1 × 10⁶ cells per well) were co-cultured with anti-CD28 antibody (1 μg/mL; Invitrogen) in anti-CD3e-coated plates. After 2 days of co-culture, cell culture supernatants were harvested, and IFN-γ, TNF-α, IL-2, IL-5, and IL-17A levels were analyzed using a cytokine-specific ELISA kit. Kits were purchased from Thermo Fisher Scientific. Finally, the harvested T cells were stained with anti-CD4 or anti-CD8 antibodies, and T cell proliferation levels were analyzed using a Life Launch Attune Nxt Flow Cytometer.

CD4⁺ T cells were isolated from human whole blood drawn. For their isolation STEM CELL CD4⁺ T cell negative selection kit (#17952) was used according to manufacturers provided protocol. Cells were treated with 10M final concertation of cell permeable CFB inhibitor or equivalent volume of vehicle control (DMSO) and activated in 96 well flat bottom plates (Greiner, #655083) coated with 1g/ml anti-CD3 antibody (clone OKT, Biolegend, #317347) and 2g/ml anti-CD28 antibody (Thermo Fisher, #16-0289-85). Following 48 hours in culture, cells were transferred into 96-well V-bottom plate and analyzed for FSC and SSC properties on BD FACS Canto. Data shown is percent of viable cells in three healthy donors.