Anti cd28

Naïve CD4⁺ CD62L⁺ T cells from BALB/c mice were isolated from fresh spleens using a mouse CD4⁺ CD62L⁺ T Cell Isolation Kit II (Miltenyi) with a MiniMacs Separator in combination with LS and MS columns according to the manufacturer’s instructions. The purity of the cells after separation was routinely > 85% as determined by flow cytometric analysis. For in vitro polarization of Th17 cells, purified naïve T cells were incubated in 24-well plates precoated with anti-CD3 (5 μg/ml), anti-CD28 (1 μg/ml), IL-2 (5 ng/ml), anti-IL-4 antibody (2 μg/ml), anti-IFN-γ antibody (2 μg/ml), transforming growth factor-β (TGF-β, 5 ng/ml), interleukin-6 (IL-6, 20 ng/ml), and IL-23 (10 ng/ml) (all from R&D Systems) with or without EgPSC-ESPs (10 μg/ml) [24 ]. After 72 h of culture, cells and supernatants were collected for further analysis.

The conversion of Tregs in vitro was investigated using CD11c⁺ DCs that were incubated with either 5 ng/mL TGFβ₂ (R&D Systems, MN, USA) or 10 µg/mL IL-10 (R&D Systems, MN, USA) for 24 h. A group without cytokine treatment was used as a control. After washing, the DCs were cocultured at 37°C and 7.5% CO₂ for 48 h with CD4⁺CD25⁻ naïve T cells obtained by negative selection (Miltenyi Biotec, Bergisch Gladbach, Germany) from spleens from C57BL6/J mice. The polyclonal conversion of Tregs was induced by stimulation with 1 µg/mL anti-CD3 (R&D Systems, MN, USA) and 2 µg/mL anti-CD28 (R&D Systems, MN, USA). After 48 h, the percentage of CD4⁺CD25⁺FoxP3⁺Ki67⁺ cells was assessed by flow cytometry.

Dose–response and timing curves were performed for each stimulus. Ficoll-separated PBMCs (4 × 10⁶ cells/well) and MDMs (5 × 10⁵ cells/well) were stimulated for 24 h with LPS (50 µg/mL), peptidoglycan (PGN) (10 µg/mL), lipoteichoic acid (LTA) (1 µg/mL), ssRNA40, a uridine-rich ssRNA analog of HIV-1 ssRNA (6,25 µg/mL) (InvivoGen, San Diego, CA, USA), interferon-γ (IFNγ) (100 U/mL), anti-CD28 (1.25 µg/mL) (R&D System, Minneapolis, MN, USA), and anti-CD3 (2.5 µg/mL) (BD Pharmigen, San Diego, CA, USA). After 24 h, the cells were harvested for flow cytometry analyses; the supernatants of a subset of 5 HIV−, 5 HIV+ untreated, and 11 HIV + cART (5 INRs and 6 FRs) patients were collected and stored for the Luminex assay.

One day before T-cell isolation, culture plates were coated with 5 μg/mL of Anti-CD3 and 1 μg/mL of anti-CD28 antibodies in sterile PBS and incubated overnight at 4°C. Lymph nodes and spleens were isolated from 8-week-old C57BL/6 female mice using a mouse naïve T-cell isolation kit (STEMCELL Technologies EasySep™, catalog number 19782, CA, USA). After isolation, the naïve T-cells were seeded into the wells with 20 ng/mL IL-2 for stimulation and collected after 24 h to assess the activation. The remaining cells were stimulated with 20 ng/mL IL-2 and 5 ng/mL TGF-β and collected after 72 h to test their differentiation. Anti-CD3, anti-CD28, IL-2, and TGF-β were purchased from R&D Systems (MN, USA). The Mouse Treg Isolation Kit (STEMCELL Technologies EasySep, catalog number 19852) was used to isolate mouse Tregs.

For in vitro suppression assays, CD4⁺ CD25^high Neuropilin⁺ Treg cells were sorted from WT and one-allele Stat5a- or Stat5b-deficient mice. Naive, CD4⁺ CD44^low CD25^- responder cells were sorted from congenic CD45.1 mice and labelled with Carboxyfluorescein succinimidyl ester (CFSE; Sigma-Aldrich). CD11c⁺ antigen presenting cells (APCs) were purified from WT mice using positive selection beads (Miltenyi Biotec). 5 x 10⁴ CD4⁺ responder cells were stimulated with soluble anti-mouse CD3ε (1 μg/ml) in round bottom 96-well plates containing 1 x 10⁴ APCs and varying numbers of Treg cells, ranging from 5 x 10⁴ (1:1 ratio) to 1.56 x 10³ (1:32 ratio). After 96 hr, cells were stained with fluorochrome-labelled anti-mouse CD4, CD45.1, and CD25. Percent suppression was calculated relative to WT controls and reflects the percentage of responder cells exhibiting at least one cell division. For 'Treg only' cultures, cells were stimulated with anti-CD3 and anti-CD28 in the presence human IL-2 (100 units/ml) for 72 hr. For iTreg differentiation, naive CD4⁺ CD44^low CD25^- cells were sorted and cultured for 72 hr in the presence anti-CD3, anti-CD28, human TGF-β (10 ng/ml; R&D Systems, Minneapolis, MN), human IL-2 (100 units/ml) and anti-mouse IL-2, IL-4 and IFN-ɣ.