Cd3 percp

Spleen cells were collected; the red blood cells were lysed with buffer containing NH₄Cl, and adjusted to 10⁶ cells/tube. CNS-extracted cells were plated at 5 × 10⁵ cells/well and stimulated with MOG (125 μg/mL) and with C. albicans (5 yeasts/1 cell). After incubation at 37°C for 48 h, cells were collected and stained. Spleen and CNS-extracted cells were blocked with rat serum 1% for 20 min to prevent nonspecific binding via Fc receptor. After Fc blocking, cells were stained with 0.2 μg of PerCP-conjugated anti-mouse CD3 and 0.25 μg of FITC-conjugated anti-mouse CD4 for 20 min at 4°C. Intracellular FoxP3 transcription factor analysis was performed only in spleen samples by using CD3-PercP, CD4-FITC plus 0.13 μg of APC-conjugated anti-mouse CD25 and 0.2 μg of PE-conjugated anti-mouse FoxP3 and staining set (eBiosciences, San Diego, CA, USA) according to manufacturer's instructions. After staining, the cells were washed, resuspended in FACS buffer, and fixed in paraformaldehyde 1%. Analysis was performed using a FACSCanto II (BD) from Bioscience Institute (Botucatu, SP, Brazil) and the data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

Cell surface staining with antibodies conjugated with fluorochromes was performed as previously described.^{26 (link)} The following anti-human antibodies conjugated with fluorochromes were purchased from eBiosciences (San Diego, CA): CD14-FITC, CD4-FITC, CD11b-PE, CD25-PE, CD3-PerCP, CD33-PercpCY5.5, HLA-DR-APC, FoxP3-APC, and isotype-matched control antibodies conjugated with fluorochrome. Intracellular staining (ICS) with anti-human FoxP3-PE was performed using the FoxP3 staining buffer set (eBiosciences, San Diego, CA) according to the manufacturer's instructions. As a heterogeneous cell population, human MDSCs could be further divided into 2 subsets, monocytic (M-MDSC, CD14⁺) and granulocytic (G-MDSC, CD14⁻/CD15⁺).^{12 (link),18 (link),20 (link)–23 (link)} Given that G-MDSCs are unavailable in Ficoll-prepared PBMCs, we set the gating strategy for M-MDSCs: CD33⁺CD11b⁺/CD14⁺HLA-DR^Low. Meanwhile, the gating strategy for Tregs was CD3⁺CD4⁺CD25⁺FoxP3⁺. Cells were collected on a FACSCalibur (BD). The data were analyzed using FlowJo software (TreeStar, San Carlos, CA). Appropriate isotype controls were used at the same protein concentration as the test antibodies, and control staining was performed during every FACS.

Splenocytes were treated with anti-CD16/CD32 and labelled with PE-conjugated DCT_180-188 peptide-loaded MHC-I tetramer (Baylor College), PerCP-CD3, and FITC-CD8α (eBioscience) for 30 minutes in FACS buffer (1% PBS, 5% fetal calf serum, 2.5% NaZ). For ELISpot analyses, the exact number of DCT-MHC-I tetramer⁺/CD8⁺ T cells were calculated from flow cytometry proportion results and added to an IFNγ ELISpot following manufacturer’s instructions (Mabtech).

Aliquots of 1 × 10⁶ NK cells were analyzed for surface markers using a panel of prediluted fluorochrome-conjugated anti-human monoclonal antibodies. FITC-CD3, PE-Cy™7-IFNγ, FITC-CD107a, PE-NKp44, and PerCP-Cy™5.5-NKG2D were purchased from BD Biosciences. PerCP-CD3, PE-CD253/TRAIL, Alexa Fluor-700-TNFα, APC-CD56, APC-NKp30, PE-Cy7-NKp46, and Alexa Fluor-700-TIM-3 were purchased from eBioscience (San Diego, CA, USA). PE-Galectin-9 was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Samples were run on a FACSAria II dual-laser 8-color cytometer (BD Biosciences, Europe), and data were analyzed with FACSDiva software 6.1.3.