Anti cd8 apc

PBMCs were isolated and cryopreserved. At the time of analysis, 5 x 10⁵ thawed PBMCs were incubated with the following monoclonal antibodies: anti-CD3 VioBright R720, anti-CD8 APC, anti-CD4 APC-Vio770, anti-CD45RA PE-Vio615, anti-CD57 Vioblue, anti-PD1 ViobrightFITC, anti-CD49d PE770 (Miltenyi Biotech, Germany), anti-CCR7 BV785 (Biolegend, USA) and FVS510 (Fixable Viability Stain 510) (BD Biosciences, USA).
After 10 min on ice in the dark and one wash with PBSA (PBS supplemented with bovine serum albumin), the cells were acquired in an LSRFortessa flow cytometer (BD Biosciences, CA, USA). Dot plots were generated using FlowJo v10.6.2 software (Tree Star, USA).

Antigen-specific T cell activation by macrophages was determined essentially as described [75 (link)]. In brief, MDMs or TAMs were loaded with 1μg/ml CEFT peptide pool (jpt Peptide Technologies, Berlin, Germany) as recall antigens and incubated with a 5-fold excess of lymphocytes for 18 h in the presence of Brefeldin A (Sigma Aldrich, Steinheim, Germany). Lymphocytes were harvested and stained with anti-CD8-APC (Miltenyi Biotec, Bergisch Gladbach, Germany) and after permeabilization with anti-IFNγ-FITC (eBioscience, Frankfurt, Germany). Flow cytometry (FACS Canto, BD Bioscience, Heidelberg, Germany) data were expressed as IFNγ+/CD8+ cells after subtracting background staining of non-stimulated controls.

Brains from 22- to 26-week-old animals were homogenized using a 5-ml Glass Tissue Grinder (A. Hartenstein). The homogenized solution was mixed with 100 % Percoll solution (GE healthcare) to a final 70 % Percoll solution and layered under a 30 % Percoll solution. After 30 min centrifugation at 500g without brake at RT, the cells were isolated from the 30–70 % layer and washed with PBS. Per staining, 100 000 cells were used. The staining was performed in FACS-PBS (PBS, 2 % FCS, 5 mM EDTA) in the presence of the FcBlocker (Miltenyi Biotec) using the following fluorescently labeled antibodies: anti-CD4-PE, anti-CD8-APC, anti-CD11b-FITC, and anti-CD45-APC (all from Miltenyi Biotec), for 15 min at RT in the dark. Samples were analyzed using a Gallios flow cytometer (Beckman Coulter). Thirty thousand events were recorded. Flow cytometry data were analyzed using the FlowJo 8.5.3 software (FlowJo, LLC). For viability testing, the Fixable Viability Dye eFluor^® 780 (eBioscience) was used according to the manufacturer’s instructions. The gating of the living cell population was performed based on the live staining; dead cell populations were excluded. The total cell number was determined utilizing bead measurement (Beckman Coulter CC Size Standard L10), and the absolute cell numbers were calculated according to the manufacturer’s instructions.

PBMCs were stimulated with 2.5 µg/mL Concanavalin A in the absence or presence of MSCs for 5 days. Then, PBMCs were collected and stained with the following antibodies: Anti-CD3-VioGreen, anti-CD19-PEVio770, anti-CD8-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), or corresponding isotype controls following the manufacturer’s instructions. Data were acquired using the MACSQuant^® analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Data were analyzed using FlowJo^TM v10.6.2 Software (BD Life Sciences, Ashland, OR, USA).