Kaluza software version 1.5a

Peripheral blood was collected in heparin coated tubes. Whole blood was either stimulated with CMV cell lysate (10 µg/mL) or control lysate (10 µg/mL, HEL-299) (both Lophius Biosciences) for six hours in the presence of anti-human CD28/CD49d (1 µg/mL, clones L293 and L25, BD Biosciences, Heidelberg, Germany). If cytokine production of antigen specific T-cells was determined, Brefeldin A (Sigma Aldrich, Munich, Germany) was added after the first two hours of incubation. Following incubation, whole blood was lysed (FACS lysing solution, BD Biosciences) and permeabilized (FACS Permeabilizing Solution 2, BD Biosciences). Cells were then stained for lineage markers (anti-human CD3, Pacific Blue, clone UCHT1, and anti-human CD4 APC, clone 13B8.2, both Beckman Coulter Krefeld, Germany; anti-CD8 APC-H7, clone SK1, BD Biosciences) and IFN-γ (anti-IFN-γ, FITC, clone 45.15, Beckman Coulter). If CD154 expression was determined on antigen-specific T-cells, whole blood was washed after six hours of incubation and then stained for the above-mentioned lineage-markers, as well as for CD154 (anti-human CD154 FITC, clone 24–31, Biolegend, San Diego, CA, USA). Whole blood was lysed (VersaLyse, Beckman Coulter) and washed twice. Samples were then measured with a flow cytometer (Navios, Beckman Coulter). Data was analyzed using Kaluza Software Version 1.5a (Beckman Coulter).

The surface marker expression of the transduced ASCs was characterized by flow cytometry. The following antibodies (all purchased from BD Biosciences) were used: CD105-Brilliant Violet-421 (BV421) (clone 266), CD90-allophycocyanin (APC) (5E10), CD73-APC (AD2), CD13-phycoerythrin-cyanin 7 (PE-Cy7) (WM15), CD45-fluorescein isothiocyanate (FITC) (2D1), CD34-APC (581), and HLA-DR, DP, DQ-BV421 (Tu39). Fixable, viable stain 780 (FVS780) (cat. number 565388) was used to discriminate live from dead cells. Upon harvest, the cells were washed in PBS twice and stained with FVS780 for 10 min. Subsequently, the cells were washed in FACS PBS (Hospital Pharmacy) containing 1% ethylenediaminetetraacetic acid (EDTA) (Hospital Pharmacy) and 10% newborn calf serum (Gibco, Thermo Fisher Scientific) and stained for 30 min at room temperature, protected from light. Finally, the cells were washed in FACS PBS and resuspended in PBS. At least 4000 cells were measured on a Navios flow cytometer and analyzed using Kaluza software version 1.5a (both Beckman Coulter). Debris, doublets, and dead cells were excluded from the analysis.

Spleen and mesenteric lymph nodes (LN) were harvested and cells were obtained by mashing over a 40 µm filter. Single cell suspensions of spleen and LN were phenotypically analyzed using a Navios multi-color flow cytometer (Beckman-Coulter, Mijdrecht, the Netherlands). Human leukocytes were identified using an antibody directed against the common human surface leukocyte marker CD45 (anti-human-CD45-KO (J33, Beckman-Coulter). For cell-surface staining of human CD4 and CD8 T cells, the following antibodies were used: anti-CD4-PC5.5 (13B8.2, Beckman Coulter), and anti-CD8-APC-AF700 (B9.11, Beckman-Coulter). For intracellular staining with anti-FOXP3-e450 (PCH101, eBioscience), cells were fixed and permeabilized by fix-perm treatment (eBioscience) according to manufacturers instruction. Data were analyzed using Kaluza software version 1.5a (Beckman-Coulter) and gates were set based on single staining and FMOs (Supplemental Fig. 1)