plates (Corning Costar) if needed. Cells were cultured at 37˚C, 95% humidity and 5%
CO2. The total number of viable cells was determined by trypan blue exclusion counting
on day 7, 14 and 21. At each time-point a sample was taken for flow cytometry analysis, and absolute numbers of DCs were calculated by multiplying the frequency of DCs with
the absolute number of total cells generated from 105 CD34+ cells.
Isolation of peripheral blood (PB)- and HSPC-DCs
PB-DCs were isolated from buffy coats (Sanquin blood bank, Nijmegen, The Netherlands) of healthy individuals after written informed consent. First, Peripheral blood mononuclear cells PBMCs were purified using Ficoll-Hypaque density centrifugation. Subsequently, non-DCs were depleted from PBMCs by negative selection with anti-CD3/CD14/CD19 magnetic beads (Becton Dickinson, Franklin Lakes, NJ, USA) or with Pan-DC enrichment kit (Miltenyi Biotech). Next the negative fraction was labeled with BDCA1, BDCA3, BDCA4 (all from Miltenyi Biotech), CD14, CD20 (both from Biolegend, CA, USA) and CD19 (Dako, Glostrup, Denmark) antibodies.
Finally, pDCs, BDCA1+mDCs and BDCA3+ mDCs were sorted on an EPICS Elite cell
sorter (Beckman Coulter, Fullerton, CA, USA) or FACS Aria (Becton Dickinson) as
BDCA4+BDCA1-CD14-CD19-, BDCA1+BDCA3-CD14-CD19-CD20- and BDCA3+BDCA1-
CD14- CD19-CD20- cells, respectively.
HSPC-DCs were sorted from the whole bulk of cultured cells at day 21 of culture. First, the cultured cells were incubated with 10-20% human serum (HS; PAA Laboratories, Austria) at 4˚C to block Fc receptors. Next, the cultured cells were stained with CD123 (Biolegend), CD14 (Beckman Coulter), BDCA2 (Miltenyi Biotech), BDCA1 and BDCA3
antibodies and sorted with the FACS Aria for pDCs (CD14-CD123hiBDCA2+ cells),
BDCA1+mDCs (CD14-CD123lowBDCA1+BDCA3- cells) and BDCA3+ mDCs (CD14-CD123low
BDCA3+BDCA1- cells). Purity of isolated PB- and HSPC-DCs was >90%.
Flow cytometry
Immunophenotypical analysis was performed by flow cytometry, where cells were first washed with PBS/0.5% BSA and subsequently stained with appropriate antibody concentrations for 30 min at 4˚C. To determine the phenotype and maturation state of DCs, following antibodies and isotype controls were used: BDCA1, BDCA2, BDCA3 (all from Miltenyi Biotech), CD83, CD123, lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56) (all from Becton Dickinson), CD11c, CD14, CD80, CD86, HLA-DR, mouse IgG1, mouse IgG2b (all from Beckman Coulter), DNGR1, CCR7, mouse IgG2a (all from Biolegend,) and CD86 (Dako) antibodies. To evaluate T cell proliferation in mixed lymphocyte reaction (MLR), cells were stained with CD3, CD8 (both from Beckman coulter) and CD4 (Biolegend) antibodies. For all analysis, live cells were gated based on forward scatter and side scatter characteristics. Additionally, in some experiments,
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SYTOX blue stain (Invitrogen, CA, USA) was used to further exclude dead cells from analysis. Furthermore, cell doublets were excluded based on signal pulse height and width. Acquisition was performed with the Coulter FC500 flow cytometer or CyAn ADP analyzer and data analysis was performed with Kaluza or CXP analysis software (all from Beckman Coulter).
DC stimulation with TLR ligands
Sorted HSPC-derived pDCs, BDCA1+mDCs and BDCA3+ mDCs were resuspended in
IMDM (Invitrogen) supplemented with 10% FCS (Integro, Zaandam, The Netherlands), 1% penicillin and streptomycin (PS; MP Biomedicals, Ohio, USA) and seeded in a 96-well round bottom plate (Corning Costar). 10 ng/ml IL-3 or 800 IU/ml GM-CSF (both from ImmunoTools) was additionally added to the medium for survival of the pDCs and mDCs, respectively. Next, pDCs were stimulated with 3.4 μg/ml CpG ODN 2216 (CpG-A) or CpG ODN 2006 (CpG-B, both from Enzo Life Sciences, NY, USA) whereas mDCs were stimulated with 20 μg/ml Poly I:C (Sigma-Aldrich, St. Louis, USA) and 5 μg/ml R848 (Enzo Life Sciences). After overnight stimulation, IFN-т, IL-6, TNF-т and IL-12 concentrations were measured in the supernatant by ELISA according to manufacturer’s instructions (Human IFN-т Module set ELISA (Bender MedSystems GmbH, Vienna, Austria); Human IL-6 ELISA Ready-Set-Go (eBioscience); PeliPair human TNF-т ELISA reagent set (Sanquin, Amsterdam, The Netherlands); IL-12 ELISA (Pierce Endogen, Rockford, IL, USA)) and phenotypical maturation was evaluated by flow cytometry. Allogeneic MLR
HSPC-pDCs and mDCs were harvested and washed after overnight stimulation with 3.4 μg/ml CpG-B or 10 μg/ml Poly I:C, respectively. Subsequently, they were co-cultured with allogeneic PBMCs from healthy donors that were labeled with 1.25 μM CFSE
(Molecular Probes Europe, Leiden, The Netherlands) as described previously.20
Cocultures were performed at a 1:10 ratio (DC:PBMCs) in IMDM supplemented with 10% FCS and 1% PS in 96-well round bottom plates in triplicate. After 4-5 days of
co-culture, supernatant was collected for cytokine analysis (Th1/Th2/Th17 cytometric
bead array, Becton Dickinson) and T cell proliferation was quantified by flow cytometry
by evaluating the CFSE dilution within the CD3+CD4+ and CD3+CD8+ T cell populations.
Antigen-specific T cell activation
CD8+ T cells specific for the minor histocompatibility antigen LRH-1 or HA-1 were
isolated from PBMCs obtained from patients that had chronic myeloid leukemia and were treated with allogeneic stem cell transplantation and pre-emptive donor
lymphocyte infusion as previously described.21, 22 Sorted HSPC-DCs from HLA-B7+ or
HLA-A2+ donors were loaded with 1 μM LRH-1 peptide (TPNQRQNVC, IHB-LUMC
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