using Ficoll-Paque density gradient centrifugation
as described (58). Single-cell suspensions were
stained per manufacturer recommendations with
different panels of antibodies (table S12) designed
to enrich for certain population for single-cell sorting and single-cell RNA sequencing (scRNA-seq)
(6). Flow cytometry and FACS sorting of PBMCs
was performed on a BD Fortessa or BD FACS
Fusion instrument, and data analyzed using
FlowJov10.1. Single cells were sorted into 96-well
full-skirted Eppendorf plates chilled to 4°C, pre-prepared with lysis buffer consisting of 10 ml of
TCL buffer (Qiagen) supplemented with 1%
b-mercaptoethanol. Single-cell lysates were
sealed, vortexed, spun down at 300 g at 4°C for
1 min, immediately placed on dry ice, and transferred for storage at –80°C. Tonsil was mechanically disrupted to obtain single-cell suspension.
Single-cell RNA sequencing
Smart-Seq2 protocol was performed on single sorted
cells as described (7, 8), with some modifications (6).
For DCs, a total of 8 × 96-well plates (768 single
DCs) were initially profiled from the same blood
draw and sort from the index volunteer and subsequent validation performed on an additional 10
healthy individuals. For monocytes, a total of four
plates were profiled (372 single monocytes and 12
population samples). An additional 975 single cells
were profiled to further characterize the CD1C+
DC subsets (n = 125), AXL+SIGLEC6+ cells (n = 372),
CD11C–CD123– compartment at day 0 (n = 164), differentiation assay outputs (n = 218), CD100hiCD34int
cells (n = 96), and BPDCN patient samples (n = 269).
Note that some of these single cells were excluded
from the analysis after applying QC filters and
analytically confirming cell type (6).
Single-cell RNA sequencing analyses
Raw sequencing data were processed as described
(59) (see tables S13 to S16 for cell identities that
accompany raw data and gene expression matrices). Briefly, short sequencing reads were aligned
to the UCSC hg19 transcriptome. These alignments
were used to estimate transcriptomic alignment
rates and were also used as input in RSEM v 1.2.1
to quantify gene expression levels (transcripts
per million; TPM) for all UCSC hg19 genes in all
samples. We filtered out low-quality cells from
our data set based on a threshold for the number
of genes detected (a minimum of 3000 unique
genes per cell for cells sequenced at HiSeq depth,
and 2000 unique genes per cell for cells sequenced
at MiSeq depth). All genes that were not detected
in at least 0.5% of all our single cells were discarded, leaving 21,581 genes for all further analyses. Data were log-transformed [log(TPM + 1)]
for all downstream analyses, most of which were
performed using the R software package Seurat
( https://github.com/satijalab/seurat; http://satijalab.
org/seurat/). See (6) for further details, including
R script used to generate clusters.
DC differentiation assay on MS5
DC differentiation assay was performed as described (23–25) with minor adaptation. Briefly, 1 ×
104 purified progenitors, DCs, and monocyte subsets were cultured in 96-well flat-bottom plates
layered with 4 × 104 murine MS5 stromal cells
(DSMZ, Germany) in the presence of human FLT3
ligand (FL; 100 ng/ml; Miltenyi Biotec), recombinant human SCF (20 ng/ml; R&D Systems),
and recombinant human granulocyte-macrophage
colony-stimulating factor (GM-CSF) (10 ng/ml;
Peprotech). MS5 stromal cells were seeded 24 hours
prior to coculture. Growth factors were replenished
on day 3 of culture. Cells were in culture for up to
7 days prior to harvesting by physical dissociation
on ice. Cells were then stained on ice either for flow
cytometry analysis (see output panel in table S12)
or single-cell index sorting of CD45+ cells for
scRNA-seq of culture output analysis.
Cytokine production measurements
Purified subsets were cultured at 5 × 103 cells per
well in 96-well round-bottom plates in the presence of LPS (100 ng/ml; Invivogen) and ODN2395
(1 mM; Invivogen) or ODN5328 (ODN2395 control,
1 m M; Invivogen), or in the presence of LPS, poly(I:C)
(25 mg/ml; Invivogen), and R848 (2.5 mg/ml; Enzo
Life Sciences). Culture supernatants were harvested
after 24 hours and analyzed using a multiplexed
cytokine assay (ProcartaPlex, eBioscience), or by
leveraging the 92 inflammatory-related protein
biomarker panel and four controls provided by
Olink Proteomics (Uppsala, Sweden) (6).
Assessing T cell stimulatory potential
DC, monocyte, and progenitor subsets were purified from peripheral blood of healthy donors by
FACS sorting (BD FACS Fusion; see table S12 for
sorting panels and antibodies). For T cell stimulatory
potential, purified DCs, monocytes, AXL+SIGLEC6+
subsets, and progenitor subset were cultured at cell
density of 5 × 104 per well. All purified cell subsets
were matured with LPS (100 ng/ml, Sigma) and R848
(2.5 mg/ml, Invivogen), or with just LPS (100 ng/ml),
for 24 hours prior to coculture with 5 × 105
CFSE-labeled allogeneic unfractionated CD3+ T cells at a
1:10 DC:T cell ratio. T cell proliferation was assessed
by measuring CFSE dilution on day 5 of culture.
Cytospin and immunostaining
Cytospin of FACS-purified cells was prepared as
described (60) using Shandon Cytospin 4 (Thermo
Scientific). Giemsa-Wright staining was performed
using Advia S60 (Siemens) and imaged using
Axioimager.Z2 microscope with Axiovision
softwarev4.8 (Carl Zeiss, Germany). Human tonsil paraffin sections were immunostained with
the antibodies anti-AXL (MM0098-2N33, Abcam),
CD123 (BR4MS, Leica Biosystems) and CD3 (LN10,
Leica Biosystems) using a Ventana Benchmark XT
Monitoring cell proliferation
PBMCs were labeled with Cell Trace Violet (CTV,
Life Technologies) according to manufacturer’s
protocol. CTV-labeled FACS-purified progenitors
and DC subsets were cultured on murine MS5
stromal cells as described above and analyzed
on day 5 to assess proliferation measured by
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