Isolation of immune cells
Primary spleen DCs were isolated as previously described (50). In brief,
organs were finely chopped and digested in the presence of DNase I
(Roche) and collagenase type 3 (Worthington Biochemicals). Intercellular
clusters were disrupted by addition of 10 mM EDTA. Light density cells
were isolated by density gradient separation in 1.077
g/cm3 Nycodenz (Nycomed Pharma). Upper fractions were
collected, washed and subject to further enrichment by resuspending in a
depletion cocktail containing the following rat anti-mouse mAbs specific
for: CD3 (KT3-1.1), CD90 (T24/31.7), red blood cells (Ter119), B220
(RA3-6B2), Ly6G (IA8). For purification of plasmacytoid DC, the upper
fractions were incubated with rat anti-mouse mAbs specific for CD3
(KT3-1.1), Thy1 (T24/31.7), red blood cells (Ter119), Ly6G (IA8) and
CD19 (ID3). Cells were incubated with antibodies, washed, and incubated
with BioMag anti-rat IgG-coupled magnetic beads (Qiagen). The
DC-enriched supernatant was recovered by magnetic separation. Where
required, DCs were sorted to purity by flow cytometry on a Becton
Dickinson Influx (Murdoch Children’s Research Institute Flow Cytometry
and Imaging facility). cDC1 were defined as CD11c+CD8+ CD11b-, cDC2:
CD11c+ CD8-CD11b+, double positive cDC1 (DP cDC1):
CD11c+ CD8+ CD11b-CD103+ CD86+, double negative cDC1
(DN cDC1): CD11c+ CD8+CD11b- CD103-CD86-, or single positive cDC1 (SP cDC1)
CD11c+ CD8+ CD11b-CD103+ CD86+.
For T cell isolation, single cell suspensions were generated from lymph
nodes. Cells were stained with rat anti-mouse mAbs specific for: CD11b
(M1/70), F4/80 (F4/80), red blood cells (TER119), Ly6G/-Ly6C (RB68C5),
MHCII (M5/114), CD45R (RA36B2) and CD4 (GK1.5) for
CD8+ T cells or CD8 (53-6.7) for
CD4+ cells. Cells were washed and incubated with
BioMag anti-rat IgG-coupled magnetic beads (Qiagen). After magnetic
depletion, the CD4+ or CD8+ T
cell-enriched supernatant was recovered. Purity, determined by flow
cytometry, was >90%.
For B cell isolation, single cell suspensions were generated from
spleens. Cells were resuspended in EDTA-BSS medium 2% (v/v) FCS and
gradient centrifugation was carried out with
Ficoll-PaqueTM PLUS (GE Healthcare). Mononuclear cells
were isolated and negative selection was carried out by incubating the
cells with FITC-conjugated antibodies against CD4 (GK1.5), Ly76 (TER119)
and CD43 (S7). Cells were washed and incubated with anti-FITC microbeads
(Miltenyi Biotec). The B cell enriched supernatant was collected after
separation with LS magnetic columns (Miltenyi Biotec). Purity,
determined by flow cytometry, was >90%.
Cells were stained with mAbs specific for CD8α (53-6.7), CD11b (M1/70),
CD11c (N418), CD19 (6D5), CD24 (M1/69), CD40 (FGK45.5), CD45R/B220
(RA3-6B2), CD80 (16-10A1), CD86 (GL1), CD135/Flt3 (A2F10), CD172
(Sirpα), CD205/DEC205 (NLDC-145), CD274/PD-L1 (10F.9G2), CD317/BST2
(927), CD370/Clec9A (7H11), H-2Kb (AF6-8815), IgD (11-26c.2a), IgM
(RMM-1), SiglecH (551), XCR1 (ZET) (all BioLegend). To obtain absolute
cell numbers, an internal microsphere counting standard (BD Biosciences)
was used. Analysis was performed with a Becton Dickinson LSRFortessa,
FlowJo and Graphpad Prism software.