Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Supplementary methods:
Cell preparation
Peripheral blood (PB) was drawn via cardiac puncture with EDTA as anti-coagulant. Single cell
suspensions from spleens and LNs were prepared by grinding the tissue through 70 µm cell
strainers (BD Biosciences). Erythrocytes in spleen and PB were lysed using NH4Cl-based lysis
buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2). Peritoneal cavity (PC) exudates
were harvested by injection and withdrawal of 5-7 ml of PBS in the peritoneal cavity (PC) and
traces of blood contaminations were removed by brief treatment with NH4Cl-based lysis buffer.
Bone marrow cells were prepared as previously described.(1) Cell counting was performed
using a Vi-CELL XR (Beckman Coulter).
Quantitative reverse transcriptase polymerase chain reaction
PC cells were harvested from 10 months or older WT (n=4) and TCL1 (n=6) mice and were
debulked from B cells using mouse CD19 microbeads (Miltenyi Biotec). Afterwards, PC
macrophages were FACS-sorted as CD45+ CD11b+ F4/80+ cells and RNA was extracted using the
RNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol. cDNA synthesis was done
using QuantiTect Reverse Transcription Kit (Qiagen) containing a oligo-d(T)20/ random hexamer
primer mix as indicated in the manufacturer’s manual. Quantitative reverse transcriptase
polymerase chain reaction (qRT-PCR) was performed using SYBR Green ROX Mix (Abgene,
Epsome, UK) for amplification and quantification in an ABI Prism 7900HT system (life
technologies). Arginase 1 (Arg1) expression was detected using the following forward (5′-
TACAAGACAGGGCTCCTTTCAG-3′) and reverse (5′-TGAGTTCCGAAGCAAGCCAA-3′) primers. After
calculating the efficiency of amplification, expression of Arg1 was quantified relative to the
mean value of two housekeeping genes: Actb (forward; 5′-AGATGTGGATCAGCAAGCAG-3′,
reverse: 5′-GCGCAAGTTAGGTTTTGTCA-3′) and B2m (forward; 5′-TTCTGGTGCTTGTCTCACTGA-3′,
reverse: 5′-CAGTATGTTCGGCTTCCCATTC-3′). Triplicate qRT-PCR reactions were carried out for
each macrophage preparation.
Intracellular phospho-protein stainings
PC cells were harvested, resuspended in PBS+0.5% BSA and stimulated for 10 min at 37°C with
10 ng/ml murine IL-10 or IL-4 (Miltenyi Biotec, Germany). Cells were afterwards fixed with 2%
PFA, permeabilized using BD permeabilization buffer III and finally stained for 30 min using
isotype control, p-STAT3 (pY705) or p-STAT6 (pY641) Alexa Flour 647 antibodies (all from BD
Biosciences).
Cytokine quantification
Serum was harvested by centrifugation of clotted blood at 2,000 g and 4°C for 20 min. IL-10,
CCL2, CCL3, GM-CSF, CXCL10 and TNF-α were measured using MILLIPLEX Mouse
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Cytokine/Chemokine Magnetic Bead Panel (Merck Millipore, Germany) according to the
manufacturer’s protocol on a Bioplex-200 System (Bio-Rad). Flt3L, CXCL9, IL-1ra and CCL4 (R&D
Systems), CXCL16 (RayBiotech) and IL-1β (eBioscience) were measured by ELISA according to
manufacturers’ protocols.
Splenocytes/bone-marrow derived macrophage co-cultures
Bone-marrow derived macrophages (BMDMs) were prepared as previously described (2)
resulting in CD11b+ F4/80+ content of more than 98%. TCL1 splenocytes were cultured at a
density of 2x106 cells/ml either alone or with 1x106 BMDM in 6-well plates in RPMI medium
supplemented with 10% fetal calf serum, 4 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL
streptomycin and 0.1% 2-mercaptoethanol. Cells were cultured at 37°C in a 5% CO2 humidified
incubator and 48 hours later viability of CD5+CD19+ cells was quantified using Annexin-V/7AAD
staining as previously described.(3) CCL2, CCL3 and CCL4 were quantified in the culture
supernatant using ProcartaPlex mouse Luminex beads (eBioscience) on a on a Bioplex-200
System (Bio-Rad).
In vivo EdU staining
Mice were injected i.p. with 0.1 mg/g EdU (5-ethynyl-2´-deoxyuridine, Life Technologies, UK).
After 20 hours, mice were sacrificed and EdU incorporation in spleen cDCs (CD11chi MHC-II+),
CLL cells (CD5+CD19+) and monocytes (Lin- CD11b+ CD11clow-int F4/80int) was analyzed after Click-
iT reaction according to the manufacturer’s instruction.
Gene expression profiling
Six weeks after AT, splenocytes from leukemic mice (n=5) were debulked from CLL cells using
mouse CD19 microbeads (Miltenyi Biotec) and the flow-through was used for FACS-sorting of
total spleen monocytes as Lin- CD11b+ F4/80int CD11clow-int MHC-IIlow cells on a BD FACSAria (BD
Biosciences). Control monocytes from WT mice were sorted accordingly and pooled from 4-5
mice per sample (n=5). The purity of sorted monocytes was ≥95%. RNA was extracted from
sorted cells using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol.
Gene expression was determined using the Illumina Mouse Sentrix-6 BeadChip arrays (Illumina,
Inc.). The quality of total RNA was checked by gel analysis using the total RNA Nano chip assay
on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). RNA
concentrations were determined using the NanoDrop spectrophotometer (NanoDrop
Technologies, Wilmington, DE).
The laboratory work was done in the Genomics and Proteomics Core Facility at the German
Cancer Research Center, Heidelberg, Germany (DKFZ). Biotin-labeled cRNA samples for
hybridization on Illumina Mouse Sentrix-6 BeadChip arrays (Illumina, Inc.) were prepared
according to Illumina's recommended sample labeling procedure based on the modified
Eberwine protocol (4). In brief, 100 ng total RNA was used for complementary DNA (cDNA)
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-
labeled cRNA according to the Illumina® Total Prep™ RNA Amplification Kit (Life Technologies).
Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was
column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µl of water.
Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer
and spectrophotometrically quantified (NanoDrop).
Hybridization was performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of
100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and
highly abundant RNAs were added, as well as mismatch control and biotinylation control
oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at
55°C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes (in
between washed with ethanol at room temperature). After blocking for 5 min in 4 ml of 1%
(wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology,
Inc., Rockford, IL), array signals were developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-
streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution.
After a final wash in E1BC, the arrays were dried and scanned. Microarray scanning was done
using an iScan array scanner. Data extraction was done for all beads individually, and outliers
were removed when >2.5 MAD (median absolute deviation). All remaining data points were
used for the calculation of the mean average signal for a given probe, and standard deviation
for each probe was calculated.
A cluster analysis using Euclidean distances and average linkage was performed to search for
unwanted grouping effects. Intensity values were normexp-by-control background corrected
and quantile normalized with control probes (5). Probes that failed to reach a detection P-value
of 0.05 on at least two arrays were filtered as not expressed. Linear regression models
approach of Smyth (6) was used to derive moderated statistics to compare WT with TCL1
monocytes. To correct for multiple testing P-values were adjusted using the method of
Benjamini and Hochberg (7) to control the false discovery rate (FDR). The analysis was done
using R, version 3.0.2, together with R/Bioconductor packages limma, version 3.18.13, and
beadarray, version 2.12.0. Dataset is available on Gene Expression Omnibus (GEO) under the
accession number GSE66858
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=yzetomgervqpzyd&acc=GSE66858).
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Supplementary Table 1: Flow cytometry antibodies and respective clones
Antibody Clone Vendor
anti-mouse CD19 eBio1D3 eBioscience
anti-mouse CD3 145-2C11 eBioscience
anti-mouse CD11b M1/70 eBioscience
anti-mouse CD11c N418 eBioscience
anti-mouse F4/80 BM8 eBioscience
anti-mouse MHC-II I-A/I-E M5/114.15.2 eBioscience
anti-mouse CD31 390 eBioscience
anti-mouse CD54 YN1/1.7.4 eBioscience
anti-mouse PDL1 MIH5 eBioscience
anti-mouse PDL2 TY25 eBioscience
anti-mouse CD86 GL-1 eBioscience
anti-mouse CD4 RM4-5 eBioscience
anti-mouse CD8a 53-6.7 eBioscience
anti-mouse CD62L MEL-14 eBioscience
anti-mouse CD44 IM7 eBioscience
anti-mouse CD124 MIL4R-M1 BD Biosciences
anti-mouse CD5 53-7.3 BD Biosciences
anti-mouse Ly6G 1A8 BD Biosciences
anti-mouse Ly6C AL-21 BD Biosciences
anti-mouse CD43 S7 BD Biosciences
anti-mouse CD117 2B8 BD Biosciences
anti-mouse TER-119 TER-119 Biolegend
anti-mouse NK1.1 PK136 Biolegend
anti-mouse CD45 30F-11 Biolegend
anti-mouse CXCR3 CXCR3-173 Biolegend
anti-mouse CD206 Polyclonal Goat IgG R&D
anti-mouse Trem-1 174031 R&D
anti-mouse F4/80 Cl:A3-1 AbD Serotec
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Supplementary Table 2: Top 10 most differential pathways between TCL1 AT and WT splenic
monocytes as obtained by Ingenuity pathway analysis
Ingenuity
Canonical
Pathways
-log
(p-value) Molecules
1
Type I
Diabetes
Mellitus
Signaling
1,32E01
IRF1,TRAF2,SOCS3,HLA-DQA1,TNFRSF1A,SOCS4,HSPD1,
NFKBIE,NFKBIA,STAT1,TNF,CD3D,HLA-DRB5,NFKBIB,
MAPK11,CD3E,NFKB1,HLA-DOA,HLA-DQB1,CD3G,PRF1,
HLA-G,HLA-DOB,NFKB2,HLA-A,MAPK13,H2-Eb2,FAS,BID,
IKBKE
2
OX40
Signaling
Pathway
1,15E01
JUN,TRAF2,H2-T24,TNFRSF4,HLA-DQA1,NFKBIE,NFKBIA,H2-
Q8,H2-K2/H2-Q9,CD3D,HLA-DRB5,NFKBIB,H2-T10,H2-
T22,CD3E,NFKB1,HLA-DOA,HLA-DQB1,CD3G,HLA-G,HLA-
DOB,TRAF5,NFKB2,HLA-A,H2-Eb2
3 CD40 Signaling 1,01E01
JUN,TANK,TRAF2,NFKB1,PIK3R6,JAK3,NFKBIE,CD40,NFKBIA,
TRAF5,NFKB2,FOS,STAT3,PIK3CA,TNFAIP3,MAPK13,NFKBIB,
MAPK11,ICAM1,IKBKE
4
Role of NFAT
in Regulation
of the Immune
Response
9,62E00
JUN,PIK3R6,HLA-DQA1,GNG10,RCAN3,NFKBIE,NFKBIA,
FOS,GNAQ,PIK3CA,LCK,CD3D,HLA-DRB5,NFKBIB,NFAT5,
CD3E,NFKB1,HLA-DOA,GNB2L1,HLA-DQB1,RCAN1,CD3G,
RAF1,CD79B,HLA-DOB,FCGR2A,NFKB2,Calm1 (includes
others),FCGR2B,H2-Eb2,GNG2,GNA15,IKBKE
5 Dendritic Cell
Maturation 9,49E00
PIK3R6,HLA-DQA1,TNFRSF1A,NFKBIE,CD40,NFKBIA,
TREM2,IRF8,IL1RN,PIK3CA,CD1D,STAT1,RELB,TNF,HLA-
DRB5,NFKBIB,MAPK11,TLR2,LTB,NFKB1,HLA-DOA,HLA-
DQB1,STAT2,HLA-DOB,FCGR2A,NFKB2,HLA-A,FCGR2B,
MAPK13,IL10,H2-Eb2,ICAM1,IKBKE
6
Activation of
IRF by
Cytosolic
Pattern
Recognition
Receptors
9,34E00
JUN,TANK,DHX58,NFKB1,STAT2,DDX58,IFIT2,NFKBIE,CD40,
NFKBIA,NFKB2,IRF7,STAT1,TNF,ISG15,NFKBIB,IL10,ZBP1,
IKBKE
7 IL-10 Signaling 9,23E00
JUN,NFKB1,SOCS3,LBP,NFKBIE,NFKBIA,FCGR2A,IL10RA,
NFKB2,FOS,CCR5,STAT3,FCGR2B,IL1RN,TNF,MAPK13,
NFKBIB,IL10,MAPK11,IKBKE
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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8 TNFR2
Signaling 9,12E00
JUN,BIRC2,TANK,TRAF2,NFKB1,NFKBIE,NFKBIA,NFKB2,FOS,
TNFAIP3,TNF,NFKBIB,IKBKE
9 iNOS Signaling 8,95E00
IRF1,JUN,NFKB1,JAK3,LBP,NFKBIE,NFKBIA,IRAK3,NFKB2,
FOS,Calm1 (includes others),STAT1,MAPK13,NFKBIB,
MAPK11,IKBKE
10
Acute Phase
Response
Signaling
8,16E00
JUN,TRAF2,CFB,TF,SOCS3,TNFRSF1A,SOCS4,IL6R,LBP,
NFKBIE,NFKBIA,FOS,C4A/C4B,STAT3,FN1,HP,IL1RN,PIK3CA,
TNF,NFKBIB,MAPK11,VWF,Saa3,NFKB1,RAF1,NFKB2,SOD2,
CEBPB,MAPK13,IKBKE
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Supplementary figure 1
Supplementary figure 1: A) Representative dot plots showing infiltration of CD11bint Ly6Chi monocytes in PC of TCL1 mice. B) Representative dot plots for the expression of CD11c, Ly6G and F4/80 on CD11bint Ly6Chi cells in TCL1 PC (n=6). C) Total PC CD11b+ F4/80+ macrophages were analyzed for expression of CD206, CD124, CD86 and PD-L2. Representative histograms are shown. D) Normalized MFI of CD206, CD124, CD86 and PD-L2 on PC macrophages in TCL1 AT mice and matched WT controls in at least 5 mice per group. E) PC cells from TCL1 and WT mice were stimulated for 10 min with 10ng/ml IL-10 or IL-4 at 37°C. Levels of p-STAT3 and p-STAT6 in CD11b+ F4/80+ cells upon IL-10 or IL-4 stimulation, respectively, were assessed. One representative example is shown.
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Supplementary figure 2
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Supplementary figure 2: Single cell suspensions of spleens were analyzed by flow cytometry.
Total DAPI-negative cells were used for the analysis. A) Gating strategy to define splenic
monocytes (Lin- (CD19- CD3- Ly6G- NK1.1-) CD11b+ F4/80int CD11clow-int MHC-IIlow SSCint), cDCs
(Lin- CD11chi MHC-II+) and macrophages (Lin- F4/80hi CD11blow). B) Representative histograms
showing CD62L and CD11c expression on TCL1 and WT spleen monocytes as determined by
flow cytometry. C) Representative histograms showing PECAM1 and ICAM1 expression on TCL1
and WT spleen monocytes as determined by flow cytometry.
Supplementary figure 3
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Supplementary figure 3:
A) Percentage of CD5+CD19+ cells and B) spleen weight following adoptive transfer of 1x107
splenocytes from leukemic TCL1 mice into young syngenic WTs (TCL1 AT). 3-4 mice were
sacrificed at the indicated time points.
4x107 splenocytes from leukemic TCL1 mice were adoptively transferred to young syngeneic
WTs (TCL1 AT) via tail vein injection and mice were sacrificed 6 weeks later. C) Splenic
monocytes were identified by flow cytometry as described in Suppl. Fig. 2 and their
percentages out of B220- cells in TCL1 AT (n=9) and matched WT (n=6) spleens are shown. Bar
plots represent percentage of inflammatory and patrolling subsets of total monocytes (D) and
total absolute counts of monocytes (E) in spleens of TCL1 AT (n=9) and matched WT (n=6) mice.
F) Proliferation of spleen cDCs in TCL1 AT mice as measured by the percentage of EdU+ cells in
3-4 mice per time point. G) Serum levels of GM-CSF and Flt3L in WT and TCL1 AT mice as
measured by multiplex bead arrays or ELISA in at least 5 mice per group. All bar plots show
means ± SEM. Statistical analysis was performed using unpaired t-test. **p<0.01, ***p<0.001.
Supplementary figure 4
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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Supplementary figure 4: Splenocytes from TCL1 mice (n=4) were either cultured alone or co-
cultured with bone marrow-derived macrophages (BMDM), and cell-free cell culture
supernatant was collected 48 hours later. Levels of A) CCL2, B) CCL3 and C) CCL4 were
quantified using ProcartaPlex multiplex beads.
Three months old age- and sex-matched WT (n=8) and CCR2-/- (n=6) mice were intraperitoneally
transplanted with 2x107 CLL cells. Graphs show spleen weight (D) and absolute counts of
CD5+CD19+ cells in spleen (E) after 6 weeks of transplantation. Results are derived from two
independent transplantation experiments.
Supplementary figure 5
Supplementary figure 5: A) EdU incorporation in splenic monocytes (Lin- CD11b+ CD11clow-int
F4/80int) in PBS Lip. (n=8) and Clod. Lip. (n=9) treated mice. B) Relative subset composition of
spleen monocytes in PBS Lip. (n=9) and Clod. Lip. (n=14) mice at day 30 based on Ly6C and
CD43 expression. C) Normalized MFI of TREM-1 expression on total splenic monocytes of PBS
Lip. (n=9) and Clod. Lip. (n=10) mice. D) Percentage of CXCR3+ cells in CD4 and CD8 T cells in
Hanna et al., In vivo targeting of skewed myeloid cells in CLL
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spleens of PBS Lip. (n=9) and Clod. Lip. (n=10) mice. All bar plots show means ± SEM. Statistical
analysis was performed using unpaired t-test. ns = not significant, *p<0.05, **p<0.01,
***p<0.001.
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