Hanna et al., In vivo targeting of skewed myeloid cells in CLL 1 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 NH 4 Cl-based lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 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 NH 4 Cl-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
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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
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
(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.
References:
1. McClanahan F, Hanna B, Miller S, Clear AJ, Lichter P, Gribben JG, et al. PD-L1 Checkpoint Blockade Prevents Immune Dysfunction and Leukemia Development in a Mouse Model of Chronic Lymphocytic Leukemia. Blood. 2015. 2. Ying W, Cheruku PS, Bazer FW, Safe SH, Zhou B. Investigation of macrophage polarization using bone marrow derived macrophages. Journal of visualized experiments : JoVE. 2013(76). 3. Schulz A, Toedt G, Zenz T, Stilgenbauer S, Lichter P, Seiffert M. Inflammatory cytokines and signaling pathways are associated with survival of primary chronic lymphocytic leukemia cells in vitro: a dominant role of CCL2. Haematologica. 2011;96(3):408-16. 4. Eberwine J, Yeh H, Miyashiro K, Cao Y, Nair S, Finnell R, et al. Analysis of gene expression in single live neurons. Proceedings of the National Academy of Sciences of the United States of America. 1992;89(7):3010-4. 5. Shi W, Oshlack A, Smyth GK. Optimizing the noise versus bias trade-off for Illumina whole genome expression BeadChips. Nucleic acids research. 2010;38(22):e204. 6. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology. 2004;3:Article3.
7. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach
to multiple testing. Journal of the Royal Statistical Society Series B, 1995; 57: 289-300.