www.sciencetranslationalmedicine.org/cgi/content/full/7/282/282ra47/DC1

Supplementary Materials for

Vitamin D–dependent induction of cathelicidin in human macrophages results in cytotoxicity against high-grade B cell lymphoma

Heiko Bruns,* Maike Büttner, Mario Fabri, Dimitrios Mougiakakos, Jörg T. Bittenbring,

Markus H. Hoffmann, Fabian Beier, Shirin Pasemann, Regina Jitschin, Andreas D. Hofmann, Frank Neumann, Christoph Daniel, Anna Maurberger,

Bettina Kempkes, Kerstin Amann, Andreas Mackensen, Armin Gerbitz

*Corresponding author. E-mail: heiko.bruns@uk-erlangen.de

Published 8 April 2015, Sci. Transl. Med. 7, 282ra47 (2015) DOI: 10.1126/scitranslmed.aaa3230

The PDF file includes:

Materials and Methods Fig. S1. M2-like phenotype of TAMs in BL. Fig. S2. Cytokine profile in lymph node sections. Fig. S3. Phenotype of generated M1 and M2 macrophages. Fig. S4. Activation of M1 and M2 macrophages after exposure to Raji targets. Fig. S5. Analysis of effector molecules secreted by macrophages. Fig. S6. Expression of TNFR1, CD95, DR4, and DR5 on BL cell lines. Fig. S7. Phagocytosis of BL cells. Fig. S8. Dose-dependent cytotoxicity of LL-37 for BL Raji cells. Fig. S9. Expression of “LL-37 receptors” on BL cell lines and proliferating B cells. Fig. S10. Cytotoxic activity of LL-37 counteracted by overexpression of BCL-2. Fig. S11. LL-37 targeting mitochondria of BL cells. Fig. S12. Z-stack analysis of colocalization of LL-37 and mitochondria. Fig. S13. Colocalization of LL-37 and mitochondria. Fig. S14. ROS induced by LL-37 in BL cells. Fig. S15. Expression of antiapoptotic BCL-2 family proteins in BL cells. Fig. S16. Cathelicidin mediating vitamin D–induced cytotoxicity of M2 macrophages. Fig. S17. Time course of macrophage-mediated cytotoxicity. Fig. S18. Phenotype of M2 macrophages after vitamin D treatment. Fig. S19. VDR, CYP27B1, and CYP24A1 expression in vitamin D–treated healthy volunteers.

Fig. S20. Cell sorting strategy to obtain TAMs and lymphoma cells. Reference (50)

Supplementary Materials (SM)

Materials and Methods

Study design

This study focused on the impact of vitamin D on macrophage-mediated cytotoxic activity

against high-grade B cell lymphomas. The objective of the study was to demonstrate why

macrophage-mediated cytotoxicity is absent in Burkitt´s lymphoma and to show that vitamin

D alters macrophages towards more cytotoxicity by the expression of cathelicidin. Therefore,

Burkitt´s lymphoma samples were analyzed by immunohistochemistry and quantitative RT-

PCR based on sample availability at our institution. Further in vitro experiments on

cytotoxicity of generated macrophages against Burkitt´s lymphoma cells demonstrated the

critical importance of vitamin D with regard to the induction of cathelicidin and provided the

rationale for the vitamin D supplementation of healthy individuals with vitamin D deficiency.

Healthy volunteers with vitamin D deficiency were treated with 40,000 units of vitamin D3

weekly over a period of 6 weeks to normalize vitamin D serum levels and to demonstrate

vitamin D-induced expression of cytotoxic effector molecules in macrophages. The treatment

of healthy volunteers was approved by the ethic committee “Ärztekammer des Saarlandes”

file 227/12. Analysis was performed after treatment of 5 healthy individuals. Analysis

included cell culture and flow cytometric analysis of peripheral blood mononuclear cells and

quantitative RT-PCR analysis before and after treatment with vitamin D. Treatment of further

healthy individuals was terminated due to significance of the results. Mechanistic studies

elucidating the effect of cathelicidin on BL cell lines were performed in vitro on cells from

healthy blood donors without blinding or randomization after informed consent. The number

of replicates of each experiment is indicated for each experiment in the respective figure

legend.

Cell culture reagents

Cells were cultured in RPMI 1640 medium (Biochrom) supplemented with glutamine (2 mM;

Sigma), 10 mM HEPES, 13 mM NaHCO3, 100 µg/ml streptomycin, 60 µg/ml penicillin (all

from Biochrom), and 10% human AB serum (PAN Biotech) (complete medium, CM). For

culture of lymphoma cell lines, human AB serum was replaced by 10% FCS (Sigma).

Concentration of 25D in human serum was 70 nmol/L, and the 1,25D3 concentration was 0.1

nmol/L, as determined by CLIA (Synlab). The same batch of human serum was used

throughout the study. For experiments in which 25D-sufficient (25D ≈ 100 nmol/L) and

deficient sera (25D ≤ 40 nmol/L) were compared, pooled human serum was collected as

described previously (12)

Cell lines

Mycoplasma detection was performed monthly in our institution on all cell lines currently in

use. All lines were repeatedly tested mycoplasma negative.

Tissue

Paraffin-embedded specimens of Burkitt´s lymphomas (n=9) and of reactive

lymphadenopathies (n=9) were selected from the archive of the Institute of Pathology,

University Hospital Erlangen, Germany. BL were classified according to the WHO

classification 2008. One case showed BCL-2 expression in single tumor cells. One case

showed some cellular pleomorphism but a marker profile reminiscent of BL and a “starry

sky” pattern, so that it was included in the group of B-cell lymphoma, unclassifiable, with

features intermediate between DLBCL and BL. Tissue was used in accordance with a general

statement of our local ethical board on the use of tissue from the archive of the Institute of

Pathology. In some cases, not enough material was available for histology or isolation of

RNA of required quality. Therefore, 8 cases were used for histology in fig. S1C and vitamin

D metabolism analysis by RT-PCR, and 7-8 cases were used for cytokine analysis (fig. S2).

Antibodies and reagents

The following antibodies were used for immunofluorescence, flow cytometry or western blot:

CD68-FITC (clone: Y1/82A, BD), CD163-PE (clone: GHI/61, eBioscience), CD15-BV510

(clone: W6D3, BioLegend), CD20-FITC (clone: 2H7, BD), lambda-PE (clone: 1-155-2, BD),

kappa-APC (clone: G20-193, BD), HLA-DR-PerCP (clone: L243, BD), CD11b-APC (clone:

M1/70.15.11.5, Miltenyi Biotec), CD206–APC (clone: 19.2, eBioscience), IRF4-PE (clone:

3E4, eBioscience), IRF5-Alexa Fluor 488 (R&D Systems), FasL-PE (clone: NOK-1,

BioLegend), Trail-PE (clone: RIK-2, BD), TNFR-FITC (clone: 16803, R&D Systems),

CD95-FITC (clone: DX2, BD), DR4-PE (clone: DJR1, eBioscience), DR5-PE (clone: DJR2-

4, eBioscience), Annexin-V-APC (BD), 7-AAD (Sigma), cathelicidin (clone: H7,

BioLegend), cathelicidin (clone: 3D11, Hycult biotech), VDR (clone: D-6, Santa Cruz),

CYP27B1 (clone: H-90, Santa Cruz), CYP24A1 (clone: H-87, Santa Cruz), p91-phox(clone:

NL7, Santa Cruz), phospho-tyrosine (clone: P-Tyr-100, Cell Signaling), p67-phox (clone:N-

19, Santa Cruz), fMLP receptor-PE (clone: 5F1, BD), P2X7R-PE (clone: H265, Santa Cruz),

IGF-1R-APC (clone: 1H7, eBioscience), pro-Survival BCL-2 Family Antibody Sampler Kit

(Cell Signaling), calreticulin (clone: DE36, Cell Signaling), HSP60 (clone: D6F1, Cell

Signaling), rabbit IgG F(ab’)2 Fragment 674 conjugate, mouse IgG F(ab’)2 Fragment 647

conjugate (all from Cell Signaling), and mouse monoclonal beta-actin (AC-15, Abcam). The

fluorescent probes were purchased from Invitrogen (MitoTracker, MitoSox, DiOC6(3)), from

Sigma (Hoechst 33258, CFSE), and from Innovagen (LL-37-TAMRA). Rituximab was

obtained from the pharmacy of Universitätsklinikum Erlangen. The antioxidant N-Acetyl-L-

cysteine (NAC), the mitogen Phorbol 12-myristate 13-acetate, pokeweed mitogen (PWM),

and the v-ATPase inhibitor concanamycin A were obtained from Sigma. Active vitamin D

(1,25D3, Biomol) and the vitamin D receptor agonist BXL-628 (Axon Medchem) were

dissolved in ethanol (Sigma) at 1 mg/ml and stored at -70°C in glass vials.

Isolation of RNA from FFPE samples and quantitative RT- PCR

From each FFPE section (10 μm thick), RNA was isolated using a standardized, fully

automated isolation method based on germanium-coated magnetic beads (XTRAKT RNA

kits, STRATIFYER Molecular Pathology GmbH) in combination with liquid-handling robot

XTRAKT SL (STRATIFYER Molecular Pathology GmbH), as previously described in detail

(50). The method involves extraction-integrated deparaffinization and DNase I digestion

steps. DNA-free total RNA was eluted with 100 µL elution buffer and stored in -80°C.

Relative transcript expression levels were measured by quantitative real-time PCR (qPCR)

with probes for 18S and GAPDH (reference genes), VDR, CYP27B1, CYP24A1, cathelicidin,

IL4, IL10, M-CSF, and IFNγ. qPCR was performed with 40 cycles of nucleic acid

amplification using QuantiFast Probe Assays for one-step qRT-PCR specifically designed for

degraded material (Qiagen) according to the manufacturer’s instructions. Standard and no-

template controls were assessed in parallel to exclude contamination. Samples were analyzed

by the Applied Biosystems 7500 Fast Real-Time PCR System and the 7500 Software Version

2.0.6 (Applied Biosystems).

To detect messenger RNA expression in generated macrophages, cells were lysed, and mRNA

was isolated from the cells using an RNeasy mini kit (Qiagen) according to the

manufacturer’s recommended protocol. Contaminating DNA was removed by DNase I

treatment. cDNA was prepared by Random Decamers (Applied Biosystems) as suggested by

the manufacturer. cDNA was analyzed by quantitative real time qPCR (ABI step one) using

the following primers (selected by Pearlprimer; synthesized by Metabion): CD68: forward 5'-

GCT ACA TGG CGG TGG AGT ACA A-3', reverse 5'-ATG ATG AGA GGC AGC AAG

ATG G-3'; CD163: forward 5'-CCA GTC CCA AAC ACT GTC CTC-3', reverse 5'-ATG

CCA GTG AGC TTC CCG TTC AGC-3'; CD206: forward 5'-TGA ACG GAA TGA TTG

TGT AGC TTT A-3', reverse 5'-CAC GTT GGA AGA CGG TTT AGA AG-3'; IL10:

forward: 5'-AAC AAG AGC AAG GCC GTG G-3', reverse 5'-GAA GAT GTC AAA CTC

ACT CAT GGC-3'; IL12: forward 5'-TGA TGG CCC TGT GCC TTA G-3', reverse 5'-GAT

CCA TCA GAA GCT TTG CAT TC-3'; IRF4: forward 5'-AGC GCA TTT CAG TAA ATG

TAA ACA CAT-3', reverse 5'-TCT TGT GTT CTG TAG ACT GCC ATC A-3'; IRF5:

forward 5'-GCC TTG TTA TTG CAT GCC AGC3', reverse 5'-AGA CCA AGC TTT TCA

GCC TGG3'; GAPDH: forward 5-TGG CAA AGT GGA GAT TGT TGC C-3, reverse 5-

AAG ATGT GTG ATG GGC TTC CCG-3; 18S: forward 5'-CGC CGC TAG AGG TGA

AAT-3', reverse 5'-CGA ACC TCC GAC TTT CGT-3'. Primer sequences for human

cathelicidin, DEFB4, CYP27B1, VDR, and h36B4 were previously reported (11).

Immunocytochemistry of FFPE samples

Tissue sections (2 µm) from BL and control tissue of reactive lymph nodes were de-

paraffinized in xylene, rehydrated with graded ethanol (100-70%), and stained on a Ventana

BenchMark Ultra stainer immunohistochemistry device (Roche). For detection, we used an

alkaline phosphatase-labeled polymer kit (Zytochem-Plus AP-PolymerKit, Zytomed Systems)

or fluorescence-labelled secondary antibodies (mouse IgG F(ab’)2 Fragment 647 conjugate,

Cell Signaling). Fast Red (Sigma) and Fast Blue (Sigma) were used as chromogens.

Quantitative evaluation of histologic sections

A computer-assisted analysis was performed on whole block sections. For image acquisition,

we applied a standard light microscope and a CCD camera or a Mirax Midi system (Zeiss) for

digital slide scanning, with Mirax viewer software (Zeiss). Numbers of positive cells (defined

as brown spots with a minimum size of >5 µm2) per mm² in the area with the densest

infiltration were evaluated using the image analysis software BZ-9000 (Keyence).

Preparation of macrophages

PBMC were isolated by density gradient centrifugation of buffy coat preparations from the

peripheral blood of healthy donors (Deutsches Rotes Kreuz). Monocytes were isolated by

adherence on plastic and cultured in the presence of GM-CSF (50 ng/ml, Berlex) to generate

M1 macrophages, or in the presence of M-CSF (50 ng/ml, R&D) to obtain M2 macrophages.

Macrophages were detached with EDTA (1 mM, Sigma) after 6 d of culture. Phenotype was

evaluated by expression of surface markers CD68, CD163, HLA-DR, CD11b, CD206, IRF4,

IRF5 by flow cytometry, and secretion of cytokines IL12, IL10, TNF after activation with

LPS (100 ng/ml for 48 h) was measured by ELISA following the manufacturer’s protocol

(DuoSet, R&D).

B cell purification

Primary B cells were obtained from healthy donor buffy coats. B cells were purified using

Ficoll-Paque (GE Healthcare) and CD19 microbeads (Miltenyi Biotec) according to the

manufacturer’s instructions, and the purity was over than 95% as determined by flow

cytometry.

Isolation of TAMs and lymphoma cells from human DLBCL bone marrow.

Bone marrow (BM) from 4 patients with DLBCL was obtained from Cambridge Bioscience.

BM was stained with CD163, CD15, and CD20 antibodies. Macrophages (CD163+/CD15-)

and lymphoma cells (CD20+) were isolated by flow cytometry (fig. S20). Purity was over

95%. Clonality of B cells was determined by kappa/lambda staining.

Vitamin D conversion assay

To detect the conversion of 25D into bioactive 1,25D3, 1 x106 M1 or M2 macrophages were

incubated with CM in a 12-well plate (24 h, 37°C). Supernatants were collected (1000 µL) on

ice, and concentration of 1,25D3 was immediately analyzed by CLIA (carried out by Synlab)

Killing assay and cytotoxicity of synthetic LL-37

M1 and M2 macrophages or freshly isolated TAMs from patients with diffuse large B cell

lymphoma were incubated in round bottom microplates (96-well) for 48 h together with BL

cells or primary lymphoma cells (effector to target ratio: 1:1, 5:1, and 10:1). In some

experiments, cells were incubated in the presence or absence of a monoclonal anti-cathelicidin

antibody (clone: 3D11, Hycult Biotech, 10 µg/ml) or an isotype control (IgG1, 10 µg/ml).

Non-adherent cells were harvested and stained with a CD11b antibody (to exclude

macrophage contamination). Viability of CD11b- cells was measured by AnnexinV and

7AAD staining (BD) according to the manufacturer’s instructions. BL cells or B cells from

healthy donors were immediately analyzed by flow cytometry (FACS Canto II, BD). To

determine cytotoxicity of synthetic LL-37, cells were incubated in round bottom microplates

(96-well) with LL-37 (1 µM, 2.5 µM, 5 µM, 10 µM) for 24 h. Cytotoxicity of LL-37 was

measured by 7AAD staining according to the manufacturer’s instructions (BD Biosciences).

Cathelicidin staining

Generated macrophages were adhered to 8-chamber slides and incubated with or without

CFSE-labeled BL Raji cells (stained with 0.5 μM carboxyfluorescein diacetate succinimidyl

ester, CFSE) for 24 hours. Cells were fixed with paraformaldehyde (4%), treated with Triton

X-100 (Sigma, 0.3%, 10 min on ice), and incubated with anti-cathelicidin (clone: H7,

BioLegend, 1:100) overnight at 4°C in a humid chamber. After incubation with anti-mouse

IgG F(ab’)2 Fragment Alexa 488 (2 hours, 4°C in a humid chamber), Hoechst 33342 (1

µg/ml) was added during the last 10 min. Slides were analyzed by z-stack sections creating up

to 10 optical slices (0.5 µm thick each) using a fluorescence microscope (Axio-Imager M2,

Zeiss) at x630 magnification.

DiOC6 and active caspase 8 staining

At low concentrations, the cell-permeable dye DiOC6 accumulates in mitochondria with intact

ΔΨm and fluoresces bright green. Mitochondria with compromised ΔΨm cannot efficiently

take up DiOC6, leading to a reduction in green fluorescence (DiOC6Dim). For the detection of

active caspase 8, we used Vybrant FAM Caspase-8 Assay Kit (Invitrogen). Active caspase 8

was detected by a fluorescent specific inhibitor (FLICA), which selectively binds to the

enzymatic reactive center of an activated caspase 8. BL cells (0.25 x 106/ml in 250 µl) were

incubated with cathelicidin (2.5 µM) for 24 h and stained with DiOC6 (5 µM) or the FLICA

reagent for 1 h at 37°C. After a washing step, cells were immediately analyzed by flow

cytometry using WinMDI 2.8 software (J. Trotter).

Extracellular flux analysis

The extracellular acidification rate and oxygen consumption of BL Raji cells were determined

using the XFe96 Extracellular Flux Analyzer and XFe96 Extracellular Flux Assay Kits

(Seahorse Bioscience) according to the manufacturer’s instructions. Briefly, cells were

washed twice and resuspended in XF Assay Medium (Seahorse Bioscience; supplemented

with sodium pyruvate (2 mM) and glucose (11 mM), pH 7.4), seeded in XF Cell Culture

Microplates (Seahorse Bioscience), and subsequently immobilized using CELL-TAK (BD

Biosciences). One hour before measurement, cells were incubated at 37°C in a CO2-free

atmosphere. First, basal oxygen consumption rate (OCR, indicator of oxidative metabolism)

and extracellular acidification rate (ECAR, indicator of glycolytic activity) were detected.

Next, OCR and ECAR response towards the application of different concentrations of

synthetic LL-37 was evaluated during a period of four hours. Untreated cells receiving only

medium served as controls.

LL-37-TAMRA and MitoSox staining

Cell lines were incubated with LL-37-TAMRA (2.5 µM) for 4 hours at 37°C, washed in pre-

warmed PBS, and mitochondria were stained with MitoTracker Green (100 µM) for 30

minutes at 37°C. Hoechst 33258 (1 µg/ml, blue) was added for nuclear staining for the final

10 min. of incubation. After a washing step, cells were immediately analyzed by fluorescence

microscopy. Cell lines were incubated with synthetic LL-37 (2.5 µM) for 24 hours in the

presence or absence of N-acetyl-L-cysteine (10 mM, NAC) at 37°C. Mitochondrial

superoxides were detected by MitoSox (100 µM), according to the manufacturer’s

instructions, and viability of the cell lines was determined by 7AAD exclusion using flow

cytometry.

Stimulation of P493-6 cells and B cells

P493-6 cells were treated with tetracycline (10 nM) for 24 hours. Cells were harvested and

cell cycle was analyzed as described above. In other experiments, cells were treated with

synthetic LL-37 (2.5 µM) for 24 h, and the viability was measured by 7AAD staining and

flow cytometry. Isolated B cells were incubated in round-bottom microplates (96-well) with

Pokeweed mitogen (10 µg/ml) for 72 h, and the cell cycle was evaluated. In some

experiments, synthetic LL-37 (2.5 µM) was added after 24 hours, and the viability was

measured after further 48 h, by 7AAD exclusion.

Cell cycle analysis

Cell cycle analysis was performed using an apoptosis, DNA damage, and cell proliferation kit

(BD Biosciences) following the manufacturer’s instructions. Briefly, cells were pulsed with

BrdU for the final hour of culture and stained with 7-AAD as well as antibodies against

H2AX and cleaved PARP, and subsequently fixed. Cells were then evaluated using multicolor

flow cytometry (FACS Canto II, BD Biosciences). First, acquired events were gated on

forward and side scatter to separate cells from debris, and the cellular events were further

analyzed based on their BrdU and 7-AAD levels. This BrdU/7-AAD-based gating strategy

allows a separation into three cell-cycle phases: S-phase (BrdU+ cells), G0G1-phase (BrdU-/7-

AAD - cells), and G2M phase (BrdU-/7-AAD+ cells).

Western Blot and Dot Blot

To lyse cells, we used modified RIPA buffer (50 mM Tris-HCl (Roth), pH 7.4; 1% NP-40

(Fluka); 0.25% sodium deoxycholate (Fluka); 150 mM NaCl (Roth); 1 mM EDTA) and

protease inhibitor cocktail tablets (Roche). Cell debris were removed by centrifugation

(14.000 rpm, 10 min), and protein content in the supernatant was determined (BCA protein

assay, Pierce). Lysates were boiled for 10 min in Laemmli sample buffer (1% (w/v) SDS

(Sigma); 4 mM urea (Roth); 80 mM Tris-HCl, pH 6.8; 0.1% (w/v) bromophenol blue

(Sigma)) and analyzed by SDS-PAGE (16%) and western blot. The membranes were

incubated with an anti-VDR antibody (1:2000), an anti-CYP27B1 or anti-actin antibody

(1:1000) (all from Santa Cruz), and incubated with donkey anti-rabbit IgG-HRP (1:1000)

(Jackson ImmunoResearch Laboratories) or anti-mouse IgG-HRP (1:5000) (Invitrogen).

Proteins were detected by chemiluminiscence (Amersham Biosciences) following the

manufacturer’s protocol.

For dot blot analysis, 1 x 105 M1 or M2 macrophages were incubated with BL Raji cells (E/T

= 5:1) for 24 h in a 24-well plate, and supernatants (500 µL) were collected thereafter. M1

macrophages were incubated with buffer as a negative control. For positive control, M1

macrophages were treated with phorbol-12-myristate-13-acetate (PMA, 100 ng/ml) for 24 h.

Supernatants were concentrated 10-fold by lyophilisation (Lyovac GT 2, Leybold-Heraeus),

and protein content in the supernatant was determined by BCA protein assay. Lysates were

loaded onto a nitrocellulose membrane, and anti-cathelicidin (clone: 3D11, Hycult Biotech,

1:500) was diluted in 5% nonfat milk and 3% BSA in PBS. After washing and incubation

with the species-specific, HRP-conjugated secondary Ab (Sigma), immunoreactive proteins

were detected by chemiluminescence (Amersham Biosciences) following the manufacturer’s

protocol.

1,25D3 or BXL-628 treatment

Generated M1 and M2 macrophages (1x105) were activated with different concentrations of

1,25D3 or BXL-628 (1x10-7, 1x10-8, and 1x10-9) for 24 hours. For the qPCR, macrophages

were treated as described above. For the cytotoxic assay, cells were harvested with EDTA,

washed, and co-incubated in round-bottom microplates (96-well) (48 h, 37°C) with BL Raji

target cells in various E/T ratios (1:1, 5:1, and 10:1). Non-adherent cells were harvested and

stained with a CD11b antibody (to exclude macrophage contamination). Viability of CD11b-

cells was determined by Annexin-V and 7AAD staining.

Vitamin D treatment of healthy volunteers

Otherwise healthy volunteers with vitamin D deficiency (< 10 ng/ml) were supplemented with

40000 IE units of vitamin D weekly. Blood was drawn from these individuals for isolation of

peripheral blood mononuclear cells before and after 6 weeks of vitamin D supplementation.

All participants gave written informed consent for blood collection. This part of the study was

approved by the local Ethic committee “Ärztekammer des Saarlandes” file 227/12.

ADCC with rituximab

Monocytes from healthy donors before and after vitamin D supplementation were isolated and

differentiated to M2 macrophages (in the presence of autologous serum, 50%) as described

above. Raji cells were incubated with rituximab (1 µg/ml) for 30 min at 4°C, washed, and co-

incubated with M2 macrophages in round-bottom microplates (96-well) (24 h, 37°C) in

various E/T ratios (1:1, 5:1, and 10:1). In experiments using frozen samples from patients

with high-grade lymphoma, isolated TAMs were treated with 1,25D3 (10-7 M) for 24 h,

washed, and incubated with autologous lymphoma cells in round-bottom microplates (96-

well) in the presence or absence of rituximab (1 µg/ml) (48 h, 37°C). Non-adherent cells were

harvested and stained with an anti-CD11b antibody (to exclude macrophage contamination).

Viability of CD11b- cells was determined by Annexin-V and 7AAD staining.

Statistical analysis

Comparisons between patients and controls were done with nonparametric Mann-Whitney U-

test, and two-tailed Student’s t-tests were used for all other figures. The results are presented

as mean ± standard error of the mean (SEM). Differences were considered significant if

p<0.05.

Figure S1

M2-like phenotype of TAMs in BL. (A) Infiltration of CD68+ cells (macrophages) or (B) CD163+ cells (M2 macrophages) was quantified in reactive lymph nodes (control, n=9) and BL (n=9). Numbers of positive cells per mm² in the area with the densest infiltration were evaluated using the image analysis software BZ-9000 (Keyence). (C) Lymph node sections of the control group or BL patients were stained with anti-CD206 (mannose-receptor, M2 marker) and anti-iNOS (nitric oxide synthase, M1 marker) (n=8). Scale bar = 200 µm.

Figure S2

Cytokine profile in lymph node sections. qPCR analysis of coding RNA for IFNγ, IL4, IL10, and M-CSF isolated from paraffin-embedded tissues (control: benign reactive lymph nodes, n=8; BL: lymph node sections of BL patients, n=7).

Figure S3

Phenotype of generated M1 and M2 macrophages. (A) Expression of surface markers CD68, CD163, HLA-DR, CD11b, CD206 and transcription factors IRF5 and IRF4 by flow cytometry. (B) ELISA quantitation of cytokines (IL12, IL10, TNF) in the supernatant after activation with LPS (100 ng/ml for 48 h) (M1 upper panel, M2 lower panel). Histograms show representative results from 5 independent experiments. The bar graphs show average concentrations (pg/ml, n=5). (C) Secretion of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) by M1 or M2 macrophages was evaluated by ELISA. Error bars show SEM (n=5).

Figure S4

Activation of M1 and M2 macrophages after exposure to Raji targets. (A) M1 and M2 macrophages were incubated with Alexa647-labeled BL Raji cells (red) (E:T = 5:1) for 4 h in an 8-chamber slide. Macrophages were stained with an anti-phospho-tyrosine antibody (green) and analyzed by fluorescence microscopy (DAPI, nuclei, blue; DIC: differential interference contrast). Photographs show representative areas from one out of four independent experiments performed with different donors. Scale bar: 10 µm. (B) M1 or M2 macrophages were incubated with BL Raji cells (E:T = 5:1) for 24 h. PMA (100 ng/ml)-exposed cells were used as a positive control. Macrophages (CD11b+) were analyzed for their CD107a expression by flow cytometry. Concentration of 25D in the medium used in A and B was ≈ 70 nmol/L. The histograms show a representative result from 3 independent experiments.

Figure S5

Analysis of effector molecules secreted by macrophages. (A) Expression of FasL and TRAIL on M1 and M2 macrophages was analyzed by flow cytometry (M1 gray, M2 black line). Histograms show representative results from 5 independent experiments. (B) Bars show average mean fluorescence intensity (MFI) of 5 independent experiments. (C) Expression of gp91-phox (subunit of the NADPH oxidase) and ROS (DCF used as an indicator for reactive oxygen) production in M1 and M2 macrophages was evaluated by flow cytometry. The graphs show average MFI of 5 independent experiments. (D) Secretion of TNF after activation of M1 or M2 macrophages with LPS (100 ng/ml for 48 h) or after co-culture with Raji targets (5:1) was quantified by ELISA (M1 gray bar, M2 black bar). (E) Effect of TNF on viability of Jurkat cells (TNF-sensitive) or Raji cells was measured by Annexin-V/7-AAD staining by flow cytometry after 24 hours. The graph shows the average result of five independent experiments.. Error bars show SEM.

Figure S6

Expression of TNFR1, CD95, DR4, and DR5 on BL cell lines. Expression of TNF receptor 1 (TNFR1), CD95 (Fas), death receptor 4 (DR4), and death receptor 5 (DR5) was analyzed by flow cytometry (specific antibody gray, isotype control black line) on BL30, BL41, BL70, and Raji cells. Histograms show representative results from 5 independent experiments.

Figure S7

Phagocytosis of BL cells. (A) M1 or M2 macrophages were cocultured with various CFSE-labeled BL cell lines for 24 hours (E:T 10:1). Macrophages (CD11b+) were harvested, and the percentage of macrophages with phagocytic activity (CD11b+ and CFSE+) was analyzed by flow cytometry. Scatter plots show representative results from 5 independent experiments. (B) Bar graphs show mean percentage of phagocytic M1 (gray) or M2 (black) macrophages (CD11b+ and CFSE+) compiled from 5 independent experiments. Concentration of 25D in the media used in A and B was ≈ 70 nmol/L. Error bars show SEM.

Figure S8

Dose-dependent cytotoxicity of LL-37 for BL Raji cells. The effect of synthetic LL-37 (black square) or a scrambled peptide control (open square) on viability of BL Raji cells was measured by Annexin-V/7-AAD staining and flow cytometry after 24 hours. The graph shows average viability combined from 5 independent experiments. Error bars show SEM.

Figure S9

Expression of “LL-37 receptors” on BL cell lines and proliferating B cells. (A) Expression of formyl peptide receptor 1 (fMLP), P2X7R, and insulin-like growth factor 1 receptor (IGF-1R) on BL Raji, BL30, BL41, BL70 and on M2 macrophages was analyzed by flow cytometry (specific antibody gray, isotype control black line). (B) Receptor expression on P493-6 cells in the presence or absence of tetracycline (10 nM) or on normal B cells after stimulation with pokeweed mitogen. Histograms show representative results from 4 independent experiments. (C) Bars show average mean fluorescence intensity (MFI) of 4 independent experiments. Error bars show SEM.

Figure S10

Cytotoxic activity of LL-37 counteracted by overexpression of BCL-2. (A) Murine lymphoma cell lines 291PC or 291PC-BCL-2 (overexpressing BCL-2) were treated with LL-37 (2.5 µM, 24 h), and viability was analyzed by flow cytometry (7-AAD exclusion). Representative histogram of 1 out of 4 independent experiments. (B) Compilation of 4 independent experiments. The graph shows the cytotoxicity of LL-37 relative to the untreated control (n=4). Error bars show SEM.

Figure S11

LL-37 targeting mitochondria of BL cells. B cells from healthy donors or BL30 or BL41 cells were incubated with synthetic LL-37-TAMRA (2.5 µM, red) for 4 h, and mitochondria were stained with MitoTracker Green (100 µM, green). Hoechst 33258 (1 µg/ml, blue) was added for nuclear staining. Scale bar: 10 µm. Photographs show representative slides from three independent experiments.

Figure S12

Z-stack analysis of colocalization of LL-37 and mitochondria. Confocal z-stack analysis indicated that LL-37-TAMRA (red) predominantly colocalizes with the mitochondria labeled with the marker HSP60 (green) in Raji cells. Z-stacks of 8.03 µm thickness (0.4 µm intervals) were acquired with a LSM 510. Scale bar: 10 µm.

Figure S13

Colocalization of LL-37 and mitochondria. Colocalization of LL-37-TAMRA (red, upper panel) or a scrambled peptide (red, lower panel, used as control) with the mitochondrial marker HSP60 (green) in BL Raji cells. Cells were treated with LL-37-TAMRA or the scrambled control peptide (both 2.5 µM). Raji cells were stained with anti-HSP60, anti-rabbit Alexa 647 antibodies, and Hoechst nuclear dye. Slides were analyzed by confocal laser microscopy and evaluated for co-localization of LL-37-TAMRA/control peptide and HSP60. Photographs show representative slides from three independent experiments. Scale bar: 10 µm.

Figure S14

ROS induced by LL-37 in BL cells. (A) Mitochondrial superoxides were detected by MitoSox (100 µM), and (B) viability of cell lines was determined by 7-AAD exclusion using flow cytometry. The histograms show representative results from 1 out of 4 independent experiments.

Figure S15

Expression of antiapoptotic BCL-2 family proteins in BL cells. Protein expression of anti-apoptotic BCL-2 family proteins in western blot analysis of various BL cell lines (BL30, BL41, BL70, and Raji). Total protein extracts from the cell lines were analyzed by western blot for BCL-2, MCL-1, BCL-xl, and β-actin expression. The blot shows a representative result from 3 independent experiments.

Figure S16

Cathelicidin mediating vitamin D–induced cytotoxicity of M2 macrophages. (A) M2 macrophages

were treated for 2 days with 1,25D3 or BXL-628 (10-7 M, 10

-8 M, and 10

-9 M), and cathelicidin (red)

expression was analyzed by fluorescence microscopy. A minimum of 100 macrophages was analyzed by fluorescence microscopy. Scale bar: 10 µm. (B) M2 macrophages were incubated for 2 days with

1,25D3 (10-7 M, control) or BXL-628 (10

-7 M, control) and subsequently cocultured with BL Raji

target cells at an E:T ratio of 5:1 for 48 h in the presence or absence of a monoclonal anti-cathelicidin antibody (10 µg/ml) or an isotype control (IgG1, 10 µg/ml). Viability of Raji cells (gated CD11b-) was determined by Annexin-V/7-AAD staining and flow cytometry. The graph shows percent killing of BL Raji cells (CD11b- and 7-AAD+) calculated relative to the untreated control (n=10). Error bars

show SEM. (C) BL Raji cells were incubated for 4 days with 1,25D3 or BXL-628 (10-7 M, 10

-8 M, 10

-9

M). Viability of the lymphoma cells was determined with Annexin-V/7-AAD staining by flow cytometry. The graph shows percent killing of BL Raji cells (Annexin-V+ and 7-AAD+ cells) relative to the untreated control (n=5). Error bars show SEM. Concentration of 25D in the media used in A-C was ≈ 70 nmol/L.

Figure S17

Time course of macrophage-mediated cytotoxicity. Raji cells were incubated alone or in the presence of M1 (A) or M2 (B) macrophages (pretreated with or without 1,25D3) at a 5:1 E/T ratio for 24, 48, 72, and 120 hours (96 well plates, round bottom). Concentration of 25D in the medium was ≈ 70 nmol/L. Viability of Raji cells (gated on CD11b-) was determined by Annexin-V/7-AAD staining with flow cytometric analysis. Error bars show the SEM. Decreasing viability in the control group (black circles, Raji alone) at 120 h was due to consumption of medium.

Figure S18

Phenotype of M2 macrophages after vitamin D treatment. In vitro generated M2 macrophages

(n=6) were treated for 48 hours with 1,25D3 (10-7 M) and analyzed for (A) the macrophage marker

CD68, (B) for M1 markers (cathelicidin, IRF5, IL12,) or (C) for M2 markers (CD163, CD206, IRF4, and IL10) by quantitative real-time PCR. Concentration of 25D in the medium was ≈ 70 nmol/L. M1 macrophages were used as a positive control for M1 markers and as a negative control for M2 markers. The graphs show the average x-fold mRNA expression of vitamin D-treated samples compared with untreated M2 macrophages from 6 independent donors. Error bars show SEM.

Figure S19

VDR, CYP27B1, and CYP24A1 expression in vitamin D–treated healthy volunteers. Monocytes were isolated before and after vitamin D supplementation, differentiated to M2 macrophages, and VDR, CYP27B1, and CYP24A1 expression was determined by quantitative real-time PCR. Concentration of 25D in the medium was ≈ 70 nmol/L.

Figure S20

Cell sorting strategy to obtain TAMs and lymphoma cells. TAMs (CD163+/CD15-) and lymphoma B cells (NHL cells, CD20+) were isolated from bone marrow of NHL patients using a FACS Aria cell sorter (BD).