Splenic Marginal Zone Granulocytes Acquire an Accentuated Neutrophil

Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-15-3486 Cancer Microenvironment and Immunology Research

Splenic Marginal Zone Granulocytes Acquire an Accentuated Neutrophil B-Cell Helper Phenotype in Chronic Lymphocytic Leukemia Marcel Gatjen€ 1, Franziska Brand2, Michael Grau3,4, Kerstin Gerlach1, Ralph Kettritz5, Jorg€ Westermann6, Ioannis Anagnostopoulos7, Peter Lenz3, Georg Lenz4,8, Uta E. Hopken€ 2, and Armin Rehm1,6

Abstract

Recruitment of tumor-associated macrophages and neutro- the phenotype of monocytes and neutrophils. Selective ablation phils (TAM and TAN) to solid tumors contributes to immuno- of either of these cell populations in mice delayed leukemia suppression in the tumor microenvironment; however, their growth. Despite tumor infiltration of these immune cells, a contributions to lymphoid neoplasms are less clear. In human systemic inflammation was not detected. Notably, in progressive chronic lymphocytic leukemia (CLL), tumor B cells lodge in CLL, splenic neutrophils were observed to differentiate toward a lymph nodes where interactions with the microenvironment B-cell helper phenotype, a process promoted by the induction of occur. Tumor cell homing stimulates proliferation, such that leukemia-associated IL10 and TGFb. Our results suggest that engagement of the B-cell receptor is important for malignant targeting aberrant neutrophil differentiation and restoring mye- progression. In the Em-Tcl1 murine model of CLL, we identified loid cell homeostasis could limit the formation of survival niches gene expression signatures indicative of a skewed polarization in for CLL cells. Cancer Res; 76(18); 1–13. 2016 AACR.

Introduction resume provided that they get access to stimuli within specific niches (4). The concept of the microenvironment as a pivotal Chronic lymphocytic leukemia (CLL) is a low-grade B-cell þ regulator of CLL antiapoptotic signaling and cell division implies tumor characterized by an accumulation of monoclonal CD5 a leading role of antigen stimulation through the B-cell receptor mature B cells in secondary lymphoid organs (SLO), bone mar- (BCR; ref. 4). Accordingly, inhibition of Bruton tyrosine kinase led row, and peripheral blood (1). Gene expression profiling (GEP) to an impressive clinical success (5). Accessory signals provided by showed that these malignant B cells resemble antigen-experienced the microenvironment, that is, those perceived by Toll-like recep- memory B cells (2, 3). Although many circulating CLL cells are in a tors (TLR; ref. 6), and B-cell growth factor receptors (7), are quiescent G –G cell-cycle phase, their proliferative capacity can 0 1 concomitantly engaged with the BCR. In addition, a strong influence of excess growth signals from the microenvironment, including B-cell–activating factor (BAFF), could already be 1Department of Hematology, Oncology and Tumorimmunology, Max- involved in clonal selection and costimulate putatively autoreac- Delbruck-Center€ for Molecular Medicine, Berlin, Germany. 2Depart- tive B cells (8). € ment of Tumor Genetics and Immunogenetics, Max-Delbruck-Center In vitro, CLL cells rapidly undergo apoptosis from which they for Molecular Medicine, Berlin, Germany. 3Department of Physics, Philipps-University Marburg, Marburg, Germany. 4Cluster of Excel- can be rescued by coculturing with stromal cells and cytokines lence EXC 1003, Cells in Motion, Munster,€ Germany. 5Department of (9). This interaction provides tumor cells with prosurvival Nephrology and Intensive Care Medicine, Experimental and Clinical signals and conversely, stromal cells are transformed toward Research Center, Charite-University Medicine Berlin, Berlin, Germany. 6Department of Hematology, Oncology and Tumorimmunology, a tumor-permissive niche. In vitro, a hallmark of lymphoma- Charite-University Medicine Berlin, Berlin, Germany. 7Institute of induced stroma polarization is the activation of the NF-kB Pathology; Charite-University Medicine Berlin, Berlin, Germany. pathway associated with an inflammatory lymphoid stroma 8Translational Oncology, Department of Medicine A, University Hos- pital Munster,€ Munster,€ Germany. phenotype (10, 11). Recently, we have shown that murine CLL cells in the transgenic Note: Supplementary data for this article are available at Cancer Research Em-Tcl1 model migrated CXCR5 dependently into B-cell follicles Online (http://cancerres.aacrjournals.org/). where they lodged adjacent to follicular dendritic cells (FDC). € M. Gatjen, F. Brand, and M. Grau contributed equally to this article. Leukemia B cells trafficked in a marginal zone (MZ) B-cell–like A. Rehm and U.E. Höpken contributed equally to this article. manner as they moved directly from the MZ sinus across the MZ- Corresponding Authors: Armin Rehm, Department of Hematology, Oncology white pulp border into the B-cell follicle (12). It remains still and Tumorimmunology, Max-Delbruck-Center€ for Molecular Medicine, Robert- unclear how tumor cells receive antigenic stimulation and second, Rossle-Str.€ 10, Berlin 13125, Germany. Phone: 4930-9406-3817; Fax: 4930-9406- what role accessory signals from the microenvironment have on € 3124; E-mail: [email protected]; and Uta E. Hopken, top of BCR activation. [email protected] Here, we defined tumor stroma as the nonmalignant doi: 10.1158/0008-5472.CAN-15-3486 tumor cell microenvironment, including mesenchymal cells, 2016 American Association for Cancer Research. vasculature, extracellular matrix, but also hematopoietic cells.

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þ Functionally, stroma forms a growth and survival niche for PBS/1% BSA. The shift of green fluorescence in Gr-1 neutrophils lymphoma B cells in SLOs. We analyzed the leukemia cell– was determined and the mean fluorescence intensity (MFI) is imposed stromal alterations along their trafficking route reported. including the splenic MZ, the B-cell follicle, and the FDC network. Adoptive tumor cell transfers or in vitro cell cultures Gene expression profiling are often skewed towards a strong inflammatory component Gene expression profiling was done as described previously and may mimic rather a tumor initiation phase. We performed (12). An extended description of the method is given in Supple- GEP on laser capture microdissected tissues obtained from mentary Data. spontaneously diseased Em-Tcl1 mice, a condition that might fl better re ect an equilibrium or escape phase in tumor pro- Gene set enrichment analysis gression (13, 14). We demonstrate a splenic tissue remodeling Gene set enrichment analysis (GSEA) was performed as predominated by the expansion and polarized differentiation described previously (17) against an integrated database con- of monocytic cells and neutrophils. This innate immune cell taining the Molecular Signature Database v3.1 (18), the Gene- fi fl in ltration was not associated with a systemic in ammation. SigDB (19) and the Staudt Lab library (20). Signatures of In established CLL, tumor-associated macrophages (TAM) human genes were translated via gene homology. Only gene exhibited an M2 polarization, whereas tumor-associated neu- signatures that displayed a significant enrichment [P < 0.005, trophils (TAN) in spleen were skewed toward a neutrophil B- false discovery rate (FDR) < 5%] as well as those that contained – cell helper like (NBH) phenotype. Selective depletion of mye- at least 20% significantly differentially expressed genes (P < loid subpopulations in mice retarded leukemia progression 0.05 by two-sample t tests when comparing leukemia B cells substantially. Innate immune cells provided additional growth with Wt stroma samples, respectively, by paired t tests when factors, but were also involved in the structural integrity of the comparing measured with hypothetical mixtures) were consid- fi spleen necessary for leukemia cell traf cking and homing. ered to represent differentially regulated pathways. Signatures with less than 10 genes were filtered out. Materials and Methods Adoptive tumor cell transfers Antibodies Recipient mice were injected intravenously with 1–2 106 Em- A comprehensive list of all antibodies is given in Supplemen- Tcl1 cells. For the depletion of neutrophils, the anti-Ly6G clone tary data. 1A8 (Biolegend) or a rat IgG2a k isotype control antibody were injected intraperitoneally. Mice Em-Tcl1 transgenic mice on a mixed C3H/C57BL/6 (B6C3F1) or In vivo in vitro C57BL/6 background (F10) were used as described previously and cell proliferation (12). C57BL/6-Gt (ROSA)26Sor/J (iDTR) Bromodeoxyuridine (BrdUrd) incorporation into proliferat- mice were bred to LysM-Cre mice to create LysM-Cre/iDTR mice ing leukemia cells was analyzed as described previously (12). m where the diphtheria toxin receptor (DTR) is expressed in macro- BrdUrd was added at 10 mol/L daily into cocultures of phages. MARCO gene-deleted mice (MARCO / ) were used as leukemia cells and isolated neutrophils (cell ratio of 1:10). described previously (15). An extended description of mice is For BAFF blockade, a recombinant mouse BAFF receptor/Fc given in Supplementary data. chimera (BAFFR-Ig) was used (R&D Systems), and for APRIL neutralization an anti-APRIL antibody (clone: Apry-1-1; Adi- Flow cytometry and cell sorting pogen) was added. Cells were blocked with CD16/32 antibody and further antibody In vivo tumor cell apoptosis detection stained and washed in FACS buffer [PBS, 0.5% w/v BSA, 0.05% þ þ (v/v) NaN ]. A flow cytometry gating strategy for leukemia cells was Apoptosis of splenic CD5 CD19 leukemia B cells was 3 fl – performed as described previously (12). Neutrophils were gated assessed with ow cytometry applying AnnexinV FITC and pro- þ þ based on SSC/FSC scatter plot, and CD11b Ly6G reactivity. pidium iodine (PI) staining (eBioscience) exactly as described by þ þ þ þ Macrophages were gated by CD11b Ly6C or CD11b F4/80 the manufacturer. staining. Data were acquired on a FACSCantoII flow cytometer and analyzed with FlowJo software (TreeStar). Cell sorting was In vivo blockade of neutrophil activation done on a FACSAria (BD Biosciences). Em-Tcl1 mice were treated over 8 days with three injections intraperitoneally of an anti-IL10 (500 mg; clone JES5-2A5, Biole- Measurement of respiratory burst gend) and anti TGF-b (500 mg; clone 1D11.16.8, BioXCell) Neutrophils from femurs and tibias were flushed with PBS and antibody. Alternatively, an anti-IL10 receptor (IL10R) antibody isolated by Percoll density gradient centrifugation. Superoxide (clone 1B1.3A, BioXCell) was applied. For mock control, a rat was measured using the superoxide dismutase-inhibitable ferri- IgG1 isotype antibody was used. cytochrome C reduction assay as described previously (16). Generation of intracellular reactive oxygen species was measured Statistical analysis by oxidation of CM-H2DCFDA (Sigma-Aldrich). Bone marrow Results are expressed as arithmetic means SEM if not other- or splenic neutrophils (1 107/mL HBSS) were loaded with wise indicated. Values of P < 0.05 were considered statistically 1 mmol/L CM-H2DCFDA for 15 minutes at 37 C. A total of 2.5 significant, as determined by the Mann–Whitney U test, the 105 cells were incubated with buffer control, PMA, or opsonized unpaired or paired Student t test, or the Wilcoxon signed rank zymosan. Reactions were stopped after 60 minutes by adding test where appropriate.

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þ þ Results follicles and adjacent MZ. GEPs from sorted CD5 CD19 Gene expression signatures reveal a predominant myeloid cell tumor cells were determined separately (12). We utilized a infiltration of the splenic stroma compartment in leukemic Em- linear biostatistical mixture model whereby tumor cell– Tcl1 mice induced changes in gene expressions of stroma cells could be Using genome-wide expression arrays, we examined relative identified selectively. We identified 502 genes that were selec- changes in gene expression from na€ve wild-type (Wt) stroma tively upregulated (P < 0.01) in tumor-bearing stroma com- compared with tumor-bearing Em-Tcl1 stroma covering B-cell pared with na€ve Wt mice (Fig. 1A). The Venn diagram

Figure 1. Expansion of Em-Tcl1 leukemia cells in spleen induces expression of genes associated with myeloid cell structure and function. A, gene expression proﬁling of tumor-bearing Em-Tcl1 splenic stroma and Wt (B6C3F1) stroma. Compared are all genes that were signiﬁcantly (P < 0.01) upregulated. B, the volcano plot depicts all upregulated genes in tumor versus Wt stroma. C, a heatmap showing differential gene expression in tumor (n ¼ 6) compared with Wt stroma (n ¼ 6). Gene expression changes are depicted according to the color scale. Depicted are genes of the signature "LIAN_LIPA_TARGETS_3M" (P 0.001 by permutation test; FDR ¼ 0.001; enrichment score ¼0.766). D, enrichment plot of gene expression signature.

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Figure 2. Increase of innate immune cells in tumor-bearing Em-Tcl1 mice. A, splenic leukocytes from Wt and Em-Tcl1 mice (B6C3F1) were differentiated by flow cytometry. The gating strategy for granulocytes CD11bþLy6Gþ, inflammatory monocytes CD11bþ Ly6Chigh, and macrophages CD11bþ Ly6Cint is shown. Representative dot plots from two–three experiments with n ¼ 4 Wt and n ¼ 5Em-Tcl1 mice are given. Numbers indicate the percentage of cell populations within the gates. B, quantification of myeloid cells (CD11bþ) in spleens, bone marrow (BM), and PBL. C, inflammatory monocytes (CD11bþLy6Chigh)and macrophages (CD11bþ Ly6Cint; D)in spleens and granulocytes (CD11bþLy6Gþ; E) were determined in spleens, bone marrow, and PBL. F, serum from Wt (n ¼ 8) and Em-Tcl1 mice (n ¼ 7) was analyzed by cytokine bead array. G, spleen sections were stained for MARCOþ MZ macrophages, MOMA-1þ metallophilic macrophages, B220þ B cells, and Ly6Gþ granulocytes (n ¼ 3–5 mice/ group). Tissues were analyzed by immunofluorescence microscopy. Scale bar, 100 mm. In B–E, P values were determined by a Mann–Whitney U test, and in F, bars indicate the mean SEM, and an unpaired Student t test was applied. , P < 0.05; , P < 0.01; , P < 0.001; n.s., nonsignificant.

illustrates the speciﬁcity of the biostatistical model to detect To gain insights into biologic processes, we used the 126 changes solely in tumor-bearing stroma because only 55 over- top upregulated genes (P < 0.001; Supplementary Table S1) lapping genes between both analysis groups were detected. as input for Gene Ontology (GO) analysis. Signiﬁcant

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Figure 3. Macrophages support Em-Tcl1 progression. A, MARCO/ (n ¼ 13) or Wt mice (B6; n ¼ 10) were transplanted intravenously with 1 106 Em-Tcl1 leukemia cells, and splenic tumor load was determined after 31–32 days (n ¼ 2 independent experiments). B, DT (n ¼ 9) or NaCl (n ¼ 8) was applied repeatedly to LysM-Cre/iDTR mice. Em-Tcl1 leukemia B cells were transferred (1 106), and CD5þCD19þ tumor cell load was assessed after 13 days (n ¼ 2 independent experiments). Macrophages were detected by CD11bþF4/80þ staining. C, spleen sections from NaCl (DT) or DT- treated (þDT) LysM-Cre/iDTR animals (n ¼ 3–5 animals per treatment group) were stained for leukemia B cells (Tcl- 1þ), MOMA-1þ, and CD11cþ (DCs; top), and for B cells (B220þ), MOMA-1þ, and MARCOþ (bottom). Scale bar, 100 mm. In A and B, unpaired Student t tests were applied. Error bars, mean SEM. , P < 0.05; , P < 0.01; , P < 0.001. D, SNARF-1-labeld leukemia cells were transferred (1 107) two days after start of DT- (n ¼ 8) or NaCl pretreatment (n ¼ 9) into LysM-Cre/ iDTR animals. After 5 hours, mice were sacriﬁced. Spleen sections were stained for MAdCAM-1þ (marginal reticular cells) and B220þ. One representative section for each group is shown. Broken white lines, MAdCAM-1 ring. Scale bar, 100 mm. E, Em-Tcl1 cells from D were counted within and outside the follicle in the MZ and RP (n ¼ 5 independent experiments). Three sections per spleen were stained; for each section 5 images of nonoverlapping areas were analyzed. Data are presented as percent reduction of total MZ/RP- localized tumor cell numbers in controls (set to 100%) versus DT- treated animals. The Wilcoxon signed- rank test was applied. Error bars, mean SEM. , P < 0.05.

overrepresentation of the terms neutrophil chemotaxis [e.g., Marco,log2(ratio) ¼ 1.8, P ¼ 2.2e5; Fig. 1B; Supple- (GO:0030593, 7/33 genes are among the top 126 upregulated mentary Table S2). genes of all 21,225 measured genes, P ¼ 8.3e10, hypergeo- GSEA conﬁrmed enriched signatures related to granulocytes, metric test), innate immune response (GO:0045087, 11/176 innate immune response, DCs, macrophages, and monocytes genes, P ¼ 8.3e9), and neutrophil aggregation (GO:0070488, (Supplementary Fig. S1A–S1J). An exemplary heatmap depicting 2/2 genes, P ¼ 3.5e5) was obtained. The top 25 upregulated the signature "LIAN_LIPA_Targets_3M" contains genes indicative genes [log2(ratio) 1.5] contained markers that are preferen- of granulocyte (e.g., Cxcr2, Ccr1, C3ar1, CD244), macrophage tially expressed by neutrophils [e.g., Mpo,log2(ratio) ¼ 1.6, (CD68, Marco, Mafb), and DC-associated (Tlr7, Spic) expression P ¼ 0.002; Ly6G,log2(ratio) ¼ 1.9, P ¼ 1.4e5], DCs [e.g., (enrichment score -0.77, P ¼ 0.001 via permutation test, FDR ¼ CD209b,log2(ratio) ¼ 2.9, P ¼ 5.9e6], and macrophages 0.001; Fig. 1C and D).

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þ Expansion and altered differentiation of myeloid cell neutrophil infiltration (MPO ) into the neoplastic MZ, a reduc- subsets tion of the MZ-red pulp (RP) demarcation, and a tight intermin- Flow cytometry analysis of myeloid leukocytes was aimed at gling between neutrophils and lymphoma B cells (Supplementary a discrimination of differentiation states depending on the Fig. S3). Thus, the murine model mimics microanatomic aspects anatomic context. We found enhanced frequencies of myeloid of granulocyte infiltration into distinct splenic compartments as þ cells (CD11b )inleukemia-bearingEm-Tcl1 mice compared seen in an indolent human lymphoma. with Wt mice in spleen, bone marrow, or peripheral blood þ (Fig. 2A and B). Total numbers of CD11b cells were increased Macrophage depletion delays Em-Tcl1 leukemia progression 2.5-fold in spleen, but not in bone marrow. An increase in The macrophage receptor with collagenous structure (MARCO; frequencies and numbers (5.5-fold) of inflammatory mono- Supplementary Table S2) confers MZ macrophages with a high þ cytes (CD11b Ly6Chigh)aswellasforpatrollingmonocytes/ capacity for particle uptake important for MZ B-cell interactions. þ macrophages (CD11b Ly6Cint; 4.9-fold) was obtained in Because Em-Tcl1 leukemia cells traffic in a MZ B-cell–like manner, spleens from Em-Tcl1 mice (Fig. 2C and D). we explored the clinical disease course in MARCO / mice after Next, we compared gene expression from in vitro differentiated adoptive cell transfer. Up to 32 days, tumor load in MARCO / M1 or M2 human monocytes with genes upregulated in Em-Tcl1 recipients was substantially lower compared with Wt animals stroma (21). Applying the Martinez and colleagues gene signa- (Fig. 3A). To deplete additional macrophage subsets, we used the ture, in tumor-exposed splenic stroma, a significant enrichment of Lysozyme M–directed myeloid-specific DT (LysM-Cre/iDTR) an M2 signature (enrichment score ¼0.61, GSEA P 0.001 by transgenic mouse strain. After DT or NaCl administration, we permutation test and P ¼ 0.001 by t test between signature adoptively transferred Em-Tcl1 leukemia B cells into LysM-Cre/ averages), was calculated (Supplementary Fig. S2). iDTR recipients. Macrophage depletion retarded substantially In spleens, bone marrow, and peripheral blood from Em-Tcl1 tumor progression in the spleen (Fig. 3B). A 3.4-fold reduction þ þ þ þ mice, an expansion in neutrophil (CD11b Ly6G ) numbers (4.6- of the spleen-resident F4/80 CD11b macrophage population þ fold) and frequencies occurred (Fig. 2E). In serum the release of was seen in DT-treated mice. Reduction of MOMA-1 and þ proinflammatory mediators (Fig. 2F) was indistinguishable MARCO MZ macrophages in DT-treated LysM-Cre/iDTR mice between Wt and Em-Tcl1 mice. GM-CSF and the proinflammatory could be confirmed (Fig. 3C). marker S100A8 remained below the detection limit. Hence, Next, we explored the possibility that splenic MZ macro- increased numbers of innate immune cells were not associated phages control leukemia cell translocation into B cell follicles. with a systemic inflammation. Labeled tumor cells were injected into LysM-Cre/iDTR mice To assess the conditions for stroma remodeling, we analyzed pretreated with DT or NaCl. Five hours later, spleens were "secreted protein acidic and rich in cysteine" (SPARC) gene stained for B-cell follicles, and with anti MAdCAM-1 to high- expression. Under autoinflammatory settings, lack of SPARC light the border between the MZ and the follicles (Fig. 3D). We resulted in a disturbed compartmentalization within SLOs quantitated the numbers of labeled leukemia cells within the þ þ (22). Here, SPARC was weakly upregulated in Em-Tcl1 spleens follicles (B220 )andoutsideinMZ(MAdCAM-1 /B220 )and ¼ ¼ compared with controls [log2(ratio) 0.7; P 0.0053]. Collec- RP. A substantial reduction by 39% in the total number of tively, the innate immune response signature of Em-Tcl1 mice was leukemia cells in the RP and MZ of DT-treated LysM-Cre/iDTR characterized by an expansion of granulocytes and monocyte compared with mice with a preserved MZ macrophage ring was subsets, but lacked serum markers for an acute inflammatory state. observed (Fig. 3E). At this early time point, only a small þ An expanded neutrophil population (Ly6G ) was seen adja- proportion of tumor cells was detectable in the follicles. Col- þ cent to the MZ (MARCO ) and in the red pulp of Em-Tcl1 mice. lectively, this indicated that macrophages regulate the retention Neutrophils were absent from the B-cell follicle, as marked by the of Em-Tcl1 leukemia cells in the MZ. þ metallophilic marginal macrophage marker MOMA-1 and þ B220 staining (Fig. 2G). Next, we chose human splenic marginal Neutrophils support CLL progression in Em-Tcl1 mice zone lymphoma (SMZL) to compare localization of granulocytes Neutrophilic granulocytes constitute a significant fraction of with the Em-Tcl1 model. SMZL and murine CLL B cells are both inflammatory cell infiltrates found in many solid cancers. They are localized in the splenic MZ, grow in an indolent manner, and also referred to as TANs and depending on their activation and share a strong dependency on BCR and TLR signaling (23). In differentiation state, anti- and protumorigenic functions have SMZL compared with normal spleen, we observed a stronger been proposed (24).

Figure 4. Depletion of neutrophils delays Em-Tcl1 leukemia growth by inhibiting cell-cycle progression. A, for the signature "Neutrophil granule constituents" the heatmap shows significantly upregulated gene expressions (P ¼ 4e5byt test between signature averages) in tumor stroma (Em-Tcl1 mice, n ¼ 6) compared with Wt stroma (n ¼ 6). Right, GSEA shows a significant enrichment of this signature at upregulated genes in Em-Tcl1 spleen (enrichment score ¼0.706; GSEA, P ¼ 0.001 by permutation test; FDR ¼ 0.002). B, overrepresentation analyses of GO terms in upregulated genes. The term "Neutrophil Chemotaxis" (GO:0030593, P ¼ 8.3e10, hypergeometric test) is given. C and D, Em-Tcl1 mice with a tumor load between 3% and 9.3% of all leukocytes in peripheral blood were treated with an anti-Ly6G antibody (n ¼ 11 mice; two independent experiments) or an isotype control (n ¼ 8) over 28 days. A representative dot plot shows gated CD11bþLy6Gþ neutrophils in C, total numbers and frequencies of splenic CD5þCD19þ tumor cells in D. E, antibody-treated Em-Tcl1 mice were injected with BrdUrd and splenic leukemia B cells were analyzed for BrdUrd uptake and AnnexinV/PI reactivity. F, Em-Tcl1 tumor cells were cocultured for 48 hours with splenic granulocytes (PMN) from Em-Tcl1 (n ¼ 7) or B6 animals (n ¼ 7; n ¼ 3 independent experiments); 25 ng/mL G-CSF was included. BrdUrd incorporation was analyzed by flow cytometry. G, CD5þCD19þ cells at day 28 after anti-Ly6G or isotype antibody treatment as in C were sorted and subjected to RT-qPCR analysis (isotype control, n ¼ 7; aLy6G treated, n ¼ 9). Gene expressions are depicted relative to Gapdh. D, an unpaired Student t test; in E and G, a Mann–Whitney U test; and in F, the Wilcoxon signed-rank test was applied. Error bars, mean SEM. , P < 0.05; , P < 0.01; , P < 0.001.

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GSEA revealed an enrichment of upregulated genes from sig- Gene expression for several chemokines (Ccl3, Ccl4, Ccl5, natures coding for granulocytes (enrichment score ¼0.706, Ccl6) and chemokine receptors (Ccr1, Ccr3, Cxcr2, Cxcr3)was GSEA P ¼ 0.001 by permutation test; P ¼ 4e5byt test between downregulated (Supplementary Fig. S5) in tumor-challenged signature averages; Fig. 4A). We confirmed the conditions for animals. Other inflammatory cytokines (IL1, Ltb)andreceptors neutrophil recruitment into leukemic spleens as indicated by the (IL6ra) were downregulated as well.Notably,thesamecytokine significant overrepresentation of the GO term "neutrophil che- and chemokine gene markers were indistinguishable when motaxis" covering chemotactic factors (S100A8, S100A9), recep- analyzed in bone marrow–derived neutrophils, indicating that tors (CXCR2), inflammatory cytokines (IL1b), and integrins microenvironmental conditions can lead to a distinct neutro- (Itgam/CD11b; Fig. 4B). phil differentiation. Splenic neutrophils were cultured for 24 We depleted neutrophils using a Ly6G-specific antibody over hours in the presence of G-CSF. The rate of apoptotic neutro- four weeks. Em-Tcl1 mice treated with the isotype antibody phils from Em-Tcl1 mice was 2.4-fold lower compared with Wt developed a 2-fold higher splenic tumor load compared with mice, as revealed by a higher frequency of AnnexinV /PI cells anti-Ly6G–treated animals (Fig. 4C and D). Disappearance of (Fig. 5E). þ þ CD11b Ly6G cells after anti-Ly6G-treatment was confirmed by We isolated neutrophils from bone marrow and used PMA flow cytometry and immunohistology (Fig. 4C and Supplemen- and zymosan that stimulate the respiratory burst via different tary Fig. S4A). In control spleens, neutrophils were found pre- signaling pathways. Release of the reactive oxygen (ROS) spe- dominantly in the RP, but also in the MZ where they tightly cies O2 was indistinguishable (Fig. 5F). Likewise, H2O2-cat- þ intermingled with leukemia B cells (SNARF-1 ). Neutrophils alyzed hydrolysis of the dye CM-H2DCGFDA into the fluores- remained excluded from the T cell and follicular B-cell zone. cent dye DCF was similar between Wt and Em-Tcl1 neutrophils Splenic-resident leukemia B cells from neutrophil-depleted (Fig. 5G). þ mice had a lower proportion of cells in S-phase (BrdUrd iso- The cytokines IL10 and TGFb to contribute to splenic neu- type-treated, 10.72% 0.6; Ly6G-treated 6.8% 0.4), but no trophil reprograming (24, 27). In GEP, Tgfb gene expression change in the apoptosis rate (Fig. 4E). Granulocyte depletion did itself was unchanged in leukemic stroma (Tgfb1–3, P > 0.05); not cause major changes in the inflammatory cytokine profile however, a strong upregulation of the TGF-b–induced gene (Supplementary Fig. S4B). Tgfbi was seen [log2(ratio) ¼ 1.3, P ¼ 0.005]. This gene encodes Next, we performed cocultures of isolated splenic neutro- an extracellular matrix protein that can bind integrins and thus, phils and Em-Tcl1 tumor cells. Neutrophils taken from either Wt confers adhesion to monocytes (28). In tumor-bearing mice, þ or Em-Tcl1 mice enhanced the BrdUrd uptake in leukemia cells TGFBi was much stronger associated with MAdCAM-1 struc- about 2-fold (Fig. 4F). However, we noted that splenic neu- tures in the B-cell follicle and MZ (Supplementary Fig. S6). For trophils in Em-Tcl1 mice outnumbered those obtained from Wt IL10, we found a strong gene upregulation in leukemia cells mice substantially (Fig. 2E). relative to Wt stroma [log2(ratio) ¼ 2.97, P ¼ 5e5], MZ B-2 Growth promoting and antiapoptotic stimuli for MZ B cells [log2(ratio) ¼ 2.99] or B-1 [log2(ratio) ¼ 2.52] lymphocytes. are often regulated via the NF-kB transcriptional pathway. When Release of IL10 from leukemia cells was 39-fold higher com- neutrophils were depleted, an obvious change of NF-kB target pared with Wt B cells. In serum from Em-Tcl1 mice, the immu- genes in leukemia cells was not seen. In the neutrophil-depleted noinhibitory cytokine was increased (mean Wt: <5pg/mL,Em- group the transcription factors early growth response 1 (Egr1) and Tcl1: 693 pg/mL; Fig. 6A and B). Tumor-bearing animals were Egr2 were significantly upregulated 1.7-fold (Egr1) and 1.6-fold treated with neutralizing anti-IL10/anti-TGFb antibodies. (Egr2; Fig. 4G). Egr1 enhances p21Waf/Cip1 promoter activity, Downregulation of CD62L and upregulation of CD16/32 indi- which results in cell-cycle arrest at the G2–M phase. At high cated that splenic neutrophils maintained their activation level expression Egr1 maintains hematopoietic stem cell quiescence in an IL10 and TGFb dependent manner (Fig. 6C). LAIR-1 as the and counteracts their migratory capacity. Egr2 seems to be main inhibitory receptor expressed on activated neutrophils crucial for defined B-cell developmental steps (25, 26). Collec- remained unaltered. tively, Egr1 and Egr2 transcriptional activities could control Next, we compared gene expression of sorted neutrophils þ þ leukemia B-cell activation, depending on the crosstalk with (CD11b Ly6G ) from Wt and Em-Tcl1 mice. The gene markers neutrophils. Ikbke, Bcl2a1a, and Mpo were chosen because they discriminate between granulocytes and granulocytic-myeloid derived suppres- Leukemia-associated neutrophils acquire a B-cell helper–like sor cells (G-MDSC; ref. 29). Neutrophils from Em-Tcl1 mice function exhibited lower gene expression for Mpo (Fig. 6D), indicating a Under physiologic conditions, a spleen-resident NBH popula- mature differentiation state. Expression of the antiapoptotic tion that receives reprograming signals from sinusoidal endothe- gene Bcl2a1a was 2-fold higher in neutrophils from Em-Tcl1 mice lial cells exists (27). TANs had a 1.5-fold higher gene expression (Fig. 6D). for Baff and a 3-fold higher April expression compared with Wt To corroborate that IL10 and TGFb led to neutrophil repro- mice (Fig. 5A). Because BAFF concentrations in serum were not graming and subsequently, leukemia expansion, we treated different (Fig. 5B), we isolated splenic neutrophils and stimulated Em-Tcl1 mice with a combination of anti-IL10R and anti-TGFb them with PMA. Induced secretion of BAFF was 2.4-fold higher in antibodies. In the anti-IL10R/anti-TGFb treatment group, the neutrophils from leukemic animals (Fig. 5C). transcription factor Egr1 was upregulated in isolated Em-Tcl1 cells, Next, we isolated granulocytes from bone marrow and cocul- indicating that tumor cells had entered cell-cycle arrest. This result tured them with Em-Tcl1 tumor cells for 48 hours. In the presence was consistent with the effects seen when neutrophils were of an inhibitory BAFF-receptor fusion protein in combination depleted (see Fig. 4G). Bcl2a1a, Baff, and April gene expressions with an anti-APRIL antibody, BrdUrd uptake in tumor cells was in neutrophils showed a tendency to fall when animals were reduced (Fig. 5D). treated with anti-IL10R/anti-TGFb antibodies.

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Figure 5. CLL-associated neutrophils provide growth factors and have an extended lifetime. A, RT-qPCR arrays were performed with neutrophils from Wt (n ¼ 3) and Em-Tcl1 mice (n ¼ 3). Expression of Baff and April are depicted relative to housekeeping genes (HKG). B, serum concentrations of BAFF (Wt, n ¼ 3; Em-Tcl1, n ¼ 4 mice). C, splenic neutrophils from Wt (n ¼ 3) and Em-Tcl1 mice (n ¼ 3) were cultured with or without PMA for 24 hours, followed by BAFF measurement. D, Em-Tcl1 tumor cells were cocultured for 48 hours with bone marrow (BM)-derived PMNs (B6) and 10 mmol/L BrdUrd. BAFF-R (10 mg/mL) and anti-APRIL (10 mg/mL) were added (n ¼ 3 independent experiments). Percentage of BrdUrdþ tumor cells is given. E, neutrophils from Wt (n ¼ 5) and Em-Tcl1 mice (n ¼ 6) were cultured in the presence of 50 ng/mL G-CSF for 24 hours. Apoptosis of Ly6Gþ granulocytes was measured by AnnexinV/PI staining. Ly6Gþ neutrophils are shown on the left; on the right, frequencies of viable (AVPI) and apoptotic cells are given (n ¼ 2 experiments). F, release of ROS from purified bone marrow neutrophils was induced with PMA and zymosan. Secreted O2 species were measured using the ferricytochrome C reduction assay. G, the respiratory burst activity in granulocytes was stimulated with PMA and zymosan (n ¼ 4Wt,n ¼ 4Em-Tcl1). The H2O2 catalyzed hydrolysis of nonfluorescent CM-H2DCFDA to fluorescent DCF was measured. Values are expressed as MFIs; n ¼ 2 independent experiments. In A–C and E–G, a Mann–Whitney U test, and in D, paired Student t test was used. Error bars, mean SEM. , P < 0.05; , P < 0.01; , P < 0.001; n.s., nonsignificant.

Collectively, IL10 and TGFb contribute to neutrophil differen- granulocytes cooperatively enhance the progression of CLL in tiation in Em-Tcl1 mice. mice. In human follicular lymphoma, the adverse immune-response signature 2 included genes associated with monocytes and DCs Discussion (30). In Em-Tcl1 mice, we validated expanded monocyte and We used GEP to identify the stromal alterations that occur neutrophilic granulocyte populations, a situation that pointed when an oncogene-driven lymphoid neoplasia lodges within to a link between inflammation and cancer (13). Increased murine spleens. In the absence of an overt proinflammatory numbers or an altered differentiation state of innate immune cytokine milieu, we showed that macrophages and neutrophilic cells is commonly observed in human chronic inflammation,

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Figure 6. Tumor cells in cooperation with the stroma microenvironment control the phenotyping skewing of neutrophils. A, IL10 content in serum from Wt (n ¼ 4) and tumor- bearing Em-Tcl1 mice (n ¼ 5). B, leukemia cells from Em-Tcl1 mice (n ¼ 3) with a splenic tumor load >50% of all leukocytes and follicular B cells from Wt mice (n ¼ 4) were cultured for 4 hours in the presence of PMA/ionomycin (þPMA). IL10 content in culture supernatant was measured by ELISA. C, Em-Tcl1 mice with a tumor load of 2.8%–8.9% in peripheral blood were treated with a combination of anti-IL10/anti-TGFb antibodies (n ¼ 5) or control antibody (n ¼ 5) over 8 days. Gated CD11bþLy6Gþ neutrophils with the indicated markers are given. Filled curve, isotype control. Quantiﬁcation of antigen expression is given as geometric MFI. D, splenic neutrophils were isolated from Wt (n ¼ 5) and Em-Tcl1 mice (n ¼ 11). For Wt, 3–4 experiments per marker and for Em-Tcl1 4–5 experiments were conducted. Gene expressions of Ikbke, Bcl2a1a,andMpo was determined by RT-qPCR, depicted are values relative to Gapdh. E, Em-Tcl1 mice were injected with anti-IL10R/TGFb antibodies (n ¼ 5) or an isotype control (n ¼ 5) as in C. Gene expression in isolated splenic CD5þCD19þ leukemia cells was determined by RT-qPCR and depicted are values relative to Gapdh. F, neutrophils isolated from the bone marrow (BM) of isotype (n ¼ 6) or anti-IL10R/anti-TGFb–treated (n ¼ 6) Em-Tcl1 mice were analyzed by RT-qPCR. Values are given relative to Gapdh.InA–F, a Mann–Whitney U test was applied. Error bars, mean SEM. , P < 0.05; , P < 0.01; n.s., nonsigniﬁcant.

which bears a substantial risk for extranodal marginal zone B-cell TNF and NF-kB signaling pathways (11). In contrast, we here lymphomas (31). analyzed stroma gene signatures under in vivo settings, which might To further define the relationship between innate immune cell better reflect systemic conditions of cellular networks. The occur- infiltration and a putative sterile inflammation in vivo, we assessed rence of an M2-skewed phenotype of TAMs indicated the gradual proinflammatory cytokines in serum from diseased mice; how- inhibition of NF-kB activity and is consistent with M2 polarization ever, these mediators were similar to controls. Recently, it was found in established tumors. On the contrary, activated NF-kBin suggested that anti PD-L1 blockade reverses a CLL-induced skew- myeloid cells is rather associated with tumor promotion in inflam- ing of myeloid cells, which was associated with the reduction of mation-associated cancer models (35). selected inflammatory cytokines (32). These effects were seen after In contrast to M1 inflammatory macrophages, macrophages adoptive tumor cell transfer and are therefore not comparable that occur in cancer tissues are polarized toward an M2 phenotype with our results from transgenic mice. (36). These cells enhance tumor progression, either through direct By unbiased GSEA, we found no obvious enrichment for tran- secretion of cytokines that sustain tumor cell proliferation, or by scriptomic signatures dependent on the proinflammatory tran- dampening the immune response through secretion of immu- scription factors NF-kB, AP-1, STATs, CEBP/b, and HIF-1a (33). nosuppressive cytokines and overexpression of T-cell–inhibitory In vitro, mesenchymal stromal cells (MSC) obtained from follicular ligands (14, 32, 37–39). In CLL, monocyte-derived nurse-like cells lymphoma–infiltrated bone marrow exhibited a higher CCL2 exist that resemble M2-polarized TAMs and support leukemia cell expression leading to recruitment of monocytes, followed by their survival in vitro (40, 41). In diffuse large B-cell lymphoma a high polarization into a TAM-like phenotype (34). Bone marrow–MSCs density of an M2-polarized TAM population correlated with an produced more neutrophil-attractant IL8 and were activated via unfavorable prognosis (42).

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The functional relevance of macrophages in human lympho- ment in leukemic spleens shapes the gene expression program of ma is still controversial (43–45). These uncertainties relate to this NBH-like subset, which does not exist in peripheral blood or in their lineage plasticity, which could be influenced by EBV bone marrow. Because of the local priming, drawing parallels to infection and cytokines, but also by the microanatomic local- human is tempting, but for ethical reasons splenic specimen from ization (46). Here, we obtained a substantial increase of CLL patients are not available. inflammatory Ly6Chigh monocytes and, on the other hand, Immunosuppressive G-MDSCs share the surface markers patrolling Ly6Cint monocyteswereexpandedaswell.Inaliver CD11b, Ly6G, and Ly6Clow with neutrophils (29). Release of model of sterile inflammation a phenotypic conversion increased amounts of ROS was shown to mediate immunosup- between Ly6Chigh and Ly6Cint occurred (47). This functional pression by G-MDSCs (54); however, in Em-Tcl1 neutrophils, ROS plasticity was controlled by IL4 and IL10, which implies that production was unaltered. Expression of two hallmark genes that Em-Tcl1 leukemia cells themselves might provide the stimuli for are either strongly overexpressed in G-MDSCs versus neutrophils M2 macrophage differentiation (13). (Mpo), or vice versa downregulated (Ikbke) was not substantially Macrophages in the spleen are strategically located to recognize different between Wt and Em-Tcl1 neutrophils. We conclude that and retain blood-borne antigens (48). We observed delayed Em- splenic neutrophils in leukemic mice exhibit a stronger overlap Tcl1 leukemia progression upon genetic deletion of the scavenger with neutrophils than with G-MDSCs. receptor MARCO and of MZ and metallophilic macrophages (15). Gene signatures obtained from Em-Tcl1 splenic stroma reflect a These results indicated that leukemia cells do not only trafficina homeostatic condition in which leukemia B-cell survival depends MZ B-cell–like pattern, but may cooperate with local macrophage on the expansion and skewed differentiation of innate immune subsets in a manner similar to benign MZ B cells. This interaction cells. Splenic neutrophils may act as tumor-instructed NBH-like regulates antigen uptake by macrophages and antigen capture by cells that provide the B-cell growth factors in excess, and thus B cells, followed by B-cell trafficking into the follicle and antigen influence leukemia B-cell ontogeny during B-cell selection and delivery onto FDCs (49). When splenic macrophages were pres- progression. ent, leukemia cells accumulated stronger in the MZ and adjacent areas as compared with macrophage-depleted animals. Our find- Disclosure of Potential Conflicts of Interest ing is consistent with the dynamics of physiologic B lymphocyte No potential conflicts of interest were disclosed. distribution in macrophage-depleted animals (50). In this sce- nario, MZ macrophages might control CXCR5/CXCL13-guided Authors' Contributions leukemia B-cell trafficking into B-cell follicles where a stronger Conception and design: R. Kettritz, G. Lenz, U.E. Hopken,€ A. Rehm FDC-dependent growth stimulus becomes accessible (12). Development of methodology: M. G€atjen, M. Grau, K. Gerlach, J. Westermann, Neutrophils exhibit substantial plasticity depending on envi- A. Rehm Acquisition of data (provided animals, acquired and managed patients, ronmental stimuli (51). Under the conditions of a tumor-prev- € b provided facilities, etc.): M. Gatjen, F. Brand, M. Grau, K. Gerlach, R. Kettritz, alent TGF milieu the differentiation of a tumor-supporting N2 J. Westermann, I. Anagnostopoulos, U.E. Hopken,€ A. Rehm polarization can occur (24). In agreement with an altered differ- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, entiation depending on early versus late tumor stages, splenic computational analysis): M. G€atjen, F. Brand, M. Grau, I. Anagnostopoulos, neutrophils in Em-Tcl1 mice exhibited downregulated genes for P. Lenz, G. Lenz, U.E. Hopken,€ A. Rehm € proinflammatory mediators (52). Thus, TANs from Em-Tcl1 mice, Writing, review, and/or revision of the manuscript: M. Gatjen, M. Grau, R. Kettritz, I. Anagnostopoulos, G. Lenz, U.E. Hopken,€ A. Rehm which carry their tumor burden for several months, share prop- Administrative, technical, or material support (i.e., reporting or organizing erties of an N2 differentiation. data, constructing databases): K. Gerlach, A. Rehm Moreover, we found enhanced gene expression for neutrophil- Study supervision: U.E. Hopken,€ A. Rehm attractant factors in Em-Tcl1 stroma, an expansion of splenic þ Ly6G neutrophils, and an extended neutrophil lifespan. Neu- Acknowledgments trophils localized to the RP and MZ, a distribution that was clearly We thank Kerstin Kruger€ (Max-Delbruck-Center,€ Berlin, Germany) for excel- distinguishable from autoinflammatory processes where neutro- lent technical assistance. phils can be located in the T-cell zone (53). Depletion of neutrophils in Em-Tcl1 mice delayed leukemia proliferation associ- Grant Support ated with the upregulation of the cell-cycle inhibitor Egr1 and the This work was supported by German Research Foundation (DFG) and in part € differentiation factor Egr2 (25, 26). by Wilhelm-Sander-Stiftung (A. Rehm and U.E. Hopken). The costs of publication of this article were defrayed in part by the payment of The functional properties of splenic neutrophils in Em-Tcl1 page charges. This article must therefore be hereby marked advertisement in mice were reminiscent of NBH cells important for MZ B-cell accordance with 18 U.S.C. Section 1734 solely to indicate this fact. antibody production (27). An important cytokine for intrasplenic reprogramming of neutrophils into NBH is IL10, which could be Received December 21, 2015; revised June 17, 2016; accepted July 6, 2016; provided by leukemia B cells. We suggest that the local environ- published OnlineFirst August 3, 2016.

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Splenic Marginal Zone Granulocytes Acquire an Accentuated Neutrophil B-Cell Helper Phenotype in Chronic Lymphocytic Leukemia

Marcel Gätjen, Franziska Brand, Michael Grau, et al.

Cancer Res Published OnlineFirst August 3, 2016.

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