CD40L TNF-Related Factors BAFF, APRIL, and Lymphocytic Leukemia

Published May 16, 2012, doi:10.4049/jimmunol.1102066 The Journal of Immunology

Stromal Endothelial Cells Establish a Bidirectional Crosstalk with Chronic Lymphocytic Leukemia Cells through the TNF-Related Factors BAFF, APRIL, and CD40L

Montserrat Cols,* Carolina M. Barra,† Bing He,* Irene Puga,† Weifeng Xu,* April Chiu,‡ Wayne Tam,x Daniel M. Knowles,x Stacey R. Dillon,{ John P. Leonard,‖ Richard R. Furman,‖ Kang Chen,* and Andrea Cerutti*,†,#

Chronic lymphocytic leukemia (CLL) is a clonal B cell disorder of unknown origin. Accessory signals from the microenvironment are critical for the survival, expansion, and progression of malignant B cells. We found that the CLL stroma included microvascular endothelial cells (MVECs) expressing BAFF and APRIL, two TNF family members related to the T cell-associated B cell-stimulating molecule CD40L. Constitutive release of soluble BAFF and APRIL increased upon engagement of CD40 on MVECs by CD40L aberrantly expressed on CLL cells. In addition to enhancing MVEC expression of CD40, leukemic CD40L induced cleavases that elicited intracellular processing of pro-BAFF and pro-APRIL proteins in MVECs. The resulting soluble BAFF and APRIL proteins delivered survival, activation, Ig gene remodeling, and differentiation signals by stimulating CLL cells through TACI, BAFF-R, and BCMA receptors. BAFF and APRIL further ampliﬁed CLL cell survival by upregulating the expression of leukemic CD40L. Inhibition of TACI, BCMA, and BAFF-R expression on CLL cells; abrogation of CD40 expression in MVECs; or suppression of BAFF and APRIL cleavases in MVECs reduced the survival and diversiﬁcation of malignant B cells. These data indicate that BAFF, APRIL, and CD40L form a CLL-enhancing bidirectional signaling network linking neoplastic B cells with the microvascular stroma. The Journal of Immunology, 2012, 188: 000–000.

hronic lymphocytic leukemia (CLL) is an incurable dis- 4). The presence of unmutated VH genes and expression of CD38 ease of unknown etiology characterized by progressive and ZAP-70 are biological variables associated with worse prog- C accumulation of clonal B lymphocytes with mature nosis. B cell intrinsic factors such as abnormal expression of morphology and phenotype (1). The clinical course of CLL is antiapoptotic NF-kB and Bcl-2 proteins and accumulation of variable and correlates with the mutational status of Ig H chain heterogeneous genomic lesions regulate the survival, expansion, variable (VH) genes and the percentage of leukemic cells and clonal evolution of CLL cells (5, 6). B cell extrinsic factors expressing CD38 and z-chain–associated protein 70 (ZAP-70) (2– such as antigenic stimulation may also be important, as leukemic B cells from unrelated groups of CLL patients express restricted Ig *Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029; VDJ genes encoding stereotyped Ag-binding VH regions (7–9). †Institut Municipal d’Investigacio´ Me`dica-Hospital del Mar, Barcelona 08003, Spain; Accessory signals from the microenvironment further contri- ‡Department of Pathology, Brigham and Women’s Hospital, Harvard Medical x bute to the pathogenesis of CLL (10, 11). Indeed, stromal cells School, Boston, MA 02115; Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY 10065; {ZymoGenetics such as nurselike cells, mesenchymal cells, follicular dendritic (a Bristol-Myers Squibb Company), Seattle, WA 98102; ‖Department of Medicine, cells (DCs), and macrophages recruit CLL cells by secreting # Weill Medical College of Cornell University, New York, NY 10065; and Institucio´ CXCL12 and CXCL13 chemokines and enhance CLL cell sur- Catalana de Recerca i Estudis Avançats, Barcelona 08003, Spain vival by releasing BAFF or BLyS and a proliferation-inducing Received for publication July 19, 2011. Accepted for publication April 13, 2012. ligand (APRIL) (12–14). These TNF family members are usu- This work was supported by a Chronic Lymphocytic Leukemia Research Center Grant from Cornell Comprehensive Cancer Center (to A. Cerutti), National Institutes ally expressed by myeloid and epithelial cells and engage BAFF-R of Health Grants R01 AI074378, P01 AI61093, U01 AI95613, and U19 096187 (BR3), transmembrane activator and CAML interactor (TACI), (to A. Cerutti), Ministerio de Ciencia e Innovacioń Grant SAF 2008-02725 (to and B cell maturation Ag (BCMA) on B cells (15–21). The A. Cerutti), Instituto de Salud Carlos III (to C.M.B.), and the Juan de la Cierva program (to I.P.). ensuing recruitment of TNFR-associated factor adaptor proteins Address correspondence and reprint requests to Dr. Andrea Cerutti, Institut Municipal leads to the activation of NF-kB, a transcription factor that reg- d’Investigacio´ Me`dica-Hospital del Mar, Barcelona Biomedical Research Park, Bar- ulates the survival, proliferation, diversification, and differentia- celona, Spain 08003. E-mail addresses: [email protected] or andrea.cerutti@mssm. tion of B cells (19, 20, 22). A similar pathway becomes activated edu after engagement of the CD40 receptor on B cells by CD40L, The online version of this article contains supplemental material. a TNF family member typically expressed by T cells but also by Abbreviations used in this article: AID, activation-induced cytidine deaminase; CLL cells (5, 23). APRIL, a proliferation-inducing ligand; CLL, chronic lymphocytic leukemia; CSR, class switch DNA recombination; DC, dendritic cell; EBM-2, endothelial basal me- CD40L, BAFF, and APRIL likely represent an important dium; EGM-2MV, endothelial growth medium; LMVEC, lymphatic microvascular component of CLL proliferation centers, which consist of pseu- endothelial cell; MVEC, microvascular endothelial cell; PoAb, polyclonal Ab; QRT- PCR, quantitative RT-PCR; siRNA, small interfering RNA; SMVEC, splenic micro- dofollicles containing focal aggregates of activated and prolifer- vascular endothelial cell; TACE, TNF-a converting enzyme; TACI, transmembrane ating CLL cells. Proliferation centers also include T cells ex- activator and CAML interactor; VEGF, vascular endothelial growth factor; VH,H pressing CD40L and stromal macrophages and nurselike cells chain variable; vWF, von Willebrand factor; ZAP-70, z-chain–associated protein 70. releasing BAFF and APRIL (13, 24). Remarkably, malignant Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 B cells shape the cellular composition of proliferation centers to

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102066 2 INTERPLAY OF BAFF, APRIL, AND CD40L IN CLL generate a CLL-supportive microenvironment. Indeed, CLL cells 5 mM, respectively (Enzo Life Sciences, Plymouth Meeting, PA), whereas release chemokines such as CCL22 to attract CD40L-expressing IKK inhibitor III (BMS-345541) was used at 1 mM (Calbiochem, San T cells and produce angiogenic molecules such as vascular en- Diego, CA). After a 3-h incubation with IKK inhibitor III (an NF-kB inhibitor) or control DMSO vehicle, CLL cells were washed twice with dothelial growth factor (VEGF) to stimulate the formation of endothelial medium and then cocultured with MVECs. microvessels (24, 25). In addition to favoring leukemic dissemi- nation, microvessels deliver survival signals to CLL cells through Flow cytometry unknown factors released by endothelial cells (26). Cells were stained with FITC-, PE-, allophycocyanin-, or cyanine-3–conju- We found that the CLL stroma contained abundant microvas- gated mouse mAbs to human CD5, CD19, CD38 (BD Pharmingen); BAFF- cular endothelial cells (MVECs) that constitutively expressed the R, BAFF, CD31 (eBiosciences, San Diego, CA); CD40, CD40L (Ancell, B cell-stimulating factors BAFF and APRIL. MVECs increased the Bayport, MN); CD54, CD102, CD144 (BioLegend, San Diego, CA); CD105 (ImmunoTools, Friesoythe, Germany); IgG, IgA, or control isotype-matched release of soluble BAFF and APRIL after engagement of CD40 reagents (Southern Biotech). Biotinylated Abs to TACI (ZymoGenetics) were on MVECs by CD40L on CLL cells. Signals from CD40 induced stained with PE-conjugated streptavidin (BD Pharmingen). A goat Ab to furin and TNF-a converting enzyme (TACE or ADAM17), which BCMA (Santa Cruz Biotechnologies, Santa Cruz, CA) was stained with an cleaved inactive BAFF and APRIL precursors into active soluble appropriate PE-conjugated secondary Ab (BD Pharmingen). Dead cells were proteins. By activating NF-kB through TACI, BAFF-R and BCMA, excluded from analysis using 7-amino-actinomycin D. Events were acquired using a FACScalibur or LSRII (BD Biosciences) and were analyzed by endothelial BAFF and APRIL delivered survival, activation, and FlowJo software (Tree Star, Ashland, OR). Ig DNA-modifying signals to CLL cells. Endothelial BAFF and APRIL also increased the expression of CD40L on CLL cells, Immunofluorescence and histology indicating that BAFF, APRIL and CD40L link malignant B cells Endothelial cell lines were grown on glass cover slides in EGM until with stromal MVECs through an integrated bidirectional signaling reaching confluence. Tissues and cells were fixed and washed as described network. Interruption of this network by specific inhibitors may be elsewhere (15) and then stained with the following unconjugated or con- useful for the treatment of CLL. jugated primary Abs to various human Ags: goat polyclonal Ab (PoAb) to IgD (Southern Biotech); mouse mAb to Pax-5 (Santa Cruz); rabbit PoAb to Pax-5 (NeoMarkers, Fremont, CA); mouse mAb to Ki-67, Factor VIII, Materials and Methods CD31, CD3, and elastase (Dako, Carlsbad, CA); rabbit PoAb to von Patients and samples Willebrand factor (vWF)(Dako); rabbit PoAb anti-APRIL (ProSci, Poway, CA); mouse mAb to CD3, CD11c, CD21, CD206 (BD Pharmingen); goat Leukemic cells were isolated from the peripheral blood of 21 CLL patients PoAb to activation-induced cytidine deaminase (AID) (Santa Cruz Bio- (Table I). The diagnosis of CLL was made according to standard criteria technologies); rabbit PoAb to furin (Santa Cruz Biotechnologies); rabbit (27). All blood samples were obtained by phlebotomy upon request of PoAb to CD63 (Novus Biologicals, Littleton, CO); mouse mAb to CD68 consent according to a protocol approved by the Institutional Review (Abcam, Cambridge, MA); mouse mAb to CD40L (Ancell); and rabbit Board of Weill Medical College of Cornell University. The VH gene PoAb to TACE (Sigma-Aldrich, St. Louis, MO). Primary Abs were labeled mutational status and Zap70 expression of each CLL sample were deter- with an appropriate Alexa 488-, 546-, or 647-conjugated or cyanine-5– mined as previously reported (2–4). Normal peripheral blood B cells were conjugated secondary Abs (Jackson Immunoresearch Laboratories, West isolated from buffy coats provided by the New York Blood Center. Frozen Grove, PA). Cell nuclei were visualized with DAPI (Boehringer Mann- tissues samples from lymph nodes, bone marrow, and spleen of 10 healthy heim, Indianapolis, IN). Coverslips were applied with SlowFade reagent controls and 10 CLL patients were obtained from tissue repositories (Molecular Probes, OR). Images were acquired with a Zeiss Axioplan 2 according to a protocol approved by the Institutional Review Board of microscope (Atto Instruments, Rockville, MD). Weill Medical College of Cornell University. Cells Cell viability and proliferation assays Normal and CLL B cells were MACSorted from PBMCs by negative selec- Cell viability was evaluated through a trypan blue exclusion test. To measure tion using a B cell isolation kit (Miltenyi Biotec, Bergish-Gladbach, Ger- apoptosis, cells were double stained with FITC-conjugated Annexin V and many). Marginal zone CD19+IgDlowCD27+ B cells, follicular naive CD19+ propidium iodide (Calbiochem) and analyzed by flow cytometry. Cell pro- IgDhighCD272 B cells, and memory CD19+IgD2CD27+ B cells were sorted liferation and/or survival were monitored using a yellow tetrazolium MTT cell from splenic mononuclear cells, using a BD FACSAria III (BD Biosciences, proliferation assay (Trevigen, Gaithersburg, MD). In this assay, dehydro- San Diego, CA). Sorting was preceded by labeling of splenic mononuclear genases expressed by metabolically active cells generate NADH and NADPH cells with FITC-, PE-, or allophycocyanin-conjugated mouse mAbs to hu- that convert MTT into formazan. Briefly, 100-ml aliquots of culture super- man CD19 (BD Pharmingen, San Diego, CA), IgD (Southern Biotech, Bir- natant were transferred into a 96-well plate and incubated overnight with 20 mingham, AL), and CD27 (BD Pharmingen). HUVECs and lymphatic mi- ml MTT reagent at 37˚C and 5% CO2, followed by a 2-h incubation with 100 crovascular endothelial cells (LMVECs) are commercially available (Lonza, ml detergent reagent. Absorbance readings were performed at 570 nm in Walkersville, MD). Splenic microvascular endothelial cells (SMVECs) were a microplate reader. Cell proliferation was further evaluated through a thy- MACSorted from enzymatically digested splenic cell suspensions stained midine incorporation assay, as described in published reports (28). The with mouse mAbs to CD31 and mannose receptor (BD Pharmingen). proliferation index was calculated according to the following formula: mean of counts per minute of triplicates with stimulus/mean of counts per minute Cultures and reagents of triplicates without stimulus. CFSE was used to trace cell division as in- structed by the manufacturer (Invitrogen, Grand Island, NY). Endothelial cells were propagated in endothelial basal medium (EBM-2) supplemented with endothelial growth medium (EGM-2MV) Single- ELISA Quots (Lonza). Endothelial cells were seeded with EBM-2 plus EGM-2MV medium in 12-well plates at a concentration of 1.5 3 104 cells per well for Human IgG, IgA, IgM, BAFF, and APRIL were measured by ELISA as 48 h prior to coculture with CLL cells. The EBM-2 plus EGM-2MV described elsewhere (29). medium was mixed with standard RPMI 1640 medium supplemented 6 with 10% FCS (1:4, vol/vol) after seeding CLL cells at 1 3 10 cells/ml. RT-PCR, quantitative RT-PCR, and Southern blot analysis This mix is referred to as endothelial medium throughout the text and was used in all cultures involving CLL cells unless specified otherwise. Con- RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA), and ditioned endothelial medium was obtained after culturing SMVECs for cDNA was synthesized as described (29). Germline Ig1-Cg1, Ig2-Cg2, and 4 d in endothelial medium. Some endothelial cell–CLL cell cocultures Ig3-Cg3 transcripts, as well as Ig1/2-Cm,Ig3-Cm,andIa1-Cm switch circle were performed in a 24-well transwell system (Corning, Lowell, MA). transcripts, were RT-PCR amplified and were hybridized with appropriate Recombinant BAFF, APRIL, and CD40L MegaLigand (Alexis Bio- radiolabeled probes by Southern blot analysis as described (15). TACI for- chemicals, San Diego, CA) were used at 100 ng/ml. TACI-Ig and CD40-Ig ward: 59-CAGACAACTCGGGAAGGTACC-39, and reverse: 59-GCCACC- decoy receptors and Fc5 or Ig (IgG1) controls (ZymoGenetics, Seattle, TGATCTGCACTCAGCTTC-39; BAFF-R forward: 59-ACCGCGGGACTG- WA) were used at 5 mg/ml. Furin convertase inhibitor (peptidyl choro- AAAATCT-39, and BAFF-R reverse: 59-CACGCTTATTTCTGCTGTTCT- methylketone) and TNF-a processing inhibitor-2 were used at 100 mM and GA-39;BCMAforward:59-GCTTGCATACCTTGTCAACTTCGATG-39, The Journal of Immunology 3 and BCMA reverse: 59-GAATCGCATTCGTTCCTTTCACTG-39;CD40 In CLL spleens, MVECs expressed CD206 (mannose receptor, forward: 59-CACACTGCCACCAGCACAA-39, and CD40 reverse: 59-GCC- a molecule highly expressed by sinusoidal vessels) and were in close TTCTTCACAGGTGCAGAT-39; AID forward: 59-AGAGGCGTGACAGT- proximity with neoplastic B cells. Similar to MVECs from normal GCTACA-39,andAIDreverse:59-TGTAGCGGAGGAAGAGCAAT-39; BAFF forward: 59-GGGAATTCGATGACTCCACAGAAAGGGAGCAGT- lymphoid tissues, MVECs from CLL tissues expressed CD54 CA-39, and BAFF reverse: 59-GGGCGGCCGCTTACAGCAGTTTCAATG- (ICAM-1), CD102 (ICAM-2), CD105 (endoglin), and CD144 (VE- CACCAAAAAA-39; APRIL forward: 59-CTGCACCTGGTTCCCATTAAC- cadherin) (Fig. 1C). Flow cytometry showed that model MVECs 39, and APRIL reverse: 59-AAGAGCTGGTTGCCACATCAC-39; TACE from healthy individuals expressed a phenotype similar to that of forward: 59-GAGGCGATTAATGCTACTTGCA-39, and TACE reverse: 59- TGGAGGCGGGCACTCA-39; furin forward: 59-GGCCTGCTCGTCCA- MVECs from CLL patients (Fig. 1B–D, Supplemental Fig. 1). CACT-39, and furin reverse: 59-TCGTCACGATCTGCTTCTCATT-39;and Indeed, UVECs, SMVECs, and LMVECs expressed CD31, CD54, b-actin forward: 59-GCTTGCATACCTTGTCAACTTCGATG-39,andb-ac- CD102, CD105, and CD144 as MVECs from CLL patients did (Fig. tin reverse: 59-GAATCGCATTCGTTCCTTTCACTG-39 were used to quan- 1D). As in SMVECs from CLL patients, SMVECs further tify specific mRNAs by RT-PCR, as previously reported (29). expressed CD206, whereas UVECs and LMVECs did not (Fig. 1D). Immunoblots Given their phenotypic similarity to MVECs from CLL tissues, SMVECs, LMVECs, and UVECs were used in a simplified coculture Equal amounts of total protein lysate, cytoplasmic extract, or nuclear extract model to establish the role of MVECs in the survival of malignant were fractioned onto a 10% NaDodSO4-polyacrylamide gel and transferred ∼ onto nylon membranes (BioRad, Hercules, CA). After blocking, membranes B cells. In the presence of SMVECs, 90% of CLL cells survived were probed with primary Abs to APRIL (ProSci), BAFF (Millipore, Bed- after 7 d of culture, as established by a canonical viability assay (Fig. ford,MA),TACE,furin,orb-actin (Santa Cruz Biotechnologies). as reported 1E). The prosurvival effect of SMVECs was partly dependent on cell- (29). Then, membranes were washed and incubated with an appropriate to-cell contact, because conditioned medium from SMVEC cultures secondary Ab (Santa Cruz Biotechnologies). Proteins were detected with an induced the survival of only 40% of CLL cells after 7 d. Of note, ECL detection system (Amersham, Little Chalfont, United Kingdom). SMVECs and conditioned medium induced stronger survival signals RNA interference than did endothelial medium or standard medium alone, which indeed induced only 30% and 5% CLL cell survival, respectively. The The following small interfering RNA (siRNA) duplexes were used after validation with three distinct siRNAs for each target gene of interest: 59- partial prosurvival activity of endothelial medium alone was likely GGAAATCAAATTCAGCTCTTT-39 siRNA to BAFF-R; 59-CAGCGGA- mediated by VEGF, a known CLL cell survival cytokine (30, 31). GTGGAGAAGTTGAA-39 siRNA to TACI (Ambion, Austin, TX); 59- Compared with endothelial medium alone, MVECs increased CLL ACCATTAAAGGACGAGTTTAA-39 siRNA to BCMA (Qiagen); and 59- survival and/or proliferation, as measured in an MTT assay in which AGGGCACCUCAGAAACAGATT-39 siRNA to CD40 (Quiagen). The metabolically active cells convert MTT into formazan (Fig. 1F). specificity of each siRNA was validated using a scrambled siRNA as control. Transfection of specific or control (scrambled) siRNA in CLL In agreement with these findings, proliferation centers from CLL cells was performed as recommended by the manufacturer (Ambion). tissues included factor VIII-positive MVECs that were in close Briefly, 5 mM siRNA was incubated with 100 ml nucleofection mix contact with actively cycling IgD-positive malignant B cells 3 6 (Amaxa, Walkersville, MD) and 100 ml CLL cell suspension (3 10 expressing the proliferation-associated nuclear molecule Ki-67 cells) for 2 min before transfection, using the Amaxa nuclefector program U-15. After 24 h, cells were seeded in 1 ml complete medium. Transfec- (Fig. 2A). The possible proliferation-inducing activity of MVECs tion efficiency was monitored after 48 h by measuring CLL cells positive was suggested by means of CFSE dilution assays, which docu- for a control GFP-expressing plasmid through flow cytometry. Specific mented the division of a fraction (from 5% to 15% of total B cells) suppression of TACI, BCMA, and BAFF-R gene expression was validated of the leukemic clone upon exposure of CLL cells to UVECs or through quantitative RT-PCR (QRT-PCR). These measurements demon- SMVECs but not endothelial medium alone (not shown). Thy- strated highly specific targeting of TACI, BCMA, or BAFF-R expression by distinct siRNAs. Transfection of CD40 or control siRNA in endothelial midine incorporation assays demonstrated induction of DNA cells was performed using a SuperFect protocol (Qiagen). Briefly, en- replication in CLL cells exposed to SMVECs but not endothelial dothelial cells were cultured on a monolayer with complete EGM until medium alone (Fig. 2B). Unlike CLL cells, nonmalignant B cells optimal confluency was reached. Then, 5 nM siRNA duplexes and 7.5 ml expressing IgM and IgD did not augment DNA replication in re- HiPerFect Transfection Reagent were diluted in 100 ml endothelial cell culture without serum. The mixture was incubated at room temperature to sponse to SMVECs, suggesting that MVECs require interaction allow formation of transfection complexes and further added to the cells with a CLL-specific factor (or factors) to induce the proliferation dropwise. Transfection efficiency was monitored by measuring the ex- and/or survival of leukemic B cells. These data show that MVECs pression of CD40 through QRT-PCR after 48 h. are a major component of the CLL stroma and indicate that MVECs deliver survival and possibly proliferation signals to CLL Statistical analysis B cells. One-way ANOVA followed by a one-tailed unpaired Student t test was used to determine statistical significance. MVECs trigger CLL cell activation and differentiation In addition to active proliferation, a fraction of the CLL clone shows Results ongoing Ig DNA remodeling via class switch DNA recombination MVECs promote CLL cell survival (CSR) (32, 33). This process is highly dependent on AID, a DNA- To elucidate the composition of the CLL stroma, we immunostained editing enzyme expressed by actively proliferating CLL cells in re- lymph node, spleen, and bone marrow tissues from CLL patients for sponse to poorly understood signals generated by the activated mi- molecules typically expressed by stromal cells. Immunohisto- croenvironment (32–37). To further elucidate the stimulating activity fluorescence showed that all CLL tissues included microvessels of MVECs on the leukemic clone, we determined whether MVECs expressing CD31 (PECAM-1) and containing factor VIII and vWF induce AID expression and CSR in CLL cells (32). RT-PCR followed coagulation proteins (Fig. 1A, 1B). MVECs from these micro- by Southern hybridization analysis showed that, compared with CLL vessels established an intimate contact with malignant B cells cells exposed to endothelial medium alone, CLL cells incubated with expressing IgD (a molecule associated with mature B cells and SMVECs induced or enhanced the expression of molecular hallmarks CLL cells) and generally were more abundant than macrophages, of ongoing CSR from IgM to IgG1/IgG2, IgG3, or IgA (15), including follicular DCs, DCs, and neutrophils expressing CD68 (Fcg re- switch circle Ig1/2-Cm,Ig3-Cm,andIa-Cm transcripts, respectively ceptor), CD21 (complement receptor 2), CD11c (integrin a X), (Fig. 2C). SMVECs also upregulated the expression of germline and elastase (a granular protease), respectively. Ig1-Cg1, Ig2-Cg2, and Ig2-Cg2 transcripts, an early event in IgM 4 INTERPLAY OF BAFF, APRIL, AND CD40L IN CLL

FIGURE 1. MVECs infiltrate CLL tissues and promote malignant B cell survival and proliferation. (A) Immunofluorescence analysis of CLL lymph node (LN; one of five cases), spleen (SP; one of three cases), and bone marrow (BM; one of three cases) tissue samples stained for CD11c, CD21, CD31, CD68, CD206, elastase, factor VIII, vWF, or CD206. DAPI (blue) counterstains nuclear DNA. Original magnification 310. (B) CLL LN and SP tissue samples (one of five and three cases, respectively) stained for IgD (green), factor VIII or CD206 (red), and DAPI (blue). Original magnification 340. (C) CLL and normal LN tissue samples (five cases in each group) stained for CD54, CD105, CD144 (red), and DAPI (blue). Origi- nal magnification 340. (D). Flow cytometric analysis of CD31, CD54, CD102, CD105, CD144, and CD206 expression by UVECs, SMVECs, and LMVECs. Gray solid profile, isotype-matched control. (E). Time course analysis of viable trypan blue-negative CLL cells (one of five cases) exposed to standard medium (SM, orange line), endothelial medium (EM, black line), conditioned medium from endothelial cell cultures (CM, blue line), or SMVECs (red line). (F) MTT proliferation assay with CLL cells from mutated (MU, left panel) or unmutated (UM, right panel) samples (five cases in each group) cocultured with endothelial medium alone (control), UVECs, or SMVECs. Results are expressed as fold induction release of MTT-derived formazan by proliferating CLL cells. Data are from one of three or five experiments yielding similar results (A–E) or summarize three different experiments (F). Error bars, SEM; *p , 0.05 (ANOVA followed by one- tailed unpaired Student t test).

to IgG1, IgG2, and IgG3 CSR, respectively (Fig. 2C). In the majority MVECs release BAFF and APRIL of cases, CLL cells constitutively expressed germline Ia-Ca tran- Myeloid and stromal cells enhance the survival of peripheral scripts, and MVECs did not further augment this expression (not B cells and under specific conditions elicit AID expression and CSR shown). by releasing BAFF and APRIL (20). These immune mediators are SMVECs also upregulated the expression of transcripts for AID also involved in the pathogenesis of CLL (13, 42–46), which in CLL cells (Fig. 2D). This in vitro finding correlated with tissue prompted us to determine the involvement of BAFF and APRIL data showing MVEC–proximal CLL cells that expressed AID in the activation of CLL cells by MVECs. ELISAs demonstrated (Fig. 2E), a hallmark of ongoing B cell activation and Ig gene constitutive BAFF and APRIL release by MVECs, including diversification (32). ELISA and flow cytometric studies showed SMVECs, LMVECs, and UVECs (Fig. 3A). Contact-dependent that SMVECs also induced secretion of class-switched IgG and exposure of MVECs to CLL cells further increased endothelial IgA as well as unswitched IgM proteins (Fig. 2F), expression of secretion of BAFF and APRIL, whereas contact-independent ex- surface IgG and IgA proteins (Fig. 2G), and upregulation of sur- posure of MVECs to CLL cells in a transwell system or exposure face CD38 (Fig. 2H), a microenvironment-inducible activation of MVECs to conditioned medium from CLL cell cultures did not molecule that activates neoplastic B cells by binding to CD31 on increment steady state BAFF and APRIL secretion (Fig. 3A). MVECs (38–41). UVECs induced similar activation and differ- Fluorescence microscopy and confocal microscopy confirmed entiation signals to CLL cells. Similar to endothelial medium the constitutive expression of BAFF and APRIL proteins by alone, a CLL cell survival factor such as the cytokine IL-4 did not MVECs, including SMVECs from CLL tissues, and further showed induce CLL cell proliferation, AID expression, or Ig secretion, that these MVECs stored BAFF and APRIL proteins in intracellular despite enhancing the viability of CLL cells (data not shown). endosomal and granular structures that also contained the tetra- These findings demonstrate that MVECs provide powerful stim- spanin protein CD63 and the coagulation proteins factor VIII and ulating signals to CLL cells, including AID-inducing and CSR- vWF (Fig. 3B, 3C). Flow cytometry and QRT-PCR assays dem- activating signals. onstrated that MVECs did not express membrane-bound BAFF The Journal of Immunology 5

FIGURE 2. MVECs promote division, activation, and differentiation of CLL cells. (A) Immunofluo- rescence analysis of CLL lymph node (LN) tissue samples (one of five cases) stained for Ki-67 (green or red), factor VIII (red), and IgD (blue or green). DAPI (blue) counterstains nuclei. Original magnification 310 (left panel)or363 (right panels). (B) [3H]Thymidine deoxyribose incorporation of B cells from CLL patients or healthy individuals (five cases in each group) cultured for 4 d with endothelial medium alone (control) or SMVECs. (C) Southern blot analysis of germline Ig1-Cg1, Ig2- Cg2, and Ig3-Cg3 transcripts, as well as Ig1/2-Cm, Ig3-Cm,andIa-Cm switch circle transcripts RT- PCR amplified from CLL cells (one of five cases with little or no constitutive CSR) cultured for 4 d with endothelial medium alone (control), UVECs, or SMVECs. b-actin is a loading control. (D)QRT- PCR analysis of AID mRNA from CLL cells (five cases) cultured for 4 d as in (C). (E) CLL LN tissue (one of five cases) stained for AID (green), factor VIII (red), and Pax5 (blue). Arrowheads point to an intercellular bridge that connects factor VIII-positive MVECs with an AID-positive CLL cell, AID being a hallmark of ongoing B cell activation. Original magnification 363. (F) ELISA of IgM, IgG, and IgA secreted by CLL cells (five cases) cultured for 7 d, as in (C). (G) Flow cytometric analysis of IgG and IgA on CLL cells (one of five cases) cultured with endothelial medium alone (red open profile) or UVECs (blue open profile) for 7 d. Gray solid profile, isotype-matched control. (H) Flow cytometric analysis of CD19 and CD38 on CLL cells (one of five cases) cultured for 7 d, as in (C). Data are from one of five experiments yielding similar results (A, C, E, G, H) or summarize three different experiments (B, D, F). Error bars, SEM; *p , 0.05 (ANOVA followed by one-tailed unpaired Student t test). RE, Relative expression as compared with freshly isolated CLL cells.

(Fig. 3D) and yet contained more BAFF and APRIL transcripts and a powerful inducer of BAFF and APRIL release by myeloid than did myeloid cell types with well-defined B cell-licensing cells (5, 15, 44). functions (20), including DCs and macrophages (Supplemental A soluble TACI-Fc5 decoy receptor, which prevents binding of Fig. 2). Compared with CLL cells exposed to endothelial medium BAFF and APRIL to B cells, decreased CLL cell survival, AID alone, CLL cells exposed to endothelial medium supplemented expression, as well as IgM and IgA secretion as induced by SMVECs with BAFF or APRIL showed increased proliferation and/or sur- or UVECs (Fig. 4C–E). In these cocultures, an Fc5 molecule was vival, AID transcription, as well as surface IgA expression and used as control (Fig. 4C–E). CLL cells transfected with siRNAs IgA secretion (Fig. 3E–I). BAFF and APRIL also increased the specifically suppressing the expression of either BAFF-R or TACI or secretion of IgM and IgG (Fig. 3I). These data indicate that BCMA receptor showed not only decreased survival but also atten- MVECs promote CLL cell survival and diversification through uated expression of AID as induced by SMVECs (Fig. 4F–H). These both contact-dependent and contact-independent mechanisms, data indicate that MVECs elicit survival and activation signals in including BAFF and APRIL. CLL cells through a mechanism that involves engagement of multiple BAFF and APRIL receptors, including BAFF-R, as well as MVECs stimulate CLL cells through BAFF and APRIL TACI and BCMA receptors. Published data show that BAFF and APRIL deliver survival signals to normal and malignant B cells through BAFF-R, plasma cell MVECs express CD40 and enhance CD40L expression on CLL survival signals through BCMA, and CSR signals through TACI cells (19, 20, 44, 45, 47–49). TACI can also deliver B cell survival and Stromal cells enhance BAFF and APRIL release in response to proliferation signals (20, 50). As shown by flow cytometry, BAFF-R various immune signals, including CD40L (15), an NF-kB–in- was detected on malignant B cells from all 21 CLL cases analyzed, ducible T cell molecule aberrantly expressed by CLL cells (5, 23, whereas TACI and BCMA were found on malignant B cells from 12 51). Given the ability of CLL cells to upregulate endothelial re- and 9 CLL cases, respectively (Fig. 4A, Table I). BAFF-R expression lease of BAFF and APRIL in a contact-dependent manner, we was generally more elevated than TACI and BCMA expression (Fig. wondered whether this upregulation involved engagement of 4A). QRT-PCR analysis demonstrated that TACI, BCMA, and CD40 on MVECs by CD40L on CLL cells. Fluorescence mi- BAFF-R transcripts increased after incubation of CLL cells with croscopy showed that factor VIII-positive MVECs from CLL or CD40L (Fig. 4B), a molecule aberrantly expressed by CLL cells nonmalignant lymphoid tissues expressed CD40 (Fig. 5A). In 6 INTERPLAY OF BAFF, APRIL, AND CD40L IN CLL

FIGURE 3. MVECs enhance BAFF and APRIL release by interacting with CLL cells. (A) ELISA of BAFF and APRIL secreted by UVECs, SMVECs, or LMVECs cultured for 7 d in the absence (left panel)orpresence(right panel) of CLL cells (three cases) or CLL cell-derived conditioned medium (CM) (three cases). Cultures were performed in plates with or without a transwell system (TW and no TW, respectively). (B) Immunofluorescence analysis of BAFF or APRIL (green), CD31, vWF, CD63, or TGN-46 (red), and DAPI (blue) in MVECs. Original magnification 340. (C) Immunofluorescence analysis of CLL lymph node (LN; five cases) and spleen (SP; three cases) tissue samples stained for BAFF or APRIL (green), factor VIII or CD206 (red), and DAPI (blue). Original magnification 340. (D) Flow cytometric analysis of BAFF on SMVECs. Gray open profile, isotype-matched control; red open profile, BAFF. (E) Time course analysis of viable trypan blue-excluding CLL cells from unmutated (UN) or mutated (MU) samples (one of five cases in each group) cultured with endothelial medium in the presence or absence of BAFF or APRIL. (F) Formazan release assay with CLL cells (five cases) cultured for 4 d, as in (D). (G) QRT-PCR analysis of AID mRNA from CLL cells (five cases) cultured for 4 d as in (D). (H) Flow cytometric analysis of surface IgA on CLL cells (one of five cases) cultured for 4 d with endothelial medium alone (red open profile) or BAFF (blue open profile). Gray solid profile, isotype-matched control. (I) ELISA of IgM, IgG, and IgA secreted by CLL cells cultured for 7 d, as in (E). Data are from one of three or five experiments yielding similar results (B–E, H)or summarize three different experiments (A, F, G,andI). Error bars, SEM; *p , 0.05 (ANOVA followed by one-tailed unpaired Student t test). RE, Relative expression compared with freshly isolated CLL cells. addition, CD40L was detected in CLL cells expressing the B cell- cases or ZAP-70–positive versus ZAP-70–negative cases (Table I). specific membrane protein IgD and nuclear protein Pax5, but QRT-PCR identified CD40L transcripts in malignant B cells from lacking the T cell-specific membrane protein CD3 (Fig. 5B). Some of both CLL cases with high CD40L protein expression and low these CD40L-expressing CLL cells also contained the proliferation- CD40L protein expression (Fig. 5D), indicating that transcription associated nuclear protein Ki-67 and seemingly occupied CLL of the CD40L gene is not always associated with surface CD40L proliferation centers (Fig. 5B). In nonmalignant lymphoid tissues, protein expression. The QRT-PCR assay was highly specific be- only some germinal center B cells expressed CD40L (Fig. 5C). cause, unlike CLL cells, nonmalignant naive, marginal zone, and These CD40L-expressing B cells had a germinal center pheno- memory B cells expressed little or no CD40L transcripts. The type, as they expressed AID but lacked IgD (Fig. 5C). expression of CD40L transcripts in CLL cells further increased Flow cytometry confirmed the expression of CD40 on MVECs upon exposure to SMVECs, but this increase did not occur after as well as the expression of CD40L on malignant B cells from some a short preincubation of CLL cells with a specific inhibitor of the CLL cases, but not on naive, marginal zone, and memory B cells NF-kB pathway (Fig. 5D). We next wondered whether CD40L on from healthy donors (Fig. 5A–D, Table I). CD40L expression on CLL cells exerted a positive feedback effect on CD40 expression malignant B cells ranged from high to low among CLL cases and by MVECs. Exposure of MVECs to recombinant CD40L induced did not seem to preferentially target unmutated versus mutated more expression of CD40 transcripts in MVECs (Fig. 5E). Simi- The Journal of Immunology 7

FIGURE 4. MVECs stimulate CLL cells by en- gaging TACI, BCMA, and BAFF-R through BAFF and APRIL. (A) Flow cytometric analysis of BAFF- R, TACI, and BCMA (red lined open proﬁle) proteins on CLL cells from UN (upper row)orMU(lower row) samples (one of four cases in each group; see Table I). Gray solid proﬁle, isotype-matched control. (B) Time course QRT-PCR analysis of BAFF-R, TACI, and BCMA mRNAs from CLL cells (three cases) exposed to CD40L. RE, relative expression compared with freshly isolated CLL cells. (C) Viable trypan blue-negative CLL cells (one of three cases) cultured in endothelial medium with or without UVECs or SMVECs and in the presence or absence of control Fc5 or TACI-Ig for 0 or 144 h. (D)QRT- PCR analysis of AID mRNA from CLL cells (three cases) cultured for 4 d in endothelial medium with SMVECs and control Fc5 or TACI-Ig. RE, relative expression compared with freshly isolated CLL cells. (E) ELISA of IgM and IgA secreted by CLL cells (three cases) cultured for 7 d, as in (D). (F) QRT-PCR of BAFF-R, TACI, and BCMA mRNAs in CLL cells (three cases) nucleofected with scrambled, BAFF-R–, TACI-, or BCMA-targeting siRNAs. RE, relative expression compared with non-nucleofected CLL cells. (G) Viable trypan blue-negative CLL cells (three cases) nucleofected with scrambled, BAFF-R–, TACI-, or BCMA-targeting siRNAs and exposed to SMVECs for 4 d. (H) QRT-PCR analysis of AID mRNA from CLL cells (three cases) treated and cultured as in (G). RE, relative expression compared with nonnucleofected CLL cells. Data are from one of eight (A) or three (C) experiments yielding similar results or summarize three different experiments (B, D–H). Error bars, SEM; *p , 0.05 (ANOVA followed by one-tailed unpaired Student t test).

larly, exposure of MVECs to CD40L-positive CLL cells upregu- inhibits engagement of CD40 by CD40L (Fig. 5F). These data lated the expression of CD40 on MVECs, but this upregulation did show that endothelial NF-kB–inducing factors upregulate CD40L not occur in the presence of a soluble CD40-Ig decoy receptor that expression on CLL cells and show that leukemic CD40L estab-

Table I. Genotypic and phenotypic features of CLL cases

ID Ig VH Zap-70 CD38 CD40L TACI BCMA BAFF-R

011 UM, VH4–31 + + + — + + 014 UM, VH1–31 +/2 —+—— + 021 MU, VH3–48 + + +/2 +/2 —+ 025 MU, VH2–5 +/2 ———+ + 031 MU, VH3–74 + — + + — + 045 UM, VH1–69 + — + + +/2 + 048 MU, VH4–4 + +/2 +++ + 051 UM, VH2–5 + — — +/2 —+ 062 N/A N/A N/A + + + + 067 N/A +/2 —+ +— + 078 MU, VH3–30 +/2 ———+ + 085 MU, VH2–5 N/A N/A — — — + 090 UM, VH3–7 — — + — — + 091 N/A N/A — + — — + 095 UM, VH3–11 — + + — — + 098 UM, VH1–69 + + +/2 ++ + 107 MU, VH6–1 + +/2 ++—+/2 112 N/A + + — — + + 123 N/A + + + +/2 —+ 126 MU, VH4–34 +/2 —+ ++/2 + 128 MU, VH1–18 + + + + — + MU, Mutated (.2% mutations compared with putative germline VDJ gene); N/A, not available; UM, unmutated (#2% mutations compared with putative germline VDJ gene); +/2, intermediate expression (,15% but .5% of the cells). 8 INTERPLAY OF BAFF, APRIL, AND CD40L IN CLL

FIGURE 5. MVECs express CD40 and up-regulate CD40L on CLL cells. (A) Upper panels, Immunofluorescence analysis of CLL lymph node (LN; five cases) or normal tonsillar (TO; five cases) tissues stained for CD40 (green), factor VIII (red), and IgD (blue). Bottom panels, Flow cytometric analysis of CD40 (red open profile) on UVECs, SMVECs, and LMVECs. Gray solid profile, isotype-matched control. Original magnification 310 (upper panels) and 340 (bottom panels). (B) Upper panels, CLL bone marrow (BM; three cases) stained for IgD or Pax5 (green), CD40L (red or blue), CD3 (yellow), and Ki- 67 (blue). Inset in large left panel shows merged IgD and CD40L. Original magnification 310 (left panels)or363 (right panels). Bottom panels, CD40L (red open profile) on CLL cells (three cases). Gray solid profile, isotype-matched control. Numbers indicate specific CLL cases. (C) Left panels, Normal tonsillar (TO; five cases) tissue stained for IgD (green), AID (red), and CD40L (blue). Original magnification 310 (upper panel)or320 (lower panel). Right panels, CD40L (red open profile) on naive, marginal zone, or memory B cells from a healthy spleen (three cases). Gray solid profile, isotype-matched control. (D) QRT-PCR of CD40L mRNA in naive, marginal zone, or memory B cells from healthy spleens (three cases), freshly isolated CLL cells (six cases, each analyzed in triplicates), or CLL cells exposed to SMVECs (two cases, each analyzed in triplicates) for 4 d. CLL cells were preincubated with control vehicle (DMSO) or an NF-kB inhibitor (IKK inhibitor III) for 3 h, washed, and then cocultured with SMVECs. Results are normalized to freshly isolated naive B cells. Numbers indicate CLL cases. (E) QRT-PCR of CD40 mRNA in UVECs, SMVECs, or LMVECs incubated with endothelial medium alone (control) or CD40L. (F) Flow cytometric analysis of CD40 on SMVECs incubated with control Ig or CD40-Ig in the presence of CD40Lhigh CLL cells (blue open profile; one of three cases) or absence of CD40Lhigh CLL cells (red open profile) for 48 h. Gray solid profile, isotype-matched control. Data are from one of six experiments yielding similar results (A–C, E, F) or summarize three different experiments (D). Error bars, SEM. lishes positive feedback by enhancing CD40 expression on site of pro-BAFF and pro-APRIL was found to be partially over- MVECs. lapping with an Ala134–Val134 motif resembling the Ala76–Val77 motif used by the disintegrin metalloprotease TACE to cleave the MVECs release BAFF and APRIL in response to CD40L pro-TNF protein (56). Thus, we hypothesized that CD40L may Considering that CD40L is a powerful inducer of BAFF and APRIL stimulate BAFF and APRIL cleavage in MVECs via both furin (15, 52), we wondered whether engagement of CD40 on MVECs convertase and TACE proteins. by CD40L on CLL cells elicits endothelial release of BAFF and Consistent with this possibility, immunoblotting showed de- APRIL. QRT-PCR and ELISA assays demonstrated that SMVECs, creased intracellular amounts of pro-BAFF and pro-APRIL pro- UVECs, and (not shown) LMVECs upregulated the expression of teins upon exposure of MVECs to CD40L (Fig. 6D). Further- BAFF and APRIL transcripts as well as the release of soluble BAFF more, ELISA showed decreased CD40L-induced release of soluble and APRIL proteins as early as 3 h after incubation with CD40L or BAFF and APRIL proteins from SMVECs exposed to either CLL cells expressing CD40L (Fig. 6A, 6B). Soluble BAFF and a peptidyl chloromethylchetone that specifically inhibits furin APRIL derive from processing of pro-BAFF and pro-APRIL pro- convertase or a hydroxamate-based inhibitor that specifically teins by furin convertase (53–55). This trans-Golgi network protease inactivates TACE (Fig. 6E). A combination of these inhibitors cleaves the membrane-proximal stalk region of pro-BAFF between completely abrogated BAFF and APRIL release by CD40L- Arg133 and Ala134 and the membrane-proximal stalk region of pro- stimulated SMVECs (data not shown). In agreement with these APRIL between Arg104 and Ala105 (Fig. 6C). Alternative processing findings, immunohistofluorescence analysis and confocal micros- of BAFF has also been reported (55). Accordingly, the furin cleavage copy showed that MVECs from CLL tissues contained abundant The Journal of Immunology 9

FIGURE 6. MVECs enhance cleavase-dependent processing of BAFF and APRIL in response to CD40L. (A) Left panel, Time course QRT-PCR analysis of BAFF and APRIL mRNAs from SMVECs incubated with CD40L. Right panel, QRT-PCR analysis of BAFF mRNA from SMVECs exposed to CLL cells with low or high CD40L expression (three cases in each group). RE, relative expression compared with unstimulated SMVECs. (B) ELISA of BAFF and APRIL from UVECs or SMVECs incubated for 0, 3, or 6 h with CD40L. (C) Furin cleavage sites (blue arrows) and putative TACE cleavage sites (green arrows) of pro-BAFF and pro-APRIL proteins. The TACE cleavage site (red arrow) of the pro-TNF protein is shown for comparison. Numbers indicate amino acid residues in the membrane-proximal stalk region of BAFF, APRIL, and TNF. (D) Immunoblot of full-length pro-BAFF and pro-APRIL proteins from total lysates of UVECs and SMVECs incubated with medium alone (control) or CD40L for 6 h. b-Actin is a loading control. (E) ELISA of BAFF and APRIL in the supernatants from SMVECs incubated with or without CD40L and in the presence or absence of vehicle (DMSO), furin inhibitor (cho- romethylketone, CMK) or TACE inhibitor (TAPI-2). (F) Immunoﬂuorescence analysis of CLL lymph node (LN; three cases) stained for TACE or furin (green), Factor VIII (red), and IgD (blue). Original magniﬁcation 310. (G) QRT-PCR of TACE and furin in UVECs and SMVECs incubated for 1 or 5 h with CD40L. Results are normalized to b-actin mRNA. RE, relative expression compared with unstimulated cells. (H) Immunoblot of TACE and furin from SMVECs incubated with CD40L for various time points. Actin is a loading control. In the middle panel, 96-, 90-, and 60-kDa bands correspond to precursor, mature, and cleaved furin proteins. (I) Viable trypan blue-excluding CLL cells (one of three cases) cultured with SMVECs, UVECs, or MVECs in the presence or absence of control Ig or CD40-Ig for 0, 24, or 48 h. (J) Viable CLL cells (one of three cases) cultured with SMVECs, nucleofected with scrambled (scr) or CD40 siRNA for 0, 24, or 48 h. Data are from one of three experiments yielding similar results (C, D, F, H–J) or summarize three different measurements (A, B, E, G). Error bars, SEM; *p , 0.05 (ANOVA followed by one-tailed unpaired Student t test).

TACE and furin proteins, which were predominantly localized in and immunoblotting showed rapid upregulation of the expres- perinuclear compartments together with APRIL and (not shown) sion of furin and TACE transcripts as well as furin and TACE BAFF (Fig. 6F, Supplemental Fig. 3). Furthermore, QRT-PCRs proteins in SMVECs or UVECs exposed to CD40L (Fig. 6G, 6H). 10 INTERPLAY OF BAFF, APRIL, AND CD40L IN CLL

The importance of CD40L in the generation of active BAFF uated the viability of CLL cells cultured in the absence of MVECs and APRIL by MVECs was further documented by experiments (Fig. 7C). These data indicate that leukemic CD40L requires en- showing that the prosurvival activity of SMVECs or UVECs on dothelial BAFF and APRIL signals for its upregulation by MVECs CLL cells decreased when SMVECs or UVECs were either made and promotes CLL cell expansion and differentiation not only by deficient of CD40 through RNA interference (Fig. 6I, Supple- inducing paracrine release of BAFF and APRIL from MVECs but mental Fig. 4A) or exposed to a CD40L inhibitor such as CD40-Ig also by stimulating CLL cells in an autocrine manner (Fig. 7D). (Fig. 6J). Accordingly, interruption of CD40 signaling in MVECs attenuated the release of BAFF and APRIL elicited by CD40L- Discussion expressing CLL cells (Supplemental Fig. 4B). These data indicate We found that stromal MVECs delivered survival, activation, Ig that MVECs enhance CLL cell survival by releasing more BAFF gene remodeling, and differentiation signals to CLL cells by re- and APRIL in response to leukemic CD40L, possibly as a result of leasing BAFF and APRIL. Engagement of CD40 on MVECs by an increased intracellular processing of BAFF and APRIL pro- CD40L aberrantly expressed on CLL cells enhanced BAFF and proteins through furin and TACE cleavases. APRIL release through induction of furin and TACE cleavases, which elicited processing of BAFF and APRIL proproteins into MVECs upregulate CD40L on CLL cells via BAFF and APRIL soluble molecules. In addition to stimulating CLL cells through Given that MVECs upregulate CD40L expression on CLL cells TACI, BCMA, and BAFF-R, endothelial BAFF and APRIL aug- via NF-kB, we hypothesized that this upregulation could involve mented leukemic expression of CD40L, suggesting that MVECs MVEC production of NF-kB–inducing factors such as BAFF and and malignant B cells establish a bidirectional crosstalk via a APRIL. Consistent with this possibility, flow cytometry showed complex signaling network involving multiple TNF family that blockade of BAFF and APRIL by TACI-Ig inhibited not only members. the upregulation of class-switched IgG and IgA isotypes but also CLL is characterized by a dynamic balance between malignant the upregulation of CD40L on CLL cells exposed to SMVECs B cells circulating in the blood and malignant B cells located in (Fig. 7A). This inhibition was specific because blockade of BAFF permissive niches of lymphoid organs (57, 58). Circulating CLL and APRIL by TACI-Ig did not decrease TACI, BCMA, and cells show resistance to apoptosis, but little or no proliferation, BAFF-R expression on CLL cells exposed to SMVECs. In whereas a significant fraction of tissue-based CLL cells show agreement with these findings, CLL cells upregulated CD40L active proliferation, suggesting that the tissue microenvironment expression on CLL cells exposed to BAFF or APRIL (Fig. 7B). provides a distinct set of signals to the leukemic clone (10, 11). In This inhibition was reversed by preincubating CLL cells with a this regard, the CLL stroma has been proposed to provide Ig- specific NF-kB inhibitor (data not shown). The functionality of driven antigenic signals by facilitating the interaction of stereo- CD40L on CLL cells was further evaluated by verifying its ability typic leukemic Igs with self-antigens exposed on apoptotic cells to deliver autocrine survival signals independently of MVECs. (59). The CLL stroma also contains nurselike cells, macrophages, Consistent with this possibility, a CD40-Ig decoy receptor atten- and follicular DCs that provide accessory survival signals, in-

FIGURE 7. MVECs upregulate CD40L expression on CLL cells through BAFF and APRIL. (A) Flow cytometric analysis of CD40L, IgG, IgA, BAFF-R, TACI, and BCMA on CLL cells (one of six cases) incubated with endothelial medium alone (red open profile; also shown in center and right panels), SMVECs plus control Fc5 (blue open profile), or SMVECs plus TACI-Ig (blue open profile) for 7 d. Gray solid profile, isotype-matched control. (B) Flow cytometric analysis of CD40L on CLL cells (one of six cases) incubated with endothelial medium alone (red open profile; also shown in center and right panels)or endothelial medium supplemented with BAFF or APRIL (blue open profile) for 7 d. Gray solid profile, isotype-matched control. (C) Viable trypan blue-excluding CLL cells (one of six cases) cultured with standard medium in the presence of control Ig or CD40-Ig for 0, 24, or 48 h. Data in (A)–(C) are from one of six experiments yielding similar results. (D) Model summarizing crosstalk between MVECs and CLL cells via CD40L, BAFF, and APRIL. The Journal of Immunology 11 cluding BAFF and APRIL (13, 14, 60, 61). Moreover, the CLL sion in MVECs, as well as TACI, BCMA, and BAFF-R expression stroma contains T cells expressing CD40L (24), a B cell-stim- in neoplastic B cells, suggesting that CD40, TACI, BCMA, and ulating factor structurally and functionally related to BAFF and BAFF-R form a CLL-enhancing signaling network that bidirec- APRIL (19, 20). tionally links the microvascular stroma with the leukemic clone. Our findings indicate that MVECs constitute a key component Consistent with this possibility, published data show that CD40L of the CLL stroma by establishing an intimate crosstalk with neo- and BAFF cooperatively enhance B cell survival (44, 65). Re- plastic B cells through BAFF, APRIL, and CD40L. CLL tissues markably, TACI and BCMA contributed as much as BAFF-R to contained MVECs that were proximal to malignant B cells the survival and activation of CLL cells exposed to MVECs. Al- expressing molecular hallmarks of ongoing proliferation and Ig though the important function of BAFF-R and TACI in nonma- gene remodeling, including Ki-67 and AID, respectively. Con- lignant and malignant B cells is well established (19, 20, 43–45, sistent with these data, MVECs stimulated leukemic cell survival, 47–49, 66–71), less is known about the function of BCMA. This activation, and differentiation, including CD38 expression, AID receptor delivers survival signals to nonmalignant plasma cells expression, CSR, and Ab production, through both contact-de- (72, 73), but growing evidence indicates its additional involve- pendent and contact-independent mechanisms, including soluble ment in B cell and plasma cell tumors (44, 45, 47, 49, 74). BAFF and APRIL, as well as membrane-bound CD40L molecules. Blockade of endothelial BAFF and APRIL by TACI-Ig or That MVECs may play a pivotal role in the pathogenesis of CLL is blockade of leukemic CD40L by CD40-Ig reduced the survival, in agreement with evidence showing that malignant B cells from activation, and diversification of CLL cells cocultured with aggressive CLL cases express CD49d, a receptor for VCAM-1 (or MVECs. The inhibitory effect of TACI-Ig on leukemic AID ex- CD106) expressed by endothelial cells (62). It is also in agree- pression as induced by MVECs may have clinical relevance, be- ment with data showing interaction of leukemic CD38 with the en- cause Ig and non-Ig gene lesions induced by dysregulated AID dothelial adhesion molecule CD31 (39) and with additional studies expression may play a role in the survival and clonal evolution demonstrating that CLL cells produce angiogenic factors such as of CLL cells (6, 33, 75). In conclusion, our findings show that VEGF and generate survival signals via VEGF receptors (25, 30). stromal MVECs establish a bidirectional interplay with CLL cells In mice, BAFF from radiation-resistant stromal cells is manda- through multiple functionally related members of the TNF family, tory for the survival of peripheral B cells (63), but the nature of including BAFF, APRIL, and CD40L. Blocking these molecules BAFF-producing stromal cells remains unclear. We found that with specific inhibitors may be beneficial for the treatment of MVECs contained more BAFF transcripts than did DCs and macro- CLL. phages and released large amounts of BAFF and APRIL, both constitutively and after activation. Tonic release of BAFF and APRIL may involve CD63-expressing endosomes widely dispersed in the Acknowledgments cytoplasm of MVECs, whereas inducible release of BAFF and We thank Lauren Tyrell (Weill Medical College of Cornell University, New York, NY) for administrative and technical assistance. APRIL may involve perinuclear Weibel–Palade bodies containing vWF and factor VIII. These endothelial storage granules release their content in response to various signals, including CD40L (64). Disclosures In agreement with this, engagement of CD40 on MVECs by CD40L S.R.D. is employed by ZymoGenetics (a Bristol-Myers Squibb company) aberrantly expressed on CLL cells augmented endothelial pro- and helped with the early development of TACI-Ig. The other authors have cessing of pro-BAFF and pro-APRIL proteins into soluble mole- no financial conflicts of interest. cules through a mechanism involving furin and TACE cleavases, which were constitutively stored in Weibel–Palade bodies. Of note, CD40L rapidly increased furin and TACE transcription and References expression by MVECs. This process may be important for 1. Chiorazzi, N., K. R. Rai, and M. Ferrarini. 2005. Chronic lymphocytic leukemia. N. Engl. J. Med. 352: 804–815. MVECs to rapidly undergo post-degranulation replenishment of 2. Damle, R. N., T. Wasil, F. Fais, F. Ghiotto, A. Valetto, S. L. Allen, furin and TACE proteins. The relative contribution of furin and A. Buchbinder, D. Budman, K. Dittmar, J. Kolitz, et al. 1999. Ig V gene mutation TACE to BAFF and APRIL cleavage remains uncertain, but one status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94: 1840–1847. possibility is that these proteases form alternative proteolytic path- 3. Hamblin, T. J., Z. Davis, A. Gardiner, D. G. Oscier, and F. K. Stevenson. 1999. ways to maximize BAFF and APRIL release by MVECs (54, 55). 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