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 Avanc¸ats, 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 Innovacio´n 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 in- hibitor) 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);