Heterogeneous Expression of Interleukin-18 and Its Receptor in B-Cell Lymphoproliferative Disorders Deriving from Naive, Germinal Center, and Memory B Lymphocytes

144 Vol. 10, 144–154, January 1, 2004 Clinical Cancer Research

Irma Airoldi,1 Lizzia Raffaghello,1 Conclusions: Dysregulated expression of IL-18 and/or Claudia Cocco,1 Roberta Guglielmino,1 IL-18R in chronic B-cell lymphoproliferative disorders may sometimes contribute to tumor escape from the host immune Silvio Roncella,2 Franco Fedeli,2 system. Claudio Gambini,3 and Vito Pistoia1 Laboratories of 1Oncology and 3Pathology, G. Gaslini Institute, 2 INTRODUCTION Genoa, Italy, and Laboratory of Pathology, St. Andrea Hospital, La ␥ Spezia, Italy Interleukin (IL)-18 or IFN- inducing factor (IGIF) is a pro-inflammatory cytokine produced by different cell types (as reviewed in Ref. 1). IL-18 is synthesized as inactive precursor ABSTRACT that is converted into the bioactive molecule after intracellular Purpose: Dysregulated cytokine/cytokine receptor ex- cleavage by caspase-1 (2, 3), or extracellular modification by pression may occur in B-cell lymphoproliferative disorders. the proteinase-3 (PR-3) serine esterase (4). Little information is available on interleukin-18 receptor IL-18 shares a number of functional properties with IL-12, (IL-18R) and IL-18 expression in normal and malignant B such as amplification of IFN-␥ secretion by T, natural killer cells. Our purpose was to investigate this issue in human (NK), and NKT cells (5–9), enhancement of granulocyte macro- naive, germinal center (GC) and memory B cells, and in phage colony-stimulating factor production by T lymphocytes their neoplastic counterparts. (10), and induction of cytotoxic activity, as well as of Fas ligand Experimental Design: We have evaluated IL-18 expres- expression, in T and NK cells (8, 11). IL-18 drives T helper (Th) sion and production in tonsil naive, GC, and memory B cells type 1 (Th1) responses, mostly in association with IL-12 (1, 5, and in their presumed neoplastic counterparts by reverse 12). Furthermore, it has been demonstrated that IL-18 can transcription-PCR and ELISA. Moreover, IL-18R␣ and ␤ promote Th2 responses in different in vitro and in vivo models expression was investigated in the same cells by reverse (7, 13). transcription-PCR, flow cytometry, and immunohistochem- Effective antitumor responses are boosted in mice by IL-18 istry. gene transfer into tumor cells or systemic administration of the Results: We found that: (a) IL-18 mRNA was expressed cytokine, especially in combination with IL-12 or IL-2 (14–20). in tonsil naive, GC, and memory B cells. Bioactive IL-18 was The antitumor activity of IL-18 is primarily mediated by NK- secreted by naive and GC, but not by memory B cells; (b) and T-cell activation (16, 18–21). IL-18R␣ and ␤ transcripts were expressed in the three The IL-18 receptor (IL-18R) is a heterocomplex composed B-cell subsets. IL-18R␣ was detected on the surface of naive, of a constitutive ligand-binding chain, designated as ␣ chain and GC, and memory B lymphocytes, and IL-18R␤ was detected originally described as IL-1 receptor-related protein (IL-1RrP; on GC and memory, but not naive, B cells; (c) mantle zone, Ref. 22), and of an inducible accessory chain, the ␤ chain, follicular, marginal zone, Burkitt lymphoma (BL), and B- originally named accessory protein like (AcPL; Ref. 23). IL- cell chronic lymphocytic leukemia (B-CLL) cells expressed 18R␣ binds the cytokine with low affinity, whereas the ␤ chain IL-18 mRNA. B-CLL and BL cells did not produce bioactive increases IL-18 binding affinity and is necessary to initiate IL-18; and (d) lymphoma B cells displayed heterogeneous signal transduction in target cells (23). IL-18R␣ is expressed on ϩ ϩ expression of either or both IL-18R chain mRNA. In con- the surface of NK cells, CD8 T lymphocytes, activated CD4 ϩ trast, B-CLL cells expressed both IL-18R chains at the T cells, and CD19 peripheral blood B lymphocytes (24), but mRNA and protein levels. information on IL-18R␤ expression is lacking. Scanty data are thus far available on expression of IL-18 and its receptor in human tumor cells. Thus, IL-18 mRNA has been detected in malignant cells from colon and ovarian carcinoma (25, 26), mantle cell lymphoma (27), and skin cancer (28). Received 7/11/03; revised 9/18/03; accepted 9/18/03. IL-18R␣ expression has been reported in different hematopoi- Grant support: Grants from Associazione Italiana per la Ricerca sul etic cell lines, part of which were derived from patients with Cancro (to V. P. and S. R.) and from Ministero della Salute (to V. P.). The costs of publication of this article were defrayed in part by the lymphoproliferative or myeloproliferative disorders, but not in payment of page charges. This article must therefore be hereby marked primary tumor cells (29). EBV infection has been shown to advertisement in accordance with 18 U.S.C. Section 1734 solely to up-regulate IL-18 expression in secondary lymphoid tissues indicate this fact. from patients with infectious mononucleosis and, at a lesser Requests for reprints: Irma Airoldi, Laboratory of Oncology, G. Gaslini Institute, Largo G. Gaslini 5, 16148 Genoa, Italy. Phone: 39- extent, posttransplant lymphoproliferative disorder (22). 010-5636342; Fax: 39-010-3779820; E-mail: laboncologia@ospedale- Previous studies have shown that abnormal expression of gaslini.ge.it. cytokine and/or their receptors in neoplastic B cells may con-

tribute to some aspects of the pathophysiology of B-cell lym- clonal antibodies (mAbs), and subsequently with immunomag- phoproliferative disorders (as reviewed in Ref. 30). netic beads coated with goat antimouse immunoglobulin Here we have investigated expression of IL-18, IL-18R␣, (Immunotech, Marseille, France; Ref. 38). Neoplastic B lympho- and IL-18R␤ in the major B-cell subsets present in secondary cytes, either from lymph nodes or peripheral blood, were puri- lymphoid tissues, i.e., naive, germinal center (GC) and memory fied by depletion of T and NK cells and of macrophages as B cells, and in their postulated malignant counterparts, based on above, and of residual normal B cells bearing the immunoglob- the hypothesis that dysregulated expression of IL-18, IL-18R␣, ulin light chain not expressed by the malignant clone using and IL-18R␤ may occur in the latter cells in relation to malig- immunomagnetic beads (38). nant transformation. Furthermore, although IL-18 production by The resulting cell fractions from tonsil contained on aver- monocytes-macrophages and dendritic cells has been well char- age 99% B cells, as assessed by staining with CD19 mAb (see acterized (1), little is known on the synthesis of this cytokine in below). The purity of B lymphoma cells was higher than 99%, the third type of professional antigen-presenting cells, i.e., B as assessed by expression of CD19 and of monotypic immuno- ϩ ϩ lymphocytes. IL-18 expression in B cells may influence their globulin light chains. CD3 T cells, CD56 NK cells, and ϩ ability to drive Th-cell differentiation after antigen presentation. CD68 macrophages were virtually absent (Ͻ1%) from these cell suspensions, as assessed by flow cytometry (see below). Naive B lymphocytes were isolated as IgDϩ cells from MATERIALS AND METHODS tonsil B lymphocyte suspensions by immunomagnetic bead ma- Patient Samples. This investigation was performed after nipulation. The IgDϪ B-cell fractions were further separated approval by a local Institutional Review Board. Surgically re- into CD38ϩ (GC) cells and CD38- (memory) cells using the moved tonsils from 10 patients with localized inflammatory same technique (39, 40). All of the above separation procedures disorders were obtained after informed consent. were performed at 4°C to prevent spontaneous apoptosis of GC Lymph node biopsies from six patients with follicular B cells. In some experiments, tonsil B cells and B-CLL cells lymphoma (FL), three patients with mantle cell lymphoma isolated from peripheral blood were cultured for 48 h (see (MCL), two patients with marginal zone lymphoma (MZL), and below). four patients with B-cell chronic lymphocytic leukemia (B- Cell Lines. A panel of Burkitt lymphoma (BL) cell lines, CLL), were obtained from the pathologists after completion of either EBVϩ (RAJI, DAUDI, and LAMD2.1) or EBVϪ all of the diagnostic procedures. Peripheral blood samples from (RAMOS, HBL2, and BJAB), were maintained in culture in 12 additional B-CLL patients were obtained after informed RPMI 1640 (Seromed) supplemented with 10% FCS (Seromed) consent. Diagnosis was established according to the criteria of mAbs for Flow Cytometry and Immunohistochemistry. the WHO classification (31). Clonal excess was assessed by the The following reagents were used for single, double, or triple ␬/␭ or ␭/␬ immunoglobulin light chain ratio, that ranged from a staining: phycoerythrin (PE)-conjugated anti-IL-18R␣ mAb minimum of 8:1 to a maximum of 20:1 in the different cases. All (R&D System, Minneapolis, MN), antihuman IL-18R␤ goat of the patients (15 males, 12 females; age range, 40–75 years) IgG (R&D System, Minneapolis, MN), PE-conjugated swine were untreated at the time of study. antigoat IgG (Caltag, Burlingame, CA), horseradish-conjugated According to the Revised European-American classifica- antigoat IgG (Santa Cruz Biotechnology, Santa Cruz, CA), tion of Lymphoid neoplasms (REAL; Ref. 32). and the subse- PE-conjugated anti-IgD mAb (Dako, Glostrup, Denmark), quent WHO classifications (31), FL originates from GC B cells, FITC-conjugated anti-IgD mAb (Caltag), tri-color-conjugated the majority of MCL cases derive from a CD5ϩ, CD19ϩ naive CD38 mAb (Caltag), FITC-conjugate CD3, CD19, CD56, and B cell homing in the follicular mantle of secondary lymphoid CD68 mAbs (Becton-Dickinson, San Jose`, CA). Controls were follicles, and MZL represents the abnormal expansion of mem- fluorochrome-conjugated, isotype-matched mAbs of irrelevant ory B cells. On the ground of recent studies, B-CLL can be specificity or goat non-immune serum (Caltag). The goat anti- dissected into different subsets according to the mutational human IL-18R␤ antiserum worked efficiently in immunohisto- status of immunoglobulin variable (IgV) region genes or to the chemistry assays with frozen, but not formalin-fixed and paraffin- expression of the CD38 or ZAP-70 markers (33–35). Although embedded, tissue sections, although it did not perform in flow the precise normal counterparts of B-CLL cells have not yet cytometry and Western blot assays. In contrast, the PE- been defined, cases with unmutated IgV genes may originate conjugated anti-IL-18R␣ mAb worked in flow cytometry ex- from a pre-GC B cell, either naive or activated, whereas those periments, but not in immunohistochemistry assays. carrying IgV gene mutations are of post-GC/memory B-cell Cells were scored using a FACScan analyzer (Becton- derivation (36, 37). Because of such heterogeneity, six periph- Dickinson, San Jose, CA) and data were processed using eral blood samples from unmutated and 6 from mutated B-CLL CellQuest software (Becton-Dickinson). The threshold line for patients were tested in this study. Analysis of IgV gene muta- assessment of percentage of positive cells was based on the tions was carried out by Dr. Franco Fais, Department of Exper- maximum staining obtained with irrelevant isotype-matched imental Medicine, University of Genova, as published previ- mAb, used at the same concentration as test mAb. Negative cells ously (33). were defined such that Ͻ1% of cells stained positive with Cell Separation and Culture. Mononuclear cells were control mAbs. Cells labeled with test antibody that were brighter isolated from tonsils on Ficoll-Hypaque density gradients and than those stained with isotypic control antibody were defined were depleted of T cells by rosetting with neuraminidase-treated as positive. Mean fluorescence intensity (MFI) values of the sheep erythrocytes. Non-T cells were deprived of macrophages isotypic control and of test mAbs were used to evaluate whether and NK cells by incubation, first with CD68 and CD56 mono- the differences between the peaks of cells were statistically

significant with respect to control. The Kolmogorov-Smirnov Tissue Studies. Frozen tissue sections were fixed in ice- test for the analysis of histograms was used, according to the cold acetone for 10 min at room temperature and were dried and CellQuest software user’s guide washed twice with Optimax Wash buffer. Endogenous peroxi- RT-PCR and Sequencing. RNA was extracted from dase activity was blocked by 30-min incubation at room tem-

freshly isolated cells using RNeasy Mini kit from Qiagen (Qia- perature with methanol containing 3% H2O2. Sections were gen GmbH, Hilden, Germany) and was subjected to reverse washed twice in Optimax Wash buffer and were incubated transcription-PCR (RT-PCR), as reported previously (41). overnight at 4°C with anti-human IL-18R␤ goat IgG at 5 ␮g/ml Primer sequences and profiles of amplification were described or with goat non-immune serum. Sections were washed three in previous studies (41), with the exception of the following: times in Optimax Wash buffer and were subsequently incubated IL-18 5Ј-GCTGAAGATGAAAACC and 3Ј-AGCTAGAAAG- with horseradish peroxide conjugate antigoat IgG (5 ␮g/ml). TATCCTTC; IL-18R␣ 5Ј-ATT ACC CTT GAC CCT TTG GG After washing in Optimax Wash buffer, peroxidase activity was and 3Ј-TCA AAC TCG GCG TTC TTC TT; IL-18R␤ 5Ј-CCT detected by incubating the sections for 6–10 min with DAKO GCC CTT CAT GGG TAG TA and 3Ј-ATC CAC TAC GAT Liquid DAB Substrate Chromogen System (DAKO). Sections TCG GTT GC. Amplification profile was 94°C for 1 min, were counterstained with Mayer’s hematoxylin (Sigma). annealing 55°C (IL-18), 47°C (IL-18R␣), 58°C (IL-18R␤) for 1 In some experiments, which were complementary to those min and extension at 72°C for 1 min. Each cycle of amplifica- carried out by immunohistochemistry, double staining of frozen tion was repeated 35 times. tonsil sections with CD19 mAb and anti-IL-18R␤ goat anti- Ten ␮l of each sample were electrophoresed through a 1% serum was performed by immunofluorescence. Sections were agarose gel containing ethidium bromide. The specificity of fixed in ice-cold acetone for 10 min at room temperature, were amplification products was checked by confirming the known dried, were washed twice with PBS, and were incubated for 20 base-pair sequence length and by sequencing. min at room temperature with blocking solution (PBS ϩ 10% Direct sequencing of PCR products was performed with the goat serum). Sections were next incubated overnight at 4°C with use of the Dye Terminator Cycle Sequencing kit (ABI PRISM; goat anti-human IL-18R␤ at 5 ␮g/ml or with goat non-immune Perkin-Elmer Applied Biosystem, Norwalk, CT). Sequences serum. After washing in PBS, sections were stained with a were resolved and analyzed on the ABI 373A Sequence Appa- PE-conjugated swine antigoat antibody for 45 min at 4°C. ratus (Perkin-Elmer Applied Biosystem). Subsequently, sections were washed three times in PBS and For semiquantitative RT-PCR analysis, nonsaturating con- were incubated with FITC-conjugated CD19 mAb for1hat ditions were used. In particular, one-fifth of the cDNA concen- 4°C. After washing in PBS and drying, sections were mounted tration tested above (i.e., 1 ␮l versus 5 ␮l) was used, and 30 with Vectashield Mounting medium containing 4Ј,6-diamidino- cycles of amplification instead of 35 were performed. 2-phenylindole (DAPI; Vector, Burlingame, CA). Digital im- Detection of IL-18 in Culture Supernatants. Tonsil ages of FITC, rhodamine, and DAPI fluorescence were acquired B-cell subsets were cultured in the presence or absence of separately by a CCD camera with highly selective filters using 6 different stimuli for 48 h at the concentration of 1 ϫ 10 cells/ml a Nikon Eclipse E1000 microscope (Nikon Instruments, Bad- in RMPI 1640 supplemented with 10% FCS. The stimuli tested howedorp, the Netherlands). were: anti-␬ (1 ␮g/ml; Southern Biotechnology Associates, Bir- Assay for Assessment of Apoptosis. Purified tonsil B mingham, Alabama) in combination with anti-␭ (1 ␮g/ml) im- lymphocytes were cultured in the presence or absence of munoglobulin light chain mAbs (Southern Biotechnology As- hrIL-18 (50 ng/ml) for 4–12 h, and GC apoptotic cells were sociates); CD40 mAb (1 ␮g/ml; Immunotech, Marseille, detected by tricolor staining for CD38, annexin V, and pro- France), alone or in combination with recombinant (r)IL-4 (10 pidium iodide by flow cytometry, using a kit according to ng/ml; Genzyme, Cambridge, MA); human recombinant the manufacturer’s instruction (Bender MedSystems, Vienna, (hr)IFN-␥ (1,000 units/ml; Boehringer Mannheim, Mannheim, Austria). Germany); hrIL-6 (100 units/ml) and hrIL-12 (kindly provided by Genetics Institute; 20 ng/ml); staphylococcus aureus (Calbiochem, La Jolla, CA; 1:10,000); and lipopolysaccharides RESULTS (Sigma-Aldrich, St. Louis, MO; 10 ng/ml). Because anti-␬, Purity of Neoplastic and Normal B-Cell Suspensions anti-␭, and CD40 mAbs were all of IgG1 isotype, an isotype- and Study Outline. Expression of the CD3␥, CD56, CD68, matched mAb of irrelevant specificity was tested as control at and CD19 genes in purified normal or neoplastic B-cell frac- the final concentration of 1 ␮g/ml. Cross-linking of surface tions was investigated by RT-PCR, to exclude that even minute immunoglobulin light chains was carried out by adding a goat amounts of contaminant cell types were present in the cell antimouse immunoglobulin antiserum (Caltag) to the B-cell suspensions. cultures containing anti-␬ and anti-␭, or isotype-matched, CD3␥ is selectively expressed in T cells, CD56 is a NK- mAbs. Purified B-CLL cells were cultured 48 h as above in the associated marker, CD68 is a specific macrophage marker and presence or absence of anti-immunoglobulin light chain mAbs CD19 expression is restricted to the B-cell lineage (41). Only under cross-linking conditions, or of CD40 mAb with or without normal or malignant B-cell suspensions that tested negative for rIL-4. BL cell lines were cultured for 48 h in medium alone. CD3␥, CD56, and CD68 mRNA expression were subjected to Culture supernatants were harvested and tested in triplicate additional studies. for IL-18 by ELISA, using a quantitative test that detects spe- Purified normal or neoplastic B cells were tested for IL-18, cifically the biological active form of IL-18 (MBL, Nagoya, IL-18R␣, and IL-18R␤ gene expression immediately after iso- Japan). The minimum threshold of this assay is 12.5 pg/ml. lation by RT-PCR. To check the correspondence between IL-18

mRNA accumulation and protein secretion, B-cell culture supernatants were tested for the presence of bioactive IL-18 by ELISA. IL-18R␣ surface expression was studied in naive, memory, and GC B lymphocytes, as well as in B-CLL cells, by flow cytometry. IL-18R␤ expression was investigated by immunohistochemistry on frozen sections from tonsils and on infiltrated B-CLL lymph nodes. Expression of the IL-18 Gene and Production of the IL-18 Protein in Human Tonsil Naive, GC, and Memory B Lymphocytes. Naive, GC, and memory B cells, freshly isolated from tonsils, were first tested for expression of the IL-18 gene. Fig. 1A shows one representative experiment in which the purity of tonsil B cells was assessed by RT-PCR, according to the protocol described in the previous paragraph. As apparent, the different B-cell fractions expressed CD19, but not CD3␥, CD56, or CD68 mRNA. Fig. 1B shows two experiments, representative of the 10 performed with superimposable results, in which the IL-18 transcript was found to be expressed in naive, GC, and memory B cells. To investigate potential differences among the three B-cell subsets in IL-18 mRNA expression, the RT-PCR conditions were modified to allow a semiquantitative evaluation of the results. Fig. 1C shows one experiment representative of the three performed with comparable results. As apparent, the intensity of IL-18 transcript bands appeared of similar intensity in naõ¬ve, GC, and memory B cells. Next, the same B-cell fractions were cultured for 48 h in the presence or absence of different stimuli (i.e., SAC, LPS, IFN-␥, IL-6, IL-12, CD40 mAb, and/or IL-4, anti-␬ and anti-␭ mAbs followed by cross-linking with goat antimouse immunoglobulin), and IL-18 was assayed in culture supernatants by an ELISA, which detects exclusively the biologically active form Fig. 1 A, expression of the GAPDH, CD3␥, CD56, CD68, and CD19 of the cytokine. CD40 mAb and IL-4 mimic in vitro T-depend- genes in naive, germinal center (GC) and memory B lymphocytes ent B-cell activation, whereas cross-linking of the B-cell recep- freshly purified from tonsil, as assessed by reverse transcription (RT)- PCR. One representative experiment of the 10 performed with similar tor mimics T-independent B-cell activation. All of the other results is shown. From left to right: Mr markers (MW); negative control stimuli allow us to assess the effects of bacterial components or (NC), represented by water in the place of cDNA; positive control (PC), pro-inflammatory cytokines on B-cell IL-18 production. represented by mononuclear cells isolated from tonsil; purified tonsil B Table 1 shows the ranges of the results obtained in eight lymphocytes (Tonsil B cells); naive B cells (Naive), GC B lymphocytes (GC), and memory B cells (Memory). On the right, the expected M of different experiments. Naive and GC B lymphocytes produced r the amplified bands. B, expression of the IL-18 gene in naive, GC, and IL-18 in the absence of stimuli, and such production was only memory B lymphocytes freshly isolated from tonsil. Two representative marginally decreased by CD40 mAb and anti-immunoglobulin experiments of the 10 performed with similar results are shown. From mAbs. GC B lymphocytes produced lower amounts of IL-18 left to right: Mr markers (MW); negative control (NC), represented by than did naive B cells (Table 1). In contrast, memory B lym- water in the place of cDNA; positive control (PC), represented by mononuclear cells isolated from tonsil; naive B cells (Naive); GC B phocytes did not produce IL-18, either unstimulated or after in lymphocytes (GC); and memory B cells (Memory). On the right, the vitro stimulation (Table 1). Notably, unfractionated tonsil B expected Mr of the amplified band. C, semiquantitative RT-PCR anal- cells released IL-18 in culture supernatants in the absence of ysis of IL-18 gene expression in naive, GC, and memory B lymphocytes stimuli (1480Ð2120 pg/ml), indicating that production of the freshly isolated from tonsil (see “Materials and Methods” for details). cytokine detected with purified naive and GC B cells was not One representative experiment of the three performed with similar results is shown. From left to right: Mr markers (MW); negative control induced by the separation procedures. (NC), represented by water in the place of cDNA; naive B cells (Naive), IL-18R␣ and IL-18R␤ Expression in Human Tonsil GC B lymphocytes (GC), and memory B cells (Memory). On the right, Naive, GC, and Memory B Lymphocytes. Fig. 2A shows the expected Mr of the amplified band. that IL-18R␣ and ␤ transcripts were consistently expressed in freshly isolated naive, GC, and memory B lymphocytes. Two representative experiments of the 10 performed with identical 2B shows one such experiment, representative of the three results are shown. performed with comparable results. As apparent, the bands Additional studies were carried out to investigate potential corresponding to IL-18R␣ and ␤ transcripts appeared fainter in differences in IL-18R␣ and ␤ mRNA expression among naive, naõ¬ve B cells than in GC or memory B cells (Fig. 2B), suggest- GC, and memory B cells, using the same semiquantitative ing that the rate of IL-18R␣ and ␤ gene transcription was lower RT-PCR technique referred to in the previous paragraph. Fig. in the former than in the latter cells.

Table 1 Production of bioactive interleukin (IL)-18 (pg/ml) by human tonsil B-cell subsetsa,b Naive Germinal center Memory Stimuli B cells B cells B cells Medium 1680Ð2120 424Ð653 Ͻ12.5 ␣␬ϩ␭ 856Ð1042 243Ð328 Ͻ12.5 ␣CD40 825Ð934 214Ð252 Ͻ12.5 ␣CD40 ϩ IL-4 1078Ð1182 285Ð318 Ͻ12.5 IgG1 isotype 1450Ð1630 387Ð541 Ͻ12.5 IL-4 1698Ð1769 290Ð320 Ͻ12.5 IL-6 1600Ð1768 362Ð486 Ͻ12.5 IL-12 1320Ð1746 334Ð380 Ͻ12.5 IFN-␥ 1462Ð2120 420Ð683 Ͻ12.5 SAC 1618Ð1676 437Ð469 Ͻ12.5 LPS 1644Ð2120 410Ð547 Ͻ12.5 a Results are ranges from eight different experiments. b Culture supernatants were harvested after 48 h. Bioactive IL-18 was detected by ELISA, as indicated in “Materials and Methods.” The minimum threshold of this assay is 12.5 pg/ml.

Next, surface expression of IL-18R␣ was investigated by flow cytometric analysis of freshly isolated tonsil B cells. Tonsil B-cell fractions were stained with anti-IgD, CD38, and anti-IL- 18R␣ mAbs. Naive B cells were identified as IgDϩ, CD38Ϫ cells; GC B cells as CD38ϩ, IgDϪ cells; and memory B cells as IgDϪ, CD38Ϫ cells. Fig. 2C shows the results obtained in three different experiments of the six performed. GC and memory B lymphocytes expressed surface IL-18R␣ (Fig. 2C) in the following ranges: 36Ð53% for GC, and 32Ð56% for memory B cells. Notably, expression of IL-18R␣ was detected both in CD38ϩϩ centro- blasts and in CD38ϩ centrocytes (not shown). From 2 to 6% of naive B cells stained for IL-18R␣ in the six experiments (Fig. 2C). Immunohistochemical studies were subsequently carried out by staining frozen tonsil sections with anti-IL-18R␤ antibodies. IL-18R␤-positive cells were detected both in the GC and in the subepithelial area, whereas staining of the follicular Fig. 2 A. Expression of the IL-18R␣ and IL-18R␤ genes in tonsil naive, germinal center (GC), and memory B lymphocytes, as assessed mantle was predominantly negative, with the exception of by 2 representative experiments of the 10 performed with similar results scanty positive cells (Fig. 3A, left and middle panels). No are shown. From left to right: Mr markers (MW); negative control (NC), staining at all was detected when non-immune goat serum was represented by water in the place of cDNA; positive control (PC), used in the place of the anti-IL-18R␤ antiserum (Fig. 3A, right represented by purified tonsil B cells incubated with rIL-12 (20 ng/ml) panel). for 24 h; naive B cells (Naive), GC B lymphocytes (GC), and memory B cells (Memory). On the right, the expected Mr of the amplified Fig. 3B, panel 1, shows DAPI staining of a frozen tonsil bands. B, semiquantitative reverse transcription (RT)-PCR analysis section in which the mantle zone and the GC of a follicle are of IL-18R␣ and IL-18R␤ gene expression in naive, GC, and memory highlighted together with the subepithelial area, in which B cells B lymphocytes freshly isolated from tonsil (see “Materials and are located (40). Serial tonsil sections were stained by immu- Methods” for details). One representative experiment of the three ␤ performed with similar results is shown. From left to right: Mr nofluorescence with CD19 mAb, anti-IL-18R antibodies, or markers (MW); negative control (NC), represented by water in the both. To better visualize coexpression of CD19 and IL-18R␤, place of cDNA; naive B cells (Naive), GC B lymphocytes (GC), and

we selected and inspected, at high magnification, microscopic memory B cells (Memory). On the right, the expected Mr of the fields enriched for CD19ϩ cells. amplified band. C, expression of IL-18R␣ on the surface of tonsil GC Fig. 3B, panel 2, shows an enlarged GC area, as detected (GC), memory cells, and naive B lymphocytes, as assessed by flow cytometry. Three representative experiments (Exp. 1,2,3) of the six by DAPI staining. Panels 3 and 4 show single staining with performed with similar results are shown. Results are expressed as CD19 and anti-IL-18R␤, respectively, whereas panel 5 shows percentage of positive cells. Purified tonsil B cells were stained with triple staining with DAPI, CD19, and anti-IL-18R␤. As appar- fluorochrome-conjugated CD38, anti-IgD, and anti-IL-18R␣ mono- ent, in the latter panel, a yellow staining developed as a conse- clonal antibodies (mAbs). GCs were detected gating on CD38ϩ, IgDϪ cells; memory B cells gating on CD3Ϫ, IgDϪ cells; and naive quence of green (panel 3) and red (panel 4) color overlap, ϩ Ϫ ϩ B cells gating on IgD , CD38 cells. Open profiles, specific indicating that virtually all CD19 cells in the GC coexpressed staining for IL-18R␣; black profiles, staining obtained with fluoro- IL-18R␤. chrome-conjugated, isotype-matched mAbs of irrelevant specificity.

Fig. 3 Expression of IL-18R␤ in frozen tonsil sections, as assessed by immunohistochemistry (A) and immunofluorescence (B). A, left panel, expression of IL-18R␤ in the germinal center (GC), the mantle zone (MZ), and the subepithelial (SE) area after peroxidase staining (ϫ10). Middle panel, higher magnification (ϫ40) of the microscopic area highlighted in the left panel shows that most GC and SE B lymphocytes display staining for IL-18R␤. In contrast, only occasional positive cells (arrowheads) are observed in the MZ. Right panel, a follicle stained with control antibody by the peroxidase reaction (ϫ10). No staining is observed in GC, SE, or MZ areas. B, 1, 4Ј,6-diamidino-2-phenylindole (DAPI) staining of a frozen tonsil section, in which MZ and GC of a follicle, together with SE, are highlighted (ϫ20). 2, enlarged GC area, as detected by DAPI staining (ϫ100). 3, single staining of this area with CD19 (green) shows diffuse positivity (ϫ100). 4, single staining of the same area with anti-IL-18R␤ (red) shows that virtually all of the cells are positive (ϫ100). 5, triple staining of the GC area with DAPI, CD19, and anti-IL-18R␤ (ϫ100). Overlap of green and red colors gives rise to diffuse yellow staining, indicating that most cells coexpress CD19 and IL-18R␤. 6, enlarged SE area, as detected by DAPI staining (ϫ100). 7, single staining of this area with CD19 (green) shows diffuse positivity (ϫ100). 8, single staining of the same area with anti-IL-18R␤ (red) shows that virtually all of the cells are positive (ϫ100). 9, triple staining of the SE area with DAPI, CD19, and anti-IL-18R␤ (ϫ100). Overlap of green and red colors gives rise to diffuse yellow staining, indicating that most cells coexpress CD19 and IL-18R␤. 10, double staining of the SE area with DAPI and a FITC-conjugated, isotype-matched irrelevant monoclonal antibodies used as control for CD19 staining.

Fig. 3B, panels 6, shows DAPI staining of an enlarged These results indicated that the complete IL-18R, com- subepithelial area, in which memory B cells settle (40). Fig. 3B, posed of the ␣ and ␤ chains, was expressed by GC and memory panels 7 and 8, show single staining of the same area with CD19 B cells, but not by naive B lymphocytes. and anti-IL-18R␤, respectively, whereas panel 9 shows triple Because GC B cells were the only subset that produced staining with DAPI, CD19, and anti-IL-18R␤. Most CD19ϩ constitutively the IL-18 protein and that expressed IL-18R on cells in the subepithelial area were found to coexpress IL-18R␤, the cell surface, they were tested for propensity to undergo as indicated by the development of a yellow staining. Panel 10 apoptosis in vitro after 4Ð12 h culture of purified tonsil B shows the negative staining, obtained with fluorochrome- lymphocytes with or without rIL-18. The proportions of apo- conjugated isotypic control of CD19 mAb, of a DAPI-counter- ptotic GC B cells that were detected as CD38ϩ, annexin Vϩ,or stained tonsil tissue section. propidium iodideϩ cells were similar in the presence and in the Finally, most CD19ϩ B cells in the follicular mantle were absence of rIL-18 at all times tested, indicating that the cytokine IL-18R␤ negative (not shown). This is consistent with the was devoid of any pro- or antiapoptotic activity (not shown). immunohistochemical results shown in Fig. 3A. Expression of the IL-18 Gene in Neoplastic B Cells from When goat non-immune serum was tested in the place of MCL, FL, and MZL. IL-18 gene expression in MCL, FL, and anti-IL-18R␤ antibodies, no staining was observed (not shown). MZL B cells was next investigated. Fig. 4A shows the purity of

genes), were cultured with medium alone, with CD40 mAb with or without rIL-4, or with anti-immunoglobulin light chain mAbs under cross-linking conditions for 48 h. IL-18 was never detected in cell culture supernatants (data not shown). Expression of the IL-18 Gene and Production of the IL-18 Protein by BL Cell Lines. Fig. 4B shows that IL-18 gene transcript was detected in six BL cell lines, which represent malignant expansions of GC B lymphocytes (32), irrespective of their EBVϩ (three cell lines) or EBVϪ (three cell lines) status. Constitutive production of the bioactive IL-18 protein was evaluated by ELISA, using supernatants from BL cell lines cultured for 48 h in the absence of stimuli. The assay was negative for all of the supernatants tested. IL-18R␣ and -␤ Gene Expression in Neoplastic B Cells from MCL, FL, and MZL. Next, the expression of IL-18R␣ Fig. 4 A, expression of CD3␥, CD56, CD68, and CD19 genes in and ␤ transcripts in freshly isolated malignant B cells from three neoplastic B lymphocytes purified from lymph nodes of patients with MCL, six FL, and two MZL cases was investigated. Variable mantle cell lymphoma (MCL), follicular lymphoma (FL), and marginal expression of IL-18R␣ and ␤, as assessed by RT-PCR, was zone lymphoma (MZL), or from peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients, as assessed by reverse tran- detected in the individual cases. Thus, B cells from two of three scription (RT)-PCR. One representative experiment is shown. From left MCL patients expressed the transcripts of both IL-18R chains, to right: Mr markers (MW); negative control (NC), represented by water whereas, in the third case, neither transcript was found. Tumor in the place of cDNA; positive control (PC), represented by mononu- cells from three of six FL patients showed expression of IL- clear cells isolated from tonsil; tumor cells from MCL, FL, MZL, and 18R␣ and ␤ mRNA. IL-18R␤ mRNA only was detected in two B-CLL. On the right, the expected Mr of the amplified bands. B, expression of the IL-18 gene in malignant B cells from two samples, of six cases, and IL-18R␣ transcript only was observed in one of each, of MCL, FL, MZL, and B-CLL, as well as from six Burkitt six cases. Malignant B cells from one of two MZL patients lymphoma (BL) cell lines, as assessed by RT-PCR. From left to right: expressed the transcripts of both IL-18R chains, whereas, in the M markers (MW); NC, represented by water in the place of cDNA; PC, r other case, mRNA of either IL18R␣ or ␤ was not detected. Fig. represented by mononuclear cells isolated from tonsil; MCL, FL, MZL, B-CLL, and BL cell lines (Raji, Daudi, LAMD2.1, Ramos, HBL2, 5A shows the results obtained with two each of representative

BJAB). On the right, the expected Mr of the amplified band. MCL, FL, and MZL cases. IL-18R␣ and IL-18R␤ Expression in B-CLL. Next, IL-18R␣ and ␤ expression was investigated at the mRNA level in the same 12 B-CLL cases already tested for IL-18 expression the malignant B-cell suspensions from one each, representative (see above). Fig. 5A shows that, in two representative B-CLL MCL, FL, and MZL cases, in which only the CD19 transcript cases (one with unmutated, the other with mutated IgV genes), was consistently found (Fig. 4A). malignant B cells expressed the transcripts of the genes encod- Fig. 4B shows IL-18 gene expression in B cells from two ing both IL-18R components. The same pattern of expression representative cases of MCL, FL, and MZL. Identical results was consistently detected in the remaining 10 B-CLL cases (not were obtained with one additional MCL and four additional FL shown). cases (not shown). In subsequent experiments, IL-18R␣ expression on B-CLL MCL B cells, the presumed counterpart of naive B lym- cells was studied by flow cytometry. Fig. 5B shows two repre- phocytes, constitutively expressed the IL-18 transcript. The sentative cases (one with unmutated, the other with mutated IgV same result was obtained with B cells from FL, which originates genes), in which IL-18R␣ was detected on the surface of ma- from GC B lymphocytes, and from MZL, the postulated coun- lignant cells. Expression of IL-18R␣ was consistently observed terpart of memory B cells (Fig. 4B). Because of the paucity of in six additional B-CLL cases, three with unmutated and three purified neoplastic B cells, production of IL-18 in culture su- with mutated IgV region genes (range of positive cells, 18Ð pernatants could not be addressed. 39%). Expression of the IL-18 Gene and Production of the Finally, immunohistochemical studies with frozen sections IL-18 Protein by B-CLL Cells. We next investigated IL-18 from four B-CLL lymph nodes were carried out to investigate gene expression and production of bioactive IL-18 by malignant expression of the IL-18R␤ protein on the surface of tumor cells. B cells purified from the peripheral blood of 12 B-CLL patients, All of these samples displayed effacement of the lymph node 6 with unmutated and 6 with mutated IgV genes. architecture. Fig. 5C shows the results obtained with two B-CLL Fig. 4A shows the purity of one representative B-CLL case, lymph nodes, representative of the four studied. As apparent, the as assessed by molecular analysis. Fig. 4B shows constitutive IL-18R␤ protein was detected on the surface of large cells, often expression of IL-18 gene transcript in B cells from 2 represent- referred to as paraimmunoblasts (42), that appeared clustered in ative B-CLL cases (one with unmutated, the other with mutated pseudofollicles. Most tumor cells with small lymphocyte mor- IgV genes), of the 12 tested with superimposable results. phology did not express IL-18R␤ (Fig. 5C). To investigate the production of bioactive IL-18 in B-CLL IL-18R␣ and IL-18R␤ Gene Expression in BL Cell culture supernatants, malignant B cells, purified from four dif- Lines. In a final series of experiments, IL-18R␣ and IL-18R␤ ferent B-CLL cases (two with unmutated, two with mutated IgV mRNA expression was investigated in the six BL cell lines. The

Fig. 5 A, expression of the IL-18R␣ and IL-18R␤ genes in malignant B cells from two samples each of mantle cell lymphoma (MCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL), and B-cell chronic lymphocytic leukemia (B-CLL), as well as from three representative Burkitt lymphoma (BL) cell lines, as assessed by reverse transcription (RT)-PCR. From left to right: Mr markers (MW); negative control (NC), represented by water in the place of cDNA; positive control (PC), represented by purified tonsil B cells incubated with recombinant interleukin (rIL-12; 20 ng/ml) for 24 h; MCL, MZL, FL, ␣ B-CLL, and BL cell lines (Raji, Ramos, BJAB). On the right, the expected Mr of the amplified bands. B, expression of IL-18R on the surface of malignant B cells from two representative B-CLL cases, as assessed by flow cytometry. Results are expressed as percentage positive cells. Purified B cells were stained with fluorochrome-conjugated anti-IL-18R␣ monoclonal antibody (mAb). Open profiles, specific staining for IL-18R␣; violet profiles, staining obtained with fluorochrome-conjugated, isotype-matched mAbs of irrelevant specificity. C, expression of IL-18R␤ in frozen B-CLL lymph nodes sections, as assessed by immunohistochemistry. Two representative cases of the four studied with similar results are shown. The lymph node architecture is effaced by neoplastic B cells. Large lymphoid cells, often referred to as paraimmunoblasts, stain for IL-18R␤ (left panel, arrows), although the bulk of lymphocytes are apparently negative (ϫ40). Paraimmunoblasts are clustered in pseudofollicles (right panel, arrows).

results obtained with one representative EBVϩ and one repre- from MCL, FL, MZL, B-CLL, and BL. Only naive and GC B sentative EBVϪ BL cell line are shown in Fig. 5A. The tran- cells produced the bioactive IL-18 protein in culture superna- scripts of the IL-18R␣ and ␤ chains were not detected in these, tants, whereas this was not the case for memory B cells, B-CLL, nor in the four remaining, BL cell lines. and BL tumor cells. Expression of IL-18R␣ and IL-18R␤ transcripts was al- DISCUSSION ways observed in tonsil naive, GC, and memory B cells. Cyto- In this study, we have investigated the expression of IL-18, fluorimetric studies showed that IL-18R␣ was expressed in GC IL-18R␣, and IL-18R␤ in human tonsil naive, GC, and memory and memory B cells but was virtually undetectable in naive B B lymphocytes, and in their presumed neoplastic counterparts. cells. Likewise, the bulk of tonsil GC and subepithelial, but not Expression of the IL-18 gene was consistently detected in follicular mantle, B cells were found, by immunohistochemistry, freshly isolated normal B-cell subsets and in malignant B cells to express IL-18R␤. The finding that the IL-18R␣ and IL-18R␤

proteins were poorly expressed in naive B cells may be related consistent expression of IL-18R␣ and -␤ mRNA, irrespective of to transcriptional and/or posttranscriptional events. Semiquanti- the IgV gene mutational status. Surface expression of IL-18R␣ tative RT-PCR experiments, indeed, suggested that the tran- was detected in B-CLL cells isolated from the peripheral blood, scriptional rate of IL-18R␣ and IL-18R␤ genes was lower in whereas IL-18R␤ protein expression was investigated in infil- naive than in GC or memory B lymphocytes. trated B-CLL lymph nodes. The latter experiments demon- Finally, variable positivity for mRNA expression of IL- strated that the majority of small B-CLL lymphocytes were 18R␣ or ␤ was found in MCL, FL, and MZL samples. B-CLL unreactive with the anti-IL-18R␤ antibody, whereas larger ma- cases showed consistent expression of IL-18R␣ and ␤ tran- lignant B cells, morphologically identifiable as paraimmuno- scripts, whereas neither transcript was detected in six BL cell blasts, displayed consistent staining. lines. A fraction of malignant B cells in the individual B-CLL IL-18R␣ and -␤ expression on the surface of B-CLL cells clones expressed IL-18R␣ and ␤ proteins. were obtained from discrete samples because of technical lim- The finding that GC B cells had the unique feature of itations of the antibodies used. However, neither chain of the producing IL-18 and expressing the two chains of the IL-18R on IL-18R was detected on the totality of malignant B cells from the cell surface, raised the possibility that the cytokine could the individual cases, suggesting that expression of the complete modulate programmed cell death in a paracrine and/or autocrine heterodimeric receptor was not clonally distributed. manner. However, no evidence in support of this hypothesis was Neither B-CLL cells nor BL cell lines produced bioactive obtained from in vitro experiments performed in the presence of IL-18, despite the consistent expression of IL-18 mRNA. Be- rIL-18. cause the B-CLL samples included mutated and unmutated Alternative functions of IL-18 produced by naive and GC cases, the negative results obtained with the latter are discordant B cells might be stimulation of immunoglobulin and IFN-␥ in relation to those observed with their possible normal coun- synthesis by the B cells themselves, as shown in human and terparts, i.e., naive B cells. Failure of BL tumor cells to produce murine models (43, 44), or interaction with other cell types IL-18 is clearly at variance with the behavior of their normal present in secondary lymphoid organs, such as T cells or fol- postulated counterparts, i.e., GC B cells. licular dendritic cells. In summary, the present study suggests that malignant B As mentioned, tonsil naive B cells displayed low-to-absent lymphocytes from different B-cell chronic lymphoproliferative surface expression of IL-18R␣ and ␤. However, we have pre- disorders display heterogeneous expression of IL-18 and of the viously demonstrated that IL-12 up-regulates the expression of two components of the IL-18R, as compared with their normal both IL-18R chains in tonsil B cells, and synergizes with IL-18 counterparts. It is tempting to speculate that deranged expres- in the induction of IFN-␥ production by IgDϩ naive B cells (43). sion of the cytokine and/or its receptor in neoplastic B cells may These latter findings, together with the results of the present in some cases contribute to the facilitating of tumor growth, e.g., study, suggest that resting naive B cells are unresponsive to by impairing antitumor effector mechanisms because of down- IL-18 but become responsive to it after interaction with IL-12, regulation of IL-18 production. e.g., in the course of an inflammatory process. Despite consistent expression of IL-18 mRNA, memory B ACKNOWLEDGMENTS cells did not produce the bioactive cytokine in culture superna- We thank Dr. Annalisa Pezzolo for help in the immunofluores- tants, possibly as a consequence of posttranscriptional events. cence experiments with tonsil tissue sections, Dr. Franco Fais for the Studies carried out in different murine models have dem- analysis of IgV gene mutations in the B-CLL samples, Marta Camoriano onstrated that B cells presenting antigenic peptides to specific T for help in immunohistochemistry studies, and Chiara Bernardini for cells do not drive generation of Th1 cells (45, 46). Although the excellent secretarial assistance. major cytokines involved in the induction of Th1 cell polariza- tion are IFN-␥ and IL-12 (47Ð49), it has been shown that also REFERENCES IL-18, especially in synergism with IL-12, participates in this 1. Nakanishi, K., Yoshimoto, T., Tsutsui, H., and Okamura, H. 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Irma Airoldi, Lizzia Raffaghello, Claudia Cocco, et al.

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