Expression of B7-2 (CD86) Molecules by Reed–Sternberg Cells Of

Expression of B7-2 (CD86) molecules by Reed–Sternberg cells of Hodgkin’s disease SW Van Gool1,2, J Delabie3, P Vandenberghe4, L Coorevits1, C De Wolf-Peeters3 and JL Ceuppens1

1Laboratory of Experimental Immunology, 4Laboratory of Experimental Hematology, Department of Pathophysiology, 2Department of Pediatrics, and 3Department of Pathology II, Catholic University of Leuven, Belgium

Ligation of CD28 on T cells with its natural ligands B7-1 (CD80) non-Hodgkin’s lymphomas. We also studied the expression of or B7-2 (CD86) provides a major costimulatory signal for T cells B7-2 in the HD-derived cell lines L428 and KM-H2 and evalu- and is of potential importance for tumor rejection. We previously reported a strong expression of B7-1 on Reed– ated its function in the human allogeneic mixed lymphocyte Sternberg cells and anaplastic large cell lymphoma cells. We reaction using these HD-derived cell lines as stimulators. report here our ﬁndings on B7-2 expression by malignant lymphomas (n = 70). B7-2 was present on the neoplastic cells of anaplastic large cell lymphoma in two of three cases studied, Materials and methods and on a subpopulation of the malignant cells in one out of four cases of follicular lymphoma. B7-2 was not expressed by the neoplastic cells of the other non-Hodgkin’s lymphomas Monoclonal antibodies (n = 32), including T cell-rich B cell lymphoma. In contrast, Reed–Sternberg cells in lymph nodes affected by Hodgkin’s Anti-B7-1 mouse monoclonal B7-2415 and anti-B7-2 mAb disease are strongly positive for B7-2 (n = 31). Evidence for a were gifts from Innogenetics (Gent, Belgium). Both mAbs functional correlate of this expression was obtained by our block ligand binding to the CD28 receptor. They were used ﬁndings that the combination of anti-B7-1 and anti-B7-2 mono- for functional studies and for immunohistochemistry. PE-lab- clonal antibodies was more effective than each separately in blocking allogeneic T cell activation (proliferation and cytokine elled anti-B7-1 (clone BB-1) and FITC-labelled anti-B7-2 secretion) by Hodgkin’s disease-derived cell lines as stimu- (clone FUN-1) were purchased from Becton Dickinson lators. The possible role of B7-1 and B7-2 expression for the (Mountain View, CA, USA) and from Pharmingen (San Diego, course and symptomatology of Hodgkin’s disease is dis- CA, USA) respectively. These mAbs were used for staining of cussed. cell lines and FACS analysis. Anti-CD40 mAb 5D12, a gift Keywords: Hodgkin’s disease; non-Hodgkin’s lymphomas; CD80; from Mark de Boer (PanGenetics, Amsterdam, The CD86; tumor immunology Netherlands), was produced as previously described.15

Introduction Immunohistochemistry

Two different B7 molecules (B7-1 or CD80 and B7-2 or CD86) The lymph node samples used in this study are listed in have been shown to be the natural ligands of CD28, a mem- Table 1. The study included tissue samples of 31 cases of HD brane receptor of T cells (reviewed in Ref. 1). B7 molecules covering the four histological subtypes, and 39 cases of non- are expressed by professional antigen-presenting cells (APC). Hodgkin’s lymphoma. All tissue samples were snap-frozen in The binding of B7 to CD28 provides a major costimulatory liquid nitrogen-cooled isospentane and stored at −70°C until signal for T cell activation after engagement of the T cell used for immunohistochemistry. Acetone-ﬁxed cryostat sec- receptor with antigen. CD28 ligation on T cells speciﬁcally tions (4 ␮m) were used. A three step ABC technique was used increases the secretion of cytokines such as interleukin (IL)-2, for immunodetection according to Hsu et al.16 The ABC com- ␣ tumor necrosis factor (TNF)- , lymphotoxin, interferon (IFN)- plex was peroxidase-conjugated (Dakopatts, Glostrup, ␥ and granulocyte–monocyte colony-stimulating factor (GM- 1 2 Denmark). Aminoethylcarbazole and H2O2 were used as per- CSF), and prevents anergy-induction of T cells. B7 transfec- oxidase substrates. Controls consisted of replacement of the tion of mouse melanoma cells induces an effective tumor- primary antibody by an irrelevant mAb of similar isotype or directed T cell-mediated cytotoxic response, by delivering the use of chromogen alone. costimulatory signals necessary for full activation of T cells against immunogenic tumor cells.3 B7 transfection in tumor cells as a potential method of immunotherapy has now been Cell lines reported by several groups.3–13 We and others have shown that B7-1 is expressed on Reed– HD-derived cell line KM-H2 was kindly provided by Dr S Sternberg cells (RS cells), the malignant cells of Hodgkin’s dis- Fukuhara (Kyoto University, Kyoto, Japan) and the HD-derived ease (HD), on follicular lymphoma cells and on large anaplas- 14 cell line L428 was obtained from Dr V Diehl (Medizinische tic lymphoma cells. Tumors grow despite the presence of Hochschule, Hannover, Germany). These cell lines have been B7-1. No information is available on in situ expression of B7- previously characterized.17,18 Both cell lines were grown in 2 in these malignancies. We therefore evaluated the RPMI-1640 medium (Gibco, Paisley, UK), supplemented with expression of B7-2 in Hodgkin’s disease and in a range of 10% fetal calf serum, 2 mML-glutamine, penicillin (100 U/ml) and streptomycin (100 ␮g/ml).

Correspondence: JL Ceuppens, Laboratory of Experimental Immu- nology, Faculty of Medicine, Onderwijs en Navorsing, Herestraat 49, B-3000 Leuven, Belgium Flow cytometric analysis of cell surface antigens SW Van Gool, J Delabie and P Vandenberghe are postdoctoral fellows of the Fund for Scientiﬁc Research, Flanders (FWO-V). Cells were suspended in 100 ␮l of PBS containing 1% bovine Received 3 October 1996; accepted 4 March 1997 serum albumin (Sigma Chemical, St Louis, MO, USA). The B7 expression on Hodgkin cells SW Van Gool et al 847 Table 1 B7 expression on lymphoma cells Cell culture for T cell proliferation

+ + B7-1( )/total B7-2( )/total T cells (5 × 104 per well) were suspended in RPMI-1640 number of cases14 number of cases (Gibco) supplemented with 2 mML-glutamine, penicillin (100 U/ml), streptomycin (100 ␮g/ml), and 10% autologous Hodgkin’s disease Nodular sclerosis 27/27 12/12 plasma, and mixed with irradiated (2500 rad) KM-H2 or L428 Lymphocyte 5/5 4/5 cells (T:RS cell ratio: 1:2 or 1:1, respectively), in the presence predominance or absence of mAbs to B7-1 or B7-2. Cultures were performed ° Mixed cellularity 10/10 10/10 at 37 C and 5% CO2, in flat-bottomed 96-well microculture Lymphocyte 5/5 4/4 plates (Costar, High Wycombe, UK) for 5 days in quadrupli- depletion cate. At the end of the culture, cells were pulsed with 1 mCi Non-Hodgkin’s 3 lymphoma of H-thymidine (specific activity 2 Ci/mmol; Amersham, B cell type Buckinghamshire, UK). Eight hours later, cells were harvested Small lymphocytic 0/2 0/2 with a multiple automated sample harvesting apparatus and (C11) radioactivity on the filter papers was counted in a liquid scin- Follicle center cell 4/4 1/4 tillation counter. Results are expressed in mean c.p.m. ± s.d. lymphoma Mantle cell 0/3 0/3 lymphoma Diffuse large cell 0/5 0/5 Cell culture for cytokine production lymphoma Immunoblastic 0/2 0/2 lymphoma One million T cells were cultured with 1 × 106 RS cells (L428) Small noncleaved 0/2 0/2 in a 24-well plate for 5 days. Supernatants were taken after 5 cell lymphoma days. IFN-␥ and IL-5 were determined with a sandwich ELISA T cell-rich B cell 0/11 0/11 technique, using combinations of unlabeled and biotin- or lymphoma enzyme-coupled mAb to different epitopes of each cytokine Histiocyte-rich B 0/5 ND ␥ cell lymphoma (IFN- : ScreeningLine obtained from Medgenics, Fleurus, T cell type Belgium; IL-5: mAb obtained from Pharmingen). Peripheral T cell 0/5 0/5 lymphoma Lymphoblastic 0/2 0/2 lymphoma Results Anaplastic large cell 5/5 2/3 lymphoma Immunohistochemical analysis of Hodgkin’s disease Lymph node samples were analyzed with immunohistochemistry using the ABC technique for the presence of B7-114 and B7-2 RS cells in all but one case of HD studied (n = 31) expressed expressing tumor cells. The cases analyzed for B7-2 expression the B7-2 molecule on their membrane (Table 1, Figure 1). The are the same as those analyzed for B7-1 expression. 31 cases studied covered the four subtypes of HD. In addition, some cytoplasmic reactivity with the anti-B7-2 antibody was also observed in most RS cells. Around the RS cells, CD28(+)T cells were observed.14 The one case which was B7-2 negative belonged to the nodular lymphocyte predominance group, and RS cells did express B7-1. cell suspension was incubated for 30 min at 4°C with mAb identifying the surface antigens of interest. When unconju- gated mAb were used, the cells were further incubated (after ′ two washes) with FITC-labelled F(ab )2 fragments of goat-anti- mouse IgG (Becton Dickinson) at 4°C for 30 min. After two washes, cells were fixed with 1% paraformaldehyde, and analysis was performed on a Facscan flow cytometer (Becton Dickinson).

T cell separation

Peripheral blood mononuclear cells of healthy donors were isolated on Ficoll–Hypaque (density 1.077) gradients. Mono- cytes were removed by cold agglutination and T lymphocytes were further purified using complement-fixing anti-natural killer cell mAb and two cycles of lympho-KWIK-T treatment (One Lambda Inc, Los Angeles, CA, USA), as previously 19 reported. The cell preparations contained more than 97% Figure 1 Hodgkin’s disease, nodular sclerosis subtype. Reed– CD3(+), and less than 1% CD16(+) cells. CD14(+) monocytes Sternberg cells express the B7-2 antigen (ABC-perioxidase labelled were not detected. staining, ×500). B7 expression on Hodgkin cells SW Van Gool et al 848 exclude all helper signals derived from autologous accessory cells. As expected, the KM-H2 and L428 cells strongly stimulated the proliferation of T cells from all donors tested (Figure 4), the mean proliferative counts in MLR with KM-H2 being 52 455 (s.e.m. = 3421, n = 3), and with L428 being 156484 (s.e.m. = 13 782, n = 4). They also strongly stimulated both Th1-type and Th2-type cytokine production, as exem- plified by IFN-␥ and IL-5 production (Figure 5). We could not detect IL-4 or IL-10 in the culture supernatants. To examine the relative contributions of B7-1 and B7-2 in this stimulating capacity, the MLR was performed in the presence or absence of mAb to B7-1 and B7-2. As shown in Figure 4, anti-B7-1 and anti-B7-2 separately partially inhibited the capacity of KM-H2 and of L428 cells to stimulate allogeneic T cell proliferation. For both KM-H2 and L428, anti- B7-1 resulted in a significant inhibition of the T cell prolifer- Figure 2 Anaplastic large cell lymphoma. Large sheet of lymphoma cells expressing the B7-2 antigen (ABC-peroxidase labelled staining, × 310).

Immunohistochemical analysis of non-Hodgkin’s lymphomas

Part of the cells of one case of follicle center cell lymphoma (out of four cases studied), and two (out of three) cases of anaplastic large cell lymphoma also expressed B7-2 (Figure 2). No reactivity was observed with the neoplastic cells in the other non-Hodgkin’s lymphomas tested, including T cell-rich B cell lymphoma. In all tissues studied, B7-2 was expressed by a highly vari- able number of inﬁltrating monocyte-derived cells inter- spersed between the malignant cells.

Allogeneic T cell stimulation with Hodgkin’s cell- derived cell lines Figure 4 B7 molecules on KM-H2 and L428 cells costimulate T cells. T cells (0.25 × 106/ml) were cultured with KM-H2 cells (0.5 × 106/ml) or L428 cells (0.25 × 106/ml) as indicated. Anti-B7-1 Flow cytometric analysis of HD-derived cell lines KM-H2 and ␮ L428 revealed strong expression of B7-1 and B7-2 (Figure 3). and anti-B7-2 mAb were used at 5 g/ml. After 5 days, proliferative response was analyzed by 3H-thymidine incorporation. Results are KM-H2 and L428 cells were used as stimulators in mixed lym- expressed as mean % of control response. Anti-B7-1 and anti-B7-2 phocyte reactions (MLR) with purified T cells from normal significantly blocked the proliferative reponse (ANOVA for KM-H2: donors as responder cells. Purified T cells were used to P = 0.0017, n = 3; for L428: P Ͻ 0.0001, n = 4).

Figure 3 Flow cytometric analysis of KM-H2 and L428 cells. Cells were stained with anti-B7-1-PE, anti-B7-2-FITC or anti-CD40 + FITC- labelled goat anti-mouse antibodies. Analysis was performed on a FACS-Sort and results are presented in histograms. Mean ﬂuorescence is indicated for each histogram. B7 expression on Hodgkin cells SW Van Gool et al 849

Figure 5 Hodgkin cells induce Th1- and Th2-type cytokine responses. One million T cells were stimulated with 1 × 106 L428 cells in 1 ml. Anti-B7-1, anti-B7-2 (both mAb used at 5 ␮g/ml) and/or CsA (400 ng/ml) were added as indicated. After 5 days, supernatants were collected. IL-5 and IFN-␥ were measured by ELISA. Results are expressed in pg/ml. Experiments with T cells from two different donors are presented.

ation (P Ͻ 0.05). The inhibition with anti-B7-2 mAb was con- ecules, including B7-1 and B7-2, by RS cells in situ, strongly sistently less marked as the inhibition with anti-B7-1 mAb, but indicate that RS cells in vivo can function as APC. was still significant when L428 cells were used as stimulators HD-derived cell lines have been shown to be potent stimu- (P Ͻ 0.05). The addition of both anti-B7-1 and of anti-B7-2 lators of a MLR and are able to present soluble antigen to T resulted in an additive and more effective blocking effect, but cells in an MHC class II-restricted fashion.23,24 To study the still failed to completely block T cell proliferation (Figure 4). role of B7 molecules in the accessory cell function of RS cells, Similarly to T cell proliferation, the production of cytokines we performed allogeneic MLR using the HD-derived cell lines could partially be blocked by the combination of both anti- KM-H2 and L428 as stimulator cells. We found that the allo- B7-1 and anti-B7-2 (Figure 5). Surprisingly, the production of geneic T cell activation induced by these cells can be partially IFN-␥ and IL-5 was only minimally blocked by anti-B7-1 blocked by the anti-B7-1 or anti-B7-2 antibodies, or their alone, and addition of anti-B7-2 mAb even resulted in an combination. Both mAb have a strong blocking activity, and enhanced cytokine production in one experiment. Finally, the can completely inhibit their respective targets on transfected combination of anti-B7-1 and anti-B7-2 mAb together with cell lines (unpublished). It is thus likely that other costimu- cyclosporin A (CsA) resulted in a complete inhibition of T cell latory molecules are also expressed on HD-derived cell lines, proliferation (not shown) and of cytokine production and that they contribute to T cell stimulatory properties of HD (Figure 5). The synergistic activity of anti-B7 and CsA has pre- cell lines. We have previously shown that KM-H2 also express viously also been shown by us for MLR reactions with EBV- ICAM-1 (CD54), HLA-DR and CD40 but not LFA-1 (CD11a), transformed B cells as stimulators.19 CD13, CD14 or LFA-3 (CD58) and that L428 also expressed CD11a, CD54, HLA-DR, CD40 and CD58 but not CD13 or CD14.14 LFA-3–CD2 and ICAM-1–LFA-1 interactions are Discussion thought to play a role in the stimulating capacity of these cell lines.14,25 Looking to potential differences between B7-1 and B7-1 expression on RS cells was first shown by us14 and later B7-2 in providing the accessory signal, we found that B7-1 confirmed by Munro et al20 and by Gruss et al.21 In this article, was the stronger CD28 ligand for costimulation. This finding we have shown that RS cells in all cases of Hodgkin’s disease is compatible with other data that B7-1 is superior to B7-2 in studied also express B7-2 molecules. Similarly, both B7 mol- inducing an antitumor immune response.10,12 ecules were detected on KM-H2 and L428 HD-derived cell Despite recent evidence indicating a heterogeneous origin lines. One previous report has also assessed the in situ of the RS cell, the accessory cell function may be character- expression of B7-2 in HD.22 The authors used a mAb FUN-1 istic of ‘Hodgkin’s syndrome’, a clinical picture with fatigue, which at that time was not yet identified as an anti-B7-2 anorexia, drenching night sweats and unexplained weight loss reagent. Our functional data obtained with HD-derived cell and fever. In all cases tested, the majority of non-neoplastic lines, as well as the expression of multiple accessory mol- T cells surrounding the RS cells expressed CD28.20 Moreover, B7 expression on Hodgkin cells SW Van Gool et al 850 RS cells themselves produce several cytokines after interaction 2 Harding FA, McArthur JG, Gross JA, Raulet DH, Allison JP. CD28- with T cells through the CD40-CD40L and CD30-CD30L mediated signalling co-stimulates murine T cells and prevents binding.26 Therefore, the clinical picture can be considered as induction of anergy in T-cell clones. Nature 1992; 356: 607–609. 3 Townsend SE, Allison JP. Tumor rejection after direct costimu- the result of a cytokine- and cell contact-dependent activation lation of CD8+ T cells by B7-transfected melanoma cells. Science network typical of HD, a disease with cytokine-producing 1993; 259: 368–370. tumor cells and surrounding reactive T cells.26 4 Chen L, Ashe S, Brady WA, Hellstro¨m I, Hellstro¨m KE, Ledbetter B7-2 was expressed by the malignant cells in two out of JA, McGowan P, Linsley PS. Costimulation of antitumor immunity three cases of anaplastic large cell lymphoma (so called Ki-1 by the B7 counterreceptor for the T lymphocyte molecules CD28 lymphoma) studied here. All three cases also expressed B7-1. and CTLA-4. Cell 1992; 71: 1093–1102. 22 5 Baskar S, Ostrand-Rosenberg S, Nabavi N, Nadler L, Freeman G, In accordance with our data, Nozawa et al using FUN-1 Glimcher L. Constitutive expression of B7 restores immunogenicity mAb, found B7-2 expression on anaplastic large cell lym- of tumor cells expressing truncated major histocompatibility com- phomas. In our series, B7-2 was expressed by a subpopulation plex class II molecules. Proc Natl Acad Sci USA 1993; 90: of neoplastic cells in one case (out of three) of follicular lym- 5687–5690. phoma. In all three cases, a subpopulation of the cells also 6 Chen L, McGowan P, Ashe S, Johnston J, Li Y, Hellstro¨mI, expressed B7-1. The expression of B7-1 and B7-2 on tumor Hellstro¨m KE. Tumor immunogenicity determines the effect of B7 cells of these lymphoma entities might indicate that these costimulation on T cell-mediated tumor immunity. J Exp Med 1994; 179: 523–532. tumor cells have retained the ability to interact with T cells. 7 Li Y, McGowan P, Hellstro¨m I, Hellstro¨m KE, Chen L. Costimu- In none of the other cases of non-Hodgkin lymphoma could lation of tumor-reactive CD4+ and CD8+ T lymphocytes by B7, a we detect B7-2 expression. Of interest is the absence of B7- natural ligand for CD28, can be used to treat established mouse 1 and of B7-2 expression on the neoplastic cells of T cell- melanoma. J Immunol 1994; 153: 421–428. rich B cell lymphomas. B7-1 and B7-2 are therefore useful 8 Baskar S, Glimcher L, Nabavi N, Jones RT, Ostrand-Rosenberg S. + + diagnostic markers for distinguishing this entity from HD, Major histocompatibility complex class II B7-1 tumor cells are 27 potent vaccines for stimulating tumor rejection in tumor-bearing which may morphologically appear very similar. The mice. J Exp Med 1995; 181: 619–629. absence of B7 molecules in these lymphomas might also be 9 Dunussi-Joannopoulos K, Weinstein HJ, Nickerson PW, Strom TB, of significance for their failure to elicit an immune response. Burakoff SJ, Croop JM, Arceci RJ. Irradiated B7-1 transduced pri- The presence of MHC class II, B7-1, B7-2, and other mary acute myelogenous leukemia (AML) cells can be used as adhesion molecules on tumor cells apparently does not auto- therapeutic vaccines in murine AML. Blood 1996; 87: 2938–2946. matically lead to the eradication of the tumor by the immune 10 Gajewski TF, Fallarino F, Uyttenhove C, Boon T. Tumor rejection system as has been suggested by several experimental sys- requires a CTLA4 ligand provided by the host or expressed on the 3–13 tumor: superiority of B7-1 over B7-2 for active tumor immuni- tems. Whether the accessory cell function of RS cells elic- zation. J Immunol 1996; 156: 2909–2917. its an autologous T cell-mediated cytotoxic response, and 11 Li YW, Hellstro¨m KE, Newby SA, Chen LP. Costimulation by whether B7 expression is essential to such a response in vivo, CD48 and B7-1 induces immunity against poorly immunogenic however, remains to be demonstrated. B7 expression on RS tumors. J Exp Med 1996; 183: 639–644. cells may also block rather than stimulate the antitumor 12 Matulonis U, Dosiou C, Freeman G, Lamont C, Mauch P, Nadler response as suggested by Leach et al.28 In this study, B7-trans- LM, Griffin LD. B7-1 is superior to B7-2 costimulation in the induction and maintenance of T cell-mediated antileukemia fected and native colon carcinoma cells were rejected only immunity: further evidence that B7-1 and B7-2 are functionally when mice were treated with anti-CTLA-4 antibodies. CTLA- distinct. J Immunol 1996; 156: 1126–1131. 4 is a second receptor for B7 molecules which is expressed 13 Baskar S, Clements VK, Glimcher LH, Nabavi N, Ostrand-Rosen- on activated T cells, and it transduces a negative signal to the berg S. Rejection of MHC class II-transfected tumor cells requires T cells.1 B7 might well provide a negative signal by preferen- induction of tumor-encoded B7-1 and/or B7-2 costimulatory mol- tial interaction with CTLA-4 on T cells rather than with CD28. ecules. J Immunol 1996; 156: 3821–3827. It would therefore be of interest to study CTLA-4 expression 14 Delabie J, Ceuppens JL, Vandenberghe P, de Boer M, Coorevits L, De Wolf-Peeters C. The B7/BB1 antigen is expressed by Reed– on the surrounding T cells in HD lymph nodes. Further study Sternberg cells of Hodgkin’s disease and contributes to the stimul- is needed to clarify the lack of in vivo antitumor immune ating capacity of Hodgkin’s disease-derived cell lines. 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