Analysis of Interleukin-27 (EBI3/p28) Expression in Epstein-Barr Virus- and Human T-Cell Leukemia Virus Type 1-Associated Lymphomas
2005; Elsevier BV; Volume: 166; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62340-1
ISSN1525-2191
AutoresFrédérique Larousserie, Émilie Bardel, Stefan Pflanz, Bertrand Arnulf, Carmen Lome‐Maldonado, Olivier Hermine, Laurence Brégeaud, Monique Perennec, Nicole Brousse, Rob Kastelein, Odile Devergne,
Tópico(s)Animal Disease Management and Epidemiology
ResumoInterleukin (IL)-27 is a novel heterodimeric cytokine of the IL-12 family that is composed of two subunits, Epstein-Barr virus (EBV)-induced gene 3 (EBI3) and p28. EBI3 is expressed at high levels in EBV-transformed B-cell lines and is induced in vitro by the EBV oncogene LMP1 in a nuclear factor (NF)-κB-dependent manner. We show here that EBI3 expression is up-regulated in human T-cell leukemia virus type 1 (HTLV-1)-infected cell lines and IL-2-dependent leukemic cells from adult T-cell leukemia/lymphoma (ATL) patients, compared to normal activated T cells. EBI3 expression was decreased in HTLV-1-transformed cells after treatment with the NF-κB inhibitor BAY11-7082 and was induced in Jurkat cells by expression of HTLV-1 wild-type Tax oncoprotein, but not by the Tax mutant M22, which is defective for NF-κB activation. In situ analysis of EBI3 and p28 expression in Hodgkin's lymphomas (HLs), in various EBV-associated lymphoproliferative disorders (LPDs) (including post-transplant LPDs and nasal-type NK/T-cell lymphomas), and in ATL showed that EBI3 was expressed by neoplastic cells in all cases of HL and of LMP1-positive EBV-associated LPD, at variable levels in ATL cases, but rarely in control T-cell lymphomas. In contrast, in all lymphomas tested, no or few tumoral cells expressed p28. Consistent with these data, no significant p28 or IL-27 expression was detected in HL-derived cell lines, or in EBV- or HTLV-1-transformed cell lines. This selective overexpression of EBI3 by transformed cells suggests that EBI3 may play a role, independently from its association to p28, in regulating anti-viral or anti-tumoral immune responses. Interleukin (IL)-27 is a novel heterodimeric cytokine of the IL-12 family that is composed of two subunits, Epstein-Barr virus (EBV)-induced gene 3 (EBI3) and p28. EBI3 is expressed at high levels in EBV-transformed B-cell lines and is induced in vitro by the EBV oncogene LMP1 in a nuclear factor (NF)-κB-dependent manner. We show here that EBI3 expression is up-regulated in human T-cell leukemia virus type 1 (HTLV-1)-infected cell lines and IL-2-dependent leukemic cells from adult T-cell leukemia/lymphoma (ATL) patients, compared to normal activated T cells. EBI3 expression was decreased in HTLV-1-transformed cells after treatment with the NF-κB inhibitor BAY11-7082 and was induced in Jurkat cells by expression of HTLV-1 wild-type Tax oncoprotein, but not by the Tax mutant M22, which is defective for NF-κB activation. In situ analysis of EBI3 and p28 expression in Hodgkin's lymphomas (HLs), in various EBV-associated lymphoproliferative disorders (LPDs) (including post-transplant LPDs and nasal-type NK/T-cell lymphomas), and in ATL showed that EBI3 was expressed by neoplastic cells in all cases of HL and of LMP1-positive EBV-associated LPD, at variable levels in ATL cases, but rarely in control T-cell lymphomas. In contrast, in all lymphomas tested, no or few tumoral cells expressed p28. Consistent with these data, no significant p28 or IL-27 expression was detected in HL-derived cell lines, or in EBV- or HTLV-1-transformed cell lines. This selective overexpression of EBI3 by transformed cells suggests that EBI3 may play a role, independently from its association to p28, in regulating anti-viral or anti-tumoral immune responses. In humans, two transforming viruses, Epstein-Barr virus (EBV) and human T-cell leukemia virus type 1 (HTLV-1), are causally associated with the development of lymphomas. EBV, a human γ-herpesvirus, efficiently transforms primary B cells, in vitro, into continuously growing lymphoblastoid cell lines (LCLs), and has been closely associated with the development of several lymphoid malignancies, including endemic Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), and lymphoproliferative disorders (LPDs) arising in immunocompromised individuals.1Rickinson A Kieff E Epstein-Barr virus.in: Knipe DM Howley PM Fields Virology. 4th ed. Lippincott/Williams & Wilkins Co., Philadelphia2001: 2575-2628Google Scholar, 2Küppers R B cells under influence: transformation of B cells by Epstein-Barr virus.Nat Rev Immunol. 2003; 3: 801-812Crossref PubMed Scopus (426) Google Scholar HTLV-1 is a retrovirus and is the etiological agent of adult T-cell leukemia/lymphoma (ATL), an aggressive malignancy of mature CD4-positive T lymphocytes.3Yoshida M Multiple viral strategies of HTLV-1 for dysregulation of cell growth control.Annu Rev Immunol. 2001; 19: 475-496Crossref PubMed Scopus (399) Google Scholar Previously, we reported the characterization of a novel EBV-induced gene, EBI3.4Devergne O Hummel M Koeppen H Le Beau MM Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40-related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar EBI3 was cloned by substractive hybridization from an EBV-infected BL cell line and was found to be highly expressed in LCLs. It codes for a soluble type 1 cytokine receptor, homologous to the p40 subunit of interleukin (IL)-12. Recently, EBI3 has been shown to associate with a new IL-12 p35-related subunit, p28, to form a novel noncovalently linked heterodimeric cytokine (EBI3/p28), named IL-27.5Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar The receptor complex for IL-27 is composed of TCCR (also called WSX-1 or IL-27R) and gp130.5Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar, 6Pflanz S Hibbert L Mattson J Rosales R Vaisberg E Bazan JF Phillips JH McClanahan TK de Waal Malefyt R Kastelein RA WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27.J Immunol. 2004; 172: 2225-2231PubMed Google Scholar Initial studies have suggested that IL-27 may play an important role in initiation of Th1 responses. This role was based on: 1) the in vitro expression profile of EBI3 and p28, as defined by quantitative reverse transcriptase-polymerase chain reaction analysis, showing that co-expression of EBI3 and p28 was mainly observed in activated macrophages and dendritic cells;5Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar 2) the ability of IL-27 to induce T-bet and IL-12Rβ2 expression in naive CD4-positive T cells,7Takeda A Hamano S Yamanaka A Hanada T Ishibashi T Mak TW Yoshimura A Yoshida H Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment.J Immunol. 2003; 170: 4886-4890PubMed Google Scholar, 8Hibbert L Pflanz S De Waal Malefyt R Kastelein RA IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells.J Interferon Cytokine Res. 2003; 23: 513-522Crossref PubMed Scopus (277) Google Scholar, 9Lucas S Ghilardi N Li J de Sauvage FJ IL-27 regulates IL-12 responsiveness of naive CD4+ T cells through Stat1-dependent and -independent mechanisms.Proc Natl Acad Sci USA. 2003; 100: 15047-15052Crossref PubMed Scopus (398) Google Scholar to stimulate their proliferation, and to synergize with IL-12 for interferon-γ production5Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar, 7Takeda A Hamano S Yamanaka A Hanada T Ishibashi T Mak TW Yoshimura A Yoshida H Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment.J Immunol. 2003; 170: 4886-4890PubMed Google Scholar; 3) the phenotype of IL-27R-deficient mice showing a defect in Th1 response initiation on infection with Listeria monocytogenes or Leishmania major.10Chen Q Ghilardi N Wang H Baker T Xie MH Gurney A Grewal IS de Sauvage FJ Development of Th1-type immune responses requires the type I cytokine receptor TCCR.Nature. 2000; 407: 916-920Crossref PubMed Scopus (342) Google Scholar, 11Yoshida H Hamano S Senaldi G Covey T Faggioni R Mu S Xia M Wakeham AC Nishina H Potter J Saris CJ Mak TW WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.Immunity. 2001; 15: 569-578Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar Consistent with this model, we observed that EBI3 and p28 were co-expressed by macrophages and macrophage-derived cells at the site of disease in human Th1-associated granulomatous diseases.12Larousserie F Pflanz S Coulomb-L'Herminé A Brousse N Kastelein R Devergne O Expression of IL-27 in human Th1-associated granulomatous diseases.J Pathol. 2004; 202: 164-171Crossref PubMed Scopus (116) Google Scholar However, subsequent studies led to reconsider this model and a possible role of IL-27 as a suppressor of T-cell activation has emerged.13Trinchieri G Pflanz S Kastelein RA The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses.Immunity. 2003; 19: 641-644Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar Indeed, two recent studies showed that IL-27R-deficient mice were capable of mounting a Th1 response to infection by Toxoplasma gondii and Trypanosoma cruzi, but exhibited increased mortality because of dysregulated T-cell activation and hyperproduction of proinflammatory cytokines.14Hamano S Himeno K Miyazaki Y Ishii K Yamanaka A Takeda A Zhang M Hisaeda H Mak TW Yoshimura A Yoshida H WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production.Immunity. 2003; 19: 657-667Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 15Villarino A Hibbert L Lieberman L Wilson E Mak T Yoshida H Kastelein RA Saris C Hunter CA The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection.Immunity. 2003; 19: 645-655Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar Taken together, these findings have suggested that IL-27 functions may be complex, and that IL-27 may, depending on the context, function either as a Th1 response co-inducer or as a negative regulator of T-cell and inflammatory responses. Previously we showed that EBI3 is induced in vitro by the EBV oncogene LMP1, and that its induction by LMP1 depends on nuclear factor (NF)-κB activation.4Devergne O Hummel M Koeppen H Le Beau MM Nathanson EC Kieff E Birkenbach M A novel interleukin-12 p40-related protein induced by latent Epstein-Barr virus infection in B lymphocytes.J Virol. 1996; 70: 1143-1153Crossref PubMed Google Scholar, 16Devergne O Cahir McFarland ED Mosialos G Izumi KM Ware CF Kieff E Role of the TRAF binding site and NF-κB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression.J Virol. 1998; 72: 7900-7908Crossref PubMed Google Scholar LMP1 is one of the nine EBV-encoded proteins expressed in latently infected B cells and plays a key role in EBV-mediated growth transformation.17Cahir McFarland ED Izumi KM Mosialos G Epstein-Barr virus transformation: involvement of latent membrane protein 1-mediated activation of NF-κB.Oncogene. 1999; 18: 6959-6964Crossref PubMed Scopus (140) Google Scholar LMP1 is also expressed in vivo in many EBV-associated LPDs, including HL, post-transplant LPD, and nasal type NK/T-cell lymphoma. In a previous study, we found that EBI3 was not expressed in EBV-positive BL, consistent with the absence of LMP1 expression in this type of lymphoma, but was expressed by Hodgkin and Reed-Sternberg (HRS) tumoral cells in most cases of HL.18Niedobitek G Pazolt D Teichmann M Devergne O Frequent expression of the Epstein-Barr virus (EBV)-induced gene, EBI3, an IL-12 p40-related cytokine, in Hodgkin and Reed-Sternberg cells.J Pathol. 2002; 198: 310-316Crossref PubMed Scopus (82) Google Scholar The expression of EBI3 in other lymphomas has not been reported. The expression profile of p28 in tumor tissues has also not been investigated. Activation of the NF-κB pathway is a common feature of transforming virus.19Hiscott J Kwon H Genin P Hostile takeovers: viral appropriation of the NF-κB pathway.J Clin Invest. 2001; 107: 143-151Crossref PubMed Scopus (514) Google Scholar Indeed, activation of NF-κB by viral proteins leads to transactivation of numerous cellular genes, including cytokines, involved in cell survival and evasion of immune response. Thus, similar to EBV-transformed B cells, HTLV-1-transformed T cells exhibit high constitutive NF-κB activity.20Mori N Fujii M Ikeda S Yamada Y Tomonaga M Ballard DW Yamamoto N Constitutive activation of NF-κB in primary adult-T-cell leukemia cells.Blood. 1999; 93: 2360-2368PubMed Google Scholar, 21Arima N Matsushita K Obata H Ohtsubo H Fujiwara H Arimura K Kukita T Suruga Y Wakamatsu S Wakamastu S Hidaka S Tei C NF-κB involvement in the activation of primary adult-T-cell leukemia cells and its clinical implications.Exp Hematol. 1999; 27: 1168-1175Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar The HTLV-1 Tax oncoprotein plays an important role in T-cell transformation through its ability to induce constitutive NF-κB activation and to dysregulate cellular gene expression. To investigate the potential role of IL-27 in viral lymphomagenesis, we further analyzed the expression of both subunits of IL-27 in EBV-associated lymphoid malignancies and extended our study to ATL. Both in situ and in vitro analyses indicated that EBI3, but no or low p28 and IL-27, is expressed by tumoral cells. This dissociated expression of EBI3 and p28 suggests that EBI3 may play a role, independently from its association with p28, to regulate anti-viral and anti-tumoral responses. KMH2, L428, and HDLM2 are EBV-negative cell lines derived from HL patients with nodular sclerosis (L428 and HDLM2) or mixed cellularity (KMH2) subtype. NC-37 is an EBV-positive BL cell line. IB4 and LCL-1, -2, -3, -4 (gift from Ellen Cahir-McFarland, Harvard Medical School, Boston, MA) are in vitro EBV-transformed lymphoblastoid cell lines. Jurkat, MOLT-4, and CEM are HTLV-1-negative T-cell lines. MT-2 and HUT-102 are Tax-positive HTLV-1-infected T-cell lines. ATL1 and ATL2 are IL-2-dependent leukemic cells derived from two different patients with acute ATL. All cell lines were maintained in RPMI 1640-Glutamax media (Invitrogen Corp.) supplemented with 10 to 20% fetal bovine serum and antibiotics. Media for leukemic cells was further supplemented with IL-2 (20 U/ml; Roche Diagnostics). To analyze fresh leukemic cells, peripheral blood mononuclear cells from five patients diagnosed with either acute (n = 4) or chronic (n = 1) ATL were purified by Ficoll-Hypaque (Amersham Biosciences) gradient centrifugation. Samples analyzed contained >70% of leukemic cells. For two of these ATL patients, tissue biopsies were available and were studied by immunohistochemistry for IL-27 expression (cases 23 and 26 in Table 3). CD3-positive T cells, CD4-positive or CD8-positive T-cell subsets were isolated from peripheral blood mononuclear cells from HTLV-1-negative donors by negative selection using magnetic beads (Miltenyi Biotec). Purity was >99.5% for CD3-positive cell selection, >97.5% for CD4-positive cell selection, and >94% for CD8-positive cell selection, as assessed by fluorescence-activated cell sorting (FACS) analysis and/or immunocytostaining. All blood samples were obtained after informed consent. Purified T cells and T-cell subsets were cultured in RPM1 1640 media supplemented with 10% fetal bovine serum, l-glutamine, and antibiotics and were stimulated for various times with phytohemagglutinin (4 μg/ml, Roche Diagnostics) and IL-2 (20 U/ml). BAY11-7082 was purchased from Calbiochem and reconstituted in dimethyl sulfoxide. Jurkat cells (5 × 106 cells per transfection) were transfected with pJFE control plasmid or pJFE plasmid encoding wild-type Tax or M22 mutant (gift from Françoise Bex, Université libre de Bruxelles, Brussels, Belgium) by electroporation with a Bio-Rad Gene Pulser Xcell electroporation system at 250 V and 500 μF at room temperature in 200 μl of Optimem media (Gibco BRL) containing DNA.Table 3Immunohistochemical Analysis of IL-27 Expression in Involved Tissues from ATL Patients and Control T-Cell Lymphomas% of positive tumoral cellsCase no.DiagnosisSiteEBI3p28Controls1AITLLymph node+−2AITLLymph node−−3PTLLymph node+−4PTLLymph node−−5PTLLymph node−−6PTLSalivary gland−−7PTLLymph node−−8PTLLymph node+−9ALCLLymph node−−10ALCLLymph node−−11ALCLLymph node−−12ALCLLymph node−ND13MFSkin−ND14MFSkin−ND15MFSkin−ND16MFSkin−NDATL17ATL, leukLymph node−−18ATL, leukSkin++19ATL, leukLymph node−ND20ATL, leukSkin−−21ATL, leukLymph node+++++22ATL, leukLymph node+++NA23ATL, leukLymph node+−24ATL, leukSkin+−25ATL, leukSkin+++++26ATL, leukSkin−−27ATL, leukLymph node++++−28ATL, leukLymph node++NI29ATL, lympLymph node+ND30ATL, lympLymph node+ND31ATL, lympLymph node−ND32ATL, lympLymph node+−33ATL, lympAbdomen+−34ATL, lympLymph node+−35ATL, lympLymph node−−AITL, angioimmunoblastic T-cell lymphoma; PTL, peripheral T-cell lymphoma, unspecified; ALCL, anaplastic large cell lymphoma; MF, mycosis fungoides; ATL, adult T-cell lymphoma/leukemia; leuk, leukemic form; lymp, lymphomatous form; ND, not done; NI, not interpretable; NA, not available.−, Virtually all cells negative;+, 1 to 5% positive cells;++, 10 to 30% positive cells;+++, >30 to 80% positive cells;++++, >80% positive cells. Open table in a new tab AITL, angioimmunoblastic T-cell lymphoma; PTL, peripheral T-cell lymphoma, unspecified; ALCL, anaplastic large cell lymphoma; MF, mycosis fungoides; ATL, adult T-cell lymphoma/leukemia; leuk, leukemic form; lymp, lymphomatous form; ND, not done; NI, not interpretable; NA, not available. −, Virtually all cells negative; +, 1 to 5% positive cells; ++, 10 to 30% positive cells; +++, >30 to 80% positive cells; ++++, >80% positive cells. Most tissues analyzed were retrieved from the files of the Department of Pathology of Necker Hospital. Lymphomas were classified according to the World Health Organization classification. We studied paraffin-embedded tissues from 14 cases of HL (7 nodular sclerosis cases, 2 lymphocyte depletion cases, 2 mixed cellularity cases, 1 unclassified case, and 2 nodular lymphocyte predominant cases), 16 cases of EBV-associated LPD (12 cases of B- or T-cell LPD, 10 of which from transplanted patients; 1 case of lymphomatoid granulomatosis; and 3 cases of NK/T-cell lymphoma, nasal type), 19 cases of ATL (12 cases of leukemic form and 7 cases of lymphomatous form), and 16 cases of control T-cell lymphomas (2 cases of angioimmunoblastic T-cell lymphoma; 6 cases of peripheral T-cell lymphoma, unspecified; 4 cases of anaplastic large cell lymphoma; and 4 cases of mycosis fungoides). All cases of HL- and of EBV-associated LPD were tested for LMP1 expression by immunohistochemistry, and when negative were further tested for EBERs by in situ hybridization to determine their EBV status. Among HLs, eight cases were negative for EBV (LMP1− and EBER−), and six cases were EBV-positive (LMP1+). Among EBV-associated LPDs, 13 cases were LMP1+, and 3 cases were LMP1− but EBER+. Diagnosis of ATL was made based on previously proposed criteria,22Shimoyama M Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma.Br J Haematol. 1991; 79: 428-437Crossref PubMed Scopus (1353) Google Scholar including clinical and biological features, the presence of anti-HTLV-1 antibodies in the serum, and the detection of HTLV-1 genome in DNA of leukemic cells. ATL patients originated from West Africa, French Guiana, and French West Indies. Seven cases of nonneoplastic lymph nodes exhibiting follicular hyperplasia of unknown origin were included as controls. All tissues analyzed were collected for histological examination and diagnosis purposes. Therefore, this study complies with the French ethical law for studies on human tissues. Immunostaining was performed on either paraffin-embedded tissue sections or acetone/methanol (1:1)-fixed cytospin preparation. For immunostaining on cytospin preparation, slides were rehydrated in Tris-buffered saline and saturated by incubation with Tris-buffered saline containing 20% normal human serum or 5% human veinoglobulins for 30 minutes. They were then incubated with the primary antibody diluted in Tris-buffered saline-0.3% bovine serum albumin for 1 hour. Binding of primary antibodies was detected using peroxidase-conjugated EnVision+ reagent (DakoCytomation). The peroxidase reaction was developed with 3′-diaminobenzidine and slides were counterstained with Harris hematoxylin. Immunostaining of paraffin sections was performed as previously described,12Larousserie F Pflanz S Coulomb-L'Herminé A Brousse N Kastelein R Devergne O Expression of IL-27 in human Th1-associated granulomatous diseases.J Pathol. 2004; 202: 164-171Crossref PubMed Scopus (116) Google Scholar by an indirect avidin-biotin peroxidase technique using ChemMate detection reagents (DakoCytomation). In double-immunostaining experiments, binding of the primary antibody in the first label was detected using peroxidase-conjugated EnVision+ reagent and diaminobenzidine (DakoCytomation) as a chromogenic substrate. Binding of the primary antibody in the second label was detected using an indirect avidin-biotin-alkaline phosphatase kit (BioGenex) and Fast blue (Sigma) as a chromogene, and slides were counterstained with methyl green. To ensure the absence of cross-reactivity between the first and second labeling steps, primary antibody was omitted or isotype-matched control antibody was used in the second label. EBI3 was detected using 2G4H6 mouse monoclonal antibody (mAb) (IgG2a)23Devergne O Coulomb-L'Herminé A Capel F Moussa M Capron F Expression of Epstein-Barr virus-induced gene 3, an interleukin-12 p40-related molecule, throughout human pregnancy: involvement of syncytiotrophoblasts and extravillous trophoblasts.Am J Pathol. 2001; 159: 1763-1776Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar at 2 μg/ml, in parallel with an isotype-matched control mAb (RPC5, IgG2a; Cappel Durham). p28 was detected using affinity-purified rabbit polyclonal anti-p28 antibodies (DNAX)12Larousserie F Pflanz S Coulomb-L'Herminé A Brousse N Kastelein R Devergne O Expression of IL-27 in human Th1-associated granulomatous diseases.J Pathol. 2004; 202: 164-171Crossref PubMed Scopus (116) Google Scholar at 1 to 3 μg/ml and normal rabbit IgG (Sigma) were used as a negative control. In some experiments, rat anti-p28 29B5 mAb (DNAX)12Larousserie F Pflanz S Coulomb-L'Herminé A Brousse N Kastelein R Devergne O Expression of IL-27 in human Th1-associated granulomatous diseases.J Pathol. 2004; 202: 164-171Crossref PubMed Scopus (116) Google Scholar was used at 10 μg/ml, in parallel to normal rat IgG (Sigma) as a negative control. LMP1 was detected using CS1 to CS4 mouse mAbs (DakoCytomation) at 4 μg/ml. CD3 mAb (clone F7.2.38) and CD8 mAb (clone C8/144B), both from DakoCytomation, were used at 10 μg/ml and 0.25 μg/ml, respectively. CD25 mAb (clone 4C9; Novocastra Laboratories Ltd.) was used at a 1:100 dilution. For immunocytostaining, the following antibodies were used: CD3 mAb (clone UCHT1, DakoCytomation) at 1.5 μg/ml, CD4 mAb (clone SK3; BD Biosciences) at 0.15 μg/ml, and CD8 mAb (clone DK25, DakoCytomation) at 0.25 μg/ml. The detection of EBERs was performed by in situ hybridization using EBER PNA probe and the PNA in situ hybridization detection system (DakoCytomation). Cells were washed in cold phosphate-buffered saline (PBS) and lysed for 1 hour on ice in lysis buffer (1% Nonidet P-40, 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 3% glycerol, 1.5 mmol/L ethylenediaminetetraacetic acid) supplemented with protease inhibitors (1 mmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml pepstatin, 1 μg/ml leupeptin). Cell lysates were centrifuged for 15 minutes at 13,000 × g to remove cell debris and protein concentration was determined using the bicinchoninic acid protein assay reagent (Pierce). Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for immunoblotting with anti-EBI3 2G4H6 mAb, mouse anti-Tax mAb (provided by John Brady, NCI, NIH, Bethesda, MD), or rat anti-p28 mAbs (clones 29B5 or 18C5, DNAX). Binding of mouse or rat mAbs was detected with horseradish peroxidase-conjugated sheep anti-mouse antibodies (Amersham Biosciences) or horseradish peroxidase-conjugated goat anti-rat antibodies (Santa Cruz), respectively. Peroxidase reaction was developed with chemiluminescence reagents (Pierce). EBI3 and IL-27 ELISA were previously described.5Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal Malefyt R Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1208) Google Scholar, 23Devergne O Coulomb-L'Herminé A Capel F Moussa M Capron F Expression of Epstein-Barr virus-induced gene 3, an interleukin-12 p40-related molecule, throughout human pregnancy: involvement of syncytiotrophoblasts and extravillous trophoblasts.Am J Pathol. 2001; 159: 1763-1776Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar EBI3 ELISA detects both free EBI3 and IL-27 (detection limit, 1 ng/ml), whereas IL-27 ELISA is specific for EBI3/p28 heterodimer (detection limit, 0.3 ng/ml). For ELISA, cells (5 to 10 × 105 per ml) were grown in RPM1 1640 complete media or in RPMI 1640 media supplemented with 1% Nutridoma (Roche Diagnostics) for 24 to 48 hours. Supernatants were then collected, spun to remove debris, and stored at −80°C. In some experiments, cell culture supernatants were concentrated using Centriprep 10 (Millipore) or Ultrafree-4 centrifugal filter unit (Millipore), before being tested by ELISA. Cells were washed in FACS buffer (PBS, 2% fetal bovine serum, 0.01% sodium azide), and incubated for 30 minutes on ice with isotype control (MOPC 141, IgG2b; Sigma) or anti-IL-27R mAb (anti-TCCR, IgG2b; R&D Systems) at 10 μg/ml in FACS buffer. Binding of mouse antibodies was detected using phycoerythrin-conjugated F(ab′)2 fragment goat anti-mouse IgG (Coulter) and a minimum of 5000 gated cells were analyzed on a FACScan. In binding assays, cells were washed in FACS buffer, and incubated with recombinant Flag-tagged IL-27 (DNAX) for 1 hour at 1 μg/ml in FACS buffer followed by incubation with M2 anti-Flag antibody (Sigma), and goat anti-mouse IgG as above. As a control, Flag-tagged IL-27 was omitted in the first step. Anti-TCCR mAb was first verified to detect cell surface IL-27R by transfection of COS7 cells with an expression vector coding for human IL-27R (gift from Hugues Gascan's laboratory, Institut National de la Santé et de la Recherche Medicale U564, Angers, France). To further investigate the expression of EBI3 and IL-27 in EBV-associated lymphoid neoplasia, 14 cases of HL (6 EBV+ and 8 EBV−) and 16 cases of various EBV-associated LPDs were analyzed by immunohistochemistry for expression of the two subunits of IL-27, EBI3 and p28, in parallel to LMP1 expression (Table 1, Table 2, and Figure 1).Table 1Immunohistochemical Analysis of IL-27 Expression in HLCase no.DiagnosisSiteLMP1 status% of EBI3+ HRS cells% of p28+ HRS cells1NSLymph nodepos++++−2NSLymph nodepos+++++3NSLymph nodeneg++++−4NSLymph nodeneg+++++5NSLymph nodeneg+++++6NSMediastinumneg++++−7NSMediastinumneg++++−8LDLymph nodepos+++++9LDLymph nodeneg++++−10MCLymph nodepos++++−11MCLymph nodepos+++++12ucLymph nodepos++++−13NLPLymph nodeneg++++−14NLPLymph nodeneg++++−NS, nodular sclerosis; LD, lymphocyte depletion; MC, mixed cellularity; uc, unclassified; NLP, nodular lymphocyte predominant.−, virtually all cells negative;+, 1 to 5% positive cells;++++, >90% positive cells. Open table in a new tab Table 2Immunohistochemical Analysis of IL-27 Expression in Various EBV-Associated LPD% of positive tumoral cellsCase no.DiagnosisSiteLMP1EBI3p281post-transplant B-cell LPDLymph node+++++++−2post-transplant B-cell LPDSmall Bowel+/++++++++++/+3post-transplant B-cell LPDRetroperitoneum+++++−4post-transplant B-cell LPDKidney+++++++−5post-transplant B-cell LPDSkin−−ND6post-transplant B-cell LPDLymph node++++++−7post-transplant B-cell LPDSmall bowel++/++++++/++++−8post-transplant B-cell LPDGingiva−−ND9post-transplant B-cell LPDTonsil++++++−10post-transplant T-cell LPDLymph node++++++−11B-cell LPDLymph node++++++++−1
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