From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Blood First Edition Paper, prepublished online January 21, 2014; DOI 10.1182/blood-2013-10-532895 B Cell Receptors Expressed by Lymphomas of Hepatitis C virus (HCV)-Infected Patients Rarely React with the Viral Proteins Patrick P. Ng1, Chiung-Chi Kuo1, Stanley Wang1, Shirit Einav1, Luca Arcaini2, Marco Paulli2, Carol S. Portlock3, Joe Marcotrigiano4, Alexander Tarr5, Jonathan Ball5, Ronald Levy1 and Shoshana Levy1 1 Stanford University Medical Center, Stanford, CA; 2University of Pavia, Pavia, Italy; 3 Memorial Sloan Kettering Cancer Center, NY, NY; 4Rutgers University, Piscataway, NJ; 5 University of Nottingham, Nottingham, UK Corresponding Author: Shoshana Levy [email protected] Phone: 650-725-6425 Fax: 650-736-1454 Short Title: HCV-induced Lymphomagenesis 1 Copyright © 2014 American Society of Hematology From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Key points: - We tested the hypothesis that B-cell lymphomas arising in HCV-infected patients express BCRs specific to the virus. - We analyzed the reactivity of these BCRs with HCV proteins using several experimental approaches, none of which supported the hypothesis. Abstract Chronic HCV infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this comes from clinical observations of tumor regressions upon anti-viral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon anti-viral treatments. We expressed the lymphoma B-cell receptors (BCRs) as soluble IgGs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region (V) gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus. 2 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Introduction Survival of B cells requires the expression of BCR, as demonstrated in knockout mice1,2 and in some patients with non-X-linked agammaglobulinemia. Lymphoma B cells undergo somatic hypermutation in their V genes, which would be expected to generate protein loss variants. However, in the various lymphoma types, the BCR is retained3, suggesting importance for lymphoma cell survival. Yet, lymphomas’ cognate antigens are not known. B-cell proliferative diseases such as mixed cryoglobulinemia (MC) and Non-Hodgkin lymphoma (B-NHL) that arise in HCV-infected patients represent a special opportunity to study antigenic drive in lymphomagenesis. First, both MC and B-NHL use a restricted V gene repertoire shared by anti-HCV envelope antibodies4,5. Second, elimination of HCV by anti-viral therapy in patients with these B-cell diseases has been associated with their regression6. Moreover, we previously identified an HCV-associated lymphoma whose BCR bound the HCV envelope protein E27. Normal B cells aimed at eliminating HCV would be expected to bind the virus via two receptors, the cognate BCR and the viral entry receptor, CD81, which is a member of a costimulatory complex with CD19/CD21. Such B cells would receive dual stimulatory signals and might undergo unchecked proliferation during chronic HCV infection. Here we tested this hypothesis by expressing BCRs from lymphomas of HCV-infected patients as soluble IgG and as membrane IgM. We included patients that had tumor regressions after anti-viral therapy8 expecting that they would be more likely to express anti-HCV BCRs. We used several methods to test the reactivity of the rescued lymphoma BCRs with viral proteins and particles. However, we found no reactivity and therefore no evidence to support the hypothesis that viral antigens drive B-cell lymphomas. Methods Patients Biopsies of patients with B-NHL and chronic HCV infection were collected at Stanford University Medical Center, Sloan Kettering Memorial Cancer Center and at the University of Pavia Medical School. Patients’ medical record numbers were de-identified and reassigned numbers. Institutional review boards at each center approved this study, 3 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. and written informed consents were obtained from all patients in accordance with the Declaration of Helsinki. V gene rescue mRNA was isolated using RNeasy (Qiagen, Valencia, CA) and cDNA amplified using SMARTer RACE (Clontech, Mountain View, CA), V region amplification used 5’ RACE and the following constant regions primers: IgM 5’-ggtggargcctgaggagacggtgacc-3’ IgG 5’- ggagsagggygccagggggaagac-3’ κ 5’-tgtgacgggcgagctcaggccctgat-3’ λ 5’-gcgtcaggcacagatagctgctggccgc-3’. Expression of lymphoma idiotypes (Ids) Amplified products were inserted into an IgG1 expression vector9, then expressed, as previously4. IgGs in the supernatant of transiently transfected COS-7 cells were quantitated by ELISA. Expression of the rescued V regions in A20 cells as membrane IgM was as previously10. HCV proteins Expression of E2661 and J6E2 were as previously7,11. HCV-E1E2 of various genotypes were encoded by pCR 3.1-UKN1B12.16, -UKN1B5.23, -UKN2A1.2, and -UKN2A2.412. The E1E2 sequences from these plasmids, and the E1E2 of the H77c strain (genotype 1a) were ligated into pCDM8 expression plasmids and transiently transfected into 293T cells. An anti-HCV ELISA kit (DIAsource, Louvain-la-Neuve, Belgium) analyzed interaction of patient IgGs with core, NS3, NS5A and NS5B proteins. Binding of rescued IgG and IgM ELISA detecting binding to HCV-E2 was as previously7. Flow cytometry was used to detect rescue IgG binding to intracellular E1E2 in permeabilized 293T cells, and the binding of A20 cell surface IgM to soluble E2. 4 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Results and Discussion The incidence of B-cell proliferative diseases, including MC and NHL is higher in HCVinfected patients than in non-infected individuals, especially in certain geographical areas, such as Italy8,13. Moreover, the regression of B-cell diseases in response to successful anti-viral therapies implies a causative link between HCV infection and B-cell proliferative diseases 6,8. Here, we aimed to validate the hypothesis that B-cell lymphomas arise from expansion of anti-viral B cells in HCV-infected patients by analyzing the reactivity of their lymphoma BCRs with HCV. Patients were diagnosed in the US and in Italy, the latter received anti-viral therapy and included oncological responders and non-responders. Analysis of V gene usage showed a restricted repertoire, specifically, usage of VH-169 and Vκ3-20 (Table 1). We sequenced the V region genes and expressed them as secreted human IgG1/κ in transfected cells. We then analyzed the reactivity of all rescued IgG1 with the soluble HCV E2 ectodomain, E2661 (genotype 1a)7 or J6E2 (genotype 2a)11. However, except for the anti-E2 mAb controls, none of the tested IgG1 reacted with E2661 (Table 1) or with J6E2 (Fig 1A). Next, we tested all the rescued IgG1s with internal HCV antigens, which are included in a diagnostic kit; however, none reacted with core, NS3, NS5A and NS5B proteins (Fig 1B). HCV is enveloped by two heterodimeric proteins, E1 and E214. We tested the possibility that the rescued IgG1s recognize the heterodimer in its native membrane-bound form by using 293T cells transfected with constructs encoding full E1E2 polypeptides12. We specifically selected E1E2 of the HCV genotypes 1b and 2a matching the infected patients genotypes, as well as E1E2 derived from HCV isolate H77 of genotype 1a. The heterodimers were expressed intracellularly, as detected by flow cytometry using anti-E1 and anti-E2 mAb (Fig 1C, top panels). However, none of the patients’ rescued IgG1 showed reactivity (Fig 1C, bottom panels). Chronic HCV infection is thought to have a causative role in MC, characterized by the benign proliferation of B cell secreting IgM with rheumatoid factor (RF) activity. Evidence also exists that implicate MC as the precursor to frank NHL. However, we did not find RF activity in any of the rescued IgG1s (data not shown). 5 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. The VH-169 gene is also repetitively used in human mAbs that react with HIV15 and with the influenza hemagglutinin (HA) protein16. Importantly, in a recent study germline VH169 expressed as surface IgM reacted with HA, while soluble versions of the mAb were unreactive. It was proposed that clustering of surface BCR on a naïve B cell increases the avidity of the otherwise low-affinity germline BCR to HA to a level sufficient to trigger B-cell activation17. We therefore expressed the lymphoma Ids as human IgMs on A20, a mouse B cell line (Fig 1D top panel) and tested their reactivity with soluble E2 proteins. However, only positive control cells expressing IgM with known anti-E2 reactivity showed binding to soluble E2661 and J6E2 proteins (Fig 1D, middle and bottom panels, respectively). In addition, these cell-surface-expressed lymphoma Id IgMs did not bind HCV-core, or NS proteins (data not shown). Lastly, we took a step further to explore whether lymphoma Ids interact with HCV proteins on an assembled virion. HCV produced in cell culture, HCVcc are associated with lipids18,19 and may contain other antigens not studied in previous experiments. However, incubation of HCVcc with A20 cells expressing surface lymphoma Id IgMs did not reduce their infectivity, whereas neutralizing anti-E2 mAbs (CBH-2 and CBH-5) blocked infection (Fig 1E). Non-neutralizing anti-E2 mAbs (CBH-4B, CBH-4G) expressed as cell surface IgMs (Fig 1E), or as soluble CBH-4G mAb (data not shown), did not block infection. This study tested the hypothesis that B cells aimed at eliminating the virus give rise to HCV-associated B-cell lymphomas. We included patients that responded to anti-viral therapy expecting them to be more likely to bind the virus. However, while confirming a restricted usage of VH-169, we did not identify a single BCR that reacted with HCV (Table 1), hence, no evidence to support the hypothesis. 6 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Acknowledgments We thank Drs. William Robinson and Jeremy Sokolove for providing RF positive and negative plasma, and technical expertise in testing RF activity. The research was supported by a grant from Stanford’s Institute for Immunity, Transplantation, and Infection. S. Einav was supported by K08 AI079406 from the National Institute of Allergy and Infectious Diseases. Authorship Contribution: P.P.N., C-C.K., and S.W. performed the experiments; P.P.N. and S.W. analyzed the results and made the figures; L.A., C.S.P, and R.L. provided biopsy specimens; J.M. provided the J6E2; A.T. and J.B. provided the plasmids encoding E1E2 glycoproteins; P.P.N., S.W., S.E., R.L. and S.L. designed the research and wrote the paper. Conflict-of-interest: The authors declare no conflict of interest. 7 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. References 1. Gu H, Kitamura D, Rajewsky K. B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound D mu protein and selection of DH element reading frames. Cell. 1991;65(1):47-54. 2. Kitamura D, Roes J, Kuhn R, Rajewsky K. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature. 1991;350(6317):423-426. 3. Gururajan M, Jennings CD, Bondada S. Cutting edge: constitutive B cell receptor signaling is critical for basal growth of B lymphoma. J Immunol. 2006;176(10):5715-5719. 4. Chan CH, Hadlock KG, Foung SK, Levy S. V(H)1-69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen. Blood. 2001;97(4):1023-1026. 5. Charles ED, Green RM, Marukian S, et al. Clonal expansion of immunoglobulin M+CD27+ B cells in HCV-associated mixed cryoglobulinemia. Blood. 2008;111(3):13441356. 6. Hermine O, Lefrere F, Bronowicki JP, et al. Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection. N Engl J Med. 2002;347(2):89-94. 7. Quinn ER, Chan CH, Hadlock KG, Foung SK, Flint M, Levy S. The B-cell receptor of a hepatitis C virus (HCV)-associated non-Hodgkin lymphoma binds the viral E2 envelope protein, implicating HCV in lymphomagenesis. Blood. 2001;98(13):37453749. 8. Peveling-Oberhag J, Arcaini L, Hansmann ML, Zeuzem S. Hepatitis Cassociated B-cell non-Hodgkin lymphomas. Epidemiology, molecular signature and clinical management. J Hepatol. 2013;59(1):169-177. 9. Reff ME, Carner K, Chambers KS, et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994;83(2):435-445. 10. Ng PP, Jia M, Patel KG, et al. A vaccine directed to B cells and produced by cellfree protein synthesis generates potent antilymphoma immunity. Proc Natl Acad Sci U S A. 2012;109(36):14526-14531. 11. Whidby J, Mateu G, Scarborough H, Demeler B, Grakoui A, Marcotrigiano J. Blocking hepatitis C virus infection with recombinant form of envelope protein 2 ectodomain. J Virol. 2009;83(21):11078-11089. 12. Tarr AW, Urbanowicz RA, Hamed MR, et al. Hepatitis C patient-derived glycoproteins exhibit marked differences in susceptibility to serum neutralizing antibodies: genetic subtype defines antigenic but not neutralization serotype. J Virol. 2011;85(9):4246-4257. 13. Sautto G, Mancini N, Clementi M, Burioni R. Molecular signatures of hepatitis C virus (HCV)-induced type II mixed cryoglobulinemia (MCII). Viruses. 2012;4(11):29242944. 14. Moradpour D, Penin F. Hepatitis C virus proteins: from structure to function. Curr Top Microbiol Immunol. 2013;369:113-142. 15. Gorny MK, Pan R, Williams C, et al. Functional and immunochemical crossreactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals. Virology. 2012;427(2):198-207. 16. Ohshima N, Iba Y, Kubota-Koketsu R, Asano Y, Okuno Y, Kurosawa Y. Naturally occurring antibodies in humans can neutralize a variety of influenza virus strains, including H3, H1, H2, and H5. J Virol. 2011;85(21):11048-11057. 8 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. 17. Lingwood D, McTamney PM, Yassine HM, et al. Structural and genetic basis for development of broadly neutralizing influenza antibodies. Nature. 2012;489(7417):566570. 18. Moriishi K, Matsuura Y. Exploitation of lipid components by viral and host proteins for hepatitis C virus infection. Front Microbiol. 2012;3:54. 19. Neveu G, Barouch-Bentov R, Ziv-Av A, Gerber D, Jacob Y, Einav S. Identification and targeting of an interaction between a tyrosine motif within hepatitis C virus core protein and AP2M1 essential for viral assembly. PLoS Pathog. 2012;8(8):e1002845. 20. Murray CL, Jones CT, Tassello J, Rice CM. Alanine scanning of the hepatitis C virus core protein reveals numerous residues essential for production of infectious virus. J Virol. 2007;81(19):10220-10231. 9 Isotype Diagnosis 101 102 103 104 105 106 107 108 109 110 111 112 121 122 123 124 125 126 127 IgM/k IgM/k Ig*/k IgM/k IgM/k IgM/k IgM/k IgM/k IgM/k IgM/k IgG/k IgM/k IgM/k IgM/k IgM/k IgM/k IgM/k IgM/k IgM/k DLBCL FL MZL DLBCL DLBCL NHL NA NA NA NA NA NA MALT MZL SMZL SMZL MALT MZL SMZL MALT MZL NHL Lymphoma response to anti-viral therapy ND ND ND ND ND ND ND ND ND ND ND ND Yes No Yes Yes Yes Yes No VH VL Accession # VH / VL HCV genotype E2661 J6E2 E1E2 NS + Core VH1-69 VH3-48 VH4-59 VH1-69 VH1-69 VH4-59 VH3-21 VH4-59 VH4-34 VH4-59 VH1-02 VH4-34 VH1-69 VH4-59 VH1-69 VH4-30 VH3-30 VH1-69 VH1-69 Vk3-20 Vk1-39 Vk3-20 Vk1D-16 Vk3-20 Vk3-15 Vk1-39 Vk3-15 Vk3-20 Vk3-20 Vk2-30 Vk3-20 Vk3-20 Vk3D-15 Vk3-20 Vk3-15 Vk1-8 Vk3-20 Vk3-20 KF895775/6 KF895777/8 KF895779/80 KF895781/2 KF895783/4 KF895785/6 KF895787/8 KF895789/90 KF895791/2 KF895793/4 KF895795/6 KF895797/8 KF895799/800 KF895801/2 KF895803/4 KF895805/6 KF895807/8 KF895809/10 KF895811/2 ND “ “ “ “ “ “ “ “ “ “ “ 2a/2c 1b 2a/2c 2a/2c 2a/2c 1b 1b Neg ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ‘’ ND “ “ “ “ “ “ Neg “ “ “ “ “ “ “ “ “ “ “ ND “ “ “ “ “ “ Neg “ “ “ “ “ “ “ “ “ “ “ ND “ “ “ “ “ “ Neg “ “ “ “ “ “ “ “ “ “ “ E2661, soluble envelope protein of HCV genotype 1a; J6E2, envelope protein of HCV genotype J6; E1E2, HCV envelope proteins expressed intracellularly. NS+Core, HCV non-structural + core proteins. DLBCL, diffuse large B cell lymphoma; FL, follicular lymphoma; MALT, mucosa-associated lymphoid tissue; MZL, marginal zone lymphoma; NHL, Non-Hodgkin lymphoma; SMZL, splenic marginal zone lymphoma; NA, not available. ND, not done; Neg, negative. Table 1 10 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Patient From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Figure legend Rescued HCV-associated lymphoma idiotypes do not react with viral proteins when expressed as soluble IgGs or as cell surface IgMs. (A) Purified J6E2 captured on lectin-coated 96-well plates was incubated with the indicated patients’ Id IgG1s, with anti-E2 mAbs CBH-4G or CBH-5, or with a human IgG1/κ isotype control. Plate-bound IgGs were detected with an HRP-conjugated anti-human IgG. (B) 96-well plates coated with HCV core, NS3 and NS5 antigens were incubated with the indicated patients’ Id IgG1s, with anti-HCV plasma, or with negative control plasma, diluted as indicated. After wash, plate-bound Igs were detected with HRP-conjugated HCV core, NS3 and NS5 antigens. (A, B) Each bar represents the mean O.D. of wells incubated with each group of Id IgGs ± standard deviation. Representative results of two experiments for each assay are shown. (C) Single-cell suspensions of 293T cells transfected with empty pCDM8 vector (filled gray) or with pCDM8 vectors encoding E1E2 of the indicated genotypes were fixed and permeabilized. The cells were then stained with the anti-E2, anti-E1, a human IgG1/κ isotype control mAb, or with a mixture of the indicated patients’ IgG1 containing 0.5 μg of each Id. Cells were then washed, stained with PE-conjugated anti-human IgG, and analyzed by flow cytometry. (D) The patients’ Ids were expressed as human IgMs on the surface of the mouse B cell line, A20. Positive controls were A20 cells expressing CBH-4B or CBH-4G. Cells were stained with FITC conjugated antihuman IgM (top panel); Cells were incubated for 1 h on ice with cell culture supernatant containing soluble E2661 or mock supernatant. Cells were then washed, stained with AlexaFluor® 647-conjugated mouse anti-E2 mAb (H53)(middle panel); Cells were incubated for 1 h on ice with soluble J6E2 or BSA, washed and further incubated for 1 h on ice with a 1:1 mixture of the anti-E2 mAbs CBH-2 and CBH-5. After wash, cells were stained with AlexaFluor® 647-conjugated anti-human IgG (bottom panel). (C, D) Cells were washed and analyzed by flow cytometry. (E) A20 cells expressing the indicated surface lymphoma patients’ Id IgM or an IgM of irrelevant specificity (SIC5); neutralizing anti-E2 mAbs (CBH2 or CBH5) or a control human IgG1 mAb were incubated with luciferase reporter HCVcc (J6/JFH(p7-Rluc2A) HCV20, titer: 6.3×105 TCID50/ml) for 1hr at 37°C. These samples were then used to inoculate naïve huh-7.5 cells. To measure infectivity, cells were lysed at 48hr and subjected to standard luciferase assays. Y-axis represents HCVcc infection relative to the A20 SIC5 control. Data represent means and s.d. (error bars). 11 From www.bloodjournal.org by guest on February 4, 2015. For personal use only. From www.bloodjournal.org by guest on February 4, 2015. For personal use only. Prepublished online January 21, 2014; doi:10.1182/blood-2013-10-532895 B cell receptors expressed by lymphomas of hepatitis C virus (HCV)-infected patients rarely react with the viral proteins Patrick P. Ng, Chiung-Chi Kuo, Stanley Wang, Shirit Einav, Luca Arcaini, Marco Paulli, Carol S. Portlock, Joe Marcotrigiano, Alexander Tarr, Jonathan Ball, Ronald Levy and Shoshana Levy Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include digital object identifier (DOIs) and date of initial publication. Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.