B-cell receptors expressed by lymphomas of hepatitis C virus (HCV)–infected patients rarely react with the viral proteins

Survival of B cells requires the expression of B-cell receptors (BCRs), as demonstrated in knockout mice^1,2  and in some patients with non–X-linked agammaglobulinemia. Lymphoma B cells undergo somatic hypermutation in their variable region (V) genes, which would be expected to generate protein loss variants. However, in the various lymphoma types, the BCR is retained,³  suggesting immunoglobulin G (IgG)'s importance for lymphoma cell survival. Yet lymphomas’ cognate antigens are not known. B-cell proliferative diseases such as mixed cryoglobulinemia (MC) and B-cell non-Hodgkin lymphoma (B-NHL) that arise in hepatitis C virus (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 antibodies.^4,5  Second, elimination of HCV by antiviral therapies in patients with these B-cell diseases has been associated with their regression.⁶  Moreover, we previously identified an HCV-associated lymphoma whose BCR bound the HCV envelope protein E2.⁷  Normal B cells aimed at eliminating HCV would be expected to bind the virus via 2 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 IgGs, suggesting their importance, and as membrane IgMs. We included patients who had tumor regressions after antiviral therapies,⁸  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.

Biopsy specimens of patients with B-NHL and chronic HCV infection were collected at Stanford University Medical Center, Sloan Kettering Memorial Cancer Center, and the University of Pavia Medical School. Patients’ medical record numbers were de-identified and reassigned numbers. The institutional review boards at each center approved this study, and written informed consent was obtained from all patients in accordance with the Declaration of Helsinki.

mRNA was isolated using RNeasy (Qiagen, Valencia, CA), cDNA was amplified using SMARTer RACE (Clontech, Mountain View, CA), and 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′

Amplified products were inserted into an IgG1 expression vector⁹  and then expressed, as was done previously.⁴  IgGs in the supernatant of transiently transfected COS-7 cells were quantitated by enzyme-linked immunosorbent assay (ELISA). Expression of the rescued V regions in A20 cells as membrane IgM was done, as previously.¹⁰ 

Expression of soluble envelope protein of HCV genotype 1a (E2₆₆₁) and envelope protein of HCV genotype 2a (J6E2) were done as previously.^7,11  HCV envelope proteins expressed intracellularly (HCV-E1E2) of various genotypes were encoded by pCR 3.1-UKN1B12.16, -UKN1B5.23, -UKN2A1.2, and -UKN2A2.4.¹²  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.

ELISA was done to detect binding to HCV-E2, as was done previously.⁷  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.

The incidence of B-cell proliferative diseases, including MC and NHL, is higher in HCV-infected patients than in noninfected individuals, especially in certain geographical areas such as Italy.^8,13  Moreover, the regression of B-cell diseases in response to successful antiviral 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 antiviral 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, and the latter received antiviral therapy and included oncological responders and nonresponders. Analysis of V-gene usage showed a restricted repertoire, specifically usage of V_H-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 monoclonal antibody (mAb) controls, none of the tested IgG1 reacted with E2₆₆₁ (Table 1) or with J6E2 (Figure 1A).¹⁴  Next we tested all of the rescued IgG1s with internal HCV antigens, which are included in a diagnostic kit; however, none reacted with core, NS3, NS5A, and NS5B proteins (Figure 1B).

HCV is enveloped by 2 heterodimeric proteins, E1 and E2.¹⁵  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 polypeptides.¹²  We specifically selected E1E2 of the HCV genotypes 1b and 2a matching the infected patients genotypes, as well as E1E2 derived from the HCV isolate H77 of genotype 1a. The heterodimers were expressed intracellularly, as detected by flow cytometry using anti-E1 and anti-E2 mAb (Figure 1C, top panels). However, none of the patients’ rescued IgG1 showed reactivity (Figure 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 implicates MC as the precursor to frank NHL. However, we did not find RF activity in any of the rescued IgG1s (data not shown).

The VH-1₆₉ gene is also repetitively used in human mAbs that react with HIV¹⁶  and with the influenza hemagglutinin (HA) protein.¹⁷  Of note, in a recent study, germline VH-1₆₉ expressed as surface IgM reacted with HA, whereas 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 activation.¹⁸  We therefore expressed the lymphoma Ids as human IgMs on A20, a mouse B-cell line (Figure 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 (Figure 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).

Finally, we took a step further to explore whether lymphoma Ids interact with HCV proteins on an assembled virion. HCVcc are associated with lipids^19,20  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 (Figure 1E). Nonneutralizing anti-E2 mAbs (CBH-4B, CBH-4G) expressed as cell surface IgMs (Figure 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 antiviral 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 there was no evidence to support the hypothesis.

The authors thank Drs William Robinson and Jeremy Sokolove for providing RF-positive and -negative plasma, and technical expertise in testing RF activity.

This study was supported by a grant from Stanford’s Institute for Immunity, Transplantation, and Infection; and the National Institutes of Health, National Institute of Allergy and Infectious Diseases (K08 AI079406) (S.E.).

Contribution: P.P.N., C.-C.K., and S.W. performed the experiments; P.P.N. and S.W. analyzed the results and created the figures; L.A., M.P., 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; and P.P.N., S.W., S.E., R.L., and S.L. designed the research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Shoshana Levy, Stanford University Medical Center, CCSR Building, Room 1105a, Stanford, CA 94305; e-mail: slevy@stanford.edu.