- Email: [email protected]
Hepatitis C virus (HCV)-driven stimulation of subfamily-restricted natural IgM antibodies in mixed cryoglobulinemia
Available online at www.sciencedirect.com
Autoimmunity Reviews 7 (2008) 468 – 472 www.elsevier.com/locate/autrev
Hepatitis C virus (HCV)-driven stimulation of subfamily-restricted natural IgM antibodies in mixed cryoglobulinemia Mario Perotti, Nadia Ghidoli, Raffaele Altara, Roberta A. Diotti, Nicola Clementi, Donata De Marco, Monica Sassi, Massimo Clementi, Roberto Burioni, Nicasio Mancini ⁎ Laboratorio di Microbiologia e Virologia, Università Vita-Salute San Raffaele, Milan, Italy Received 15 January 2008; accepted 12 March 2008 Available online 10 April 2008
Abstract Hepatitis C virus (HCV) infection has been closely related to mixed cryoglobulinemia (MC). During HCV infection, cryoglobulins derive from the restricted expression of few germline genes as VH1-69, a subfamily highly represented in antiHCV humoral response. Little is known about the self-reacting IgM component of the cryoprecipitate. In the present study, the IgM/K repertoire of an HCV-infected cryoglobulinemic patient was dissected by phage-display on well-characterized antiHCV/E2 VH1-69-derived monoclonal IgG1/Κ Fab fragments cloned from the same patient. All selected IgM clones were shown to react with the anti-HCV/E2 antibodies belonging to VH1-69 subfamily. More than 60% of selected clones showed a bias in VH gene usage, restricted to two VH subfamilies frequently described in autoimmune manifestations (VH3-23; VH3-21). Moreover, all selected clones showed an high similarity (N 98.5%) to germline genes evidencing their natural origin. A possible hypothesis is that clones belonging to some subfamilies are naturally prone to react against other VH gene subfamilies, as VH 1-69. An antigen-driven stimulation of these subfamilies, and their overexpression as in HCV infection, could lead to a breaking of humoral homeostatic balance exposing the patients to the risk of developing autoimmune disorders. © 2008 Elsevier B.V. All rights reserved. Keywords: HCV; Cryoglobulinemia; Lymphoma; Autoimmunity
Contents 1. Introduction . . . . . . . . . . . . . . . . . . 2. Cloning strategies of autoreactive IgM Fabs . 3. Sequence analysis of IgM heavy chains . . . 4. Binding characteristics of selected IgM Fabs . 5. IgM natural antibodies and cryoglobulinemia . Take-home messages . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
469 470 470 470 470 471 472
⁎ Corresponding author. Laboratorio di Microbiologia e Virologia, Università “Vita-Salute” San Raffaele, DIBIT2, via Olgettina 60, 20132, Milano, Italy. Tel.: +39 02 2643 4195, +39 328 2898085 (Mobile); fax: +39 02 2643 4288. E-mail address: [email protected] (N. Mancini). 1568-9972/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2008.03.008
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
1. Introduction Mixed cryoglobulinemia (MC) is a systemic smallvessel vasculitis characterized by the oligoclonal expansion of B cells expressing autoantobodies (cryoglobulins), usually IgM, that become insoluble at temperatures below 37 °C and that are endowed with rheumatoid factor (RF) activity. MC may be considered a benign lymphoproliferative disorder that in some occasions can evolve into an overt lymphoma [1]. Many evidences suggest that MC is strongly correlated to HCV infection: (I) 50%–90% of patients with MC harbor HCV [2], (II) cryoglobulins are detected in 40– 60% of HCV carriers [3] and (III) the antiviral therapy induces significant remissions in HCV-associated MC [4]. Even if the link between cryoglobulinemia and HCV infection is well established in terms of epidemiological evidences, the causative role of the pathogen remains
469
to be elucidated. Many pathogenic mechanisms have been postulated [5], and several lines of evidence show that a clonal expansion of B cells expressing antibodies against HCV can play a crucial role in cryoglobulinemia [6–8]. Indeed, the restricted V gene usage (VH1-69) shared by the anti-HCV antibody response directed against surface E2 glycoprotein (HCV/E2) and by HCVrelated cryoglobulins and B-cell lymphoma clones, together with an antibody mutation pattern suggestive of a germinal center derivation, strongly suggest that HCV-associated cryoglobulinemia and lymphomas could be two sequential phases of a B-cell expansion responding to a common stimulus. In this study, in order to investigate the potential role of anti-HCV/E2 antibodies in the genesis of cryoglobulinemia, we tested whether the immune repertoire of a patient with HCVassociated MC contains IgM clones able to react specifically against anti-HCV/E2 antibodies belonging to VH1-69 subfamily.
Fig. 1. Frequency of VH subfamilies of IgM Fab clones selected on VH1-69 anti-HCV/E2 Fab pool (a) and on human standard IgG-derived Fabs (b). Panning of the combinatorial library was performed as described [13,14]. High-binding ELISA plates (Costar) were coated with a pool of anti-HCV/ E2 Fabs encoded by VH1-69 gene (e20, e137 e301 and e509) and with a pool of human standard IgG-derived Fabs (300 ng/well). The reactivity in ELISA of purified IgM Fab clones encoded by VH3-21 (c) and Vh3-23 (d) subfamilies on different antigens is also reported. Purified IgM Fabs were tested at concentration ranging from 6.25 µg/ml to 50 µg/ml. Means and standard errors of two replicate assays are reported.
470
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
2. Cloning strategies of autoreactive IgM Fabs A phage-display library containing human antibodies of IgM/K isotype was selected on a pool of well characterized IgG1/K anti-HCV/E2 Fabs, belonging to VH169 subfamily, and derived from the same HCV-infected cryoglobulinemic patient. Generation, purification, characterization and definition of biological activities of antiHCV/E2 Fabs have been described elsewhere [9–14]. The phage-display selection procedure (panning) yielded a 100-fold enrichment of eluted IgM-bearing phage on anti-HCV/E2 Fabs. 35 IgM clones were then tested in ELISA in order to confirm their reactivity on the same pool of anti-HCV Fabs used for library screening. The same library containing IgM/K antibodies was also panned on IgG Fabs derived from human standard IgG. Also in this case, the panning showed a 100-fold enrichment of eluted phage, and 35 clones were tested in ELISA against standard IgG Fabs. 3. Sequence analysis of IgM heavy chains The sequences of the heavy chain genes were determined for all IgM Fabs able to bind the anti-HCV/E2 Fab pool, leading to the to identification of 18 different IgM clones. As far as the heavy chain is concerned, 11 out of 18 IgM clones (61%) belonged only to two VH subfamilies, VH3-23 (39%) and VH3-21 (22%) (Fig. 1a). Interestingly, this bias was not observed among clones selected on standard IgG-derived Fabs, with only 1 clone belonging to VH3-23 and no clones derived from VH3-21 (Fig. 1b). Moreover, the reported bias was not described in the original unpanned IgM/K library, with only 2.5% of clones deriving from VH3-23 or VH3-21 genes (data not shown). The mutational pattern of selected anti-HCV/E2 IgM Fabs was also investigated, showing for almost all clones a natural origin evidenced by the high homology (N 98.4%, mean: 99.5%; 99.1 b 95%CI b 99.8) to the germline counterpart (Table 1). 4. Binding characteristics of selected IgM Fabs In order to evaluate the binding characteristics of selected clones, VH3-23 and VH3-21-derived IgM Fabs were purified by immunoaffinity as previously described [15], and were tested in ELISA against several different antigens (Fig. 1c–d). Fabs derived from VH321 showed features of polyreactivity, while Fabs bearing an heavy chain encoded by VH3-23 were not polyreactive, recognizing only IgG1-derived Fab fragments (Fig. 1d). The latter Fabs were tested individually for binding to monoclonal antibodies derived from different
Table 1 Sequence analysis of heavy chains of VH3-23 and VH3-21-derived IgM Fabs selected on anti-HCV/E2 Fabs Antibody V gene
D gene
J Homology R:S p value gene (%) mutations
M1 M3 M4 M9 M11 M14 M17 M5 M10 M12 M31
D6-19 D1-26 D1-26 D3-3 D6-6 D6-6 D6-25 D6-6 D1-7 D2-2 D3-3
J3 J4 J3 J3 J3 J6 J5 J4 J3 J4 J6
FR CDR FR V3-23 V3-23 V3-23 V3-23 V3-23 V3-23 V3-23 V3-21 V3-21 V3-21 V3-21
100 99 99.6 100 99.6 99.21 98.4 100 99.22 99.21 100
0:0 1:0 0:0 0:0 1:0 0:2 3:0 0:0 0:1 0:2 0:0
0:0 0:0 2:0 0:0 1:0 0:1 1:0 0:0 0:0 0:0 0:0
– 0.67 0.07 – 0.6 0.08 0.6 – 0.19 0.08 –
CDR – 0.55 0.008 – 0.56 0.64 0.2 – 0.5 0.64 –
The VDJ gene rearrangements are shown for each clone. (R:S) ratio between replacement (R) and silent (S) mutations is reported. p-value is calculated by multinomial analysis.
germline genes showing a preferential binding to Fabs derived from VH1-69 (data not shown). 5. IgM natural antibodies and cryoglobulinemia Epidemiological and experimental evidences suggest a strong link between HCV infection, cryoglobulinemia and B-cell lymphoma. However the mechanisms that lead to clonal expansion of B-cells expressing autoantibodies endowed with rheumatoid factor activity, remain to be elucidated [16–19]. Several studies have described in the cryoprecipitate IgM antibodies that cross-react with different HCV antigens, including NS3, E2 and core proteins [[3,20,21], but this cannot exclude that anti-HCV antibodies can be bound by RF IgM and subsequently trapped in the cryoprecipitate together with antibodies directed against other antigens. Under this perspective, in the present study we describe the dissection of the IgM/K repertoire of a patient with HCV-associated MC, and the molecular cloning of antibody fragments able to recognize a pool of well characterized anti-HCV/E2 antibodies. This approach allowed the selection of IgM antibodies belonging to two subfamilies (VH3-23 and VH3-21) able to bind human IgG antibodies. More in details, the VH323 subfamily did not evidence polyreactivity features (as VH3-21), but showed a binding bias toward VH1-69derived IgG1 Fabs. Sequence analysis showed that all clones have a high homology to the germline, evidencing that they are antibodies naturally present in the human repertoire. These data suggest that VH3-23 IgM Fabs described here may be naturally prone to recognize some conserved regions of specific VH subfamilies, as
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
VH1-69, the VH gene described to be preferentially used in humoral response against HCV/E2. Overall, the results of the present study suggest a new model of an infection-driven lymphoproliferative disorder. In particular, the E2-driven stimulation of the immune system may cause the expansion of specific B cells expressing antibodies encoded by VH1-69, a VH gene subfamily recognized by some natural IgM antibody subfamilies. As a consequence, circulating immune complexes are promptly formed. Moreover the E2-driven stimulation and the cross-linking of B-cell receptor by autoantibodies may allow a chronic activation and a clonal expansion of anti-HCV/E2 B cells. Furthermore the intrinsic genetic instability of B-cells during somatic hypermutation and class-switching processes may favor genetic aberrations responsible for prolonged B cell survival. This process could lead the lymphoproliferation to become independent of antigenic stimulation, exposing the patients to the risk of developing a frank B cell malignancy.
[5]
[6]
[7]
[8]
[9]
[10]
Take-home messages • HCV infection is strongly associated with mixed cryoglobulinemia (MC), a benign lymphoproliferative disorder characterized by the expansion of B cell clones secreting antibodies encoded by restricted germline genes, as VH1-69, a VH family highly represented in anti-HCV humoral response. • Human immune repertoire shows natural antibodies belonging to distinct subfamilies, as VH3-23, that are prone to bind epitopes on restricted VH antibody subfamilies, such as VH1-69. • HCV can represent a triggering factor of MC, by an overexpression of VH1-69 antibodies that are bound by natural IgM antibodies.
[11]
[12]
[13]
[14]
References [15] [1] Monti G, Pioltelli P, Saccardo F, Campanini M, Candela M, Cavallero G, et al. Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias. Arch Intern Med 2005;165:101–5. [2] Agnello V. The aetiology of mixed cryoglobulinaemia associated with hepatitis C virus infection. Scand J Immunol 1995;42:179–84. [3] Ferri S, Dal Pero F, Bortoletto G, Bianchi FB, Lenzi M, Alberti A, et al. Detailed analysis of the E2–IgM complex in hepatitis C-related type II mixed cryoglobulinaemia. J Viral Hepatitis 2006;13: 166–76. [4] Saadoun D, Resche-Rigon M, Thibault V, Piette JC, Cacoub P. Antiviral therapy for hepatitis C virus-associated mixed cryoglo-
[16]
[17]
[18]
471
bulinemia vasculitis: a long-term follow up study. Arthritis Rheum 2006;54:3696–706. Landau DA, Saadoun D, Calabrese LH, Cacoub P. The pathophysiology of HCV induced B-cell clonal disorders. Autoimmun Rev 2007;6:581–7. Carbonari M, Caprini E, Tedesco T, Mazzetta F, Tocco V, Casato M, et al. Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis. J Immunol 2005;174:6532–9. Chan CH, Hadlock KG, Foung SK, Levy S. VH1-69 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;97:1023–6. De Re V, De Vita S, Marzotto A, Rupolo M, Gloghini A, Pivetta B, et al. Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 2000;96:3578–84. Bugli F, Mancini N, Kang CY, Di Campli C, Grieco A, Manzin A, et al. Mapping B cell epitopes of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from phage display libraries. J Virol 2001;75:9986–90. Burioni R, Bugli F, Mancini N, Rosa D, Di Campli C, Moroncini G, et al. Non-neutralizing human antibody fragments against hepatitis C virus E2 glycoprotein modulate neutralization of binding activity of human recombinant fabs. Virology 2001;288: 29–35. Burioni R, Mancini N, Carletti S, Perotti M, Grieco A, Canducci F, et al. Cross-reactive pseudovirus-neutralizing anti-envelope antibodies coexist with antibodies devoid of such activity in persistent hepatitis C virus infection. Virology 2004;327:242–8. Burioni R, Matsuura Y, Mancini N, Tani H, Miyamura T, Varaldo PE, et al. Diverging effects of human recombinant anti-hepatitis C virus (HCV) antibody fragments derived from a single patient on the infectivity of a vesicular stomatitis virus/HCV pseudotype. J Virol 2002;76:11775–9. Burioni R, Plaisant P, Manzin A, Rosa D, Delli Carri V, Bugli F, et al. Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant Fab fragments. Hepatology 1998;28: 810–4. Perotti M, Mancini N, Diotti RA, Tarr AW, Ball JK, Owsianka A, et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol 2008;82:1047–52. Barbas CF, Bjorling E, Chiodi F, Dunlop N, Cababa D, Jones TM, et al. Recombinant human Fabs neutralize human type 1 immunodeficiency virus in vitro. Proc Natl Acad Sci U S A 1992;89:9339–43. Ferri C, Antonelli A, Mascia MT, Sebastiani M, Fallahi P, Ferrari D, et al. HCV-related autoimmune and neoplastic disorders: the HCV syndrome. Dig Liver Dis 2007;39(Suppl 1):S13–21. Ferri C, Antonelli A, Mascia MT, Sebastiani M, Fallahi P, Ferrari D, et al. B-cells and mixed cryoglobulinemia. Autoimmun Rev 2007;7:114–20. Saadoun D, Landau DA, Calabrese LH, Cacoub PP. Hepatitis Cassociated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation. Rheumatology 2007;46: 1234–42.
472
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
[19] Zignego AL, Giannini C, Ferri C. Hepatitis C virus-related lymphoproliferative disorders: an overview. World J Gastroenterol 2007;13:2467–78. [20] Simula MP, Caggiari L, Gloghini A, De Re V. HCV-related immunocytoma and type II mixed cryoglobulinemia-associated autoantigens. Ann N Y Acad Sci 2007;1110:121–30.
[21] De Re V, Sansonno D, Simula MP, Caggiari L, Gasparotto D, Fabris M, et al. HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations. Leukemia 2006;20:1145–54.
Update on Sjögren's syndrome autoimmune epithelitis: from classification to increased neoplasias Sjogren's syndrome is a chronic inflammatory process primarily affecting females during the fourth and fifth decades of life. Sjogren characterizes by exocrine glands inflammation causing kerato-conjuctivitis sicca and xerostomia and less commonly by extraglandular manifestations. The extraglandular manifestation of Sjogren syndrome can be divided to two subsets. One group constitute of lung, interstitial kidney disease and liver damage as a result of lymphocytic invasion of epithelial tissues causing "autoimmune epithelitis". The second group includes vasculitis, neuropathy and glomerulonephritis as a result of immune complex disease. Recently Tzioufas AG and Voulgaerlis M. (Best Pract Res Clin Rheumatol. 2007; 21:989-1010) reviewed the clinical picture, diagnostic procedures, treatment options, differential diagnosis and risk factors for malignancy transformation of Sjogren syndrome. Non-Hodkins lymphoma (NHL) occurs in approximately 5% of Sjogren patients. Two main categories of NHL are associated with Sjogren, in the majority of patients indolent extranodal (mucosal) marginal zone lymphomas is diagnosed. The second variant is high-grade aggressive lymphomas, such as de novo or secondary diffuse large B cell disease which is only occasionally encountered. The presence of palpable purpura, low C4 and mixed cryoglobulines constitute the main prediction factors for development of lymphoma and other comorbidities, thus Sjogern patients especially those who present with risk factors should be closely followed.
Autoimmunity Reviews 7 (2008) 468 – 472 www.elsevier.com/locate/autrev
Hepatitis C virus (HCV)-driven stimulation of subfamily-restricted natural IgM antibodies in mixed cryoglobulinemia Mario Perotti, Nadia Ghidoli, Raffaele Altara, Roberta A. Diotti, Nicola Clementi, Donata De Marco, Monica Sassi, Massimo Clementi, Roberto Burioni, Nicasio Mancini ⁎ Laboratorio di Microbiologia e Virologia, Università Vita-Salute San Raffaele, Milan, Italy Received 15 January 2008; accepted 12 March 2008 Available online 10 April 2008
Abstract Hepatitis C virus (HCV) infection has been closely related to mixed cryoglobulinemia (MC). During HCV infection, cryoglobulins derive from the restricted expression of few germline genes as VH1-69, a subfamily highly represented in antiHCV humoral response. Little is known about the self-reacting IgM component of the cryoprecipitate. In the present study, the IgM/K repertoire of an HCV-infected cryoglobulinemic patient was dissected by phage-display on well-characterized antiHCV/E2 VH1-69-derived monoclonal IgG1/Κ Fab fragments cloned from the same patient. All selected IgM clones were shown to react with the anti-HCV/E2 antibodies belonging to VH1-69 subfamily. More than 60% of selected clones showed a bias in VH gene usage, restricted to two VH subfamilies frequently described in autoimmune manifestations (VH3-23; VH3-21). Moreover, all selected clones showed an high similarity (N 98.5%) to germline genes evidencing their natural origin. A possible hypothesis is that clones belonging to some subfamilies are naturally prone to react against other VH gene subfamilies, as VH 1-69. An antigen-driven stimulation of these subfamilies, and their overexpression as in HCV infection, could lead to a breaking of humoral homeostatic balance exposing the patients to the risk of developing autoimmune disorders. © 2008 Elsevier B.V. All rights reserved. Keywords: HCV; Cryoglobulinemia; Lymphoma; Autoimmunity
Contents 1. Introduction . . . . . . . . . . . . . . . . . . 2. Cloning strategies of autoreactive IgM Fabs . 3. Sequence analysis of IgM heavy chains . . . 4. Binding characteristics of selected IgM Fabs . 5. IgM natural antibodies and cryoglobulinemia . Take-home messages . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
469 470 470 470 470 471 472
⁎ Corresponding author. Laboratorio di Microbiologia e Virologia, Università “Vita-Salute” San Raffaele, DIBIT2, via Olgettina 60, 20132, Milano, Italy. Tel.: +39 02 2643 4195, +39 328 2898085 (Mobile); fax: +39 02 2643 4288. E-mail address: [email protected] (N. Mancini). 1568-9972/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2008.03.008
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
1. Introduction Mixed cryoglobulinemia (MC) is a systemic smallvessel vasculitis characterized by the oligoclonal expansion of B cells expressing autoantobodies (cryoglobulins), usually IgM, that become insoluble at temperatures below 37 °C and that are endowed with rheumatoid factor (RF) activity. MC may be considered a benign lymphoproliferative disorder that in some occasions can evolve into an overt lymphoma [1]. Many evidences suggest that MC is strongly correlated to HCV infection: (I) 50%–90% of patients with MC harbor HCV [2], (II) cryoglobulins are detected in 40– 60% of HCV carriers [3] and (III) the antiviral therapy induces significant remissions in HCV-associated MC [4]. Even if the link between cryoglobulinemia and HCV infection is well established in terms of epidemiological evidences, the causative role of the pathogen remains
469
to be elucidated. Many pathogenic mechanisms have been postulated [5], and several lines of evidence show that a clonal expansion of B cells expressing antibodies against HCV can play a crucial role in cryoglobulinemia [6–8]. Indeed, the restricted V gene usage (VH1-69) shared by the anti-HCV antibody response directed against surface E2 glycoprotein (HCV/E2) and by HCVrelated cryoglobulins and B-cell lymphoma clones, together with an antibody mutation pattern suggestive of a germinal center derivation, strongly suggest that HCV-associated cryoglobulinemia and lymphomas could be two sequential phases of a B-cell expansion responding to a common stimulus. In this study, in order to investigate the potential role of anti-HCV/E2 antibodies in the genesis of cryoglobulinemia, we tested whether the immune repertoire of a patient with HCVassociated MC contains IgM clones able to react specifically against anti-HCV/E2 antibodies belonging to VH1-69 subfamily.
Fig. 1. Frequency of VH subfamilies of IgM Fab clones selected on VH1-69 anti-HCV/E2 Fab pool (a) and on human standard IgG-derived Fabs (b). Panning of the combinatorial library was performed as described [13,14]. High-binding ELISA plates (Costar) were coated with a pool of anti-HCV/ E2 Fabs encoded by VH1-69 gene (e20, e137 e301 and e509) and with a pool of human standard IgG-derived Fabs (300 ng/well). The reactivity in ELISA of purified IgM Fab clones encoded by VH3-21 (c) and Vh3-23 (d) subfamilies on different antigens is also reported. Purified IgM Fabs were tested at concentration ranging from 6.25 µg/ml to 50 µg/ml. Means and standard errors of two replicate assays are reported.
470
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
2. Cloning strategies of autoreactive IgM Fabs A phage-display library containing human antibodies of IgM/K isotype was selected on a pool of well characterized IgG1/K anti-HCV/E2 Fabs, belonging to VH169 subfamily, and derived from the same HCV-infected cryoglobulinemic patient. Generation, purification, characterization and definition of biological activities of antiHCV/E2 Fabs have been described elsewhere [9–14]. The phage-display selection procedure (panning) yielded a 100-fold enrichment of eluted IgM-bearing phage on anti-HCV/E2 Fabs. 35 IgM clones were then tested in ELISA in order to confirm their reactivity on the same pool of anti-HCV Fabs used for library screening. The same library containing IgM/K antibodies was also panned on IgG Fabs derived from human standard IgG. Also in this case, the panning showed a 100-fold enrichment of eluted phage, and 35 clones were tested in ELISA against standard IgG Fabs. 3. Sequence analysis of IgM heavy chains The sequences of the heavy chain genes were determined for all IgM Fabs able to bind the anti-HCV/E2 Fab pool, leading to the to identification of 18 different IgM clones. As far as the heavy chain is concerned, 11 out of 18 IgM clones (61%) belonged only to two VH subfamilies, VH3-23 (39%) and VH3-21 (22%) (Fig. 1a). Interestingly, this bias was not observed among clones selected on standard IgG-derived Fabs, with only 1 clone belonging to VH3-23 and no clones derived from VH3-21 (Fig. 1b). Moreover, the reported bias was not described in the original unpanned IgM/K library, with only 2.5% of clones deriving from VH3-23 or VH3-21 genes (data not shown). The mutational pattern of selected anti-HCV/E2 IgM Fabs was also investigated, showing for almost all clones a natural origin evidenced by the high homology (N 98.4%, mean: 99.5%; 99.1 b 95%CI b 99.8) to the germline counterpart (Table 1). 4. Binding characteristics of selected IgM Fabs In order to evaluate the binding characteristics of selected clones, VH3-23 and VH3-21-derived IgM Fabs were purified by immunoaffinity as previously described [15], and were tested in ELISA against several different antigens (Fig. 1c–d). Fabs derived from VH321 showed features of polyreactivity, while Fabs bearing an heavy chain encoded by VH3-23 were not polyreactive, recognizing only IgG1-derived Fab fragments (Fig. 1d). The latter Fabs were tested individually for binding to monoclonal antibodies derived from different
Table 1 Sequence analysis of heavy chains of VH3-23 and VH3-21-derived IgM Fabs selected on anti-HCV/E2 Fabs Antibody V gene
D gene
J Homology R:S p value gene (%) mutations
M1 M3 M4 M9 M11 M14 M17 M5 M10 M12 M31
D6-19 D1-26 D1-26 D3-3 D6-6 D6-6 D6-25 D6-6 D1-7 D2-2 D3-3
J3 J4 J3 J3 J3 J6 J5 J4 J3 J4 J6
FR CDR FR V3-23 V3-23 V3-23 V3-23 V3-23 V3-23 V3-23 V3-21 V3-21 V3-21 V3-21
100 99 99.6 100 99.6 99.21 98.4 100 99.22 99.21 100
0:0 1:0 0:0 0:0 1:0 0:2 3:0 0:0 0:1 0:2 0:0
0:0 0:0 2:0 0:0 1:0 0:1 1:0 0:0 0:0 0:0 0:0
– 0.67 0.07 – 0.6 0.08 0.6 – 0.19 0.08 –
CDR – 0.55 0.008 – 0.56 0.64 0.2 – 0.5 0.64 –
The VDJ gene rearrangements are shown for each clone. (R:S) ratio between replacement (R) and silent (S) mutations is reported. p-value is calculated by multinomial analysis.
germline genes showing a preferential binding to Fabs derived from VH1-69 (data not shown). 5. IgM natural antibodies and cryoglobulinemia Epidemiological and experimental evidences suggest a strong link between HCV infection, cryoglobulinemia and B-cell lymphoma. However the mechanisms that lead to clonal expansion of B-cells expressing autoantibodies endowed with rheumatoid factor activity, remain to be elucidated [16–19]. Several studies have described in the cryoprecipitate IgM antibodies that cross-react with different HCV antigens, including NS3, E2 and core proteins [[3,20,21], but this cannot exclude that anti-HCV antibodies can be bound by RF IgM and subsequently trapped in the cryoprecipitate together with antibodies directed against other antigens. Under this perspective, in the present study we describe the dissection of the IgM/K repertoire of a patient with HCV-associated MC, and the molecular cloning of antibody fragments able to recognize a pool of well characterized anti-HCV/E2 antibodies. This approach allowed the selection of IgM antibodies belonging to two subfamilies (VH3-23 and VH3-21) able to bind human IgG antibodies. More in details, the VH323 subfamily did not evidence polyreactivity features (as VH3-21), but showed a binding bias toward VH1-69derived IgG1 Fabs. Sequence analysis showed that all clones have a high homology to the germline, evidencing that they are antibodies naturally present in the human repertoire. These data suggest that VH3-23 IgM Fabs described here may be naturally prone to recognize some conserved regions of specific VH subfamilies, as
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
VH1-69, the VH gene described to be preferentially used in humoral response against HCV/E2. Overall, the results of the present study suggest a new model of an infection-driven lymphoproliferative disorder. In particular, the E2-driven stimulation of the immune system may cause the expansion of specific B cells expressing antibodies encoded by VH1-69, a VH gene subfamily recognized by some natural IgM antibody subfamilies. As a consequence, circulating immune complexes are promptly formed. Moreover the E2-driven stimulation and the cross-linking of B-cell receptor by autoantibodies may allow a chronic activation and a clonal expansion of anti-HCV/E2 B cells. Furthermore the intrinsic genetic instability of B-cells during somatic hypermutation and class-switching processes may favor genetic aberrations responsible for prolonged B cell survival. This process could lead the lymphoproliferation to become independent of antigenic stimulation, exposing the patients to the risk of developing a frank B cell malignancy.
[5]
[6]
[7]
[8]
[9]
[10]
Take-home messages • HCV infection is strongly associated with mixed cryoglobulinemia (MC), a benign lymphoproliferative disorder characterized by the expansion of B cell clones secreting antibodies encoded by restricted germline genes, as VH1-69, a VH family highly represented in anti-HCV humoral response. • Human immune repertoire shows natural antibodies belonging to distinct subfamilies, as VH3-23, that are prone to bind epitopes on restricted VH antibody subfamilies, such as VH1-69. • HCV can represent a triggering factor of MC, by an overexpression of VH1-69 antibodies that are bound by natural IgM antibodies.
[11]
[12]
[13]
[14]
References [15] [1] Monti G, Pioltelli P, Saccardo F, Campanini M, Candela M, Cavallero G, et al. Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias. Arch Intern Med 2005;165:101–5. [2] Agnello V. The aetiology of mixed cryoglobulinaemia associated with hepatitis C virus infection. Scand J Immunol 1995;42:179–84. [3] Ferri S, Dal Pero F, Bortoletto G, Bianchi FB, Lenzi M, Alberti A, et al. Detailed analysis of the E2–IgM complex in hepatitis C-related type II mixed cryoglobulinaemia. J Viral Hepatitis 2006;13: 166–76. [4] Saadoun D, Resche-Rigon M, Thibault V, Piette JC, Cacoub P. Antiviral therapy for hepatitis C virus-associated mixed cryoglo-
[16]
[17]
[18]
471
bulinemia vasculitis: a long-term follow up study. Arthritis Rheum 2006;54:3696–706. Landau DA, Saadoun D, Calabrese LH, Cacoub P. The pathophysiology of HCV induced B-cell clonal disorders. Autoimmun Rev 2007;6:581–7. Carbonari M, Caprini E, Tedesco T, Mazzetta F, Tocco V, Casato M, et al. Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis. J Immunol 2005;174:6532–9. Chan CH, Hadlock KG, Foung SK, Levy S. VH1-69 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;97:1023–6. De Re V, De Vita S, Marzotto A, Rupolo M, Gloghini A, Pivetta B, et al. Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 2000;96:3578–84. Bugli F, Mancini N, Kang CY, Di Campli C, Grieco A, Manzin A, et al. Mapping B cell epitopes of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from phage display libraries. J Virol 2001;75:9986–90. Burioni R, Bugli F, Mancini N, Rosa D, Di Campli C, Moroncini G, et al. Non-neutralizing human antibody fragments against hepatitis C virus E2 glycoprotein modulate neutralization of binding activity of human recombinant fabs. Virology 2001;288: 29–35. Burioni R, Mancini N, Carletti S, Perotti M, Grieco A, Canducci F, et al. Cross-reactive pseudovirus-neutralizing anti-envelope antibodies coexist with antibodies devoid of such activity in persistent hepatitis C virus infection. Virology 2004;327:242–8. Burioni R, Matsuura Y, Mancini N, Tani H, Miyamura T, Varaldo PE, et al. Diverging effects of human recombinant anti-hepatitis C virus (HCV) antibody fragments derived from a single patient on the infectivity of a vesicular stomatitis virus/HCV pseudotype. J Virol 2002;76:11775–9. Burioni R, Plaisant P, Manzin A, Rosa D, Delli Carri V, Bugli F, et al. Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant Fab fragments. Hepatology 1998;28: 810–4. Perotti M, Mancini N, Diotti RA, Tarr AW, Ball JK, Owsianka A, et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol 2008;82:1047–52. Barbas CF, Bjorling E, Chiodi F, Dunlop N, Cababa D, Jones TM, et al. Recombinant human Fabs neutralize human type 1 immunodeficiency virus in vitro. Proc Natl Acad Sci U S A 1992;89:9339–43. Ferri C, Antonelli A, Mascia MT, Sebastiani M, Fallahi P, Ferrari D, et al. HCV-related autoimmune and neoplastic disorders: the HCV syndrome. Dig Liver Dis 2007;39(Suppl 1):S13–21. Ferri C, Antonelli A, Mascia MT, Sebastiani M, Fallahi P, Ferrari D, et al. B-cells and mixed cryoglobulinemia. Autoimmun Rev 2007;7:114–20. Saadoun D, Landau DA, Calabrese LH, Cacoub PP. Hepatitis Cassociated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation. Rheumatology 2007;46: 1234–42.
472
M. Perotti et al. / Autoimmunity Reviews 7 (2008) 468–472
[19] Zignego AL, Giannini C, Ferri C. Hepatitis C virus-related lymphoproliferative disorders: an overview. World J Gastroenterol 2007;13:2467–78. [20] Simula MP, Caggiari L, Gloghini A, De Re V. HCV-related immunocytoma and type II mixed cryoglobulinemia-associated autoantigens. Ann N Y Acad Sci 2007;1110:121–30.
[21] De Re V, Sansonno D, Simula MP, Caggiari L, Gasparotto D, Fabris M, et al. HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations. Leukemia 2006;20:1145–54.
Update on Sjögren's syndrome autoimmune epithelitis: from classification to increased neoplasias Sjogren's syndrome is a chronic inflammatory process primarily affecting females during the fourth and fifth decades of life. Sjogren characterizes by exocrine glands inflammation causing kerato-conjuctivitis sicca and xerostomia and less commonly by extraglandular manifestations. The extraglandular manifestation of Sjogren syndrome can be divided to two subsets. One group constitute of lung, interstitial kidney disease and liver damage as a result of lymphocytic invasion of epithelial tissues causing "autoimmune epithelitis". The second group includes vasculitis, neuropathy and glomerulonephritis as a result of immune complex disease. Recently Tzioufas AG and Voulgaerlis M. (Best Pract Res Clin Rheumatol. 2007; 21:989-1010) reviewed the clinical picture, diagnostic procedures, treatment options, differential diagnosis and risk factors for malignancy transformation of Sjogren syndrome. Non-Hodkins lymphoma (NHL) occurs in approximately 5% of Sjogren patients. Two main categories of NHL are associated with Sjogren, in the majority of patients indolent extranodal (mucosal) marginal zone lymphomas is diagnosed. The second variant is high-grade aggressive lymphomas, such as de novo or secondary diffuse large B cell disease which is only occasionally encountered. The presence of palpable purpura, low C4 and mixed cryoglobulines constitute the main prediction factors for development of lymphoma and other comorbidities, thus Sjogern patients especially those who present with risk factors should be closely followed.