Immunological Investigations, 2015; 44(4): 400–410 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2015.1015682

IgG Subclasses and Isotypes of VH4-34 Encoded Antibodies Neelima M. Bhat, Mihir A. Kshirsagar, Marcia M. Bieber, and Nelson N. H. Teng Division of Gynecologic Oncology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California, USA VH4-34 gene encoded autoantibodies are elevated in systemic lupus erythematosus (SLE) and in other diseases associated with B-cell hyperproliferation/dysfunction. One of the autoantigens recognized by VH4-34-encoded antibodies are branched/linear poly N-acetyl lactosamine chains. Since the anti-carbohydrate response in humans is dominated by the IgG2 subclass, here we tested whether VH4-34 encoded IgG showed similar subclass segregation. Serum samples from SLE, infectious mononucleosis, nasopharyngeal carcinoma and hepatitis-C were analyzed. Levels of VH4-34-encoded IgM and IgA isotypes were also tested. VH4-34-IgM and IgA were elevated in all four clinical conditions. VH4-34-IgG was detected in the IgG1 and IgG3 subclass but not in the IgG2 and IgG4 subclass. Interestingly, VH4-34-IgG3 was also detected in serum samples of normal healthy adults. These observations are discussed in context of the VH4-34 gene regulation. VH4-34 repertoire development is of interest since it is the only human VH gene profoundly overrepresented in the naı¨ve repertoire but counterselected for antibody secretion. VH4-34 B-cell could thus become a unique tool to inspect germinal center independent/dependent pathways of subclass and isotype-specific antibody secretion. Keywords 9G4, autoantibodies, HCV, IM, SLE

INTRODUCTION VH4-34 B lymphocytes can comprise up to 10% of all mature B-cells in normal human adults despite their potential for producing autoantibodies. Although overrepresented in the naive immune repertoire at the B-cell receptor [BCR] stage, secreted VH4-34 encoded antibodies are low to undetectable in the sera of healthy adults (Kraj et al., 1995; van Vollenhoven et al., 1999). In fact, VH434 B lymphocytes are steered away from entering germinal centers in normal healthy adults and maintain a profile of anergic B-cells (Pugh-Bernard et al., 2001). In selective infections, such as, infectious mononucleosis (IM) VH4-34 encoded antibodies of the IgM isotype are readily detected in patient sera (Chapman et al., 1993; Roelcke, 1989). In diseases such as systemic lupus erythematosus (SLE), VH4-34 antibodies of the IgG isotype are detected in 450% of SLE patients (Isenberg et al., 1993; van Vollenhoven et al., 1999). The operative mechanism that prevents entry of VH4-34 B-lymphocytes into

Correspondence: Dr Neelima M. Bhat, HH333, Division of Gynecologic Oncology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305, USA. E-mail: [email protected]

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germinal centers in healthy adults is defective in SLE patients (Cappione et al., 2005) leading to the secretion of isotype-switched autoantibodies. Besides IM and SLE, other conditions associated with VH4-34 antibody secretion are AIDS, cold agglutinin disease, hepatitis C (HCV) and nasopharyngeal carcinoma (NPC) (Bhat et al., 2004; Gertz, 2005; Silberstein et al., 1991). The common link among these conditions is aberrant lymphoproliferation and association with lymphotropic viruses. VH4-34 encoded Abs from HIV patients neutralizes viral infection in vitro (Alcena et al., 2013), suggesting a benefit in maintaining this unique autoreactivity. Assuming an advantage in maintaining VH4-34 self-reacting lymphocytes in the preimmune naı¨ve repertoire at the BCR stage, its regulation thus represents a puzzle in the interplay between protective immune response and autoimmunity. Hence secretion of VH4-34 encoded antibodies has been monitored to study the interplay between beneficial anti-pathogen response and breakdown in tolerance leading to autoimmunity, particularly in SLE (Milner et al., 2005). One of the autoantigens recognized by VH4-34 encoded antibodies are the i/I antigens, linear/branched poly N-acetyl lactosamine determinants, present on cord and adult RBCs, respectively, and an i-like antigen on human B-lymphocytes (Bhat et al., 1993; Grillot-Courvalin et al., 1992; Pascual et al., 1992). Despite the abundance of i-antigen on fetal RBC, the VH4-34 BCR bearing B-cells are positively selected in human fetal immune repertoire but strictly regulated such that secreted immunoglobulin is detected only in selective clinical conditions. The anti-i/anti-B-cell reactive VH4-34 antibodies range from high to low binders, with the high binders demonstrating B-cell cytotoxicity via membrane pertubations. The high B-cell binders identified to date are IgM in isotype. Capitalizing on its B-cell toxicity, a VH4-34-IgM has recently shown a favorable early response in a phase 1 trial for treatment of acute lymphoblastic leukemia (Liedtke et al., 2012). The IgG subclass response is known to correlate with the biochemical nature of antigens. IgG1 and IgG3 subclass are dominant against T-cell dependent protein antigens, such as tetanus toxoid (Hamilton, 1987; Hammarstrom & Smith, 1986; Nahm et al., 1987). On the other hand, T-cell independent antigens such as several encapsulated bacteria generate primarily an IgG2 anticarbohydrate response (Booth et al., 2006; Drake-Lee et al., 2003; Rispens et al., 2013; Umetsu et al., 1985). IgG antibodies to trisaccharide (a-GalNAC-(13)b-gal(1-2)a-Fuc) blood group A/B antigens on glycoproteins are also predominantly IgG2 (Kumpel et al., 1996). Since one of the natural ligands of VH4-34 encoded antibodies are carbohydrate moieties on B lymphocytes and RBC, we tested whether there was a selective bias of an IgG subclass in clinical disease, where VH4-34 antibodies are detected in patient serum.

MATERIALS AND METHODS Specimens Sera from 20 HCV, 14 NPC, 14 IM and 35 SLE patients were analyzed for VH434 IgG subclass and immunoglobulin isotype. Serum samples were selected from previous studies where total VH4-34 encoded antibody was measured by an inhibition ELISA. Patients positive for total VH4-34 antibody (4than 25% inhibition value) were selected for this study (Bhat et al., 2004; van

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Vollenhoven et al., 1999). Sera were stored frozen at 70  C as previously described. Blood specimens were obtained with the approval of the Committee for the Protection of Human Subjects at Stanford University. A total of 33 female and 33 male normal adult serum samples were analyzed as controls (Innovative Research, Novi, MI, Bioreclamation, Hicksville, NY and laboratory personnel). ELISA for VH4-34 Encoded IgM, IgA and IgG Subclass The anti-idiotypic antibody, 9G4, a rat IgG that specifically recognizes human VH4-34 encoded antibodies has been described in detail previously (Potter et al., 1994). Nunc ELISA plates were coated with purified 9G4 overnight at 10 mg/ml. The plates were blocked with BSA/milk and washed with PBS/Tween. Sera were serially diluted starting at 1:1000 for IgG1 and 1:200 for IgM, IgA, IgG3 and IgG4. Binding was detected with secondary antibodies conjugated with horseradish peroxidase. The substrate for color development was Azinobis-3ethylbenzthiazoline-sulfonic-acid, diammonium-salt (ABTS). Anti-IgG1-HRP (clone-4E3), anti-IgG3-HRP (clone-HP6050) and anti-IgG4-HRP (clone-6025) were from Southern Biotechnology, Birmingham, AL, anti-IgA-HRP from Invivogen, San Diego, CA, and anti-IgM-HRP was obtained from Invitrogen, Carlsbad, CA. VH4-34 derived IgG3 mAb-C7 (Bron et al., 1984) and VH4-34derived IgM mAb-216 (Bhat et al., 2002) were included as standards on plates detected with anti-human IgG3 and anti-human IgM, respectively. Since no VH4-34 controls were available for IgA, IgG1, and IgG4, the assay was standardized between plates with two control normal adult sera. OD reading for each patient sample was chosen according to titration of the two standard sera run on each plate. The two control standard sera were also analyzed on IgG3 and IgM plates. Levels greater than the normal mean + 2SD were considered elevated for each isotype and subclass. This assay gives a relative value of VH434 isotype in each serum specimen. High levels of VH4-34-IgG in serum can compete with elevated VH4-34-IgM and vice versa, so the amount of detection each isotype or subclass remains an approximation, proportional to their respective levels. Immunoglobulin standards human IgG1, IgG2, IgG3, and IgG4 were obtained from Athens Research and Technology, Athens, GA. These antibodies were used to confirm the absence of cross-reaction of HRP-labeled four secondary antibodies among IgG subclasses. To avoid false positives, wherein rheumatoid factor would bind rat IgG-9G4 non-specifically, all sera were assayed for reactivity to rat IgG (Sigma-Aldrich, St. Louis, MO) and rat IgG2a (EBiosciences, San Deigo, CA). Rheumatoid factor was not detected in normal controls or in sera from patients. Detection of VH4-34-derived IgG2 A standard sandwich ELISA described above could not be used, since mAb9G4, a rat IgG2, cross-reacted with mouse-derived anti-human IgG2 antibodies. No rat-absorbed anti-human IgG2 was commercially available. To circumvent this issue, we purified IgG2 antibodies from serum samples. Briefly, biotin-conjugated anti-human IgG2 antibody (Clone G18-21, #555874, Becton-Dickinson, San Jose, CA) and avidin-agarose beads (Pierce, Rockford, IL) were rocked overnight, washed 3 with PBS and then incubated with sera

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diluted 1:150 in PBS. Human IgG2 antibodies were eluted with 35 ml of standard low pH elution buffer and the eluant diluted to a final volume of 250 ml with PBS to pH 7.0. The amount of IgG2 eluted for each serum sample was analyzed by standard sandwich ELISA. Average IgG2 elution was 4 mgs/ ml, with some samples as high as 10 mgs/ml. Presence of VH4-34 fraction in the purified IgG2 eluant was tested by an inhibition assay with minor modifications (van Vollenhoven et al., 1999). Briefly, plates were coated with purified VH4-34-IgM at 5 mgs/ml, 50 ml per well. IgG2 purified samples (200 ml) were incubated with 9G4 (50 ml of 1.8 mg/ml) for 20 min at RT before transferring 50 ml to VH4-34-IgM coated 96-well plates. The amount of 9G4 bound to the coated VH4-34-IgM was detected using peroxidase-labeled anti-rat IgG (Caltag (Invitrogen), South San Francisco, CA). The VH4-34 encoded antibodies in IgG2 fraction will compete with coated VH4-34-IgM for 9G4 binding, leading to a range in color development depending upon the amount of VH4-34 present in the IgG2 fraction. Control VH4-34-IgG3 and IgM were run as positive controls for inhibition. C7, a monoclonal VH4-34-IgG3, gave a range of EC50 inhibition at 0.6 mgs/ml (mean from five independent titrations). The assay thus has the sensitivity to detect as low as 400 ngs/ml of VH4-34-encoded IgG. Flow Cytometry Human buffy coat samples from healthy donors (n = 4) were obtained from Stanford Blood Bank with the approval of the Committee for the Protection of Human Subjects at Stanford University. Whole blood was stained at room temperature with antibodies, cells were fixed and red cells lysed using CalLyse (GAS010, CalTag, Burlingame, CA) and analyzed on LSR (Becton-Dickinson [BD], San Jose, CA) with data management using Cell Quest (BD) or FloJo (Treestar) software. The reagents were APC-conjugated CD19 (BD Pharmingen, San Diego, CA, #555415), PE-conjugated anti-human IgG BD 34165X, PE-conjugated anti-human G1, G2, G3 and G4 (Southern Biotech, Birmingham, AL, #9052-09, #9080-09, #9210-09, #9200-09, respectively) PEconjugated anti-human IgA (Mitenyi Biotech, San Diego, CA, #130-093-128), PE-conjugated anti-human IgD (BD Pharmingen, #562024), V421-conjugated anti-human IgM (BD Pharmingen, #562618), PE-conjugated anti-human IgE (Biolegend, San Diego, CA, #325506), Cy7-APC conjugated anti-human CD3 (Biolegend, San Diego, CA, #300425). Purified 9G4, the VH4-34 anti-idiotypic antibody, was conjugated to Alexa-fluor-488 (Life Technologies, Carlsbad, CA, #A20181) using standard procedures. Statistical Analysis Significance was measured using unpaired non-parametric Mann–Whitney test performed with Prism Software version 6.0 d (Graph Pad Software, Inc., San Deigo, CA). p values of 50.05 were considered significant.

RESULTS AND DISCUSSION VH4-34 IgG subclasses and IgM and IgA levels were assayed in 14 NPC, 14 IM, 20 HCV, 35 SLE patients and 66 healthy adults (Figure 1). VH4-34 IgM and IgA was elevated in about 50% patients in all four diseases (Figure 2).

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Figure 1. Relative levels of VH4-34 encoded IgM and IgA isotype and IgG subclasses in clinical diseases associated with VH4-34 secretion. The dotted line represents the normal adult mean + 2SD calculated from the ELISA values of 66 normal adults (33 females and 33 males). Patients were considered positive for secreted antibody above this line. Satistical significance of the medians compared by Mann–Whitney test were as follows: p50.0001 for IgM and IgA isotypes in all four clinical conditions compared to normal healthy subjects; p50.02 for IgG1 in SLE compared to normal controls. The IgG1 was not significantly different in other conditions compared to healthy controls. Isotype IgG2 and IgG4 were not significantly different in any condition compared to control.

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Figure 2. Percentage of patients positive for VH4-34 encoded IgM and IgA and IgG subclasses. Since IgG3 was detected in normal adult sera, elevation of Ig secretion in disease state could not be applied for IgG3 secretion in comparison to normal. NA: non-applicable.

Although the physiological antigen recognized by VH4-34 antibodies is a RBCassociated carbohydrate ligand, it is interesting that IgG2 the second largest IgG subclass known to encode anti-carbohydrate response against blood group antigens was not represented. Instead, the VH4-34 response is skewed toward a classical anti-protein IgG1/IgG3 subclass. In particular, VH4-34 IgG1 levels were high in HCV and SLE, diseases associated with immune dysfunction, but not in an acute infection as IM, where the VH4-34 response is predominantly IgM. Secretion of isotype switched VH4-34 Abs in SLE is explained by their evasion of the censoring mechanism operative in healthy follicles (Cappione et al., 2005). Our study shows that policing of VH4-34 B-cells away from GC-reactions is also defective in HCV. In IM, a widespread disease caused by EBV, the secreted VH4-34 response is transitory, disappearing with the resolution of the disease and limited to the multivalent IgM and IgA class. No manifestation of long-term clinical autoimmunity is linked with brief secretion of high-avidity VH4-34 antibodies approaching 400 mgs/ml. In majority of IM, patient’s entry into the long-lived IgG1 compartment is avoided. In context of our observation that germline encoded VH4-34 IgMs are the best B-cell binders with linked cytotoxicity; it is possible that high-avidity VH4-34 IgM and IgA antibodies may be induced to counter the B-cell lymphoproliferation associated with the disease (Bhat et al., 1997). In fact, binding/painting of B-lymphocytes by VH4-34 encoded Igs is observed in vivo in IM, SLE and HIV patients (Bhat et al., 2002; Kobie et al., 2012). Alternatively, it could be a disease-specific anti-viral response. However, VH4-34 encoded fraction in IM patients does not bind EBV antigens (VCA, EAD, and EBNA-1) routinely used to detect infection (Bhat et al., 2004). The proteins used in these anti-EBV kits may not possess the appropriate glycosylation necessary for VH4-34 ligand recognition. Thus, it remains to be seen whether the VH4-34 response in IM is EBV-specific or immunoregulatory. A disease-specific response is seen in HIV infection, where neutralizing Abs are identified within the VH4-34 antibody fraction (Alcena et al., 2013; Kobie et al., 2012).

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Many polysaccharide epitopes induce a T-independent immune response characterized primarily by the presence of germ line-encoded IgM, lack of isotype switching and no affinity maturation. Regarding IgA, this isotype is the first line of immune protection at mucosal surfaces and similar to IgM, its class switch is also known to occur in a T-independent manner (Bergqvist et al., 2010; Cerutti, 2008; Mantis & Forbes, 2010). Barring the rare case of myeloma (Froyland et al., 2007), this is the first report of infection induced secretion of VH4-34 IgA. The detection of only VH4-34-IgG3 in normal healthy sera was unexpected particularly since very low total VH4-34 antibodies are detected in healthy adults. VH4-34-IgG3 in healthy adults may have previously escaped detection, since IgG3 represents 510% of total IgG and/or polyclonal antibodies used to detect total IgG may not have bound this minor subclass. Since VH4-34 B-cells are barred from entering GC-follicles in normal healthy adults (Pugh-Bernard et al., 2001), it is possible that VH4-34 IgG3 is secreted in a GC-independent manner. Such pathways have been suggested for antibody secretion and also memory B-cell formation (Berkowska et al., 2011; Takemori et al., 2014), akin to rodent B1 and marginal zone cells that provide recall responses against encapsulated bacteria (Sanz et al., 2008). VH4-34 IgG3 BCR bearing B-cells were detected by anti-idiotypic mAb 9G4 in peripheral blood of healthy adults (n = 4, Figure 3B). IgG3 subclass predominant GC-independent CD27-IgG+ B lymphocytes have been previously identified in human peripheral blood with phenotypic similarity to hairy cell leukemia (Fecteau et al., 2006); a malignancy with biased VH4-34 BCR expression (Arons et al., 2009). It remains to be seen if the VH4-34 IgG3 BCR bearing cells are a vestige of a previous infection thus representing a diseasespecific response. A detailed analysis, such as CD27, CD38, CD138 expression, of this small percentage of cells will help to elucidate the differentiation pathway and regulation of these class switched VH4-34 BCR bearing B-cells in healthy adults. The majority of the VH4-34 BCR cells were in the IgMlowIgDhigh fraction (Figure 3A). The expression of IgM on these cells may be considered to range from low to negligible and are reminiscent of the autoreactive anergic IgDhigh B(ND) cells that bind HepG2 and DNA (Andrews & Wilson, 2010; Duty et al., 2009). Other laboratories have also shown that IgMlow/IgDhigh/9G4+ve cells are anergic and give weak response to BCR stimulation (Cappione et al., 2005; Sabouri et al., 2014; Zheng et al., 2004). One theory suggests that their selection into IgDhigh compartment limits their availability for Ig secretion preventing the autoimmunity associated with the secreted protein (Koelsch et al., 2007). Since secreted VH4-34 IgM is not detected in healthy adult serum, it is possible that IgDhigh9G4+ B-cells are counter-selected from GC-independent IgM secretion and post IM-infection are found in this anergic sink for autoreactive B-cells. The antigenic specificities of VH4-34 encoded Abs can be broadly classified into three categories, auto-cell binding (adult RBC I-antigen, fetal RBC i-antigen, B-lymphocytes), autoantigens (DNA, cardiolipin, apoptotic ligands) and exoantigens (LPS, lipid A, allogeneic RhD). Interestingly, VH4-34 Abs from HIV-infected patients with the ability to neutralize HIV retained anti-i/ anti-B-cell reactivity but did not display autoantigen reactivity

Figure 3. Phenotype of VH4-34 BCR B-lymphocytes in human peripheral blood. (A) B-lymphocytes within the mononuclear population were gated using CD19 and distribution of 9G4+ B-cells with respect to IgM and IgD expression is shown. 495% of VH4-34 BCR bearing cells are in the IgMloIgDhi fraction of CD19+ B-cells. The cells that do not fall within the defined gates (the IgMmediumIgDmedium population) were negative for 9G4 B-cells. (B) 9G4+ B-cells were identified within total B-lymphocytes (CD19+) and expression of immunoglobulin isotype and subclass on CD19+9G4+ BCR bearing B-cells shown. An isotype control for 9G4, FITC-conjugated rat IgG2a, did not separate CD19+ cells in two populations (data not shown). Of the total B-cells, 2.7% cells were 9G4+; of which 95% were in the IgMlo/intIgDhi subgroup and 3.2% in the IgG3 subclass. Similar expression profile seen in three other independently derived peripeheral blood samples, where 493% of B-cells were in the IgMlo/intIgDhi subgroup and 2–4% in the IgG3 subclass.

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(DNA, cardiolipin, apoptotic ligands) commonly seen in SLE patients (Alcena et al., 2013). This dichotomy of autoantigen recognition by VH4-34 encoded Abs may be reflective of the tolerance mechanism operative in the two clinical conditions. In IM and HIV infection, secretion of VH4-34 Ab may constitute a pathogen-induced protective immune response and not simply a breakdown in tolerance as in SLE. Thus, it appears that with progression of immune dysfunction, the isotype of VH4-34 Abs changes from IgM/IgA to IgG1 with a concomitant change in ligand binding from anti-i/anti-B specificity to other autoantigens. It remains to be seen whether there is an advantage in maintaining this unusual autospecificity in the human pre-immune repertoire at the BCRstage. It is possible that the isotype/subclass of VH4-34 encoded antibodies may lead to clues towards its regulation. It is intriguing that VH4-34 IgG3 is detected in normal healthy controls despite their exclusion from germinal centers. In conclusion, the VH4-34 gene may provide a unique handle to study Fc-region selection and GC-dependent and -independent pathways of antibody secretion in normal and dysfunctional immune responses.

DECLARATION OF INTEREST This work was supported by Malloy Research Gift Fund, Division of Gynecologic Oncology, Department of OB/GYN, Stanford University Medical Center. The authors declare no competing financial interests.

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IgG Subclasses and Isotypes of VH4-34 Encoded Antibodies.

VH4-34 gene encoded autoantibodies are elevated in systemic lupus erythematosus (SLE) and in other diseases associated with B-cell hyperproliferation/...
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