Abstract
Idiotypes (Ids) are unique epitopes of Ab V regions and can trigger anti-Id immune responses, but immunization with several nonadjuvanted isologous IgG mAbs has induced tolerance to their Ids. We immunized non–lupus-prone mice with 11 allotype “a” of IgG2a (IgG2aa) and 4 IgG2c nonadjuvanted, isologous mAbs purified from serum-free medium. Of five IgG2aa mAbs with specificity for nucleosomes, the repeating histone-DNA subunit of chromatin, four elicited an IgG1 anti-mAb response and one mAb was nonimmunogenic. In contrast, none of six IgG2aa mAbs with unknown specificity triggered anti-mAb responses. The data suggested a link between immunogenicity and specificity for nucleosomes. One anti-nucleosome IgG2aa mAb, termed 3F7.A10, copurified with self-histones and was a potent immunogen for BALB/c mice. The response against IgG2aa 3F7.A10 was CD4+ Th cell–dependent, dominated by the IgG1 subclass, and Id specific. Ultracentrifugation converted the purified 3F7.A10 mAb into a weak immunogen, suggesting that the mAb had formed immunogenicity-enhancing immune complexes (ICs) with nucleosomal Ags during cell culture. BALB/c mice injected with viable MHC-incompatible 3F7.A10 hybridoma cells grown in serum-free medium mounted strong anti-Id responses. TLR9-deficient mice responded significantly weaker to Id-3F7.A10 than did TLR9-sufficient mice, suggesting that the cognate BCR efficiently internalizes the Id in an IC with nucleosomes. Passive transfer of IgG2aa 3F7.A10 to BALB/c mice with high titers of IgG1 anti-3F7.A10 led to glomerular deposits of IgG1/IgG2a complexes. The immunogenicity of Id-3F7.A10 raises the possibility that diverse Ids of nucleosome-specific Abs form ICs with nucleosomes released from dying cells and elicit spontaneous formation of anti-Id Abs in vivo.
Introduction
Idiotypes (Ids) are unique epitopes of Ab V regions (1–3). Ids of isologous (nonallogeneic) mAbs can be immunogenic and serve as tumor-specific Ag, suggesting a therapeutic benefit of anti-Id responses (4–6). Therefore, various ways of artificially enhancing the immunogenicity of myeloma mAbs were explored such as 1) emulsification in CFA (4, 5, 7), 2) CFA combined with polymerization of mAb alone or together with a non-self carrier protein (8, 9), and 3) incorporating a hapten-conjugated mAb in immune complexes (ICs) with anti-hapten Abs (10). By these measures, most mAbs elicited Id-specific Ab responses. However, the phosphorylcholine-specific myeloma IgA mAb T15 was an exception, and because the Id of T15 is abundant in BALB/c mice, the minimal responsiveness to T15 was attributed to tolerance (7).
Subsequent studies revealed that the Th response of BALB/c mice to the λ2 L chain of myeloma IgA mAb M315 was restricted by MHC-linked genes that mapped to I-Ed (11, 12), and the Th targeted a neoepitope derived from somatic mutations expressed in CDR3 of the L chain (13–15). Work with Id-specific T cell hybridomas derived from mice immunized with two CFA-adjuvanted isologous IgG1, κ mAbs showed that all T hybridomas reacted selectively with mutated Vκ-chain peptides presented by MHC class II (MHCII) and none recognized unmutated versions of the same L chains (16). Mice immunized with a CFA-adjuvanted unmutated version of the same mAb were Th nonresponders (17). With regard to an isologous anti-chromatin mAb derived from a lupus-prone strain, none of the T hybridomas responded to germline-encoded sequences; however, a few hybridomas recognized a peptide from the somatically generated and highly variant CDRH3 (18). Collectively, the observations suggested that CD4+ T cells are tolerant to germline gene-encoded Ids.
These findings raised the question: how immunogenic are native mAbs in the absence of adjuvant? This issue is relevant for insight into the potential role of Ids as endogenous self-antigens. In 1970, Iverson and Dresser (19) showed that infusion of a nonadjuvanted isologous IgG2a myeloma mAb prevented anti-Id responses to subsequent immunization with an adjuvanted form of the mAb, indicating that it was tolerogenic. Taking advantage of a hybridoma clone secreting immunogenic IgM, λ2 (called IgMId3) specific for the hapten trinitrophenyl (TNP) (20), we cloned three isotype switch variants producing Id3 associated with IgG1, IgG2a, and IgG2b (21). Unlike their immunogenic IgMId3 ancestor, the Id3-bearing IgG variants failed to elicit anti-Id3 responses. Furthermore, when immunization with nonadjuvanted IgMId3 mAb was preceded by infusions of the nonimmunogenic variants, the anti-Id responses were shut down by IgG2a and significantly diminished by IgG1 and IgG2b, indicating that the variants were tolerogenic (21). However, Id3 acquired immunogenicity when the switch variants were emulsified in CFA or purified from medium containing FBS (21). Thus, under physiologic circumstances, Id3 was immunogenic when borne by IgM and tolerogenic when associated with IgG.
This prompted us to examine the immunogenicity of the IgM repertoire, and we generated 73 BALB/c-derived IgM mAbs against the non-self hapten TNP, all of which were purified from serum-free medium (22). Of these, four IgM, λ2 mAbs (5.5%) elicited strong IgG1 anti-Id responses controlled by MHC-linked genes. Three of these were germline gene encoded, and one had a single somatic mutation in V region of Ig H chain (VH) codon 50. These observations suggested that, although rare, natural proteins of the primary IgM repertoire can be immunogenic without an adjuvant.
We here extend our observations to allotype “a” of IgG2a (IgG2aa) and IgG2c auto-mAbs specific for nucleosomes, the repeating histone-DNA subunit of chromatin. The signals from surface BCRs and endosomal TLR9 play a critical role for the predominance of nucleosome/DNA-reactive autoantibodies in murine models of lupus (23–26). We reasoned that, unlike the IgG anti-TNP mAbs, IgG anti-nucleosome auto-mAbs can become multivalent by forming ICs with self-nucleosomes released in vivo and thereby acquire immunogenicity by crosslinking anti-Id BCRs of B cells and low-affinity FcγRs of APCs far more extensively compared with monomeric IgG. This might be followed by transport of the ICs to an endosomal compartment and activation of TLR9 by nucleic acids from DNA of nucleosomes, culminating in an Ab response against the Id of the anti-nucleosome Ab and formation of Id/anti-Id ICs.
We initially tested the idea by measuring the IgG1 responses to a panel of 15 isologous nonadjuvanted IgG2a and IgG2c mAbs purified from serum-free medium. Of these mAbs, eight were nucleosome specific and stained HEp-2 cell nuclei homogeneously, six had unknown specificity, and one reacted with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). During this work, we identified an IgG2a anti-nucleosome mAb that was a strong immunogen, and further studies were centered on this mAb.
Materials and Methods
Mice
BALB/c, C57BL/6 (B6), (BALB/c × C57BL/6)F1 (CB6F1), and C.B-17 mice (IgCHb congenics on the BALB/c genetic background) were purchased from Taconic Biosciences. C.B10-H2b mice (MHC H2b congenic BALB/c) were purchased from The Jackson Laboratory and Harlan Europe. BALB/c mice were also bred at our facilities. TLR9-deficient (TLR9−/−) mice, originally generated by Akira and colleagues (27), were provided on a BALB/c genetic background by Dr. A. Marshak-Rothstein and Dr. E. Lien (University of Massachusetts Medical School) and bred at our facilities. Both male and female TLR-deficient mice were used in experiments. The mice were housed in a minimal disease unit at the Department of Comparative Medicine of our institution. Animal care was in accordance with good animal practice as defined by national legislation and institutional guidelines, and all animal work was approved by the Oslo University Hospital Animal Care and Use Committee.
Derivation of hybridoma mAbs of this study
An overview of the 15 hybridoma clones is shown in Table I; all except clone 15 were obtained from mice that had not been intentionally immunized. Clones 1–8 derive from a 10-mo-old female C.B10-H2b mouse; they were first selected for IgG2a production and then screened by ELISA for anti-nucleosome activity. Clones 9–11 derive from a 15=mo-old female C.B10-H2b mouse and were screened for IgG2a anti-polynucleosome activity. Clones 12–14 producing IgG2a of allotype “b” (also known as IgG2c) derive from (NZB × BXSB)F1 lupus mice and are directed at nucleosomes (28). Clone 15 (IgG2c, λ1 S43.10) derives from a B6 mouse immunized with NP-streptococci (29); it is a subclone of S43 that has lost the Ig loci of the fusion partner. Clones 1–6 have unknown specificity. Clones 1–11 were generated by fusing splenocytes from naive C.B10-H2b mice with the BALB/c-derived myeloma cell fusion partner Ouri (a minor variant of X63-Ag8.653) followed by the dropwise addition of polyethylene glycol (Roche). Hybridomas were selected with hypoxanthine/aminopterin/thymidine-supplemented RPMI 1640 (Sigma-Aldrich) and cloned by limiting dilution.
Clone . | Clone Name . | IgCH/L . | Nucleosome ELISAa . | HEp-2 Stainingb . |
---|---|---|---|---|
1 | 1D9.E10.F2 | IgG2a, κ | 0.06 | − |
2 | 8G6.H6.B5 | IgG2a, κ | 0.06 | − |
3 | 9E6.H7.A3 | IgG2a, κ | 0.06 | − |
4 | 10H12.G3.H7 | IgG2a, κ | 0.06 | − |
5 | 16E7.H8.G1 | IgG2a, κ | 0.12 | − |
6 | 16F7.E10.E3 | IgG2a, κ | 0.06 | − |
7 | 4E9.E5.E8 | IgG2a, λ2 | 0.36 | + |
8 | 16D8.C10.A7 | IgG2a, κ | 1.37 | 3+ |
9 | 3F7.A10 | IgG2a, κ | 1.40 | 3+ |
10 | 4E3.G1 | IgG2a, κ | 1.34 | 3+ |
11 | 5A12.A3 | IgG2a, κ | 1.36 | 3+ |
12 | XE3 | IgG2c, κ | 1.30 | 3+ |
13 | XG1 | IgG2c, κ | 1.23 | 3+ |
14 | XG3 | IgG2c, κ | 1.33 | 3+ |
15 | S43.10 | IgG2c, λ1 | 0.12 | − |
Clone . | Clone Name . | IgCH/L . | Nucleosome ELISAa . | HEp-2 Stainingb . |
---|---|---|---|---|
1 | 1D9.E10.F2 | IgG2a, κ | 0.06 | − |
2 | 8G6.H6.B5 | IgG2a, κ | 0.06 | − |
3 | 9E6.H7.A3 | IgG2a, κ | 0.06 | − |
4 | 10H12.G3.H7 | IgG2a, κ | 0.06 | − |
5 | 16E7.H8.G1 | IgG2a, κ | 0.12 | − |
6 | 16F7.E10.E3 | IgG2a, κ | 0.06 | − |
7 | 4E9.E5.E8 | IgG2a, λ2 | 0.36 | + |
8 | 16D8.C10.A7 | IgG2a, κ | 1.37 | 3+ |
9 | 3F7.A10 | IgG2a, κ | 1.40 | 3+ |
10 | 4E3.G1 | IgG2a, κ | 1.34 | 3+ |
11 | 5A12.A3 | IgG2a, κ | 1.36 | 3+ |
12 | XE3 | IgG2c, κ | 1.30 | 3+ |
13 | XG1 | IgG2c, κ | 1.23 | 3+ |
14 | XG3 | IgG2c, κ | 1.33 | 3+ |
15 | S43.10 | IgG2c, λ1 | 0.12 | − |
OD405nm. The mAbs were tested at 10 μg/ml.
The mAbs were tested at 10 μg/ml; the staining was homogeneous nuclear.
Growth conditions of hybridomas
Following cloning, hybridoma cells were adapted to grow at 5% CO2 in DMEM/F-12 (1:1) plus GlutaMAX (Life Technologies/Life Sciences) supplemented with a synthetic serum replacement that was protein-free except for 0.5 μg/ml final concentration of recombinant human insulin (30). In this study, this medium is referred to as serum-free. The serum replacement, known as RenCyte, was previously marketed by Medi-Cult (now Origio) and was a gift of its inventor, the late Dr. K. Bertheussen. Antibiotics or FBS were not added to the medium.
Purification of mAbs
The serum-free hybridoma cell culture was cleared by centrifugation (5000 rpm, 1 h) and the supernatant was passed through protein A–Sepharose 4 (GE Healthcare) that was washed successively with PBS, 3 M NaCl in 0.01 M PO4 (pH 7) and PBS. The mAbs were eluted with 0.1 M CH3COOH, dialyzed overnight against PBS, passed through sterile 0.2-μm filters, and stored sterile at 4°C. The IgG mAb concentration was measured at 280 nm with a NanoDrop 1000 spectrophotometer (Thermo Scientific). The OD260nm/OD280nm ratio of the purified mAbs generally ranged from 0.53 to 0.59. This ratio is 0.57 and 1.06 for a protein containing 0 and 5% nucleic acid, respectively (31). Thus, the nucleic acid content of the purified mAbs was below the limit of detection by this assay. One preparation of mAb IgG2aa 3F7.A10 was also examined for DNA level using Quant-iT PicoGreen dsDNA reagent and kits (Invitrogen) and found to contain <1 pg of DNA/56,000 pg of mAb. This preparation was immunogenic for BALB/c mice. Endotoxin levels in the mAb preparations were <0.078 European units/100 μg of mAb, as measured with the Limulus amebocyte lysate chromogenic method (Associates of Cape Cod). The purity of mAbs was monitored by reducing SDS-PAGE.
Immunizations
The mice were immunized i.p. with 20 μg of sterile, purified mAb in 100 μl of PBS with no added adjuvant. Blood was collected from a leg vein.
Other Abs
Mouse allotype “e” of IgG2a (IgG2ae) anti–MHC class I-Ad (clone MK-D6 ATCC HB-3), mouse IgG2c anti-mouse IgG2aa (clone 8.3 ATCC TIB-148), and rat IgG2b anti-CD4 (clone GK1-5 ATCC TIB207) were obtained from American Type Culture Collection and adapted to grow in serum-free medium. Carboxyfluorescein-N-hydroxysuccinimide ester–conjugated mAb 8.3 was prepared as per the protocol of Roche Applied Science. Rat IgG2a anti-mouse IgE (clone EM95) was a gift of Z. Eshhar. Mouse IgG1 anti-mouse IgG2c (clone G12-47/30) was obtained from K. Rajewsky. Biotinylated polyclonal goat Abs against mouse IgG1, IgG2a, IgG2b, IgG2c, and IgE, as well as Cy3-conjugated goat anti-mouse IgG1, were purchased from SouthernBiotech. Purification of rat IgG mAbs was carried out with protein G (Amersham Biosciences) affinity columns.
Chicken mononucleosome and polynucleosome preparations
The procedure of Ausio et al. (32) was followed for preparing soluble nucleosomes from chicken red cell nuclei. Mononucleosomes were generated as described previously using 100 U of micrococcal (staphylococcal) nuclease (Worthington)/100 OD260nm U of DNA for 15 min at 37°C (33). Mononucleosomes were stored at −70°C. Polynucleosomes were prepared using 10 U of the nuclease/100 OD260nm U of DNA for 5 min at 37°C. The digestion was terminated by adding EDTA to a final concentration 10 mM, and the suspension was chilled on ice and centrifuged at 12,000 rpm for 5 min at 4°C. The pellet was resuspended in 0.25 mM EDTA, stirred gently for 1 h at 4°C, and centrifuged at 10,000 rpm for 20 min. The supernatant was collected, NaN3 was added to a final concentration of 0.02%, and the polynucleosomes were stored at 4°C without further purification. By agarose electrophoresis of nucleosomes digested with proteinase K (1 mg/ml, 56°C, 1 h), the DNA of mononucleosomes formed a broad band averaging ∼160 bp, and polynucleosome DNA ranged in size from ∼200 bp to >4 kb, as shown in Fig. 5Aa and 5Bb, respectively. The nucleosome concentration was expressed as amount of DNA per milliliter.
ELISA for polyclonal IgG1 responses against mAbs
MaxiSorp Nunc immunoplates were coated overnight in a refrigerator with purified IgG2a or IgG2c mAb (5 μg/ml) in 0.05 M carbonate buffer (pH 9.6). The wells were washed twice with PBS/0.05% Tween 20, blocked with 1% BSA in PBS for 1 h at 37°C, and washed twice. Sera, usually diluted 1:100 in PBS containing 0.5% BSA and 0.02% Tween 20 (referred to as diluent), were added in duplicate to the wells, incubated for 1 h, and wells were washed three times. Bound IgG1 Abs were detected by incubation for 1 h with biotinylated goat anti-IgG1 diluted 1:4000, and the wells were washed three times and incubated for 30 min with AP-conjugated streptavidin (catalog no. RPN1234V1; Amersham) diluted 1:4000. After washing three times, the reactions were developed with p-nitrophenyl phosphate (p-NPP) substrate (Sigma-Aldrich) in diethanolamine/MgCl2 for 30 min, and OD405nm was measured in a Victor3 multilabel plate reader (PerkinElmer).
Cloning of an IgE-producing class switch variant of hybridoma 3F7.A10
A double Ab sandwich ELISA was used to identify wells containing spontaneously class-switched hybridoma cells (34). In brief, 1000 hybridoma cells per well were seeded in eleven 96-well tissue culture plates (Costar 3595) and allowed to grow until the cells covered about half of the well bottom. Culture medium from the wells was transferred to microtiter plates precoated with EM95 anti-mouse IgE capture mAb and incubated for 1 h. After washing, the wells were incubated sequentially with biotinylated goat anti-mouse IgE detection Ab, AP-conjugated streptavidin, and p-NPP. Cells from the well with the highest OD405nm were collected and cloned by limiting dilution using the same double Ab sandwich assay to identify IgE-containing wells. The clone produced anti-polynucleosome IgE but no detectable anti-polynucleosome IgG2a as measured by ELISA. The switched clone was adapted to grow in serum-free medium, and the cleared culture medium was passed through an affinity column of EM95 anti-IgE mAb coupled to Sepharose 4B. Bound IgE was eluted with 4 M urea in 0.1 M CH3COOH, dialyzed against PBS, sterile filtered, and stored at 4°C.
ELISA for polyclonal IgG1, IgG2a, and IgG2b activity against IgE 3F7.A10
To wells coated with the IgE class switch variant of 3F7.A10 (5 μg/ml) in 0.05 M carbonate buffer (pH 9.6) were added 1:100 dilutions of sera from BALB/c mice (n = 7) immunized with IgG2aa 3F7.A10. The wells were tested for bound IgG1, IgG2a, and IgG2b by incubating for 1 h with the appropriate biotinylated goat Abs diluted 1:4000, followed by AP-streptavidin (1:4000). OD405nm was measured after incubation with p-NPP for 30 min. The wells were washed three times between each step.
ELISA for mAb activity against nucleosomes
This has been described previously (33). Briefly, wells of MaxiSorp Nunc immunoplates were coated overnight at 4°C with 5 μg/ml mononucleosomes in PBS, washed twice with PBS/0.05% Tween 20, blocked for 1 h in PBS containing 1% BSA, and washed twice. Serial dilutions of IgG2aa or IgG2c mAb were added and incubated for 1 h, bound mAb was detected with biotinylated goat anti-IgG2a or anti-IgG2c diluted 1:4000 followed by AP-conjugated streptavidin diluted 1:4000 and p-NPP, and OD405nm was recorded.
Nucleosome competition ELISA
In this assay, nucleosomes compete with polyclonal IgG1 anti-3F7.A10 for binding to plate-coated IgG2aa 3F7.A10. To MaxiSorp wells coated with 5 μg/ml IgG2aa anti-nucleosome 3F7.A10 were added various concentrations of nucleosomes (the competitors) in 25 μl of diluent followed by 25 μl of a fixed dilution of individual antisera containing IgG1 Abs against the Id of 3F7.A10. After 2 h at room temperature, plates were washed three times with PBS/0.05% Tween 20, bound IgG1 was detected with biotinylated goat anti-IgG1 followed by AP-streptavidin and p-NPP, and OD405nm was measured. As a control for nonspecific inhibition of IgG1 binding by the nucleosome competitors, we used an in-house BALB/c antiserum containing IgG1 Abs against an IgG2ae anti–MHC class I-Ad mAb (clone MK-D6). The percentage inhibition of IgG1 binding was calculated as:(ODwithout nucleosomes − ODwith nucleosomes) × 100/ODwithout nucleosomes.
Treatment of mice with CD4-specific mAb GK1.5
BALB/c mice were injected i.p. with the purified CD4-specific rat IgG2b mAb GK1.5, 300 μg on day −1 and 100 μg on day 6 and day 15. Control mice received saline because isotype-matched rat IgG2b is immunogenic for mice and is therefore not an ideal control.
HEp-2 cell anti-nuclear Ab assay
HEp-2 cell–coated slides (Inova) were overlaid with 15 μl of IgG2aa or IgG2c mAbs (10 μg/ml) for 30 min, washed in PBS for 5 min, and bound mAbs were stained for 30 min with FITC-labeled goat anti-IgG2a (SouthernBiotech) diluted 1:300 or Alexa Fluor 488–coupled G12-47/30 anti-IgG2c, respectively. After a final wash, the slides were mounted in polyvinyl alcohol.
Generation of in vivo ICs
For renal studies, BALB/c mice that had been immunized three to four times with 20 μg of IgG2aa 3F7.A10 and were challenged i.p. 7–11 times (three times weekly) with 200–300 μg of 3F7.A10. Free IgG2aa 3F7.A10 in serum was measured by ELISA by its affinity for plate-coated polynucleosomes as described above. A standard curve was generated with known concentrations of purified IgG2aa 3F7.A10. The detection limit was 60 ng/ml 3F7.A10. The same sera were tested for IgG1 anti-3F7.A10 titers in ELISA-wells coated with IgG2aa-3F7.A10. Serial dilutions of sera were added in duplicate to the wells, incubated for 1 h, and bound IgG1 Abs were detected with biotinylated goat anti-IgG1, AP-streptavidin, and p-NPP substrate.
Direct immunofluorescence histology
Pieces of kidney were embedded in OCT compound and frozen in liquid nitrogen. Tissues were cut into 5-μm-thick sections that were attached to Superfrost Plus glass slides (Thermo Scientific), dried overnight, and fixed for 10 min in acetone. The sections were stained for 30 min with an Ab mixture (Cy3-conjugated goat anti-mouse IgG1 and carboxyfluorescein-N-hydroxysuccinimide ester–conjugated mouse mAb 8.3 IgG2c anti-IgG2aa) diluted in PBS supplemented with 1% BSA, washed for 2 min in PBS, and mounted in polyvinyl alcohol. Sections were analyzed with an epifluorescence microscope (Nikon Eclipse E1000M).
Nano liquid chromatography–tandem mass spectrometry
SDS-PAGE of IgG2aa 3F7.A10 revealed four Coomassie-stained bands near the 15 kDa marker, designated bands 3, 4, 5, and 6 (see “IgG2aa 3F7.A10 copurifies with core histones” in 23Results). These bands were cut out separately, reduced (10 mM DTT, 1 h, 95°C), alkylated (55 mM iodoacetic acid, room temperature, 45 min), and subjected to trypsin (modified mass spectrometry [MS]–grade Promega) in-gel digestion overnight at 37°C. The peptides were analyzed by nano liquid chromatography–tandem MS with a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer interfaced with an EASY-Spray ion source (Thermo Fisher Scientific) followed by database searching using the Mascot search engine (Matrix Science).
Ultracentrifugation of 3F7.A10 mAb
Two milliliters of purified 3F7.A10 mAb (2.7 mg/ml) was centrifuged at 30,000 rpm (100,000 × g) for 150 min in a thin wall polyallomer tube using an SW 55 Ti rotor and a Beckman Optima LE-80K ultracentrifuge. Only the 3F7.A10 mAb contained in the top 0.7 ml of the tube (1.9 mg/ml) was examined for immunogenicity.
Experimental design
In this study, we examined Ab responses of normal mice to nonadjuvanted mAbs. To avoid responses against IgG CH-region allotypes (35), the IgCH allotype of the mAb (the immunogen) and of the immunized mouse were shared. Thus, IgG2a mAbs of allotype “a” (referred to as IgG2aa) were tested for immunogenicity in IgCHa haplotype homozygous BALB/c mice. IgG2a mAbs of allotype “b” (IgG2ab, also known as IgG2c) were tested for immunogenicity in homo- or heterozygous IgCHb haplotype mice. This approach ensures tolerance to the IgCH region and typically directs the responses at Ab V regions (Ids). To increase the chance of obtaining hybridomas secreting IgG2aa mAbs specific for nucleosomes, hybridomas were derived from aged female C.B10-H2b mice (previously known as BALB.B mice). Beginning at ∼9 mo of age, females of these H2b-congenic BALB/c mice can develop increased serum levels of HEp-2+ anti-nuclear Ab (ANA) and IgG2a anti-nucleosome Abs but their mortality rate is not increased (33). All mAbs were purified from a cell culture medium that was protein free except for 0.5 μg/ml human recombinant insulin. This was important because BSA amplifies the immunogenicity of Ids by providing non-self carrier epitopes for Th cells (20). To avoid unphysiologic stimuli and admixture with foreign proteins, adjuvants and DNase were not added to the mAbs.
Statistical analysis
Statistical analyses were performed with GraphPad Prism software version 4 using the unpaired two-tailed t test. The p values < 0.05 were considered significant.
Results
IgG2aa 3F7.A10 copurifies with core histones
The 15 mAbs examined in this study were divided into two groups (Table I). The first group consisted of seven mAbs that were negative for HEp-2 ANA activity and did not bind nucleosomes significantly. Six of these mAbs (clones 1–6) had unknown specificity and one (clone 15) bound the non-self hapten NP.
The second group of eight mAbs (clones 7–14) bound nucleosomes by ELISA. They also stained HEp-2 cell nuclei homogeneously; a typical example is shown for mAb 3F7.A10 (Fig. 1C, top panel). Thus, they were auto-mAbs. SDS-PAGE showed that mAb 3F7.A10 contained, in addition to the IgH- and IgL-chain bands, four bands near the 15 kDa marker (Fig. 1A). These bands were detected in mAb 3F7.A10 only (Fig. 1A, Supplemental Fig. 1). They were termed band 3 (which was weak), band 4, band 5, and band 6 and aligned with histones H3, H2B, H2A, and H4, respectively (Fig. 1B). MS confirmed that the respective bands contained histones H3, H2B, H2A, and H4 (Supplemental Table I). We conclude that mAb 3F7.A10 is unique among the eight nucleosome-specific mAbs, because it bound the charged histones even at a NaCl concentration as high as 3 M.
mAb 3F7.A10 copurifies with core histones. (A) SDS-PAGE of eight purified IgG2aa mAbs. Note that 3F7.A10 contains additional bands near the 15 kDa marker. (B) SDS-PAGE of IgG2a 3F7.A10 (lane 1) and total histones (lane 2). The four bands near the 15 kDa marker in lane 1 align with the core histone bands H3, H2B, H2A, and H4 in lane 2. (C) Indirect immunofluorescence staining of HEp-2 cell nuclei by IgG2aa mAbs at 10 μg/ml. Top image, 3F7.A10; middle image, 4E9.E5.E8; bottom image, 1D9.E10.F2 of unknown specificity (original magnification, ×400). Data are representative of two independent experiments.
mAb 3F7.A10 copurifies with core histones. (A) SDS-PAGE of eight purified IgG2aa mAbs. Note that 3F7.A10 contains additional bands near the 15 kDa marker. (B) SDS-PAGE of IgG2a 3F7.A10 (lane 1) and total histones (lane 2). The four bands near the 15 kDa marker in lane 1 align with the core histone bands H3, H2B, H2A, and H4 in lane 2. (C) Indirect immunofluorescence staining of HEp-2 cell nuclei by IgG2aa mAbs at 10 μg/ml. Top image, 3F7.A10; middle image, 4E9.E5.E8; bottom image, 1D9.E10.F2 of unknown specificity (original magnification, ×400). Data are representative of two independent experiments.
Dose-response analysis of HEp-2+ mAbs for plate-coated nucleosomes
Of the eight mAbs that stained HEp-2 cell nuclei, seven mAbs reached 50% maximal binding to mononucleosomes at a concentration of ∼10–20 ng/ml and maximal binding at 200–600 ng/ml (Fig. 2). The exception was mAb 4E9.E5.E8, which bound nucleosomes weakly by ELISA (Fig. 2); however, it stained HEp-2 nuclei homogeneously consistent with specificity for chromatin (Fig. 1C, middle panel). The mAb 1D9.E10.F2 with unknown specificity did not stain (Fig. 1C, bottom panel).
ANA-positive mAbs bind to plate-coated mononucleosomes. Data are representative of two independent experiments.
ANA-positive mAbs bind to plate-coated mononucleosomes. Data are representative of two independent experiments.
IgG1 Ab responses against nonadjuvanted IgG2c and IgG2aa mAbs with or without anti-nucleosome activity
We hypothesized that there is an association between anti-nucleosome activity and immunogenicity of isologous mAbs. To test this, mice were immunized i.p. with 20 μg of adjuvant-free mAb twice weekly for a total of five injections. The responses were analyzed by ELISA with the plate-coated mAb used for immunization.
We examined four IgG2c mAbs, of which three (XE3, XG1, XG3) have strong anti-nucleosome and HEp-2 ANA activities, and one (S43.10) is specific for the non-self hapten NP. Because the genetic background and MHCII alleles can have a strong influence on immune responses to isologous mAbs in mice (11, 36), we immunized three strains: B6 (H2b/b), CB6F1 (H2b/d), and C.B-17 (H2d/d). These strains carry one (CB6F1) or two (B6 and C.B-17) IgCHb haplotypes and are very likely tolerant to the “b” allotype of IgG2c.
The responses to IgG2c mAbs are shown in Fig. 3A. The mAb XE3 elicited weak responses in C.B-17 mice, but was nonimmunogenic for B6 and CB6F1 mice. The mAb XG1 provoked a weak response in two out of four B6 mice but was nonimmunogenic for CB6F1 and C.B-17 mice. None of the strains responded to mAb XG3 or to the hapten-specific mAb S43.10. The data suggested that IgG2c anti-nucleosome mAbs can be weak immunogens in these mice.
IgG1 responses elicited by nonadjuvanted IgG2c and IgG2aa mAbs. (A) Responses of three mouse strains against four isologous IgG2c mAbs. (B) IgG2aa mAbs against nucleosomes can elicit IgG1responses in BALB/c mice. Each mouse received 20 μg of adjuvant-free mAb i.p. twice weekly for a total of five injections and was bled 1 wk after the last injection. The sera were diluted 1:100 and examined by ELISA for IgG1 Abs against the homologous plate-coated mAb. Data are mean OD405nm values of ELISAs. The ANA HEp-2 activities of the mAbs are indicated as 3+, +, or −. Each symbol represents one mouse. Comparison between two groups was performed with a Student t test. p < 0.001.
IgG1 responses elicited by nonadjuvanted IgG2c and IgG2aa mAbs. (A) Responses of three mouse strains against four isologous IgG2c mAbs. (B) IgG2aa mAbs against nucleosomes can elicit IgG1responses in BALB/c mice. Each mouse received 20 μg of adjuvant-free mAb i.p. twice weekly for a total of five injections and was bled 1 wk after the last injection. The sera were diluted 1:100 and examined by ELISA for IgG1 Abs against the homologous plate-coated mAb. Data are mean OD405nm values of ELISAs. The ANA HEp-2 activities of the mAbs are indicated as 3+, +, or −. Each symbol represents one mouse. Comparison between two groups was performed with a Student t test. p < 0.001.
The responses of BALB/c mice against 11 IgG2aa mAbs are shown in Fig. 3B. We split the mAbs into two groups: the ones that have anti-nucleosome activity (denoted by + or 3+ in the figure) and the other mAbs that have no anti-nucleosome activity (denoted by −). Of six IgG2aa mAbs with unknown specificity and no anti-nucleosome activity, none elicited IgG1 Ab responses. In contrast, of five mAbs with anti-nucleosome and HEp-2 ANA activity, four were immunogenic and one (mAb 5A12.A3) was nonimmunogenic. The results revealed a link between anti-nucleosome activity and immunogenicity of isologous IgG2aa mAbs (p < 0.001, t test), suggesting that IC formation with nucleosomes plays a role for the immunogenicity of the mAbs.
BALB/c responses against soluble nonadjuvanted IgG2aa 3F7.A10: time-course, dose dependence, and IgG subclasses
Of the anti-nucleosome mAbs described above, IgG2aa 3F7.A10 was a very potent immunogen (Fig. 3B), and further studies were therefore centered on this mAb.
The responses of BALB/c mice (n = 2) to three weekly i.p. injections of 20 μg of IgG2aa 3F7.A10 are shown in Fig. 4A. One mouse displayed a day 7 primary IgG1 response, and the Ab level of both mice increased after repeated immunization. When the dose of mAb 3F7.A10 was lowered to 4 μg per injection administered on days 0, 3, 6, 13, and 18, one in four mice displayed a clear day 13 (OD405nm = 0.45) and a strong day 25 (OD405nm = 1.49) IgG1 response (data not shown); the others did not respond.
Both purified IgG2aa 3F7.A10 and its hybridoma cells elicit responses specific for the mAb 3F7.A10. (A) IgG1 responses of BALB/c mice elicited by three i.p. injections of 20 μg of mAb. Arrows indicate immunizations. Serum dilution 1:100. (B) The IgG1 subclass dominates the response of mice immunized with the mAb. The ELISA wells were coated with an IgE class switch variant of mAb 3F7.A10. Serum dilution 1:100. *p < 0.05, ***p < 0.0001, t test. (C) The response against IgG2aa mAb 3F7.A10 is idiotype specific. Shown is titration of the IgG1 activity of one BALB/c antiserum against four plate-coated IgG2aa mAbs, including the homologous immunogen 3F7.A10. (D) The response against IgG2aa 3F7.A10 is CD4+ Th cell–dependent. BALB/c mice were treated with anti-CD4 mAb GK1.5 or saline and immunized i.p. with 20 μg of IgG2aa 3F7.A10 on days 0, 7, and 16. The day 23 IgG1 response is shown. Serum dilution 1:100. ***p < 0.0001, t test. (E) Ultracentrifugation converts IgG2aa 3F7.A10 from a strong to a weak immunogen. BALB/c mice (n = 6) were immunized i.p. weekly three or four times with 20 μg of mAb. Serum dilution 1:100. Data are pooled from two independent experiments as indicated by filled and open symbols. (F) Injected viable 3F7.A10 hybridoma cells trigger IgG1 anti-3F7.A10 responses. Arrows represent cell injections. Mice nos. 1, 2, and 3 received 7 × 106 cells and mice nos. 8 and 9 received 0.5 × 106 cells per injection. Serum dilution 1:100. Data are pooled from two independent experiments as indicated by filled and open symbols.
Both purified IgG2aa 3F7.A10 and its hybridoma cells elicit responses specific for the mAb 3F7.A10. (A) IgG1 responses of BALB/c mice elicited by three i.p. injections of 20 μg of mAb. Arrows indicate immunizations. Serum dilution 1:100. (B) The IgG1 subclass dominates the response of mice immunized with the mAb. The ELISA wells were coated with an IgE class switch variant of mAb 3F7.A10. Serum dilution 1:100. *p < 0.05, ***p < 0.0001, t test. (C) The response against IgG2aa mAb 3F7.A10 is idiotype specific. Shown is titration of the IgG1 activity of one BALB/c antiserum against four plate-coated IgG2aa mAbs, including the homologous immunogen 3F7.A10. (D) The response against IgG2aa 3F7.A10 is CD4+ Th cell–dependent. BALB/c mice were treated with anti-CD4 mAb GK1.5 or saline and immunized i.p. with 20 μg of IgG2aa 3F7.A10 on days 0, 7, and 16. The day 23 IgG1 response is shown. Serum dilution 1:100. ***p < 0.0001, t test. (E) Ultracentrifugation converts IgG2aa 3F7.A10 from a strong to a weak immunogen. BALB/c mice (n = 6) were immunized i.p. weekly three or four times with 20 μg of mAb. Serum dilution 1:100. Data are pooled from two independent experiments as indicated by filled and open symbols. (F) Injected viable 3F7.A10 hybridoma cells trigger IgG1 anti-3F7.A10 responses. Arrows represent cell injections. Mice nos. 1, 2, and 3 received 7 × 106 cells and mice nos. 8 and 9 received 0.5 × 106 cells per injection. Serum dilution 1:100. Data are pooled from two independent experiments as indicated by filled and open symbols.
IgG2a and IgG2b are included in the response to IgG2aa 3F7.A10
To determine whether the responses also involved IgG subclasses other than IgG1, antisera from mice (n = 7) immunized with IgG2aa 3F7.A10 were tested for binding to plate-coated IgE 3F7.A10 produced by a spontaneous class switch variant of hybridoma 3F7.A10. As shown in Fig. 4B, IgG1 is the dominant subclass with lesser contributions of IgG2a and IgG2b. The binding to the IgE class switch variant indicates that the humoral response against IgG2aa 3F7.A10 targets the mAb V regions (Ids).
The IgG1 response against IgG2aa 3F7.A10 is Id specific
Antisera from BALB/c mice (n = 4) immunized with IgG2aa 3F7.A10 were tested for IgG1 activity against 11 plate-coated IgG2aa mAbs (see Table I). All four antisera diluted 1:300 displayed strong IgG1 responses against 3F7.A10 (OD405 = 1.41, 1.94, 1.78, and 1.58; data not shown). Two of the four antisera (nos. 5 and 6) cross-reacted weakly with mAbs 4E3.G1 and 5A12.A3, which are nucleosome specific and derive from the same C.B10-H2b mouse as 3F7.A10. Titration of the IgG1 activity of antiserum no. 5 is shown in Fig. 4C. The antiserum bound strongly to the homologous Ag (3F7.A10), cross-reacted weakly with 5A12.A3 and minimally with 4E3.G1, and did not bind to 16D8.C10.A7. The remaining seven plate-coated IgG2aa mAbs did not bind IgG1 of any of the four antisera (data not shown). The results confirm that the IgG1 response is specific for the Id (VH plus V region of Ig L chain domains) of 3F7.A10.
Antisera against mAb 3F7.A10 do not contain IgG1 anti-histones or anti-nucleosomes
Because purified IgG2aa 3F7.A10 contains mouse core histones, it was possible that the BALB/c response against 3F7.A10 was histone specific. We tested sera from BALB/c mice (n = 5) immunized four times with 20 μg of nonadjuvanted 3F7.A10. The sera had high IgG1 titers (2700–24,300) against plate-coated IgG2a 3F7.A10. In contrast, the titers against plate-coated calf thymus histones and chicken mononucleosomes were <100 (data not shown). This result indicates that the mouse histones that copurified with mAb 3F7.A10 were nonimmunogenic for normal BALB/c mice.
The IgG1 response against IgG2aa 3F7.A10 is CD4+ Th cell–dependent
The dominance of the IgG1 subclass (Fig. 4B) suggested that the immune response of BALB/c mice to IgG2a 3F7.A10 depended on Th cells. We therefore treated BALB/c mice (n = 4) i.p. with the purified CD4+ Th cell–depleting rat IgG2b mAb GK1.5, 300 μg on day −1 and 100 μg on days 6 and 15; the control mice (n = 3) received saline. The treated mice were immunized i.p. with 20 μg of nonadjuvanted IgG2aa 3F7.A10 on days 0, 7, and 16. The sera were collected on days 23 and 44, diluted 1:100, and analyzed for IgG1 anti-3F7.A10 activity by ELISA.
No response was detectable on day 23 in mice treated with GK1.5. In contrast, sera from saline-treated control mice contained strong IgG1 activity against 3F7.A10 (Fig. 4D). The day 44 responses were quantitatively very similar except that one GK1.5-treated mouse had converted to a weak responder (OD405nm = 0.33) (data not depicted). We conclude that the Ab response against Id-IgG2aa 3F7.A10 is CD4+ Th cell–dependent.
Ultracentrifugation converted mAb 3F7.A10 into a weak immunogen
The fact that mAb 3F7.A10 copurified with core histones indicated that it had bound nucleosomal Ags during hybridoma culture and formed ICs. To find out whether putative ICs enhanced Id immunogenicity, 2 ml of purified IgG2a 3F7.A10 (2.7 mg/ml) was deaggregated by ultracentrifugation (100,000 × g) for 150 min, and mAb contained in the top 0.7 ml of the tube (1.9 mg/ml) was used for immunization (37). BALB/c mice (n = 6) received three or four weekly i.p. injections of 20 μg of centrifuged 3F7.A10 in PBS, and sera were harvested 1 wk following each injection, diluted 1:100, and analyzed by ELISA.
Two mice (nos. 78 and 81) displayed a clear IgG1 response against 3F7.A10, one mouse (no. 83) was a weak responder, and three mice were nonresponders (Fig. 4E). Overall, these responses were much weaker than those elicited by noncentrifuged 3F7.A10 (compare Fig. 4A and 4E). This might be accounted for by depletion of immunogenic aggregates consisting of ICs of mAb 3F7.A10 with nucleosomal Ags.
Injected viable MHC-incompatible 3F7.A10 hybridoma cells trigger responses to Id of 3F7.A10
To examine whether the immunogenicity of mAb 3F7.A10 was an artifact of affinity purification, we injected viable and washed 3F7.A10 hybridoma cells (grown in serum-free medium) i.p. into nonirradiated naive BALB/c mice. The cells served as an endogenous source of IgG2a 3F7.A10. The IgG1 responses against plate-coated IgG2aa 3F7.A10 were measured by ELISA. None of the mice developed signs of ascites or sickness. Because the hybridoma is the result of a C.B10-H2b/b splenocyte fused with a BALB/c (H2d/d)-derived fusion partner, the hybridoma cells are H2b/b plus H2d/d. During this experiment, therefore, the hybridoma had most likely been killed owing to MHC incompatibility.
The IgG1 anti-3F7.A10 responses of BALB/c mice (n = 5) are shown in Fig. 4F.
Mice nos. 1, 2, and 3 received 7 × 106 cells, and mice nos. 8 and 9 received 0.5 × 106 cells per injection. Mouse no. 1 was a nonresponder. Mouse no. 2 received two injections and displayed a weak day 9 Ab response that had become very strong by days 17 and 27. Mouse no. 3 received three injections and had no response on day 9, a weak response on day 17, and a strong response by day 27. The day 27 antisera of mice nos. 2 and 3 did not cross-react with an unrelated plate-coated IgG2aa mAb (16E7.H8.G1), indicating that the antisera were specific for Id-3F7.A10 (data not shown). Two additional mice (nos. 8 and 9) received four injections of 0.5 × 106 cells. They were clear responders by days 27 and 35 (Fig. 4F). Thus, following injections of washed hybridoma cells, four out of five BALB/c mice mounted robust IgG1 responses against Id-3F7.A10.
To determine whether mice harbored free IgG2aa 3F7.A10 produced by the injected hybridoma cells, sera (diluted 1:50) from days 0, 9, and 17 were tested for IgG2a activity against plate-coated polynucleosomes, using known concentrations of IgG2a 3F7.A10 as standard. We found that serum of mouse no. 2 (the strongest responder) contained 400 ng/ml 3F7.A10 on day 9 (data not shown), indicating that the transferred cells produced nucleosome-specific IgG2a. By day 17, the serum level was <60 ng/ml, suggesting that in vivo–released mAb 3F7.A10 was efficiently neutralized and/or cleared by polyclonal anti-3F7.A10 Abs. Sera from the other mice were negative for anti-nucleosome activity (data not shown).
We conclude that i.p. injection into BALB/c mice of viable MHC-incompatible hybridoma cells producing IgG2aa 3F7.A10 can elicit IgG1 responses against the Id of 3F7.A10. The results suggest that the immunogenicity of IgG2aa 3F7.A10 is not an artifact of affinity purification from hybridoma culture supernatants.
Nucleosomes compete with polyclonal anti-3F7.A10 Abs for binding to plate-coated IgG2aa 3F7.A10
As reported above, several lines of evidence indicate that the 3F7.A10 anti-nucleosome mAb elicits Abs that target the Id (VH plus V region of Ig L chain domains). To determine whether mononucleosomes (average DNA size of ∼160 bp) (Fig. 5Aa) and polynucleosomes (DNA size of 200 to >4000 bp) (Fig. 5Ab) compete with polyclonal IgG1 anti-Id Abs for binding to plate-coated IgG2aa 3F7.A10, we examined antisera from BALB/c mice (n = 3) immunized with IgG2aa 3F7.A10. As a control for nonspecific competition by nucleosomes we used a BALB/c antiserum against mAb MK-D6 combined with plate-coated MK-D6, which is an IgG2ae mAb directed at the I-Ad MHCII molecule.
Nucleosomes compete with polyclonal IgG1 anti-3F7.A10 for binding to plate-coated IgG2aa 3F7.A10. (A) Size of DNA of mononucleosomes [(a) lane 1, 123-bp ladder; lane 2, mononucleosomes]. Size of DNA of polynucleosomes [(b) lane 1, 1-kb ladder; lane 2, polynucleosomes]. (B) Polynucleosomes compete with polyclonal IgG1 anti-IgG2aa 3F7.A10 of BALB/c antisera (n = 3) but do not compete with polyclonal IgG1 anti-IgG2ae MK-D6 (specific for MHC class I-Ad). (C) Compared to polynucleosomes in (B), mononucleosomes compete ∼500-fold less effectively with polyclonal IgG1 anti-3F7.A10. Data are representative of two independent experiments.
Nucleosomes compete with polyclonal IgG1 anti-3F7.A10 for binding to plate-coated IgG2aa 3F7.A10. (A) Size of DNA of mononucleosomes [(a) lane 1, 123-bp ladder; lane 2, mononucleosomes]. Size of DNA of polynucleosomes [(b) lane 1, 1-kb ladder; lane 2, polynucleosomes]. (B) Polynucleosomes compete with polyclonal IgG1 anti-IgG2aa 3F7.A10 of BALB/c antisera (n = 3) but do not compete with polyclonal IgG1 anti-IgG2ae MK-D6 (specific for MHC class I-Ad). (C) Compared to polynucleosomes in (B), mononucleosomes compete ∼500-fold less effectively with polyclonal IgG1 anti-3F7.A10. Data are representative of two independent experiments.
In the nucleosome concentration range of 0.16–500 μg/ml, all three inhibition curves were clearly steeper for polynucleosomes relative to mononucleosomes, indicating more efficient competition by the former. At the maximal polynucleosome concentration of 500 μg/ml, binding of IgG1 of the three antisera (nos. 5, 6, and 15) to plate-coated IgG2aa, 3F7.A10 was inhibited 54, 57, and 65%, respectively (Fig. 5B). When the antisera were tested with the equivalent concentration of mononucleosomes, binding was inhibited 24, 29, and 29% (Fig. 5C). Neither polynucleosomes nor mononucleosomes inhibited binding of polyclonal IgG1 anti–MK-D6 to plate-coated IgG2ae MK-D6 (Fig. 5B, 5C).
We conclude that 1) nucleosomes compete specifically and dose-dependently with polyclonal IgG1 anti-3F7.A10, indicating that part of the IgG1 response targets the ligand (nucleosome) binding site; 2) polynucleosomes are ∼500-fold stronger competitors compared with mononucleosomes, probably owing to their higher antigenic valency and superior cross-linking ability; and 3) even polynucleosomes inhibited incompletely. The latter result suggests that a subset of polyclonal IgG1 Abs against Id-3F7.A10 Abs targets Id determinants located outside the ligand binding site. Separation of the Id and ligand-binding site has been reported before (38).
TLR9 deficiency weakens the IgG1 response against Id-3F7.A10
To determine whether the DNA-binding TLR9 has a role in responses of BALB/c mice against the Id of nucleosome-specific IgG2aa 3F7.A10, TLR9-deficient (TLR9−/−) (n = 10) and wild-type (WT; n = 6) BALB/c mice were immunized i.p. four times with 20 μg of purified nonadjuvanted IgG2aa 3F7.A10, and IgG1 responses against 3F7.A10 were measured after one, three, and four immunizations. WT mice mounted significantly stronger responses than did TLR9-deficient mice (Fig. 6A). The antisera did not cross-react with an unrelated plate-coated IgG2aa mAb (4E9.E5.E8) (Fig. 6B), confirming that the responses were Id specific. We conclude that ligation of TLR9 is not required for the immunogenicity of Id-3F7.A10, but the data strongly suggest that signals from endosomal TLR9 significantly amplify the response against Id-3F7.A10. This is consistent with the notion that the anti-Id BCR recognizes an Id/nucleosome IC, resulting in efficient cointernalization of nucleosomes and activation of TLR9.
TLR9 deficiency weakens the IgG1 response against IgG2aa 3F7.A10. (A) The responses of TLR9-deficient BALB/c mice (n = 10) against IgG2aa 3F7.A10 are significantly weaker than are those of WT mice (n = 6). *p < 0.05 (day 25), *p < 0.05 (day 37), t test. (B) The antisera against 3F7.A10 do not cross-react with a plate-coated unrelated mAb IgG2aa 4E9.E5.E8. The mice were immunized i.p. with 20 μg of mAb on days 0, 11, 18, and 30. Sera were collected on the days indicated and diluted 1:100. Each symbol represents one mouse.
TLR9 deficiency weakens the IgG1 response against IgG2aa 3F7.A10. (A) The responses of TLR9-deficient BALB/c mice (n = 10) against IgG2aa 3F7.A10 are significantly weaker than are those of WT mice (n = 6). *p < 0.05 (day 25), *p < 0.05 (day 37), t test. (B) The antisera against 3F7.A10 do not cross-react with a plate-coated unrelated mAb IgG2aa 4E9.E5.E8. The mice were immunized i.p. with 20 μg of mAb on days 0, 11, 18, and 30. Sera were collected on the days indicated and diluted 1:100. Each symbol represents one mouse.
Passive transfers of mAb IgG2aa 3F7.A10 result in circulating ICs
To determine whether polyclonal IgG1 Abs against mAb 3F7.A10 can generate circulating ICs, BALB/c mice (n = 7) were immunized i.p. three or four times with 20 μg of 3F7.A10 to initiate immune responses, followed by repeated i.p. challenges with 200–300 μg IgG2a 3F7.A10 administered three times per week. One naive control mouse received a single injection of 200 μg of 3F7.A10. The amount of mAb per challenge and the number of challenges are shown in Fig. 7.
Immune mice clear transferred IgG2aa 3F7.A10 rapidly. Following three to four injections of 20 μg of IgG2a 3F7.A10, the mice (n = 7) were challenged i.p. three times weekly with the indicated number of doses of IgG2a 3F7.A10. One naive control mouse received a single injection of 200 μg of IgG2a 3F7.A10 ∼24 h before blood collection. Shown are titers of endogenous polyclonal IgG1 anti-3F7.A10 (filled bars) and levels of transferred IgG2a 3F7.A10 anti-nucleosome mAb (open bars). The IgG1 anti-3F7.A10 titers were measured with plate-coated 3F7.A10 by ELISA. The IgG2a anti-nucleosome levels (representing 3F7.A10 injected 2 or 24 h before) were measured with plate-coated polynucleosomes; a standard curve was generated with known concentrations of IgG2a 3F7.A10. Asterisks indicate below level of detection (bld) (<60 ng/ml).
Immune mice clear transferred IgG2aa 3F7.A10 rapidly. Following three to four injections of 20 μg of IgG2a 3F7.A10, the mice (n = 7) were challenged i.p. three times weekly with the indicated number of doses of IgG2a 3F7.A10. One naive control mouse received a single injection of 200 μg of IgG2a 3F7.A10 ∼24 h before blood collection. Shown are titers of endogenous polyclonal IgG1 anti-3F7.A10 (filled bars) and levels of transferred IgG2a 3F7.A10 anti-nucleosome mAb (open bars). The IgG1 anti-3F7.A10 titers were measured with plate-coated 3F7.A10 by ELISA. The IgG2a anti-nucleosome levels (representing 3F7.A10 injected 2 or 24 h before) were measured with plate-coated polynucleosomes; a standard curve was generated with known concentrations of IgG2a 3F7.A10. Asterisks indicate below level of detection (bld) (<60 ng/ml).
The challenged mice, but not the naive mouse, harbored high serum titers (8000–600,000) of polyclonal IgG1 anti-3F7.A10 (Fig. 7, gray bars, left y-axis). In contrast, only one mouse (no. 4) (and the naive mouse) had detectable IgG2a anti-nucleosome activity representing mAb IgG2aa 3F7.A10 injected ∼24 h earlier (Fig. 7, open bars, right y-axis). Furthermore, the level of IgG2a anti-nucleosome mAb found in mouse no. 15 at 2 and 24 h after injection was 2400 and <60 ng/ml, respectively. Thus, in contrast to the naive mouse, six out of seven mice immune against mAb 3F7.A10 neutralized and/or cleared a dose of 200–300 μg IgG2a 3F7.A10 from the circulation within 24 h, indicating formation of high avidity IgG1/IgG2a ICs.
IgG2aa 3F7.A10 challenges can lead to IgG1/IgG2a deposits in renal glomeruli
The kidneys of the mice described above were harvested ∼24 h after the last i.p. challenge. By then, mice nos. 4, 5, and 6 had received seven challenges of 200 μg, nos. 15 and 17 had received 10 challenges of 300 μg, and nos. 38 and 39 had received 11 challenges of 200 μg. The naive mouse received a single dose of 200 μg of 3F7.A10.
Immunofluorescence histology revealed that the glomeruli of the naive mouse did not contain detectable IgG1 or IgG2a deposits (data not shown). Given that the serum level of IgG2aa 3F7.A10 of the naive mouse ∼24 h after the single injection was ∼7 μg/ml (Fig. 7), this indicated that mAb IgG2aa 3F7.A10 by itself does not bind to glomeruli of normal BALB/c mice in vivo. In contrast, scattered glomeruli of five mice (nos. 5, 15, 17, 38, and 39) contained granular mesangial deposits of IgG2a (green) and IgG1 (red), which stained yellow when merged, indicating colocalization of the two IgG subclasses (Fig. 8). The glomeruli of mice nos. 4 and 6 were negative for IgG1 and IgG2a deposits (data not shown). These observations indicate that the glomerular deposits contained endogenously produced polyclonal IgG1 anti-Id 3F7.A10 bound to the passively transferred mAb IgG2aa 3F7.A10.
Transfer of IgG2a 3F7.A10 to immune mice generates colocalizing glomerular IgG1/IgG2a deposits. The mice were the same as those studied in Fig. 7. Mice nos. 4, 5, and 6 received seven i.p. challenges of 200 μg, nos. 15 and 17 received 10 challenges of 300 μg, and nos. 38 and 39 received 11 challenges of 200 μg of IgG2a 3F7.A10. The kidneys were harvested ∼24 h after the last challenge. Original magnification ×400. Fluos, carboxyfluorescein-N-hydroxysuccinimide ester.
Transfer of IgG2a 3F7.A10 to immune mice generates colocalizing glomerular IgG1/IgG2a deposits. The mice were the same as those studied in Fig. 7. Mice nos. 4, 5, and 6 received seven i.p. challenges of 200 μg, nos. 15 and 17 received 10 challenges of 300 μg, and nos. 38 and 39 received 11 challenges of 200 μg of IgG2a 3F7.A10. The kidneys were harvested ∼24 h after the last challenge. Original magnification ×400. Fluos, carboxyfluorescein-N-hydroxysuccinimide ester.
Discussion
We have previously described a nonadjuvanted isologous anti-hapten (TNP) mAb, the Id of which was immunogenic when expressed as IgM but tolerogenic when joined to IgG1, IgG2a, or IgG2b (21). In this study, we extended these observations to IgG auto-mAbs against nucleosomes. The data strongly suggested a link between anti-nucleosome activity and immunogenicity of IgG2a mAbs. This idea was supported by the IgG2aa anti-nucleosome mAb 3F7.A10, which elicited potent CD4+ Th cell–dependent IgG1 anti-Id responses in BALB/c mice.
Soluble protein Ags, including Ids of Abs, are stronger immunogens when they have polymerized either chemically (8, 9) or by forming ICs with IgG Abs (10, 39). In our present model, we hypothesized that the seven mAbs with unknown or anti-hapten specificity are not likely to generate ICs following injection in mice. In contrast, IgG2a or IgG2c mAbs directed at nucleosomes presumably form ICs with nucleosomes or nucleosome-containing cell debris released from dying cells, thereby acquiring enhanced capacity to cross-link low-affinity FcγRs of APCs and anti-Id BCRs of B cells. In fact, IgD BCRs have recently been reported to be specialized for responses to polyvalent Ags (40). Furthermore, polymerized Ag in ICs improves presentation of peptide/MHCII on B cells and other APCs resulting in amplified CD4+ Th cell responses (41–44). Taken together, these mechanisms likely augment IgG1 responses to Ids of anti-nucleosome mAbs, including mAb 3F7.A10.
Additionally, generation of ICs with nucleosomes might boost anti-Id responses through triggering of TLR9, an endosomal receptor for CpG DNA, that leads to activation of dendritic cells (DCs) (45) and B cells (46). In these ICs, the Id targets nucleosomes for uptake in Id-specific B cells whereas Fc regions of the mAbs direct nucleosomes for internalization in DCs via FcγRs, leading to accumulation of nucleosomal DNA in these cells and enhanced activation of TLR9. Indeed, we found that the intentionally induced IgG1 responses against the Id of IgG2aa 3F7.A10 were significantly stronger among TLR9-sufficient relative to TLR9-deficient BALB/c mice. The results strongly suggested a role for TLR9 activation elicited by self-derived DNA in responses against Id-3F7.A10. Our model resembles the original dual BCR/TLR signaling paradigm of Leadbetter et al. (26) except that the target B cells of the present study has anti-Id instead of rheumatoid factor (anti-self IgG2a Fc) activity.
We observed that the IgG1 responses against nonadjuvanted Id-3F7.A10 were CD4+ Th cell–dependent, required small amounts of immunogen, reached high titers, and effectively neutralized or cleared passively transferred IgG2aa 3F7.A10 from the circulation. These data indicate that the polyclonal IgG1 Abs had high avidity, suggesting that Id-3F7.A10 is recognized similar to a non-self Ag. According to this notion, the Id may harbor somatic point mutations in its V regions as reported for many anti-DNA and anti-nuclear auto-mAbs (47–50). If so, B and Th cells specific for Id-3F7.A10 likely target unique mutation-derived Id neoepitopes (see 1Introduction) expressed in amounts that are too low for induction of tolerance. Hence, Id-3F7.A10 may resemble a conventional foreign protein Ag that does not require TLR9 signals for activation. Consistent with this idea, IgG responses against Th-dependent foreign soluble protein Ags exhibited small differences between WT mice and mice with defects in MyD88/TLR signaling (51, 52). This contrasts with activation of B cells bearing a low-affinity rheumatoid factor BCR specific for autologous IgG2a, which is highly dependent on TLR9 signals (25, 26).
It should be recalled, however, that our immunizations were all carried out with soluble mAbs in the absence of adjuvants, when APCs might express too weak costimulatory activity for effective activation of CD4+ Th cells (53). As a result, presentation of Id –peptide/MHCII complexes to Th cells may be inefficient owing to lower levels of MHCII and of the costimulatory molecules CD80 and CD86. In this context, signals from TLR9 might compensate, at least in part, for the lack of added adjuvant, thus accounting for why TLR9-sufficient mice responded significantly stronger to Id-3F7.A10 than did mice deficient in TLR9.
After a wash of the affinity column with 3 M NaCl, IgG2aa 3F7.A10 was the only one among the eight anti-nucleosome mAbs that copurified with the core histones H2A, H2B, H3, and H4. This raised the question: did the bound self-histones contribute to the strong immunogenicity of IgG2aa 3F7.A10? Given that the IgG1 responses against Id-3F7.A10 were CD4+ Th cell–dependent, it is possible that the Th cell–activating peptide/MHCII determinants did not derive from the Id itself but instead derived from the bound histones. If this is the case, it implies that Th cells had lost tolerance to histone peptide/MHCII epitopes. Such putative histone-specific Th cells might cooperate with B cells specific for Id-3F7.A10 as well as with putative B cells recognizing the bound histones. We addressed this question by examining antisera from mice with high IgG1 Ab titers against IgG2a 3F7.A10. However, we could not demonstrate IgG1 activity against plate-coated nucleosomes or total histones, suggesting that tolerance to histones was maintained in these non–lupus-prone mice. This result is consistent with the failure of rabbits to respond to immunization with nonadjuvanted calf thymus histones, indicating that histones are poor immunogens (54, 55).
In this study, we hypothesize that injected IgG anti-nucleosome mAbs bind released nucleosomal Ags in vivo and form ICs that drive Ab production against Ids. However, the purified mAb 3F7.A10 had bound histones during hybridoma culture in vitro, demonstrating that the mAb had already complexed during production in vitro. To explore the role of nucleosomal Ag in the development of an anti-Id response, IgG2aa 3F7.A10 was ultracentrifuged (100,000 × g, 150 min) to deplete potential ICs (37). Administration of the centrifuged preparation to six mice elicited substantially diminished Ab responses (compare Fig. 4A and 4E), indicating depletion of ICs that had formed in vitro. Nonetheless, two of the six mice were clear responders, suggesting that, after injection in these two mice, the centrifuged mAb generated fresh immunogenic ICs with nucleosomes released in vivo.
Nucleosomes have been detected in the plasma of normal BALB/c mice at a young age (4–6 wk old) but not at an older age (24–26 wk old) (56). Nucleosomal Ags are also exposed at the surface of apoptotic cells (57, 58). Furthermore, given that strong BCR signals trigger apoptosis in germinal center B cells (59–61), nucleosomes may also be displayed and form ICs with anti-nucleosome Abs within the microenvironment of germinal centers where immune responses are initiated. We hypothesize that these endogenous sources of nucleosomal Ags, perhaps along with the potent APC function of the CD11c-expressing B cell subset (62), explain why ultracentrifuged Id-3F7.A10 was immunogenic in some mice. Moreover, to account for the anti-Id responses against ultracentrifuged mAb 3F7.A10, we suggest that the expression of endogenously released nucleosomes available for IC formation can vary and might be limiting in many but not all adult nonlupus mice (56, 63). In this context, we observed that four out of five BALB/c mice injected with MHC-incompatible 3F7.A10 hybridoma cells mounted robust IgG1 responses against Id 3F7.A10 (Fig. 4F). We envisage that viable hybridoma cells provided prolonged endogenous secretion of the nucleosome-specific mAb while, in parallel, rejected cells released nucleosomal Ags. Thus, ICs generated in vivo through these two processes might account for the strong Id-specific Ab responses of this experiment.
The danger model proposes that the release of intracellular molecules from distressed or injured cells (called damage-associated molecular patterns) contributes to adaptive immune responses (64) (reviewed in Ref. 65). Histones released from dying cells can act as danger signals that provoke IL-1β secretion and sterile inflammation, resulting in activation of DCs and augmented Ag presentation (66). However, inflammation triggered by free histones is inhibited by treatment with anti-histone IgG (67–69). Hence, the tight association of core histones and IgG2aa 3F7.A10 may have prevented generation of histone-mediated danger signals.
Following repeated i.p. passive transfer of 200–300 μg mAb IgG2aa 3F7.A10 into seven BALB/c mice with high titers of polyclonal IgG1 anti-3F7.A10, we found evidence of rapid clearance and/or neutralization of the mAb indicative of circulating ICs containing high-avidity IgG1 Abs. Furthermore, we demonstrated granular deposits of colocalizing IgG1 and IgG2a in renal glomeruli of five out of the seven challenged mice. These findings are consistent with deposition of preformed IgG1/IgG2a ICs. In this experimental model, polyclonal IgG1 Abs directed at the Id of an anti-nucleosome auto-mAb can be a major component of renal IC deposits. Possibly, this can be a significant pathogenic mechanism in lupus nephritis (70).
In an immune electron microscopy-based study, 200 μg of nonadjuvanted IgG2a or IgG2b anti-dsDNA mAbs was injected i.v. eight times during a 4-wk period into normal BALB/c mice (71). This resulted in mesangial accumulation of electron-dense structures reported to contain both mAbs and chromatin fragments, and the authors proposed that the anti-DNA mAbs had bound to nucleosomes in the circulation and deposited in the mesangium (71). They found that the recipient mice were negative for polyclonal IgM directed at the Ids of anti-DNA mAbs. However, polyclonal IgG1 anti-Id Abs were not investigated, so IgG1/IgG2a ICs may also have been generated in that study. This is because, first, the anti-DNA mAbs were purified from medium containing 20% FCS. We have shown that most mAbs purified from FCS-containing cultures elicited robust IgG1 anti-Id responses that depended on a carrier effect mediated by complex formation of mAbs with BSA in the cell culture medium (20). This artifact can be avoided by purifying mAbs from serum-free hybridoma culture medium (20–22). Second, the mAbs derived from (NZB × NZW)F1 (BWF1) lupus mice are IgCH allotype “e”, unlike BALB/c mice, which are IgCH allotype “a” (35).
ANAs, including Abs against nucleosomes, are commonly found in healthy humans and mice (33, 72–74). In this study, we report evidence of a significant link between anti-nucleosome activity and immunogenicity, and we describe an IgG2aa anti-nucleosome auto-mAb called 3F7.A10, which copurified with histones and elicited CD4+ Th cell–dependent IgG anti-Id response in normal BALB/c mice. We hypothesize that nucleosomal Ags released in vivo can cross-link the mAb, thus converting the Id into a potent multivalent Ag bound to nucleosomes. We envisage that this complex is internalized into Id-specific B cells through cognate surface BCRs and into other APCs via low-affinity FcγRs, thus providing optimal conditions for contact between endosomal TLR9 of these cells and nucleosomal DNA. This mechanism may explain why the humoral response to the Id of 3F7.A10 is enhanced by signals from TLR9. The interplay between Ids and nucleosomes might lead to spontaneous Ab responses against Ids of anti-nucleosome Abs, formation of Id/anti-Id ICs, and tissue injury.
Acknowledgements
We thank Dr. Ann Marshak-Rothstein and Dr. Egil Lien for providing TLR9−/− mice, Dr. Astrid Tutturen for performing mass spectrometry, and Anne Pharo for analysis of endotoxin levels.
Footnotes
This work was supported by funding from the Oslo University Hospital and Odd Fellow Norway (to K.H.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
References
Disclosures
The authors have no financial conflicts of interest.