Idiotype-Specific Th Cells Support Oligoclonal Expansion of Anti-dsDNA B Cells in Mice with Lupus

This information is current as Kristin Aas-Hanssen, Ane Funderud, Keith M. Thompson, of September 30, 2021. Bjarne Bogen and Ludvig A. Munthe J Immunol published online 15 August 2014 http://www.jimmunol.org/content/early/2014/08/15/jimmun ol.1400640 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2014/08/15/jimmunol.140064 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 30, 2021 *average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 15, 2014, doi:10.4049/jimmunol.1400640 The Journal of Immunology

Idiotype-Specific Th Cells Support Oligoclonal Expansion of Anti-dsDNA B Cells in Mice with Lupus

Kristin Aas-Hanssen,* Ane Funderud,* Keith M. Thompson,* Bjarne Bogen,*,† and Ludvig A. Munthe*

Systemic lupus erythematosus (SLE) is marked by a Th cell–dependent hyperresponsiveness, with frequent germinal center reactions and hypergammaglobulinemia. The specificity of Th cells in lupus remains unclear, but B cell idiotypes (Ids) have been suggested. A hallmark is the presence of anti-dsDNA, mutated IgG with a preponderance of arginines in CDR3 of the Ig variable H chain (IgVH). B cells can present V region–derived Id peptides on their MHC class II molecules to Id-specific Th cells. We show that Id-specific Th cells support the proliferation of anti-dsDNA Id+ B cells in mice suffering from systemic autoimmune disease with SLE-like features. Mice developed marked clonal expansions of B cells; half of the IgVH sequences were clonally related. Anti-

dsDNA B cells made up 40% of B cells in end-stage disease. The B cells expressed mutated IgVH with multiple arginines in CDR3. Hence, Downloaded from Id-driven –B cell collaboration supported the production of classical anti-dsDNA Abs, recapitulating the characteristics of such Abs in SLE. The results support the concept that Id-specific Th cells may trigger the development of SLE and suggest that manipulation of the Id-specific T cell repertoire could play a role in treatment. The Journal of Immunology, 2014, 193: 000–000.

ystemic lupus erythematosus (SLE) is a debilitating sys- the positively charged arginine can bind to the phosphodieseter

temic autoimmune disease thatisassociated withgeneticand backbone, as well as donate up to five H bonds (9–12). http://www.jimmunol.org/ S environmental influences, with involvement of multiple Autoreactive B cells experience sustained signaling through their organs, such as the skin and kidneys (1, 2). SLE is marked by a Th cell– BCR, become anergic, and eventually undergo apoptosis, as was de- dependent B cell hyperresponsiveness, with frequent germinal center scribed for anti-dsDNA B cells (13). Nevertheless, such cells may reactions, hypergammaglobulinemia, and high levels of highly be rescued, activated, and expanded if provided with Th cell help mutated, affinity-matured IgG autoantibodies (1–6). Anti- dsDNA (13), a feature that also was found for anergic B cell responses of Abs are a serological hallmark of SLE that mediate, at least in part, other autospecificities (14, 15). However, in contrast to B cells, it is the nephritis that marks the course of this disease in both humans unclear what specificity Th cells may have in cognate interactions and mice (1, 2). with dsDNA-specific B cells. Ab idiotypes (Ids) are a plausible In other mouse models of lupus, MRL/lpr and BWF1 mice candidate because both lupus-prone mice (16–19) and SLE patients by guest on September 30, 2021 spontaneously develop anti-dsDNA and nephritis; however, anti- (20–22) have clearly measurable Th cell responses toward patho- dsDNA autoantibodies also can be induced [e.g., by vaccinating genic anti-dsDNA Abs, suggesting that Id determinants play a role non–lupus-prone strains with dsDNA and adjuvants (7, 8)]. In in pathogenesis. In such experiments, APCs, such as dendritic cells, both spontaneous and induced models, the anti-dsDNA Ig variable present somatically mutated V-region determinants (Id peptides) to H chain (IgVH) regions often express basic amino acids like ar- Th cells. Further, anti-Id Th cell responses increased with disease ginine (R) and lysine (K), or asparagine (N), in Ag-binding CDR severity, and disease was aggravated by injection of Id peptide (17). and FR3 regions (9). The presence of several arginines in CDR3 It has been clear for some time that somatically mutated Id pep- is relatively rare (10, 11) but is important for DNA binding; tides can serve as cognate Ags for Th cells (23, 24) and that this re- sponsiveness is restricted by tolerance to germline V regions (25, 26).

*Centre for Immune Regulation, Department of Immunology, Institute of Clinical Moreover, it was shown that individual B cells can present endoge- Medicine, Oslo University Hospital, University of Oslo, 0424 Oslo, Norway; and nous Id peptide on MHC class II molecules to Th cells and that such † K.G. Jebsen Centre for Influenza Vaccine Research, Department of Immunology, Id+ B cells can collaborate with Id-specific Th cells (27–30). In this Oslo University Hospital, University of Oslo, 0424 Oslo, Norway interaction, B cells can undergo the germinal center reaction, pro- Received for publication March 25, 2014. Accepted for publication July 21, 2014. vided that ligands bind the BCR (31). Such Id-driven Th cell–B cell This work was supported by The Research Council of Norway, Functional Genomics (FUGE), Project 17538, the University of Oslo, and UNIFOR (grants from the Vivi collaboration can also cause expansion of autoreactive B cells, se- Irene Hansen Foundation, the Gertrude and Jack Nelson Foundation, the Signe and cretion of autoantibodies (26, 31), and autoimmune disease (32, 33). Albert Bergsmarken Foundation, and the Henrik Homan Foundation). In the latter studies, mice were double transgenic (DTG): B cells Address correspondence and reprint requests to Dr. Kristin Aas-Hanssen, Prof. Bjarne expressed a transgenic (TG) l2 L chain (Id+), and T cells ex- Bogen, and Prof. Ludvig A. Munthe, Centre for Immune Regulation, Department of d Immunology, Institute of Clinical Medicine, University of Oslo and Rikshospitalet, P.O. pressed an MHC class II (I-E ), Id peptide–restricted TCR trans- Box 4950, Nydalen, 0424 Oslo, Norway. E-mail addresses: kristin.aas-hanssen@medisin. gene.Inthesemice,weobservedamarked,butincomplete,negative uio.no (K.A.-H.), [email protected] (B.B.), and [email protected] (L.A.M.). selection of Id-specific , a progressive expansion of low- The online version of this article contains supplemental material. frequency Id-specific T cells, an ongoing collaboration between Id- specific Th cells and Id+ B cells, hypergammaglobulinemia, and Abbreviations used in this article: ANA, anti-nuclear Ab; DTG, double transgenic; Id, idiotype; IgVH, Ig variable H chain; IMGT, International ImMunoGeneTics; autoantibodies, including high titers of anti-nuclear Abs (ANAs). In NCBI, National Center for Biotechnology Information; SLE, systemic lupus erythe- this study, we show clonal expansion of anti-dsDNA B cells in such matosus; TG, transgenic. mice, finding a marked oligoclonality, with clonal expansions of Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 B cells with mutated VH regions and arginine-rich CDR3 regions.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400640 2 Id-DRIVEN ANTI-dsDNA AUTOANTIBODIES

Materials and Methods T7 promoter of the pGEM-T Easy vector (GATC Biotech). At least four Mice colonies were sequenced for each sample. Mice were TG for both the l2315 Ig L-chain derived from the MOPC315 IgVH sequence processing and analysis myeloma, as well as an ab TCR transgene specific for the Id(l2315) d The following control data sets were downloaded from the National Center peptide presented on I-E MHC class II molecules (32, 33). The TCR for Biotechnology Information (NCBI) Web site (http://www.ncbi.nlm.nih. l 315 specificity is toward the 2 CDR3 motif that includes three replacement gov/nuccore/): control VH sequences from BALB/c mice, retrieved with mutations (CALWFRNHFVFGG) (23, 24). the search term “V region immunoglobulin heavy chain Balb”; sequences 3 + DTG F1 mice were from TCR transgenic (homozygous females) Id derived from splenic L2-TG mice IgG+ B cells, as deposited (35); anti- transgenic (homozygous males); all offspring are DTG. Alternatively, to DNA hybridomas from BWF1 mice, as deposited (36); and neonatal liver generate littermate controls (Fig. 1), offspring from TCR transgenic B cell IgVH sequences, as deposited (37). 3 + (hemizygous females) Id transgenic (hemizygous males) were ana- Sequences were analyzed with the International ImMunoGeneTics . lyzed. Both TG strains are on a BALB/c background ( 20 backcrosses). (IMGT)/HighV-QUEST version 1.1.2 or IMGT/HighV-QUEST version The Norwegian Animal Research Authority approved the experiments. 3.2.30 tools (http://imgt.cines.fr) and compared with the IMGT/V-QUEST reference directory release: 201310-4 (March 14, 2013) (38). IgVH region analysis family identification and clonality analysis were performed using the statistics module of IMGT/HighV-QUEST. Statistics were reported only Mice were weighed and monitored for disease, and hind leg saphenous vein for unique sequences (38). Translated amino acid sequences were analyzed blood samples were drawn every 10 d for autoantibody analysis. Diluted further in Excel (Microsoft); amino acids in individual positions were serum samples were analyzed for the presence of anti-dsDNA Abs or other counted with the “countif” function. Phylogenetic trees were constructed autoantibodies, as previously described (32, 33), HEp-2 cells (Immuno and visualized with the iTOL program (http://itol.embl.de). Data sets were Concepts) and Crithidia luciliae (Binding Site) served as substrates; bound compared with the two-sided Fisher exact test, two-sided x2 test, or the autoantibodies were stained with Alexa Fluor–488/546 goat anti-mouse Wilcoxon signed-rank test. Downloaded from IgG [F(ab9)2], Alexa Fluor–488/546 goat anti-mouse IgG2a, and Alexa Fluor–488/546 goat anti-mouse IgG1 (Molecular Probes). Hybridomas Analysis of Ag-driven selection of IgVH sequences were screened by an ELISA-based measurement of Abs toward confluent ethanol-fixed HEp-2 cell monolayers. Bound Abs were detected by anti- Clonally related sequences in the green and blue families of mouse 21 were mouse IgG (Fc-specific) peroxidase conjugate (Sigma-Aldrich). tested for evidence of Ag-driven selection in IgVH sequences with Base- line Version 1.1, focused selection statistics (http://selection.med.yale.edu/ Flow cytometry and cellular assays. Fetal calf DNA (Sigma-Aldrich) was baseline/) as described (39). digested into 500-bp fragments (DNase) and labeled with Alexa Fluor http://www.jimmunol.org/ 488 with the ULYSIS Nucleic Acid Labeling Kit (Molecular Probes), according to the manufacturer’s protocol. CpG 2006–fluorescein (tlrl- Results 2006f) with human TLR9-binding motifs was from InvivoGen. Cell Anti-dsDNA autoantibodies are frequently expressed in DTG suspensions were incubated for 10 min at 37˚C with DNase I from mice bovine pancreas (Sigma-Aldrich), washed four times, stained with DNA, and counterstained with fluorochrome-coupled anti-B220 (RA3-6B2; We analyzed sera from DTG mice (32, 33) and found IgG1 and Southern Biotech), anti-CD19 (1D3), and anti-CD4 (RM4-5; both from BD IgG2a anti-dsDNA autoantibodies from age 6 wk (Fig. 1A). High- Pharmingen). DNA–Alexa Fluor 488 and CpG 2006–fluorescein were titer autoantibodies stained nuclei with a predominantly homoge- tested on murine anti-dsDNA lymphoma (positive control; A. Funderud, nous pattern, and anti-dsDNA autoantibodies were found toward K. Aas-Hanssen, B. Bogen, and L.A. Munthe, unpublished observations) and A20 murine B cell lymphoma cells (negative control, ATCC TIB-208). the kinetoplast of C. luciliae (Fig, 1B, 1C). In addition, DTG mice by guest on September 30, 2021 Cells were fixed with paraformaldehyde before acquisition. DTG spleno- had variable autoantibodies of lower titer toward a range of other cytes or BALB/c controls were cultured with Th2 cells from Id-specific autoantigens, including cytosolic Ags (Fig. 1B, 1C) and extracellular TCR-TG SCID mice, as described (31). BrdU was added on day 3, and Ags (data not shown) (32, 33). This feature resembles that found in cells were stained on day 5 with the BrdU-APC Staining Kit (BD), in- cluding surface stains for B220, CD4, and CD19. Cells were acquired on SLE patients who often have autoantibodies with other autospeci- a FACSCalibur (BD) and analyzed with FlowJo X software (TreeStar). The ficities, in addition to high-titer anti-dsDNA. Thus, an interindividual Mann–Whitney U test was used to compare groups. variance was seen in addition to the dominant anti-dsDNA reactivity, a finding that was reminiscent of human SLE serology. Cells and hybridomas Spleens, lymph nodes, kidneys, and blood were collected from euthanized DNA-specific B cells are of a high frequency in DTG mice and mice, and cell suspensions and serum samples were made as previously can be activated by Id-specific Th cells described (32, 33). Hybridomas were generated by mixing splenocytes We proceeded to investigate whether B cells in DTG mice could bind with OURI cells (variant of X63-Ag8.653), followed by the drop-wise . addition of polyethylene glycol (Roche). Hybridomas were selected with DNA. B cells from spleens and lymph nodes of 115-d-old DTG hypoxanthine/aminopterin/thymidine-supplemented RPMI 1640 medium mice with symptoms of disease were isolated in the presence of DNase, (Sigma-Aldrich) and cloned by limiting dilutions. Clones were screened washed, and tested for binding to Alexa Fluor 488–conjugated for autoantibodies, as above. Sixteen hybridomas were further subcloned. dsDNA or CpG 2006–fluorescein (the latter with human TLR9- Amplification of IgVH sequences binding motifs). An increased frequency of splenic and lymph node B cells from DTG mice bound dsDNA (4 and 7% respectively) or mRNA isolation was performed on washed pelleted cells with a Dynabeads  mRNA DIRECT Kit (Life Technologies), according to the manufacturer’s CpG 2006 DNA ( 17%) (Fig. 2A, 2B, Supplemental Fig. 1). Id- recommendations. Reverse transcription was performed using a First- specific Th cells stimulated the blastogenesis and proliferation of Strand cDNA Synthesis Kit (Amersham Biosciences), according to the Id+ B cells from DTG mice (Fig. 2C, Supplemental Fig. 1), including manufacturer’s protocol. Reverse transcription was performed using the the proliferation of the dsDNA-specific subset of B cells (Fig. 2D). Not I-d(T)18 primer (First-Strand cDNA Synthesis Kit; Amersham Bio- sciences). cDNA was amplified by PCR using PfuTurbo DNA Polymerase Sequencing of IgVH from DTG mice reveals an increased (Stratagene) and a mixture of 59 H-chain FR1 region degeneracy primers, mutation rate, as well as oligoclonal expansions of B cells together with a mixture of 39 H-chain constant (C)-region primers (34) (Sigma-Genosys). The PCR products were run on a 1.5% agarose gel, and To investigate the clonal relationship of the observed anti-dsDNA gel-purified products of predicted size (400 bp) (QIAquick Gel Extrac- B cells, we selected anti-dsDNA hybridomas and compared IgVH tion Kit; QIAGEN) were ligated into pGEM-T Easy vector (Promega) and sequences of hybridomas with those derived from lymphoid organs used to transform One Shot chemically competent E. coli TOP10 cells (Life Technologies). Plasmid DNA was prepared from overnight cultures from the same mice. A total of 55 hybridomas was generated by (Wizard Plus SV Minipreps DNA Purification System; Promega), and conventional means from three mice undergoing systemic auto- colonies found to contain an insert were sequenced using a primer to the , selecting IgG1 and IgG2a producers that gave bright The Journal of Immunology 3

FIGURE 1. Development of anti-dsDNA Abs in DTG mice. Sera from DTG mice were compared with sera from singly TG (TCR-TG or Id+-TG) and non-TG mice. (A) Homogenous ANA titers of IgG1, IgG2a in sera obtained from DTG mice and littermates as a function of age (n . 7/group). (B) Frequency of positive sera of the indicated specificity. ANA, anti-cytoplasmic, and anti-dsDNA (C. luciliae) specificities are indicated. (C) Staining pattern of autoantibodies from DTG mice. The dominant anti-nuclear stain is shown (upper left) compared with control sera (BALB/c, upper right)(upper panel, original magnification 3400). Examples of anti-kinetoplast stain (C. luciliae) of IgG1 or IgG2a anti-dsDNA are indicated (lower panel, original mag-

nification 31000). Variant nuclear and cytoplasmic staining patterns of HEp-2 cells by sera from DTG. (i) Cytoskeleton + coarse nuclear; (ii) course/ Downloaded from homogenous nuclear + nuclear dots; (iii) homogenous nuclear + nuclear dots + cytoskeletal stain; (iv) homogenous nuclear + endoplasmic reticulum/ Golgi stain. signals. The majority of these hybridomas secreted ANA auto- from BALB/c mice; and IgVH derived from IgG+ anti-DNA with a homogenous nuclear pattern (data not shown). hybridomas from BWF1 mice, as published (36). We found that

Sixteen hybridomas derived from mouse numbers 21, 15, and 5 the mutation rate in the DTG sequences was increased and sig- http://www.jimmunol.org/ were subcloned, and the VH was sequenced. IgVH sequences were nificantly different from L2-TG, BALB/c, and anti-DNA hybrid- obtained from lymph nodes and spleens of mice 21 and 5. Only omas (Fig. 3A, p , 0.0001, Wilcoxon signed-rank test). a few sequences were obtained from mouse 15. To investigate clonality, we plotted VH family usage in DTG and Altogether, .600 IgVH sequences were obtained; 176 of these L2-TG mice and found that DTG mice had a skewed VH family were unique. Analyzing the V regions (excluding CDR3), we usage (Fig. 3B). These families also were increased compared found that these 176 sequences had an average of 9.6 mutations with the BALB/c dataset [note that distribution of L2-TG differs (Fig. 3A). We compared these sequences with data sets down- from the distribution of BALB/c, presumably related to the pre- loaded from the NCBI Web site: IgG IgVH from an Id+l2TG ponderance of marginal zone B cells in L2-TG mice (35, 37)]. strain (on the BALB/c background) generated by another group These results suggested that the sequences from DTG mice could by guest on September 30, 2021 (35), called L2-TG in this article; productive IgVH sequences be oligoclonal. To confirm this, a clonality analysis was performed

FIGURE 2. Quantification of anti-dsDNA B cells in DTG mice and collaboration with Id-specific Th2 cells. (A) Binding of fluorochrome-labeled DNA or CpG 2006 DNA to B220+ B cells of DTG mice compared with BALB/c control. Ungated spleen cells, DNA-Alexa Fluor 488 versus B220 expres- sion (upper panels). Ungated lymph node (LN) cells. CpG 2006–fluorescein versus B220 expres- sion is shown (lower panels). (B) Frequency of DNA- and CpG 2006–binding B220+ B cells from lymph nodes or spleens of DTG (d) or BALB/c (s) mice. Data are mean + SEM (**p , 0.008, Mann–Whitney U test). (C) Proliferation (BrdU incorporation) versus size (Fsc, left panels)or versus l2 L chain expression (right panels) in gated CD19+ B cells from a DTG mice mouse (age 24 wk) that was cultured or not with Id-specific Th2 cells. (D) Proliferation (BrdU incorporation) versus DNA binding in gated splenic B220+ B cells cul- tured or not with Id-specific Th2 cells. DTG (1) through DTG (4) correspond to mice shown in Supplemental Fig. 1B. DTG (1) through DTG (3) were between 24 and 25 wk old. DTG (4) was 42 wk old (end-stage disease). See also Supplemental Fig. 1. 4 Id-DRIVEN ANTI-dsDNA AUTOANTIBODIES Downloaded from http://www.jimmunol.org/

FIGURE 3. Global analysis of IgVH sequences in DTG mice compared with controls. All nonrepeat (unique) IgVH sequences from DTG mice and controls were subjected to the IMGT/HighV-QUEST and statistics module test. Control data sets: L2-TG denote B cell IgG VH sequences from adult singly l2315-TG mice, as downloaded from the NCBI Web site and described in (35); BALB/c IgVH sequences were retrieved from NCBI; and anti-DNA IgG VH sequences were derived from IgG+ hybridomas of BWF1 mice, as published (36). (A) Sequences in data sets with the indicated numbers of replacement mutations in unique sequences identified by IMGT/HighV-QUEST analysis. The total numbers of mutations (mut) and average mutations per sequence

(mut/seq) in each data set are shown. (B) Distribution of V gene usage in the DTG sequences compared with the L2-TG and BALB/c control data sets. Data by guest on September 30, 2021 are only shown for V genes represented in the DTG or L2-TG data set. IMGT nomenclature is used. The corresponding NCBI segment names of the most frequent families (in parenthesis): IGHV1S81 (J558.33), IGHV1-14 (J558.47), IGHV1-69 (J558.40), and IGHV1-7 (J558.45). (C) Analysis of clonal re- latedness in DTG sequences compared with control data sets, as defined by CDR3 sequences and CDR3 lengths. Only unique sequences that can be attributed to a single V gene are shown. White bars represent sequences that have unique V gene sequences, as well as unique CDR3 sequences. Black bars represent members that have different V gene sequences but identical CDR3 (in terms of nucleotide sequence) (i.e., these sequences are clonally related sets). Controls: neonatal L2-TG IgVH sequences were derived from singly l2315-TG mice (37). BALB/c sequences correspond to the BALB/c data set in (A).

(IMGT/V-high, statistics module). This module analyzes the se- CDR3 sequence was plotted from position 3 in Wu-Kabat–like plots quences that can only be attributed to single alleles (excluding se- (Fig. 4A). The anti-dsDNA hybridomas expressed one to four ad- quences that can belong to more than one allele). A total of 47% of ditional arginines (average 2.5) in their CDR3s. This feature was not the 137 DTG single-allele sequences that were analyzed in this found in the negative-control data sets (Fig. 4A). When analyzing statistics module were clonally related; these VDJ sequences the sequences from the lymph node and spleen, similar results were belonged to sets that had identical CDR3 sequences but differed in found; the sequences had, on average, 1.1 additional CDR3 argi- the V regions (Fig. 3C). Neonatal L2-TG sequences were chosen nines compared with 0.3 arginines in BALB/c sequences (Fig. 4B, as negative controls (representing sequences prior to any Ag se- data not shown). These CDR3 arginines had similar CDR3 posi- lection and clonal evolution); this data set, as well as the unbiased tional distributions as did the hybridomas from the same mice, BALB/c sequences, had minimal clonal relatedness. a phenomenon that was especially clear in mouse 5. The BALB/c control sequences had one additional R in CDR3 of every third Anti-dsDNA–specific Abs express multiple arginines in CDR3s sequence, whereas 2 Rs were found in ,0.1% of the sequences The analysis above demonstrated that nearly half of the sequences (2/2231). In comparison, two or more CDR3 arginines were in the DTG data set could be grouped into a few clonally related found in 34% (54/161) of the DTG lymph node/spleen/kidney families of B cells. In terms of the anti-dsDNA specificity, positively sequences (Fig. 4C, p , 0.0001, two-sided x2 test). charged amino acids and, especially, arginines (Rs) are frequent in anti-dsDNA mAbs, representing a hallmark for these Abs (9). CDR3 Anti-dsDNA B cells are markedly expanded in mice undergoing junctions most often begin with the amino acids cysteine (C), ala- Id-driven T–B collaboration nine (A), and arginine (R) (CAR): AR are the first two CDR3 amino Arginine-rich CDR3 sequences are hallmarks of anti-dsDNA acids. Discounting this first arginine (in position 2), a single addi- specificity, and very few (,0.1%) of the BALB/c sequences had tional R in CDR3 may be sufficient for binding to dsDNA, and two or more additional CDR3 arginines. Therefore, we chose at multiple substitutions to R were shown to have an additive effect least two additional CDR3 arginines as a cutoff to identify prob- on affinity (9). Disregarding this first CAR arginine, the DTG able anti-dsDNA–reactive families, which were verified by clonal The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ FIGURE 4. Analysis of CDR3 sequences of anti-dsDNA hybridomas and tissue B cells from DTG mice. CDR3 sequences from DTG mice and controls (as in Fig. 3) were analyzed, and amino acids were quantified. The V segment terminates in a cysteine, usually followed by an alanine (position 1 of CDR3) and an arginine, position 2 (i.e., C-A1R2). Amino acids were counted from position 3. (A) The percentage of amino acids (N, asparagine; R, arginine; K, lysine) in the indicated CDR3 positions. CDR3 N, R, K in anti-dsDNA hybridomas obtained from DTG mice 21, 5, and 15 (left panels). CDR3 from anti-dsDNA hybridomas derived from BWF1 mice with lupus disease [positive control (36)], CDR3 from BALB/c IgVH data set (negative control), and CDR3 from L2-TG mice (negative control) (right panels). (B)N,R,KofCDR3regions,asin(A), obtained from lymph nodes and spleen B cells of DTG mice 21 and 15. (C)Numberofarginines/ CDR3 sequences of the lymph node/spleen B cells and anti-dsDNA hybridomas derived from mice 21, 5, and 15 and controls, as in (A).

relatedness to anti-dsDNA hybridomas. Hence, we identified clon- members with CDR3 coding two or three arginines (mean 2.1), by guest on September 30, 2021 ally related families with anti-dsDNA–binding properties. Phylo- and it included six hybridomas (with verified anti-dsDNA speci- genetic trees were generated for all unique sequences in the ficity) in addition to the sequences from the lymphoid organs. The mice. We identified two large families in mouse 21 (Fig. 5A, second family (green family in Fig. 5) had 18 members and did Supplemental Fig. 2). The first, colored blue (blue family), had 22 not include hybridomas, but it had as many as two or three argi-

FIGURE 5. Phylogenetic tree of IgVH sequences generated from hybridomas and tissues of DTG mouse 21. (A) Phylogenetic tree generated from unique IgVH sequences obtained from DTG mouse 21. Blue or green color indicates that these sequences share the same junctions and are classified as belonging to the blue family and green family. A family in the right part of the phylogenic tree has CDR3 sequences with the YYGS motif (indicated). (B) Sequences with two or more arginines in CDR3 ($2R, total percentage, and number of sequences are indicated) identified the clonally related blue and green families, as in (A), five single-member families in mouse 21 (“Other”) (left panel), as well as the red family in mouse 5 (right panel). (C) Examples of similarities between anti-dsDNA VH CDR3 sequences obtained from DTG mice HVH-15-4, SVH-5-392, SVH-5-505, and LNVH-21-675 (blue/red families as in- dicated), as well as from lupus-prone mice reported in the literature. Sequences AAB49106.1 and AAB49043.1 are derived from BWF1 mice (36); the lower right example is from BALB gld/gld (AAO60126.1). See Supplemental Fig. 2 for enlargement of the phylogenetic tree in (A) and identification of single-member families with at least two arginines in CDR3. See Supplemental Table I for IgVH junction analysis. HVH, hybridoma IgVH; LNVH, lymph node IgVH; SVH, spleen IgVH. 6 Id-DRIVEN ANTI-dsDNA AUTOANTIBODIES nines (average 2.8) in the CDR3 sequences, strongly suggesting anti-dsDNA reactivity. Some of these members also were rei- dentified [i.e., found repeatedly (n = 17)], suggesting clonal ex- pansion of B cells with identical VH. These two families (green + blue) represented 33% of the unique (40/120) sequences, thus demonstrating a marked clonal expansion. In addition, we found five other sequences with more than two arginines, resulting in an anti-dsDNA total of 37% (45/120) in this mouse (Fig. 5B, Supplemental Fig. 2). Further, mouse 21 had a clonally related family (19/120), with the conserved CDR3 motif YYGSS asso- ciated with anti-DNA/nucleosome binding (40). This family ex- pressed YYYGSSR (Fig. 5A, left panel; Supplemental Fig. 2). In mouse 5, a family called red contained 16 members (including three anti-dsDNA hybridomas) and lymph node/spleen sequences with three or four arginines in CDR3 (Fig. 5B). We found similarities between CDR3 of our anti-dsDNA VH sequences and previously published sequences from lupus-prone mice. Examples are shown in Fig. 5C. Downloaded from The clonally expanded, dominant anti-dsDNA B cell clones are derived from B cells pre-expressing arginine in CDR3 FIGURE 6. Analysis of mutations in CDRs of anti-dsDNA B cells in DTG mice. CDR1, CDR2, and CDR3 sequences from the anti-dsDNA blue, The clonal relationships of sequences within the blue, green, and red green (mouse 21), and red family (mouse 5), as well as sequences from families were confirmed by VDJ analysis, where members had hybridomas of mouse 15, were analyzed for mutations affecting the various identical D and J genes and had the same CDR3 length (Supplemental amino acids. Shown is mutation rate to amino acids/sequence in the DTG mice or in the BALB/c control data set. The amino acids are arranged

Table I). The blue and red families had nontemplated nucleotide http://www.jimmunol.org/ additions, allowing the D gene segment to be read in an altered according to their frequency in CDR3 of the DTG sequences (top panel). reading frame, introducing one arginine to CDR3 (both with argi- nine in position 5), an event that probably was acquired in the bone identified by IgVH sequencing, were clonally related. Anti-dsDNA marrow during VDJ rearrangement. Somatic mutation accounted B cells accounted for up to 30% of the B cells (detected by flow for a second arginine in position 7 in all members of the blue family. cytometry) and nearly 40% of the IgVH (clonally related families Members of the red family had two additional arginines (position 4 with more than two arginines in CDR3). Anti-dsDNA B cells were and 6) due to somatic mutation (NCBI submission #1691092). stimulated to proliferate by Id-specific Th cells. The B cells expressed mutated -switched IgG with high levels of argi- Analysis of mutations reveals a selection of arginines in CDR3 nines in CDR3, and evidence was found for Ag-driven selection of by guest on September 30, 2021 and evidence of an Ag-driven response V-region sequences. CDR3 had sequence homologies to anti-DNA Using the hybridomas to identify anti-dsDNA B cells in DTG mice, CDR3 sequences from classical mouse models of lupus. we found that these IgVH sequences were characterized by increased Arginine amino acids in CDR3 characterizes anti-dsDNA Abs numbers of arginines in CDR3. On average, these B cells had 1.7 (10, 11). The arginine side chain can form bidentate interactions arginines caused by somatic mutation (72 mutations to arginines with G–C bp (12), as well as electrostatic interactions with compared with 88 mutations to other amino acids, Fig. 6A). This phosphate groups. One additional CDR3 arginine can be sufficient very high mutation rate to arginine was significantly different from for DNA binding (11). The results obtained demonstrate that the mutation to arginine in CDR3 of BALB/c controls (101 muta- Id-driven T cell–B cell collaboration could support clonal expan- tions to R, 1247 mutations to other amino acids; BALB/c versus sion of arginine-rich classical cationic anti-dsDNA Ab–expressing DTG, p , 0.0001, two-sided Fisher exact test). B cells by two mechanisms: selection of B cells with a priori It was of interest whether the B cells with arginine-rich CDR3 arginines in CDR3 and further expansion of such B cells, fol- also expressed mutations to arginine or other amino acids in CDR1 lowed by . The clonal families either and CDR2. Fig. 6 shows the frequency of amino acid mutations in had alternative reading frames of the D region, resulting in a the anti-dsDNA DTG data set compared with BALB/c control CDR3 arginine (red and green families), or used a D region (NCBI Web site). In general, lower mutation rates were observed coding for one arginine (blue family). Clonal expansion was accom- for CDR1 and CDR2, but mutations to arginines also were panied by further mutations to additional arginines. increased compared with BALB/c in CDR1 and CDR2. To test Our previous studies demonstrated that B cell Ag presentation whether these mutations were evidence of selection within the V (of Id) in itself is insufficient for the generation of the germinal region (disregarding CDR3), we used Bayesian estimations of Ag- center reaction: B cells also require BCR ligands for the response driven selection in Ig sequences (39). We found that clonally re- (31). Contingent upon the presence of DNA, naive anti-dsDNA lated members of the blue and green families had evidence of B cells with one or two CDR3 arginines could be helped by the significant positive selection, showing higher than expected re- low-frequency Id-specific Th cells in the mice to isotype switch placement mutations in CDR1 and CDR2 compared with FR to IgG and accumulate one or two additional CDR3 arginines by regions (p = 0.03 for both). The red family, with fewer members, somatic hypermutation. Reciprocally, it is possible that such Th had the same tendency, but the data did not reach significance. cell–B cell collaboration could play a central role in the expansion of the Id-specific Th cells. The early debut (6 wk) of anti-dsDNA Discussion Abs may be explained by such Th cell–B cell collaboration. In In this study, we demonstrate a marked clonal expansion of pro- comparison, MRL/lpr mice also develop anti-dsDNA responses totypical anti-dsDNA B cells in DTG mice developing a systemic from week 6 (average 9–10 wk) (41), whereas BWF1 mice autoimmune disease with SLE-like features. Nearly 50% of B cells, seroconvert later, from 4 mo of age (7). The Journal of Immunology 7

SLE has been associated with incomplete clearance of apop- anti-dsDNA B cells of normal mice (50), as well as BWF1 mice totic cells. In fact, the level of nucleosomes in the circulation is (17, 18), contain Id CD4+ T cell . Further, Id-specific significantly increased in patients with active disease. Hence, CD4+ T cells have been found in humans suffering from SLE nucleosomal material, including dsDNA, is accessible for BCR (20–22). ligation (42). However, B cells that are stimulated through the The results demonstrate that Id peptides have the potential to BCR in the absence of T cell help develop into anergic cells and constitute Th cell Ag in anti-dsDNA B cell responses in lupus. apoptose (reviewed in Ref. 43). Ag-specific Th cells can negate Another line of evidence implicated Id-specific Th cells in lupus: it anergy and support conventional immune responses, including was suggested that pathogenic autoreactive Id+ anti-DNA Abs are germinal center reactions, development of plasma cells, and au- regulated and counteracted by anti-Id Abs. In fact, early studies toantibody secretion (13–15, 44). Hence, anti-dsDNA B cell demonstrated that Id+ anti-DNA Abs in healthy individuals were responses are dependent on help from Th cells, and both Th2 blocked by anti-Id Abs and that titers of such anti-Id Abs corre- cells and follicular Th cells have been directly linked to SLE lated negatively with disease progression (51–53). A similar (5, 6, 44). phenomenon was described more recently for anti-GAD65 auto- Yet, because Th cells cannot be activated in a cognate manner antibodies in type 1 diabetes (54). How are such results related to by DNA, the drive for this Th cell–B cell interaction requires an Id-specific Th cells? It is increasingly clear that Abs are conven- additional level of complexity. Conceptually, anti-dsDNA B cells tional Ags for Th cell and B cell immune responses (55, 56). Thus, can either present peptide derived from endocytosed DNA-bound anti-Id B cells that can bind a particular Ab (e.g., an Id+ anti- nuclear self-proteins or present endogenously derived (B cell–in- dsDNA Ab) internalize this Id+ Ab and may elicit help from Id- trinsic) peptides to Th cells. A third possibility is a combination of specific Th cells, the same Th cells that initially could have de- Downloaded from these two concepts: Th cells could cross-react to cationic peptides livered Id-dependent help to the anti-dsDNA Id+ B cell (55, 56). In derived from DNA-binding proteins, regardless of derivation, in- these terms, pathogenic Abs are counter-balanced by Id-driven cluding both nuclear self-proteins and anti-DNA Ids (see later help of anti-Id B cells. Further studies into such circuits are discussion). warranted in SLE patients. In the first situation, endocytosed DNA-binding nuclear proteins, such as histone determinants, could serve as Th cell Ag (45). In the Acknowledgments http://www.jimmunol.org/ second situation, Th cell help of anti-dsDNA B cells would be We thank Hilde Omholt and Peter O. Hofgaard for technical help and dependent upon presentation of endogenous secretory pathway Marianne Frøyland for advice on methods; the Department of Comparative Ags. Examples include self-proteins derived from the secretory Medicine, Oslo University Hospital, for animal care; and Inger Øynebra˚ten pathway, such as unique Ids of the B cell clone (27, 28, 46), as and Peter O. Hofgaard for critical reading of the manuscript. shown in this study, presentation of peptides derived from viral proteins synthesized by B cells (47), or alloantigens in chronic Disclosures graft-versus-host models of SLE that stimulated oligoclonal The authors have no financial conflicts of interest. expansion of arginine-rich anti-dsDNA B cells (48). In this study, anti-DNA B cells bind DNA while receiving allospecific by guest on September 30, 2021 T cell help. References The current results demonstrate that Id-specific Th cells can 1. Lipsky, P. E. 2001. Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity. Nat. Immunol. 2: 764–766. reproduce classical anti-dsDNA B cell responses. However, it is 2. Vinuesa, C. G., I. Sanz, and M. C. Cook. 2009. Dysregulation of germinal interesting to note that DNA-associated proteins and anti-dsDNA centres in autoimmune disease. Nat. Rev. Immunol. 9: 845–857. 3. Kotzin, B. L. 1996. Systemic lupus erythematosus. Cell 85: 303–306. Abs (CDR3) both have positively charged DNA-binding motifs. 4. Shlomchik, M. J., J. E. Craft, and M. J. Mamula. 2001. From T to B and back It is possible that the seemingly dissimilar Th cell specificities again: positive feedback in systemic autoimmune disease. Nat. Rev. Immunol. for cationic CDR3 Ids and cationic DNA-binding peptides could 1: 147–153. 5. Vinuesa, C. G., M. C. Cook, C. Angelucci, V. Athanasopoulos, L. Rui, constitute networks of molecular mimics for Th cells in SLE. For K. M. Hill, D. Yu, H. Domaschenz, B. Whittle, T. Lambe, et al. 2005. A RING- example, histone H4(71-94) was described to be a Th cell type ubiquitin ligase family member required to repress follicular helper T cells in lupus mice and in SLE patients. This peptide contains the fol- and . Nature 435: 452–458. 6. Charles, N., D. Hardwick, E. Daugas, G. G. Illei, and J. Rivera. 2010. lowing DNA-associated motif: HAKRKTVTAMD (45). The se- and the T helper 2 environment can promote the development of lupus nephritis. quence is comparable to CDR3 from several previously described Nat. Med. 16: 701–707. 7. Theofilopoulos, A. N., and F. J. Dixon. 1985. Murine models of systemic lupus anti-DNA CDR3 peptides: for example CARRRTGTAYY [Lx- erythematosus. Adv. Immunol. 37: 269–390. 163, submitted by (49)]. If Th cells respond to cationic Id pep- 8. Gilkeson, G. S., J. P. Grudier, D. G. Karounos, and D. S. Pisetsky. 1989. In- tides of such B cells, it could be hypothesized that established Th duction of anti-double stranded DNA antibodies in normal mice by immuniza- tion with bacterial DNA. J. Immunol. 142: 1482–1486. cell responses potentially cross-react to peptide mimics from other 9. Radic, M. Z., and M. Weigert. 1994. Genetic and structural evidence for DNA-binding proteins, including histones. selection of anti-DNA antibodies. Annu. Rev. Immunol. 12: 487–520. In terms of this hypothesis, it is notable that most DNA-binding 10. Shlomchik, M., M. Mascelli, H. Shan, M. Z. Radic, D. Pisetsky, A. Marshak- Rothstein, and M. Weigert. 1990. Anti-DNA antibodies from autoimmune mice proteins are ubiquitously expressed; T cell tolerance mecha- arise by clonal expansion and somatic mutation. J. Exp. Med. 171: 265–292. nisms are likely to exert a strong tolerizing pressure to delete 11. Radic, M. Z., J. Mackle, J. Erikson, C. Mol, W. F. Anderson, and M. Weigert. 1993. Residues that mediate DNA binding of autoimmune antibodies. J. histone/DNA-binding protein-specific T cells. In contrast, Ids are Immunol. 150: 4966–4977. unique, private proteins of a particular B cell clone. Thus, bone 12. Seeman, N. C., J. M. Rosenberg, and A. Rich. 1976. Sequence-specific recog- marrow emergent B cells with anti-dsDNA–binding V regions nition of double helical nucleic acids by proteins. Proc. Natl. Acad. Sci. USA 73: 804–808. and positively charged CDR3 could fortuitously encounter rare 13. Seo, S. J., M. L. Fields, J. L. Buckler, A. J. Reed, L. Mandik-Nayak, S. A. Nish, Th cells specific for the unique Id of that B cell. B cells express R. J. Noelle, L. A. Turka, F. D. Finkelman, A. J. Caton, and J. Erikson. 2002. The relatively high levels of Id+ Ig; Id peptides were among the first to impact of T helper and T regulatory cells on the regulation of anti-double- stranded DNA B cells. Immunity 16: 535–546. be eluted from MHC class II molecules of B cells (46). Hence, 14. Cook, M. C., A. Basten, and B. Fazekas de St Groth. 1998. Rescue of self- B cells process and present their endogenous BCR as short Id reactive B cells by provision of T cell help in vivo. Eur. J. Immunol. 28: 2549–2558. peptides on MHC class II to T cells that may recognize V-region 15. Fulcher, D. A., A. B. Lyons, S. L. Korn, M. C. Cook, C. Koleda, C. Parish, Id peptides (27–30). It was shown that V regions derived from B. Fazekas de St Groth, and A. Basten. 1996. The fate of self-reactive B cells 8 Id-DRIVEN ANTI-dsDNA AUTOANTIBODIES

depends primarily on the degree of antigen receptor engagement and availability 36. Krishnan, M. R., N. T. Jou, and T. N. Marion. 1996. Correlation between the of T cell help. J. Exp. Med. 183: 2313–2328. amino acid position of arginine in VH-CDR3 and specificity for native DNA 16. Knupp, C. J., A. H. Uner, C. Korthas, and J. Gavalchin. 1993. Characterization of among autoimmune antibodies. J. Immunol. 157: 2430–2439. IdLNF1-specific T cell clones from the (NZB x SWR)F1 murine model for 37. Kretschmer, K., H. Engel, and S. Weiss. 2002. Strong antigenic selection shaping systemic lupus erythematosus. Clin. Immunol. Immunopathol. 68: 273–282. the immunoglobulin heavy chain repertoire of B-1a in lambda 17. Singh, R. R., V. Kumar, F. M. Ebling, S. Southwood, A. Sette, E. E. Sercarz, and 2(315) transgenic mice. Eur. J. Immunol. 32: 2317–2327. B. H. Hahn. 1995. T cell determinants from autoantibodies to DNA can up- 38. Li, S., M. P. Lefranc, J. J. Miles, E. Alamyar, V. Giudicelli, P. Duroux, regulate autoimmunity in murine systemic lupus erythematosus. J. Exp. Med. J. D. Freeman, V. D. Corbin, J. P. Scheerlinck, M. A. Frohman, et al. 2013. 181: 2017–2027. IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity 18. Singh, R. R., and B. H. Hahn. 1998. Reciprocal T-B determinant spreading and next generation repertoire immunoprofiling. Nat. Commun. 4: 2333. develops spontaneously in murine lupus: implications for pathogenesis. Immu- 39. Yaari, G., M. Uduman, and S. H. Kleinstein. 2012. Quantifying selection in high- nol. Rev. 164: 201–208. throughput Immunoglobulin sequencing data sets. Nucleic Acids Res. 40: e134. 19. Feng, F., C. J. Silvin, N. C. Fiore, M. L. Stoll, K. E. Price, P. S. Shanley, 40. Tsao, B. P., F. M. Ebling, C. Roman, N. Panosian-Sahakian, K. Calame, and A. E. Silverstone, and J. Gavalchin. 2012. 17b-Estradiol (E-2) administration to B. H. Hahn. 1990. Structural characteristics of the variable regions of immu- male (NZB 3 SWR)F₁ mice results in increased Id(LN)F₁-reactive memory noglobulin genes encoding a pathogenic autoantibody in murine lupus. J. Clin. T-lymphocytes and accelerated glomerulonephritis. Lupus 21: 288–301. Invest. 85: 530–540. 20. Williams, W. M., N. A. Staines, S. Muller, and D. A. Isenberg. 1995. Human 41. Mandik-Nayak, L., S. J. Seo, C. Sokol, K. M. Potts, A. Bui, and J. Erikson. 1999. T cell responses to autoantibody variable region peptides. Lupus 4: 464–471. MRL-lpr/lpr mice exhibit a defect in maintaining developmental arrest and 21. Dayan, M., R. Segal, Z. Sthoeger, A. Waisman, N. Brosh, O. Elkayam, E. Eilat, follicular exclusion of anti-double-stranded DNA B cells. J. Exp. Med. 189: M. Fridkin, and E. Mozes. 2000. Immune response of SLE patients to peptides 1799–1814. based on the complementarity determining regions of a pathogenic anti-DNA 42. Rumore, P. M., and C. R. Steinman. 1990. Endogenous circulating DNA in monoclonal . J. Clin. Immunol. 20: 187–194. systemic lupus erythematosus. Occurrence as multimeric complexes bound to 22. Mendlovic, S., Y. Shoenfeld, R. Bakimer, R. Segal, M. Dayan, and E. Mozes. histone. J. Clin. Invest. 86: 69–74. 1988. In vitro T-cell functions specific to an anti-DNA idiotype and serological 43. Cambier, J. C., S. B. Gauld, K. T. Merrell, and B. J. Vilen. 2007. B-cell anergy: markers in patients with systemic lupus erythematosus (SLE). J. Clin. Immunol. from transgenic models to naturally occurring anergic B cells? Nat. Rev. 8: 178–187. Immunol. 7: 633–643. Downloaded from 23. Hannestad, K., G. Kristoffersen, and J. P. Briand. 1986. The T re- 44. Linterman, M. A., R. J. Rigby, R. K. Wong, D. Yu, R. Brink, J. L. Cannons, sponse to syngeneic lambda 2 light chain idiotopes. Significance of individual P. L. Schwartzberg, M. C. Cook, G. D. Walters, and C. G. Vinuesa. 2009. Follicular amino acids revealed by variant lambda 2 chains and idiotope-mimicking helper T cells are required for systemic autoimmunity. J. Exp. Med. 206: 561–576. chemically synthesized peptides. Eur. J. Immunol. 16: 889–893. 45. Datta, S. K. 2003. Major peptide autoepitopes for nucleosome-centered T and 24. Bogen, B., B. Malissen, and W. Haas. 1986. Idiotope-specific T cell clones that B cell interaction in human and murine lupus. Ann. N. Y. Acad. Sci. 987: 79–90. recognize syngeneic immunoglobulin fragments in the context of class II mol- 46. Rudensky AYu, P., B. K. Preston-Hurlburt, J. al-Ramadi, Rothbard, and ecules. Eur. J. Immunol. 16: 1373–1378. C. A. J. Janeway, Jr. 1992. Truncation variants of peptides isolated from MHC

25. Bogen, B., Z. Dembic, and S. Weiss. 1993. of specific thymo- class II molecules suggest sequence motifs. Nature 359: 429–431. http://www.jimmunol.org/ cytes by an immunoglobulin idiotype. EMBO J. 12: 357–363. 47. Hunziker, L., M. Recher, A. J. Macpherson, A. Ciurea, S. Freigang, H. Hengartner, 26. Snyder, C. M., K. Aviszus, R. A. Heiser, D. R. Tonkin, A. M. Guth, and and R. M. Zinkernagel. 2003. Hypergammaglobulinemia and autoantibody in- L. J. Wysocki. 2004. Activation and tolerance in CD4(+) T cells reactive to an duction mechanisms in viral infections. Nat. Immunol. 4: 343–349. immunoglobulin variable region. J. Exp. Med. 200: 1–11. 48. Sekiguchi, D. R., R. A. Eisenberg, and M. Weigert. 2003. Secondary heavy chain 27. Weiss, S., and B. Bogen. 1989. B-lymphoma cells process and present their rearrangement: a mechanism for generating anti-double-stranded DNA B cells. endogenous immunoglobulin to major histocompatibility complex-restricted J. Exp. Med. 197: 27–39. T cells. Proc. Natl. Acad. Sci. USA 86: 282–286. 49. Kalinina, O., C. M. Doyle-Cooper, J. Miksanek, W. Meng, E. L. Prak, and 28. Weiss, S., and B. Bogen. 1991. MHC class II-restricted presentation of intra- M. G. Weigert. 2011. Alternative mechanisms of receptor editing in autoreactive cellular antigen. Cell 64: 767–776. B cells. Proc. Natl. Acad. Sci. USA 108: 7125–7130. 29. Munthe, L. A., J. A. Kyte, and B. Bogen. 1999. Resting small B cells present 50. Brosh, N., M. Dayan, M. Fridkin, and E. Mozes. 2000. A peptide based on the endogenous immunoglobulin variable-region determinants to idiotope-specific CDR3 of an anti-DNA antibody of experimental SLE origin is also a dominant

CD4(+) T cells in vivo. Eur. J. Immunol. 29: 4043–4052. T-cell epitope in (NZBXNZW)F1 lupus-prone mice. Immunol. Lett. 72: 61–68. by guest on September 30, 2021 30. Snyder, C. M., X. Zhang, and L. J. Wysocki. 2002. Negligible class II MHC 51. Abdou, N. I., R. Suenaga, M. Hatfield, M. Evans, and K. M. Hassanein. 1989. presentation of B cell receptor-derived peptides by high density resting B cells. J. Antiidiotypic antibodies against anti-DNA antibodies in sera of families of lupus Immunol. 168: 3865–3873. patients. J. Clin. Immunol. 9: 16–21. 31. Munthe, L. A., A. Os, M. Zangani, and B. Bogen. 2004. MHC-restricted Ig V 52. Williams, R. C., Jr., C. C. Malone, G. R. Huffman, F. Silvestris, B. P. Croker, region-driven T-B lymphocyte collaboration: B cell receptor ligation facilitates E. M. Ayoub, and S. Massengill. 1995. Active systemic lupus erythematosus is switch to IgG production. J. Immunol. 172: 7476–7484. associated with depletion of the natural generic anti-idiotype (anti-F(ab9)2) 32. Munthe, L. A., A. Corthay, A. Os, M. Zangani, and B. Bogen. 2005. Systemic system. J. Rheumatol. 22: 1075–1085. autoimmune disease caused by autoreactive B cells that receive chronic help 53. Tzioufas, A. G., and J. G. Routsias. 2010. Idiotype, anti-idiotype network of from Ig V region-specific T cells. J. Immunol. 175: 2391–2400. autoantibodies: pathogenetic considerations and clinical application. Auto- 33. Zangani, M., H. Carlsen, A. Kielland, A. Os, H. Hauglin, R. Blomhoff, immun. Rev. 9: 631–633. L. A. Munthe, and B. Bogen. 2009. Tracking early autoimmune disease by 54. Oak, S., L. K. Gilliam, M. Landin-Olsson, C. To¨rn, I. Kockum, bioluminescent imaging of NF-kappaB activation reveals pathology in multiple C. R. Pennington, M. J. Rowley, M. R. Christie, J. P. Banga, and C. S. Hampe. organ systems. Am. J. Pathol. 174: 1358–1367. 2008. The lack of anti-idiotypic antibodies, not the presence of the corre- 34. Wang, Z., M. Raifu, M. Howard, L. Smith, D. Hansen, R. Goldsby, and sponding autoantibodies to glutamate decarboxylase, defines type 1 diabetes. D. Ratner. 2000. Universal PCR amplification of mouse immunoglobulin gene Proc. Natl. Acad. Sci. USA 105: 5471–5476. variable regions: the design of degenerate primers and an assessment of the 55. Jacobsen, J. T., E. Lunde, V. Sundvold-Gjerstad, L. A. Munthe, and B. Bogen. effect of DNA polymerase 39 to 59 exonuclease activity. J. Immunol. Methods 2010. The cellular mechanism by which complementary Id+ and anti-Id anti- 233: 167–177. bodies communicate: T cells integrated into idiotypic regulation. Immunol. Cell 35. Kretschmer, K., A. Jungebloud, J. Stopkowicz, B. Stoermann, R. Hoffmann, and Biol. 88: 515–522. S. Weiss. 2003. Antibody repertoire and gene expression profile: implications for 56. Jacobsen, J., O. A. Haabeth, A. A. Tveita, K. W. Schjetne, L. A. Munthe, and different developmental and functional traits of splenic and peritoneal B-1 lym- B. Bogen. 2014. Naive idiotope-specific B and T cells collaborate efficiently in phocytes. J. Immunol. 171: 1192–1201. the absence of dendritic cells. J. Immunol. 192: 4174–4183.