Germinal center checkpoints in B cell tolerance in 3H9 transgenic mice

Elahna Paul1,3, Johannes Lutz3, Jan Erikson4 and Michael C. Carroll1,3

1 Department of Pediatrics, Harvard Medical School, 2 Division of Nephrology, Children’s Hospital and 3 Center for Blood Research, Boston, MA 02115, USA 4 Wistar Institute, Philadelphia, PA 19104, USA

Correspondence to: E. Paul, Center for Blood Research, 800 Huntington Avenue, Boston, MA 02115, USA. E-mail: elahna.paul{at}tch.harvard.edu
Transmitting editor: R. Geha


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Regulation throughout B cell maturation and activation prevents autoreactive B cells from entering germinal center (GC) reactions. This study shows that a subset of autoreactive B cells in VH3H9µ IgH transgenic mice escapes these serial checkpoints and proceeds into splenic GC. GC B cells isolated from these mice all express the transgenic VH3H9µ heavy chain, some co-express light chains that yield an anti-dsDNA specificity and some have somatic mutations, consistent with their GC origin. Nonetheless, B cell tolerance is ultimately preserved as serum titers of anti-dsDNA antibodies are not elevated. These observations suggest that those autoreactive GC B cells that escaped earlier checkpoints and possibly also those cells that acquire autoreactivity de novo by mutating their antigen receptor are arrested within the splenic GC before differentiating further into antibody-secreting plasma cells.

Keywords: anti-DNA antibody, autoimmunity, somatic mutation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
VH3H9µ mice, transgenic for the rearranged VDJCµ encoding an anti-DNA Ig heavy chain, use a variety of mechanisms to avoid production of anti-DNA autoantibodies. Extensive analysis of VH3H9µ hybridomas has revealed that the VH3H9µ heavy chain can pair with many light chains to form autoantibodies specific for dsDNA (13). Ig{lambda}1 is one of the endogenous light chains capable of pairing with VH3H9µ to generate an anti-DNA autoantibody (4,5) and it accounts for >80% of the {lambda} repertoire in vivo (6). In VH3H9µ transgenic mice, Ig{lambda} can be used as a surrogate marker of anti-DNA B cells, thereby offering an opportunity to track an autoreactive subset of B cells within the total B cell repertoire (6).

The overall frequency of Ig{lambda} B cells is increased in VH3H9µ transgenic mice relative to wild-type animals; however, there are no anti-DNA antibodies in the serum of transgenic BALB/c animals (3,5,6). The autoreactive B cells that are inactive in vivo are also hypoproliferative to lipopolysaccharide (LPS) stimulation in vitro (79). Follicular exclusion is the histological correlate of this functional anergy where Ig{lambda} B cells accumulate at the interface of the T cell and B cell zones of the splenic white pulp (6,10). Whether anergized or lacking cognate T cell help (6,8,9,11,12), these cells were previously presumed unable to participate in germinal center (GC) reactions.

This study shows that some anti-DNA B cells begin, but do not complete, GC reactions in VH3H9µ transgenic mice. Splenic GC in unmanipulated transgenic animals contain autoreactive B cells that have eluded the pre-GC regulatory network. A single-cell RT-PCR and sequencing approach confirms that some of these Ig{lambda} B cells retain the canonical VH3H9µ heavy chain, and presumably encode anti-DNA autoantibodies. In light of absent serum autoantibodies, the very presence of autoreactive B cells within GC implicates a tolerogenic checkpoint that can regulate autoreactive B cells after entry into GC reactions, but before terminal differentiation into antibody-secreting cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female mice on a mixed background (BALB/c, 129, C57BL/6) with or without the VH3H9µ transgene (3) were bred at the specific pathogen-free Warren Alpert Animal Facility of Harvard Medical School and studied at 24–26 weeks of age. MRL/lpr mice (Jackson Laboratory, Bar Harbor, ME) were included as positive controls where indicated.

Antibody reagents
mAb specific for CD16/CD32 (2.4G2), Ig{lambda}1 (R11-153), Ig{kappa} (187.1), IgM (R6-60.2), B220 (RA3-6B2), Thy1.2 (53-2.1) CD3 (145-2c11) and CD11b (M1/70) were from BD PharMingen (San Diego, CA). Additional reagents included peanut agglutinin (PNA)–FITC (EY Laboratories, San Mateo, CA), biotinylated PNA (Vector, Burlingame, CA), biotinylated goat anti-mouse Ig{lambda} (Southern Biotechnology Associates, Birmingham, AL), streptavidin–AMCA (Molecular Probes, Eugene, OR), streptavidin–alkaline phosphatase (Sigma, St Louis, MO) and anti-FITC–horseradish peroxide (Roche Molecular Biochemicals, Indianapolis, IN).

Immunohistochemistry
Spleens embedded in Tissue-Tek OCT compound (Miles, Naperville, IL) were cryosectioned and acetone fixed. Fc receptors were blocked and tissue sections were then stained for B220 or CD3 and for Ig{lambda}, Ig{lambda}1 or PNA. Color was developed with Fast Blue and AEC (Sigma), and slides were mounted with Crystal mount (Biomedia, Foster City, CA). Measurement of GC size and total white pulp area was performed using Openlab image-processing software (Improvision, Quincy, MA). A minimum white pulp area of 5 x 106 µm2 was measured per spleen.

dsDNA ELISA
Sera diluted 1:100 were incubated in microtiter wells coated with dsDNA, as described previously (13). Bound antibodies were detected with phosphatase-conjugated goat anti-mouse IgM or IgG (Sigma). Samples were tested in duplicate for dsDNA-specific binding (ODS) and for background binding (ODB) to wells coated only with diluent. A control panel of MRL/lpr sera on each plate was used to calculate the DNA binding index of each sample: DNA binding index = sample (ODS – ODB)/mean of control (ODS – ODB).

FACS sort
Splenic tissue mechanically dissociated into single-cell suspensions was blocked with anti-CD16/CD32 and then stained with fluorochrome-conjugated reagents. GC B cells were identified as PNAhi, B220+, CD11b or as PNAhi, B220+, IgM+, Thy1.2, or as PNAhi, IgM+, Ig{lambda}+, Ig{kappa}. Single GC B cells were sorted into 96-well microtiter plates for PCR (Fisher Scientific, Pittsburgh, PA).

Single-cell RT-PCR
Reverse transcription of mRNA from single GC B cells was performed using random primers (Invitrogen, Carlsbad, CA). PCR amplification of the expressed heavy and light chain Ig genes from single-cell cDNA was accomplished in two rounds with three sets of semi- or fully nested primers, as follows. IgH message was amplified in sequential rounds of PCR using a 5' VH primer which binds to most VH gene families [VH: GGG AAT TCG AGG TGC AGC TGC AGG AGT CTG G (14)] and two nested 3' Cµ primers [Cµ first strand: AGG GGG CTC TCG CAG GAG ACG AGG; Cµ second strand: AGG GGG AAG ACA TTT GGG AAG GAC]. Light chain message was amplified in two rounds of PCR using either a universal 5' V{kappa} primer which binds to the FR3 region of most V{kappa} gene families [V{kappa}: GGC TGC AGS TTC AGT GGC AGT GGR TCW GGR AC (15)] and two nested 3' C{kappa} primers [C{kappa} first strand: ACA CTC ATT CCT GTT GAA GCT CTT; C{kappa} second strand: TGT AAA ACG ACG GCC AGT TCT AGA TGG TGG GAA GAT GGA (16)] or using two pairs of fully nested Ig{lambda} primers [V{lambda} first strand: ATG GCC TGG ACT TCA CTT ATA CTC T; C{lambda} first strand: GCA GGA GAC AAA CTC TTC TCC ACA; V{lambda} second strand: CAG GCT GTT GTG ACT CAG GAA TCT; C{lambda} second strand: TGT AAA ACG ACG GCC AGT GAG CTC CTC AGA GGA AGG TGG AAA (16)] which can amplify Ig{lambda}1, 2 and 3, but not Ig{lambda}X. First-round PCR amplifications were performed with 3 µl of cDNA in 20 µl reactions for 30 cycles at 94°C x 40 s, 56, 50 or 60°C x 30 s (for IgH or Ig{kappa} or Ig{lambda} respectively) and 72°C x 40 s. First-round product was further amplified in a second round of 35 cycles at 94°C for 30 s, 65, 50 or 60°C x 20 s (for IgH or Ig{kappa} or Ig{lambda} respectively) and 72°C x 30 s. This amplification algorithm yields IgH, Ig{kappa} and Ig{lambda} fragments of 400, 180 and 380 bp respectively.

Sequence analysis
Separated by agarose gel electrophoresis, PCR products were isolated using gel extraction spin columns (Bio-Rad, Hercules, CA) and sequenced with an ABI Prism 3100 sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer‘s instructions. Online resources for sequence analysis included NCBI IgBlast [www.ncbi.nlm.nih.gov/igblast (17)], IMGT DNAPlot [www.dnaplot.de (18)] and H. G. Zachau’s Ig{kappa} database [www.med.uni-muenchen.de/biochemie/zachau/k.htm (19)]. Two VH3H9µ hybridoma cell lines, L17 and H26 (6,9), were used to estimate the error rate of the RT-PCR and sequencing process. Single hybridoma cells sorted into 96-well PCR plates were processed as above. Heavy chain sequences determined for 27 unique cells (total of 9000 bp) contained no base pair deviations from the canonical VH3H9µ sequence.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Follicular exclusion and GC
Follicular exclusion, the histological correlate of B cell anergy first described in IgHEL/sHEL double-transgenic mice (10), was also observed in splenic sections of VH3H9µ transgenic mice on the BALB/c genetic background (6). Unlike normal B cells that migrate into B cell zones and initiate GC reactions upon activation, autoreactive Ig{lambda} B cells in VH3H9µ mice accumulate along the T/B border of the splenic follicle (6). Similarly, spleens from naive 6-month-old female VH3H9µ mice on the mixed genetic background used in the current study were evaluated by immunohistochemistry to localize the Ig{lambda} subset of anti-DNA splenocytes. Staining of serial sections for Ig{lambda} and B220 or Ig{lambda} and CD3 confirms that the follicular exclusion of autoreactive B cells is also appreciable in these mice (Fig. 1a and b). Interestingly, in addition to their pattern of follicular exclusion, some Ig{lambda} cells are also distributed as dense aggregates within B cell follicles of these spleens. Serial sections stained with PNA demonstrate that the Ig{lambda}-rich clusters are GC (Fig. 1c). Morphometric analysis of tissue sections co-stained with PNA and B220 suggests that the splenic GC physiology is grossly intact in these animals. The relative amount of splenic white pulp devoted to GC activity without intentional immunization is equivalent in transgenic mice and their non-transgenic littermates (4.5 versus 5.7% respectively, P = 0.4; Fig. 2).



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Fig. 1. Serial sections from a representative VH3H9µ transgenic spleen were stained for follicular microarchitecture. Panels (a: blue = {lambda}; red = B220) and (b: blue = {lambda}; red = CD3) demonstrate that many Ig{lambda} B cells accumulate at the T–B interface. Serial section (c) suggests that some of these B cells are within GC (blue = PNA; red = B220) and panel (d: blue = {lambda}-1; red = B220) shows that these GC B cells express the Ig{lambda}1 light chain, known to encode anti-DNA antibodies in conjunction with the VH3H9µ heavy chain. The arrow points to the same {lambda}-rich GC in each serial section and the arrowhead points to the central arteriole of this splenic follicle.

 


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Fig. 2. Morphometric measurement of splenic GC activity was performed in 13 wild-type (open diamonds) and 15 VH3H9µ transgenic (filled diamonds) female mice at 20–25 weeks of age. The area of splenic white pulp devoted to GC activity is expressed as a percentage on the y-axis and the mean value for each group is indicated (NS; P = 0.4).

 
The co-localization of Ig{lambda} and PNA staining implies that some autoreactive B cells in VH3H9µ transgenic mice escape arrest at the T/B interface and participate in GC reactions. To address the possibility that a non-autoreactive subset of Ig{lambda} B cells (i.e. not {lambda}1) was disproportionately represented in these GC, splenic sections were also stained specifically for Ig{lambda}1. Comparable to the pan-Ig{lambda} staining displayed in Fig. 1(a–c), most GC from VH3H9µ transgenic animals also contain Ig{lambda}1-bearing cells (Fig. 1d). These histological findings suggest that the poorly understood tolerogenic checkpoint represented by follicular exclusion fails to universally exclude all autoreactive B cells from the B cell follicle.

Autoantibody titers
Identification of Ig{lambda}1 B cells in splenic GC raised the question whether B cell tolerance was perhaps impaired in the genetic background of these VH3H9µ transgenic mice. Autoreactive B cells completing GC reactions differentiate into memory and/or antibody-secreting cells, eventually producing autoantibody that should be detectable in the serum. Surprisingly, measurement of serum autoantibodies by DNA ELISA reveals that these VH3H9µ animals are as tolerant as their non-transgenic littermates (Fig. 3). These data indicate that 6-month-old VH3H9µ mice are still tolerant to DNA, despite Ig{lambda}1 expression in the GC.



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Fig. 3. An ELISA specific for IgM anti-dsDNA was performed on serum samples from 13 wild-type (open diamonds) and 17 VH3H9µ transgenic (filled diamonds) female mice at 20–25 weeks of age. DNA binding is expressed on the y-axis; MRL/lpr control sera have a binding index of 1.0 (see Methods). The mean value for each group is indicated (NS; P = 0.1).

 
IgH expression in GC
A molecular approach was used to examine IgH gene expression in GC to address the possibility that the Ig{lambda} cells had stopped expressing the VH3H9µ transgene and hence were no longer autoreactive. PNA+ GC B cells from 6-month-old VH3H9µ females were subjected to single-cell sorting strategies designed to yield either Ig{lambda}+ cells or a mixture of Ig{lambda} and Ig{kappa} expressors. Single-cell IgH message was amplified by RT-PCR using intron-flanking primers: the nested 3' oligomers are specific for the 5' region of the Cµ gene segment and the 5' universal VH primer can amplify message from all VH gene families, whether transgenic or endogenous (14). Amplified products were identified by nucleotide sequence analysis.

All of the VH gene products amplified from five individual mice had VDJ junctional identity to the VH3H9µ transgene. Each of the 159 cells analyzed expressed the transgene and none expressed an endogenous µ heavy chain. These findings contrast to an analysis of VH3H9µ transgenic mice deficient in complement component C4, which were studied for other purposes. In those animals, 8% of GC B cells express endogenous and not transgenic µ heavy chains (data not shown), demonstrating that the RT-PCR method can amplify endogenous alleles from VH3H9µ transgenic B cells. With verification that the sorted Ig{lambda}+ B cells retain the VH3H9µ transgene and maintain heavy chain allelic exclusion, one can surmise that a reasonable proportion of the Ig{lambda} GC B cells detected by immunohistochemistry also retain the transgene and are therefore autoreactive.

Mutations in the VH3H9µ transgene
The observation that 100% of GC B cells in VH3H9µ mice express the transgene is consistent with previous hybridoma analyses of VH3H9µ splenocytes (2,5,20). However, unlike the hybridoma data that exhibit no hypermutation, 23 of 159 (14.5%) VH3H9µ alleles expressed in primary GC B cells have mutated away from the canonical transgenic sequence (Fig. 4). In contrast to hybridoma studies using polyclonal stimulation to capture a repertoire of splenic B cells that may or may not have been activated in vivo, the single-cell approach exclusively targets GC B cells and thereby enriches not only for B cells that are activated in vivo, but also for those undergoing somatic hypermutation in GC reactions. Moreover, these nucleotide differences are probably derived in vivo rather than being an experimental artifact, because single-cell analysis of two distinct VH3H9µ hybridoma cell lines revealed only the canonical transgene sequence in a total of 27 independently amplified transcripts (P < 0.03 by Fisher’s exact test).



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Fig. 4. Nucleotide sequences of 23 mutated transgenic heavy chains expressed in GC B cells are arrayed beneath the canonical VH3H9µ gene sequence. Nucleotides are displayed from codon 10 of FR1 to codon 12 of CDR3, corresponding to the most 5' and 3' point mutations, respectively. Point mutations conferring amino acid replacements are indicated in upper case; silent mutations are indicated in lower case; dashes denote nucleotide identity. The complementarity-determining regions are shaded and the arrow in CDR3 indicates the beginning of JH4.

 
Mutational analysis is feasible because only one full-length copy of the VH3H9µ transgene is inserted in the genome of the VH3H9µ mouse strain (Radic, pers. commun.). Twenty-three of the 159 VH3H9µ transcripts sequenced from GC B cells had a total of 44 bp changes from the canonical VH3H9µ sequence: one transcript had n = 6 mutations, one had n = 4, four had n = 3, five had n = 2 and 12 transcripts each had a single mutation (Fig. 4 and Table 1). The mutations have a transition bias with a transition-to-transversion ratio of 1.44 and a majority of these base pair changes confer amino acid replacements (37 of 44; 82%). These observations are consistent with somatic hypermutation rather than randomly generated PCR artifact, possibly reflecting antigen-driven selection in the GC reaction (2123).


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Table 1. Mutation distribution in the mutated VH3H9µ and V{lambda}1 transcripts
 
In the absence of antigen-binding data, one can only speculate on the effects of these mutations. Overall, the amino acid substitutions do not alter the net charge of the predicted IgH polypeptide and as such do not preclude binding to DNA. Analysis of the VH3H9 heavy chain and mutational variants has previously identified specific amino acid residues important for DNA reactivity, such as the acquired arginine at position 53 (24). This Arg53 is retained by all of the GC B cells sampled in this study, possibly suggesting that the heavy chain’s potential for DNA binding is also retained. Similarly, additional acquisition of arginines by VH3H9 at positions 56 and 76 enhances DNA binding (24). No GC B cells acquiring these potentially pathogenic changes are detectable in the population sampled, consistent with maintenance of peripheral tolerance.

Ig{kappa} light chain usage in GC B cells
Both {kappa} and {lambda} light chain gene sequences were obtained from sorted GC B cells. Importantly, none of the 121 cells examined were found to express more than a single Ig light chain (90 Ig{kappa}; 31 g{lambda}) nor was dual {kappa}/{lambda} expression detectable by FACS analysis (data not shown). Given the large number of germline genes in the V{kappa} locus, a detailed mutational analysis of the expressed Ig{kappa} genes is cumbersome. Instead, V{kappa} mRNA was amplified principally to delineate V{kappa} gene family and J{kappa} gene usage in the GC B cell population. Sequences from 90 Ig{kappa}-expressing B cells are summarized in Table 2. While most V{kappa} gene families are represented in this population of primary GC B cells, the frequency of genes used from each gene family differs from hybridoma analyses of VH3H9µ mice (1,2,5,25,26). In addition, similar to some of the VH3H9µ hybridoma panels (5), but not others (2), the GC B cell population was not strongly skewed towards the downstream J{kappa}4 and J{kappa}5 gene segments, thus providing little evidence of secondary light chain rearrangements (Table 2). Distinct selection biases are almost certainly introduced when different methods—single-cell RT-PCR of sorted GC B cells versus hybridoma studies—are used to sample the B cell repertoire. Furthermore, discrepancies in mouse age may contribute to the apparent repertoire differences between this study and previously published accounts.


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Table 2. Ig{kappa} gene family usage in VH3H9 B cells
 
Ig{lambda} light chain expression and mutation
Unlike the Ig{kappa} gene locus, the small size of the Ig{lambda} gene family facilitates analysis of somatic mutations in Ig{lambda}-expressing B cells. In addition, hybridoma studies demonstrating that VH3H9 heavy chain binds DNA when paired with unmutated V{lambda}1J{lambda}1 or with V{lambda}2J{lambda}1 light chains (57) can help predict the DNA reactivity of the GC B cells. Ig{lambda} transcripts from 31 GC B cells of four mice were analyzed after GC B cells were FACS sorted for Ig{lambda} surface expression. A subset of these cells had also been FACS sorted against surface Ig{kappa} to avoid cells co-expressing both light chain isotypes. A number of Ig{lambda} transcripts with distinct potentials for DNA binding were detected (Table 3).


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Table 3. Ig{lambda} gene usage
 
Co-expression of the VH3H9µ heavy chain was confirmed in 12 of 24 V{lambda}1J{lambda}1 GC B cells, and eight of these 12 VH3H9µ/{lambda}1 cells had no somatic mutation in either chain and thus likely retained their specificity for DNA. These findings suggest that B cells expressing autoreactive BCR are present in the splenic GC of ‘non-autoimmune’ VH3H9µ mice.

Eight of the 24 V{lambda}1J{lambda}1 rearrangements had multiple point mutations (Fig. 5 and Table 1). Not only do these mutations display a transition bias with a transition-to-transversion ratio of 1.55, but the pattern of particular nucleotide substitutions correlates well with somatic mutations of V{lambda}1 expressed in wild-type mice (23). The pattern and frequency of point mutations substantiates the interpretation that these B cells are indeed of GC origin and in some cases have undergone several rounds of cell division (21,22,27). The antigen driving their amplification and selection in vivo cannot be deduced from these data, and whether these B cells have enhanced DNA reactivity or have mutated away from their original autospecificity cannot be asserted one way or another.



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Fig. 5. Nucleotide sequences of eight mutated V{lambda}1 light chains expressed in GC B cells are arrayed beneath the V{lambda}1 and J{lambda}1 germline (GL) gene sequences. Nucleotides are displayed from codon 9 of FR1 to codon 11 of CDR3. Point mutations conferring amino acid replacements are indicated in upper case; silent mutations are indicated in lower case; dashes denote nucleotide identity. The complementarity-determining regions are shaded and the arrow in CDR3 indicates the beginning of J{lambda}1. Asterisks mark the Ig{lambda} chains co-expressed with VH3H9µ heavy chains.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
VH3H9µ transgenic mice are considered non-autoimmune because their B cells do not produce significant serum titers of anti-DNA autoantibodies The regulatory mechanisms achieving this B cell tolerance remain incompletely characterized. Initially, histological evaluation localized the VH3H9µ/{lambda}1 population of autoreactive B cells to the T/B interface of the splenic follicle (6). Using a similar histological approach, the present study further identifies a subset of potentially autoreactive {lambda}1 B cells within splenic GC of VH3H9µ transgenic mice. Single-cell sorting of PNA+ splenocytes was used to isolate primary GC B cells whose heavy and light chain Ig gene transcripts were then amplified by RT-PCR. Sequence analysis of the expressed Ig genes in these GC B cells confirms that at least some of them express the canonical VH3H9µ/{lambda}1 anti-DNA BCR, unmutated, while others display heavy and/or light chain mutations consistent with somatic hypermutation. Anti-DNA ELISA confirmed that the 6-month-old mice remained free of circulating autoantibodies, implicating a tolerogenic checkpoint within the GC itself.

The unmutated VH3H9µ/{lambda}1 GC cells were presumably autoreactive in their initial Ig gene configuration, before induction of somatic hypermutation in the GC. In retrospect, these cells may correspond to the small percentage of Ig{lambda} splenocytes previously shown to be cycling in VH3H9µ transgenic BALB/c mice (9). How these autoreactive cells eluded substantial tolerogenic checkpoints to enter GC and start cycling remains a matter of speculation. Central tolerogenic mechanisms include receptor editing to alter the antigenic specificity of the developing B cell and/or clonal deletion (20). Peripheral tolerance, maintained directly by induction of B cell anergy or indirectly in absence of antigen-specific T cell help, regulates autoreactive B cells that escape negative selection in the bone marrow (6,7,28). Perhaps T cell help in the follicle is enough to activate small numbers of bystander B cells in a non-cognate manner, particularly when the B cell repertoire is skewed by a transgenic Ig heavy chain (11,12). Alternatively, modest variation in local concentrations of autoantigen may diversify BCR occupancy enough to permit a small, yet detectable, fraction of antigen-experienced B cells to proceed into GC reactions (29,30). Finally, anti-DNA hybridomas co-expressing non-autoreactive endogenous heavy chains or two distinct light chains have been recovered from non-autoimmune mice, implying that some autoreactive B cells may escape tolerance by co-expressing non-autoreactive BCR (2,5,7,20,3133). Although no evidence of either heavy or light chain allelic inclusion was found in primary GC B cells, the absence of such data does not formally exclude this possibility.

Once B cells begin a GC reaction, what prevents autoreactive VH3H9µ/{lambda}1 B cells from progressing into the plasmacyte or memory B cell compartments? We hypothesize that these cells have escaped follicular exclusion only to encounter another tolerogenic checkpoint within the GC itself. In some instances, hypermutation in the initially autoreactive VH3H9µ/{lambda}1 B cells can probably disrupt their original specificity, thereby enabling some to differentiate further. Nevertheless, as suggested by the notable absence of circulating anti-DNA antibodies, those cells retaining their autospecificity—whether via silent mutations, conservative replacements or no mutation at all—never exit the GC reaction.

Negative selection within GC has been implicated in the anti-Ars system using mice expressing transgenic BCR with dual specificities for DNA and for Ars. These animals remain self-tolerant even after immunization with the hapten Ars. Although autoreactive B cells can be detected within splenic GC of these mice, they do not enter the memory compartment and nor do they secrete autoantibody (34). Mechanisms proposed to mediate negative selection of GC B cells include antigen excess (35) and/or cognate T cell insufficiency (36). For example, it is clear that supplemental T cell help can activate VH3H9µ B cells to produce autoantibody both in vitro (6,9,28) and in vivo (11,12), indicating that non-physiologic T cell help can override endogenous mechanisms important for B cell tolerance.

An alternative explanation is offered by the artificial constraints of the VH3H9µ system where the transgenic Ig gene is inserted in an unknown genomic location. Survival of the transgenic B cell may be compromised if and when heavy chain class switching is initiated in GC, resulting in irreversible chromosomal damage. While this possibility could justify the absence of IgG autoantibodies, it cannot account for the absence of IgM anti-DNA in the serum. Moreover, autoreactivity can clearly arise de novo via somatic hypermutation even in the course of a normal GC reaction. It may be that the normal regulatory mechanism important for quenching such peripheral autoreactivity may also regulate those autoreactive VH3H9µ cells that slip past pre-GC checkpoints. The nature of this putative GC checkpoint remains to be clarified.

The current study complements prior hybridoma experiments and bypasses some potential limitations of the hybridoma technology. Separating the primary GC B cells from bulk splenocytes focuses on a subset of cells that have presumably been activated in vivo. Additionally, this approach avoids selection biases introduced by ex vivo activation protocols and by the fusion methodology itself. As a result, not only have we found anti-DNA B cells within splenic GC of tolerant mice, but we have also discovered a high rate of VH3H9µ somatic mutations. The very presence of somatic mutations in the VH3H9µ, as well as in the Ig{lambda} genes, supports the contention that these are indeed GC B cells. Furthermore, not only have these autoreactive cells escaped anergy as heretofore understood, but some must have been in GC for several rounds of cell division in order to accumulate their relatively high frequency of mutations. Interestingly, somatic hypermutation of the VH3H9µ transgene has not been observed in hybridomas derived from non-autoimmune transgenic mice. Initially, this freedom from hypermutation implied that the transgene had inserted into a locus relatively sheltered from normal Ig gene hypermutation. The current sequence analysis, however, indicates that explicit selection of primary GC B cells by cell sorting has allowed identification of a B cell subset that has been overlooked in hybridoma approaches.

In summary, despite the absence of circulating anti-dsDNA antibodies, B cells bearing the canonical autoreactive BCR VH3H9µ/{lambda}1 can be found in splenic GC of VH3H9µ transgenic mice. These anti-DNA B cells not only escape negative selection in the marrow, but they receive adequate stimulation in the periphery to escape follicular exclusion and enter GC reactions. This progression beyond the T/B interface suggests that the pre-GC tolerance mechanisms are leaky. Nevertheless, the VH3H9µ/{lambda}1 cells do not participate in a humoral immune response, suggesting that GC and/or post-GC tolerogenic checkpoints are important to the regulatory network safeguarding mice against autoreactive B cell activation.


    Acknowledgements
 
This work was funded by NIH grants HD38749 (M.C.C.) and K08-AI01841 (E.P.).


    Abbreviations
 
GC—germinal center

LPS—lipopolysaccharide

PNA—peanut agglutinin


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ibrahim, S. M., Weigert, M., Basu, C., Erikson, J. and Radic, M. Z. 1995. Light chain contribution to specificity in anti-DNA antibodies. J. Immunol. 155:3223.[Abstract]
  2. Radic, M. Z., Erikson, J., Litwin, S. and Weigert, M. 1993. B lymphocytes may escape tolerance by revising their antigen receptors. J. Exp. Med. 177:1165.[Abstract]
  3. Erikson, J., Radic, M. Z., Camper, S. A., Hardy, R. R., Carmack, C. and Weigert, M. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349:331.[CrossRef][ISI][Medline]
  4. Radic, M. Z., Mascelli, M. A., Erikson, J., Shan, H. and Weigert, M. 1991. Ig H and L chain contributions to autoimmune specificities. J. Immunol. 146:176.[Abstract/Free Full Text]
  5. Roark, J. H., Kuntz, C. L., Nguyen, K. A., Caton, A. J. and Erikson, J. 1995. Breakdown of B cell tolerance in a mouse model of systemic lupus erythematosus. J. Exp. Med. 181:1157.[Abstract]
  6. Mandik-Nayak, L., Bui, A., Noorchashm, H., Eaton, A. and Erikson, J. 1997. Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T–B interface of the splenic follicle. J. Exp. Med. 186:1257.[Abstract/Free Full Text]
  7. Roark, J. H., Bui, A., Nguyen, K. A., Mandik, L. and Erikson, J. 1997. Persistence of functionally compromised anti-double-stranded DNA B cells in the periphery of non-autoimmune mice. Int. Immunol. 9:1615.[Abstract]
  8. Nguyen, K. A., Mandik, L., Bui, A., Kavaler, J., Norvell, A., Monroe, J. G., Roark, J. H. and Erikson, J. 1997. Characterization of anti-single-stranded DNA B cells in a non-autoimmune background. J. Immunol. 159:2633.[Abstract]
  9. Mandik-Nayak, L., Seo, S., Eaton-Bassiri, A., Allman, D., Hardy, R. R. and Erikson, J. 2000. Functional consequences of the developmental arrest and follicular exclusion of anti-double-stranded DNA B cells. J. Immunol. 164:1161.[Abstract/Free Full Text]
  10. Cyster, J. G., Hartley, S. B. and Goodnow, C. C. 1994. Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire [see Comments]. Nature 371:389.[CrossRef][ISI][Medline]
  11. Sekiguchi, D. R., Jainandunsing, S. M., Fields, M. L., Maldonado, M. A., Madaio, M. P., Erikson, J., Weigert, M. and Eisenberg, R. A. 2002. Chronic graft-versus-host in Ig knockin transgenic mice abrogates B cell tolerance in anti-double-stranded DNA B cells. J. Immunol. 168:4142.[Abstract/Free Full Text]
  12. Seo, S. J., Fields, M. L., Buckler, J. L., Reed, A. J., Mandik-Nayak, L., Nish, S. A., Noelle, R. J., Turka, L. A., Finkelman, F. D., Caton, A. J. and Erikson, J. 2002. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 16:535.[ISI][Medline]
  13. Paul, E., Pozdnyakova, O. O., Mitchell, E. and Carroll, M. C. 2002. Anti-DNA autoreactivity in C4-deficient mice. Eur. J. Immunol. 32:2672.[CrossRef][ISI][Medline]
  14. Kantor, A. B., Merrill, C. E., Herzenberg, L. A. and Hillson, J. L. 1997. An unbiased analysis of VH–D–JH sequences from B-1a, B-1b, and conventional B cells. J. Immunol. 158:1175.[Abstract]
  15. Yamagami, T., ten Boekel, E., Schaniel, C., Andersson, J., Rolink, A. and Melchers, F. 1999. Four of five RAG-expressing JC{kappa}–/– small pre-BII cells have no L chain gene rearrangements: detection by high-efficiency single cell PCR. Immunity 11:309.[ISI][Medline]
  16. Seidl, K. J., MacKenzie, J. D., Wang, D., Kantor, A. B., Kabat, E. A. and Herzenberg, L. A. 1997. Frequent occurrence of identical heavy and light chain Ig rearrangements. Int. Immunol. 9:689.[Abstract]
  17. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389.[Abstract/Free Full Text]
  18. Giudicelli, V., Chaume, D., Mennessier, G., Althaus, H. H., Muller, W., Bodmer, J., Malik, A. and Lefranc, M. P. 1998. IMGT, the international ImMunoGeneTics database: a new design for immunogenetics data access. Medinfo 9:351.[Medline]
  19. Thiebe, R., Schable, K. F., Bensch, A., Brensing-Kuppers, J., Heim, V., Kirschbaum, T., Mitlohner, H., Ohnrich, M., Pourrajabi, S., Roschenthaler, F., Schwendinger, J., Wichelhaus, D., Zocher, I. and Zachau, H. G. 1999. The variable genes and gene families of the mouse immunoglobulin {kappa} locus. Eur. J. Immunol. 29:2072.[CrossRef][ISI][Medline]
  20. Gay, D., Saunders, T., Camper, S. and Weigert, M. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177:999.[Abstract]
  21. Bemark, M., Sale, J. E., Kim, H. J., Berek, C., Cosgrove, R. A. and Neuberger, M. S. 2000. Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) or recombination-activating gene (RAG)1 activity. J. Exp. Med. 192:1509.[Abstract/Free Full Text]
  22. Toellner, K. M., Jenkinson, W. E., Taylor, D. R., Khan, M., Sze, D. M., Sansom, D. M., Vinuesa, C. G. and MacLennan, I. C. 2002. Low-level hypermutation in T cell-independent germinal centers compared with high mutation rates associated with T cell-dependent germinal centers. J. Exp. Med. 195:383.[Abstract/Free Full Text]
  23. Jacobs, H., Fukita, Y., van der Horst, G. T., de Boer, J., Weeda, G., Essers, J., de Wind, N., Engelward, B. P., Samson, L., Verbeek, S., de Murcia, J. M., de Murcia, G., te Riele, H. and Rajewsky, K. 1998. Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice. J. Exp. Med. 187:1735.[Abstract/Free Full Text]
  24. Radic, M. Z., Mackle, J., Erikson, J., Mol, C., Anderson, W. F. and Weigert, M. 1993. Residues that mediate DNA binding of autoimmune antibodies. J. Immunol. 150:4966.[Abstract/Free Full Text]
  25. Prak, E. L., Trounstine, M., Huszar, D. and Weigert, M. 1994. Light chain editing in {kappa}-deficient animals: a potential mechanism of B cell tolerance. J. Exp. Med. 180:1805.[Abstract]
  26. Li, H., Jiang, Y., Prak, E. L., Radic, M. and Weigert, M. 2001. Editors and editing of anti-DNA receptors. Immunity 15:947.[ISI][Medline]
  27. Berek, C., Berger, A. and Apel, M. 1991. Maturation of the immune response in germinal centers. Cell 67:1121.[ISI][Medline]
  28. Noorchashm, H., Bui, A., Li, H. L., Eaton, A., Mandik-Nayak, L., Sokol, C., Potts, K. M., Pure, E. and Erikson, J. 1999. Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process. Int. Immunol. 11:765.[Abstract/Free Full Text]
  29. Cook, M. C., Basten, A. and Fazekas de St Groth, B. 1997. Outer periarteriolar lymphoid sheath arrest and subsequent differentiation of both naive and tolerant immunoglobulin transgenic B cells is determined by B cell receptor occupancy. J. Exp. Med. 186:631.[Abstract/Free Full Text]
  30. Fulcher, D. A., Lyons, A. B., Korn, S. L., Cook, M. C., Koleda, C., Parish, C., Fazekas de St Groth, B. and Basten, A. 1996. The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help. J. Exp. Med. 183:2313.[Abstract]
  31. Li, Y., Li, H. and Weigert, M. 2002. Autoreactive B cells in the marginal zone that express dual receptors. J. Exp. Med. 195:181.[Abstract/Free Full Text]
  32. Chen, C., Prak, E. L. and Weigert, M. 1997. Editing disease-associated autoantibodies. Immunity 6:97.[ISI][Medline]
  33. Chen, C., Radic, M. Z., Erikson, J., Camper, S. A., Litwin, S., Hardy, R. R. and Weigert, M. 1994. Deletion and editing of B cells that express antibodies to DNA. J. Immunol. 152:1970.[Abstract/Free Full Text]
  34. Notidis, E., Heltemes, L. and Manser, T. 2002. Dominant, hierarchical induction of peripheral tolerance during foreign antigen-driven B cell development. Immunity 17:317.[ISI][Medline]
  35. Shokat, K. M. and Goodnow, C. C. 1995. Antigen-induced B-cell death and elimination during germinal-centre immune responses. Nature 375:334.[CrossRef][ISI][Medline]
  36. Reed, A. J., Riley, M. P. and Caton, A. J. 2000. Virus-induced maturation and activation of autoreactive memory B cells. J. Exp. Med. 192:1763.[Abstract/Free Full Text]