Light chain editors of anti-DNA receptors in human B cells

Human L chain repertoire includes editor L chains that appear to regulate anti-DNA B cells and shape the B cell repertoire.

The antibody-combining site is formed by the interaction of the variable regions of the H and L chains; hence, replacement of either V changes the specificity of an antibody (Gay et al., 1993;Radic et al., 1993a;Tiegs et al., 1993). A characteristic feature of anti-DNA antibodies is that DNA contact is mediated primarily through the positively charged amino acid residue Arg in H-chain complementarity determining regions (CDRs; Jang et al., 1998;Radic et al., 1989Radic et al., , 1993bShlomchik et al., 1990). Hence, these Arg-containing H chains bind DNA regardless of most L chains (Ibrahim et al., 1995). However, several L chains in mouse can modify or veto DNA binding when paired with anti-DNA H chains (Li et al., 2001). These L chains are called editors and share functionally consequential structural characteristics such as a low isoelectric point (pI) and negatively charged aspartate residues (Asp) in CDRs (Li et al., 2001).
Editor L chains were originally discovered in anti-DNA H-chain transgenic models (Gay et al., 1993;Radic et al., 1993a). The majority of peripheral B cells bearing anti-DNA transgenic H chains were paired with editor L chains, indicating that only those B cells that edited their receptors were allowed to migrate to the periphery. Editing of anti-DNA receptors also influences the endogenous mouse repertoire; antibodies which use editor L chains have characteristics of lupus anti-DNA antibodiesnamely, a high frequency of H chains with Args in H3 (H3-Arg) (Kalinina et al., 2011).
Here, we studied the human antibody repertoire to determine whether it contains L chains similar to the mouse editors, whether such human L chains silence or modify anti-DNA activity, and whether antibodies that express these L chains have a high frequency of H chains with H3 Arg relative to the total human antibody repertoire. We used mouse editor L chain sequences as a guideline for identifying human editor L chains. We focused on the prominent Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans.  CDR2, and (4) in VkL4/18a CDR2. Not all human L chains listed in the table, but only those that were found in our survey.
sequence characteristics of editors-namely, the number and location of Asps in L-chain CDRs. Using this criterion, we identified several potential human editor L chains. We tested the ability of the human editors to modify DNA binding by H chains derived from monoclonal anti-DNA antibodies and found that they can silence anti-DNA H chains from both humans and mice. Because the human H3s are generated by mechanisms similar to those in mice, we reasoned that the human endogenous H-chain repertoire might also include H3-Args. If editing of anti-DNAs is a tolerance mechanism which operates in humans, then we would expect that these Arg-containing H chains are preferentially expressed with human editor light chains. This is indeed the case. This finding provides an understanding of how tolerance to DNA is maintained and, importantly, how the B cell repertoire is shaped in humans.

Human anti-DNA L chain editors
Human light chains similar to mouse editors were identified using the Ig Blast database (Table 1). Most, but not all, of these human L chains have Asps in CDRs (Table 1) and a low pI (Fig. 1). The location of Asps is the same in some of the mouse and human editor L chains, such as mouse V38c and VX and their respective human homologues VO8/O18 and V5-1 (Table 1). Our results are consistent with the observation that Asps in L chain CDR regions are particularly important for editing of anti-DNA H chains (Radic et al., 1993b;Jang et al., 1996). The importance of CDR Asps for editing was originally shown in studies of the mouse L chain editors (Gay et al., 1993;Radic et al., 1993a;Li et al., 2001). Substitution of any of the L chain CDR1 (L1) Asp by alanine in the L chain of an anti-DNA antibody resulted in increased 10-and 100-fold in 3H9 forward mutants 3H9/56R and 3H9/56R/76R (Radic et al., 1993b;Seal et al., 2000;Li et al., 2001;Chen et al., 2006). These anti-DNA H chains were expressed in mature B cells only when associated with editor L chains (Li et al., 2001(Li et al., , 2004. To test whether the putative human editor L chains can edit 3H9 and its derivative H chain 3H9/56R, we examined the anti-double stranded DNA (dsDNA) and ANA activities of hybrid antibodies, where these H chains were expressed with each of four putative human editor L chains: VO8/O18, VO2/O12, VL11, and V5-1 (Fig. 2). All four L chains vetoed the 3H9 H chain binding to dsDNA, and two L chains, VO2/O12 and V5-1, decreased the affinity of 3H9/56R for DNA (Fig. 2).
Given the resemblance between the immune systems of man and mice, editors may be important in humans. Some human H chains have Args in their CDRs; hence, these H chains may have anti-DNA properties. We expressed four apparent anti-DNA human H chains with H3 Args and two mouse anti-DNA H chains 3H9 and 3H9/56R (Table 2) in combination with L chains that lack editor properties (i.e., VA27, VB3, and V5-6) in 293 HEK cells. 11 out of 18 hybrid antibodies exhibited dsDNA or ANA activity (Fig. 2, B and D). We also cotransfected the same human H chains with the putative human editor L chains and examined the anti-dsDNA and ANA activities of the recombinant antibodies (Fig. 2). The DNA and ANA activities of human H chains DNA binding (Jang et al., 1998). The ability of Asp in L2 to interact with Arg in a H3 was revealed in the crystal structure of an anti-poly-Q antibody (Li et al., 2007).
Asp in L chain CDRs is neither necessary nor sufficient for editing; we found human L chain editors without Asps that can edit, demonstrating that editing is not restricted to the presence of Asps. For example, the human L chain, VO2/ O12, homologous to mouse editor L-chain V12-46, does not have CDR Asps but does veto DNA-binding and antinuclear antibody (ANA) activity of anti-DNA H chains (Table 1 and Fig. 2). Conversely, a homologue of the mouse editor VX, human V5-6, has two Asps in L2 but does not veto DNA or ANA activity of either mouse or human H chains with H3-Args (Table 1 and Fig. 2). Human V5-6 and mouse VX have a similar VL sequence, including the two Asps in L2 (also found in V5-1), but are dissimilar in L3. Both VX and its closest human homologue V5-1 have long L3s that contain Asp in site 96, whereas V5-6 has a short L3 and no L3 Asp. As shown for VX junctional variants, L3 is of paramount importance for editing (Radic et al., 2013).

L chains veto and modify DNA binding
The affinity of an antibody H chain for DNA depends mainly on the number and location of Args, as has been shown by mutagenesis; substitution of the 3H9 H-chain Arg with the germline-encoded VH amino acid glutamine abolishes DNAbinding, whereas addition of Args enhances DNA binding by  L chains can modify the avidity and the specificity of anti-DNA antibodies We tested the ANA activity of the expressed antibodies described above. ANA activity correlated with DNA binding for 32 out of 35 expressed antibodies in our survey (Fig. 2). The ANA staining pattern varied with L chain usage; 3H9/56R  Table 2). (B and D) dsDNA binding curves (B) and ANA staining (D) for three human L chains (V5-6, VB3, and VA27, and for some experiments VA17) which lack an editor signature, as expressed with the H chains used in testing the putative editor L chains. The hybrid antibodies were tested for reactivity to dsDNA by ELISA. The initial antibody concentration was 10 µg/ml, with four consecutive 1:3 dilutions for ELISA (A and B). Means of at least three independent experiments are plotted, with error bars representing the standard error. For testing of ANAs by HEp2 fluorescence (ANA, C and D), antibodies were used at 50 µg/ml. Bars, 2.0 µm. The ANA staining patterns were defined according to Bradwell and Hughes (2007;C and D). Data are representative of three to five independent experiments.
Args to that of the same L chains that were expressed with H chains without H3 Arg and found a higher frequency of distal J usage in editor L chains associated with H chains which have H3 Args (Table 3; P = 0.075,  2 test). When we performed the same calculations for L chains that do not have editor properties, we did not find any change in J usage between L chains that were expressed with H3 Arg H chains and H chains without H3 Arg (unpublished data).

Lambda editors
The mouse lambda locus includes an efficient editor, VX, which has high sequence homology to the human V5 lambda family. We isolated human V5 cells using a mouse anti-VX mAb (Fig. 4), and we sorted single B cells from the CD20 + VX + population (Fig. 4, right) and sequenced the L chain genes. Most of these B cells (80%) expressed L chains of the V5 family. The expression of individual genes from the V5 family in VX + sorted population was variable. V5-1, the L chain in the V5 family closest to VX, is underrepresented in the human repertoire; we found only one productive rearrangement and no nonproductive ones in 226 V5 cells. Given that the V5 family comprises 1-2% of all B cells as identified by staining (Fig. 4), the frequency of V5-1 expression is estimated to be 1 in 17,000 B cells. For V5-2, we found only three productive and two nonproductive rearrangements. L chains are predominantly associated with endogenous H chains with H3 Args (putative anti-DNAs; Kalinina et al., 2011). We hypothesize that this bias is the result of receptor editing. Here, we performed this analysis in humans; we isolated 268 single peripheral blood B cells from healthy donors and sequenced the H and L chain genes expressed in each B cell. We then compared the H chains with the editor V L chains VO8/O18, VO2/O12, and VL11 to those with non-editor V L chains (Table 1). The VH family distribution in B cells expressing the editor L chains was similar to that of B cells expressing non-editor L chains (Fig. 3). The VH3 family was expressed by >55% of B cells, followed by the VH4 family, which was expressed in >20%, and the VH1 family, which was expressed by 15%, and the expression of the VH5 and VH6 families was <5% of analyzed B cells. This distribution is the same as reported earlier (Brezinschek et al., 1997). Thus, the sorted population of peripheral B cells was representative of a human B cell repertoire. However, the B cells expressing editor L chains exhibited a significantly higher frequency of H chains with Args in H3 (60%) as compared with B cells expressing non-editor L chains (40%; P < 0.005,  2 test; Fig. 3 B).
Another feature of the edited antibodies of the mouse is a high frequency of distal J genes (Radic et al., 1993a). We compared the frequency of different J genes in editor L chains that were expressed in combination with H chains with H3   (L2) and Asp 96 (L3). These Asps are conserved between mouse and human editors ( Table 1). The crystal structures of VX antibodies (Li et al., 2007) show that Asp 60 interacts with H3 Arg. Conversely, the position of H-chain Args can also determine the efficiency of editing. Some Args, such as the Arg 76 in VH framework region 3, cannot be accessed by L chain; hence, antibodies that include a framework region Args in addition to CDR Args are only partially edited by any of the L chain editors.

L chain can modify the specificity of anti-DNA antibodies
Certain editor L chains veto DNA binding, thereby changing the specificity to an unknown antigen. Mouse editor V21D efficiently vetoes DNA binding and ANA activity of 3H9/56R H chain, but V38c paired with 3H9/56R acquires new auto-specificities, including binding to Sm/RNP and to the Golgi apparatus (Khan et al., 2011;Kishi et al., 2012). Another 3H9/56R/L chain combination, 3H9/56R/VX, binds both DNA and MBP (Doyle et al., 2006). Here, we found that ANA staining patterns depend on human L chain usage and can shift from the nuclear homogeneous to the nucleolar pattern. Similarly, Wardemann et al. (2004) have shown that L chain exchange in certain antibodies changes the ANA patterns. Therefore, human L chains can modify both affinity and specificity of autoreactive antibodies.

Receptor editing in vivo
Receptor editing has been convincingly demonstrated in mice, and here we address whether receptor editing is effectively used to regulate anti-DNA B cells in humans. In mice, we showed that endogenous H chains which contain H3 Args are preferentially associated with editor L chains (Kalinina et al., 2011). Here, we compared sequences of H chains expressed with putative editor human L chains to human L chains lacking editor properties and found that antibodies expressing editor human L chains have a higher frequency of H chains with H3 Args (60%) compared with antibodies expressing non-editor L chains (40%; P > 0.004,  2 test; Fig. 3).
The frequencies of V5-4 and V5-6 genes are 1 in 800 and 1 in 85 B cells, respectively. These data are in agreement with the analysis of VL gene expression in a large number of healthy individuals using a L-chain microarray chip, on which neither V5-1 nor V5-2 were detected (Schoettler et al., 2012). V5-1 L chain was detected on the L-chain microarray chip analysis of B cells from SLE patients (Schoettler et al., 2012). Low V5-1 and V5-2 expression could be explained by the stop codon at the 3 end of these V genes. This restricts the possibility of an in-frame rearrangement to just one register. Also, V5-1 is most proximal to the JC genes in the lambda locus (Kawasaki et al., 1997); therefore, this V gene will be deleted by most lambda rearrangements. Alternatively, V5-1 might be edited because it binds proteins such as myelin basic protein (MBP: Galin et al., 1996a,b). A majority (202 out of 226) of the B cells from the CD20 + VX + population expressed the V5-6 L chain. This L chain has high homology to the mouse editor VX, except for the H3 region, but as we showed by in vitro expression experiments, V5-6 does not silence anti-DNA or ANA activity when paired with mouse anti-DNA H chains or with human H chains with Args in H3 (Fig. 2, B and D). A comparative analysis of frequencies of H chains with H3 Args in antibodies that express V5-6 to that of antibodies expressing V editor L chains also indicates that V5-6 belongs to the non-editor group. In antibodies expressing V5-6, the percentage of H chains with H3 Args was lower (33%) than that of antibodies expressing V editor L chains (60%; P > 0.005, Chi-Square test; Table 4).

DISCUSSION
The Human L chain repertoire includes L chains that can modify DNA binding of H chain A comparison of human and mouse L chain sequences identified human L chains that resemble the mouse editor L chains.
To test whether these human L chains can function as editors to modify the DNA-binding capacity of an H chain, we expressed them in association with the mouse H chains known to bind DNA (Ibrahim et al., 1995). These human L chain editors reduce the affinity for DNA of mouse anti-DNA H chains. They also decrease the DNA binding of human H chains with H3 Args (Fig. 2). The degree to which DNA affinity is reduced  (Sanchez et al., 1991). Human peripheral blood B cells (right) were stained with Phycoerythrin/Cy7 anti-CD20 and Alexa Fluor 647 anti-X monoclonal antibodies (Sanchez et al., 1991). approved by the University of Chicago, Institutional Review Board (protocol 14801B), and informed consent was obtained from all participants. Mononuclear cells were isolated from peripheral blood using Lymphocyte Separation Medium (Corning) according to the manufacturer's instructions. Purified mononuclear cells were stained with anti-human antibodies FITC-labeled anti-Ig (BD), PE-labeled anti-Ig (BD), PE-Cy7-labeled CD20 (BD), and Alexa Fluor 647-conjugated anti-VX monoclonal antibody (10C5), specific for the mouse VX gene segment (provided by P.-A. Cazenave; Sanchez et al., 1991). Single cell sorting was performed as described in Wardemann et al. (2003) with slight modifications. Single cells were sorted on a FACSAria II (BD) into 96-well PCR plates containing 4 µl lysis solution (0.5× PBS containing 10 mM dithiothreitol and 8.4 U RNase Inhibitor [New England Biolabs, Inc.]); plates were immediately frozen on dry ice and stored at 80°C. Single B cells expressing V genes (CD20 + V + ) and VX + B cells (CD20 + VX + ) were sorted separately.
cDNA synthesis, PCR, and sequencing. For cDNA synthesis, RNA from single cells was reverse transcribed at 37°C for 55 min with 150 ng random hexamer primer (Integrated DNA Technologies), 0.5 µl dNTP mix (10 mM each, Fermentas), 1 µl of 0.1 M dithiothreitol (Invitrogen), 0.5% (vol/vol) Nonidet P-40, 5.6 U RNase inhibitors (New England Biolabs, Inc.), and 50 U Superscript III reverse transcription (Invitrogen) in a total volume of 14 µl. Heavy and light chain genes from single cells were amplified by PCR from cDNA using two rounds of reactions with previously published nested primers (Smith et al., 2009; 40 µl reactions included 10 pmol of each primer and 0.4 µl of JumpStart Taq DNA polymerase [Sigma-Aldrich]). Aliquots of second PCR products were purified by incubation with 10 U alkaline phosphatase, calf intestinal (CIP; New England Biolabs, Inc.), and 20 U exonuclease I (New England Biolabs, Inc.) in 1× NEB 3 buffer for 15 min at 37°C and sequenced using the reverse primers at the University of Chicago sequencing core. VH, DH, JH, VL, and JL usage was identified using IgBLAST.
Generation of recombinant antibodies. The recombinant antibodies were generated as described previously (Smith et al., 2009). In brief, PCR products of particular VH, V, and V genes were cloned into IgG1, Ig, or Ig expression vectors, respectively. Several expressed V (A27, L11, O8/O18, and O2/O12) and V (5-6) genes were provided by the Patrick Wilson laboratory (University of Chicago, Chicago, IL). Equimolar amounts of expression vectors containing the desired IgH or IgL genes were cotransfected into the 293 cell line using the polyethylenimine, linear, mol wt-25,000 (PEI) transfection reagent (Polysciences, Inc.). The recombinant antibodies were purified from the cell supernatant 5 d after transfection using Pierce Protein A Trisacryl Resin (Thermo Fisher Scientific) and concentrated with Amicon Ultra Centrifugal Filters (Millipore) according to the manufacturer's instructions. Antibody concentrations were determined using a NanoDrop Spectraphotometer ND-1000 (NanoDrop Technologies).
ELISA for dsDNA binding. Antibody concentrations were adjusted to 10 µg/ml, and four consecutive 1:3 dilutions in blocking solution (1% BSA in PBS) were prepared. ELISA assays were performed as described previously (Radic et al., 1993a). Immulon 4 HBX ELISA plates (Thermo Fisher Scientific) were coated with 10 µg avidin D (Vector Laboratories) in PBS at 4°C overnight, blocked in PBS with 1% BSA at RT for 2 h, and washed with PBS with 0.05% Tween (PBS-T). Biotinylated dsDNA was bound to avidin-coated plates at 37°C for 1.5 h. Diluted antibodies were applied for 1.5 h at 37°C, plates were washed three times, and DNA-Ab complexes were detected with alkaline phosphataseconjugated anti-human IgG (H+L) Ab (Bio-Rad Laboratories). After absorption for 90 min, plates were washed and the remaining anti-dsDNA Abs were quantified using AP substrate (Sigma-Aldrich). The OD was determined at 405 nm.
ANA assay. Antibody concentrations were adjusted to 50 µg/ml to test for antinuclear antigen binding using a HEp-2 ANA kit (Bion Enterprises, LTD) according to the manufacturer's instructions. Slides were examined on a DMR fluorescence microscope (Leica). Images were captured using a 63×/1.40-0.60 oil objective (Leica) and a 10×/25 ocular (Leica), with a Retiga 200R camera (Q Imaging) and Q Capture Pro (Media Cybernetics) imaging software.
Because of the correlation between DNA binding and H3 Args, we infer that the observed difference in the H chain repertoire is a result of receptor editing in humans as well as in mice. Another indicator of receptor editing is a bias of JL usage to J3/J4/J5 of the  L chain editors (Radic et al., 1993a) and we found that the editor L chains expressed with H chains with H3 Args have a trend toward a higher frequency of J3/ J4/J5 rearrangements (Table 3).

Conservation of editors
The conservation of editor L chains between mouse and human is remarkable and argues for the importance of these L chains. VX has little homology (<33% amino acid sequence identity) to either mouse  or  V genes, suggesting that VX, V, and V diverged at the same time (Sanchez et al., 1990). Nevertheless, VX is conserved across species (Sanchez et al., 1990); mouse VX and human V5-1 are 70% identical (Sanchez et al., 1991). Moreover, V5-1 and VX share unusual characteristics such as a long L3 region and a stop codon just 3 of the V coding region. The mouse anti-VX mAbs cross-react with V5-expressing B cells, which allows us to calculate the frequency of the human V5 (VX-like) family expression in the mature B cell repertoire; this low frequency is also found for VX in mice (Fig. 4). The V5-1 and the mouse VX also share specificity. The mouse editor VX alone and in combination with most H chains binds MBP (Galin et al., 1996a) and it was shown that a human IgM/V antibody, that is recognized by an anti-VX mAb, is also MBP-reactive (Noerager et al., 2001).
The putative human editor VO8/O18 shares properties with the mouse editor V38c both in sequence homology and editing properties. Moreover, studies of Ig L chain amyloidosis documented that VO8/O18 L chains form amyloidogenic proteins (Connors et al., 2007). Mouse V38c antibodies were shown to be secreted in the form of extracellular aggregates called spherons that stain with the amyloid-selective dye thioflavin T (Khan et al., 2011). The similarities between mouse and human editors indicate that these editors may perform similar functions.

Conclusions
Our data demonstrate that the human L chain repertoire includes editor L chains as do mice and presumably other species. Our finding that human editor L chains associate with H chains which have an elevated frequency of H3 Args suggests that humans use receptor editing to regulate anti-DNA B cells. We also show here that receptor editing shapes the B cell repertoire. Editor L chains rescue expression of anti-DNA H chains, and likewise anti-DNA H chains rescue some editor L chains. Some antibodies that express editor L chains, such as mouse VH7183/ VX, are frequently used in broadly neutralizing antiviral antibodies (Lee et al., 2008). Another antibody with the same editor L chain MW1/VX is reactive to poly-Q repeats (Li et al., 2007). Therefore, editing of self-reactive antibodies is important for both protective immunity and tolerance.

MATERIALS AND METHODS
Single B cell isolation and sorting. Samples of peripheral blood were collected from 5 healthy donors (22-50 yr old). The use of human blood was Statistical analysis. P-values for Ig gene repertoire analysis and arginine content in H3 were calculated using the  2 test.