Abstract
While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig remains unknown. In B cells, IgD is expressed together with IgM as transmembrane receptor for antigen through alternative splicing of long primary VHDJH-Cμ-s-m-Cδ-s-m RNAs, which also underpin secreted (s)IgD. IgD is also expressed through class switch DNA recombination (CSR), as initiated by AID-mediated double-strand DNA breaks (DSBs) in Sμ and σδ, and resolution of such DSBs by a still unknown mechanism. This synapses Sμ with σδ region DSB resected ends leading to insertion of extensive S-S junction microhomologies, unlike Ku70/Ku86-dependent NHEJ which resolves DSB blunt ends in CSR to IgG, IgA and IgE with little or no microhomologies. Our previous demonstration of a novel role of Rad52 in a Ku70/Ku86-independent “short-range” microhomology-mediated synapsis of intra-Sμ region DSBs led us to hypothesize that this homologous recombination DNA annealing factor is also involved in short-range microhomology-mediated alternative endjoining (A-EJ) recombination of Sμ with σδ. We found that induction of IgD CSR by selected stimuli downregulated Zfp318 (the suppressor of Cμ-s-m transcription termination), promoted Rad52 phosphorylation and Rad52 recruitment to Sμ and σδ, leading to Sμ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52−/− B cells aborted IgD CSR in vitro and in vivo and dampened the specific IgD antibody response to OVA. Further, Rad52 knockdown in human B cells virtually abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and lupus patients. Thus, Rad52 effects CSR to IgD through microhomology-mediated A-EJ and in concert with Zfp318 modulation. This is a previously unrecognized, critical and dedicated role of Rad52 in mammalian DNA repair that provides a mechanistic underpinning to CSR A-EJ.
IgD has been an enigmatic antibody class for many years, despite being evolutionarily ancient and highly conserved across species1–6. As primordial as IgM, IgD appeared in cartilaginous fishes, amphibians and occurs in fishes, rodents, cattle and humans2, 7. As an example, in Xenopus, the Igδ exon cluster is in the same position, immediately 3′ of the Igμ locus, as it exists in mammals7. In mice and humans, IgD is expressed primarily as a transmembrane IgD receptor together with IgM with identical antigen specificity on naïve mature B cells in the form of BCR. IgD also exists as a secreted antibody. In humans, circulating IgD occurs at concentrations up to more than two-thousand folds greater than IgE (10-250 μg/ml vs. ∼0.1 μg/ml), the rarest peripheral blood Ig class. IgD are secreted by IgM−IgD+ plasmablasts and plasma cells differentiated from B cells in lymphoepithelial organs in aerodigestive mucosae, including palatine and pharyngeal tonsils. IgM−IgD+B cells and plasma cells can also be found in the lachrymal, salivary and mammary glands3. In addition to existing as free molecule, IgD can occurs on the surface of innate effector cells, including basophils, mast cells and monocytes1, 8, 9. IgD bound to these cells would enhance immune surveillance and exert proinflammatory and antimicrobial effects1, 8, 9. These include triggering basophils to secret IL-4, IL-5 and IL-13 upon antigen engagement or attenuating basophil or mast cell allergic degranulation induced by IgE co-engagement1. Thus, IgD would contribute to mucosal homeostasis by endowing effector cells with reactivity to microbial commensals and pathogens5, 6.
Identifying the stimuli and molecular mechanisms that underpin IgD expression is important to understand the regulation of IgD secretion throughout the body. The immediately proximal location and unique integration of Cδ and Cµ gene loci in the same transcriptional unit allow these two Ig isotypes to be coordinately regulated in transcription10, 11. In naive mature B cells, (membrane) mIgM and mIgD are co-expressed by alternative splicing of long primary transcripts consisting of rearranged VHDJH exons and downstream Cµ and Cδ exons (VHDJH-Cµ-s-m-Cδ-s-m). Alternative splicing of the same long primary VHDJH-Cµ-s-m-Cδ-s-m transcripts also leads to expression of (secreted) sIgM and sIgD2, 8. Transcription of long primary VHDJH-Cµ-s-m-Cδ-s-m RNA requires the zinc finger ZFP318 repressor of transcriptional termination, which obliterates the effect of the transcriptional termination sites (TTS) intercalated between the Cμ and Cδ exon clusters10, 11 (Fig. 1a). IgD can also be expressed through class switch DNA recombination (CSR), by which IgM+IgD+B cells juxtapose VHDJH DNA from the Cµ (IgM) to the Cδ (IgD) exons cluster, giving rise to VHDJH-Cδm RNA transcripts and IgM−IgD+B cells1, 5, 8, 9, 12 (Fig. 1b). In human and mouse nasopharyngeal and intestinal lymphoid tissues, a significant proportion of mucosal B cells class-switch to IgM−IgD+B cells, which subsequentially differentiate to plasmablasts and plasma cells1, 3, 5, 6. Generally, CSR to IgD (Cδ) is a less frequent event than CSR to IgG (Cγ), IgA (Cα) or IgE (Cε), perhaps a reflection among other factors of the peculiar structure of the pseudo-switch σδ region lying immediately upstream of Cδ exons. Compared to the canonical Sμ, Sγ, Sα and Sε regions lying 5’ of the respective Igμ, Igγ, Igα and Igε loci, σδ is shorter and contain differing motifs of nucleotide (nt) repeats2, 5, 8, 13, 14. These would provide an unconventional substrate for AID-mediated insertion of DSBs, possibly more prone to end-resection and generation of single-strand overhangs for Sμ-σδ recombination, which leads to expression of post-recombination VHDJH-Cδ RNA transcripts2, 8, 13–15.
Unlike CSR to IgG, IgA and IgE, the mechanism of CSR to IgD remains unknown. Recombination involving Sμ DSB ends with DSB ends in downstream Sμ, Sγ, Sα or Sε region is effected by non-homologous end-joining (NHEJ), one of the two major DNA DSB repair pathways, the other being homologous recombination (HR)16, 17. HR accurately repairs resected (staggered) DSB ends using a sister chromatid as a homologous single-strand template during cell cycle S-G2. It critically effects error-free DSB repair in somatic cells and helps orchestrate chromosome segregation in meiosis. In contrast to HR, NHEJ is a homology-independent error-prone process. It synapses blunt or virtually blunt DSB ends that lack substantial joining complementarity to form “direct” junctions, predominantly in G1 but also throughout the whole cell cycle16. NHEJ requires Ku70/Ku86 and in CSR mediates efficient long-range synapses of Sμ DSB ends with Sγ, Sα and Sε DSB ends, leading to IgG, IgA and IgE15. The finding, however, that reduction or deletion of Ku70/Ku86 led to reduced but still substantial CSR to IgG1 and IgG3 supported the existence of an alternative CSR end-joining (A-EJ) pathway18–20. This, like HR, would join resected DSB ends, thereby giving rise to S-S junctions with microhomologies. Unlike HR, however, the A-EJ pathway juxtaposes DSB overhangs to be joined without using a homologous template as a guide. Rather, it utilizes differing extents of sequence complementarity (homology) between the upstream and downstream resected DSB overhangs to align the to-be DNA junctions21. As we have shown, HR factor Rad52 competes with Ku70/Ku86 for binding to S region DSB ends and synapses DSB ends by A-EJ through microhomology-mediated end-joining (MMEJ)20, as inferred from increased NHEJ-mediated IgG, IgA and IgE CSR events with even fewer S-S junction microhomologies in Rad52−/− B cells in vivo and in vitro20. This together with the increased CSR to IgD in B cells lacking 53BP1, which protects S regions DSB ends from resection and facilitates long-range NHEJ to IgG, IgA and IgE22–24, as well as other findings of ours showing reduced intra-Sμ DSB short-range rejoining in Rad52−/− B cells20 led us to hypothesize that by annealing to single-strand resected DSB ends, Rad52 mediates CSR to IgD through A-EJ involving short-range Sμ-σδDSB recombination.
To test the hypothesis that Rad52 synapses Sµ with σδ DSB ends for IgD CSR, we first set up to define the stimuli that consistently induce CSR to IgD in mouse and human B cells. We then used such stimuli in mouse Rad52−/−B cells and RAD52 siRNA knockdown human B cells together with molecular genetic methods to determine the impact of Rad52 deficiency as well as Rad52 phosphorylation on Sµ-σδ DNA recombination and IgD expression. We validated our findings by analyzing specific IgD antibody and total IgD titers in mouse blood, lungs and gut, as well as recombined Sµ-σδ DNA sequences in mouse spleen, mesenteric lymph nodes (MLNs) and Peyer’s patches as well as human tonsil B cells. We adapted chromatin immunoprecipitation (ChIP) assays to analyze the recruitment of Rad52/RAD52 to the σδ region in mouse and human B cells induced to undergo CSR to IgD, in which we also analyzed regulation of VHDJH-Cδ transcription. We found that different stimuli induced IgD expression by alternative splicing of long VHDJH-Cµ-s-m-Cδ-s-m RNA transcripts or by Sµ-σδ CSR. Further, we determined the expression of IgD by CSR to be related to Zfp318-mediated repression of the TTS integrated in Cμ-Cδ loci. We also correlated Sµ-σδ CSR with IgD secretion and plasma cell differentiation. Finally, we analyzed B cell Rad52 phosphorylation in lupus patients and lupus-prone mice and correlated it with CSR to IgD involving extensive microhomologies and somatic mutations in Sµ-σδ junctional sequences, as well as the occurrence of high levels of anti-nuclear antigen IgD autoantibodies. Our findings show that Rad52 mediates CSR to IgD through microhomology-mediated A-EJ and in concert with Zfp318 modulation. This is a previously unrecognized, critical and dedicated role of Rad52 in an essential DNA repair process in mammals.
Results
Definition of stimuli that induce Sµ-σδ CSR in mouse and human B cells
Toward testing our hypothesis that Rad52 mediates CSR to IgD, we first determined the stimuli that induce IgM+IgD+ B cells to undergo Sµ–σδ recombination. In these B cells, mIgD and sIgD and IgM are expressed by alternative splicing of long primary VHDJH-Cµ-s-m-Cδ-s-m mRNAs – the Cδ locus is located immediately downstream of the Cµ locus in the same transcriptional unit, allowing these two loci be coordinately regulated at the transcriptional level1, 2, 4, 6 (Fig. 1a). As CSR can be induced in a T-dependent or T-independent antibody fashion15, 25, we used CD40 ligand CD154 (for mouse and human B cells), TLR4 ligand LPS (mouse B cells) and TLR9 ligand CpG (human B cells) in conjunction with differing cytokines and/or BCR-cross-linking to induced CSR to IgD. Recombined Sµ–σδ, Sµ– Sγ1, Sµ–Sγ3, Sµ–Sα and Sµ–Sε DNAs were detected by specific nested PCRs followed by positive identification of amplified DNA by blotting and hybridization with specific DNA probes (Fig. 1 inset), complemented by sequencing of the junctional Sµ-σδ or Sµ-SX DNA. Of all stimuli used, only LPS or CD154 plus IL-4 induced CSR to IgD in mouse B cells (Fig. 2a), and only CpG plus IL-2 and IL-21, or CD154 plus IL-4 or IL-15 and IL-21 induced CSR to IgD in human B cells. IgD CSR was also detected in vivo in tonsil B cells (Fig. 2b). The effectiveness of the stimuli that did not induce CSR to IgD was verified by the respective induction of the expected Sµ–Sγ1, Sµ–Sγ3, Sµ–Sα or Sµ–Sε DNA recombination. (IgG1, IgG3, IgA or IgE) (Fig. 2a) – no CSR to IgD, IgG, IgA or IgE occurred in Aicda−/− B cells. In all cases, CSR was further confirmed by detection of post-recombination Iμ-Cγ1, Iμ-Cγ3, Iμ-Cα and Iμ-Cε transcripts at 72 h of culture – as post-recombination Iμ-Cδ transcripts are indistinguishable from germline Iμ-Cδs-m RNA transcripts and consistent with high levels of the latter in naïve B cells, Iμ-Cδ amplification products were less abundant in class-switched IgD than naïve B cells (Figs. 1, 2c). Thus, only select stimuli induce CSR to IgD in mouse and human B cells.
Sµ-σδ junctions are enriched in microhomologies and abetted by somatic mutations in mouse and human B cells
The mechanisms effecting CSR S-S synapses can leave a S-S junctional signature20, 26. As we previously showed, Rad52 mediates A-EJ of resected DSB ends by juxtaposing overhangs with nucleotide complementarities, thereby giving rise to Sμ-Sx DNA junctions with microhomologies20. Next generation sequencing of more than 100,000 recombined Sμ-Sx DNA junctions from mouse and human B cells in vitro and/or in vivo showed that Sµ–σδ junctions contained significantly more microhomologies (p <0.01) than Sµ–Sγ1 or Sµ–Sα DNA junctions (representative frequencies and lengths of microhomologies in human and mouse B cells are depicted in Fig. 3a; representative human and mouse intra-σδ and junctional Sμ-Sx sequences are depicted in Fig. 4 and Extended Data Figs. 1,2), indicating that a MMEJ21 synaptic process underpinned Sµ–σδ junction formation. In both human and mouse B cells, the microhomologies in Sµ–σδ junctions were significantly more extensive than those in Sµ– Sγ1 and, to a lesser extent, Sµ–Sαjunctions (Fig. 4, Extended Data Figs. 1,2). As one example, in human tonsil B cells, 100% of analyzed Sμ-σδ junctions contained microhomologies, consisting of 2 to 13 nucleotides (mean = 6.30), while only 21% of Sµ–Sγ1 junctions contained microhomologies, consisting of 1 to 6 nucleotides (mean = 0.72) (Fig. 3a). Interestingly, there were a few common S-S sequences shared by recombined Sµ–σδ DNA junctions in human tonsil B cells and blood naïve B cells stimulated in vitro by CpG plus IL-2 and IL-21, suggesting that Sµ and σδ DSB hotspots underpin Sµ–σδ in DNA recombinations. A high frequency of microhomologies was also evident in the synaptic repair process of intra-σδ DSBs, evocative of what we showed in intra-Sμ DSBs20. Consistent with the greatest occurrence of microhomologies in Sµ–σδ junctions, Sμ is better suited for complementary DNA single-strand annealing with σδ than Sγ1 or, to a lesser extent, Sα (mouse) or Sα1 (human), based on various numbers and contexts of these DNA regions discrete motifs, such as [Gn]AGCT repeats (Sµ, Sγ and Sα) or AGCTGAGCTG repeats (Sμ and σδ), as revealed by Pustell Matrix dot-plot analysis (Fig. 3b). Finally, Sµ–σδ DNA junctions were associated with somatic point-mutations. These were more frequent in the σδ area than Sμ area abetting the Sµ–σδ junction (e.g., 0.559 x 10−2 vs. 0.973 x 10−2 change/base in mouse spleen B cells in vivo and 1.251 x 10−2 vs. 1.985 x 10−2 change/base in mouse B cells stimulated by LPS plus IL-4 in vitro) (Fig. 3c). Thus, the high frequency of microhomologies in Sµ–σδ junctions supports a role of Rad52 in mediating CSR to IgD.
Rad52 is critically required for Sµ–σδ recombination
Having established that LPS or CD154 pus IL-4 induced CSR to IgD in mouse B cells, we used these stimuli and Rad52−/− B cells together with appropriate controls (LPS alone, LPS plus TGF-β and RA, CD154 or CD154 plus TGF-β and RA) and the same approach used in the experiments of Fig. 2 to investigate whether or not Rad52 was required for CSR to IgD. LPS plus IL-4 and CD154 plus IL-4 failed to induce Sµ–σδ recombination in Rad52−/− B cells, while either treatment efficiently induced Sμ-Sγ1 and Sμ-Sε recombinations in the same Rad52−/− B cells, and Sµ–σδ recombination in Rad52+/+ B cells (Fig. 5a) – in Rad52−/− B cells, LPS and LPS or CD154 plus TGF-β and RA induced CSR to IgG3 and IgA, respectively. As expected, CSR to IgD as well as IgG, IgA and IgE was ablated in Aicda−/− B cells. Finally, the failure of Rad52−/− B cells to undergo CSR to IgD was associated with significantly decreased secretion of IgD (Fig. 5b). Thus, Rad52 is critical for Sµ–σδ DNA recombination and seemingly important for IgD secretion.
Rad52 is required to mount a specific IgD antibody response
We determined the role of Rad52 in supporting a specific IgD antibody response by immunizing Rad52−/− and Rad52+/+ mice with OVA (20 μg in alum, i.p., 3 times). Rad52−/− mice showed no Sµ–σδ recombination in spleen, mesenteric lymph nodes (MLNs) or Peyer’s patch B cells (Fig. 6a). The lack of CSR to IgD was specific, as B cells in such mice showed Sµ–Sγ1 and Sµ– Sα DNA recombinations as B cells in Rad52+/+ mice, which also underwent Sµ–σδ recombination. In Rad52−/− mice, Sµ–Sγ1 and Sµ–Sα DNA junctions showed fewer and shorter microhomologies than in Rad52+/+ mice (Fig. 6b, Extended Data Figs. 2,3), a reflection of involvement of Rad52 in CSR to isotypes other than IgD20. Rad52−/− mice showed significantly decreased total and/or OVA-specific IgD in circulating blood, bronchoalveolar lavage (BALF), feces (free or bound to fecal bacteria), and IgD-producing cells in MLNs and lamina propria, as compared to their Rad52+/+ counterparts (Fig. 6c-h). This contrasted with the normal or elevated total and OVA-specific IgM, IgG1 and IgA levels in the same Rad52−/− mice, as predicted based on our previous findings27. Thus, Rad52 is required to mount an efficient antigen-specific class-switched IgD response.
Rad52 is modulated and phosphorylated by IgD CSR-inducing stimuli, and it is recruited to Sμ and σδ
We analyzed Rad52, Ku70, Ku86 and Aicda transcripts as well as respective Rad52, Ku70, Ku86 and AID proteins, including phosphorylated Rad52 (p-Rad52 has been shown to display enhanced ssDNA annealing activity28), in B cells induced to undergo CSR to IgD. Mouse B cells stimulated by LPS plus IL-4 and human B cells stimulated by CD154 plus IL-4 and IL-21 increased Ku70/Ku86 and Ku70/Ku86 expression at 24-48 h concomitant with significantly greater expression of Aicda and AID, which was nearly undetectable at time 0, while somewhat downregulating Rad52 and Rad52. Rad52 protein, however, was progressively phosphorylated within the same time range (Fig. 7a-c). Further supporting its role in CSR to IgD, Rad52 was recruited to Sμ, σδ (and Sγ1) in B cells stimulated by LPS plus IL-4, which induced Sμ-σδ (and Sμ-Sγ1) DNA recombination, but not by stimuli that did not induce Sµ–σδ recombination, i.e., LPS alone or LPS plus TGF-β and RA, as shown by chromatin immunoprecipitation (ChIP) using an anti-Rad52 Ab – the specificity of the ChIP Rad52 recruitment assay being emphasized by the lack of chromatin immunoprecipitation in Rad52−/− B cells (Fig. 7d,e). Recruitment of Rad52 but not Ku70/Ku86 to σδ in CSR to IgD, as induced by LPS plus IL-4, contrasted with that of Ku70/Ku86 to Sγ3 and Sα regions as induced in CSR to IgG3 and IgA (Fig. 7f), a possible reflection of the competition of these HR and NHEJ elements for binding to S region DSB ends20. Notably, LPS plus IL-4 induced recruitment of Rad52 but not Ku70/Ku86 to σδ, while inducing mostly Ku70/Ku86 recruitment to Cγ1, consistent with the efficient LPS pus IL-4 induction of CSR to IgG1, mediated mainly by NHEJ20. Thus, Rad52 expression and, importantly, Rad52 phosphorylation are modulated by IgD CSR-inducing stimuli.
Stimuli that induce Sμ-σδ DNA recombination downregulate ZFP318/Zfp318 and lead to IgD secretion
Next, we addressed the expression of mIgD and sIgD and its regulation by stimuli inducing CSR to IgD. Resting B cells expressed mIgD and mIgM, but little or no sIgD or sIgM, reflecting high levels of VHDJH-Cδm and VHDJH-Cμm trancripts and low levels of VHDJH-Cδs and VHDJH-Cμs transcripts (Fig. 8a). Induction of CSR to IgD (by LPS or CD154 plus IL-4) resulted in loss of virtually all mIgD, emergence of VHDJH-Cδs transcripts together with VHDJH-Cμs transcripts and significant IgD secretion (Fig. 8a-b). By contrast, application of IgD CSR non-inducing stimuli (LPS plus TGF-β and RA) to similar naive IgM+IgD+B cells resulted in partial loss of mIgD, no change in VHDJH-Cδm transcripts and marginal IgD secretion (Fig. 8a-b). The changes in VHDJH-Cδm, VHDJH-Cδs transcripts, mIgD and sIgD brough about by IgD CSR-inducing stimuli paralleled the downregulation of Zfp318 transcripts and Zfp318 protein – Zfp318 represses the TTS that mediates alternative transcriptional VHDJH-Cμ/VHDJH-Cδ termination, thereby allowing for long-range transcription throughout VHDJH-Cµ-s-m-Cδ-s-m DNA (Fig. 8c-e). Zfp318 downregulation was specific to IgD CSR, as it did not occur in response to IgA CSR-inducing stimuli (LPS plus TGF-β and RA). ZFP318 downregulation concomitant with decreased mIgD expression and increased IgD secretion was reproduced in human B cells submitted to IgD CSR-inducing stimuli (CpG plus IL-2 and IL-21) but not IgD CSR non-inducing stimuli (CpG plus IL-4 and IL-21) (Fig. 8,f). Similarly, ZFP318 transcripts and ZFP318 protein were downregulated in human B cells undergoing IgD CSR in vivo, as in tonsils (Fig. 8,g). Zfp318 downregulation was independent and likely preceding expression of AID or Rad52, as revealed by virtual absence of Zfp318 transcripts in LPS plus IL-4-induced Aicda−/− B cells, Rad52−/− B cells and Rad52+/+ B cells, all of which lost mIgD expression as compared to similar B cells stimulated by IgA CSR-inducing stimuli (LPS plus TGF-β and RA) (Fig. 8h-j). Thus, the stimuli that specifically induce CSR to IgD downregulate ZFP318/Zfp318 independently of AID or Rad52 expression and prior to Sμ-σδ DNA recombination.
RAD52 knockdown reduces Sμ-σδ DNA recombination and IgD secretion in human B cells
The high frequency of microhomologies in Sμ-σδ junctions of human tonsil B cells in vivo and human B cells induced to undergo CSR to IgD in vitro (Figs. 3a,4,6b, Extended Data Figs. 1-5) suggested to us that RAD52 also mediates Sμ-σδ DNA recombination in human B cells. We purified naïve IgM+IgD+B cells from peripheral blood of 3 healthy subjects and knocked down using RAD52-specific siRNAs RAD52 transcripts and RAD52 protein by up to 75% and 95%, respectively. In these B cells, Sμ-σδ recombination, as induced by CpG plus IL-2 and IL-21, was virtually abolished, while AICDA or AID expression and Sμ-Sγ1 recombination were not altered (Fig. 9a-c). The reduced Sμ-σδ DNA recombination in RAD52 knockdown human B cells was associated with decreased expression of VHDJH-Cδs transcripts, without significant alteration of VHDJH-Cδm transcripts (Fig. 9d). The critical role of RAD52 in human CSR to IgD was emphasized by RAD52 recruitment to Sμ and σδregions in human naïve B cells induced to undergo CSR to IgD (by CpG plus IL-2 and IL-21) in vitro, human tonsil (IgD+) B cells undergoing CSR to IgD in vivo, but not in unstimulated naïve IgD+IgM+ B cells (Fig. 9e). Thus, Rad52 critically mediates CSR to IgD through Sµ–σδ recombination in human B cells.
Sμ-σδ DNA recombination leads to IgD plasma cell differentiation
To determine whether the substantial IgD production we observed only upon induction of CSR to IgD (Figs. 5b,6c-h,8b,f) would be associated with plasma cell differentiation, we analyzed human IgM+IgD+B cells induced to undergo CSR to IgD by CpG plus IL-2 and IL-21. More than 13% of these B cells became mIgM−intracellular IgD+ compared to about half of their counterparts stimulated by CpG plus IL-4 and IL-21 and not switching to IgD (Fig. 10a). More than 90% of the IgM−IgD+B cells emerging from CpG plus IL-2 and IL-21 simulation expressed BLIMP-1 and almost 60% were CD27+CD38+ versus about 10% of the IgM−IgD+B cells from CpG plus IL-4 and IL-21 expressing BLIMP-1 and less than 12% being CD27+CD38+. Among mouse IgM+IgD+B cells induced to undergo CSR to IgD by LPS plus IL-4, about 25% expressed intracellular IgD. All these B cells also expressed Blimp-1 and 70% or more acquired CD138 (Fig. 10b). By contrast, among IgM+IgD+B cells induced to undergo CSR to IgA by LPS plus TGF-β and RA, about 50% expressed intracellular IgD but virtually none expressed Blimp-1 or acquired surface CD138. The relevance of IgD CSR to sustained IgD secretion was suggested by analysis of 3 human myelomas, two IgD and one IgA. Both IgD myelomas displayed Sµ–σδ DNA, but not Sµ–Sα DNA recombination (Fig. 10c). Conversely, the IgA myeloma showed Sµ–Sα, but not Sµ–σδ DNA recombination. Thus, IgD+B cells emerging by CSR would are prone to differentiate into IgD-secreting plasmablasts/plasma cells for sustained IgD secretion. And such IgD+B cells may function as precursors of neoplastic IgD+ transformants.
B cell Rad52 phosphorylation, increased CSR to IgD and IgD autoantibodies in systemic autoimmunity
Serum IgD have been suggested to increase in patients with inflammatory autoimmune diseases, such as systemic lupus erythematosus (SLE)29 and rheumatoid arthritis30, and in hereditary autoinflammatory syndromes, most notably the hyper-IgD syndrome (HIDS)31–34. While in healthy humans, many B cells make IgD that react with components of the self35, we found patients with systemic lupus to display significantly higher levels of circulating IgD, including IgD specific for nuclear antigens, than their healthy subject controls (Fig. 11a,d). This possibly reflected the higher level of B cell Rad52 and/or p-Rad52 expression in such lupus patients (Fig. 11k). Similarly, we found lupus-prone MRL/Faslpr/lpr mice to show far higher levels of IgD than their wildtype C57BL/6 counterparts in serum, feces and BALF as well as increased IgD-coated bacteria in feces, a reflection of high levels of VHDJH-Cδs transcripts in bone marrow, spleen, MLNs and Peyer’s patches B cells as well as increased numbers of IgD+ B cells in lamina propria, MLNs and Peyer’s patches (Fig. 11a-f). In MRL/Faslpr/lpr mice, the elevated IgD levels reflected increased IgD-producing cells and increased Sμ-σδ DNA recombination in bone marrow, spleen, MLNs and Peyer’s patches (Fig. 11g). Increased CSR to IgD in MRL/Faslpr/lpr was associated with high levels of p-Rad52 expression, greater frequency and length of microhomologies in Sµ–σδ as compared to Sµ-Sγ1 and Sµ-Sα junctional sequences, as well as with a high frequency of somatic point-mutations in areas abetting Sμ-Sδ DNA junctions (Fig. 11h-k and Extended Data Fig. 6). Thus, high levels of B cells expressing p-Rad52 are associated with high levels of IgD and IgD autoantibodies to nuclear antigens in lupus patients and in lupus-prone MRL/Faslpr/lpr mice. In these mice, Sμ-σδ DNA recombination events involving high frequency of junctional microhomologies occur in B cells of different body districts, giving rise to high levels of IgD autoantibodies locally and systemically.
Discussion
The mechanism of CSR to IgG, IgA and IgE are quite well understood, as mediated by Ku70/Ku86-dependent NHEJ, although occurrence of a “residual” IgM to IgG CSR in B cells lacking Ku70/Ku86 expression has suggested the existence of a Ku70/Ku86-independent A-EJ synaptic mechanism18–20. Mice lacking 53BP1, in which NHEJ-dependent CSR to IgG, IgA and IgE was significantly decreased – 52BP1 protects resection of DSB ends, thereby skewing the synaptic process toward NHEJ – showed increased CSR to IgD and increased circulating IgD levels, suggesting that the short-range Sµ–σδ CSR was mediated by a 53BP1-independent synaptic process involving resected DSB ends and entailing a high frequency of Sµ–σδ junctional microhomologies22, 23. This together with our previous demonstration that Rad52 plays a central role in synapsing intra-Sμ region resected DSB ends as well as c-Myc/IgH locus translocations also involving resected DSB ends, both processes entailing significant junctional microhomologies, prompted us to hypothesize that Rad52 mediates the A-EJ process that synapses Sµ and σδ DSB with complementary overhangs in CSR to IgD20. Here, we demonstrated that Rad52 mediates CSR to IgD in mouse and human B cells (Extended Data Fig. 7), thereby unveiling a previously unknown, critical and dedicated role of this HR factor in mammalian DNA repair.
We have provided here unequivocal evidence that Rad52 is critical for CSR to IgD in vitro and in vivo, in mouse and human B cells. In mouse Rad52−/− B cells, Sμ-σδ DNA recombination was ablated and IgD secretion greatly reduced. Similarly, in RAD52 knockdown B cells from healthy subjects, Sμ-σδ DNA recombination was virtually abrogated and IgD secretion greatly decreased – as expected1, 5, Sμ-σδ DNA recombination could not occur in the absence of AID, which introduces DSBs in σδ as it does in Sμ, Sγ, Sα or Sε. In mouse Rad52−/− B cells and human RAD52 knockdown B cells, decreased post-recombination VHDJH-Cδs transcripts resulted in reduced IgD secretion, which occurred in presence of unaltered transmembrane VHDJH-Cδm transcript levels, at least within the first 72 hours from CSR induction. Interestingly, the stimuli that selectively induced CSR to IgD modulated the overall levels of Ku70/KU70, Ku86/KU86 and Rad52/RAD52 transcripts while significantly upregulating Aicda/AICDA in mouse and human B cells. This was concomitant with induction of AID and moderate decrease in Rad52 protein, which, in fact, was increasingly phosphorylated at Tyr104. Rad52 Tyr104 phosphorylation has been shown to boost Rad52-mediated DNA single-strand annealing and is possibly effected by c-ABL kinase28. Rad52 involvement in CSR to IgD was further emphasized by recruitment of this protein to σδ region (in addition and necessarily to Sμ) in vivo in human tonsil IgD+ B cells, as well as in vitro, in mouse and human naïve B cells induced to undergo CSR to IgD, but no or only marginally in similar B cells undergoing CSR to IgG3 or IgA. Instead, these B cells recruited Ku70/Ku86 to Sγ3 and Sα, consistent with the major contribution of NHEJ to CSR to IgG3 and IgA.
Rad52 is a member of the eponymous epistasis group for DSB repair that shows strong evolutionary conservation17, 36. In Saccharomyces cerevisiae, Rad52 is a key element of the HR pathway, and its deletion or mutation impairs DNA DSB repair37, 38. Indeed, yeast Rad52 is a recombination mediator and a facilitator of annealing of complementary DNA single-strands39, 40. It functions as a cofactor of Rad51, which forms nucleoprotein filaments with single-strand DNA and promotes strand pairing, by overcoming the inhibitory effect of replication protein A (RPA)41. By contrast, Rad52 mutation or even deletion results in no obvious abnormalities in viability or functions in mammalian cells. As we have shown, Rad52−/− mice displayed no significant alteration of immune system elements, including B cells20, possibly owing to the presence of mammalian gene paralogues, such as BRCA2 and RAD51, which by encoding functions related to Rad52, can compensate for the absence of this factor42. Human BRCA2 functions as a recombination mediator by facilitating RAD51 nucleoprotein filament formation40, 43–45. Nevertheless, human BRCA2 cannot facilitate annealing of RPA-coated DNA, a function that Rad52 carries out efficiently in the absence of BRCA246. This together with Rad52 involvement in DSB repair at stalled or collapsed replication forks points at a unique role of Rad52 in catalyzing single-strand annealing in homology-directed DNA repair in human cells47–49.
Our identification of Rad52 as essential in IgD CSR Sμ-σδ synapses provides, to the best of our knowledge, the first demonstration of a critical and dedicated role of this factor in mammalian DNA repair. The short-range Rad52-mediated Sμ-σδ recombination of resected DSB ends adds to the other Rad52-mediated short-range DSB recombination we recently uncovered: intra-Sμ region DSB recombination20. This, like Sμ−σδ synapsing, engages resected DSB ends and yields significant junctional nucleotide microhomologies20. In this function, as in CSR to IgD, Rad52 is not fungible in mouse or human B cells. Our identification of Rad52 as the critical element in Sμ-σδ synapsis also sheds light on the mechanistic nature of the CSR A-EJ DSB repair pathway (originally referred to as A-NHEJ19). As per our current findings, the CSR A-EJ pathway uses HR Rad52 to synapse upstream and downstream DSB overhangs by a MMEJ process but does not require a homologous template as a guide, as the HR pathway does. The DNA polymerase θ has been suggested to contribute to A-EJ21. Our previous findings, however, did not support a role of this polymerase in Rad52-mediated intra-Sμ DSB recombination or Sμ−Sγ1, Sμ−Sγ3, Sμ−Sγ2a/Sγ2c and Sμ−Sα recombination (CSR to IgG1, IgG3, IgG2a/IgG2c and IgA)20. Finally, while (MMEJ) A-EJ functions as a back-up pathway in cells defective in NHEJ or HR, it also synapses DSB ends in cells that are competent for both NHEJ and HR50, as exemplified by microhomologies in S-S junctions in a proportion of B cells that class-switched to IgG, IgA and IgE, as well as the disappearance of such microhomologies upon Rad52 ablation20.
As we showed here, Rad52 works in concert with Zfp318 to modulate IgD expression through an interplay of alternative RNA splicing and DNA recombination, the latter after AID intervention. Zfp318 represses the TTS intercalated between the Cμ and Cδ exons within the Ighμ/Ighδ loci transcriptional complex unit10, 11, thereby allowing for transcription of long primary VHDJH-Cµ-s-m-Cδ-s-m RNA. Zfp318, however, would also simultaneously allow for the continuous expression of primary Iµ-Cµ-s-m-Cδ-s-m RNA transcripts. In fact, albeit possibly more abundant, hence their predominant detection in our specific PCR assays, Iµ-Cδ transcripts – in secretory or membrane form – are identical in sequence to their post-recombination Iµ-Cδ counterparts (Fig. 1a,b). During B cell development, Zfp318 expression closely parallels mIgD expression10, 11. Indeed, consistent with its repression of the TTS intercalated between the Cμ and Cδ exons complex, the Zfp318 protein is expressed during the transition from immature IgM+IgD− to mature IgM+IgDhi B cell10, 11. As we showed here, naïve mature B cells which express high levels of mIgD also express high levels of Zfp318 transcripts and Zfp318 protein. In these B cells, stimuli that induced Sμ-σδ DNA recombination, yielding primary VHDJH-Cδ-s-m RNA transcripts, also induced profound downregulation of Zfp318 transcripts and Zfp318 protein, suggesting that relieving Zfp318-mediated TTS repression is a prerequisite for Sμ-σδ DNA recombination to unfold. Conversely, as we also showed here, naïve mature IgM+IgD+ B cells submitted to stimuli that induced CSR to isotypes other than IgD, such as IgA (by LPS plus IL-4 and RA), further upregulated Zfp318 transcripts and Zfp318 protein, concomitant with no Sμ-σδ recombination, thereby allowing for massive expression of mIgD rather than sIgD.
The role of Zfp318 as gene transcription regulator is highly specific for IgD, as genome-wide transcriptome analysis of B cell Zfp318-deficient (Vav-Cre dependent deletion) mice identified Sva as the only other gene altered in expression11 – interestingly, Sva is also involved in alternative splicing, albeit outside the IgH locus51. Zfp318 would be under the control of 5’AMP-activated protein kinase (Ampk). This is phosphorylated by Lbk152, whose signaling triggers the B cell GC reaction. Indeed, Lbk1’s failure to activate Ampk or Ampk loss specifically muted Zfp318 expression and IgD transcription52. In contrast, activation of Ampk by phenformin impaired GC formation52, likely by heightening Zfp318 expression, possibly in addition to other mechanisms. This would result in increased expression of primary VHDJH-Cµ-s-m-Cδ-s-m RNA transcripts but not Sμ-σδ DNA recombination, suggesting that CSR to IgD is one of the multiple and complex events inherent to GC formation. This is triggered by naturally occurring generally microbial stimuli, as in tonsil GCs and GCs or other secondary lymphoid formations in aerodigestive mucosae1, 5, 6. Consistent with the contention that Ampk mediates the regulation of Zfp318 as well as the contrasting impact of IgD CSR-inducing (LPS plus IL-4) and non-inducing stimuli (LPS plus TGF-β and RA) on expression of Zfp318, stimulation of both human and mouse cells by LPS has been shown to result in dephosphorylation/inactivation of Ampk, while similar cell stimulation by TGF-β resulted in rapid phosphorylation/activation of this protein kinase53.
In our experiments, stimuli that induced CSR to IgD (e.g., LPS plus IL-4 in mouse B cells, and CD154 plus IL-2 and IL-21 in human B cells) also downregulated Zfp318 expression which, in turn, reduced VHDJH-Cδm transcript level and mIgD, while greatly increasing VHDJH-Cδs transcripts and sIgD. This argues for CSR to IgD to be critical for significant IgD secretion. Indeed, stimuli that induced Sμ-σδ recombination and IgD secretion also induced plasmablast/plasma cell differentiation, as shown by Blimp-1 and C38+CD27+ expression in human B cells, and Blimp-1 and CD138+ in mouse B cells. A similar outcome was not produced by stimuli that did not induce Sμ-σδ recombination and IgD secretion in mouse or human B cells. Thus, while alternative splicing of long primary VHDJH-Cµ-s-m-Cδ-s-m RNA transcripts in B cells that have not undergo CSR would make some contribution to the overall level of IgD production in vivo, CSR to IgD is likely required for substantial and sustained IgD production, as secreted by plasmablasts/plasma cells or by neoplastic transformants, such as IgD myeloma cells. The limited IgD amounts detected in supernatants of mouse or human B cells primed by stimuli that induced high levels of mIgD but not Sµ–σδ synaptic recombination would result from translation of alternative spliced long primary VHDJH-Cµ-s-m-Cδ-s-m RNA transcripts as well as some “shedding” of mIgD.
Bacteria and viruses have been suggested to play an important role in driving CSR to IgD, generally through stimulation of TLRs in gut and respiratory lymphoid tissues, and mesenteric lymph nodes, possibly leading to emergence of plasmablasts and plasma cells secreting IgD1, 3–5, 51, 54–56. Circulating IgD are increased in patients with frequent respiratory infections or chronic lung inflammation suggesting a protective role for this Ig isotype1, 3, 5, 56. Our findings support the notion that both T-dependent (CD154) and T-independent (TLR ligands) stimuli induce Sμ-σδ DNA recombination, in combination with various cytokines1, 4–6. Interestingly, although we previously showed that BCR-signaling synergizes with TLR-signaling for induction of AID and CSR to IgG and IgA25, BCR signaling did not synergize with TLR7 or TLR9 signaling to induce CSR to IgD, as shown by the lack of Sμ-σδ DNA recombination in B cells stimulated by CpG or R848 plus IL-4 and anti-Igδ Ab. In the in vivo T-dependent antibody response to OVA, ablation of CSR to IgD (Rad52−/− mice) resulted in reduced levels of total and specific IgD in circulating blood and BALF, decreased total and/or bacteria-bound IgD in feces as well as decreased numbers of IgD+ B cells lamina propria and MLNs, a privileged site of IgD CSR12. As predicted by our previous findings20, the overall decreased IgD levels in Rad52−/− mice were associated with increased IgG1 and IgA as well as greatly decreased frequency and lengths of microhomologies in Sμ-Sγ1 and Sμ-Sα junctions. This reflected the lack of Rad52 contribution to the synaptic process underpinning such junctions as well as the lack of Rad52 competition with Ku70/Ku8620, which resulted solely in Ku70/Ku86-mediated NHEJ, a process that limits microhomologies to 0-3 nt16.
Information on the contribution of IgD to autoimmunity is scant and contradictory. Self-antigen-binding and mostly polyreactive IgD occur in healthy subjects, much like IgM or even IgG and IgA do35, 57–60. High levels of IgD have been reported in rheumatoid arthritis patients and thought to possibly be implicated in the pathogenesis of the disease30. mIgD expression, however, has been speculated to exert an inhibitory effect on B cell autoreactivity, as suggested by elevated autoantibody production, increased deposition of immune complexes in kidneys and severe nephritis in lupus-prone C56BL/6lpr mice with deletion of the Igδ locus61, 62. Our findings showed total and self-reactive IgD (dsDNA, histone, RNP/Sm or RNA and ANAs) to be elevated in the circulation of lupus patients and lupus-prone MRL/Faslpr/lpr mice. The latter displayed higher levels of IgD in serum, BALF and feces, than their wildtype C57BL/6 counterparts. Such high IgD levels reflected CSR recombinations that included Sμ-σδ junctions with extensive microhomologies and high frequency of somatic mutations in the DNA areas abetting Sμ-σδ junctions. Such IgD CSR occurred in different districts, such as bone marrow, spleen, MLNs and Peyer’s patches, and were reflected in the IgD+B cells in those districts. This together with the B cell high levels of p-Rad52 and the low levels Zfp318 indicated that in murine and likely human lupus, IgD autoantibodies stem from extensive B cell Sμ-σδ recombination rather than alternative splicing of primary VHDJH-Cµ-s-m-Cδ-s-m RNA transcripts. Our findings do not suggest a “protective” role of IgD in autoimmunity61, 62, while supporting a role of CSR to IgD in systemic lupus autoantibody responses.
Collectively, our data outline a critical and dedicated role of Rad52 in mediating the synapsis of Sμ with σδ DSB resected ends. They also provide the first demonstration of Rad52 as a critical element in the poorly understood contribution of A-EJ to the resolution of DSBs in nonmalignant cells. In malignant B cells, Rad52 is involved in DNA recombination events that give rise to DNA deletions and translocations. As we previously showed, Rad52 ablation reduced the frequency of c-Myc/IgH translocations in mouse p53−/− B cells by more than 70%, with the residual translocations containing limited microhomologies20. Whether Rad52 intervention extends to other modalities of A-EJ in neoplastic and non-neoplastic lymphoid mammalian cells remains to be determined. The importance of this newly unveiled and essential function of Rad52 is further emphasized by our demonstration that this highly conserved HR element is critical for CSR to IgD in both mouse and human B cells. This together with the further reduction of the physiologically moderate microhomologies in Sµ–Sγ1, Sµ–Sγ3, Sµ–Sα and Sµ– Sε junctions in Rad52−/− B cells (current data and refs.18–20) solidifies the role of Rad52 as critical mediator of the A-EJ backup pathway underpinning the residual CSR to IgG, IgA and IgE in the absence of Ku70/Ku86 proteins20. Our findings also showed how stimuli that induce Sμ-σδ recombination coordinate Rad52 function, as enabled by phosphorylation, with downregulation of Zfp318, unique repressor of the TTS intercalated between the Cμ and Cδ loci, whose activity allows transcription throughout VHDJH-Sμ-Cµ-s-m-σδ-Cδ-s-m and Iµ-Cµ-s-m-σδ-Cδ-s-m. Further, they indicate that CSR to IgD is required for sustained IgD secretion and possibly a prerequisite for IgD plasma cell differentiation. They also add new and significant information to a potential role of CSR to IgD, as promoted by Rad52 phosphorylation, in systemic autoimmunity. Finally, they provide important new molecular information to approach the virtually unexplored mechanistic underpinning of hyper-IgD syndrome, a relatively rare but a severe autoinflammatory disease associated with mevalonate kinase deficiency (due to MVK recessive mutations) and exorbitant levels of IgD31, 32, 63.
Author contributions
Y. Xu and H. Zhou performed experiments; G. Post provided myeloma samples; H. Zan designed and performed experiments, analyzed data, supervised the work and wrote the manuscript; P. Casali planned the study, designed the experiments, analyzed the data, supervised the work and wrote the manuscript.
Methods
Mice
Rad52−/− mice were generated by replacing exon 3 of the Rad52 gene with positive selection marker neomycin, as driven by the phosphoglycerate kinase (PGK) promoter, and an upstream mouse sequence functioning as a transcription terminator (Dr. Albert Pastink, Leiden University, Leiden, The Netherlands)36. Rad52−/− mice were backcrossed to C57/BL6 mice for more than six generations. No full length or truncated Rad52 protein was produced from the disrupted allele36. Rad52−/− mice were viable and fertile, and showed no gross abnormalities. Aicda−/− mice (C57BL/6 background)64 were obtained from Dr. Tasuku Honjo (Kyoto University, Kyoto, Japan). C57BL/6 and MRL/Faslpr/lpr mice were purchased from Jackson Laboratory (Bar Harbor, Maine). All mice were housed in pathogen-free conditions. Both male and female mice aged 8-12 weeks were used for the experiments. The Institutional Animal Care and Use Committees (IACUC) of the University of Texas Health Science Center at San Antonio approved all animal protocols.
Mouse B cells and CSR induction in vitro
Naïve IgM+IgD+B cells were isolated from spleens of 8–12-week-old C57BL/6, Rad52−/− or Aicda−/− mice as described25. B cells were resuspended in RPMI 1640 medium with 10% FBS (FBS-RPMI), 50 mM β-mercaptoethanol and 1x antibiotic-antimycotic mixture (15240-062; Invitrogen) and stimulated with LPS (4 μg/ml) from Escherichia coli (055:B5; Sigma-Aldrich), CD154 (1 U/ml; obtained from membrane fragments of baculovirus-infected Sf21 insect cells25), CpG ODN 1826 (1.0 μM; Eurofins Genomics) or R848 (1.0 μM; Medkoo) plus nil, IL-4 (5.0 ng/ml; R&D Systems) and/or TGF-β (2.0 ng/ml; R&D Systems) and retinoic acid (RA, 10 nM) or anti-BCR Ab (anti-δ mAb-dextran, 30 ng/ml; Fina Biosolutions). Mouse B cells were cultured in FBS-RPMI at 37°C in 48-well plates for 24, 48, 72 and 96 h.
Human B cells and CSR induction in vitro
Naïve IgM+IgD+B cells were purified by negative selection using the EasySepTM human naive B cell enrichment kit (19254; StemCell Technologies) from healthy subject PBMCs, following manufacturer’s instructions. Tonsillar IgD+ B cells were isolated from human tonsil cells by positive selection using biotin-anti-human IgD mAb (clone IA6-2; 348212, Biolegend) and MagniSort™ Streptavidin Positive Selection Beads (MSPB-6003-74, Thermo Fisher Scientific). Naïve B cells were stimulated with CD154 (10 U/ml) or CpG ODN 2395 (1.0 μM; Eurofins Genomics) plus nil, IL-2 (20 ng/ml; BioLegend), IL-4 (20 ng/ml; R&D Systems), IL-15 and/or IL-21 (50 ng/ml; R&D Systems). Human B cells were cultured in FBS-RPMI at 37°C in 48-well plates for 24, 48, 72, 96 and 120 h.
Flow cytometry
For surface staining, mononuclear cells were reacted with VF-anti-CD19 mAb (75-0193-0100, Tonbo), PE-anti-IgM mAb (clone RMM1, 406507, BioLegend), and FITC anti-mouse IgD mAb (clone 11-26c.2a, 405704, BioLegend) and 7-AAD. For intracellular staining, cells were stained with anti-CD19 mAb (Clone 1D3; Tonbo) and fixable viability dye eFluor® 450 (FVD 450, eBiosciences) followed by incubation with the BD Cytofix/Cytoperm buffer at 4°C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4°C. Cells were then stained with anti-Zfp318 Ab (AAS23325C, Antibody Verify; labeled with FITC using iLink™ Antibody Labeling Kits, ABP Biosciences). FACS analysis was performed on single cell suspensions. In all flow cytometry experiments, cells were appropriately gated on forward and side scattering to exclude dead cells and debris. Cell analyses were performed using a LSR-II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (TreeStar). All experiments were performed in triplicates.
Fluorescence microscopy
Fluorescence microscopy of tissues. To analyze IgM and IgD-producing cells in the lamina propria and PPs, the intestine was folded into a “Swiss-roll”, fixed with PFA (4%), and embedded in paraffin. Ten μm sections were cut and heated at 80 °C to adhere to the slide, washed four times in xylene for 2 min, dehydrated two times with 100% ethanol for 1 min, two times with 95% ethanol for 1 min and washed two times in water for 1 min. Antigens were unmasked using 2 mM EDTA in 100 °C for 40 mins followed by a cooling step at 25 °C on the bench top, 3 times washing with 1x TBS and blocking using 10% BSA for 15 min. Slides were again washed 3 times with 1x TBS and stained with FITC–anti-IgD mAb (clone 11-26c.2a; 405713, BioLegend), PE goat-anti-mouse-IgM mAb (406507, BioLegend) for 2 h in a dark moist chamber. After washing 3 times with Triton X-100 (0.1%) in TBS, slides were air dried, and cover slips were mounted with ProLong® Gold Antifade Reagent with DAPI (Invitrogen). Fluorescence images were captured using a 10x objective lens with a Zeiss Axio Imager Z1 fluorescence microscope. To analyze IgD-producing cells in MLNs, 10 µm MLN sections were prepared by cryostat and loaded onto positively charged slides, fixed in cold acetone and stained with FITC–anti-IgD mAb (405704, BioLegend), or PE goat-anti-mouse-IgM mAb (406507, BioLegend), respectively, for 1 h at 25 °C in a moist chamber. Cover slips were then mounted using ProLong® Gold Antifade Reagent using DAPI (Thermo Fisher), before examination with a fluorescence microscope.
Fluorescence microscopy of B cells. B cells were suspended at 105 cells/100 µl in FCS-RPMI. Pre-labeled slides were then placed into Cytofunnels and ran with 50 µl of FCS-RPMI in order to wet the Cytofunnel paper. Cells were then placed again into the Cytofunnel and spun at 800 RPM for 3 min using a Cytospin™ 4 Cytocentrifuge (Thermo Fisher). For intracellular visualization of IgM, IgD, CD138 and Blimp-1 proteins, cells were fixed with methanol for 15 min and washed 3 times in PBS-Tween 20. Cells were then blocked in 10% BSA for 15 min and stained with 1:20 APC-anti-mouse IgD Ab (clone 11-26c.2a; 405713, BioLegend), FITC-anti-mouse IgM mAb (11-5790-81, Thermo Fisher), overnight in a dark moist chamber.
Detection of free and bacterial-bound antibodies
Titers of serum, BALF or fecal total IgD, IgM, IgG1 and IgA and OVA-binding IgD, IgM, IgG1 and IgA were measured using specific ELISAs, as we described20, 25, 65, 66. Total IgD in in vitro culture supernatants of stimulated human and mouse B cells or in serum, BALF or feces were measured by dot blotting with serially two-fold diluted samples.
Bacteria-bound IgD and IgA were detected in feces by flow cytometry, as we described27. Feces (10 mg) were suspended in 100 μl 1x PBS (filtered through 0.2 μm filter), homogenized and centrifuged at 400 × g for 5 min to remove large particles. The supernatant was then centrifuged at 8000 g for 10 min to remove non-bound antibodies (in supernatant). The bacterial pellet was suspended in 1 ml of PBS with 1% (w/v) BSA. After fixation with 7.2% formaldehyde for 10 min at room temperature, bacteria were washed with PBS, and stained with FITC– anti-IgD mAb (clone 11-26c.2a; 405713, BioLegend) or FITC-anti-IgA mAb (C10-3, BD Biosciences) on ice for 30 min, washed with PBS, and further resuspended in 1 x PBS containing 0.2 μg ml-1 DAPI for flow cytometry analysis. All events that stained with DAPI were considered as bacteria.
S-S region DNA recombinations and S region somatic mutations
Genomic DNA was prepared from human or mouse B cells using QIAmp DNA Mini Kit (Qiagen), or from paraffin-embedded human IgD or IgA myeloma tissue sections (obtained from the University of Arkansas for Medical Science) using Quick-DNA™ FFPE Kit (Zymo Research). Recombined Sμ–σδ, Sμ–Sγ1, Sμ–Sα and Sμ–Sε DNA were amplified by two sequential rounds of specific PCR using Phusion™ high-fidelity DNA polymerase (Thermo Scientific™) and nested oligonucleotide primers67 (Supplementary Table 1). The first and second rounds of PCR were performed at 98 °C for 30 sec, 58 °C for 45 sec, 72 °C for 4 min (30 cycles). Amplified DNA was fractionated through 1.0% agarose, blotted onto Hybond-N+ membranes (GE Healthcare) and hybridized to biotin-labeled Sμ and σδ, Sγ1, Sαor Sε specific probes. Detection was performed using the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific) according to the manufacturer’s instructions. For sequence analysis of the recombined DNA, PCR products were purified using a QIAquick PCR purification kit (Qiagen). The amplified library was tagged with barcodes for sample multiplexing, and PCR was enriched and annealed to the required Illumina clustering adapters. High-throughput 300–base pair (bp) paired-end sequencing was performed by the UTHSCSA Genome Sequencing Facility using the Illumina MiSeq platform. S-S junctions and somatic mutations in the S regions were analyzed by sequence alignment as performed by comparing PCR products sequences with germline Sµ and σδ, Sγ1 or Sαsequences using National Center for Biotechnology Information BLAST (www.ncbi.nih.gov/BLAST).
RT-PCR and quantitative RT-PCR (qRT-PCR)
For quantification of mRNA, germline IH-CH, post-recombination Iμ-CH and mature VHDJH-CH transcripts, RNA was extracted from 0.2-5.0 x 106 cells using either Trizol® Reagent (Invitrogen) or RNeasy Plus Mini Kit (Qiagen). Residual DNA was removed from the extracted RNA with gDNA eliminator columns (Qiagen). cDNA was synthesized from total RNA with the SuperScript™ IV First-Strand Synthesis System (Thermo Fisher) using oligo-dT primer. Transcript expression was measured by qRT-PCR with the appropriate primers (Supplemental Table 1) using a Bio-Rad MyiQ™ Real-Time PCR Detection System (Bio-Rad Laboratories) to measure SYBR Green (IQ™ SYBR® Green Supermix, Bio-Rad Laboratories) incorporation with the following protocol: 95°C for 15 sec, 40 cycles of 94°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec. Data acquisition was performed during 72°C extension step. Melting curve analysis was performed from 72°C-95°C. Mature VHDJH-Cμm, VHDJH-Cμs, VHDJH-Cδm and VHDJH-Cδs transcripts were analyzed by semi-quantitative PCR using serially two-fold diluted cDNA.
Western blotting
B cells were lysed in Laemmli buffer. Cell extracts containing equal amounts of protein (50-100 µg) were fractionated through SDS-PAGE (6%). The fractionated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) overnight (30 V/90 mA) at 4 °C. After blocking and overnight incubation at 4 °C with anti-AID antibody (H-80, Santa Cruz), anti-Ku70 antibody (A0883, Abclonal), anti-Ku86 antibody (A5862, Abclonal), anti-Rad52 antibody (H-300, Santa Cruz Biotechnology), anti-phospho-Rad52 antibody (Y408472, Applied Biological Materials Inc.) or anti-β-Actin mAb (2F1-1, BioLegend), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. After washing with TBS– Tween 20 (0.05%), bound HRP-conjugated antibodies were detected using Western Lightning Plus-ECL reagents (PerkinElmer Life and Analytical Sciences).
ChIP and qPCR
ChIP assays were performed as previously described68–70. Human or mouse B cells (1.0 x 107) were treated with formaldehyde (1% v/v) for 10 min at 25°C to crosslink chromatin, washed once in cold PBS with protease inhibitors (Roche) and resuspended in lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 0.1% w/v SDS and protease inhibitors, pH 8.0). Chromatin was fragmented by sonication (DNA fragments of about 200 to 1,000 bp in length), pre-cleared with protein A agarose beads (Pierce) and incubated with agarose conjugated anti-Rad52 mAb (clone F-7; sc-365341 AC, Santa Cruz Biotechnology) at 4°C overnight. Immune complexes were washed and eluted (50 mM Tris-HCl, 0.5% SDS, 200 mM NaCl, 100 µg/ml proteinase K, pH 8.0), followed by incubation at 65°C for 4 h. DNA was purified using a QIAquick PCR purification kit (Qiagen). The Sμ or σδ region DNA was amplified from immunoprecipitated chromatin by qPCR using appropriate primers (Supplemental Table 1). Data were normalized to input chromatin DNA and depicted as relative abundance of each amplicon.
RAD52 knockdown in human B cells
The human RAD52-specific siRNA oligo duplex (TT320001, Locus ID 5893) and non-effective Trilencer-27 Flurescent-labeled transfection control siRNA duplex (SR30002) were obtained from Origene Technologies. The siRNA duplexes were used to transfect purified human naïve B cells using the Human B Cell NucleofectorTM Kit (VPA-1001, LONZA). Transfected B cells were then stimulated with CpG ODN 2395 plus IL-2 and IL-21 for 96 h before genomic DNA extraction for analysis of Sμ-σδand Sμ-Sγ1 DNA recombination. Expression of RAD52 and AICDA transcripts were analyzed by qRT-PCR using specific primers 24 h after transfection. Expression of RAD52, phosphorylated-RAD52, AID and β-ACTIN proteins were analyzed by immune-blotting 24 h after transfection.
High-throughput mRNA-Seq
RNA was isolated from cells using the Directzol RNA Microprep Kit (Zymogen Research), according to manufacturer’s instructions and as previously described66. RNA integrity was verified using an Agilent Bioanalyzer 2100 (Agilent). Next generation RNA-Seq for mRNA and non-coding RNA was performed by the Genome Sequencing Facility at University of Texas Health Science Center San Antonio Greehey Children’s Cancer Research Institute. High-quality RNA was processed using an Illumina TruSeq RNA sample prep kit v2 or TruSeq Small RNA Sample Prep kit following the manufacturer’s instructions (Illumina). Clusters were generated using TruSeq Single-Read Cluster Gen. Kit v3-cBot-HS on an Illumina cBot Cluster Generation Station. After quality control procedures, individual mRNA-Seq or small RNA-Seq libraries were then pooled based on their respective 6-bp index portion of the TruSeq adapters and sequenced at 50 bp/sequence using an Illumina HiSeq 3000 sequencer. Resulting reads were checked by assurance (QA) pipeline and initial genome alignment (Alignment). After the sequencing run, demultiplexing with CASAVA was employed to generate the Fastq file for each sample. All sequencing reads were aligned with their reference genome (UCSC mouse genome build mm9) using TopHat2 default settings, and the Bam files from alignment were processed using HTSeq-count to obtain the counts per gene in all samples. Quality control statistical analysis of outliers, intergroup variability and distribution levels, were performed for statistical validation of the experimental data.
Statistical analysis
Statistical analysis was performed using Excel (Microsoft) or Prism® GraphPad software. P-values were determined by paired and unpaired Student’s t-tests; and P-values <0.05 were considered significant.
IRB for use of human tissues and peripheral blood as well as IACUC for use of mice
For the use of DNA procured from formalin fixed paraffin embedded tissues obtained from the University of Arkansas for Medical Science, the study was reviewed by the University of Arkansas for Medical Sciences Institutional Review Board (IRB) which determined that this project is not human subject research as defined in 45 CFR 46.102. Human B cells were purified from PBMCs of healthy subject buffy coats obtained from South Texas Blood and Tissue Center, San Antonio, Texas, under the Healthy Volunteer Blood Donor Program and lupus patients B cells were purified PBMCs obtained under the Long School of Medicine IRB HSC 20140234H Class switching, somatic hypermutation and plasma cell differentiation in B cells. Mouse and mouse B cell studies were performed under the Long School of Medicine IACUC 20200019AR Somatic hypermutation, class switch DNA recombination and plasma cell differentiation in antibody and autoantibody responses.
Acknowledgements
We thank Dr. Patrick M. Sung for reviewing this manuscript. We also would like to thank Amanda Fisher, Dr. Justin B. Moroney, Dr. Helia N. Sanchez and Dr. Huoqun Gan for their help in some experiments. This work was supported by NIH grants R01 AI 079705, T32 AI138944, R01 AI 105813 and the Lupus Research Alliance Target Identification in Lupus Grant 641363 to P.C.