Fig 2: Rad52 deletion ablates in vivo Sµ–sd DNA recombination and reduces IgD production.Rad52+/+ and Rad52-/- mice were immunized with OVA in alum i.p. a Recombined Sµ–sd, Sµ-S?1, and Sµ-Sa DNAs in spleen, mesenteric lymph nodes (MLNs), and Peyer’s patches (PPs) B cells, as analyzed by nested PCR using forward Iµ and reverse Cd, S?1, or Sa primers, respectively, followed by Southern-blotting using specific Sµ, sd, S?1, or Sa probe, as indicated. Data are one representative of three independent experiments yielding comparable results. b Sµ–sd, Sµ–S?1, and Sµ–Sa junctional DNAs were amplified by nested PCR and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in Sµ–sd, Sµ-S?1, and Sµ-Sa junctional DNAs are shown by violin plots. Each symbol represents a unique sequence (n = 45 per group). c–f Titers of total IgD in serum, BALF, and feces, as analyzed by dot-blotting using rat anti-mouse IgD mAb—titers of total IgM, IgD, IgG1, and IgA as well as OVA-binding IgM, IgD, IgG1, and IgA as analyzed by specific ELISAs. Each dot represents datum from one individual mouse (n = 5–8 per group, as indicated). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (unpaired two-tailed t-test). No adjustments were made for multiple comparisons. g Bacteria-bound IgD and IgA in feces as analyzed by flow cytometry. h IgM, IgD, and IgA positive cells in MLNs and lamina propria as visualized by fluorescence microscopy. Scale bar = 100 µm. Data in g, h are representative of three independent experiments. Source data are provided as a Source Data file.

Fig 3: Flagellin administration alters the intestinal microbiota towards a lower pro-inflammatory state. 4-week old C57BL/6 J Wild Type mice were purchased from The Jackson Laboratory and housed for two weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 µg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Fecal microbiota composition was analyzed using Illumina sequencing of the V4 region of 16 S rRNA genes. a–b Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix at a day -14 and b day 56 (post stabilization, post immunization). c LEfSe analysis was performed in order to investigate microbiota taxa that were significantly altered by immunization at day 56 (post stabilization, post immunization), with green and red colors highlighting taxa significantly more abundant in PBS- and flagellin-treated mice, respectively. d Percentage of IgA±-coated bacteria in PBS- and FliC-treated mice, wherein the IgA- and IgA+ gates were determined follows appropriate SSC-A/FSC-A gating of SytoBC+ cells in wild-type and µMT mice. e Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of IgA-coated bacteria. f Alpha diversity rarefaction using the Chao1 index of IgA-coated bacteria. g Taxa summarization of IgA-coated bacteria. In a and b, categories were compared and statistical significance of clustering were determined via Permanova. Data are the means ±S.E.M. Significance was determined using t test (*p = 0.05). (N=4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

Fig 4: Ggta1 deletion enhances IgA responses to the gut microbiota.(A) Relative binding of IgA in the serum of Ggta1+/+ (n = 10) and GF Ggta1+/+ (n = 5) mice to fecal extract from Ggta1+/+ mice, and Ggta1-/- (n = 6) and GF Ggta1-/- (n = 6) mice to fecal extract from Ggta1-/- mice; one experiment. (B) Relative binding of IgA in the serum of GF Ggta1+/+ (n = 7) and GF Ggta1-/- (n = 10) mice to cecal extract from Ggta1-/- mice at indicated time-points after colonization with cecal extract from Ggta1-/- mice; two experiments. (C) Median Fluorescence Intensity (MFI) of aGal+ bacterial strains stained with BSI-B4 lectin relative to unstained control; seven experiments. (D) Representative flow cytometry plots showing bacteria stained for IgA and aGal in the small intestinal content of Ggta1+/+ (n = 5) and Ggta1-/- (n = 11) mice; four independent experiments. (E) Quantification of aGal+, IgA+, and IgA+aGal+ bacteria in the same samples as in (D). (F) Concentration of anti-aGal IgM in serum of Ggta1+/+ (n = 2), Ggta1-/- (n = 12), Igha-/-Ggta1+/+ (n = 10), and Igha-/-Ggta1-/- (n = 12) mice before and after streptomycin treatment, two experiments. (G) Concentration of anti-aGal IgG, in the same mice as (F). Symbols (A, B, E, F, G) are individual mice. Red bars (A, B, E, F, G) correspond to mean values. Error bars (A, B, C, E, F, G) correspond to SD. p Values in (A, B, F, G) calculated using Kruskal-Wallis test using Dunn’s multiple comparisons test and in (E) using Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

Fig 5: p-Rad52 expression, CSR to IgD and antinuclear antigen IgD autoantibodies in lupus patients and mice.a Serum total and double-strand DNA (dsDNA)-, RNA-, histone-, or RNP/Sm-binding IgD in healthy human subjects and systemic lupus erythematosus (SLE) patients, as analyzed by specific ELISAs. Each dot represents the datum from one individual human subject. Mean ± SEM of 6–10 healthy subjects or SLE patients are depicted. *p < 0.05 (unpaired two-tailed t-test). b Human and mouse antinuclear autoantibodies (ANAs), as visualized by indirect immunofluorescence microscopy on HEp-2 cells that were incubated with serum from a healthy human subject, an SLE patient, a C57BL/6 mouse, or a MRL/Faslp/lpr mouse, as revealed by FITC-labeled rat mAb to human or mouse IgD. Scale bar = 50 µm. c Total IgD in serum, feces and BALF as analyzed by dot-blotting, and concentrations of IgD autoantibodies to dsDNA or histone in serum of C57BL/6 and MRL/Faslpr/lpr mice, as analyzed by specific ELISAs. Each dot represents datum from one individual mouse. Data are mean ± SEM of 3–9 mice, as indicated. **p < 0.01, ***p < 0.001 (unpaired two-tailed t-test). d IgD concentrations in serum, feces and BALF from C57BL/6 and MRL/Faslpr/lpr mice, as analyzed by dot-blots. Shown are dot-blots from one C57BL/6 and one MRL/Faslpr/lpr mouse, representative of 3–9 C57BL/6 and MRL/Faslpr/lpr mice. e Bacteria-bound IgD and IgA in feces from C57BL/6 and MRL/Faslpr/lpr mice, as analyzed by flow cytometry. f Expression of VHDJH-Cdm, VHDJH-Cds, VHDJH-Cµm, and VHDJH-Cµs transcripts in bone marrow (BM), spleen and mesenteric lymph nodes (MLNs), as analyzed semi-quantitative RT-PCR by serial two-fold dilutions of cDNA templates. Shown are RT-PCR data from one C57BL/6 mouse and one MRL/Faslpr/lpr mouse, representative of three C57BL/6 and three MRL/Faslpr/lpr mice. g IgD+ B cells in lamina propria, MLNs and Peyer’s patches (PPs) of C57BL/6 and MRL/Faslpr/lpr mice, as visualized by fluorescent microscopy. Scale bar = 100 µm. h Recombined junctional Sµ–sd, Sµ–S?1, and Sµ–Sa DNAs in bone marrow, spleen, MLNs, and PPs B cells from C57BL/6 and MRL/Faslpr/lpr mice as analyzed by specific nested PCR using forward Iµ and reverse Cd, S?1 or Sa primers, followed by Southern-blotting using indicated probes. Data are representative of three independent experiments. i Sµ–sd, Sµ–S?1 and Sµ–Sa junctional DNAs in non-immunized MRL/Faslpr/lpr mice, as amplified by nested PCR and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in Sµ–sd, Sµ–S?1, and Sµ–Sa junctional DNAs are rendered by violin plots. Each dot represents a unique sequence (n = 45 per group). j Somatic point-mutations in Sµ and sd regions abetting recombined Sµ-sd DNA junctions in IgD class-switched spleen B cells from three MRL/Faslpr/lpr mice. Mutations were identified in a 48–506 nt stretch of Sµ or sd regions in unique Sµ–sd DNA recombination sequences. Each dot represents an individual sequence. ns: not significant (unpaired two-tailed t-test). Box and whiskers plots show the median, quartiles, maximum, and minimum of mutation frequencies in Sµ and sd regions. In pie charts, the size of slices denotes the proportion of sequences with the same number of mutations and the gray hue denotes the number of point-mutations per sequence. Center of pie shows the total number of independent sequences analyzed. Below the pie charts is the overall mutation frequency (change/base). k Expression of phosphorylated Rad52 (p-Rad52), Rad52 and ß-Actin proteins in peripheral blood B cells from healthy human subjects and SLE patients as well as B cells from C57BL/6 mice and MRL/Faslpr/lpr mice, as analyzed by specific Western blotting using rabbit anti-p-Rad52 Ab or anti-ß-Actin mAb—p-Rd52 (Y104) Ab detected endogenous levels of Rad52 protein only when phosphorylated at tyrosine 104. l Expression of ZFP318 and AICDA transcripts in B cells from healthy human subjects and SLE patients (left panel), as well as Zfp318 and Aicda transcripts in MLNs from C57BL/6 and MRL/Faslpr/lpr mice (right panel), as analyzed by specific qRT-PCR. Data are mean ± SEM of three healthy human subjects, three SLE patients, three C57BL/6 mice, and three MRL/Faslpr/lpr mice. *p < 0.05 (unpaired two-tailed t-test). No adjustments were made for multiple comparisons. Source data are provided as a Source Data file.