Lrba-deficient mice were (6, 17) engineered at Taconic Artemis (Köln, Germany) (official allele designation: Lrbatm1.1Kili MGI: 5796558; laboratory designation: Lrba2) by constitutive deletion of the coding exon 3. This deletion introduced a frameshift mutation that was predicted to produce a truncated protein that was prone to degradation. Consequently, a shift in the reading frame occurred after amino acid 149 (5% of the coding sequence), rendering the expression of Lrba undetectable by Western Blot analysis.

The mice were bred and maintained in a C57BL/6 N gene pool under pathogen-free conditions at the animal facilities of the Research Centre and Advanced Studies (CINVESTAV, Mexico City, Mexico). Genotyping was performed using PCR amplification of genomic DNA extracted from splenocytes or tail samples using the following primer combinations: a sense primer for exon 3 (Ex3F: 5′-GAAAGTTGACAGTATGATTGCAGG-3′) paired with a wild-type-specific reverse primer in exon 4 (Ex4R: 5′-CATTGTCCTTTATCTCCTTGAA-3′), or a combination of Ex3F and a reverse primer for intron 4 (Int4R: 5′-CTAAGGAGGATGGCTCTAACC-3’).

This study was reviewed and approved by the CINVESTAV Ethics Committee and all the mice were maintained according to the Institutional Animal Guidelines for Animal Care and Experimentation (protocol number: 145–15, UPEAL-CINVESTAV-IPN).

Ninety-six-well plates were coated with 5 ng/well of the capture antibody, anti-mouse IgG, IgA, or anti-IgM (SouthernBiotech, Birmingham, AL, USA) and incubated overnight at 4°C. The plates were blocked with 1% milk in 1X PBS containing 0.05% Tween for 2 h. Samples, including sera or fecal supernatants diluted at a 1:100 ratio in PBS, were added to the wells and incubated for 1 h at 37°C. Subsequently, biotinylated antibodies specific for either IgM (Southern Biotech, 102008), IgG (Southern Biotech, 103005), or IgA (Southern Biotech, 104008) were diluted at 1:1000 and added to the plates, followed by incubation for 1 h at 37°C. Finally, streptavidin was added and coupled with horseradish peroxidase (HRP; Abcam, Cambridge, UK, ab7403) diluted at 1:5000. Tetramethylbenzidine was added, and the reaction was stopped with 0.2 M phosphoric. The absorbance was measured at 450 nm using an ELISA reader (Microplate, Sunrise™).

Splenocytes were stained with anti-CD19 BV421, anti-CD23 PerCPCy5.5, anti-CD21 PE, and anti-IgM PECy7 (all from Biolegend, San Diego, CA, USA) to detect Transitional 1, Transitional 2, Follicular, and Marginal Zone B cells. Briefly, B cells were incubated with a mix of antibodies for 30 minutes, washed, and fixed with 1% paraformaldehyde. For B1 cells, a peritoneal exudate was obtained, and cells were stained with anti-CD19 BV421, anti-CD21 PE, and anti-CD5 PerCPCy5.5 (all from Biolegend). After incubation, cells were washed and fixed with 1% paraformaldehyde. Data was acquired using a FACS LSRFortessa™ (Beckton Dickinson, Franklin Lakes, NJ, USA). Data analysis was performed using FlowJo v10.10 (Beckton Dickinson). Percentages for each subpopulation were determined, and the total number was calculated using the total cell counts obtained from either the spleens or the peritoneal exudates.

Secondary organs, including the spleen, mesenteric lymph nodes, PP, and small intestine were obtained and placed in a tissue preservation medium (Leica, Wetzlar, Germany). They were then frozen in liquid nitrogen and stored at -70°C until use. Histological sections of 5–6 μm were mounted on slides coated with poly-L-lysine, fixed in cold acetone for 15 min, and stored at -20°C after drying at room temperature.

Histological sections were rehydrated with 0.2% BSA in 1X PBS, blocked with Power Block Universal Blocking Reagent (BioGenex, CA, USA), and incubated for 1 h with anti-IgD (Becton Dickinson, 553438), anti-CD138 (Becton Dickinson, 553712), and biotinylated anti-IgA antibodies (eBioscience, San Diego, CA, USA, 13–5994-84). Subsequently, three washes with 0.2% BSA were performed, and anti-rat Alexa Fluor 594 secondary antibodies (Life Technologies, Carlsband, CA, USA, A21209) were added to detect anti-IgD and anti-CD138 antibodies, followed by incubation for 1 h at room temperature. Streptavidin coupled with Alexa Fluor 488 (Invitrogen, Waltham, MA, USA, 511223) was incubated with biotinylated primary antibodies for 30 min at room temperature. After staining, glass slides with histological sections were washed three times with 1× PBS, covered with coverslips, and mounted with 90% glycerol to keep the histological sections hydrated. Images were acquired using an Olympus BX51 microscope equipped with an Olympus U-CMAD3 camera and Olympus RFL-T epifluorescence lamp (Olympus, Tokyo, Japan) with 10× lenses. The analysis was performed using Image-Pro-Plus 7.0 (Media Cybernetics, Rockville, MD, USA) and Fiji ImageJ v2.7.0 software (18).

The spleens were obtained from the mice and disaggregated to create suspensions in 1× PBS. Cells were adjusted to 1×10⁶ cells in 50 μL of 1× PBS and stained with anti-TGFβRI PE (R&D, Minneapolis, USA, FAB5871P) at a 1:300 dilution, anti-TGFβRII (Abcam, ab61213) at a 1:100 dilution, and anti-CD19 BV605 (BD Pharmingen, 563148) at a 1:300 dilution, and viability dye with fixation eFLUORTM 450 (eBioscience) was added. The mixtures were incubated for 30 min. Subsequently, a secondary antibody for anti-TGFβRII, anti-rabbit coupled to Alexa Fluor 488 (Invitrogen, A21206), was added, and the cells were fixed with 1% paraformaldehyde.

For intracellular detection, after fixation with 4% PFA for 10 min, the cells were washed with 1× PBS. Blocking with 10% goat serum was performed for 30 min, followed by washing with 1× PBS. The cells were then permeabilized with a BD Perm/Wash™ (Becton Dickinson) for 10 min, followed by a final wash. Lymphocytes were then incubated with anti-TGFβRI PE and anti-TGFβRII for 1 h at 37°C. Subsequently, cells were washed with PBS and incubated with secondary antibodies against rabbit Alexa Fluor 488 in the case of the cell suspension with anti-TGFβRII. After the designated time, the cells were wash with 1X PBS was performed.

Spleens were obtained from Lrba-/- and wild-type (WT) mice, disaggregated, and resuspended in 1x PBS. Subsequently, the MojoSort™ Mouse CD19 Nanobeads kit (BioLegend, San Diego, CA, USA) was used to enrich B cells, following the manufacturer’s instructions.

Purified B cells were adjusted to 3x10⁶ cells, resuspended in PBS, and placed in 1.5-mL tubes. Subsequently, 10 ng/mL of recombinant TGFβ1 (BioLegend, 763102) was added, and the cells were incubated at 37°C for 5, 15, and 30 min. Unstimulated cells served as controls. After stimulation, the cells were processed for immunoblotting.

The cells were lysed with 100 μL of lysis buffer (Cell Signaling Technologies, Danvers, MA, USA), containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyruvate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin). Protease inhibitors (Roche, Mannheim, Germany) were added and the mixture was incubated for 30 min. The mixture was then centrifuged at 14,000 rpm for 15 min at 4°C. The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Samples were heated at 95°C for 5 min in Laemmli buffer (containing 1.0M Tris base pH 6.8, 10% SDS, 20% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue) and separated using 10% SDS-PAGE.

The proteins were transferred onto nitrocellulose membranes and blocked with 5% milk in TBS-Tween for 1 h at room temperature. Subsequently, the membranes were washed thrice for 10 min each with TBS-Tween, after which primary antibodies were added according to the specific assay requirements.

Lrba and TGFβRI subunits were detected either in the immunoprecipitates, or total protein extracts were determined through immunoblotting, following standard procedures. Briefly, 20 μg of protein extracts from B cells were loaded onto a gradient gel 8–15% polyacrylamide (Bio-Rad, Hercules, CA, USA). A 10% polyacrylamide gel was used to detect the phosphorylated SMADs. The proteins were transferred to a nitrocellulose membrane for 1.5h at 100 V in standard Tris-glycine buffer with 20% methanol. The membranes were incubated overnight at 4°C with agitation using anti-Lrba, anti-TGFβRI (GeneTex, Zeeland, MI, USA, GTX134290), anti-TGFβRII, anti-phospho-SMAD2 (Cell Signaling, 138D4), anti-total SMAD 2/3 (Abcam, ab202445), and anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA). Subsequently, the membrane was washed, and a secondary antibody coupled to HRP was added and incubated for 1 h with agitation at room temperature. Finally, chemiluminescence detection was performed using Super Signal West Femto Maximum Sensitivity substrate on a ChemiDoc XRS (Bio-Rad), and images were acquired with a ChemiDoc™ (Bio-Rad).

The previously purified B cells were adjusted to 1x10⁶ cells in 50 μL of 1× PBS and fixed with 4% PFA for 15 min. They were then washed with 1× PBS, followed by a 30-min blocking step with 10% goat serum. After another wash with 1× PBS, the fixed cells were permeabilized with BD Perm/Wash™ (Becton Dickinson, 51–2091 KZ) for 10 min. Following a second wash, the lymphocytes were incubated with goat anti-LRBA (Santa Cruz Biotechnology, sc-164907), rabbit anti-TGFβRI, or rabbit anti-TGFβRII antibodies for 1 h at 37°C. Subsequently, the cells were washed with PBS and incubated with secondary antibodies: anti-goat-Alexa Fluor 488 (Invitrogen, A11055), anti-rabbit-Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA, St Louis, MO, USA, 711–585-152), and DAPI (Sigma-Aldrich, 10236276001) for 1 h. Finally, the cells were washed and adhered to glass coverslips treated with poly-L-lysine (Sigma-Aldrich, P8920) for 1 h at 37°C. The preparations were mounted on slides using the VECTASHIELD mounting medium (Vector Laboratories, Newark, CA, USA, H-1000).

Images were captured using a TCS SP-8 microscope (Leica Microsystems) at 63× magnification. Images were acquired using Leica LAS X software (Leica Microsystems). Co-localization analysis of samples with dual staining was conducted using Mander’s correlation coefficient for at least 50 cells from three independent experiments. Data analysis and Mander’s correlations were calculated using Fiji Image J software v2.7.0.

Results are presented as means ± standard deviation. Unless otherwise specified, non-parametric tests were used for statistical analysis, with p values <0.05 considered significant.

The IgA levels in Lrba-/- were explored in serum and feces, we found elevated IgA levels in Lrba-/- mice. We observed higher levels of IgA in both the serum and feces of Lrba-/- mice at baseline, regardless of whether the mice were young (10 weeks) or old (12 months), compared with those in WT mice. We also observed higher levels of circulating IgG in young Lrba-deficient mice than that in WT mice ( Figure 1 ). As Burnett et al. previously reported increased IgG2b levels, we determined this isotype in the sera from these mice; however, as shown in Supplementary Figure 1 , similar IgG2b levels were observed in both mice. These results suggest potential alterations in B cell pathways that are essential for IgA production in Lrba-deficient mice. In addition, the mechanisms underlying the induction of the other isotypes were unaffected.

After observing elevated IgA levels in Lrba-deficient mice, we assessed whether there was an alteration in the development of the spleen and B1 cell subpopulations in the peritoneal cavity. The spleen populations were not affected by the absence of Lrba. However, upon analyzing B1 lymphocyte subpopulations in the peritoneal cavity, we observed a decrease in both B1a and B1b subpopulations ( Figures 2A, B ).

Normal B cell differentiation in the spleen and altered levels of B1a and B1b cells in the peritoneal cavity have led to an unclear source of IgA. To determine the lymphoid organs that predominantly produce IgA, we analyzed the mesenteric lymph nodes (MLN), PPs, and small intestine to detect the presence of IgA+ B cells and IgA+ plasma cells.

In the histological sections corresponding to the spleen and MLN, we observed a trend toward a higher number of IgA+ B cells and a more substantial presence of IgA+ plasma cells in both organs of Lrba-deficient mice ( Figures 3A, B ).

Smaller germinal centers were observed in the PP of Lrba– mice than that in WT, but with an evident size increase for these organs ( Figures 4A, B ). Additionally, a lower number of IgA+ B cells was counted in these organs, which contrasts with the results obtained in the spleen and MLN; however, upon determining the number of IgA+ plasma cells, we observed similar numbers in both mouse groups ( Figure 4C ). Analysis of the number of PPs in the small bowel of mice and measurement of the width and height of these organs confirmed a significantly higher number and size in Lrba-/- mice ( Figure 4D ). Considering the predominance of PPs in Lrba-/- mice, these lymphoid organs can be considered important sources of IgA.

The small intestine was analyzed, as it is one of the sites where IgA+ plasma cells reside, produce, and secrete IgA. Notably, significantly higher number of IgA+ plasma cells were observed in Lrba-/- mice ( Figures 5A, B ).

Considering these results, the high levels of IgA in the serum and feces of Lrba-deficient mice could be explained by the increased number of IgA-producing plasma cells observed in all anatomical sites evaluated.

We analyzed aspects of the signaling pathway, including TGFβRI and TGFβRII expression in B cells. As shown in Figure 6A , Lrba-/- B cells have lower total TGFβRI and TGFβRII expression. As TGFβR is a membrane receptor that undergoes continuous recycling, we sought to determine if there was a difference in expression on the cell surface or intracellularly in B cells.

Surface and intracellular expression of TGFβRI is depicted in Figure 6B . There were no differences in the surface expression of this protein in B cells between the WT and Lrba-deficient mice. However, when analyzing the intracellular expression, we detected significantly lower expression of TGFβRI within the B cells of Lrba-deficient mice. Similarly, there were no differences in surface expression of TGFβRII, and a significant reduction in intracellular expression was also detected.

After observing reduced intracellular expression of TGFβR in B cells from Lrba-deficient mice, SMAD2 phosphorylation was analyzed. Protein extracts were obtained from unstimulated purified splenic B cells or after stimulation with TGFβ1 at different time points (5, 15, and 30 min).

Notably, SMAD2 phosphorylation was significantly increased in unstimulated Lrba-/- B cells ( Figure 7 ), whereas stimulation with the recombinant cytokine slightly induced SMAD2 phosphorylation, which remained similar to the basal levels throughout the entire kinetics ( Figures 7A, B ). This result contrasts with B cells from Lrba+/+ mice, where initial phosphorylation was low, and it gradually increased with TGFβ1 stimulation. These data suggest that Lrba participates in controlling the activation of TGFβR.

We analyzed whether TGFβR colocalizes with Lrba. Figures 8A and B show that Lrba co-localizes with both TGFβRI and II, respectively, with an approximate Mander’s correlation coefficient of around 0.7 ( Figure 8 ). Co-immunoprecipitation experiments were also performed; however, the Lrba antibody did not sufficiently immunoprecipitate this protein, and only a weak signal was observed in both Lrba and TGFβRII immunoprecipitates ( Supplementary Figure 2 ). Collectively, these results suggest that Lrba is involved in regulating TGFβR activation.