Antibodies and reagents used in this work including NF-κB p50 (E-10), phospho NF-κB p50 (Ser336) (A-8), RELA/NF-κB p65 (A-12), phospho-RELA/NF-κB p65 (Ser536) (27. Ser536), NFKBA/IKb alpha (H-4), beta Actin (C4), Plcγ2 (B-10) and Nur77 (C-5) were obtained from (Santa Cruz Biotechnology, CA, USA). Phospho-IκBα (Ser32/36) (5A5) mouse mAb #9246 was procured from (Cell Signaling Technology, MA, USA). Purified Anti-Human Btk antibody and BD Pharmingen Purified Mouse anti-Btk (Y551)/ItkY511 (24a/BTK) was purchased from BD Transduction Laboratories (NJ, USA), PE anti-Plcγ2 Phospho (Tyr759) Recombinant Antibody (QA20A56) from Biolegend (CA, USA), and Plcγ2 inhibitor U73122 (Merck Millipore, MA, USA). Additionally, PE anti mouse-Cd21/Cd35 Monoclonal Antibody (4E3), PerCP/Cy5.5 anti-mouse Cd44(IM7); (eBioscience, San Diego, CA, USA), goat anti-Mouse IgG (H+L) Secondary Antibody, Goat IgG anti-mouse Alexa Fluor 594 (Invitrogen), and Propidium Iodide (Thermo Scientific, MA, USA) were used. Polyclonal Anti-LRBA/BGL antibody was purchased from Abcam, Cambridge, UK). PE anti-mouse I-A^b Antibody (AF6-120.1), APC anti-mouse/human Cd45R/B220 Antibody (RA3-6B2), and PerCP/Cyanine5.5 anti-mouse Cd24 Antibody (M1/69) were purchased from BioLegend (CA, USA). AffiniPureTM F(ab’)2 Fragment Goat Anti-Mouse, μ chain specific (referred to as Anti-IgM), secondary antibodies Peroxidase AffiniPure™ Goat Anti-Mouse IgG (H+L) and Peroxidase AffiniPure™ Goat Anti-Rabbit IgG (H+L) were obtained from Jackson ImmunoResearch Laboratories Inc®, PA, USA. DAPI was purchased from Sigma-Aldrich (St. Louis, MO).

Lrba^-/- mice were kindly donated by PhD Manfred W. Kilimann from the Max Planck Institute, Germany (26, 27). C57BL/6 Lrba^-/- and Lrba^+/+ mice were maintained in germ-free installations at the animal facility of the Centro de Investigacion y de Estudios Avanzados (CINVESTAV) according to the institutional animal guidelines for animal care and experimentation (Protocol number 0145-15, UPEAL-CINVESTAV-IPN). These mice have a deletion in exon four that drives the absence of the protein. For the experiments described here, the mice oscillated between 8 and 12 weeks of age. The mice were genotyped before the experiments were performed as described previously (26).

Mice were euthanized by cervical dislocation and spleens were obtained. The sizes and weights of the spleens were measured. Then, the spleens were disaggregated, and erythrocytes were lysed using a homemade solution (NH4Cl 0.15 M, KHCO3 10 mM, EDTA 100 Mm) for 5 min at 25 °C. After cell counting, the samples were prepared for the assays described below.

B cell subpopulations were determined using flow cytometry. Total splenocytes were stained with anti-B220-APC, anti-Cd21-PE, and anti-Cd23-PerCP/Cy5.5 and anti-IgM APC-Cy7 antibodies, incubated for 30 min, and washed once with PBS. For analysis using FlowJo (Becton Dickinson), B220-positive cells were gated among lymphocytes, and Cd23, Cd21, and IgM levels were used to determine the proportions of T1 (Cd23-Cd21^lowIgM+), T2 (Cd23+Cd21^hiIgM+), follicular B cells (Cd23+^low Cd21^lowIgM^low) and Marginal zone (MZ) B cells (Cd23+^low Cd21^highIgM+). Finally, absolute numbers for each subpopulation were calculated based on the percentages obtained for each B cell subpopulation. Data were acquired on a Northern Lights spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA).

Splenocytes were incubated with anti-mouse PerCP/Cy5.5-Cd44, PE anti-mouse I-A^b, and APC anti-B220 (Biolegend) for 30 minutes to detect activation marker expression, then washed once with PBS. Data were acquired using a FACs Aria I flow cytometer (Beckton Dickinson). Median fluorescence intensities (MFI) for Cd44 and I-A^b were obtained using FlowJo software.

Proliferation assays were conducted using CellTrace™ CFSE (Life Technologies, Carlsbad, CA, USA). The splenocytes were harvested at a final concentration of 1 × 10⁶ cells and stained with CFSE at 0.5 μM. The stained splenocytes were then cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) (Gibco, NY, USA), penicillin-streptomycin 1X (Sigma-Aldrich®, MO, USA), and 100 ng/mL of recombinant murine IL-4 (Biolegend, CA, USA). Cells were plated in 24-well cell culture plates at a concentration of 2.5 × 10⁵ cells per well and stimulated with anti-IgM at a final concentration of 10 μg/ml. After 96 h of incubation, the cells were stained with anti-B220-APC and data acquisition was performed using a FACs Aria instrument (Becton Dickinson). Proliferation was analyzed from viable (propidium iodide negative) B220+ B cells.

Total splenocytes (5 × 10⁵ cells/ml) were cultured in 24-well cell culture plates for 4, 12, or 20 h. Anti-IgM stimuli were added at a concentration of 10 μg/mL. After incubation, cells were stained with anti-B220-APC and propidium iodide to evaluate cell viability. Analysis was performed using FlowJo software (Becton Dickinson).

B cell assays were conducted using samples enriched for B cells through negative selection with magnetic beads, employing the MojoSortTM Mouse Pan B Cell Isolation Kit and following the manufacturer’s instructions (Biolegend®, CA, USA). Briefly, cells were resuspended at a concentration of 1.5 × 10⁸ cells/mL in a handmade buffer for sorting B cells, consisting of PBS with 0.1% bovine serum albumin and 0.5 μM EDTA. Subsequently, 10 μl of the Biotin-Antibody cocktail from the MojoSortTM Mouse Pan B Cell Isolation Kit (Biolegend®, CA, USA) was added, and the mixture was incubated on ice for 15 min. After incubation, the cells were washed once and Streptavidin Nanobeads were added and incubated for another 15-min incubation. The cells were washed again, and B cells were isolated by incubating the cell suspension three times for 10 min each in a MojoSort™ Magnet (Biolegend®, CA, USA). The purified B cells were then resuspended in PBS at a concentration of 10 × 10⁶ cells/mL and stimulated with Anti-IgM (10 μg/mL) for 10 and 20 min at 37°C. For inhibition assays, cells were treated similarly, however, they were preincubated with 0.25 μM U73122 for 2 h, and p50 phosphorylation was determined.

After stimulation, the cells were centrifuged and pellets were obtained. Whole-cell lysate was prepared by adding 100 μl of Cell Lysis Buffer 1× (Cell Signaling Technologies®, MA, USA) along with the complete ULTRA tablets’ protein inhibitor cocktail (ROCHE®, Switzerland). The cells were centrifuged at 17200 × g at 4°C for 10 min, and supernatants were collected. Protein concentration was determined using a DC™ Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). The lysates were then mixed with 20 μl of Laemmli buffer 6X added with 5% β-mercaptoethanol was added, and the mixture was boiled at 95°C.

To evaluate the expression and/or phosphorylation of signaling proteins after BCR activation, samples were separated on a 12% acrylamide gel, and electrophoresis was performed for 3 h at 80V. Proteins were transferred to PVDF membranes for 25 min at 25V using a Trans-Blot Turbo Transfer System (Bio-Rad). The PVDF membranes were blocked for 30 min in a solution of 3% fat-free milk in TBS-Tween 0.1%. Primary antibodies including Btk, Plcγ2, p50, pSer336 p50, p65, pSer536, p65, IκBα, and pSer32/36 IκBα, diluted at 1:500, and β-Actin, diluted at 1:2000, were incubated overnight. Secondary antibodies (anti-mouse IgG-HRP or anti-rabbit IgG-HRP, diluted 1:3000) were added and incubated for 90 min. The membranes were washed three times after incubation with 1% TBS-Tween 0.1% in 5% fat-free milk for 10 min.

For protein detection, SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific®, MA, USA) was used, and membrane visualization was performed using the ChemiDocTM XRS+ imaging system, densitometric analysis was performed using the ImageLab™ Software (Bio-Rad®, CA, USA).

Splenocytes were stained with APC anti-B220 for 30 minutes, fixed, and permeabilized with BD Phosflow™ Fix Buffer I and Perm Buffer III (Becton Dickinson), and following manufacturer’s instructions. After permeabilization, the cells were incubated overnight with PE anti-pY759 Plcγ2. The cells were then washed with PBS containing 1% FCS and acquired using a flow cytometer. MFI was calculated for each sample, and an index of expression was obtained by dividing the MFI of pY759 Plcγ2 positive cells by the MFI of negative cells.

For p65 staining, 5 × 10⁵ splenocytes were stimulated with anti-IgM (10μg/mL) for 5 and 15 min, as described previously. After washing with PBS, cells were fixed with 4% paraformaldehyde in PBS for 15 min at 4°C. Then, cells were permeabilized with 0.2% Triton X-100 for 10 min at room temperature and blocked with 10% goat serum in PBS for 30 min at 37°C. Subsequently, cells were incubated with anti-p65 antibody diluted 1:150 and incubated for 1 h at 4°C. After incubation, cells were stained with anti-mouse IgG Alexa Fluor 596 (1:750) and DAPI (1:1000) for 30 min at 4°C.

Cells were mounted on coverslips previously coated with 0.01% Poly L-lysine (Sigma-Aldrich®, MO, USA) and incubated for 1 h at 37°C. The coverslips were then washed with PBS and mounted on slides using the Vectashield mounting medium (Vector Laboratories Inc., CA, USA).

For Nur77 detection, enriched B cells were lysed as described previously, and protein extracts were loaded into 12% acrylamide/bis-acrylamide gels and transferred onto nitrocellulose membranes. Procedures for Western blotting and protein detection were described in section 2.8.

An LSM 710-Live Duo Scan confocal microscope (Zeiss, Germany) was used to visualize the cells. Subsequently, images were analyzed using FIJI software (28). The co-localization test and Colocalization Finder Plugin were used to determine the nuclear localization of p65 and Pearson’s correlations.

Subpopulation proportions, proliferation, and viability were statistically analyzed using the Mann–Whitney U test. Densitometric data from the immunoblots were obtained using ImageLab™ software (Bio-Rad). The amount of total or phosphorylated proteins was normalized by calculating the ratio of total protein or phosphorylated protein to housekeeping proteins. Statistical differences were determined using the Mann-Whitney U test.

For nuclear localization of p65, colocalization with DAPI was determined, and Pearson’s coefficient was obtained using the FIJI software (28), where a coefficient close to one indicates strong nuclear localization of p65. Statistical analysis of variance (ANOVA) was performed to compare the nuclear localization of p65 in basal and BCR-crosslinked cells from both wild type (WT) and Lrba^-/- mice. All statistical analyses were conducted using GraphPad Prism software version 8.0 for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com).

In humans, LRBA deficiency results in an extended phenotype that includes recurrent infections, autoimmunity, lymphoproliferation, and splenomegaly. Although Lrba^-/- mice appeared to have a healthy phenotype, evidence of splenomegaly was observed in this study. The weights of the spleens were measured ( Figure 1A ). The spleen weight of Lrba^-/- mice was higher (0.1048 ± 0.009 g) than Lrba^+/+ mice (0.0715 ± 0.0178 g), suggesting splenomegaly. Total splenocytes were obtained as previously described. In addition to the macroscopic data of the spleen (size and weight), the total splenocyte count was higher in Lrba^-/- mice (179.9 ± 50.6 × 10⁶ cells) than in WT mice (127.5 ± 25.04 × 10⁶ cells) as shown in Figure 1A .

Splenocytes were immediately stained with anti-Cd23, anti-Cd21, anti-IgM, and anti-B220 antibodies. There were no differences in the B cell proportion or total counts ( Supplementary Figure 1A ). The proportions of transitional 1 (T1), transitional 2 (T2), marginal zone (MZ), and follicular B cell subpopulations were determined by flow cytometry ( Figure 1B ). T1 cell proportions were lower in Lrba^-/- mice (10.26 ± 2.93%) than in WT mice (23.98 ± 6.69%), which was statistically significant. T2 cell proportions were similar between both strains, with values of (14.24 ± 1.55%) for Lrba^-/- and (12.56 ± 3.97%) for Lrba^+/+. Follicular B cell proportions were similar in Lrba^-/- mice (44.31 ± 10.35%) Lrba^+/+ mice (32.37 ± 8.3%). Finally, there MZ cells are higher in Lrba^-/- mice (9.37 ± 2.07%) versus Lrba^+/+ mice (5.01 ± 3.44%), Figure 1B .

Absolute numbers of T1 B cells were lower in Lrba^-/- mice (4.16 ± 1.86) than in Lrba^+/+ mice (12.96 ± 5.33). Also, the numbers of MZ cells were higher in Lrba^-/- mice (4.68 ± 1.63) than in Lrba^+/+ mice (2.27 ± 1.63). No differences were observed with the total number of T2 (7.31 ± 2.85) in Lrba^-/- mice and (6.04 ± 3.2) in Lrba^+/+ mice; follicular B cells (23.66 ± 12.02) in Lrba^-/- mice and (15.52 ± 7.22) in WT mice ( Figure 1B ).

When immature B cells enter the spleen, a second negative selection occurs in T1 cells. Therefore, BCR signaling at this stage of differentiation drives cell death. Following this line of thought, the differences in T1 proportions in the absence of Lrba suggest a possible altered response to BCR cross-linking. Proliferation was measured to determine if Lrba^-/- B cells responded to BCR stimulation. Interestingly, the proportion of proliferating B cells stimulated with anti-IgM was lower in Lrba^-/- B cells (22.56 ± 12.8%) than in WT mice (40.34 ± 15.88%). These differences were significant ( Figure 2A ). In contrast, Ki67 expression was measured as a marker for in situ proliferation, interestingly increased Ki67 expression was observed in Lrba^-/- B cells ( Supplementary Figure 1B ).

Increased expression of activation markers, as CD44 and I-A^b were detected in B cells from Lrba^-/- mice. In the case of CD44, MFI was 409 ± 73.3 for Lrba^-/- and 296 ± 66.86 for Lrba^+/+ B cells, while for I-A^b expression, MFI was 1768 ± 506.6 for Lrba^-/- and 542.8 ± 230.9 for Lrba^+/+ B cells ( Figure 2B ).

Lower proliferative responses detected in Lrba^-/- mice could be the result of altered cell survival, as previously reported for B cells from LRBA-deficient patients (1). Therefore, B cell survival assays were performed on unstimulated and BCR-stimulated cells over time. The proportion of Propidium Iodide (PI)-negative cells was calculated within the B220+ gate ( Figure 2C ). At time 0 or under unstimulated conditions, B cells from both mice showed similar survival proportions (85.54% ± 1.9 for Lrba^-/- and 86.57% ± 2.96 for WT). After 4 h of culture and BCR crosslinking, survival was significantly higher in Lrba^-/- mice (93.27% ± 2.55) compared to that (85.55% ± 5.23) in WT mice. A similar trend was observed at 12 h of BCR activation: the proportion of viable B cells in Lrba^-/- mice was 84.07% ± 7.65 compared to 71.46% ± 7.48 observed in B cells from the WT counterpart. Interestingly, after 20 h of stimulation, Lrba^-/- B cell survival drastically decreased (62.26% ± 5.84) compared to WT B cell survival (69.21% ± 2.64) ( Figure 2C ). These data suggested that the low proliferative response after 96 h of stimulation may be due to reduced cell survival in response to BCR activation in B cells from Lrba^-/- mice.

The inadequate proliferative and survival responses to BCR crosslinking suggested an inadequate response in this pathway. BCR signaling begins with the phosphorylation of ITAM motifs and activates kinases such as Syk and Lyn. Following this, Btk is phosphorylated, leading to the activation of Plcγ2. The evaluation of proximal BCR signaling protein expression revealed increased basal expression of Btk and Plcγ2; these results were statistically significant when densitometric analysis using β-actin as a control was performed ( Figure 3A ).

Diacylglycerol production after Plcγ2 activation has a direct impact on NF-κB activation. NF-κB is composed of IκBα, p50, and p65. Expression of IκBα and p65 was similar between Lrba^-/- and Lrba^+/+ B cells. However, p50 expression increased in Lrba^-/- B cells ( Figure 3A ). The expression of IκBα, p50, and p65 was similar in total splenocytes ( Supplementary Figure 2 ). Increased expression of proximal BCR signaling molecules suggests that this signaling pathway is altered. Therefore, we explored the phosphorylation status of NF-κB.

Plcγ2 phosphorylation was explored; however, as shown in Figure 3B , a tendency towards increased phosphorylation for Plcγ2 was observed (14.4 ± 3.69 for Lrba^-/- , and 8.41 ± 4.72 for Lrba^-/- ), but the differences were not significant ( Figure 3B ), similar data was obtained for Btk phosphorylation, Figure 3C .

NF-κB is a family of transcription factors broadly expressed in different cell types, and importantly, in immune responses with significance in BCR signaling. Following the finding that proximal BCR signaling molecules were overexpressed, the impact of such high expression on the phosphorylation status of NF-κB components was explored. Notably, higher levels of phosphorylated p50 and IκB proteins were detected in basal conditions in Lrba^-/- B cells, Figure 3C . These data indicated an exacerbated activated state without BCR crosslinking. However, p65 phosphorylation under basal conditions was similar to Lrba^+/+ B cells. IκBα, p50, and p65 phosphorylation were similar in total splenocytes ( Supplementary 2B ), suggesting that the abnormalities are exclusive to B cells. Treatment with the Plcγ2 inhibitor U73122 notably reduced the levels of p50 phosphorylation ( Figure 3D ).

We next explored whether NF-κB components could be further activated when the BCR is cross-linked. Purified B cells were obtained by negative selection and activated for 10 or 20 min with anti-IgM. Consistent with previous results, in unstimulated cells ( Figure 4A ), IκBα and p50 were over phosphorylated in Lrba^-/- B cells under basal conditions. Importantly, after 10 min of stimulation with anti-IgM, no increase in the levels of phosphorylation of IκBα, p50, and p65 was detected, in contrast to the observed response in Lrba^+/+ B cells ( Figure 4A ). When the phosphorylation of IκBα, p50, and p65 was explored after 20 min of activation with anti-IgM, the phosphorylation remained at similar levels in Lrba^-/- B cells than the unstimulated conditions; meanwhile, the levels of phosphorylation of these molecules in the Lrba^+/+ B cells returned to basal values ( Figure 4A ). These data suggest that Lrba deficiency correlates with spontaneous B cell activation; however, such cells are unable to respond appropriately to BCR stimulation.

NF-κB activation culminates in the entry of the transcription factor formed by p50/p65 into the nucleus, inducing transcription of genes important for survival, proliferation, and activation of B cells. Until now, IκBα and p50 have shown increased basal phosphorylation levels, and no further activation is observed in BCR-crosslinked B cells. However, p65 does not exhibit this behavior as it has low phosphorylation levels in unstimulated or BCR-crosslinked B cells. We wondered whether the high levels of p50 phosphorylation, the cellular location of p65 was affected. To investigate this, the nuclear localization of p65 in splenocytes was explored using confocal microscopy. Interestingly, higher nuclear colocalization of p65 was observed in Lrba^-/- cells under unstimulated conditions. After five min of BCR crosslinking, nuclear p65 significantly increased in both Lrba^-/- and WT mice. However, 15 min after activation, nuclear p65 levels remained unchanged in both mice ( Figures 4B, C ). The Pearson’s correlation analysis confirmed that these results were significant ( Figure 4C ). This data is consistent with the increased phosphorylation status of NF-κB in non-BCR activated Lrba^-/- B cells.

In addition, BCR crosslinking after 5 and 15 min induced p65 nuclear localization in WT splenocytes; however, in Lrba^-/- mice, the increase in the nuclear localization of p65 was less pronounced ( Figure 4C ).

Chronic B cell stimulation by BCR has been associated with Nur77 induction. Here, we evaluated Nur77 expression to show that the spontaneous activation of BCR signaling in Lrba^-/- mice originates from a chronic process. As shown in Figure 4D , Nur77 was overexpressed in B cells from these mice.