Suppression of Class Switch Recombination to IgA by RASA2 and RASA3 through Inhibition of TGF-β Signaling

The role of Abs in adaptive immunity is critical, because they function to bind to and neutralize pathogens and mobilize the cellular immune response to combat infections. Somatic hypermutation (SHM) and class switch recombination (CSR) are both secondary Ab diversification processes that contribute to humoral immunity. SHM is a process that enhances Ab affinity, whereas CSR leads to the production of different Ab isotypes (e.g., IgG, IgA, IgE). Activation-induced cytidine deaminase (AID) facilitates SHM and CSR processes by catalyzing targeted mutations in the variable regions of Ab genes and inducing DNA breaks to promote recombination that leads to isotype switching (1–4). These processes begin with AID catalyzing the deamination of deoxycytidine to deoxyuridine within Ig genes (5–7).

The small GTPase RAS undergoes oscillation between an active GTP-bound state and an inactive GDP-bound state. RAS serves as a crucial mediator in cytokine-dependent signaling pathways in erythropoiesis (8, 9) and thrombopoiesis (10, 11), playing a central role in transmitting signals to multiple effector pathways that govern cell proliferation, differentiation, and survival. Additionally, RAS holds significance in the development of B- and T-lymphocyte lineages (12). Oncogenic RAS mutations are present in around 30% of human cancers overall, with a notable prevalence in myeloid malignancies (13).

RAS p21 protein activator 2 (RASA2) and RAS p21 protein activator 3 (RASA3) are members of a RAS GTPase-activating protein 1 (GAP1) family (14). RASA2 and RASA3 are two closely related members of this family, sharing structural and functional similarities (15) but exhibiting distinct patterns of expression and activity (14). RASA2 plays a role in regulating cell proliferation, differentiation, and metabolism (14, 16, 17). Mutations or depletion of RASA2 can lead to increased RAS activation in various cancer types (16, 18, 19), suggesting its involvement in cancer initiation and progression. RASA2 was discovered as a new target for immunotherapy that enables human primary T cell proliferation (17). Depleting RASA2 heightened Ag sensitivity in both conventional and chimeric Ag receptor-T cells, resulting in enhanced rejection of tumors. RASA3 is another member of the Ras-GAP1 family with dual GAP activity for RAP1 and RAS (14, 20, 21). RASA3 plays a critical role in erythropoiesis and megakaryopoiesis (22). The absence of RASA3 resulted in changes to T cell adhesion, migration, and T cell–dependent Ab responses (21). However, their exact mechanisms of action are not fully understood, although recent evidence suggests that RASA2 may modulate TGF-β signaling, as well as RAS signaling (17). Because TGF-β is a cytokine that promotes CSR to IgA, it is possible that RASA2/RASA3 may modulate B cell responses and regulate CSR.

CRISPR Cas9 was previously employed in CH12F3–2 (CH12) cells, a mouse B cell lymphoma line that undergoes switching from IgM to IgA upon exposure to a combination of anti-CD40, IL-4, and TGF-β (CIT) (23). Guide RNAs (gRNAs) that target the Rasa2 and Rasa3 genes were decreased in IgA-ve CH12 cells, suggesting that these two gene products inhibit CSR. Indeed, knockout of either Rasa2 or Rasa3 in CH12 cells increased CSR. This effect was mediated through TGF-β receptor signaling as Rasa2^−/− and Rasa3^−/− CH12 cells had increased canonical but decreased noncanonical TGF-β signaling. Hence, RASA2 and RASA3 are novel regulators of TGF-β receptor signaling in B cells and regulate class switch recombination to IgA.

CH12F3-2 (CH12) cells were cultured in RPMI 1640 medium (catalog no. 350-000-CL, Wisent) supplemented with 10% FBS, penicillin/streptomycin, and 50 μM β-mercaptoethanol. HEK293T cells were cultured in DMEM (catalog no. 319-005-CL, Wisent) supplemented with 10% FBS and penicillin/streptomycin in a 5% CO₂ at 37°C.

Lenti-puro-guide vector was purchased from (Addgene, catalog no. 52963). All oligonucleotides and primers were generated using Benchling and purchased from Invitrogen; they are listed in Supplemental Table I.

Lentivirus generation was performed as mentioned before (23). Briefly, to generate lentiviruses, HEK 293T cells were transduced with LentiGuide-Puro (Addgene, catalog no. 52963) expressing single-guide RNA targeting the Rasa2 and Rasa3 genes (Supplemental Tables I and II). After 48 h, virus-containing supernatants were harvested and filtered through a 22-μm filter prior to the addition of 10 μg/ml polybrene and 20 mM HEPES. Supernatant was added to 5 × 10⁵ CH12 cells, and a spinfection was performed at 800g for 1 h at room temperature. After 24 h, CH12 cells were transferred into complete RPMI 1640 medium containing 1 μg/ml puromycin for selection of transfected cells and maintained expression of the construct. After 3 d, transduced cells were selected in 0.5 μg/ml puromycin for 5 d and then moved to puromycin-free medium to recover before proceeding with subcloning. Rasa2^−/− and Rasa3^−/− CH12 clones were isolated by subcloning the bulk cultures. Knockout clones were validated through sequencing, RT-qPCR, and Western blotting.

Splenic B cells were purified using a mouse B cell isolation kit (StemCell Technologies). Rasa2^−/− and Rasa3^−/− B cells were generated via lentiviral transduction with lenti-puro-guide vectors containing single-guide RNA. Transduced cells were selected with 0.5 μg/ml puromycin for 4 d and stimulated with 50 μg/ml LPS (Sigma-Aldrich) combined with 1 ng/ml TGF-β. The cells were collected on day 4 poststimulation and stained with anti–IgA-PE (SouthernBiotech, catalog no. 1040-09) and anti–IgM-APC (eBioscience, catalog no. 17-5790-82) to assess CSR to IgA.

CH12 cells were seeded at 0.5 × 10⁵ cells/ml in complete RPMI 1640 medium and stimulated with CIT mixture (2 μg/ml of α-CD40, 10 ng/ml of IL-4, and 1 ng/ml of TGF-β) and individual agents for 24, 48, and 72 h. Unstained and unstimulated conditions were run in parallel for each sample. Cells were harvested at the indicated timepoints, stained with anti–IgA-PE (SouthernBiotech, catalog no. 1040-09), and analyzed by flow cytometry (BD FACSymphony instrument). The data represent percentages of IgA⁺ of stimulated conditions minus unstimulated percentages of IgA⁺ backgrounds.

CH12 cells were grown and seeded at 5 × 10⁴ cells/ml in 24-well tissue culture plates. CH12 cells were harvested and seeded in a similar way. Cell counts were performed daily in duplicate after seeding, using a hemocytometer, trypan blue staining, and light microscopy. For the CFSE assay, CH12 cells were stained with 5 μM CFSE (CellTrace, Life Technologies) for 20 min at 37°C, following the manufacturer’s instructions. After incubation, the stained cells were cultured in complete RPMI 1640 medium containing 1 ng/μl TGF-β for 3 d. The cells were harvested 3 d after CFSE incorporation and analyzed by flow cytometry using a BD FACSymphony instrument, and the data were analyzed using FlowJo V10 software.

CH12 cells were seeded in a flat-bottomed 96-well plate (Greiner, Kremsmünster, Austria) at 1 × 10⁴ cells/100 μl of medium per well and incubated with and without 1 ng/ml of TGF-β. Cell viability was determined by CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI). CellTiter-Glo reagent (100 μl) was added to 100 μl of cell suspension in a 96-well plate. After mixing on an orbital shaker to induce cell lysis for 2 min, the plate was incubated at room temperature for an additional 10 min. Luminescence readings were determined using a SpectraMax i3 (MiniMax 300 imaging cytometer).

APC-conjugated annexin V apoptosis detection kit (eBioscience, catalog no. 88-8007-72) was used following the manufacturer’s instructions. CH12 cells were seeded at 1 × 10⁵ cells/ml in complete RPMI 1640 medium in the presence or absence of 1 ng/ml of TGF-β stimulation. The cells were collected at 48 h and stained for annexin V-APC and propidium iodide. The cells were analyzed by flow cytometry on a BD FACSymphony instrument and FlowJo V10 software.

Western blotting was performed as before (24). Harvested cells were resuspended in radioimmunoprecipitation assay lysis buffer supplemented with protease and phosphatase inhibitors. The cell lysates were incubated on ice for 15 min and centrifuged at 15,000g for 10 min at 4 °C to remove debris prior to use. CH12 cells were treated with and without 1 ng/ml of TGF-β for 3 d prior to harvesting pellet. The following Abs were used: anti-RASA3 (Invitrogen, catalog no. PA5-30445), anti-AID (Invitrogen, catalog no. 39-2500), anti–α-tubulin (Abcam, catalog no. ab4074), anti–phosphor-SMAD2/3 (Invitrogen, catalog no. PA5-36028), anti–phosphor-(S473)-AKT (Cell Signaling Technology, catalog no. 9271S), anti–phosphor-(T185, Y187)- ERK1/2 (Invitrogen, catalog no. 44-680G), and anti–phosphor-(T180, Y182)-p38 MAPK (Cell Signaling Technology, catalog no. 9211S). Goat anti-rabbit IgG and goat anti-mouse IgG HRP-conjugated Abs (Cell Signaling) were used at a dilution of 1:1,000, and blots were developed with chemiluminescent HRP substrate-ECL reagent (ThermoFisher) using iBright 1500 imager (Invitrogen). ImageJ software (National Institutes of Health, Bethesda, MD) was used to perform densitometry analysis.

TRIzol (Life Technologies)-chloroform extractions of total RNA from cell pellets were performed under the manufacturer’s protocol. RNA was resuspended in UltraPure DNase/RNase-free distilled water (ThermoFisher, catalog no. 10977015), Maxima H Minus reverse transcriptase (ThermoFisher) was used to generate cDNA. Amplification of cDNA was achieved using PowerTrack SYBR Green Master Mix (Applied Biosystems), and the gene-specific primers listed in Supplemental Table I were used for qPCR with the CFX96 Touch real-time PCR detection system and CFX Manager 3.0 (Bio-Rad).

Biological replicates were processed in the flow cytometer. CH12 cells were stimulated with the CIT mixture and individual agents for indicated timepoints prior to flow cytometry, cell viability, annexin V apoptosis assay, and immunoblot analysis. The data were acquired using the BD FACSymphony flow cytometer instrument, and then the data were analyzed using FlowJo V10 software.

Sanger sequencing was performed at The Center for Applied Genomics (TCAG) in Toronto, Ontario, Canada.

All experiments were performed in duplicate/triplicate, and the data are expressed as means ± SEM. One-way ANOVA and two-way ANOVA were performed to determine statistical differences among sample groups using the GraphPad Prism version 9.00 (GraphPad Software, San Diego, CA). p values were considered statistically significant if *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Our previous work showed that gRNAs targeting Rasa2 and Rasa3 are decreased in abundance in IgA-negative CIT-stimulated CH12 cells (Fig. 1A) (23). These results suggest that RASA2 and RASA3 inhibit CSR. The expression levels of Rasa2 and Rasa3 are high across a range of immune cell types, including B cells, specific T cell subsets, NK cells, and monocytes (Supplemental Fig. 1A, 1B). To confirm the involvement of RASA2 and RASA3 in CSR, two gRNAs targeting each gene were used to generate knockouts in CH12 cells. By using two different gRNAs, this minimized the possibility that the results were due to off-target Cas9 mutagenesis. CH12 cells transduced with lentiviruses encoding these gRNAs and selected in bulk had increased IgA CSR compared with cells transduced with control gRNAs (Fig. 1B). Using these same gRNAs, we infected purified splenic B cells (Supplemental Fig. 1C) and induced CSR to IgA using LPS + TGF-β and selected for transduced cells with puromycin. Indeed, we observed a ∼3-fold increase in IgA CSR in B cells expressing the gRNAs targeting the Rasa2 and Rasa3 genes (Fig. 1C).

We next isolated Rasa2- and Rasa3-knockout CH12 clones (Supplemental Fig. 2). A RT-qPCR analysis revealed reduced Rasa2 and Rasa3 mRNA expression in the knockout clones, compared with WT CH12 clones (Supplemental Fig. 3A, 3B). Western blot analysis showed no RASA3 protein in Rasa3^−/− CH12 clones (Supplemental Fig. 3C), with no effect on RASA3 levels in Rasa2^−/− CH12 clones (Supplemental Fig. 3D). There is no reliable anti-RASA2 Ab. Rasa2^−/− and Rasa3^−/− CH12 clones had increased IgA CSR in CH12 cells compared with WT clones at various timepoints after CIT treatment (Fig. 1D, 1E). These results show that RASA2 and RASA3 suppress IgA CSR in CH12 and primary mouse B cells.

RASA2 and RASA3 might suppress CSR by negatively regulating signaling through CD40R, IL-4R, and/or TGF-βR, which are used to stimulate CSR in CH12 cells. To test whether signaling through any of these receptors is increased in Rasa2^−/− and Rasa3^−/− CH12 cells, we assessed the effect of individual cytokine reagents (IL-4, anti-CD40, and TGF-β) on CSR in CH12 cells. TGF-β was revealed to be the major signaling pathway responsible for IgA CSR in CH12 cells (Fig. 2A, 2B). Indeed, gRNAs targeting both Tgf-βr1 and Tgf-βr2 are enriched in the CRISPR/Cas9 screen in CIT-stimulated IgA-ve CH12 cells (Fig. 1A) (23). Together, these results suggest that CH12 cells are essentially a TGF-βR reporter cell. Importantly, CSR was increased in response to TGF-β in both Rasa2^−/− and Rasa3^−/− cells but not in response to IL4 or anti-CD40 (Fig. 2A, 2B). Furthermore, increasing TGF-β concentrations increased IgA CSR more in Rasa2^−/− and Rasa3^−/− CH12 cells than in WT clones (Fig. 2C, 2D). These data suggest that RASA2 and RASA3 negatively regulate TGF-βR signaling, a signaling pathway that promotes CSR to IgA.

To determine the reason for the increased CSR in Rasa2^−/− and Rasa3^−/− CH12 cells, we assessed whether AID protein levels were increased in response to TGF-β. Western blot analysis revealed a significant increase in AID protein expression in TGF-β–treated Rasa2^−/− and Rasa3^−/− CH12 cells compared with controls (Fig. 3A, 3B). Additionally, the expression of AID was further increased with increasing dosage of TGF-β (Fig. 3C). Similarly, Rasa2^−/− CH12 cells, and less so Rasa3^−/− CH12 cells, exhibited increased Aicda mRNA expression compared with WT CH12 cells upon TGF-β stimulation (Fig. 3D, 3E).

Because CSR levels are also affected by germline transcripts, we assessed whether Iμ and Iα transcripts were affected by RASA2 and RASA3 deletion. Upon TGF-β stimulation, Iμ expression was observed to be decreased, whereas Iα expression was observed to be increased in Rasa2^−/− and Rasa3^−/− CH12 cells compared with controls (Fig. 4; Supplemental Fig. 4).

Because reduced cell proliferation results in reduced CSR (25–28), we assessed whether RASA2 and RASA3 deletion affects cell proliferation and viability. Unstimulated Rasa2^−/− and Rasa3^−/− CH12 cells had slightly reduced proliferation and viability compared with controls (Supplemental Fig. 5A–C). We also observed increased apoptosis in unstimulated Rasa2^−/− and Rasa3^−/− CH12 cells (Supplemental Fig. 5D). However, we did not observe any such defects upon TGF-β treatment (Supplemental Fig. 5). Together, these data suggest that the increased CSR in CH12 cells upon RASA2 and RASA3 deletion was due to increased AID expression and Iα transcription downstream of TGF-β signaling.

TGF-β signaling occurs through two known pathways: the canonical SMAD TGF-β pathway and the noncanonical TGF-β pathways that proceed through the AKT and MAPK (29, 30) among other mediators. To investigate whether RASA2 and RASA3 interfere with any of these signaling pathways, Western blot analyses were carried out on distinct molecular mediators of SMAD and non-SMAD pathways. TGF-β stimulation increased the protein levels of phosphorylated SMAD3, but not phosphorylated SMAD2, in both Rasa2^−/− and Rasa3^−/− CH12 cells compared with controls (Fig. 5A, 5B). This result suggests that RASA2 and RASA3 may be involved in negatively regulating the canonical TGF-β signaling pathway. For the noncanonical TGF-β signaling pathway, we found that phosphorylation levels of AKT and p38 MAPK were decreased in both Rasa2^−/− and Rasa3^−/− cells compared with WT (Fig. 5C, 5D), although there were no changes in ERK phosphorylation (Supplemental Fig. 6). Overall, these findings suggest that RASA2 and RASA3 negatively regulate the canonical TGF-β signaling pathway that proceeds through SMAD3 but positively regulate the noncanonical TGF-β signaling pathway.

TGF-β plays a pivotal role in regulating diverse biological processes, including cell growth, differentiation, autophagy, apoptosis, epithelial–mesenchymal transition, angiogenesis, inflammation, and immunity (31–35). TGF-β mediates these biological functions through two primary pathways: the canonical SMAD-dependent pathway and the alternative non–SMAD-dependent pathway. The initiation of TGF-β signaling occurs when TGF-β binds to its serine and threonine kinase receptors, namely type II and type I receptors, located on the cell membrane. In B lymphocytes, TGF-β1 is crucial for restraining proliferation and Ab secretion, influencing B cell differentiation, survival, and promoting the development of IgA-secreting plasma cells (36–38). Consistent with this, mice lacking TGF-β receptor type II in B cells exhibit heightened B cell responsiveness, an expanded population of B1 cells, increased Ab production, but with a specific defect in IgA Abs (39). This underscores TGF-β’s critical role in modulating B cell responses.

Both RASA2 and RASA3 belong to the GAP1 family, sharing structural and functional similarities, and are expressed across various immune cell types, including B cells, NK cells, T cell subsets, and monocytes. Despite their widespread expression, the specific roles of RASA2 and RASA3 in regulating B cell function and CSR had not been investigated until this study. Our study shows that RASA2 and RASA3 are negative regulators of IgA CSR by modulating TGF-β signaling. Upon TGF-β stimulation, SMAD2/SMAD3 dimers, along with SMAD4, are recruited to the IgH Iα promoter, initiating the transcription of germline Iα-Cα, marking the initial step in the progression of CSR toward IgA (40–45). Our findings revealed that the absence of RASA2 and RASA3 in B cells results in heightened germline Iα levels, indicating a crucial role for RASA2 and RASA3 in the early phases of IgA CSR. The increased IgA CSR in Rasa2^−/− and Rasa3^−/− CH12 cells was likely due to elevated activation of the canonical signaling protein SMAD3. Indeed, overexpression of SMAD3 in the presence of TGF-β1 selectively increased both surface IgA expression and IgA production in mouse B lymphocytes (42). SMAD2/3 and SMAD4 are crucial mediators of TGF-β signaling, with SMAD2 and SMAD3 having distinct roles (46–49). Canonical TGF-β signaling is inhibited by SMAD6 and SMAD7 through the prevention of SMAD2 phosphorylation or by interfering in SMAD2/4 complexes (50–52). It is tempting to speculate that RASA2 and/or RASA3 positively regulates SMAD6/7 function, such that in the absence of RASA2/3, SMAD6/7 activity is diminished, and canonical TGF-β signaling is increased.

TGF-β also activates an alternative signaling pathway involving RAS and RHO GTPases, as well as MAPKs (53, 54). Our work further found that Rasa2^−/− and Rasa3^−/− CH12 cells showed a notable decrease in the activation of noncanonical signaling proteins (e.g., phosphorylation of AKT and p38 MAPK) compared with WT cells. These results suggest that RASA2/3 promotes this alternative noncanonical TGF-β signaling. Previous studies have shown that deletion of RASA2 in T cells following TCR stimulation leads to elevated levels of RAS-GTP, MEK, and ERK phosphorylation—key components of the MAPK pathway—and increased effector cytokine levels compared with control-edited T cells (17). However, RASA2 ablation did not affect MAPK signaling or alter T cell activation, proliferation, or viability in the absence of TCR stimulation, indicating that the impact of RASA2 deletion on T cell activities depends on activation stimuli. RASA3 plays crucial roles in T cell adhesion and migration by negatively modulating the binding of LFA-1 to its ligand, ICAM-1 (21).

These findings suggest that RASA2 and RASA3 suppress canonical TGF-β signaling pathway and promote noncanonical TGF-β signaling pathway. Through canonical and noncanonical pathways, TGF-β plays diverse roles in regulating cell growth, differentiation, cell motility, apoptosis, angiogenesis, extracellular matrix production, and immune response. Our results underscore the potential regulatory role of RASA2 and RASA3 in controlling B cell responses through these pathways. Further investigation of in vivo mouse models, specifically targeting B cells, would offer valuable insights into the functions of RASA2 and RASA3 within the complexities of an intact organism. This approach holds potential for translational research, paving the way for preclinical investigations into immune disorders, fibrosis, and cancer that are affected by TGF-β (55, 56).

The authors have no financial conflicts of interest.

We are thankful to the members of the Martin lab for helpful comments.