Card11 E134G, K215M mutant, and Card11 KO C57BL/6 mice were generated as described previously (25). All mice were maintained and bred in a specific pathogen-free facility. Mouse experimental protocols were approved by the Institutional Biomedical Research Ethics Committee of the Shanghai Institute of Nutrition and Health Science, Chinese Academy of Sciences.

The Ethics Board of Children’s Hospital, Fudan University, approved all the patient-related studies. Whole blood samples were obtained from patients and healthy controls using vacutainer tubes containing EDTA anticoagulant. Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation with Human Lymphocyte Separation Medium (Dakewe Biotech), followed by red blood cell lysis buffer (Biolegend) treatment. Isolated PBMCs were tested by flow cytometric analysis or cryopreserved in media containing 90% heat-inactivated fetal bovine serum (FBS) and 10% DMSO.

Mouse spleen and thymus were collected and mashed through a 40-µm cell strainer to generate single-cell suspensions, followed by red blood cell lysis. Cells were then blocked with Fc receptor antibody (Cat. BE0307, Bio X cell) for 15 minutes at 4°C before staining with fluorochrome-conjugated surface antibodies: Viability Dye (Cat. 65-0865-14), CD45.1 (clone: A20, BioLegend), CD45.2 (clone: 104, BioLegend), CD4 (clone: GK1.5, Biolegend), CD8 (clone: 53-6.7, BioLegend), CD25 (clone: PC61, BD), CD69 (clone: H1.2F3, BD). After surface staining, cells were fixed with fixation/permeabilization buffer (Cat.00-5123-43, eBioscience) and then permeabilized and stained in permeabilization buffer (Cat. 00-8333-56, eBioscience). Antibodies for intracellular staining are FoxP3 (clone: FJK-16s). Flow cytometric analysis was performed using a Gallios flow cytometer. Data were processed and analyzed through FlowJo software.

Equal amounts of protein from each sample were denatured in the loading buffer by heating for 20 minutes at 100°C. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8%-10% polyacrylamide bio-tris gels and transferred to nitrocellulose membranes. After transfer, nitrocellulose membranes were blocked in 5% milk for up to 1 hour at room temperature, followed by incubation with primary antibodies against phosphor-AKT (T308), phosphor-AKT (S473), phosphor-FOXO1, total FOXO1, total AKT, and mouse anti-actin (Cell Signaling Technology) at 4°C overnight. Membranes were washed with PBST and then incubated with HRP-conjugated secondary antibodies. Proteins were visualized and quantitated by the Bio-Rad ChemiDoc XBS System.

Naïve CD4⁺ T cells were purified using the MojoSort™ Mouse Naïve CD4⁺ T cell isolation kit (Cat. 480033, Biolegend) following the manufacturer’s protocol. Naïve CD4⁺ T cells were then activated and differentiated by plating at a density of 1x10⁶ cells per well on coated anti-CD3 (2µg/mL, Cat.BE0001-1, BioXcell) and anti-CD28 (2µg/mL, Cat. BE0015-1, BioXcell) 48-well plates for four days in 37 °C incubators with 5% CO₂ with IL-2. IL-12 and anti-IL-4 were added to growth media to promote Th1 differentiation. IL-4, anti-IFN-γ, and anti-IL-12 were added to growth media to promote Th2 differentiation. TGFβ, IL-6, IL-1β, IL-23, anti-IL-4, anti-IL-12, and anti-IFN-γ were added to growth media to promote Th17 differentiation. TGFβ, anti-IL-4, anti-IL-12, and anti-IFN-γ were added to growth media to promote iTreg differentiation. After differentiation, cells were collected, washed with PBS, and analyzed by flow cytometry.

Isolated PBMCs were transfected with WT and mutated CARD11 plasmids, respectively, by using the Human T Cell Nucleofector™ Kit (Cat.VPA-1002, Lonza). Phosphor-AKT (T308) in CD4⁺ T cells was detected by flow cytometry after 24 hours.

HEK-293T cells were transfected with NF-κB reporter plasmids together with EV (Empty Vector), WT CARD11, and mutation plasmids, respectively, using polypropyleneimine (PEI). 24 hours after transfection, NF-κB induced GFP fluorescence in transfected HEK-293T cells was detected by flow cytometry.

CD25^high Tregs from CD45.2⁺ WT, heterozygous E134G, and homozygous K215M mutant mice were purified by flow cytometry sorting. Total CD4⁺ T cells of CD45.1⁺ WT mice were purified with the MojoSort™ Mouse CD4⁺ T cell isolation kit and stained with CFSE. CD45.1⁺ and CD45.2⁺ antigen-presenting cells were treated with mitomycin (20µmol/mL). Then, all the cells above were cocultured with anti-CD3 (1µg/mL) and IL-2 (10ng/mL) for 4 days. CFSE fluorescence intensity was analyzed by flow cytometry.

Mouse Foxo1 cDNA was cloned into an MSCV-IRES2-EGFP retroviral vector. Retrovirus was made by transfection of HEK-293T cells. BM cells were collected from the femurs and tibiae of 6- to 8-week-old donor mice. After red blood cell lysis, hematopoietic stem cells were enriched using the Lineage Cell Depletion Kit (Miltenyi Biotec). Hematopoietic stem cells were cultured in chemically defined serum-free medium X-VIVO 10 with gentamicin (Lonza) supplemented with L-glutamine, β-mercaptoethanol (50 mM), mouse recombinant stem cell factor (50 ng/mL), IL-6 (20 ng/mL), IL-3 (10 ng/mL), FLT-3L (10 ng/mL), and IL-7 (10 ng/mL) (PeproTech) for 24 hours. Then, cocultured cells were transduced by spin infection with retroviral supernatant. Cells were incubated for another 24 hours before intravenous tail injection into WT recipient mice, which were irradiated at 9 Gy for at least 12 hours before adoptive transfer. 6 weeks after transfer, chimeras were analyzed by flow cytometry.

Two-tailed t-tests were used for two-data group comparisons, and multiple t-test statistical significance used the Holm-Sidak method for multiple comparisons. Statistical tests were run using GraphPad software. SEM was reported for all experiments, and P<0.05 was considered statistically significant.

To better understand the pathogenic role of CARD11 mutations, we established various Card11 transgenic mouse models with corresponding genetic modifications (25). The E134G mutant mice were used to investigate the pathogenesis of BENTA disease (24). And the K215M mutant mimics the DLBCL (19). Finally, the Card11 KO mice represent the PID patients without CARD11 ( Supplementary Figure 1A ). We compared the morphology of the thymus and spleens of WT, E134G, and K215M mutant mice ( Supplementary Figures 1B, C ). There was no significant difference in thymus size. However, the spleen of the K215M mutant mice is larger than that of the WT and E134G mutant mice. That may be due to the aberrant B cell activation and proliferation in K215M mutant mice, as reported before (25). Then, we checked the Treg development in the thymus of those distinct Card11 transgenic mouse models. In Card11 KO thymus, the tTreg population was significantly decreased, as in the previous report ( Figure 1A ). This dramatic Treg reduction also led to less mature Tregs in the KO spleen ( Figure 1B ). We then explored the Treg development under the CARD11 mutant background. The E134G mutant was identified in BENTA disease patients and autonomously induced NF-κB activation (24). The K215M mutant was an oncogenic mutant from germinal center type (GCB)-DLBCL, which activates downstream NF-κB at the same level as WT CARD11 (19). Therefore, those two mutations are not LOF mutants.

We found that CARD11 mutations significantly changed Treg development in the thymus. The E134G mutant abolished the Treg generation in the thymus and left almost no CD25⁺ FoxP3⁺ population in the CD4⁺ SP population ( Figure 1C ). On the contrary, the Treg in the thymus of K215M mutant mice was significantly increased, and reached about twice the quantity of Tregs in WT mice ( Figure 1D ). The tTreg development interrupted by the CARD11 mutants also changed the mature Treg population in the secondary lymphoid tissue. The mature splenic Tregs in E134G mutant mice were dramatically reduced to almost none, compared to around 10% of total CD4⁺ T cells in WT spleens ( Figure 1E ). By contrast, the splenic Treg percentage in K215M mutant mice was increased to three times as many as that in WT spleens ( Figure 1F ). To better mimic the genetic conditions in patients whose CARD11 mutations occurred in one allele, we further evaluated the tTreg development in heterozygous Card11 mutant mice ( Figures 1G–J ; Supplementary Figures 1D-G ). The single allele E134G mutant still reduced the Treg populations in the thymus and spleen compared to WT mice, but the reduction is milder than that in the homozygous mutant mice ( Figures 1G, H ). The same pattern was observed in K215M heterozygous mutant mice, with mildly increased Treg in the thymus and spleen, compared to WT and homozygous mutant mice ( Figures 1I, J ). The same patterns of Treg absolute counts in the spleen and thymus of Card11 mutant and KO mice were observed ( Supplementary Figures 1H–M ). Finally, we compared the suppressive function of Tregs carrying distinct Card11 mutants. Since there are very few Tregs in E134G homozygous mutant mice, we used the Tregs from E134G heterozygous mutant mice in this in vitro suppression assay. Tregs purified from WT, heterozygous E134G, and homozygous K215M mutant mice had the same suppressive function on the proliferation of conventional WT CD4⁺ T cells ( Figure 1K ).

Our results demonstrated that the GOF CARD11 mutant, E134G, represents an overactivated NF-κB but still disrupts Treg generation in the thymus. Furthermore, another rare oncogenic mutant, K215M, inducing standard NF-κB activation, increases the Treg population. After comparative analyses of all those Card11 transgenic mouse models, we concluded that CARD11 regulates Treg development in an NF-κB independent manner.

Given the crucial role of CARD11 in TCR signal transduction, we next investigate whether the CARD11 mutation will affect CD4⁺ T cell early activation or differentiation. We purified naïve CD4⁺ T cells followed by anti-CD3/CD28 agonist antibody stimulations in vitro. After 12 hours of antibody incubation, the WT and CARD11 mutant CD4⁺ T cells were successfully activated. E134G and K215M mutant cells had the same level of CD25 and CD69 upregulation compared to WT cells ( Figure 2A ). Next, we investigated the proliferation of CD4⁺ T cells with various genetic backgrounds. WT CD4⁺ T cells show apparent cell division after 48 hours, and the same level of proliferation was observed in E134G and K215M mutant CD4⁺ T cells ( Figure 2B ). A cell cycle assay by staining proliferative cells with DNA dye, Dapi, suggested that E134G mutant CD4⁺ T cells have equal quantities of cells at the G1, S, and G2 phases compared to WT cells. K215M mutant CD4⁺ T cells also had equivalent populations at the G1 and G2 phases but showed fewer cells at the S phase than WT cells ( Figures 2C–E ). We compared the cell viability by staining activated CD4⁺ T cells with Annexin-V and Cell-Live dye. Both CARD11 mutants showed no impact on cell death compared to WT CARD11 ( Figures 2F, G ).

After activation, CD4⁺ T cells differentiated into different helper T cells under distinct immune microenvironments. We evaluated the differentiation potential of WT and CARD11 mutant CD4⁺ T cells under distinctively skewed conditions toward Th1, Th2, Th17, and iTreg, respectively. E134G mutant cells show defective Th1 differentiation ( Figure 2H ). K215M mutant cells have enhanced Th1 differentiation potential ( Figure 2I ). For the Th2 cells, both the E134G and K215M mutants increased their differentiation, and the K215M mutant had the best Th2-induction potential ( Figures 2J, K ). The E134G mutant also downregulated Th17 differentiation, but the level was minimal ( Figure 2L ). The K215M mutant does not affect Th17 cell differentiation compared to the WT control ( Figure 2M ). Finally, iTreg differentiation did not change in E134G mutant CD4⁺ T cells, but K215M mutant cells show reinforced iTreg differentiation ( Figures 2N, O ).

In summary, CARD11 mutants have no impact on CD4⁺ T cell activation, proliferation, or apoptosis but partially change CD4⁺ T helper cell differentiation potential.

Previous studies demonstrated that CARD11 mutations impact B cell development and differentiation through the AKT/FOXO1 signal axis rather than the NF-κB pathway (25). Therefore, we wonder whether the same regulatory mechanism impacts T cells, especially tTreg development. We purified naïve CD4⁺ T cells from WT and Card11 mutant mice, followed by in vitro stimulation with anti-CD3/CD28 antibodies. Then, we evaluated the downstream TCR signal transduction by measuring the phosphorylation of critical signal proteins at indicated time points. Phosphorylated FOXO1 was detected in WT CD4⁺ T cells after 15 minutes of TCR stimulation. The E134G mutant cells showed increased AKT activation, reflected by stronger phosphorylation at T308 and S473 amino acids. Reinforced AKT activation further exacerbates FOXO1 phosphorylation, which will destabilize FOXO1 protein and interrupt downstream target gene expression ( Figure 3A ). By contrast, the K215M mutant represented an opposite influence on signal transduction, in which phosphorylation of AKT and FOXO1 after TCR stimulation was alleviated ( Figure 3B ).

To confirm that CARD11 mutants changed the AKT/FOXO1 signal pathway in vivo, we collected the CD4⁺ SP T cells from the thymus and directly measured the AKT phosphorylation by flow cytometry. We found CD4⁺ SP thymocytes in E134G mutant mice showed elevated phosphorylation levels of AKT ( Figure 3C ). On the contrary, the K215M mutant CD4⁺ SP thymocytes had reduced AKT and FOXO1 phosphorylation ( Figure 3D ). These data demonstrate that the E134G and K215M mutants have the opposite impact on the AKT signal axis in CD4⁺ T cells.

Finally, we tested whether the aberrant AKT signal pathway in E134G mutant CD4⁺ T cells was responsible for the defective tTreg development. As the critical downstream target of the activated AKT signal pathway, FOXO1 is phosphorylated and degraded by the proteasome (26). We applied bone marrow (BM) chimeric mouse models to overexpress exogenous FOXO1 in E134G-mutated hematopoietic stem cells by retroviral transduction. After 8 weeks of reconstitution, we collected the thymus from chimeric mice and evaluated the tTreg development. Extra FOXO1 did rescue the tTreg development defects in E134G mutant cells ( Figure 3E ). These findings support the idea that CARD11 mutants regulate the development of tTreg independently of NF-κB.

Although transgenic mice with BENTA-associated CARD11 mutations show tTreg development defects, whether the BENTA patients represent the same phenotype is unclear. Therefore, we set up a small PID cohort to recruit patients carrying CARD11 mutations and verify the functional effects of those mutations on the Treg lineage.

According to the clinical and lab investigations, we finally confirmed 5 PID patients whose symptoms and genetic modifications were similar to those previously reported in CARD11 mutant patients ( Figure 4A ; Table 1 ). P1 in this cohort had a CARD11 heterozygous mutation at G126D located in the Latch domain (27), where most BENTA-associated CARD11 mutants were initially discovered. The G126D mutant was reported in one BENTA patient published before (28) and induced an autonomously activated NF-κB pathway (27). Mutations in the other four patients are at the C-terminal of the CARD11 protein. The P2 (Q679L) and P3 (S694L) mutations are in the PDZ domain. P4 (R848C) and P5 (R1104Q) mutants are in the SH3-GUK domain. Although all five patients had an elevated percentage of transitional B cells, a type of immature B cells ( Figure 4B ; Table 2 ), only P1 represents the B cell lymphocytosis phenotype that is frequently observed in BENTA patients ( Table 2 ). The other four patients have normal quantities of B cells in their peripheral blood ( Table 2 ). Patients have an average naïve T cell population in their PBMC, indicating normal conventional T cell development. All the other immune cell types are in the normal range ( Table 2 ). After PMA treatment to mimic the antigen stimulations, CD4⁺ T cells from one healthy control and patient P4 showed upregulated AKT S473 phosphorylation, and cells from P4 showed higher AKT activation ( Figure 4C ). It recapitulates the hyper-activated AKT signaling in CD4⁺ T cells from E134G mutant mice. Then, we collected the peripheral blood from 3 patients (P1, P2, and P4) and evaluated their Treg population by flow cytometry. Healthy control samples always showed that 5-10% of the total CD4⁺ T cells are FoxP3⁺ CD25⁺ Tregs. However, all 3 patients have less Tregs in their peripheral blood ( Figure 4D ; Table 2 ). These results suggest that patients with pathogenic CARD11 mutations also have defective Treg populations, as in mouse models. We also evaluated AKT T308 phosphorylation and NF-κB activation by transfecting cells with patient-derived CARD11 mutant plasmids. In contrast to WT, overactivated phospho-AKT (T308) was observed in human T cells transfected with CARD11 mutant plasmids ( Figures 4E, F ). However, there was no significant difference in CARD11 mutants (P2-P5) induced NF-κB activation in a GFP-based reporter assay compared to WT CARD11 ( Figure 4G ).

Tregs play an essential role in preventing autoimmune pathology in vivo. Since CARD11 mutants in patients cause Treg development defects, we wonder whether autoimmune syndromes are present. P1 with the G126D mutation is a typical BENTA patient. Like other reported BENTA patients, P1 did not show general autoimmunity but had hemophagocytic syndrome, which is also associated with an overactivated immune system ( Table 2 ). Patients P2, P3, and P4 represented atopic syndrome as observed in CADINS patients with LOF CARD11 mutations (22, 23). P2 showed itching of the eyes, edema of the eyelids, and rubbed eyes repeatedly exposed to pollen, which developed into atopic dermatitis (recurrent itchy skin of the scalp, limbs, and trunk). P3 had recurrent coughing and sneezing. The symptoms were significantly relieved after treatment with glucocorticoids and H1 receptor antagonists. Repeated sneezing, a runny nose, and thickening of the nasal mucosa characterized P4. The symptoms were relieved after treatment with glucocorticoids and leukotriene antagonists ( Table 2 ). We re-checked our mouse models since BENTA patients did not show general autoimmune syndromes. We found that the CARD11 E134G mutant did cause severe inflammatory dermatitis, reflected by severe lymphocyte infiltration and thickened dermal epidermis when those mice reached six months of age. However, no such phenotype was observed in colon and lung tissues ( Figure 4H ). It suggests that GOF CARD11 mutants also cause abnormal dermatological phenotypes, as do LOF CADINS mutants. Those data indicate that the compromised Treg generation may be the reason for atopy. However, only the skin-specific autoimmune responses indicated that the dermal immune microenvironment is the most sensitive to the tTreg decrease.

In conclusion, CARD11 regulates tTreg development through its NF-κB-independent function. These new findings extend our knowledge about the critical role of CARD11 in immune cell lineage determination and functional differentiation.