Clinical and laboratory data of patients and peripheral blood samples of APDS patients were obtained from Children’s Hospital of Fudan University (P1-P17) and University of South Florida (USF) and Johns Hopkins All Children’s Hospital (JHACH) at St. Petersburg of Florida (P18-P24). Patients 1, 5, 7, 13 and 16 were analyzed for B cell subset distribution and B cell survival, activation and differentiation ( Figures 1 – 5 and Supplemental Figure 1 ). P18-P24 were analyzed for the effects of the targeted therapy on B cell subset changes and serum IgM levels ( Figure 6 and Supplemental Tables 1 and 2 ). Approval for this study was obtained from the human research ethics committee of Children’s Hospital of Fudan University (2019–048) and USF/JHACH [USF-Pro00025693 (PI: J.W.), JHMI-IRB00175372 (PI: J.W.)]. All participants of this research have signed an informed consent.

Single-cell suspensions from peripheral blood were incubated with an Fc block™ (BD Biosciences) first to block Fcγ receptor and then stained with the following Abs: APC-H7-anti-CD3 (clone SK7, BD Biosciences), FITC-anti-CD4 (clone RPA-T4, BD Bioscience), APC-anti-CD8a (clone RPA-T8, Biolegend), anti-CD45RA (clone HI100, BD Bioscience), anti-TCR αβ (clone T10B9.1A-31, BD Bioscience), anti-TCRγδ (clone B1, BD Bioscience), APC-anti-CD19 (clone HIB19, Biolegend), FITC-anti-CD20 (clone 2H7, Biolegend), APC/Cy7-anti-CD27 (clone O323, Biolegend), PE-anti-IgD (clone IA6-2, BD Biosciences), FITC-anti-IgM (clone MHM-88, Biolegend), PE/Cy7-anti-IgG (clone G18-145, BD Biosciences), PE-anti-CD43 (clone CD43-10G7, Biolegend), PE/Cy7-anti-CD38 (clone HIT2, Biolegend), CD27-V450 (clone M-T271, BD Biosciences), IgD-BV510 (clone IA6-2, BD Biosciences), FITC-anti-CD80 (clone L307.4, BD Biosciences), PE-anti-CD86 (clone IT2.2, BD Biosciences), PE-anti-HLA-DR (clone G46-6, BD Biosciences), PE-Cy7-anti-CD69 (clone FN50, BD Bioscience) and PE-anti-CD40L (clone 24-31, Biolegend). The proportion of CD3⁺CD4⁺ (CD4⁺ T), CD3⁺CD4⁺CD45RA⁺CD27⁺ (naïve CD4⁺ T), CD3⁺CD4⁺CD45RA^-CD27⁺ (Central memory CD4⁺ T), CD3⁺CD4⁺CD45RA^-CD27^- (Effector memory CD4⁺ T), CD3⁺CD8⁺ (CD8⁺T), CD3⁺CD8⁺CD45RA⁺CD27⁺ (naïve CD8⁺ T), CD3⁺CD8⁺CD45RA^-CD27⁺ (Central memory CD8⁺ T), CD3⁺CD8⁺CD45RA^-CD27^- (Effector memory CD8⁺ T), CD3⁺CD8⁺CD45RA⁺CD27^- (Terminal effector memory CD8⁺ T), CD3⁺CD4^-CD8^- (double negative T, DNT), CD19⁺ (B), CD19⁺IgD⁺CD27^- (naïve B), CD19⁺IgD⁺CD27⁺ (MZB), CD19⁺IgD^-CD27⁺ (memory B), IgG⁺ switched memory B, IgM⁺ non-switched memory B, CD19⁺IgD^-CD27^- (DNB), IgG⁺ DNB, IgM⁺ DNB, CD19⁺CD20⁺CD38^hiCD27^hi (pre-PB), CD19⁺CD20⁺CD27⁺CD43⁺ (B-1) B and CD20^-CD38^hi (PB) cells was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) using FlowJo software.

Naïve B cells were isolated from freshly isolated peripheral blood using MojoSort Human Naïve B Cell Isolation Kit (Biolegend) together with biotinylated anti-human IgG beads to remove IgG⁺ cells. Purified naïve B cells were cultured in RPMI 1640 containing 5×10⁻⁵ M 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin, supplemented with 10% heat-inactivated fetal bovine serum (all from Thermo Fisher Scientific). F(ab’)₂ anti-human IgM Ab (α-IgM, 2.5 μg/ml; Southern Biotech), CpG ODN 2006 (CpG, 0.6 μM; Sangon Biotech), recombinant human IL-2 (IL-2, 10 ng/ml; R&D), IL-4 (20 ng/ml; R&D) and SA-CD40L (0.2 μg/ml) were freshly added. The SA-CD40L is a tetrameric form of human CD40L generated in house (16). Human naïve B cells were seeded at 1.5×10⁵ cells/ml density in 96-well plates in a final volume of 0.2 ml. All the cells were cultured at 37°C in humidified air and 5% CO₂. Naïve B cells were cultured for two days for survival and activation assays, and six days for CSR and antibody secretion assays. For the six-day culture, half of the medium was removed on day 3 and replaced with fresh medium supplemented with SA-CD40L, α-IgM, CpG, IL-2 or IL-4.

B cell viability was determined by 7-amino-actinomycin D (7-AAD, Biolegend) staining followed by flow cytometry analysis. B cell activation was determined by their expression of costimulatory molecules including CD80, CD86, HLA-DR and CD69, and by analyzing their forward scatter (FSC), which represents cell sizes. Ig gene CSR was assessed by determining the frequency of IgG⁺ cells. Differentiation of naïve B cells to PB was assessed by their acquisition of the CD38^hiCD20^- phenotype.

FACS-sorted naive B and DN B cells were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and 5×10⁻⁵ M 2-mercaptoethanol and supplied with TLR7 agonist R848 (1 μg/mL, Invivogen), BAFF (10 ng/mL, R&D Systems, Bio-Techne), IL-21 (10 ng/mL, R&D Systems, Bio-Techne), IL-2 (50 ng/mL, R&D Systems, Bio-Techne), IFN-γ (20 ng/mL, Peprotech) and goat F(ab′)₂ anti-human IgM (10 μg/mL, SouthernBiotech). Four days later, the supernatants were collected and the quantity of IgM and IgG Abs was analyzed by ELISA as described (17).

IgM and IgG levels in the culture supernatants were measured by standard ELISA as described (17). Briefly, 96-well ELISA plates (Costar) were coated with goat anti-human Ig (Cat#2010-01, Southern Biotech), blocked with 1% BSA in blocking buffer and incubated with serially diluted culture supernatants and standards. After washing, the biotin-conjugated anti-human IgG (Cat#2040-08, SouthernBiotech) or anti-human IgM biotin (Cat#2020-08, Southern Biotech) was added, followed by Avidin-HRP (Lot: B152022, Biolegend). The HRP-activity was visualized with TMB substrate reagents (Invitrogen) and OD₄₅₀-OD₅₇₀ was measured by spectrophotometer (Bio-Rad). The amount of Ig was calculated according to the standard curve.

Data obtained from flow cytometry were analyzed with FlowJo 7.6 Software (Treestar, Woodburn, OR), and exported to Excel spreadsheets. Results are presented as mean ± standard deviation (SD). Data were analyzed using t-test or linear regression test (P < 0.05 was considered statistically significant) by GraphPad Prism program (GraphPad Software, San Diego, CA, USA). *P < 0.05; **P < 0.01; ***P < 0.001.

A total of 24 APDS patients were studied ( Table 1 ). Twenty-one patients carried the hot spot E1021K (c.3061G>A) mutation, and three patients, P13, P7 and P23, carried the previously described E1025G (c.3074A>G), Y524N (c.1570T>A) and E81K (c.241G>A) mutations, respectively. All of these missense mutations were described as GOF in previous studies (10, 18, 19). B cell counts were decreased in 20 patients (83%) whereas serum IgM levels were higher than the upper limit of the normal range in 20 patients (87%) ( Table 1 ). We have recently established the reference ranges of extended T/B cell subsets in Chinese children based on age and gender (20). In 21 out of 24 patients, fresh peripheral blood was available for lymphocyte subpopulation typing ( Table 2 ). Reduced proportions of naïve CD4⁺ T cells and naïve CD8⁺ T cells were observed in most of the patients. More than half of the patients showed relatively increased proportions of CD4⁺ (n=15, 75%) and CD8⁺ (n=16, 80%) effector memory T cells. The frequency of memory B cells was decreased in 9 of 24 (38%) patients. The proportions of transitional B cells were increased in 19 of 21 (90%) patients and of PB cells were increased in 12 of 19 (63%) whereas the proportions of naive B cells were decreased in 11 of 21 (52%) patients ( Table 2 ).

PBMC from 5 patients with 3 different PIK3CD mutations (patients 1, 5 and 16 with E1021K; patient 13 with E1025G; patient 7 with Y524N) and 4 HC were further analyzed for the proportions of different B cell subsets ( Figure 1 ). Four patients (except patient 7) had received rapamycin treatment at the time of analysis. Figure 1A shows the gating strategies. No differences were observed in the proportions of naïve B (CD19⁺IgD⁺CD27^-), MZB (CD19⁺IgD⁺CD27⁺) and B-1 B (CD20⁺CD27⁺CD43⁺) cells between the patients and HC ( Figure 1B ). The proportions of class-switched (IgG⁺) cells were significantly reduced in the patients (5.20 ± 5.63% vs. 15.30 ± 1.25% in HC) ( Figure 1C , left panel). While the frequencies of total memory B (CD19⁺IgD^-CD27⁺) were comparable between the patients and HC ( Figure 1C , 2nd panel), the frequencies of IgG⁺ memory B cells (IgG⁺CD19⁺IgD^-CD27⁺) were significantly lower in patients (4.40% ± 3.42%) compared with HC (42.60 ± 7.47%) ( Figure 1C , 3rd panel), which is consistent with the decreased IgG⁺ cells ( Figure 1C , left panel) and previous studies (21, 22). We found significantly higher frequencies of the IgM⁺ memory B cells (IgM⁺CD19⁺IgD^-CD27⁺) in the patients (36.78% ± 18.90%) compared with HC (10.49 ± 9.44%) ( Figure 1C , right panel), which had not been described previously. In agreement with a previous study (21) was the finding that the proportions of pre-PB (CD20⁺CD38^hiCD27^hi) and PB (CD20^-CD38^hi) were increased ( Figure 1D ) although the differences did not reach statistical significance. We also noted an increased proportion of the CD27^-IgD^- double negative B (DNB) cells in the patients (20.92 ± 10.65% vs. 7.54 ± 2.45% in HC, P <.05) ( Figure 1D , right).

A previous study showed that B cells in APDS patients were in an activated state, as suggested by their elevated pAKT levels relative to B cells from HC (21). Since B cell activation is frequently associated with increased cell size, we next analyzed the sizes of naïve B and DNB cells ( Figure 2A ). Indeed, we found that naïve B cells of the patients had significantly larger cell sizes, as revealed by their increased forward scatter (FSC) and higher percentages of the FSC^high population than B cells from HC ( Figure 2B , left and middle panels). Naïve B cells in the patients contained higher proportions of 7AAD⁺ dead cells than those in HC ( Figure 2B , right panel), although the difference did not reach statistical significance. Moreover, DNB from the patients had much larger cell sizes than those from HC (P <.001) ( Figure 2C , left panel). The majority of the DNB cells from the patients were IgM⁺, which was in striking contrast to those from HC that were mostly IgG⁺ ( Figure 2C ). Such DNB cells have recently been shown to be derived from activated naïve B cells and are precursors of PC (23).

To analyze intrinsic defects in B cell function, we purified naïve B cells and analyzed their survival and activation under various in vitro culture conditions. We used MojoSort B cell purification kit and also included biotinylated anti-human IgG-conjugated beads to remove IgG⁺ cells. The purified B cells were mostly IgD⁺ as shown in Supplemental Figure 1 . While freshly isolated PBMC from the patients contained an increased proportion of dead naïve B cells than those from HC ( Figure 2B , right panel), purified naïve B cells from the patients and HC had similarly high viability ( Figure 3B , day 0), possibly because the dead cells had been removed during the purification processes. We cultured purified naïve B cells for 2 days in medium alone or in the presence of α-IgM (BCR signal), CpG2006 (TLR9 signal), α-IgM + CpG + IL-2 (T cell-independent stimuli, TI), or SA-CD40L plus IL-4 (T cell-dependent stimuli, TD). As shown in Figure 3B , naïve B cells from the patients had significantly lower viability than those from HC when cultured in the presence of α-IgM Abs (48.37 ± 5.94% vs. 74.08 ± 9.98% in HC). Live cell counts were decreased in parallel but without statistically significant difference ( Figure 3C ). These observations suggest that B cells from the patients had an intrinsic defect in antigen-triggered survival. Although naïve B cells from the patients had larger sizes than those from HC ( Figure 2B ), they expressed similar levels of CD80, CD86, HLA-DR and CD69 as naïve B cells from HC ( Figure 3D ). Patients’ B cells cultured in medium alone had elevated levels of the costimulatory molecule CD80 and the early activation marker CD69 than did HC B cells ( Figure 3E ). After the stimulation, they expressed higher levels of CD86 but downmodulated CD69 expression ( Figures 3G – I ). These observations suggest that the patients’ B cells exhibit enhanced activation.

To analyze abnormalities in B cell differentiation into PC, we next cultured purified naïve B cells for 6 days in medium alone or in the presence of various stimuli. In medium alone, B cells from both the patients and HC died during the 6 day-culture ( Figures 4A, B ). In the presence of α-IgM, the majority of the patients’ B cells died while ~30% of the HC B cells were still alive ( Figures 4A, B ), consistent with the results of the 2-day culture ( Figure 3B ). When cultured with CpG alone or TI stimuli, patients’ B cells also showed significantly reduced viability ( Figure 4A ) and a dramatic decrease in the live cell numbers ( Figure 4B ). The viability and number of live B cells were not different between the patients and HC after 6 day-culture with TD stimuli ( Figures 4A, B ). TD stimuli are known to induce CSR. As shown in Figures 4C, D , the frequency of IgG⁺ cells among CD19⁺ B cells in the patients was greatly reduced compared to HC (1.69 ± 0.52% in the patients vs. 8.80 ± 2.24% in HC). These results are in agreement with a recent study (22) showing that B cells from APDS patients failed to generate IgG⁺ PB in response to CD40L + IL-21 and indicated that elevated activation of PI3K pathway inhibited Ig gene CSR in B cells.

PB are precursors of PC with shorter life span and are mainly produced by T cell-independent antigen stimulation in the blood circulation (24). Stimulation of B cells with TLR9 ligand CpG + IL-2 was reported to induce PC generation in human B cells (25, 26). We then cultured purified naïve B cells with CpG + α-IgM + IL-2 (TI stimuli) for 6 days ( Figure 4E ), which induced efficient PB differentiation accompanied by extensive B cell expansion ( Figure 4G ). We found that B cells from HC and the patients generated a similar proportion of the CD20^lowCD38^high PB ( Figure 4F ), suggesting that PIK3CD GOF did not affect naïve B cell differentiation into PB induced by TI stimuli. However, because the cell viability and the live cell counts were both reduced in the patients compared with HC ( Figures 4A, B ), the numbers of PB were lower than those generated by HC B cells ( Figure 4G ). Consistently, PB from the patients secreted lower amounts of IgG ( Figure 4H ) and IgM ( Figure 4I ) than those from HC, although the former did not reach statistical significance. We further calculated antibody secretion on a per PB basis by dividing the amount of IgG and IgM in the culture supernatants by the total number of PB shown in Figure 4G . We found that PB from HC and the patients secreted comparable amounts of antibodies ( Figures 4J, K ). Stimulation with CD40L + IL-4 (TD stimuli) was not as efficient as TI stimuli in inducing PB differentiation ( Figures 4E – G ) and B cells from HC and the patients generated similar percentages ( Figure 4F ) and numbers ( Figure 4G ) of PB. Collectively, while the patients’ B cells are impaired in CSR, they can differentiate into PB and secrete comparable levels of antibodies as HC B cells.

To explore the pathogenicity of the DNB in the patients, we sorted naïve B ( Figure 5A ) and DNB ( Figure 5B ) cells from a HC and a patient (P13) and cultured them under mixed stimuli (TLR7 agonist R848 + BAFF + IL-21 + IL-2 + IFN-γ + F(ab′)₂ anti-human IgM) for 4 days. We found that while DNB cells from the HC and the patient secreted comparable amounts of IgG ( Figure 5C , right), only the patient’s DNB cells secreted detectable levels of IgM ( Figure 5C , left). In contrast, naïve B cells from both the HC and the patient produced neither IgM nor IgG ( Figure 5 ). These results are consistent with the higher frequency of IgM⁺ DNB cells in the patients than in the HC ( Figure 2B , right panel). Moreover, we found that the serum IgM levels positively correlated with the percentages of the DNB cells in 13 APDS patients who were analyzed for the DNB population ( Figure 5D ).

Rapamycin and leniolisib are used as targeted therapeutic drugs for APDS patients. We then compared B cell phenotypes in three patients (P19, P22, P23) who had not received the treatment and five patients (P18, P20-P22, P24) who had received the treatment. Among these, one patient (P22) was analyzed before and after the treatment. Compared with HC, the untreated patients had significantly increased proportions of transitional B and IgM⁺ DNB and decreased proportion of IgG⁺ DNB cells, which were partially normalized in the patients with targeted therapy ( Figure 6 ), Although statistically not significant, the increased percentage of PB and elevated levels of serum IgM were also partially ameliorated ( Figure 6 ). As shown in Supplemental Table 1 , P22 had improved AIHA with Coombs test turning negative after the treatment. Consistently, the targeted therapy partially normalized B cell phenotypes in P22, as revealed by the decreased proportions of transitional B, IgM⁺ memory B and IgM⁺ DNB populations and the increased proportions of IgG⁺ memory B and IgG⁺ DNB cells ( Supplemental Table 2 ).