Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS–independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.
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We thank S. Kapnick for discussions and protocol suggestions; K. Mao for help with gut ‘Swiss roll’ preparation; S. Wincovitch for help with bright-field microscopy; S. Anderson and M. Kirby for cell sorting; L. Perez for sharing protocols; M. Yan for help with autoantibody arrays; F. Sallusto (Institute for Research in Biomedicine, Bellinzona, CH) for reagents; and S. Crotty for helpful discussions. Supported by the US National Institutes of Health (funds from intramural programs of the National Human Genome Research Institute and National Institute of Allergy and Infectious Diseases; and R01 AI102888-01A1 to M.O.L.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a, Schematic representation of Pik3cdE1020K/+ generation. After crossing to ZP3-Cre mice to remove the neoR cassette, the point mutation and one LoxP site in the upstream intron remain. (b, d-h) Wild-type and Pik3cdE1020K/+ mice were analyzed at steady state between 2 and 4 months of age. b, Cellularity of resting lymph node (n=5 per group). c, Representative contour plots of CD44 and CD62L on CD4+ T cells in the spleen of 1-year-old mice. d, Numbers of CD93+CD138— transitional B cells in the spleen (percent of B220+CD19+); representative flow staining and histograms of percentages and numbers of T1, T2, T3 and IgM— CD23— B cells (gated on B220+CD19+CD93+CD138—) (wild-type n=3, Pik3cdE1020K/+ n=4). e, Representative FACS plots of surface IgM and IgD (gated on B220+CD19+ B cells in the spleen) and relative histograms (wild-type n=8 Pik3cdE1020K/+ n=7). f, Illustrative contour plots for CD21 and CD23 to identify MZ, FO, and CD21—CD23— B cells in the spleen (percent of B220+CD19+). Bar graph on the right (wild-type n=6, Pik3cdE1020K/+ n=5). (g-h) Analysis of peritoneal cavity (n=3 per group). g, Cellularity of peritoneal lavage; representative flow staining and cell counts of CD3+ T and CD19+ B cells. h, Representative contour plots and cell counts of B1 (CD23—) and B2 (CD23+) B cells gated on CD19+ cells as in (g); on the right, representative histograms and numbers of B1a (CD5+) and B1b (CD5—) gated on B1 B cells. Data in (b-f) are representative of three, and data in (g,h) of two, independent experiments. Data are represented as mean ± SEM with each dot indicating one mouse. Significance analyzed by Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001
Supplementary Figure 2 Baseline immune response of wild-type and Pik3cdE1020K/+ mice in lymph node and spleen.
a, Representative flow plots and histogram of BCL-6 and CXCR5 staining for GC-TFH, pre-TFH and non-TFH cells (gated on CD4+B220— T cells in the spleen) (n=6 per group). b, Frequency of PD-1+CXCR5+Foxp3— TFH cells (percent of CD4+B220— T cells) in the popliteal lymph node of 2 and 4-month-old mice at steady state. c, Representative flow plots and cell counts of CD4+ follicular T cells: CXCR5+PD-1+ (left panel) and CXCR5+ICOS+ (right panel) in 1-year-old mice in the spleen. d, Frequency of GL-7+FAS+ GC B cells (percent of B220+CD19+ B cells) in the popliteal lymph node of 2 and 4-month-old mice at steady state (b,d, 2 month-old, n=5 per group; 4 month-old, wild-type n=8, Pik3cdE1020K/+ n=7). e, Representative flow plots and cell counts of GL-7+FAS+ GC B cells (percent of B220+CD19+ B cells, left panel), IgD+ B cells (percent of B220+CD19+ B cells, right panel), and CD138+B220int/lo plasma cells (of live cells, lower panel) in 1-year-old mice in the spleen (c, e, wild-type n=3, Pik3cdE1020K/+ n=5). Data are representative of two independent experiments. Data are represented as mean ± SEM. Significance analyzed by Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001
Wild-type and Pik3cdE1020K/+ mice were immunized subcutaneously, at different age (2-months, 4-months, 1-year-old), with NP-OVA in alum in the hock and sacrificed on day+10. Analyses performed on draining (dLN) and resting (rLN) popliteal lymph nodes. a, Analyses of 2-month-old mice: frequency of PD-1+CXCR5+Foxp3— TFH cells (percent of CD4+B220— T cells, left panel) and GL-7+FAS+ GC B cells (of B220+CD19+ B cells, right panel) in rLN and dLN (n=5 per group). (b-c) Analyses of 4-month-old mice. b, cellularity of rLN and dLN (n=6 per group). c, Numbers of NP— and NP+ GC B cells in dLN (wild-type n=6, Pik3cdE1020K/+ n=5). d, Analyses of 1-year-old mice: percentage and numbers of NP+ GC B cells in dLN (wild-type n=7, Pik3cdE1020K/+ n=9). e, Analyses of 2-month-old mice: percentage of NP+ GC B cells and ratios between numbers of NP+ and NP— GC B cells in dLN (n=5 per group). f, Analyses of 4-month-old mice: ELISA for serum IgG1 (wild-type n=8, Pik3cdE1020K/+ n=6). (g-m) Analyses based on Figure 3g. g, Proportion of FDC networks with a GC having greater than 10 BCL-6+ cells (wild-type rLN, n=3; wild-type dLN, n=2; Pik3cdE1020K/+ rLN and dLN n=3). Each symbol represents an entire LN section corresponding to 5-8 FDC networks. h, Area of BCL-6+ GCs per FDC region. Each symbol corresponds to a GC (wild-type rLN, n=3; wild-type dLN, n=5; Pik3cdE1020K/+ rLN, n=13; Pik3cdE1020K/+ dLN n=6). i, Histo-cytometric representation of GCs from wild-type dLN depicted in Figure 3g, II. The distribution of PD-1+CD4+ T cells within the LZ and DZ of the GC was determined using the density of CD35 and BCL-6 to mark the LZ and DZ, respectively. j, Percentage of TFH cells (PD-1+CD4+ surfaces) in the DZ as determined by histo-cytometry. k, Normalized numbers of TFH cells (identified as in j) per DZ GC area (BCL-6+CD35—). Each symbol corresponds to a GC (j,k, wild-type dLN, n=4; Pik3cdE1020K/+ rLN, n=12; Pik3cdE1020K/+ dLN n=6). IF analyses are representative of 2-3 independent lymph nodes analyzed. l, Representative FACS plots and histograms of DZ (CXCR4hiCD86lo) and LZ (CXCR4loCD86hi) gated on NP+ GC B cells (wild-type n=8, Pik3cdE1020K/+ n=7). m, Percentage (of live cells) and numbers of CD4+B220—CXCR5—PD1—Foxp3+ Treg cells, and CD4+B220—PD-1+CXCR5+Foxp3+ TFR cells in rLN and dLN (wild-type n=8, Pik3cdE1020K/+ n=7). Data in (a-f, l, m) are representative of two independent experiments. Shown is the mean ± SEM. Significance analyzed by Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001
(a-b, c, j) Experimental setting described in Figure 4a. a, Representative CD44 histogram on wild-type and Pik3cdE1020K/+ OT-II cells and endogenous CD4+ T cells. b, Representative flow plots and histograms of GC-TFH, pre-TFH and non-TFH cells (gated on transferred OT-II cells in the spleen) (wild-type n=7, Pik3cdE1020K/+ n=6). c, Intracellular staining for IL-21 after in vitro re-stimulation with PMA/Ionomycin on FACS sorted wild-type or Pik3cdE1020K/+ OT-II non-TFH cells (PD1—CXCR5—CD4+B220—) and TFH cells (PD1+ CXCR5+CD4+B220—), isolated on day +7 post immunization, as described in Fig. 4a (pool of wild-type OT-II (n=7) and Pik3cdE1020K/+ OT-II cells (n=6)). d, Analysis of IgG supernatant on in vitro culture between wild-type B cells and wild-type or Pik3cdE1020K/+ polyclonal TFH cells stimulated with anti-IgM and anti-CD3 for 7 days (pool of 4-5 mice per group). e, Frequency of endogenous PD-1+CXCR5+ of CD4+ B220— T cells in wild-type mice adoptively transferred with wild-type or Pik3cdE1020K/+ OT-II cells and treated with isotype control (wild-type OT-II n=5, Pik3cdE1020K/+ OT-II n=4), or anti-ICOS-L (wild-type OT-II n=4, Pik3cdE1020K/+ OT-II n=5) (experiment described in Fig. 4d). f, Experiment outline of (g, h). Naïve wild-type and Pik3cdE1020K/+ mice received isotype control (wild-type n=5, Pik3cdE1020K/+ n=5), or anti-ICOS-L (wild-type n=6, Pik3cdE1020K/+ n=5) on day 0 (i.v.), +2, +4, +6 (i.p.) and were sacrificed on day+7 post treatment. g, Representative contour plots and histogram of endogenous PD-1+CXCR5+ T cells (of CD4+B220—). h, Histogram of FAS+GL-7+ GC B cells (of B220+CD19+ B cells). i, Analysis of CD69 and CD25 on wild-type and Pik3cdE1020K/+ naïve OT-II cells activated in vitro with CD11c+ DC and OVA323-339 for 20 h (pool of 2-3 mice per group). j, Frequency of FAS+GL-7+ GC B cells (of B220+CD19+ B cells) related to Fig. 4a (wild-type n=7, Pik3cdE1020K/+ n=6). Data are representative of two independent experiments. Data are expressed as mean ± SEM with each dot indicating one mouse. Significance analyzed by Mann-Whitney U test. *P < 0.05; **P < 0.01
a, Related to Figure 5a. Frequency of wild-type OT-II and wild-type OT-II TFH cells (PD-1+CXCR5+CD4+B220—) in wild-type hosts together with wild-type (n=8) or mutant (n=7) MD4 B cells immunized i.p. with HEL-OVA 323-339 in alum. b, Schematic experimental layout for panels (c-f). Wild-type OT-II (CD45.1+) and wild-type (n=6) or Pik3cdE1020K/+ (n=8) polyclonal B cells (CD45.2+) were adoptively transferred into MD4 hosts (CD45.1/2+) and immunized i.p. with NP-OVA in alum. Analysis in the spleen on day+8. c, Frequency of transferred polyclonal CD45.2+B220+CD19+ B cells (of live cells). d, Numbers of transferred CD45.2+B220+CD19+GL-7+FAS+ GC B cells. e, Frequency of polyclonal CD138+B220int/lo plasma cells/blasts (of transferred CD45.2+ cells). f, Representative contour plots of frequency of NP+ GC B cells within transferred B cells, histogram of NP+ GC B cells, and ratio between numbers of NP+ and NP— GC B cells. g, Analysis of CD86 and CD69 on wild-type and Pik3cdE1020K/+ follicular (FO) MD4 B cells activated in vitro with HEL for 20 h (pool of 2-3 mice per group). h, Analysis of in vitro differentiated plasma cells (BLIMP-1-YFP+CD138+) generated from FO B cells stimulated with LPS and IL-4 or IL-21 for 3 days (gated on live B cells) (pool of 2-3 mice per group). i, Experimental outline of mixed BM chimeras between different ratios (20:80 n=6, 80:20 n=10, 50:50 n=2) of wild-type (CD45.1+) and Pik3cdE1020K/+ (CD45.2+) bone marrow cells, transferred into irradiated wild-type hosts (CD45.1/2+). j, Frequency of B220+CD19+GL-7+FAS+ GC B cells within wild-type and Pik3cdE1020K/+ cells, and live GC B cells (Annexin— viability dye—). k, FO B cells were kept in vitro without stimulation and analyzed for viability at different time points (wild-type open circles, mutant closed circles). l, m, FO B cells were activated in vitro for 3 days with the indicated stimuli and assessed for CTV dilution and viability. n, FO B cells were stimulated in vitro with LPS+IL-4, treated with or without CAL-101 (PI3Kδ inhibitor) and analyzed for CTV dilution and viability on day+3 (k-n, pool of 2-3 mice per group). Data are representative of 2 (a, b-g, i-k, n), and 3 (h, l, m) independent experiments. Data are expressed as mean ± SEM with each dot indicating one mouse. Significance analyzed by Mann-Whitney U test. **P < 0.01
(a-b) Wild-type and Pik3cdE1020K/+ sera were analyzed at 14-16 weeks of age. a, ELISA for serum ANA-IgM (n=5 per group). b, Serum IgM autoantibody array chip is shown; a colorimetric representation of relative autoantibody reactivity in each sample and for each self-antigen is shown based on the scale (0-3) depicted on top of the heat map. The sera of 3 wild-type and 11 Pik3cdE1020K/+ were analyzed, and sera from 2 lupus-prone MRL/NZM mice were used as a positive control. Data in (a) are representative of 2 independent experiments. Data in (b) have been obtained from one experiment examining sera from multiple litters. Data are expressed as mean ± SEM with each dot indicating one mouse. Significance analyzed by Mann-Whitney U test. *P < 0.05
Supplementary Figure 7 Disorganized GCs in Pik3cdE1020K/+ mLN, and microbiome composition of wild-type and Pik3cdE1020K/+ mice.
a, Confocal immunofluorescence images from the mLNs of naïve wild-type and Pik3cdE1020K/+ mice (scale bar 200 μm left panel; 30 μm enlargements). White dotted line denotes the boundary between the B cell follicle and T cell zone and is based on B220 staining (not shown). CD35 staining intensity was used to mark the light zones (LZ) and dark zones (DZ) of the GC (BCL-6+). Images are representative of mLNs from 2 mice per group from 2 separate experiments. b, Numbers of GCs per mLN (n=1 per group). c, Histo-cytometry was used to quantity the distribution of TFH cells in DZ as described in Supplementary Figure 3f. The percentage of cells within the DZ gate is shown. Each symbol refers to a GC. d, Normalized numbers of TFH cells (identified as in c) per DZ GC area (BCL-6+ CD35—) (c, d, wild-type n=9, Pik3cdE1020K/+ n=19) e, Bar plots of baseline microbiota profile by 16s rRNA sequencing in wild-type (n=8) and Pik3cdE1020K/+ (n=6). Relative family-level abundances of fecal samples prior to sorting based on IgA are shown. f, Principal Coordinates Analysis (PCoA) plot shown using the Canberra beta diversity metric, performed on baseline microbiota profiles (wild-type n=8; Pik3cdE1020K/+, n=6). Significance of clustering based on genotype (wild-type vs. Pik3cdE1020K/+ mice) was assessed using PERMANOVA (P=0.46). Data in (e, f) have been obtained from 3 independent experiments. Shown is the mean ± SEM. Differences between groups were compared with Mann-Whitney U test. *P < 0.05
Supplementary Figures 1–7
Baseline characterization of T and B cell populations in wild-type and Pik3cdE1020K/+ mice at 2 and 4 months of age
List of autoantibodies significantly increased in Pik3cdE1020K/+ compared to wild-type mice
List of taxa targeted by IgA in Pik3cdE1020K/+ versus wild-type mice
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Preite, S., Cannons, J.L., Radtke, A.J. et al. Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity. Nat Immunol 19, 986–1000 (2018). https://doi.org/10.1038/s41590-018-0182-3
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