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Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency

Abstract

The p110δ subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report fourteen patients from seven families who were heterozygous for three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ). These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and viremia due to cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV). Strikingly, they had a substantial deficiency in naive T cells but an over-representation of senescent effector T cells. In vitro, T cells from patients exhibited increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR, enhanced glucose uptake and terminal effector differentiation. Notably, treatment with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely 'rescued' the in vitro T cell defects and improved the clinical course.

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Figure 1: A previously unknown primary immunodeficiency characterized by the accumulation of T cells and B cells with lymphoid nodules and defective memory cell populations.
Figure 2: Heterozygosity for a gain-of-function mutation in PIK3CD in all affected patients results in hyperphosphorylation of Akt.
Figure 3: PBMC populations from patients include virus-specific CD8+ T cells but do not proliferate after stimulation of the TCR in vitro because of terminal differentiation and senescence.
Figure 4: T cells from patients exhibit normal TCR signaling responses but elevated mTOR activity and glucose uptake in vitro.
Figure 5: In vivo treatment with rapamycin improves disease phenotypes in vitro and in vivo.
Figure 6: Proposed model for the effects of activating substitutions (red asterisk) in p110δ.

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Protein Data Bank

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Acknowledgements

We thank the referring physicians, as well as the patients and families. Supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, Clinical Center of the US National Institutes of Health (C.L.L., H.S.K., J.E.N., M.B., J.S., W.O., V.K.R., A.A., A.A.H., K.N.O., T.A.F., S.P., S.M.H., J.I.C., M.J.L., G.U.), the National Human Genome Research Institute of the US National Institutes of Health (F.Z., J.L.C., P.L.S.), the Frederick National Laboratory for Cancer Research of the US National Institutes of Health (HHSN261200800001E), the National Health and Medical Research Council of Australia (E.K.D., U.P., S.G.T.), Cancer Council NSW (S.G.T.), the Cancer Institute NSW (U.P.), the Research Foundation-Flanders, Belgium (L.M.) and the National Institute of General Medical Sciences (C.L.L. and R.Z.). The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

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C.L.L did experiments, analyzed data and developed and wrote the manuscript; H.S.K., F.Z., E.K.D., U.P., D.T.A., L.M. and J.L.C. did experiments and analyzed data; J.E.N. analyzed genomic DNA sequencing and bioinformatics, discovered candidate genes and analyzed protein structure; M.B. analyzed p110 structure; J.S. did experiments and analyzed genomic DNA sequencing and data; W.O. did experiments; D.M.F. supervised research and data analysis; V.K.R. evaluated patients and collected data; T.P.A. and J.I.C. provided patient access, clinical data, samples and advice; A.A. evaluated patients and collected and analyzed data; A.A.H. coordinated patient access, data collection and analysis; L.R.F. evaluated and prepared data from clinical imaging studies; K.N.O. evaluated patients and collected and analyzed data; T.A.F. supervised research and data analysis and provided advice; S.P. did histological and immunohistochemical analyses of patient samples; S.M.H. supervised research and data analysis and provided advice; J.B.O planned and supervised whole-exome sequencing experiments; S.G.T. planned and supervised experiments, analyzed data, provided advice and prepared the manuscript; P.L.S. planned and supervised experiments, analyzed data and provided advice; M.J.L. supervised research and data analysis, provided advice and prepared the manuscript; G.U. coordinated research efforts, supervised research work and data analysis, and prepared the manuscript; and all authors discussed and revised the manuscript.

Corresponding author

Correspondence to Gulbu Uzel.

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Additional information

Note added in proof: Another study has now independently described a cohort of patients with the E1021K substitution of p110δ48.

Integrated supplementary information

Supplementary Figure 1 Lymphadenopathy, mitogen response, effector cells, and serum Ig assessment.

(a) Computerized Tomography (CT) images demonstrating lymphadenopathy (arrows) in patient F.II.1 and absence of spleen (removed surgically). (b) Three dimensional surface rendered image with cut plane at level of porta hepatis showing lymph node masses surrounding the portal vein and adjacent vasculature (darker red mass within white oval). (c) 3H-Thymidine incorporation in counts per minute (CPM) to assess proliferation of PBMCs after three days in response to the indicated stimulus. Each symbol represents an individual healthy control subject (Ctrl, n = 10) or patient (Pt, n = 10). Small horizontal lines indicate mean (± s.d.). *P < 0.0001, **P = 0.0002, ***P < 0.0001, ****P < 0.0001 (Mann-Whitney test). (d) Cumulative data for multiple healthy controls (Ctrl, n = 8) and patients (Pt, n = 9) showing % CCR7 negative cells among CD8+ T cells in the peripheral blood. Small horizontal lines indicate mean (± s.d.). *P < 0.0001 (unpaired t-test). (e) IgM (left) and IgA (right) serum concentrations as a function of the patient's age. Dotted lines indicate the upper and lower boundaries of the normal range from healthy subjects. Data are representative of ten independent experiments with one patient each (c), four independent experiments (d) or two to ten separate measurements over time for each of the nine indicated patients.

Supplementary Figure 2 Patient B cell defects.

(a) The proportion of total B cells (i.e., CD20+ cells) within the lymphocyte population of healthy controls (Ctrl, n = 6) compared to patients (Pt, n = 7). Small horizontal lines indicate mean (± s.d.). (b, c) Expression of (b) CD5 on transitional and naïve B cells and of (c) surface IgG and IgA on memory B cells from healthy controls (red histogram) and patients (blue histogram) was determined by flow cytometry. (d) CFSE-labeled naïve B cells were cultured with CD40L, CD40L+CpG, CD40L+anti-Ig or CD40L together with IL-4, IL-10, or IL-21 for 5 days before being assessed for dilution of the dye in healthy control (Ctrl, red) and patient (Pt, blue) samples. (e) Expression of AICDA mRNA was determined by quantitative PCR after stimulation of naïve B cells with the indicated stimulus. (f,g) Secretion of IgM, IgG, and IgA by stimulated naïve (f) and memory (g) B cells was determined by ELISA. Values for healthy controls (Ctrl) and patients (Pt) are given as mean ± sem of replicate cultures. *P = 0.03, **P = 0.005 (unpaired t-test). Data are representative of four independent experiments (b,c), two independent experiments (e), three independent experiments (f) or two independent experiments (g) or are from five independent experiments (a) or one experiment (d).

Supplementary Figure 3 Alignment of amino acid sequence of human p110α (PIK3CA) with that for human p110δ.

Residues mutated in our patients are shown in yellow, cyan and magenta.

Supplementary Figure 4 Hyperphosphorylation of Akt and intact association of mutant p110δ with p85α.

(a) Quantification of band intensities for the immunoblot shown in Fig. 2d in which serum-starved, activated T cells were assessed for p110δ, Akt, p-Akt (S473), and β-tubulin in patients D.II.1, D.II.2, and E.1 compared to three healthy controls (Ctrl) with 10 min anti-CD3 stimulation (+) or not (−). Total Akt values were normalized to β-tubulin abundance, then the ratio of p-Akt (S473) to normalized Akt was graphed. Small horizontal lines indicate mean (± s.d.). *P = 0.0002, **P = 0.01, ***P = 0.008 (unpaired t-test). (b) Summary of p-Akt (S473) by flow cytometry comparing mean fluorescence intensity (MFI). Basal p-Akt (S473) in paired (i.e., cultured for the same duration) T cell blasts from healthy controls (Ctrl, n = 5) and patients (Pt, n = 5) (left) or induced p-Akt (S473) after 10 min stimulation with anti-CD3 in T cell blasts from Ctrl (n = 3) and Pt (n = 3) (right, with mean (± s.d.)) are shown. *P = 0.02 (paired t-test), **P = 0.03 (unpaired t-test). (c) Immunoblots for the indicated proteins using PBMC lysates from patients with mutant p110δ or healthy controls (Ctrl) after stimulation with beads coated with anti-CD3 and anti-CD28 for 10 min (+) or not (?). (d,e) Representative histograms (left) and cumulative MFI data (right) for flow cytometry staining for p-Akt (T308) in patient (Pt, n = 3) versus healthy control (Ctrl, n = 3) T cell blasts, gating on CD4+ or CD8+ cells. Small horizontal lines indicate mean (± s.d.). *P = 0.03 (unpaired t-test). (f) Quantification of band intensities for the immunoblot shown in Fig. 2e together with data from two additional independent experiments. Numbers indicate the relative ratio (WT set to 1.0) of p-Akt (S473) to total Akt normalized for Flag expression. Small horizontal lines indicate mean (± s.d.). *P = 0.05, **P = 0.009 (unpaired t-test). (g) Immunoblot for p-Akt (S473) and β-tubulin on lysates from the human H9 T cell line overexpressing WT or the indicated mutant p110δ. (h) Immunoprecipitates (IP) of empty vector (EV), wild-type (WT), or mutant p110δ fused to a Flag epitope in HEK293T cells overexpressing p85α were blotted with antibodies against p85α, the Flag epitope, β-actin or immunoglobulin heavy chain (IgH). Data are from one experiment with three patients (a) or three independent experiments (f) or are representative of three independent experiments (b,d,e), four independent experiments with one patient each (c) or two independent experiments (g,h).

Supplementary Figure 5 Patient CD8+ T cells are hyperactivated with characteristics of enhanced effector function.

(a) Cumulative data for MHC class I-peptide tetramer (Tet) stains for EBV lytic (Ctrl n = 4 and Pt n = 4) and EBV latent antigens (Ctrl n = 3 and Pt n = 3). (b) Flow cytometry of gated EBV lytic and latent antigen-specific CD8+ T cells, stained for CCR7 and CD45RA. The percent of total events is shown in quadrants. (c) CD38 expression on the populations identified in (a) for patient G.1 (blue) compared to those for a healthy control (Ctrl, red). (d) Cumulative data for intracellular IFN-γ production in CD8+ T cell blasts from healthy controls (Ctrl, n = 3) versus patients (Pt, n = 4). Small horizontal lines indicate mean (± s.d.). P = 0.09 (unpaired t-test). (e) Cumulative data for granzyme B expression in CD4+ or CD8+ T cell blasts stimulated with low-dose, immobilized anti-CD3. Small horizontal lines indicate mean (± s.d.). *P = 0.003, **P = 0.01 (unpaired t-test). (f) Total LAMP-1 levels in activated CD8+ T cells from patients A.1 and G.1 compared to healthy control (Ctrl). (g) Cytolysis of P815 targets by anti-CD3-mediated redirected lysis for the indicated patients (Pt, n = 4) and healthy controls (Ctrl, n = 4). (h) Composite data for % CD57+ CD8+ T cells in patient (Pt, n = 4) versus healthy control (Ctrl, n = 4) PBMCs. Small horizontal lines indicate mean (± s.d.). *P = 0.002 (unpaired t-test). Data are from three (latent) or four (lytic) independent experiments (a) or three independent experiments (h) or are representative of four independent experiments (b,c,g) or two independent experiments (d,e,f).

Supplementary Figure 6 Expression of PD-1 on CD3+ cells in peripheral blood.

PBMCs were gated on CD3+ cells and assessed for CD45RA versus PD-1 expression in indicated patients (Pt, n = 7) and healthy controls (Ctrl, n = 2 adult and n = 2 pediatric). Data are representative of four independent experiments.

Supplementary Figure 7 Normal expression of signaling molecules in patient T cell blasts.

(a) Immunoblots for PTEN, PKC , p110δ, p-Erk, total Erk, and β-tubulin in T cell lysates from patients E.1 and F.II.1 or healthy control (Ctrl) cells stimulated with anti-CD3 for the times indicated in minutes (min). (b) Immunoblot for the PH domain-containing ARF6, p27kip1, and β-tubulin in indicated patient (n = 5 samples) and healthy control (Ctrl, n = 5 samples) T cell blasts after 10-min stimulation with anti-CD3 (+) or not (−). Data are from one experiment with two (a) or five (b) different patient samples.

Supplementary Figure 8 Increased, glucose- and amino acid-dependent basal S6 phosphorylation and glucose uptake in patient T cell blasts.

(a) Cumulative flow cytometric analysis of mean fluorescence intensity (MFI) for stimulated and unstimulated, permeabilized patient (Pt) or healthy control (Ctrl) T cell blasts after staining for p-S6 (S235, S236). Paired healthy control and patient blasts (i.e., cultured for the same duration) are connected by a line. *P = 0.04 (paired t-test). (b) Data from the same experiment as that shown in Fig. 4c but where cells were first rested in PBS for 2 hr before flow cytometric analysis of p- S6 (S235, S236) in unstimulated T cell blasts from patients G.1, D.II.1, and D.II.2 compared to healthy controls (Ctrl). (c) Glucose uptake by flow cytometric analysis of 2-NBDG fluorescence (linear MFI) after a 20-minute incubation period in glucose-starved T cell blasts from healthy controls (Ctrl, n = 3) and patients (Pt, n = 3). Paired healthy control and patient blasts (i.e., cultured for the same duration) are connected by a line. *P = 0.03 (paired t-test). Data are from three independent experiments (a) or one experiment with three different patients (b) or are representative of five independent experiments (c).

Supplementary Figure 9 CT images comparing hepatosplenomegaly and lymphadenopathy in patient A.1 before and after treatment with rapamycin.

Coronal reformations at the level of the tracheal bifurcation reveal more severe hepatosplenomegaly, hilar and mediastinal adenopathy, and splaying of tracheal bifurcation before (a) compared to after (b) rapamycin treatment.

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Lucas, C., Kuehn, H., Zhao, F. et al. Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency. Nat Immunol 15, 88–97 (2014). https://doi.org/10.1038/ni.2771

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