Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder
- Department of Physiology, David Geffen School of Medicine, UCLA, United States
- Leeds Institute of Medical Research, University of Leeds, United Kingdom
- Division of Haematology and Immunology, Leeds Institute of Medical Research, University of Leeds, United Kingdom
- Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Switzerland
- Department of Clinical Immunology and Allergy, St James's University Hospital, United Kingdom
- Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, United Kingdom
- National Institute for Health Research-Leeds Biomedical Research Centre and Leeds Institute of Rheumatic and Musculoskeletal Medicine, Wellcome Trust Brenner Building, St James's University Hospital, United Kingdom
Figures
Figure 1 with 1 supplement
Clinical and immunological features of index patient.
(A) A computerized tomography scan of the chest showing area of cylindrical bronchiectasis at the basal segment of the left lower lobe. (B) TCR repertoire as assessed by T receptor …
Figure 1—figure supplement 1
T helper (TH) TH1, TH2, and TH17 peripheral blood phenotyping.
(A) T-helper (Th) 1, Th2 and Th17 cell distribution among CD4+ T cells in the peripheral blood. Grey bars healthy controls (N = 6), blue bars patient (two separate evaluations). (B) Representative …
Figure 2
A rare biallelic mutation in CRACR2A segregates with the disease.
(A) Pedigree showing the index case and p.[R144G/E300*] and p.E278D CRACR2A variant carriers. The circles and squares represent female and male members, respectively. Black shading shows affected …
Figure 3
Patient B cell responses to T cell-dependent (TD) stimulation.
(A) Schematic representation of the culture conditions used to assess plasma cell differentiation in vitro. PBs – Plasmablasts, PCs – Plasma cells (B) Representative flow plots showing CD38 and …
Figure 4 with 1 supplement
Patient T cell responses to T cell receptor stimulation.
(A) Representative flow plots showing expression of IFN-γ in human PBMCs from a healthy donor and the patient. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hr and cultured for …
Figure 4—figure supplement 1
Expression of ORAI1 and STIM1 in PBMCs from healthy control and patient.
(A) Representative immunoblot showing expression of indicated proteins in PBMC lysates from control and patient. (B) Relative transcript expression of indicated genes from PBMCs of healthy control …
Figure 5 with 1 supplement
Cytokine expression profiles in T cells expressing individual allelic mutations in CRACR2A.
(A) Representative flow plots showing expression of IL-2 in control or CRACR2A KO Jurkat T cells stably expressing FLAG-tagged WT CRACR2A, CRACR2AE278D (E278D), or CRACR2AR144G/E300* (DM) after …
Figure 5—figure supplement 1
Expression profile of individual allelic mutants of CRACR2A in different cell types.
(A) Lysates of HEK293 cells stably expressing N-terminally FLAG-tagged WT or indicated mutants of CRACR2A were immunoblotted with anti-FLAG antibody. (B) Representative immunoblot showing endogenous …
Figure 6
SOCE and JNK phosphorylation in T cells expressing individual allelic mutations in CRACR2A.
(A) Representative traces showing averaged SOCE from control (48 cells) or CRACR2A-KO Jurkat T cells (KO) transduced with an empty vector (55 cells) or those encoding FLAG-tagged WT CRACR2A (45 …
Figure 7
Biochemical interactions and localization profiles of individual allelic mutations in CRACR2A.
(A) Immunoprecipitation for detection of binding between WT and indicated mutants of CRACR2A (E278D, or DM) with ORAI1/STIM1. Lysates of HEK293T cells expressing FLAG-tagged CRACR2A in the presence …
Author response image 1
Levels of intracellular Ca2+ are measured in primary T cells by exchanging Ca2+-free solution with that containing 2 mM CaCl2.
Copied from PMID: 22586105.
Tables
Table 1
Immunological characteristics of patient with variants in EFCAB4B.
| Date/year | 2005 | 2011 | 2017 | 2019 | 2021 (Feb) | 2021 (Nov) | Ref | |
|---|---|---|---|---|---|---|---|---|
| Total lymphocyte count | 2,880 | 990 | 1,201 | 1,572 | 1,472 | 1,268 | 1000–2800 cells/µl | |
| CD3+ T cells | 2,505 | 733 | 891 | 990 | 952 | 846 | 700–1200 cells/µl | |
| CD4+ T cell | Total | 216 | 235 | 148 | 165 | 122 | 112 | 300–1400 cells/µl |
| CD27+ CD45RA+ (naïve)% | 20.92 | 12.65 | 25.5 | 14–65% | ||||
| CD27+ CD45RA- (memory)% | 42.34 | 47.04 | 42.9 | 15–52% | ||||
| CD27-CD45RA- (memory effector) % | 36.01 | 37.67 | 20.5 | 8–35% | ||||
| CD27+ CD45RA- | 2.64 | 11.1 | 0–22% | |||||
| CD25+ CD127- (Treg)% | 0.73 | 1.6 | 2.9 | 3.5 | 3–10% | |||
| CD8+ T cells | Total | 2080 | 475 | 738 | 840 | 772 | 689 | 200–900 cells/µl |
| CD27+ CD45RA+ (naïve) | 17.69 | 31.3 | 1.5–65.5% | |||||
| CD27+ CD45RA- (memory) | 25.16 | 19.1 | 3.5–28.6% | |||||
| CD27- CD45RA- (memory effector) | 16.09 | 11.1 | 0.7–72.6% | |||||
| CD27- CD45RA+ (effector) | 41.07 | 38.6 | 1.6–53% | |||||
| CD19+ (B cells) | Total | 245 | 102 | 73 | 77 | 87 | 71 | 100–500 cells/µl |
| CD27- IgM+ IgD + (naïve) % | 63.1 | 89.18 | 67.7 | 78 | 44–84% | |||
| CD24hi CD38hi (Transitional) % | 6.7 | 10.3 | 8.2 | 11 | 2–14% | |||
| CD27+ IgM + IgD+ (non-switched) % | 15.4 | 3.69 | 22.5 | 11 | 5–32% | |||
| CD27+ IgM- IgD- (switched memory) % | 14.7 | 7.15 | 5.7 | 5 | 5–33% | |||
| Plasmablasts % | 0.2 | 0.4 | 1.1 | 1 | 0.2–5% | |||
| CD56+ CD16+ (NK cells) | 144 | 139 | 290 | 486 | 429 | 340 | 90–600 cells/µl | |
| Immunoglobulin profile | ||||||||
| IgG | 1.4 | 8.4 | 10.3 | 8.3 | 9.9 | 6–16 g/l | ||
| IgA | < 0.06 | < 0.06 | < 0.06 | < 0.06 | < 0.06 | 0.8–4 g/l | ||
| IgM | < 0.05 | < 0.05 | < 0.05 | < 0.05 | < 0.05 | 0.5–2 g/l | ||
| IgE | < 0.2 | 1–120 ku/l | ||||||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | CRACR2A | NCBI | Gene ID: 84,766 | |
| Strain, strain background (Escherichia coli) | DH5α | Thermo fisher Scientific | Cat# 18265017 | |
| Cell line (Homo sapiens) | Jurkat E6-1 T cells | ATCC | Not tested for mycoplasma | Cat# TIB-152 |
| Cell line (Homo sapiens) | HEK293T | ATCC | Not tested for mycoplasma | Cat# CRL-3216 |
| Cell line (Mus musculus) | CD40L-expressing L cell fibroblasts | Diehl et al., 2008 | Irradiated at 50 Gy for 50 minutes prior to use | |
| Cell line (Mus musculus) | M2-10B4 bone marrow stromal cells | Lemoine et al., 1988 | Irradiated at 57 Gy for 57 minutes prior to use | |
| Transfected construct (Homo sapiens) | Primers for plasmid construction | This paper | Supplementary file 1 | |
| Biological sample (Homo-sapiens) | Primary human peripheral blood mononuclear cells | This paper | Isolated from the patient and healthy controls | |
| Antibody | Anti-human Phospho JNK (mouse monoclonal) | Cell Signaling Technologies | Cat# 9,255 | Flow cytometry (1:100) |
| Antibody | Anti-Human STIM1(rabbit) | Cell Signaling Technologies | Cat# 5,668 S | WB (1:5000) |
| Antibody | Anti-His-tag(rabbit) | Cell Signaling Technologies | Cat# 12,698 S | WB (1:5000) |
| Antibody | Anti-FLAG tag(mouse monoclonal) | Millipore Sigma | Cat# F3040 | WB (1:5000) |
| Antibody | Anti-human ORAI1(rabbit polyclonal) | Millipore Sigma | Cat# AB9868 | WB (1:5000) |
| Antibody | Anti-Vav1(rabbit) | Cell Signaling Technologies | Cat# 2,502 | WB (1:2000) |
| Antibody | Anti-β-actin(mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-47778 | WB (1:2500) |
| Antibody | anti-human CD3 antibody (mouse monoclonal) | Bio X Cell | Clone OKT-3 | 1 μg/ml |
| Antibody | Anti-human CD28 antibody (mouse monoclonal) | Bio X Cell | Cat. #: BE0921Clone CD28.2 | 1 μg/ml |
| Antibody | Anti-CD4-FITC(mouse monoclonal) | eBioscience | Clone OKT-4 | FACS5 μl/test |
| Antibody | Anti-IFN- gama-PE-Cy7 | eBioscience | Clone 45.B3 | FACS5 μl/test |
| Antibody | Ant-IL2-PE | eBioscience | Cat# MQ1-17H12 | FACS5 μl/test |
| Antibody | Anti-human,CD19-BV421(mouse monoclonal) | BD | Cat. #: 562,440Clone HIB19 | FACS5 μl/test |
| Antibody | Anti-human, CD27-PE (mouse monoclonal) | BD | Cat. #: No:555,441Clone: M-T271 | FACS20 μl/test |
| Antibody | Anti-human,IgD-FITC(mouse monoclonal) | BD | Cat. #: 561,490Clone IA6-2 | FACS5 μl/test |
| Antibody | Anti-human,IgM-Perc-cy5.5(mouse monoclonal) | BD | Cat. #: 561,285Clone G20-R7 | FACS5 μl/test |
| Antibody | Anti-human, CD24-FITC (mouse monoclonal) | BD | Cat. #: 555,427Clone ML-5 | FACS20 μl/test |
| Antibody | Anti-human,CD38-Pecy7(mouse monoclonal) | BD | Cat. #: 335,825CloneHB-7 | FACS5 μl/test |
| Antibody | Anti-human,CD3-V500(mouse monoclonal) | BD | Cat. #: 561,416Clone UCHT-1 | FACS5 μl/test |
| Antibody | Anti-human, CD4-BV421(mouse monoclonal) | BD | Cat. #: 562,424Clone RPA-T4 | FACS5 μl/test |
| Antibody | Anti-human, CD25-Pecy7monoclonal | BD | Cat. #: 335,824Clone 2A3 | FACS5 μl/test |
| Antibody | Anti-human, CD127-PrcP Cy5.5(mouse monoclonal) | BD | Cat. #: 560,551Clone HIL-7R-M21 | FACS5 μl/test |
| Antibody | Anti-human, FoxP3- Alexa 488(mouse monoclonal) | BD | Cat. #: 566,526Clone 2632/E7 | FACS5 μl/test |
| Antibody | Anti-human, CD196-PE(mouse monoclonal) | BD | Cat. #: 559 562Clone 11A9 | FACS10 μl/test |
| Antibody | Anti-human,CD-183-APC(mouse monoclonal) | BD | Cat. #: 550,967 | FACS20 μl/test |
| Antibody | Polyclonal F(ab′)two goat anti-human IgM/IgG/IgA | Jackson ImmunoResearch | Cat. #: 109-006-129 | 10 μg/ml |
| Antibody | Anti-human CD138-APC(mouse monoclonal) | Miltenyi Biotech | Cat. #: 130-117-395 | FACS2 μl/test |
| Recombinant DNA reagent | FGllF | This paper | N/A | |
| Recombinant DNA reagent | pmCherry-N1 | Clontech | Clontech plasmid#632,523 | |
| Recombinant DNA reagent | pMD2.G | Addgene | Addgene plasmid #12,259 | |
| Recombinant DNA reagent | psPAX2 | Addgene | Addgene plasmid #12,260 | |
| Recombinant DNA reagent | pLentiCas9- blasticidin | Addgene | Addgene plasmid #52,962 | |
| Recombinant DNA reagent | pLentiguide-puro_ hCRACR2Asg#1 | This paper | Details in Supplementary file 1 | |
| Recombinant DNA reagent | pLentiguide-puro_ hCRACR2Asg#2 | This paper | Details in Supplementary file 1 | |
| Recombinant DNA reagent | pLentiguide-puro_ hCRACR2Asg#3 | This paper | Details in Supplementary file 1 | |
| Recombinant DNA reagent | pLentiguide-puro_ hORAI1sg | This paper | Details in Supplementary file 1 | |
| Recombinant DNA reagent | FG11F CRACR2A WT | This paper | Details in Supplementary file 1 | |
| Recombinant DNA reagent | FG11F CRACR2AE278D | This paper | Details in Supplementary file 1 | |
| Recombinant DNA reagent | FG11F CRACR2AR144G | This paper | Details in Supplementary file 1 | |
| Recombinant DNA reagent | FG11F CRAC R2AR144G,E300* | This paper | Details in Supplementary file 1 | |
| Sequence- based reagent | SureSelect XT Human All Exon V5 | Agilent | ||
| Sequence- based reagent | CRACR2AExon 6_F | This paper | ATGATTCCT GGCAGGTGAGA | |
| Sequence- based reagent | CRACR2AExon 6_R | This paper | ATTCCAGTG CAGGGACCAG | |
| Sequence- based reagent | CRACR2AExon 9_F | This paper | GGCCCTGATG TTGAGTAGGT | |
| Sequence- based reagent | CRACR2AExon 9_R | This paper | GTGAATGGC AGGGAAAGTGG | |
| Sequence- based reagent | CRACR2AExon 10_F | This paper | AAACAAGGT GAGGCCAGGG | |
| Sequence- based reagent | CRACR2AExon 10_R | This paper | AGCCCAAAT CCTCTT TTCACAG | |
| Peptide, recombinant protein | IL-2 | Roche | Cat. #: HIL2-RO | |
| Peptide, recombinant protein | IL-21 | Peprotech | Cat. #: 200–21 | 50 ng/ml |
| Peptide, recombinant protein | IL-2 | Peprotech | Cat# 200–02 | 20 units/ml |
| Commercial assay or kit | aCD3 plates | Corning | Cat. #:354,725 | 10 ng/ml |
| Commercial assay or kit | B cell isolation kit | Miltenyi Biotech, | Cat. #:130-091-151 | 100 U/ml |
| Commercial assay or kit | QIAamp DNA Blood kit | Qiagen | Cat. #:61,104 | |
| Commercial assay or kit | Human IgG ELISA Quantitation Set | Bethyl Laboratories Inc, | Cat. #: E80-104 | |
| Commercial assay or kit | Human IgM ELISA Quantitation Set | Bethyl Laboratories Inc, | Cat. #: E80-100 | |
| Commercial assay or kit | MagniSort human naïve CD4+ T cell enrichment kit | Thermofisher Scientific | Cat. #: 8804-6814-74 | |
| Commercial assay or kit | FOXP3/ Transcription Factor staining Buffer set | Thermofisher Scientific | Cat. #: 2229155 | |
| Chemical compound, drug | PHA-L | Sigma | Cat. #:L-4144 | |
| Chemical compound, drug | 3HThymidine | Perkin Elmer | Cat. #: 027001 | |
| Chemical compound, drug | LymphoPrep | Axis Shield | Cat. #:1114547 | |
| Chemical compound, drug | 7-AAD-PerCP-Cy5 | BD | Cat. #: 559,925 | FACS5 μl/test |
| Chemical compound, drug | Fura 2-AM | Thermofisher Scientific | Cat# F1221 | |
| Chemical compound, drug | Brefeldin A | Thermofisher Scientific | Cat# 00-4506-51 | |
| Chemical compound, drug | Thapsigargin | EMD Millipore | Cat# 80055–474 | |
| Chemical compound, drug | Phorbol 12- myristate 13- acetate (PMA) | EMD Millipore | Cat# 5.00582.0001 | |
| Chemical compound, drug | Ionomycin | EMD Millipore | Cat# 407,951 | |
| Chemical compound, drug | Polybrene | Millipore Sigma | Cat# TR-1003 | |
| Chemical compound, drug | Puromycin | Invivogen | Cat# ant-pr-1 | |
| Chemical compound, drug | Blasticidin | Invivogen | Cat# ant-bl-05 | |
| Chemical compound, drug | Poly-D-Lysine | Thermo Fisher Scientific | Cat# A003E | |
| Chemical compound, drug | Fixable Viability Dye eFluor 780 | eBioscience | Cat# 65-0865-14 | 1 μl/ml of cells |
| Software, algorithm | FlowJo v10 | TreeStar | ||
| Software, algorithm | Slidebook software | Intelligent Imaging Innovations, Inc | ||
| Software, algorithm | OriginPro | Originlab | ||
| Software, algorithm | Image J | NIH | ||
| Software, algorithm | Fluoview FV10i Confocal Microscope | Olympus | ||
| Software, algorithm | Fluoview software | FlowJo, LLC | ||
| Software, algorithm | Exome sequence analysis (various) | Please see methods section | ||
| Other | LAS-3000 LCD camera | FujiFilm | ||
| Other | ECM 830 electroporator | BTX | ||
| Other | BD Fortessa flow cytometer | BD Biosciences | ||
| Other | Cytoflex LX flow cytometer | Beckman Coulter | ||
| Other | HiSeq 3,000 | Illumina |
Additional files
-
Supplementary file 1
List of all the sgRNAs, and primers used for cDNA sub cloning and qRT-PCR in this study.
- https://cdn.elifesciences.org/articles/72559/elife-72559-supp1-v1.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/72559/elife-72559-transrepform1-v1.docx
-
Source data 1
Source data for all figures.
Zip folder containing raw and uncropped images for Western blots and Excel spreadsheets of quantitation.
- https://cdn.elifesciences.org/articles/72559/elife-72559-supp2-v1.zip
