Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations

William Rae; John M Sowerby; Dorit Verhoeven; Mariam Youssef; Prasanti Kotagiri; Natalia Savinykh; Eve L Coomber; Alexis Boneparth; Angela Chan; Chun Gong; Machiel H Jansen; Romy du Long; Giorgia Santilli; Ilenia Simeoni; Jonathan Stephens; Kejia Wu; Marta Zinicola; Hana Lango Allen; Helen Baxendale; Dinakantha Kumararatne; Effrossyni Gkrania-Klotsas; Selma C Scheffler Mendoza; Marco Antonio Yamazaki-Nakashimada; Laura Berrón Ruiz; Cesar Mauricio Rojas-Maruri; Saul O Lugo Reyes; Paul A Lyons; Anthony P Williams; Daniel J Hodson; Gail A Bishop; Adrian J Thrasher; David C Thomas; Michael P Murphy; Timothy J Vyse; Joshua D Milner; Taco W Kuijpers; Kenneth G C Smith

doi:10.1126/sciimmunol.abn3800

. Author manuscript; available in PMC: 2026 Sep 9.

Published in final edited form as: Sci Immunol. 2022 Aug 12;7(74):eabn3800. doi: 10.1126/sciimmunol.abn3800

Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations

William Rae ^1,², John M Sowerby ^1,^2,^#, Dorit Verhoeven ^3,^4,^#, Mariam Youssef ^5,^#, Prasanti Kotagiri ^1,², Natalia Savinykh ⁶, Eve L Coomber ⁷, Alexis Boneparth ⁵, Angela Chan ⁵, Chun Gong ⁸, Machiel H Jansen ^3,⁴, Romy du Long ⁹, Giorgia Santilli ¹⁰, Ilenia Simeoni ^11,¹², Jonathan Stephens ^11,¹², Kejia Wu ¹³, Marta Zinicola ¹⁰, Hana Lango Allen ^12,¹⁴, Helen Baxendale ¹⁵, Dinakantha Kumararatne ¹⁶, Effrossyni Gkrania-Klotsas ^14,¹⁷, Selma C Scheffler Mendoza ¹⁸, Marco Antonio Yamazaki-Nakashimada ¹⁸, Laura Berrón Ruiz ¹⁹, Cesar Mauricio Rojas-Maruri ²⁰, Saul O Lugo Reyes ¹⁹, Paul A Lyons ^1,², Anthony P Williams ²¹, Daniel J Hodson ⁸, Gail A Bishop ^22,^23,²⁴, Adrian J Thrasher ^10,²⁵, David C Thomas ²⁶, Michael P Murphy ^2,²⁷, Timothy J Vyse ¹³, Joshua D Milner ⁵, Taco W Kuijpers ^3,^4,^†, Kenneth G C Smith ^1,^2,^†,^*

¹Cambridge Institute of Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK

²Department of Medicine, University of Cambridge School of Clinical Medicine, University of Cambridge, Cambridge, UK

³Emma Children’s Hospital, Amsterdam University Medical Center (AUMC), University of Amsterdam, Department of Pediatric Immunology, Rheumatology and Infectious Diseases, Amsterdam, Netherlands

⁴Amsterdam University Medical Center (AUMC), University of Amsterdam, Department of Experimental Immunology, Amsterdam Infection & Immunity Institute, Amsterdam, Netherlands

⁵Department of Pediatrics, Columbia University Irving Medical Center, New York, NY, USA

⁶NIHR Cambridge BRC Cell Phenotyping Hub, Department of Medicine, University of Cambridge, UK

⁷Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK

⁸Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

⁹Amsterdam University Center (AUMC), University of Amsterdam, Department of Pathology, Amsterdam, Netherlands

¹⁰UCL Great Ormond Street Institute of Child Health, London, UK

¹¹Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK

¹²NIHR Bioresource - Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, UK

¹³Department of Medical and Molecular Genetics, King’s College London, London, UK

¹⁴MRC Epidemiology Unit, University of Cambridge, Cambridge, UK

¹⁵Cambridge Centre for Lung Defence, Papworth Hospital, Cambridge, UK

¹⁶Department of Clinical Biochemistry and Immunology, Addenbrooke’s Hospital, Cambridge, UK

¹⁷Department of Infectious Diseases, Cambridge University Hospital NHS Trust, Cambridge UK

¹⁸Clinical Immunology Service, National Institute of Pediatrics, Secretariat of Health, Mexico City, Mexico

¹⁹Immune Deficiencies Laboratory, National Institute of Pediatrics, Secretariat of Health, Mexico City, Mexico

²⁰Pathology Department, National Institute of Pediatrics, Secretariat of Health, Mexico City, Mexico

²¹Wessex Investigational Sciences Hub, Faculty of Medicine, University of Southampton, Southampton, UK

²²Department of Microbiology and Immunology, The University of Iowa, Iowa City, IA, USA

²³Department of Internal Medicine, University of Iowa, IA, USA

²⁴Veterans Affairs Medical Center, Iowa City, IA, USA

²⁵Great Ormond Street Hospital for Children, NHS Foundation Trust, London, UK

²⁶Department of Immunology and Inflammation, Centre for Inflammatory Diseases, Imperial College London, London, UK

²⁷MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

Correspondence to: Professor Kenneth G. C. Smith, Cambridge Institute of Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK, CB2 OAW kgcs2@cam.ac.uk Tele: +44 1223 336 849

^†

co-senior authors

Contributed equally.

PMCID: PMC7619461 EMSID: EMS217723 PMID: 35960817

Abstract

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a central regulator of immunity. TRAF3 is often somatically mutated in B cell malignancies, but its role in human immunity is not defined. Here, in five unrelated families, we describe an immune dysregulation syndrome of recurrent bacterial infections, autoimmunity, systemic inflammation, B cell lymphoproliferation and hypergammaglobulinemia. Affected individuals each had monoallelic mutations in TRAF3 that reduced TRAF3 expression. Immunophenotyping showed patients’ B cells were dysregulated, exhibiting increased NF-κB2 activation, elevated mitochondrial respiration, and heightened inflammatory responses. Patients had mild CD4⁺ T cell lymphopenia, with a reduced proportion of naïve T cells but increased regulatory T cells and circulating T follicular helper cells. Guided by this clinical phenotype, targeted analyses demonstrated that common genetic variants, which also reduce TRAF3 expression, are associated with an increased risk of B cell malignancies, systemic lupus erythematosus, higher immunoglobulin levels, and bacterial infections in the wider population. Reduced TRAF3 conveys disease risks by driving B cell hyperactivity via intrinsic activation of multiple intracellular proinflammatory pathways and increased mitochondrial respiration, with a likely contribution from dysregulated T cell help. Thus, we define monogenic TRAF3 haploinsufficiency syndrome, and demonstrate how common TRAF3 variants impact upon a range of human diseases.

Introduction

Inborn Errors of Immunity (IEI) often present with a complex clinical picture including predisposition to infection, autoimmunity and malignancy. The genetic investigation of monogenic IEIs has provided unique insights into how individual genes control human immunity, and how their perturbation gives rise to pathology, and can both inform treatment of individual patients and provide a valuable resource for drug target discovery (1, 2). In parallel, population-wide studies of the impact of common genetic variants on disease susceptibility often implicate immune genes. Integrating rare and common variant genetic studies to understand the impact of individual genes on immune function at the population level represents an opportunity for the field.

Tumor necrosis factor alpha receptor-associated factor 3 (TRAF3) is one of a family of cytoplasmic proteins that control signal transduction from different receptor families, including the tumor necrosis factor receptors (TNFRs) and Toll-like receptors (TLRs). TRAF3 serves as a constitutive negative regulator of the alternative NF-κB pathway, and has a potential role in many immune cell types, but defects in it have only been studied in detail in cell lines and mouse models (3). While genome-wide association studies (GWAS) have linked common variants at the TRAF3 locus with several immune traits and autoimmune and atopic conditions (4–8), and the TRAF3 variant p.R118W (rs143813189-T) has been reported in association with herpes simplex encephalitis in a single case (9), TRAF3’s role in humans is yet to be fully determined. Here we describe the breadth of impact on human immunity, and mechanism of action, of rare and common genetic variants in TRAF3.

Results

Patient clinical phenotypes consisting of infection and autoimmunity

We investigated nine individuals from five unrelated families with childhood-onset immune diseases and recurrent infections (Fig. 1A and Table 1). All patients had suffered recurrent ear and sinopulmonary infections, including pneumonias from encapsulated bacteria Streptococcus pneumoniae and Haemophilus influenza, resulting in early-onset bronchiectasis in several individuals (fig. S1A-E). Patients A1 and E1 both underwent left lower lobectomy with histology showing bronchiectasis and follicular bronchiolitis with associated massive lymphoid hyperplasia, with a prominent lymphocytic infiltrate comprising T cells (CD4⁺>CD8⁺), B cells and numerous plasma cells (Fig. 1B and fig. S1F).

Table 1. Clinical phenotypes of patients.

	Family A	Family B				Family C		Family D	Family E
Patient	A1	B1	B2	B3	B4	C1	C2	D1	E1
Age, gender	40, M	10, F	15, M	47, F	71, F	15, F	50, M	15, M	20, M
Bacterial Infections	Recurrent sinopulmonary infections, Recurrent otitis media	Recurrent sinopulmonary infections, Recurrent otitis media Lymphadenitis, Pulmonary NTM	Recurrent otitis media, Recurrent sinusitis	Recurrent sinopulmonary infections, Lymphadenitis Peritonitis	Recurrent sinopulmonary infections	Recurrent sinopulmonary infections, Lymphadenitis	Recurrent sinopulmonary infections	Recurrent otitis media	Recurrent otitis media, Recurrent sinopulmonary infections, Pulmonary NTM
Lymphadenopathy	Splenomegaly GI lymphoid hyperplasia Pulmonary	Pulmonary, Splenomegaly	UNK	Splenomegaly	UNK	Pulmonary	UNK	Tonsils, Adenoids, Peripheral	Cervical, Inguinal, Pulmonary Splenomegaly
Respiratory	Bronchiectasis	Bronchiectasis	-	Bronchiectasis	Bronchiectasis	Bronchiectasis	-	-	Bronchiectasis, Follicular bronchitis.
Immune dysregulation	Enteropathy, Sicca syndrome	-	-	Sjögren’s syndrome	Sjögren’s syndrome, Vasculitis	Celiac disease	Atopic dermatitis, Allergic sensitization	Hashimoto’s thyroiditis, Systemic juvenile inflammatory arthritis Food and drug allergy, Atopic dermatitis Allergic sensitization	Vasculitis, Antiphospholipid syndrome, Atopic dermatitis
Immunoglobulins (g/L)	IgG 9.4↔ IgA <0.01↓ IgM <0.2*↓ IgE <2 IU/ml Polysaccharide responses ↓ Tetanus abs 0.21 IU/ml	IgG 13.5↑ IgA 1.6↔ IgM 2.3↔ IgE 6.5IU/ml Polysaccharide responses ↓ Tetanus abs 42.5 IU/ml	IgG 18.2↑ IgA 3.7↔ IgM 2.8↑ Polysaccharide responses ↓	IgG 19.5↑ IgA 2.8↔ IgM 6.3↑ Polysaccharide responses UNK	IgG 11.4↔ IgA 1.6↔ IgM 2.5↔ Polysaccharide responses UNK	IgG 17.3↑ IgA <0.01↓ IgM 1.5↑ IgE 116IU/ml Polysaccharide responses ↓ Tetanus abs 4.7 IU/ml	IgG 16.9↑ IgA 4.5↑ IgM 1.8↔ Polysaccharide responses UNK	IgG 19.5↑ IgA 4.8↑ IgM 2.0↑ IgE 2477 IU/ml↑ Polysaccharide responses UNK Tetanus abs 0.8 IU/ml	IgG 31.6↑ IgA 0.44 IgM 0.82 IgE 2.07 IU/ml Polysaccharide responses ↓
Autoantibodies	Anti-Ro	-	-	Anti-Ro, Anti-La	Anti-Ro, Anti-La, ANCA PR3	TTG IgG	UNK	Anti-TPO, Anti-Tg, Thyroid stimulating antibodies	Anticardiolipin, B₂ glycoprotein, Lupus anticoagulant, ANCA MPO / PR3

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Clinical and immunological phenotypes of the patients. Immunoglobulin level normal ranges based on clinical laboratory age adjusted values. ↔ = normal, ↑ = raised, ↓ = low. NTM = non-tuberculous mycobacteria, ANCA = anti-neutrophil cytoplasmic antibodies, TTG = tissue transglutaminase, TPO = thyroid peroxidase, Tg = thyroglobulin, UNK = unknown, - = not present, * = on immunoglobulin replacement

Lymphadenopathy was observed in multiple patients and lymph node histology showed granulomatous inflammation with no pathogens identified. Elective splenectomy due to massive splenomegaly was performed in patient B3, and histology showed numerous reactive lymphoid follicles with large germinal centers. T and B cell immunohistochemistry indicated normal follicular architecture was preserved with no evidence of abnormal clonality (using kappa/lambda staining), malignancy or extramedullary hematopoiesis (Fig. 1C). Immune dysregulation was prevalent; three patients were diagnosed with Sjögren’s syndrome and suffered sicca symptoms, each with anti-Ro52 antibodies and characteristic B cell hyperplasia (fig. S1G). In addition to their Sjögren’s syndrome, patient B4 developed an ANCA-positive vasculitis with glomerulonephritis which was treated with cyclophosphamide and rituximab, resulting in persistently absent B cells. Immune dysregulation was also prominent in patient D1, with substantial atopic disease, multiple food and drug allergies, elevated serum IgE, as well as autoimmune thyroid disease, and an autoinflammatory phenotype of systemic juvenile inflammatory arthritis with markedly raised ferritin and IL-18, managed with IL-1 receptor blockade. Patient E1 first presented in infancy with recurrent sinopulmonary bacterial infections and pneumonia, complicated by empyema, leading to bronchiectasis. Diagnoses of systemic autoimmunity and follicular bronchitis led to immunosuppressive therapy with prednisolone, hydroxychloroquine, and thalidomide, with concurrent antimicrobial treatments. Long term antimicrobial prophylaxis, immunoglobulin replacement, and lobectomy improved the patient’s infection burden. Further clinical details on these patients are found in the patient case summaries in the Supplementary Materials.

TRAF3 haploinsufficiency in patients

Genomic investigation revealed heterozygous premature stop codon mutations in TRAF3 in all patients, segregating with disease consistent with a pattern of autosomal dominant inheritance (Fig. 1E). In A1 the de novo single nucleotide change c.1275C>G in TRAF3 resulted in the premature stop-gain p.Y425X, and in affected individuals of family B the nucleotide deletion c.1066Gdel caused the frameshift p.S356PfsX in TRAF3. Patients C1 and C2 each had a four nucleotide insertion in TRAF3, c.339_340TAGA, resulting in the p.Q114X stop-gain mutation, and D1 had a de novo c.487C>T causing the stop-gain mutation p.R163X in TRAF3. In patient E1, as in patient A1, the TRAF3 variant c.1275C>G p.Y425X was identified (Fig. 1F). Each mutation caused a reduction of both TRAF3 RNA and protein expression, consistent with haploinsufficiency (Fig. 1G, H). TRAF3 protein undergoes constitutive ubiquitination and proteasomal degradation via TRAF2-cIAP1/2, with reduction in TRAF2 or cIAP1/2 resulting in increased TRAF3 (10, 11). We hypothesized that the observed >50% reduction of TRAF3 protein may be a consequence of increased ubiquitin-mediated proteasomal degradation of residual TRAF3 from the wild-type allele due to a reduced ratio of TRAF3:TRAF2-cIAP1/2. Consistent with this, treatment with a proteasome inhibitor (MG132) increased TRAF3 expression to nearer the expected 50% of control (fig. S2). Therefore, in these families with evidence of an autosomal dominant inherited immune dysregulatory syndrome, genetic investigation identified heterozygous high-impact TRAF3 mutations resulting in TRAF3 haploinsufficiency (TRAF3^HI).

Dysregulation of immune cells in TRAF3^HI patients

The features of this syndrome suggested widespread immune dysregulation. Total and CD4⁺ T cell numbers were reduced in TRAF3^HI patients, with reduced naïve cells and increased proportions of memory T cells (Fig. 2A-C and fig. S3A). As observed in T cell-specific Traf3-deficient mice (12), regulatory T cells (T_reg) were increased in TRAF3^HI patients (Fig. 2D and fig. S3B). CD8⁺ T cell numbers were preserved, but the proportion of naïve CD8⁺ T cells was reduced (Fig. 2E,F and fig. S3C). T cell proliferation in response to anti-CD3/CD28 was normal (fig. S3D), but there was reduced production of interleukin (IL)-17 and interferon (IFN)-γ following CD3/CD28 stimulation of patient T cells (Fig. 2G).

Assessment of additional cytokines showed production of the T-helper (T_H)2 cell cytokines IL-4, IL-13, and IL-10 was comparable with controls (Fig. 2G), as was IL-2 (albeit in a single patient: fig. S3E). Reductions in IFN-γ and IL-17 but preserved T_H2-associated cytokines are consistent a mild impairment of T cell receptor signaling strength in TRAF3^HI patients, as was observed in T cell-specific Traf3-deficient mice (13), resulting in a relative increased propensity to T_H2 differentiation (14). Circulating T follicular helper (cT_FH) cells were increased in TRAF3^HI patients (Fig. 2H), with increased T_FH effector mRNA levels and surface ICOS expression (Fig. 2I,J). Circulating T follicular regulatory (cT_FR) cells were reduced (Fig. 2K), as also seen in T_reg-specific Traf3-deficient mice (15). Together increased T_FH and reduced T_FR would be likely to promote aberrant germinal center responses and immunoglobulin production. Therefore, a distinct pattern of T cell abnormalities was present in TRAF3^HI patients, which might support exaggerated B cell responses.

Polyclonal increased serum immunoglobulins were seen in most TRAF3^HI patients, but responses to polysaccharide vaccines were impaired and autoantibodies were prevalent (Table 1).Total B cell numbers were normal (Fig. 3A), yet the proportion of class-switched memory cells was reduced across both IgG and IgA isotypes (Fig. 3B, C), while CD19⁺CD21^lowCD38^-Tbet^high B cells, associated with autoimmune disease (16), were increased (Fig. 3D). TRAF3^HI patients had normal numbers of circulating marginal-zone like B cells, and marginal zones appeared histologically normal in one patient (Fig. 1C), unlike mice (see (17) and Discussion). Patients had increased antibody-secreting cells in blood (Fig. 3E), and their B cells showed increased proliferation and induction of plasmablast formation upon stimulation with TLR9 agonists and IL-2 (Fig. 3F). In patient-derived B lymphoblastoid cell lines (BLCLs), both TLR7 and TLR9 expression were increased, consistent with a more pronounced activation state than in control BLCLs, and TLR9 stimulation resulted in enhanced production of TNFα (fig. S4). B cell receptor (BCR) repertoire analysis showed increased usage of IGHV4-34 and IGHV4-59, immunoglobulin heavy chain genes previously associated with SLE, vasculitis, Sjögren’s syndrome, and lymphoma (fig. S5A-E) (18, 19). Thus, the clinical and immune phenotypes of patients with TRAF3^HI were consistent with an immune dysregulatory syndrome involving T cells and hyperactive and autoreactive B cells.

Fig. 3 — (A) Bar graph of B cell numbers (CD19⁺ cells) in *TRAF3*^HI patients (Pts, n=8) and controls (Ctrls, n=10). (B) Proportions of marginal zone-like (CD19⁺CD27⁺IgM⁺) and class-switched memory B cells (CD19⁺CD27⁺IgM^-)in Pts (n=7) and Ctrls (n=10). P values calculated by Mann Whitney U test. (C) Plots of the proportions of IgM and class-switched memory IgG⁺, IgA⁺ B cells in a Ctrl and Pts. (D) Flow cytometry plots of CD19⁺ CD21^lowCD38^-Tbet⁺ B cells, and graph of percentages in Pts (n=6) and Ctrls (n=10). (E) Plots of antibody secreting cells (ASCs) (CD19+CD27⁺⁺CD38⁺⁺) from Ctrl and C2, and bar graph of the ASCs percentage of total CD19⁺ B cells in Ctrls (n=10) and Pts (n=7). P values calculated by Mann Whitney U test. (F) FACS plots of B cell proliferation and plasmablast differentiation a Ctrl and in C2 by CD38⁺ upregulation and CFSE dilution from culture of PBMCs, corrected for B cell number, unstimulated or stimulated with CpG (200ug/ml) and IL-2 (50 U/ml) for 6 days.

Graph of the percentages of CFSE^low CD38⁺ B cells in Ctrls (n=10) and Pts (n=6) after 6 days of stimulation of PBMCs. Graphs show mean ± s.e.m. P value by two-way unpaired t test.

We then explored myeloid cells in TRAF3^HI patients, as Traf3^flox/flox/LysM^cre myeloid cell-specific Traf3-deficient mice showed increased inflammatory cytokines after TLR stimulation in macrophages, and increased T-dependent antibody responses (20). Monocyte populations varied between patients (A1, B1, C1), with a possible increase of non-classical inflammatory (CD14^dimCD16⁺⁺) monocytes (fig. S6A). Circulating dendritic cells in TRAF3^HI patients were broadly comparable to controls and, in the one patient where functional studies were possible, enhanced TNFα and IL-6 were observed in monocytes and dendritic cells (fig. S6B-D). These data raise the possibility that a dysregulated myeloid compartment may contribute to the clinical phenotype of TRAF3^HI patients.

Functional impacts in TRAF3 haploinsufficient B cells

To understand the molecular mechanisms underlying the TRAF3^HI syndrome, we focused on B cells, given the prominence of B cell hyperactivity in patients, and on the nuclear factor-kappa beta 2 (NF-κB2) pathway, shown to be negatively regulated by TRAF3 in B cell malignancies (21). TRAF3^HI patient-derived BLCLs showed increased NF-κB inducing kinase (NIK) protein, phosphorylated NF-κB2 p100, and the active NF-κB2 p52 subunit, together indicative of constitutive NF-κB2 activation (Fig. 4A), which was correctable with re-expression of TRAF3 (fig. S7A-D). The use of BLCLs is a limitation of this study, as EBV immortalization can impact upon NF-κB pathway activation via LMP-1, and the B cell subset from which BLCLs are derived could vary between TRAF3^HI patients and controls. For discussion of this issue, and results showing that LMP-1 expression and B cell origins are similar between patients and controls, see fig. S8.

Fig. 4 — (A) Western blots of NIK, phosphorylated NF-κB2 p100 (p-p100), NF-κB2 p100 and p52 from whole cell BLCL lysates from controls (Ctrls) and *TRAF3*^HI patients (Pts), and bar graphs of quantifications of western blots from patients (n=3) and controls (n=9). Bar graphs show mean ± s.e.m. P values by two-way unpaired t test. (B) Gene set enrichment of the TNF – NFKB pathway in Pts’ naïve B cells (CD19⁺CD27^-). (C) Flow histograms of intranuclear NF-κB2 in Ctrls (n=4) and Pts (n=2) gated on naïve B cells (CD19⁺CD27^-) from PBMCs unstimulated or with BAFF 100ng/ml for 24 hrs. (D) Histograms of CD38 and HLA-DR expression on B cells (CD19⁺) from PBMCs, corrected for B cell number, stimulated with anti-IgM (5ug/ml) and IL-4 (50ng/ml) for 72 hrs. (E) Histograms of phosphorylated ERK and AKT in CD19⁺ B cells from PBMCs stimulated with anti-IgM for 7 minutes. (F) OCR of Ctrl BLCLs (n=3) with empty vector transduction (EV), Pt BLCLs (n=3) with EV, and *TRAF3*⁺ wild-type transduced Pt BLCLs (n=3) with sequential administration of oligomycin (Oligo), carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and rotenone/antimycin A (Rot/AA). Line graph shows mean ± s.e.m. 3 biological samples each with 5 replicates. (G) Activity fluorescence decay curve of assessment of cytochrome c oxidase activity from mitochondria of A1 and Ctrl BLCLs, with bar graph of cytochrome c oxidase activity normalized against controls for Ctrls (n=3) and Pts (n=3) BLCLs. Bar chart shows mean ± s.e.m. P value calculated by two-way unpaired t test. (H) Transmission electron microscopy images of mitochondria morphology in EV transduced control BLCLs (Ctrl EV), EV transduced *TRAF*^HI- BLCLs from B1, and B1 BLCLs transduced with wild type *TRAF3*⁺ expressing vector. Images representative of 2 experiments. (I) Western blot of NF-κB2 p100 and p52, and PGC1α in control BLCLs transduced with either mock short hairpin (shRNA) or *NFKB2* shRNA. (J) OCR in the mock shRNA or *NFKB2* shRNA transduced BLCLs treated with oligo, FCCP and Rot/AA at timepoints as indicated. Line graph of mean ± s.e.m. from 5 replicates.

Further functional studies focused on primary patient B cells. The transcriptomes of sorted naïve and memory B cells from TRAF3^HI patients showed upregulation of the TNF-NF-κB and CD40 pathway gene sets respectively, consistent with state of increased basal NF-κB2 activation (Fig. 4B and fig. S7E). It should be noted that in TRAF3^HI patients this memory (CD19⁺CD27⁺) population will contain fewer class-switched memory B cells (CD19⁺IgM^- CD27⁺) which may contribute to the observed differences. B cell activation factor (BAFF) is an important regulator of peripheral B cell survival via NF-κB2 (22). Consistent with this, TRAF3^HI patients’ naïve B cells showed heightened NF-κB2 responses to BAFF (Fig. 4C). Together these data indicate that the NF-κB2 pathway is hyperactive in TRAF3^HI patient B cells, and these enhanced responses to BAFF in B cells may be an important factor for the risk of developing autoimmunity, as was evident in BAFF-transgenic mice (23).

Despite the increase in NF-κB2, stimulation of TRAF3^HI patient B cells with anti-IgM/anti-CD40/IL-21 or anti-CD40/IL-21 did not result in convincing differences in plasmablast formation or increased IgG production compared with controls (fig. S9), which might be explained by redundancy between TRAF3 and TRAF2 in CD40 signaling (24). Recently, it has been reported that TRAF3 is a negative regulator of BCR signaling, with Traf3-deficient murine B cells showing elevated intracellular signaling cascades after BCR stimulation (25, 26). In TRAF3^HI patient B cells, stimulation with anti-IgM/IL-4 resulted in increased CD38 and HLA-DR expression (Fig. 4D), and in increased phosphorylation of ERK and AKT, both consistent with enhanced BCR signaling (Fig. 4E). Furthermore, TRAF3^HI patient B cells showed upregulated gene expression signatures associated with BCR signaling (fig. S10). Therefore, as with Traf3-deficient murine B cells, TRAF3^HI patient B cells appear to have increased BCR responsiveness.

In B cells TRAF3 regulates proinflammatory pathways in addition to NF-κB2 and, consistent with this, patients’ B cells demonstrated upregulation of ‘inflammatory response’ and ‘JAK – STAT3’ gene signatures (fig. S11A-D), and showed enhanced STAT3 phosphorylation in response to IL-6, and perhaps to IL-10 at later timepoints (fig. S11E-H). The enhanced response to IL-6 may contribute to the hypergammaglobulinemia in TRAF3^HI patients.

TRAF3^HI patient B cells also showed upregulation of the mitogen associated protein kinase (MAPK) - activator protein-1 (AP-1) transcription factor gene signatures (fig. S12A, B), reinforcing that B cell hyperactivation in human TRAF3 haploinsufficiency is driven by dysregulation of a number of proinflammatory pathways due to the pleiotropic effects of TRAF3 in B cells (fig. S12C).

Mitochondrial respiration in TRAF3 haploinsufficient B cells

B cell activation is associated with increased mitochondrial respiration (27), and patient BLCLs showed an increased oxygen consumption rate (OCR), indicative of elevated respiration, which was reduced by TRAF3 re-expression (Fig. 4F). Consistent with this increased OCR, both COX II (a component of the mitochondrial electron transport chain complex IV) expression and cytochrome c oxidase activity were increased in patients (Fig. 4G and fig. S13A, B). This increase in mitochondrial respiration was accompanied by altered mitochondrial morphology, with patient BLCLs showing darkened and thickened mitochondrial cristae by electron microscopy, which normalized following re-expression of wild-type TRAF3 (Fig. 4H). Re-expression of wild-type TRAF3, or silencing of NFKB2, reduced both these metabolic abnormalities and the expression of the mitochondrial co-transcriptional regulator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), the master regulator of mitochondrial biogenesis (28) (Fig. 4I, J and fig. S13C). Thus, reduced TRAF3 leads to an increase in mitochondrial respiration, and demonstrates a link between NF-κB2 and mitochondrial capacity in B cells.

Common variants in TRAF3 impact on disease risks

We sought to use the relatively discrete clinical features of this TRAF3^HI syndrome to focus genetic analyses, reducing multiple testing corrections, and allowing us to further define the impact of common TRAF3 variants on human disease. We thus analyzed associations between TRAF3 variants and bacterial infections, SLE, Sjögren’s syndrome, celiac disease and immunoglobulin levels, also adding B cell malignancy as it occurs in 10% of cases of Sjögren’s syndrome (29) and as somatic loss-of-function TRAF3 variants are frequent in myeloma and diffuse large B cell lymphoma (30, 31). Meta-analysis of GWAS identified previously unreported association at the TRAF3 locus with myeloma and non-Hodgkin’s lymphoma (rs753490592-C(T)₁₄, P = 2.02×10^-7, β -0.098) (Table S1 and fig. S14A).

Mendelian randomization analysis with whole blood expression quantitative trait loci (eQTL) data showed colocalization with TRAF3 cis-eQTLs (top eQTL P_SMR = 1.64×10^-2, P_eQTL = 1.27×10^-142, β_eQTL = 0.21) that reduce TRAF3 expression (Fig. 5A, fig S14B, and Table S2). Reduced TRAF3 expression also correlated with increased risk of B cell malignancy and worse patient survival in myeloma (P = 7.9×10^-4) (Fig. 5B). Meta-analysis of SLE GWAS found association at the TRAF3 locus (rs12436513-A, P = 4.79×10^-8, β = -0.102), with associated variants colocalizing with a TRAF3 cis-eQTL in CD4⁺ T cells (Table S1 and fig. S14C, D). Common TRAF3 variants also associated with immunoglobulin levels (rs12887521-A, P =7.42×10^-160, β = 0.061), and consistent with this, the lead variant is a TRAF3 eQTL in EBV-transformed B lymphoblastoid cell lines (BLCLs) (32) (Table S1, Fig. 5C, and fig. S14E). Therefore, common variants in TRAF3 are associated with an increased risk of polygenic diseases, and these risk-associated loci convey reduced TRAF3 expression.

Fig. 5 — (A) Inverse correlation of whole-blood *TRAF3* eQTL effect size on *TRAF3* expression and effect size from myeloma and non-Hodgkin’s lymphoma GWAS meta-analysis. (B) Kaplan-Meier survival curves for myeloma patients with high or low *TRAF3* expression in myeloma cells, and time (days) from diagnosis. (C) Funnel plots of the effect of rs12887521 eQTL zygosity on *TRAF3* expression in BLCLs from GTEx v8 (32). (D) Western blot and bar graphs of quantifications of TRAF3 and NF-κB2 p100 and p52 subunits in sorted B cells (CD19⁺) from healthy volunteers wild-type for TRAF3 p.R118 (Ctrls, n=5) and healthy volunteers heterozygous for TRAF3 p.R118W (n=5). Bar graphs show mean ± s.e.m. P values for TRAF3 and p52 by two-sided t test, P value for p100 by Mann-Whitney U test. (E) Flow cytometry plots of CD27 and IgM expression on B cells in a TRAF3 p.R118 wild-type (Ctrl) and p.R118W carrier. (F) Flow cytometry plots of CD27 and CD38 on B cells each with bar graphs of controls (n=5) and p.R118W carriers (n=5). MZ-like, marginal zone-like (CD19⁺CD27^-IgM⁺); ASC, antibody secreting cells (CD19⁺CD27⁺⁺CD38⁺⁺). Bar graphs show mean ± s.e.m. P values calculated by two-sided unpaired t test.

Previously, a single case carrying the heterozygous TRAF3 missense variant NM_145725.3:c.352C>T:p.R118W (rs143813189-T) was reported in association with herpes simplex encephalitis (HSE) via a dominant-negative effect on protein expression (9). This TRAF3 variant is now recognized to have a prevalence of approximately 1 in 200 to 250 in Europeans (33–35). The UK Biobank (34) contains 2,383 individuals heterozygous, and 4 homozygous, for TRAF3 p.R118W, none of whom had documented HSE despite HSV IgG serology showing similar HSV exposure to those without p.R118W (fig. S15A-D). However, given the relatively low number of HSE cases in this analysis, in an n+1 model analysis with the ‘next’ R118W carrier encountered having suffered HSE, the results would show that R118W conferred an insignificant but increased odds ratio (OR)= 4.17 [95% CI 0.59 – 30.2] P = 0.16. Thus, while heterozygosity for TRAF3 R118W is not a highly penetrant cause of HSE, it cannot be excluded that carrier status may convey a small increased risk. Cell transfection with TRAF3 p.R118W showed reduced TRAF3 expression (fig. S15E), and B cells from healthy volunteers heterozygous for TRAF3 p.R118W showed a trend towards reduced TRAF3 expression and an increase in NF-κB2 (Fig. 5D), together with reduced marginal zone-like B cells and increased antibody-secreting cells (Fig. 5E,F). The majority of the latter were IgM negative, as were those wild-type for TRAF3 p.R118 (fig. S16). While not associated with HSE in large cohorts, p.R118W was associated with increased immunoglobulin levels (OR = 1.02 P = 4.57×10^-4), bacterial infection (OR = 1.15 P = 5.10×10^-3), SLE (OR = 2.46 P = 7.7×10^-4), and myeloma and non-Hodgkin’s lymphoma (OR = 1.91, P = 2.38×10^-7) (Table S1 and fig. S15F-K). Thus a range of common variants at the TRAF3 locus reduce TRAF3 expression and increase the risk of autoimmune disease and B cell malignancy, suggesting they operate through pathological mechanisms shared with the higher penetrance TRAF3^HI syndrome.

Somatic loss of TRAF3 in malignant B cells

Somatic loss of TRAF3 is a frequent finding in myeloma, with loss-of-function mutations reported in up to ~20% of cases (36). We wondered whether the functional impacts observed in germline TRAF3^HI were also seen after somatic loss of TRAF3, providing a malignant cell survival advantages, and corroborating observations made in TRAF3^HI patient B cells. The TRAF3-deficient myeloma-derived U266 line shows constitutive NF-κB2 activation, and transduction with either wild-type TRAF3, or NFKB2 silencing, reversed this (Fig. 6A-C), while also reducing cell proliferation, increasing cell death, and reducing U266 cell OCR (Fig. 6D-G). Furthermore, targeting the NF-κB2 pathway, with either the NIK inhibitor SMI1 or the IKKα/β inhibitor BMS-346641, had similar effects (Fig. 6H-K). This increased metabolic respiration in B cells with reduced TRAF3 may further promote B cell malignancies in vivo, as it is reported that increased metabolic rates also augment EBV B cell transformation (37). Alongside increased OCR in TRAF3^HI patient BLCLs, increased GLUT1 expression was seen, consistent with the enhanced metabolic rates observed (fig. S17). While we cannot draw conclusions on the functional impact of all identified somatic TRAF3 mutations in B cell malignancies, those that are demonstrated to be loss-of-function (21) appear to have effects on B cells that are concordant with those associated with germline TRAF^HI-causing variants. This in turn suggests that, in human B cell malignancies with somatic loss-of-function TRAF3 mutations, targeting NIK and the NF-κB2 pathway may offer a therapeutic approach.

Fig. 6 — (A) Sanger sequencing fluorescent traces from a control BLCL line and U266 cells showing insertion of a deoxythymidine (red arrow) leading to a stop-codon. (B) Bar graph of relative gene expression of *TRAF3*, normalized to wild-type *TRAF3* from control BLCLs, in U266 cells transduced with an empty vector (EV), *NFKB2* shRNA (*NFKB2* KD), or wild-type *TRAF3* (TRAF3+). (C) Western blot of TRAF3, NF-κB2 p100 and p52 in U266 cells transduced with either mock shRNA, *NFKB2* shRNA, or wild-type *TRAF3*⁺ expressing vector. (D) Line graph of U266 cell death percentages, (E) U266 cell proliferation, (F) U266 cell numbers over time from selected stably transduced colonies, and (G) OCR measurement of U266 cells transduced with either mock shRNA, *NFKB2* shRNA or wild-type *TRAF3*⁺ over time with administration of oligomycin (Oligo), carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and rotenone and antimycin A (Rot/AA). Representative of two independent experiments with 3 replicates each. Line graphs show means ± s.e.m. (H) U226 percentage cell death, (I) U266 cell proliferation, and (J) total U266 cell numbers over time when treated with Ctrl (DMSO), or NIK inhibitor SMI1 10 μM, IKKα/β inhibitor BMS-345541 10 μM, or bortezomib 5 nM. (K) OCR measurement of U266 cells pre-treated for 6 hours with SMI1, BMS-345541, or bortezomib. Representative of two independent experiments. Line graphs shows means ± s.e.m. from 3-5 replicates for each condition and timepoint.

Discussion

We define the monogenic syndrome of TRAF3^HI in humans. The predominant findings of hypergammaglobulinemia, lymphadenopathy, splenomegaly and associated histological findings, and autoantibody-associated diseases, point to a prominent role for hyperactive B cells in the pathology of human TRAF3^HI. Reduced TRAF3 in B cells resulted in activation of inflammatory intracellular pathways, including those involving NF-κB2, MAPK and STAT3, and increased mitochondrial respiration. These alterations culminated in immune dysregulation, with predisposition to bacterial infection and autoimmune disease. We demonstrate that common germline disease-associated TRAF3 variants act in a similar way, through reducing TRAF3, albeit to a lesser extent than observed in the monogenic disease, explaining how both common and rare germline TRAF3 variants convey disease risks.

Sjögren’s syndrome was the most common autoimmune manifestation in the TRAF3^HI cohort. Primary Sjögren’s syndrome patients are reported to have increased IL-17 (38, 39), yet TRAF3^HI patients have low IL-17 production, perhaps due to the increased NIK-dependent activation of NF-κB2, a similar mechanism to suppression of T_H17 differentiation by second mitochondrial-derived activator of caspase (SMAC) mimetics (40). Sjögren’s syndrome is comprised of different endotypes associated with differing underlying disease biology (41, 42), so rather than representing a conflict, it is perhaps not surprising that different clinical or immune defects may occur between Sjögren’s syndrome driven by TRAF^HI compared to ‘conventional’ polygenic disease. The observation that TRAF3^HI is associated with both Sjögren’s syndrome and exuberant B cell responses, supports recent evidence for B cell-targeted therapy in primary Sjögren’s syndrome such as anti-BAFF monoclonal antibodies (42).

Although only present in 3/9 TRAF3^HI patients, allergy and atopic disease present a possible association with TRAF3^HI, given patients demonstrate a relative increase of T_H2 cytokine responses. Moreover, common variants at the TRAF3 locus are associated with risk of atopy (8, 43, 44), with the top SNP associations at the TRAF3 locus annotated as TRAF3 whole blood eQTLs, associated with reduced TRAF3 gene expression (44, 45). Thus, while only present in a minority of the patients here, atopic disease may be an additional clinical feature of TRAF3^HI.

Phenotyping of the TRAF3^HI patients has allowed us to delineate the systemic effects of reduced TRAF3, and has highlighted the similarities and differences between a human TRAF3 haploinsufficient state and cell-specific complete lack of TRAF3 in mouse models. Human TRAF3^HI most closely recapitulates the B cell-specific knockout mouse model. While whole-body Traf3-deficient mice show early-onset runting, loss of peripheral white blood cells and death by ten days of life (46), development of mice with cell specific Traf3 deficiencies allowed dissection of the roles of TRAF3 in different cell types. Traf3-deficient B cell mice develop lymphoid hyperplasia, hypergammaglobulinemia, autoimmune disease and B cell malignancies as they age (17), with these Traf3-deficient B cells demonstrating enhanced survival, increased TLR responses, and metabolic alterations (17, 36). T cell-specific Traf3-deficient mice have shown TRAF3 is required for optimal T cell receptor signaling and T cell immunity (13), but also functions as a negative regulator of IL-2 signaling and thereby T_reg differentiation, resulting in an increase in T_reg numbers, as also observed in TRAF3^HI patients (12). Furthermore, akin to TRAF3^HI patients, T_reg-specific Traf3-deficent mice show reduced T_FR cells, a concomitant increase in T_FH cells, and increased germinal center formation with enhanced IgG production (15). It may appear somewhat incongruous that TRAF3^HI patients had polyclonal hypergammaglobulinemia with reduced circulating class-switched memory B cells. Antigen-activated B cells can differentiate into either plasma cells or memory cells – and control of each outcome is distinct (47, 48). A reduction of memory B cells with normal/raised immunoglobulins has been reported in other IEIs (49), which could be driven by increased plasma cell differentiation of either naïve or memory B cells. The enhanced activity of several signaling pathways in TRAF3^HI patient B cells, in particular increased BCR signaling, could drive such differentiation to antibody secreting cells in vivo. T cell help is also likely to influence this balance, with the dysregulation of T cells in TRAF3^HI patients, particularly increased cT_FH cells and reduced cT_FR, likely to contribute to B cell hyperactivity and disease. Thus, the phenotype of human TRAF3 haploinsufficiency appears to be predominantly driven by B cell hyperactivity, but with T cell dysregulation also likely to play an important role.

Proportions of marginal zone-like B cells were normal in TRAF3^HI patients and reduced in TRAF3 p.R118W carriers. These observations are unexpected, as marginal zone B cells were increased in B cell-specific Traf3-deficient mice (17), and BAFF and NF-κB2 play a role in the development and survival of marginal zone B cells in mice (50). One reason for his apparent discrepancy might lie in fundamental differences between the marginal zones of mice and humans. Murine marginal zone B cells are primarily neither isotype-switched nor somatically mutated, while those in humans are thought to be derived from and reflect memory B cells (51, 52). Increased B cell activation resulting from reduced TRAF3 might have distinct impacts on these very different cells, perhaps driving expansion in the more “naive-like” mouse cells, but plasma cell differentiation of the memory-derived human marginal zone cells. Differences in TRAF3 expression may also play a complex role – with TRAF3 expression being partially reduced in many cell types in human TRAF3^HI and R118W individuals, while its absence is complete but confined to a single cell type in B cell-specific Traf3-deficient mice. These conflicting observations underline the importance of studying immunology in humans as well as mouse models.

The increased mitochondrial respiration resulting from reduced TRAF3 was evident in both the BLCLs of TRAF3^HI patients and the TRAF3-deficient plasmacytoid B cell U266 cells with increased OXPHOS. In both cell types re-expression of TRAF3 could reduce OCR. Recently, TRAF3 has been reported to control B cell survival under stress by mobilizing to the mitochondria where it interacts with mitochondrial fission factor, triggering mitochondria-dependent apoptosis (53). It was also noted that TRAF3-deficient B cells displayed increased mitochondrial respiration, and larger, more abundant mitochondria (53) - similar to our observations in TRAF3^HI BLCLs. Thus, reduced TRAF may provide further survival advantages to B cells by both reducing apoptosis and increasing mitochondrial respiration, increasing the risk of B cell malignancy.

TRAF3^HI revealed a biological link between TRAF3, NF-κB2, and enhanced mitochondrial biogenesis in B cells, driving B cell hyperactivity and consequently a syndrome of immune dysregulation, autoimmunity and systemic inflammation. Clinical features of the syndrome enabled a genetic exploration of the impact of TRAF3 variants across a spectrum of allele frequencies, and demonstrated that more frequent TRAF3 variants, which reduce TRAF3 expression, increase the risks of B cell malignancy, infections and autoimmune disease in the general population.

Materials and Methods

Study design

This study was designed to investigate adult and pediatric patients with signs and symptoms suggestive of immunodeficiency and/or autoimmunity. All studies had ethical and regulatory committee approvals. All participants or parents/guardians provided written informed consent and assent. Details of patient genetic investigations and TRAF3 common variant analyses are in the Supplementary Materials.

Cell immunophenotyping

For flow cytometry, PBMCs from patients and controls were stained with fluorochrome conjugated antibodies described in table S5 with gating strategies as in Figures and fig. S18. FACS was performed on LSRFortessa X-20 (BD Biosciences) and acquisition was performed with FACS Diva software (BD Bioscience). Further analysis was performed with FlowJo software (LLC). Further details, including conjugated antibody details, RNA-seq, BCR sequencing, Western blot, and electron microscopy methods, are described within the Supplementary Materials and Methods.

B cell stimulation and proliferation

PBMCs were labelled with CFSE 0.5 μM in PBS for 10 minutes at 37°C under constant agitation. PBMCs were corrected for B cell number of 20,000/well and were then cultured in 48-well flat bottom plates for 6 days at 37°C 5% CO₂ in complete RPMI with CpG oligodeoxynucleotide 2006 (200 μg/ml; InvivoGen) and human IL-2 (50 U/ml; R&D Systems), or saturating anti-IgM, anti-CD40, and IL-21 (25 ng/ml0, or anti-CD40 and IL-21 (25 ng/ml). B cell proliferation was measured using CFSE dilution and regulation of CD38. PBMCs, corrected for B cell number, were stimulated with anti-IgM (5 μg/ml) and IL-4 (1 μg/ml) for 72 hrs and then analyzed for CD38 and HLA-DR expression.

Cytokine analysis

PBMCs corrected for T cell number were simulated with anti-CD3/CD28 beads (Miltenyi Dynabeads) for 6 days, then supernatants were analyzed for IFN-γ, IL-17, IL-4, IL-13 and IL-10 by ELISA. For IL-2 in patient A1, PBMCs corrected for T cell number were stimulated with anti-CD3/CD28 beads (Miltenyi Dynabeads) for 48 hrs then supernatant was analyzed by ELISA. BLCLs were incubated in complete RPMI with TLR9 ligand ODN2006 (5μM; InvivoGen). Samples were incubated at 37°C 5% CO₂ for 48 hours then supernatants were removed from cells and analyzed with V-plex Proinflammatory Cytokine Assay (Meso Scale Discovery) according to the manufacturer’s instructions. Samples were run in triplicate and mean result calculated. For monocyte and dendritic cell cytokine analysis, PBMCs were incubated in complete RPMI with lipopolysaccharide (500 ng/ml; InvivoGen) for 5 hours and supernatants analyzed by ELISA.

Cell transduction and transfection

For lentiviral transduction, BLCLs or U266 cells were plated into a 24-well plate in complete RPMI with 5 μg/ml polybrene at a cell concentration of 10⁶/ml. Lentiviral particles of TRAF3 (TRAF3 NM_14725 Human Untagged Clone, cat#SC309131, Origene) in a lentiviral construct, or lentiviral particles containing NFKB2 shRNA (cat# sc-29409-V, Santa Cruz Biotechnologies) or lentiviral particles containing mock shRNA (cat#sc-108080, Santa Cruz Biotechnologies) were added, mixed, and incubated at 37°C for 16 hrs. Cells were then washed and re-plated in complete RPMI at 5×10⁵cells/ml and incubated for 48 hrs. Following this, puromycin selection was performed in escalating concentrations of puromycin to a maximum of 2 μg/ml until stable colonies were established. Transduction knockdowns or protein re-expressions were checked by Western blot for protein expression for every culture. For transfection, BLCLs or U266 cells transfections were performed with Lipofectamine 3000 (Invitrogen, cat#L3000001) according to the manufacturer’s instructions. Cells were incubated in Opti-MEM reduced serum media. TRAF3 (NM_14275) Human tagged ORF clone pCMV6-Entry (Origene, cat#RC210417) was altered to remove the Myc-DDK tag by inserting a stop codon after the ORF. For p.R118W the nucleotide at position 352 within the ORF was altered C>T. For Y425X the nucleotide at position 1274 within the ORF was altered C>G. Following transfection cells were incubated in complete RPMI for 48 hours prior to cell lysis. Further methods for B cell EBV immortalization and U266 cell experiments are described in the Supplementary Materials.

Metabolic assessments

BLCLs were plated in Agilent Seahorse XFe96 Cell Culture microplates at 2×10⁵ cells/well in XF96 RPMI Seahorse media supplemented with glucose 1 μM, pyruvate 1 μM and L-glutamine 1 μM (all Agilent). Cells were then treated sequentially with reagents according to the Mitochondrial Stress Test Kit (Agilent) or the Glycolytic Stress Assay (Agilent) manufacturer’s instructions. Oligomycin was used at a final concentration of 1.5 μM, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) 1μM, rotenone and antimycin 0.5 μM, etomoxir (cat# E1905, Sigma) 4 μM, and 2-deoxy-d-glucose 50 mM. For experiments with cell pre-treatments, cells were incubated with either control dimethyl sulfoxide (DMSO) (cat# D8418, Sigma), BMS-345541 10 μM (cat# B9935, Sigma) or SMI1 (cat# HY-112433, MedChemExpress) for 16 hours then recounted immediately prior to plating for the assay. BMS-345541 is an IKKα (IC50 = 4 μM) and IKKβ (IC50 = 0.3 μM) inhibitor (54). Analysis was performed on a XF96 Seahorse (Agilent). Cytochrome c oxidase activity was assessed with the Complex IV Human Enzyme Activity Microplate Assay Kit (cat# ab109909, Abcam). The assay was performed according to the manufacturer’s instructions and all samples were run in triplicate for each of the three biological patient samples and controls. Analysis was performed on a CLARIOstar Plus (BMG Labtech).

Supplementary Material

Supplementary Checklist

EMS217723-supplement-Supplementary_Checklist.pdf^{(265.9KB, pdf)}

Supplementary Data S1

EMS217723-supplement-Supplementary_Data_S1.zip^{(37.8KB, zip)}

Supplementary Materials

EMS217723-supplement-Supplementary_Materials.pdf^{(3.4MB, pdf)}

One-Sentence Summary.

Reduced TRAF3 in humans results in B cell hyperactivity and T cell defects predisposing to infections, autoimmunity and malignancy.

Acknowledgments

We acknowledge the participation of all Primary Immunodeficiency Consortium for the National Institute for Health Research (NIHR) Bioresource, which is part of the NIHR Bioresource, with a full list of members included in the Supplementary Material. This research was supported by the NIHR Cambridge Biomedical Research Centre (BRC) and the Cambridge NIHR BRC Cell Phenotyping Hub. The NIHR Cambridge BRC is a partnership between Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge, funded by the NIHR. This research was supported by the NIHR BRC based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London.

The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. We thank the Cambridge Advanced Imaging Centre for help and support with electron microscopy. This research has been conducted using the UK Biobank Resource under Application Number 53026. We want to acknowledge the participants and investigators of the Finngen study. Full acknowledgements for dbGaP datasets are included in the Supplementary Material. We thank GeneMatcher for enabling the identification of additional patients. We are grateful to all the participants who have been part of the EPIC-Norfolk project and to the many members of the study teams at the University of Cambridge who have enabled this research.

Funding

W.R. was supported by a Wellcome Trust PhD for Clinicians Fellowship (216382/Z/19/Z). M.Z. was supported by the NIHR Blood and Transplant Research Unit, University College London (2104-10074). E.G-K. is funded by an NIHR Greenshoots Award. C.G. was supported by a fellowship from the Kay Kendall Leukaemia Fund (KKLF1398).A.J.T. is supported by the Wellcome Trust (217112/Z/19/Z) and the NIHR BRC at Great Ormond Street Hospital for Children NHS Foundation Trust. M.P.M. is supported by the Medical Research Council UK (MR_U105663142) and by a Wellcome Trust Investigator award (110159/A15/Z). D.J.H. was supported by a Senior Research Fellowship from CRUK (RCCFEL/100072) and receives core support from the Wellcome Trust to the Wellcome – MRC Cambridge Stem Cell Institute (203151/Z/16/Z). K.G.C.S. was supported by the MRC (Programme grant MR/L019027) and is a Wellcome Investigator. The EPIC-Norfolk study (DOI 10.22025/2019.10.105.00004) has received funding from the MRC (MR/N003284/1, MC-PC_13048, MC-UU_12015/1). The NIHR Bioresource is funded by the NIHR (RG65966). The ‘Primary Immunodeficiency: mechanism and diagnosis via integrative clinical immunogenomics’ (INTREPID) collaboration is funded by a Wellcome Trust Collaborative Award (219506/Z/19/Z).

Footnotes

Author contributions: W.R. and K.G.C.S. conceived and designed the study; W.R., J.M.S., D.V., M.Y., P.K., N.S., E.L.C., G.S. C.G., I.S., J.S., H.L-A., designed and performed the experiments; W.R., J.M.S, D.V., M.Y., P.K., E.L.C., C.G., K.W., C.M.R-M., E.G-K., D.C.T., T.J.V, J.D.M. and K.G.C.S. performed the data analysis; W.R., D.V., A.B., A.C., H.B., D.K., S.C.S.M., M.A.Y-N., L.B.R., S.O.L.R., A.P.W., and T. W.K., cared for the patients and collected clinical data; W.R., A.J.T., J.D.M., T.J.V. and K.G.C.S. provided funding; W.R., G.A.B., J.D.M., and K.G.C.S. wrote the paper; all authors provided input into the drafting and editing of the paper.

Competing interests: K.G.C.S. is a co-founder/consultant with PredictImmune and Rheos Medicines and on the Scientific Advisory Board of GSK. None of these associations are of direct relevance to this publication. The other authors declare that they have no competing interests.

Data and Materials Availability

Genomic sequencing from the individuals presented here can be accessed by application to the NIHR Bioresource Data Access Committee (dac@bioresource.nihr.ac.uk). Transcriptomic data from this study has been deposited in the in the European Genome-phenome Archive (EGA) with data accession number EGAD00001008966, under the study number EGAS00001006341. Data from the UK Biobank is available by application to the UK Biobank. All data used from dbGaP datasets are available by application to the NCBI dbGaP as detailed in the Supplementary Materials. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.

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Supplementary Materials

Supplementary Checklist

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Supplementary Data S1

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Supplementary Materials

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Data Availability Statement

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PERMALINK

Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations

William Rae

John M Sowerby

Dorit Verhoeven

Mariam Youssef

Prasanti Kotagiri

Natalia Savinykh

Eve L Coomber

Alexis Boneparth

Angela Chan

Chun Gong

Machiel H Jansen

Romy du Long

Giorgia Santilli

Ilenia Simeoni

Jonathan Stephens

Kejia Wu

Marta Zinicola

Hana Lango Allen

Helen Baxendale

Dinakantha Kumararatne

Effrossyni Gkrania-Klotsas

Selma C Scheffler Mendoza

Marco Antonio Yamazaki-Nakashimada

Laura Berrón Ruiz

Cesar Mauricio Rojas-Maruri

Saul O Lugo Reyes

Paul A Lyons

Anthony P Williams

Daniel J Hodson

Gail A Bishop

Adrian J Thrasher

David C Thomas

Michael P Murphy

Timothy J Vyse

Joshua D Milner

Taco W Kuijpers

Kenneth G C Smith

Abstract

Introduction

Results

Patient clinical phenotypes consisting of infection and autoimmunity

Fig. 1. TRAF3 haploinsufficiency in patients.

Table 1. Clinical phenotypes of patients.

TRAF3 haploinsufficiency in patients

Dysregulation of immune cells in TRAF3HI patients

Fig. 2. T cell immunophenotype in TRAF3HI patients.

Fig. 3. B cell immunophenotyping in TRAF3HI patients.

Functional impacts in TRAF3 haploinsufficient B cells

Fig. 4. Enhanced inflammation and metabolism in patient B cells.

Mitochondrial respiration in TRAF3 haploinsufficient B cells

Common variants in TRAF3 impact on disease risks

Fig. 5. Impacts of common variation at the TRAF3 locus associated with immune diseases.

Somatic loss of TRAF3 in malignant B cells

Fig. 6. Impacts of somatic loss of TRAF3.

Discussion

Materials and Methods

Study design

Cell immunophenotyping

B cell stimulation and proliferation

Cytokine analysis

Cell transduction and transfection

Metabolic assessments

Supplementary Material

One-Sentence Summary.

Acknowledgments

Funding

Footnotes

Data and Materials Availability

References and Notes

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Dysregulation of immune cells in TRAF3^HI patients

Fig. 2. T cell immunophenotype in TRAF3^HI patients.

Fig. 3. B cell immunophenotyping in TRAF3^HI patients.