key: cord-0876409-p9ee9xzw
authors: Kutukculer, Necil; Seeholzer, Thomas; O’Neill, Thomas J.; Graß, Carina; Aykut, Ayca; Karaca, Neslihan Edeer; Durmaz, Asude; Cogulu, Ozgur; Aksu, Guzide; Gehring, Torben; Gewies, Andreas; Krappmann, Daniel
title: Human immune disorder associated with homozygous hypomorphic mutation affecting MALT1B splice variant
date: 2020-08-25
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.07.034
sha: 5c2e8410de91adcb038f6003531705f47dd61c32
doc_id: 876409
cord_uid: p9ee9xzw

nan

We identified the first human hypomorphic germline MALT1 mutation selectively disrupting NF-κB activation by the MALT1B splicing isoform in T cells, leading to a complex primary immune disorder with signs of immune deficiency and autoimmunity.

Identifying genetic mutations associated with human primary immune disorders provides unique opportunities to dissect the pathophysiological relevance of genes for the human immune system and may guide therapeutic approaches for the affected patients.

T and B cell antigen receptor (TCR/BCR) ligation on lymphocytes triggers activation of canonical NF-κB signaling, which is critical for mounting an adaptive immune response.

The CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex acts as the gatekeeper for antigen receptor-induced NF-κB activation. In activated T cells, MALT1 serves a dual role as a scaffold and a protease. While recruitment of the ubiquitin ligase TRAF6 to MALT1 triggers NF-κB signaling, the MALT1 protease cleaves distinct substrates to modulate T cell effector functions. Germline mutations in all CBM components have been associated with a phenotypically diverse set of human primary immunodeficiencies termed 'CBM-opathies' 1 .

To date, nine patients have been identified comprising six different mutations in the MALT1 gene that are linked to combined immune deficiency (CID) 1, 2 . Importantly, all known mutations cause loss-of-function due to absence or strong reduction of MALT1 protein expression. Here we describe the first case of a severe immune syndrome caused by a homozygous hypomorphic mutation in the human MALT1 gene.

A 19-year-old Turkish girl of parents who are first-degree cousins presented with a history of recurrent chronic purulent otitis and secondary hearing-poorness at admission. She was treated and hospitalized several times for bronchopneumonia. Physical examination J o u r n a l P r e -p r o o f diagnosed a widespread seborrheic dermatitis on the scalp, psoriasis on the hands and feet extensor regions, severe warts on the hands and bilateral axillary and cervical lymphadenomegaly. High-resolution chest tomography revealed superior segmental linear atelectasis with bronchiectasis in the right lung lower lobe. In axillary and retropectoral regions 2.7x12mm conglomerate lymph nodes were determined. In abdominal ultrasonography, splenomegaly and grade 1 hydroureteronephrosis in the right kidney as well as multiple lymphadenopathies in the epigastric and para-aortic regions were found. An excisional biopsy revealed follicular hyperplasia of an enlarged lymph node, but malignancy was excluded. Scalp biopsies showed hyperplasia in squamous epithelium and psoriasis with perivascular lymphocyte infiltration in the dermis, diagnosed as psoriasis with cutaneous symptoms.

Laboratory examinations ( Figure   E1A ), but a change in CD4+ to CD8+ T cell numbers in the patient (~0.8/1 ratio) compared to healthy controls (~2/1 ratio) indicates altered immune function ( Figure E1B ). There was a slight tendency to more activated CD69-positive CD4+ T cells in peripheral blood of the patient ( Figure E1C ). Patient CD3+ T cells were able to proliferate in vitro in response to PHA treatment ( Figure E1D ).

Since clinical features indicated a primary immune deficiency with additional signs of autoimmunity, targeted next-generation sequencing was performed using a primary immune deficiency research panel comprising 264 genes. We identified a heterozygous missense The MALT1 c.2418G>C exchange occurs directly within the C-terminal evolutionarily conserved TRAF6 binding motif 2 (T6BM2) of MALT1 ( Figure 1C and E5) . Recruitment of TRAF6 via T6BMs to MALT1 is essential for NF-κB activation, but is not directly connected to MALT1 protease activation in T cells 4 . While MALT1A contains two functional T6BMs (T6BM1 and T6BM2), MALT1B lacks T6BM1 encoded by the alternatively spliced exon 7 and thus encodes only the T6BM2, which is mutated in the patient ( Figure 1C) . Indeed, just like MALT1 T6BMs destruction by E/A replacement 4 , the MALT1B E795D or MALT1A E806D patient mutation abolishes or diminishes TRAF6 binding, respectively. Stronger J o u r n a l P r e -p r o o f T6BM2 ( Figure E6 ).

In the context of MALT1A, we now mutated the C-terminal T6BM2 in combination with the T6BM1, to show that both sites are required for full TRAF6 recruitment ( Figure E6 ) antibodies to mimic TCR/CD28 co-stimulation and the cytokine TNFα (Figure E7C and D) .

As expected, TNFα-triggered NF-κB activation was not affected by MALT1 loss or mutations ( Figure 1G and H) . Whereas the E806D mutation in MALT1A did not decrease NF-κB activation after P/I or TCR/CD28 co-stimulation, the identical E795D mutation in MALT1B abolished NF-κB activation in response to T cell activation. Human blood CD4+ T cells express ~5-fold higher levels of MALT1B over MALT1A 4 . To mimic such a scenario in vitro, we co-transduced MALT1 KO Jurkat T cells with untagged MALTB E795D and increasing amounts of ST-tagged MALT1A WT or E806D ( Figure 1I) . Indeed, low expression of MALT1A WT or E806D was unable to rescue defective TCR/CD28-and P/Iinduced NF-κB activation in the presence of MALT1B E795D. MALT1A E806D was even more severely compromised in rescuing NF-κB signaling, demonstrating that the relative expression of MALT1A E806D and MALT1B E795D in patient T cells strongly influences the severity of the hypomorphic MALT1 c.2418G>C loss-of-function mutation.

We describe a patient-derived homozygous MALT1 c.2418G>C transversion, which leads to a unique hypomorphic MALT1 alteration that selectively disrupts signaling of the MALT1B isoform ( Figure 1J) . The immune pathology is manifested by symptoms of immune deficiency such as chronic ear and bronchial infections and at the same time autoimmunity as evident from skin psoriasis, lymphadenopathies and autoantibodies. We Informed consent was obtained from the patient and her parents.

Targeted next-generation sequencing (TNGS) was performed using an Ion S5™ Sequencer.

The Ion AmpliSeq™ Primary Immune Deficiency (PID) Research Panel comprising 264 genes was used and analysis of mutations within 264 PID genes was done with Ion reporter software. The potential functional impacts of the identified variants were assessed using VarSome.

To purify PBMCs for flow cytometry and Western blot analysis, blood was drawn from donors into Lithium-Heparin tubes and centrifuged (300 x g without brake, 10 min, RT). 

The CBMopathies-A Rapidly Expanding Spectrum of Human Inborn Errors of Immunity Caused by Mutations in the CARD11-BCL10-MALT1 Complex

Novel MALT1 Mutation Linked to Immunodeficiency, Immune Dysregulation, and an Abnormal T Cell Receptor Repertoire

The Pyrin Inflammasome in Health and Disease

Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells

Combined immunodeficiency associated with homozygous MALT1 mutations

An allosteric MALT1 inhibitor is a molecular corrector rescuing function in an immunodeficient patient

CARD14/CARMA2 Signaling and its Role in Inflammatory Skin Disorders

Serum immunoglobulin(IgG, IgM, IgA) and IgG subclass concentrations in healthy children: a study using nephelometric technique

Age and gender leucocytes variances and references values generated using the standardized ONE-Study protocol

U/ml penicillin/ streptomycin) and transfection of HA-TRAF6 (pEF vector) and MALT1-FlagStrepII constructs (pHAGE vector) using calcium phosphate precipitation. Cells were lysed in co-immunoprecipitations (co-IP) buffer (25 mM HEPES (pH 7.5), 150 mM NaCl, 0.2% NP-40, 10% glycerol, 1 mM DTT, 10mM sodium fluoride, 8 mM β-glycerophosphate, 300 µM sodium vanadate and Roche protease inhibitor cocktail)

Jurkat T cell lines were grown in RPMI medium supplemented with 10 % FCS and 100 U/ml

MALT1A E806D, MALT1B, MALT1B E795D were cloned into pHAGE-h∆CD2-T2A backbone vector. For this, lentiviruses were produced in HEK293T cells. In brief, 2x 10 6 HEK293T cells were seeded in a 10 cm 2 dish in 8 ml DMEM medium one day prior to transfection. The following day, HEK293T cells were transfected with 1 µg of the lentiviral envelope plasmid pMD2.G (Addgene #12259

Trono), and 2 µg MALT1 transfer plasmid (pHAGE-h∆CD2-T2A-MALT1

After 3 days, the supernatant containing the virus particles was removed and sterile filtered (0.45 µM) before adding virus with 8 µg/ml polybrene to 5x10 5 MALT1-deficient Jurkat T cells. After 24 h cells were washed with PBS (three times) and re-suspended in 1 ml fresh RPMI medium. After two weeks, transduction efficiency was assessed by analyzing surface expression of h∆CD2 in flow cytometry with anti-CD2-APC (RPA-2.10, 1:400, invitrogen)

MALT1B E795D as determined by FACS after P/I, aCD3/CD28, or TNFa stimulation. Gate depicts the EGFP-positive cells used for quantification in Figure

We thank Caspar Ohnmacht for technical support. Work in Turkey was supported by the Jeffrey Modell Foundation (JMF). Work by DK was supported by the Deutsche Forschungsgemeinschaft (DFG) SFB 1054/A04 and SFB 1335/P07.