A Homozygous mucosa-associated ly 1 mphoid tissue 1 (MALT1) mutation in a family with 2 combined immunodeficiency

Abstract

BACKGROUND

Combined immunodeficiency (CID) is characterized by severe recurrent infections with normal numbers of T and B lymphocytes, but deficient cellular and humoral immunity. Most cases are sporadic, but autosomal recessive inheritance has been described. In the majority of cases, the cause of CID remains unknown.

OBJECTIVE

To identify the genetic cause of CID in two siblings, the products of a first-cousin marriage, who suffered from recurrent bacterial and candidal infections with bronchiectasis, growth delay, and early death.

METHODS

We performed immunologic, genetic, and biochemical studies in the two siblings, their family members and healthy controls. Reconstitution studies were performed using T cells from Malt1^−/− mice.

RESULTS

The numbers of circulating T and B lymphocytes were normal, but T cell proliferation to antigens and antibody responses to vaccination were severely impaired in both patients. Whole genome sequencing (WGS) of one patient and her parents, followed by DNA sequencing of family members and healthy controls, revealed the presence in both patients of a homozygous missense mutation in MALT1 that resulted in loss of protein expression. Analysis of T cells that were available on one of the patients revealed severely impaired IκBα degradation and IL-2 production after activation, two events that depend on MALT1. In contrast to wild type human MALT1, the patients' MALT1 mutant failed to correct defective NF-κB activation and IL-2 production in MALT1 deficient mouse T cells.

CONCLUSIONS

An autosomal recessive form of CID is associated with homozygous mutations in MALT1. Should future patients found to be similarly affected they should be considered as candidates for allogeneic hematopoietic cell transplantation.

INTRODUCTION

Classical severe CID (SCID) results from mutations in genes that are critical for T cell, or both T and B cell development. These genes include the recombinase activating genes RAG1 and RAG2, which are critical for the rearrangement of the T cell receptor (TCR) and B cell receptor (BCR) genes, as well as the IL-7Rα and IL-2Rγ genes, which encode the IL-7 receptor subunits critical for thymocyte survival.¹ Combined immunodeficiency (CID) is characterized by a normal number, but severely impaired function of T and B lymphocytes. CID has been associated with homozygous mutations in ORAI-I and STIM-1,^{2, 3} which are necessary for calcium influx, an essential event in mature T cell activation. Most cases of CID are sporadic, but autosomal recessive inheritance is common in countries with a high incidence of consanguinity. In the majority of cases, the cause of CID remains unknown.

MALT1 (mucosa associated lymphoid tissue lymphoma-translocation gene 1) is a caspase-like cysteine protease essential for NF-κB activation downstream of antigen receptors, other receptors with immune-receptor tyrosine-based activation motifs, and some G-protein coupled receptors.⁴ MALT1 is recruited and activated through B-cell lymphoma 10 (BCL-10) and scaffolding proteins containing a caspase-recruitment domain (CARD) and a coiled-coil domain, such as caspase recruitment domain membrane-associated guanylate kinase protein (CARMA) 1/CARD11, CARMA3/CARD10, or CARD9. T cell activation via the TCR or mitogenic stimuli such as phorbol myristic acid (PMA) and ionomycin (IO) results in activation of protein kinase C θ (PKCθ), which phosphorylates CARMA1.⁵ This enables recruitment of BCL-10 and MALT1, followed by oligomerization of the CARMA1/BCL-10/MALT1 (CBM) complex. The CBM complex recruits TRAF6 which activates the IκB kinase (IKK) complex that phosphorylates the inhibitor IκBα, resulting in its ubiquitination and degradation by the proteasome. This frees the p50 and p65/RelA subunits of NF-κB from IκBα, permitting NF-κB to undergo nuclear translocation and initiate gene transcription for cellular activation (Fig 1).

Figure 1. Schematic representation of the NFkB activation following ligation of the TCR.

TCR ligation causes SRC kinase and Zap-70 dependent activation of protein kinase C θ (PKCθ), which phosphorylates CARMA1/CARD11. This enables recruitment of BCL-10 and MALT1, followed by oligomerization of the CARMA1/BCL-10/MALT1 (CBM) complex. The CBM complex activates TRAF6, which results in the K63 ubiquitinylation of NEMO and activation and the IKK complex. Activated IKK2 phosphorylates the inhibitor IκBα, resulting in its K48 ubiquitination and degradation by the proteasome. This frees the p50 and p65/RelA subunits of NF-κB from IκBα, permitting NF-κB to undergo nuclear translocation and initiate gene transcription necessary for cellular activation, including IL-2.

Studies in Malt1-null mice have demonstrated that MALT1 is essential for T cell activation and IL-2 production in response to TCR ligation and is required for TCR-induced NF-κB activation.⁶ MALT1 is dispensable for B cell activation via the BCR, but is important for NF-κB activation by the B-cell-activating factor receptor in B cells, which is critical for marginal zone B cell development.⁷ In MALT1 lymphomas, a t(11;18)(q21;q21) chromosomal translocation results in the formation of an oncogenic fusion protein that combines the N-terminus of the cellular inhibitor of apoptosis 2 (cIAP2) with the C-terminus of MALT1.^8,9 The cIAP2 N-terminus enables spontaneous oligomerization of the cIAP2-MALT1 fusion protein, thereby constitutively activating the transcription factor NF-κB.

We report a homozygous point mutation in MALT1 in two siblings who died from CID. The mutation results in loss of MALT1 expression. The MALT1-deficient T cells failed to degrade IκBα or produce IL-2 following T cell activation. Experiments in the murine Malt^−/− model showed that only wild-type MALT1, and not the mutated human MALT1 gene found in the patients, could restore IκB$\underset{.}{\alpha }$degradation and IL-2 production in response to T cell activation.

METHODS

Study Participants

We enrolled 14 members of a consanguineous Lebanese family in the study as shown in Fig. 2A. Flow cytometry and proliferation studies were performed prior to the patients' death on freshly obtained PBMCs as part of routine clinical care. A limited number of viable frozen PBMCs and of T cells grown in PHA and IL-2 were available on Patient (Pt) 1, but none on Pt 2. Only banked genomic DNA was available on Pt 2. Blood samples were obtained for DNA analysis from 12 of their relatives, who were all healthy. The participants provided written informed consent on forms approved by local ethics committees and by the Institutional Review Board at Children's Hospital, Boston. DNA samples from 150 healthy Lebanese donors were used as controls.

Figure 2. Family pedigree of the patients and T cell proliferation to mitogens and antigens.

A. Family pedigree. Numbers refer to members studied. B, C. Response of PBMCs from the patients (Pt) to mitogens (B), and antigens (C) expressed as percentage of the proliferation of PBMCs from two healthy adult controls (C). Values represent mean± SE of two independent determinations. * P<0.05, *** P<0.001.

Genetic Analysis

Homozygozity mapping was performed using the Affymetrix genome-wide human SNP array 6.0 and analyzed using Birdsuite version 1.4 and PLINK version 1.07, using default parameters as previously described.¹⁰ WGS was performed on banked genomic DNA from Pt 1 and both parents through Complete Genomics, Inc. WGS rather than whole exome sequencing was performed so that analysis of structural variations could be done if no candidate single nucleotide variants were identified. Analysis of WGS data was performed using MolBioLib.¹¹ Sanger sequencing on banked genomic DNA available from both patients, their family members and controls was used to verify the mutation in MALT1 and to confirm that this mutation was not present in an ethnically matched control population.

Cell culture

Isolation of PBMCs and stimulation with mitogens and antigens were performed as part of routine clinical care as previously described.¹² Briefly, PBMCs (1×10⁶ cells/ml) were stimulated with PHA, Con A, pokeweed mitogen (PWM) and anti-CD3 mAb (OKT3), (Sigma) for 64 hours, and with the antigens tetanus toxoid (TT) and diphtheria toxin (DT) (National Institute for Biological Standards and Control) and candida (Greer Labs) for six days. Proliferation was assayed by measuring ³H-thymidine incorporation added for the last 16 hours of culture. PHA-stimulated PBMCs (PHA T blasts) from Pt 1 and controls were maintained in culture in the presence of recombinant (r) IL-2 and were cryofrozen after 12–14 days.

Flow Cytometry

Standard flow cytometric methods were used for staining of cell-surface and intracytoplasmic proteins. For IL-2 expression, PHA T cells were washed and were stimulated with either medium or PMA (32nM, Sigma) plus Ionomycin (IO, 0.5μM, Sigma) in the presence of Brefeldin A (eBioscience) for 16 hours. For IκBα, degradation, thawed PHA T cells were rested 4 hours before stimulation with PMA (162nM) and IO (0.5μM) for 15 minutes. Staining was performed according to the manufacturer's suggestions. Anti-human monoclonal antibodies to the following molecules, with the appropriate isotype-matched controls were used for staining: CD3 (HIT3a), CD4 (RPA-T4), CD8 (RPA-T80, CD19 (HIB19), CD56 (NCAM16.2), IL-2 (MQ1-17H12), and IκBα (25/IκBα/MAD-3) (BD Pharmingen). Data collected with a LSRFortessa cell analyzer (BD Biosciences) were analyzed with FlowJo software (TreeStar).

RT-PCR analysis of MALT1 mRNA levels

RNA was extracted from PHA T-blasts by TRIzol (Invitrogen) and was reverse transcribed by Supercript II RT (Invitrogen) according to manufacturer's instructions. RT-PCR was performed using the following primers: for MALT1, forward- 5'-ATGTCGCTGTTGGGGGACCCGCTA-3' and reverse- 5'-GGAGGTCATTTTTCAGAAATTCTGAGCCTGTCAG-3'; and for the housekeeping gene GAPDH, forward- 5'-GGGAAGGTGAAGGT- 3' and reverse- 5'-CTGATGATCTTGAGG CTGTTG- 3'.

Immunoblotting

Lysates from PHA T-blasts were separated by SDS-PAGE gel and transferred to nitrocellulose membranes. Specific proteins were detected using a rabbit anti-MALT1 antibody (# 2494S, Cell Signaling) and was reprobed with anti-actin (Chemicon International) as loading control.

Retroviral reconstitution of Malt1^−/− CD4⁺ T cells with human MALT1

CD4⁺ T cells were MACS-purified from spleen and lymph nodes of Malt1^−/− mice, previously described in Ref. 6, and stimulated with plate-bound anti-CD3 (5 μg/ml, 145-2C11, eBioscience) and soluble anti-CD28 (2.5 μg/ml, 37.51, eBioscience) antibodies for 48 hours. Retrovirus was produced in Phoenix cells after transfection with bicistronic pMSCV-Thy1.1 constructs containing wild type (WT) and mutant forms of human MALT1. For infection, supernatants were harvested after 48 hours and filtered before spin infection of activated Malt1^−/− CD4⁺ T cells in the presence of Polybrene (4 μg/ml). Subsequently, infected Malt1^−/−CD4⁺ T cells were expanded for 72 hours in medium supplemented with IL-2 (10 ng/mL, R&D Systems). For IκBα degradation, cells were fixed with paraformaldehyde at various time points after stimulation and stained for CD4 and Thy1.1. After permeabilization with methanol, cells were stained using mouse anti- IκBα antibody (L35A5, CST) and anti-mouse IgG1 FITC (eBioscience) and subsequently were analysed with a FACS Canto II (BD). For measurement of intracellular IL-2, cells were harvested, thoroughly washed and then rested before restimulation with PMA (300nM) and IO (400nM) for 5 hours in the presence of Brefeldin A (10 μg/ml, Sigma). Before fixation and permeabilization, cells were stained with anti-CD4 PE and anti-Thy1.1 APC antibodies (eBioscience) as well as with a live/deadfixable cell stain (Invitrogen). Intracellular stains were performed using anti-IL2 PE-Cy7 antibody and Foxp3 intracellular staining kit (eBioscience). Analysis was performed using FlowJo Software (Treestar) and gates were set on live CD4⁺ Thy1.1^high and Thy1.1^low T cells, respectively.

Statistical analysis

Student t-test was used for statistical analysis.

RESULTS

Cinical presentation

Pt 1 and her younger male sibling, Pt 2, were the products of a first-cousin marriage in a highly consanguineous family (IV.1 and IV.2 in Fig 2A). Both patients experienced recurrent pulmonary infections since 4 months of age with resultant bronchiectasis. Bronchial lavage cultures taken at different times grew Pseudomonas, Streptoccus pneumoniae, and Candida albicans (C. albicans) from Patient 1 and Haemophilus influenzea (H. influenzae), Klebsiella pneumoniae, and Staphyloccus aureus from both patients. Both patients developed mastoiditis, chronic aphthous ulcers, cheilitis, and gingivitis. Pt 1 suffered from pneumococcal meningitis at 6 months and H. influenzae meningitis at 15 months of age. Endoscopy revealed esophagitis, gastritis and duodenitis in both patients. Small intestinal biopsy revealed villous atrophy in Pt 1 and increased numbers of intraepithelial lymphocytes in Pt 2. C. albicans was cultured from the duodenal biopsy and the stools in Pt 2. Cytomegalovirus was repeatedly recovered from the urine of both patients. Blood was not tested for the presence of CMV. Growth was delayed, but neurologic development was normal in both patients. Despite intravenous immunoglobulin (IVIG) replacement therapy, both patients had persistent infections and expired from respiratory failure. Pt 1 died at age 13.5 years in 2007, and Pt 2 died at age 7 years in 2002.

Lymphocyte phenotyping and functional immunologic studies

Serum immunoglobulin levels determined prior to institution of IVIG therapy in Pt 1 at ages 1 and 2 years and in Pt 2 at age 1 year were found to be normal. Both patients had group A+ red blood cells, but no detectable anti-hemagglutinin B antibodies. Anti-tetanus antibody titers were non-protective in both patients and antibodies to all pneumococal serotypes tested (3, 7F, 9N, and 14) were undetectable, despite a history of vaccination with tetanus and unconjugated pneumococcal polysaccharide (Pneumovax) vaccines.

Absolute lymphocyte counts were repeatedly found to be normal in both patients (data not shown). FACS analysis of PBMCs, performed on two occasions during the patient's life, revealed normal percentages of CD3⁺, CD4⁺ and CD8⁺ T cells, and of CD19⁺ B cells (Table 1 ¹³). Analysis of naive and memory T cells was performed once on CD4⁺ cells from Pt 1 at age 9 and revealed a distribution of CD4⁺CD45RA⁺ cells (68%) and CD4⁺CD45RO⁺ cells (38%), which was normal for age. B cell subsets were not examined; their analysis in the deceased patients' frozen PBMCs was precluded by the limited numbers of cells available. The percentage of CD56⁺ NK cells was decreased in Patient 1 (Table 1), but was also decreased in the healthy mother (data not shown). PBMCs from the patients had impaired proliferation to Con A and PWM, and to a lesser degree to PHA, compared to normal controls (Fig 2B). The patients' PBMCs proliferated poorly to anti-CD3 mAb, TT, diphtheria toxoid (DT) and Candida antigens (Fig. 2B, C). T cell activation markers and the ability of IL-2 to correct the defective T cell proliferation were not examined. Delayed cutaneous hypersensitivity to Candida antigen was absent in both patients.

Table 1.

Phenotypic analysis of lymphocytes.

	Patient 1	Patient 2	Healthy controls*
Age (yrs)	4	2.25	2 – 6
% of lymphocytes
CD3+	80	80	56 – 84%
CD3+CD4+	39	53	28 – 52%
CD3+CD8+	34	17	14– 30%
CD19+	19	11	13 – 37%
CD56+	0.4	5	4 – 17%

The analyses shown were performed at age 4 in Pt 1 and age 2.25 in Pt 2. Similar results were obtained on a second occasion when the patients were aged 9 yrs and 5 yrs respectively.

^*Based on values in Ref. 13

Homozygous mutation in MALT1 in the patients

Since this novel primary immunodeficiency affected two patients of both genders with consanguineous parents, the disease was most likely transmitted through an autosomal recessive mode of inheritance. Microarray analysis of fourteen family members (Fig. 2A) identified two regions of homozygosity present exclusively in the two patients: a 3.2 Mb interval on chromosome 17 and a 10 Mb interval on chromosome 18. Whole genome sequencing (WGS) of Pt 1 and her two parents revealed a total of 36 coding/splice non-synonymous variants that were homozygous in the patient, heterozygous in both parents. Of these, three mutations were on an autosomal chromosome and novel (i.e. not found in the US National Center for Biotechnology Information database (db) of single nucleotide polymorphism (SNP) and 1000 Genome databases). These mutations were in MALT1, NCOA5, which encodes a coregulator for estrogen receptors, and TRIO, which encodes a guanine nucleotide exchange factor. Only the missense mutation in MALT1 (c. 266G>T) resided in one of the regions of homozygosity, specifically on chromosome 18. Sanger sequencing of genomic DNA confirmed that both patients were homozygous for the mutation and that both parents were heterozygous (Fig. 3A). Seven of nine other family members studied were heterozygous for the mutation and two did not carry the mutation (Fig. 2A). In addition to its absence in the dbSNP and 1000 genome databases, the mutation was not found in 150 healthy ethnically matched controls, indicating that it is not a common polymorphism. The mutation results in an amino acid (aa) change from serine to isoleucine at position 89 in MALT1 CARD domain (MALT1^S89I, Fig. 3B), and is predicted to be deleterious by both SIFT (score 0) and Polyphen (score 0.916). The mutated residue is highly conserved among multiple species (Fig. 3C). Crystal structure of the CARD domain of MALT1 (aa 29–126) revealed that the mutated Ser⁸⁹ residue is buried in the core of this domain;¹⁴ mutation of this residue to isoleucine would disrupt the structure of the domain and render the mutant protein susceptible to degradation. Given the patients' impaired lymphocyte proliferation studies and the results of the genetic studies, we considered MALT1 to be the most likely candidate because of its critical role in lymphocyte activation.

Figure 3. MALT1 mutation in the patients.

A. Electrophoregram depicting the c.266G>T mutation in Pt 1 and her father. B. Genomic organization of MALT1 (top) and protein structure (bottom) of MALT1. Blue boxes represent exons. The mutation in exon 2 is shown. C. Alignment of MALT1 homologues. D. RT-PCR analysis of MALT1 mRNA in PHA T blasts. E. Immunoblot analysis of MALT1 in lysates of PHA T cell blasts. Data are representative of two experiments.

MALT1 mRNA levels and protein expression in the patient

The impact of the mutation on the expression of MALT1 mRNA and MALT1 protein was examined in PHA T-blasts available from Pt 1. No viable cells or cDNA were available from Pt 2. RT-PCR analysis revealed comparable levels of MALT1 mRNA in T cells from Pt 1, her father, and a healthy control (Fig 3D). In contrast, immunoblotting of T cell lysates with a rabbit antibody directed against the carboxy terminal of MALT1 revealed no detectable MALT1 protein in the patient, while MALT1 was readily detectable in cells from her father and the healthy control (Fig 3E). These results suggest that the mutant MALT1 is rapidly degraded.

Effect of the homozygous MALT1 S89I mutation on IκBα degradation and IL-2 expression following T cell activation

T cells from Malt1^−/− mice have impaired IκBα degradation and IL-2 expression after activation with PMA/IO and anti-CD3+anti-CD28.⁶ IκBα degradation following stimulation of PHA T cell blasts with PMA/IO was impaired in the patient, as evidenced by intracellular FACS analysis (Fig 4, left panels). Furthermore, intracellular IL-2 expression in PMA/IO-stimulated T cells was severely reduced in the patient compared to healthy control (Fig 4, right panels). The deficient IL-2 expression is consistent with the critical role NF-κB activation plays in driving IL-2 gene transcription.¹⁵

Figure 4. Impaired IκBα degradation and IL-2 expression in the patient's T cells.

Intracellular FACS analysis of IκBα degradation (left panels) and intracellular IL-2 expression (right panels) in gated CD3⁺ T cells following stimulation of PHA T cell blasts from Pt 1 and a control with PMA+IO. Data are representative of two experiments.

The patient's mutant MALT1 cDNA fails to rescue the defective activation of T cells from Malt^−/− mice

To determine the effect of the S89I mutation on MALT1 function, we examined whether expression of MALT1^S89I could rescue the NF-κB signaling defect in T cells deficient in MALT1. Because only limited numbers of PBMCs and PHA T cell blasts were available from Pt 1, and none from Pt 2, and both patiens were deceased, reconstitution experiments were conducted in primary CD4⁺ T cells from Malt1^−/− mice. Malt1^−/− Thy1.2 positive T cells were retrovirally infected with a bicistronic vector that encodes for the Thy1.1 surface marker and either MALT1^S89I or WT human MALT1 (hMALT1). As expected, non-infected T cells from Malt1^−/− mice failed to degrade IκBα or express IL-2 in response to stimulation with PMA/IO (Fig 5). Thy1.1 positive cells from T cell cultures transduced with retrovirus encoding WT hMALT1 degraded IκBα and expressed IL-2 in response to stimulation with PMA/IO, indicating that WT hMALT1 can rescue the signaling defect in murine Malt1^−/− T cells. In contrast, the retrovirus encoding the mutant MALT1^S89I failed to rescue the signaling defect in Malt1^−/− T cells, demonstrating that this mutation abrogates NF-κB signaling (Fig 5).

Figure 5. Failure of the MALT1 mutant to reconstitute IκBα degradation and IL-2 expression in T cells from Malt1^−/− mice.

A. Intracellular FACS analysis of IκBα and IL-2 in PMA+IO stimulated CD4⁺ T cells from Malt1^−/− mice that were uninfected, or transduced with human WT or mutant MALT1 and Thy1.1. Gated CD4⁺Thy1.1^high and CD4⁺Thy1.1^low cells were analyzed. The fluorescence intensity of Thy1.1^high cells was comparable in cells transduced with mutant and WT MALT1. Data are representative of two experiments. B. Kinetic analysis of IκBα degradation post-stimulation with PMA+IO.

DISCUSSION

Several lines of evidence support the notion that the mutation in MALT1 was responsible for the immunodeficiency in the patients. First, MALT1 was the only gene with a non-synonymous novel mutation within the regions of homozygosity shared by the patients. Second, the mutation abolished MALT1 protein expression. Third, like T cells from Malt1^−/− mice, T cells from the patient studied had impaired IκBα degradation and IL-2 expression after PMA/IO stimulation. Fourth, retroviral transduction of the mutant hMALT1^S89I, unlike that of the wild type MALT1, failed to correct the signaling defect in T cells from Malt1^−/− mice. Finally, the patients share clinical and laboratory findings with a recently described patient with a loss of function mutation in CARMA-1/CARD11, another member of the CBM complex.¹⁶ The shared findings include normal number of circulating T and B cells, poor antibody responses, decreased T cell proliferation to anti-CD3, and impaired IκBα degradation and IL-2 expression following T cell stimulation with PMA+IO. In addition, the CARD 11 deficient patient had virtually absent memory B cells and impaired interferon-γ secretion by NK cells.

Several features are shared between the patients and MALT1 deficient mice. These include the normal number and subset distribution of peripheral T cells and their defective proliferation to mitogens and anti-CD3.⁶ The numbers of circulating B cells in the patients were normal, as are the numbers of follicular B cells in the spleens of Malt1^−/− mice. The severely reduced antibody response to pneumococcal polysaccharide vaccine and the absence of natural isohemagglutin antibodies in the patients are consistent with the severe reduction in Malt1^−/− mice of marginal zone B cells and peritoneal B1 cells, which produce antibodies to type II T-independent antigens, such as pneumococcal polysaccharides,¹⁷ and IgM natural antibodies respectively.¹⁸ The limited numbers of PBMCs available on Pt 1 and the lack of availability of EBV-transformed B cells from either patient precluded us from examining whether BAFFR signaling is impaired in our patients' B cells, as in B cells from Malt1^−/− mice.⁷ Both patients had normal serum immunoglobulin levels, in contrast to Malt1^−/− mice whose serum immunoglobulin levels are low. This difference is likely explained by non-specific polyclonal B cell activation in the chronically infected patients but not in the infection-free Malt1^−/− mice housed under pathogen-free conditions.

The patients we report demonstrate the impact of MALT1 deficiency on host defense against environmental pathogens. The spectrum of susceptibility to infectious organisms has not been evident from studies of Malt1^−/− mice because they are reared in specific pathogen-free conditions. Defective T cell activation by antigens likely played a critical role in the patients' susceptibility to opportunistic infections with CMV and C. albicans. Recently, a mutation in CARD11, a member of the CBM complex which functions immediately upstream of MALT1, has been reported in a patient with hypogammaglobulinemia and Pneumocystis jirovecii pneumonia.¹⁶ This patient had only subtle changes in the T-cell compartment, whereas B cell differentiation was blocked at the transitional stage. The present report, together with these findings, indicates the importance of the CBM complex in host defense.

MALT1, by associating with CARD9 and Bcl-10, links the β-glucan receptor dectin-1 and the α-mannan receptor dectin-2, which are important for innate immunity against fungi,¹⁹ to NF-κB activation in DCs and macrophages.^20,21 Patients with mutations in CARD9 suffer from fungal infections, but have not been reported to suffer from other opportunistic infections.²² MALT1 is also essential for dectin-1- and dectin-2-driven production of the Th17 polarizing cytokines IL-1β and IL-23p19 by DCs in response to C. albicans.²³ The IL-17 response is critical for host protection against infection with C. albicans, as mutations in IL-17 and IL-7Rα are associated with recurrent and persistent candidiasis.²⁴ MALT1 has been recently shown to be important for the Th17 response in mice.²⁵ Thus, MALT1 deficiency in DCs and macrophages may have contributed to the susceptibility of the patients to infection with C. albicans by interfering with IL-17 production.

Taken together, the clinical, immunologic and biochemical findings strongly suggest that MALT1 deficiency in humans causes autosomal recessive CID. This novel immunodeficiency syndrome demonstrates the importance of MALT1 in T cell activation, expands the phenotype of disease associated with mutations in components of the CBM complex,^16,22,26 and highlights the importance of screening for mutations in genes encoding other members of the CBM complex and related signaling proteins in CID patients.

Clinical implications: A search for mutations in MALT1 should be performed in patients with combined immunodeficiency.

Abbreviations

BCR

B cell receptor

BCL-10

B-cell lymphoma-10

CARD

Caspase-recruitment domain

CARMA

Caspase recruitment domain membrane-associated guanylate kinase protein

cIAP2

Cellular inhibitor of apoptosis 2

CID

Combined immunodeficiency

DT

Diphtheria toxin

IκBα

Inhibitor of NFκBα

IO

Ionomycin

MALT1

Mucosa associated lymphoid tissue lymphoma-translocation gene 1

NFκB

Nuclear factor κB

PMA

Phorbol myristic acid

PWM

Pokeweed mitogen

SNP

Single-nucleotide polymorphism

TCR

T cell receptor

TT

Tetanus Toxoid

WGS

Whole genome sequencing