Mistuned NF-κB signaling in lymphocytes: lessons from relevant inborn errors of immunity

Summary

Inborn errors of immunity (IEIs) continuously remind us that multiple checks and balances are built into the adaptive immune system to maintain homeostasis, ensuring effective pathogen defense without causing inadvertent immunopathology, autoimmunity, or lymphomagenesis. The nuclear factor of κB (NF-κB) family of transcription factors serve a vital role in the immune system, inducing scores of genes responsible for lymphocyte survival, proliferation, differentiation and effector function. In recent years, the discovery and characterization of IEIs that impact NF-κB activity have illuminated the importance of carefully tuning this pathway to ensure effective immune defense without hyperinflammation and immune dysregulation. Here we examine several illustrative cases of IEIs that arise from pathogenic mutations encoding NF-κB inducers, regulators, and NF-κB family components themselves, illuminating how these genes ensure normal adaptive immune system function by maintaining a “Goldilocks effect” state in NF-κB pathway activity.

This review discusses human inborn errors of immunity caused by genetic variants that disrupt normal NF-κB signaling, primarily in T and B lymphocytes. The discovery and investigation of these disorders underscores how mistuned NF-κB signaling can contribute to both immune deficiency and dysregulation.

Graphical Abstract

Graphical Abstract.

Introduction

Inborn errors of immunity (IEI) represent a group of ~430 genetic disorders that result in increased susceptibility to infections, autoimmune disorders, autoinflammatory diseases, allergy, and/or malignancy [1–3]. While some IEIs simply present as pure primary immune deficiencies (PIDs) with specific and/or recurrent infections, other gene defects result in broader phenotypic spectra of immune dysregulation better classified as primary immune regulatory disorders (PIRDs) [4]. Accessible next-generation sequencing (NGS) technology has enabled rapid identification of novel genetic variants, making the diagnosis of once rare disorders more commonplace, with an aggregated prevalence between 1/1000 and 1/5000 individuals [5]. Monogenic germline mutations can result in loss of protein expression and/or loss-of-function (LOF, amorphic/hypomorphic) or gain-of-function (GOF, hypermorphic), often inherited as autosomal recessive (AR) or dominant (AD) traits, respectively. A heterozygous variant may also trigger AD disease by exhibiting dominant-negative (DN) interference or haploinsufficiency. Studies linking molecular mechanisms of pathogenesis to the broad range of clinical outcomes associated with different monogenic disorders have helped establish the foundations of human immunology, revealing non-redundant roles for specific genes in immune function, and exposing avenues for targeted therapeutics. Herein we explore the crucial role of NF-κB in lymphocyte function and immune homeostasis, informed by IEIs caused by mutations that perturb an otherwise finely tuned signaling pathway.

The NF-κB family is a complex master regulator of gene transcription in hematopoietic cells, held in latency in the cytoplasm until the engagement of a relevant receptor (e.g. pattern recognition receptor (PRR), TNF superfamily, antigen receptor (AgR)) triggers nuclear translocation, and transcriptional activity. Originally discovered as a nuclear factor binding near the κ light-chain gene in B cells [6], active NF-κB binds to -κB consensus sites found in gene promoters and enhancers. Dimeric NF-κB complexes are assembled from five monomeric subunits comprising the Rel transcription factor family: NFKB1/p105/p50, NFKB2/p100/p52, RELA/Rel A (p65), RELB/Rel B, and REL/c-Rel [7]. These proteins can homo- and hetero-dimerize to create 15 different NF-κB complexes that control the transcription of many genes responsible for cell survival, proliferation, and immune function. Each subunit contains a 300 amino acid Rel homology domain (RHD) that allows for the sequence-specific DNA binding, dimerization, and inhibitory protein binding, as well as a nuclear localization sequence (NLS) [6, 8, 9]. However, p100 and p105 do not contain transactivation (TA) domains, and therefore must dimerize with other REL proteins to drive transcriptional activity [6, 9]. The canonical NF-κB pathway utilizes p105/p50, Rel A(p65), and c-Rel, whereas the alternative pathway utilizes p100/p52 and Rel B [7] (Fig. 1A). Although numerous IEIs have been linked to gene variants affecting both pathways and summarized in detail elsewhere [6], this review primarily focuses on both PIDs and PIRDs linked to defects in canonical NF-κB signaling and their effects on adaptive immunity (Table 1).

Figure 1:

(A) Schematic diagram of canonical (e.g. TCR) versus alternative (e.g. CD40) NF-κB signaling pathways in lymphocytes. (B) Illustration of ‘Goldilocks’ principle of tuned NF-κB signaling to maintain immune homeostasis. Too little or too much NF-κB signaling in leukocytes can manifest in specific gene defects which give rise to primary immune deficiency (PID) and/or primary immune regulatory disorder (PIRD) phenotypes.

Table 1:

Clinical and laboratory abnormalities found in monogenic IEIs associated with reduced or excessive NF-κB signaling in lymphocytes

Reduced NF-κB signaling
Gene	Protein	Effect	Inheritance	Major clinical presentation	Other clinical symptoms	Laboratory abnormalities
NFKB1	P105/P50	LOF	AD (haploinsuff)	CVID	Respiratory/GI infections, autoimmunity, lymphoproliferation	Low Ig, low CS/memory B cells, low Tregs
NFKB2	P100/P52	LOF	AD	CVID, adrenal insufficiency	Autoimmunity	Low Ig, low CS/memory B cells
RELB	RELB	LOF	AR	CID	Autoimmunity, FTT	Impaired T/B cell development, dysplastic thymus
NFKBIA	IκB	GOF	AD	EDA-ID	Recurrent pyogenic infections	Dysgammaglobulinemia, low memory T cells, poor T cell Ag responses
IKBKG	NEMO	LOF	XL	EDA-ID	IP, lethal herpesvirus infections, autoinflammation	Low Ig, low NK cell function, poor inflammatory cytokine production
IKBKB	IKKβ	LOF	AR	SCID		Poor T/B cell Ag responses, low memory T/B cells
CARD11	CARD11 CARMA1	LOF	AR	SCID	P. jirovecii pneumonia	Low Ig, low memory T/B cells, low Tregs, poor T/B cell Ag responses
CARD11	CARD11 CARMA1	DN	AD	Severe atopic disease	Recurrent respiratory/cutaneous viral infections, autoimmunity	Low Ig, low CS/memory B cells, poor T cell Ag responses, Th2 skewing, high IgE, eosinophilia
BCL10	BCL10	LOF	AR	SCID	Recurrent respiratory viral infections, oral candidiasis	Low memory T/B cells, absent Tregs, poor T/B cell Ag responses
MALT1	MALT1	LOF	AR	CID + IPEX-like	Eczema, enteropathy, FTT, periodontal disease	Very low Tregs, poor T cell responses to Ag

Excessive NF-κB signaling
IKBKB	IKKβ	GOF	AD	Mild CID	Inflammation, epithelial defects	Lymphopenia, low Ig, increased/normal Tregs, increased T cell Ag response
CARD11	CARD11 CARMA1	GOF	AD	Polyclonal B cell lymphocytosis	Splenomegaly, mild recurrent infections,	Low CS/memory B cells, low T cell responses to Ag
TNFAIP3	A20	LOF	AD (haploinsuff)	Behcet-like autoinflammation	Retinal scarring, oral ulcers, skin abscesses, fevers	Elevated inflammatory cytokines (e.g. IL-1β), Th9/17 skewing

Abbreviations: AD: autosomal dominant; Ag: antigen; AR: autosomal recessive; CID: combined immunodeficiency; CS: class-switched; CVID: common variable immunodeficiency; EDA-ID: anhidrotic ectodermal dysplasia with immunodeficiency; FTT: failure to thrive; IPEX: immune dysregulation, polyendocrinopathy, enteropathy, X-linked); XL: X-linked.

CARD11 (caspase recruitment domain family member 11) encodes a scaffold protein primarily expressed in lymphocytes that bridges AgR ligation with the activation of downstream signaling pathways including NF-κB, c-Jun N-terminal kinase (JNK), and mechanistic target of rapamycin (mTOR) [7, 10–13]. Proximal AgR triggers assembly of the ‘CBM complex’ comprised of CARD11, BCL10 (B cell lymphoma 10), and MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), a pivotal regulator of lymphocyte proliferation and effector function [11, 14]. The importance of the CBM complex in T/B cell function was experimentally established when lymphocytes from mice deficient in BCL10, MALT1, or CARD11 all displayed impaired NF-κB activation, survival, proliferation, and cytokine secretion. These abnormalities were later confirmed by the discovery of human patients carrying pathogenic mutations in each component, manifesting in both immunodeficiency and/or immune dysregulation phenotypes collectively referred to as ‘CBM-opathies’ [15]. In recent years, our group and others have described a rapidly expanding group of IEIs linked to impactful CARD11 variants, all of which impact canonical NF-κB signaling.

The CARD11 scaffold is built from modular domains that allow for AgR-triggered BCL10 interaction (CARD domain), oligomerization (LATCH, coiled-coil), and higher order multimerization at the plasma membrane (membrane associated guanylate kinase (MAGUK) domain). Proximal B-cell receptor (BCR) and T-cell receptor (TCR) signaling is highly symmetrical, culminating in the phosphorylation of a complex inhibitory linker domain that normally retains CARD11 in an inactive state in resting lymphocytes [16, 17]. This derepression converts CARD11 to an open conformation allowing for pre-associated BCL10-MALT1 units to bind and oligomerize to initiate CBM complex formation (Fig. 1A) [18–21]. This dynamic, filamentous signalosome then acts as a recruiting platform for downstream signaling partners including the LUBAC (linear ubiquitin chain assembly complex) and an E3 ligase, TRAF6 [13, 22–24]. The LUBAC complex facilitates the physical recruitment of the IKK complex (IKKα-IKKβ-IKKγ) through IKKγ (also known as NEMO or NF-κB essential modifier), a noncatalytic regulatory subunit of the complex, and two kinase subunits IKKα and IKKβ, to ubiquitinated MALT1 [7, 25, 26]. TRAF6 polyubiquitinates MALT1 further by adding K63-linked ubiquitin chains, which recruit the TAB2/3-TAK1 complex to phosphorylate and activate the neighboring IKK complex partners IKKα/β [22, 23, 27–29]. IKK-mediated phosphorylation triggers proteasomal degradation of IκBα, exposing the NLS sequence on Rel family proteins to release NF-κB dimers into the nucleus. p105 is processed and partially degraded to a p50 subunit that can interact with other Rel family members (e.g. p65) and translocate to the nucleus to stimulate gene transcription (Fig. 1A) [6]. Extensive studies of both murine models and IEIs (including CBM-opathies) underscore this NF-κB transcriptional program must be carefully calibrated to ensure lymphocyte homeostasis, including negative feedback loops that promptly return NF-κB to latency.

The NF-κB dependent gene tumor necrosis factor alpha-induced protein 3 (TNFAIP3) encodes A20, one such important negative regulator of NF-κB signaling that restricts excessive inflammation [30]. A20 expression may be induced in numerous cell types by proinflammatory pathways that activate NF-κB [30]. Indeed, genome-wide association studies (GWAS) linked single nucleotide polymorphisms (SNPs) in TNFAIP3 to various autoimmune inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and inflammatory bowel disease (IBD) [31–34]. A20 was initially discovered to be a ubiquitin-editing enzyme, responsible for modulating the TNF signaling cascade through cleavage of K63-ubiquitin chains and addition of K48-ubiquitin to degrade participating proteins and terminate signaling [35, 36]. In T cells, A20 can cleave K63-linked ubiquitin chains attached to MALT1 via its deubiquitinase (DUB) activity to disrupt and downregulate NF-κB signaling (Fig. 1A) [37].

In this review, we primarily focus on the consequences of ‘mistuned’ NF-κB signaling in lymphocytes by examining illustrative IEIs that arise from mutations in NF-κB genes themselves (NFKB1/2), as well as key upstream activators (CBM complex genes, IKBKB, NFKBIA) and a crucial downstream regulator (TNFAIP3). We will review our current understanding of the genetics and pathophysiology associated with each disorder, resulting in clinical phenotypes ranging from pure immunodeficiency to complex immune dysregulation. In doing so, we reference relevant biochemical studies and murine models and highlight critical knowledge gaps that invite further mechanistic studies.

Too little NF-κB signaling

NFKB1 LOF

Monoallelic mutations in NFKB1 usually manifest as common variable immunodeficiency (CVID), one of the most commonly diagnosed IEI associated with increased susceptibility to respiratory infections, sometimes accompanied by other sequalae characteristic of a PIRD [38]. CVID is a clinically and genetically heterogeneous disorder linked to variants in at least 22 genes, although ~90% of CVID-like conditions remain genetically undefined [39]. CVID is characterized by hypogammaglobulinemia (with low IgG, IgM, and IgA), impaired antibody production (including after vaccination), and abnormal lymphocyte phenotypes including low class-switched memory B cells and regulatory T cells (Tregs), impaired natural killer (NK) cell maturation and cytotoxicity, and aberrations in T and B cell maturation and function [3, 40–45]. Milder specific antibody deficiencies restricted to IgA or IgG subclasses have been noted in relatives with or without clinical symptoms. Indeed, incomplete clinical penetrance (~70%) and age-dependent phenotypic severity has been reported, with autoimmunity also noted in a subset of patients [46]. Until recently, the mode of autosomal dominant inheritance associated with NFKB1-driven CVID was poorly understood. However, a recent report shed light on this mechanism by utilizing an NF-κB reporter assay that specifically tested p65:p50 heterodimer-dependent transcription [47]. Among 60 predicted LOF NFKB1 variants tested in this assay, 80% were confirmed LOF/hypomorphic with no evidence of DN activity, strongly suggesting haploinsufficiency as the explanation for AD NFKB1 deficiency. LOF haploinsufficiency results in partial reduction of both p105 and p50 in resting lymphocytes, with severely reduced p105 phosphorylation after stimulation. In the aforementioned study, all deleterious mutations were found in the RHD, where they were shown to disrupt dimerization, nuclear localization, DNA binding, and interaction with IκB proteins. However, mutations have been reported throughout the protein, including those localized to the C-terminus degron that disrupt p105 phosphorylation and processing to p50 [48]. These heterozygous LOF mutations have been shown to affect protein stability, subunit phosphorylation, and nuclear translocation, which likely contributes to variability in clinical phenotypes [49].

NFKB2 LOF

Although we focus here on canonical NF-κB signaling associated with AgR-signaling in lymphocytes, a growing collection of deleterious mutations identified in both NFKB2 and RELB illuminate how impaired alternative NF-κB signaling also manifests in CVID-like phenotypes in humans [50, 51]. NFKB2 deficient patients also suffer from recurrent respiratory infections in early childhood, but ~50% also develop pituitary hormone deficiencies and central adrenal insufficiency, distinguishing this IEI from NFKB1 deficiency [52]. Autoimmune manifestations affecting skin, hair, and nails are also observed in some patients; breakdown of tolerance affecting the endocrine system has been postulated but not proven. Recently discovered RELB-deficient patients share similar infectious susceptibility and humoral immune defects (despite normal serum Ig), consistent with a CID involving impaired T and B cell differentiation and reduced thymic output [53]. Reduced BAFF-R and CD40-induced alternative NF-κB signaling may account for poor B cell maturation, whereas the mechanism explaining thymic dysplasia and a skewed oligoclonal T cell compartment remains mysterious. Homozygous RELB LOF mutations result in total absence of RELB expression, whereas various LOF (−/+ DN activity) and GOF NFKB2 mutations have been described, often with incomplete penetrance [50, 54]. Indeed, two people harboring the same NFKB2 mutation can present differently, highlighting gaps in our understanding of alternative NF-κB signaling and the influence of other genetic and environmental modifiers—a common theme among many IEIs discussed here. These NFKB2 and RELB mutations have variable detrimental effects on T- and B-cell development, activation, proliferation, and survival [50, 53, 55]. Interestingly, NFKB1 and NKFB2 mutations typically manifest with similar CVID-like symptoms despite the divergent pattern of LOF mutations observed. While NFKB1 mutations affect residues throughout the p105/p50 protein, NFKB2 mutations are typically localized to the p100 degron, interrupting the ability of p100 to be degraded and converted to p52 therefore decreasing alternative NF-κB signaling [51, 56]. The inability to degrade p100 can also lead to increased binding to p65 and dominant interference of canonical NF-κB signaling as well, which may help to explain overlapping defects in lymphocyte differentiation and function [57]. Indeed, LOF mutations in MAP3K14 (NIK) cause CID due to defects in both canonical and alternative NF-κB signaling [58].

NFKBIA GOF

Among the IκB family, IκBα (encoded by NFKBIA) is the only member to date linked to a human monogenic IEI. Since 2003, 20 patients have been identified with heterozygous GOF NFKBIA mutations, resulting in autosomal dominant inheritance [59]. Patients typically present with profound CID, recurrent pyogenic and mycobacterial infections, and dysgammaglobulinemia, with most suffering from anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). Most NFKBIA GOF mutations are either missense variants that disrupt the phosphorylation of residues Ser32 and Ser36, or N-terminal protein truncations removing these serine residues altogether. Although IκBα protein is expressed, these mutations abrogate stimulation-induced phosphorylation and Ub-mediated degradation, enhancing inhibitory function by remaining constitutively bound to NF-κB [60]. Patient T cells respond poorly to antigens, resulting in no detectable memory T cells despite significant naive lymphocytosis. Impaired TLR, TNFR, and CD40 signaling are also observed. Patients harboring one of four mutations resulting in early stop codons presented early in life with EDA-ID and similar infectious manifestations. Hematopoietic stem cell transplantation (HSCT) remains the only viable option for correcting hematopoietic deficits in these patients [59].

IKBKG LOF

Notably, X-linked EDA-ID can also arise from hemizygous hypomorphic mutations in NEMO/IKBKG, manifesting in similar infection susceptibility with milder T cell immunodeficiency relative to NFKBIA GOF patients [61]. NEMO is critical for relaying signals downstream of AgRs, TNFR, TLR3, and RIG-I-like receptors (RLRs), resulting in activation of both NF-κB and IRF3. Similar to other genes discussed herein, the phenotypic breadth of clinical symptoms varies depending on the location and potency of IKBKG mutations, ranging from PID- to PIRD-like presentations [62]. Whereas N-terminal deletions disrupt IKKα/β association to drive incontentia pigmenti, CID, and lethal herpesvirus infections, C-terminal deletions can also trigger hyperactive TLR/TNFR signaling and autoinflammatory disease due to abrogated recruitment of the negative regulator A20 (see below) [63, 64]. Intriguingly, a new PIRD dubbed NEMO deleted exon 5 autoinflammatory syndrome (NDAS) was recently linked to alternative splicing mutations that specifically remove exon 5, which contributes to differential signal dysregulation from RLRs and TNF in dermal fibroblasts versus peripheral blood mononuclear cells (PBMC) [65].

IKBKB LOF

IKBKB encodes IKKβ, a key subunit of the IKK complex responsible for driving canonical NF-κB activity. Two homozygous IKBKB LOF variants have been reported since 2013, including a missense mutation (Y395H) and a homozygous G duplication in exon 13, resulting in a frameshift starting at amino acid 432 (Gln432Profs*62). Patients harboring these variants presented early in life with bacterial, viral, fungal, and mycobacterial infections. Despite normal T and B cell development, AgR-induced responses are severely impaired, resulting in a severe combined immunodeficiency (SCID) phenotype [66, 67]. Without IKKβ, normal T/B cell activation, proliferation, and differentiation into effector and memory cells are blocked, explaining an overabundance of naïve cells. Eight patients received HSCT, but only three survived [67]. PBMCs from Y395H carriers showed degradation of IKKβ protein with normal levels of IKKα and IKKγ, resulting in reduced canonical NF-κB activation [68]. Gln432Profs*62 results in premature stop codon and results in complete loss of IKKβ protein. In these patients’ cells, IKKα and NEMO/IKKγ also showed decreased protein levels despite normal mRNA expression, implying IKKβ has some stabilizing effect on the IKK complex. Importantly, patient T- and B cells showed decreased responses to stimulation through TLRs, cytokine receptors, and mitogens as well, highlighting that both innate and adaptive immune cells are impacted in this PID [69].

CBM deficiency: SCID

Before human CBM variants were characterized, several groups demonstrated that murine knockouts of Card11, Bcl10, or Malt1 all resulted in profound T and B cell activation defects and panhypogammaglobulinemia despite normal lymphocyte development. More recently discovered CBM deficiencies in CID patients largely mirror these PID-like phenotypes. Five cases of germline homozygous LOF mutations in CARD11 have been reported in humans [70–73]. All patients were born to consanguineous parents and presented early in life with severe respiratory tract infections and pneumonia typically caused by Pneumocystis jirovecii. CARD11 mutations were localized to either the CC or MAGUK domains including Phe902_Glu946del, Gln945*, Cys150*, and Arg837* [72], often preserving the expression of a truncated but non-functional protein. Despite normal B and T cell frequencies, patients displayed increased numbers of transitional B cells, decreased (class-switched) memory B cells, predominant naïve T cells with severely reduced Tregs, and pan-hypogammaglobulinemia. As expected, patient lymphocytes exhibited diminished AgR-induced activation including decreased p65 phosphorylation and impaired IκBα degradation, decreased IL-2 production, and poor T cell proliferation. In one case, a somatic reversion (Cys150Leu) partially restored CARD11 function in a proportion of conventional T cells, manifesting in Omenn syndrome [70]. A handful of patients described with BCL10 or MALT1 deficiency (due to AR LOF variants) present with a highly similar CID phenotype, as well as immune dysregulation marked by GI and respiratory tract inflammation, likely driven by a striking death of Tregs [15]. In fact, MALT1 deficiency resembles the PIRD known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome (e.g. eczema, enteropathy, and failure to thrive), with periodontal disease included as an unusual phenotypic twist. Curative HSCT is recommended for any CBM-deficient patient, with a relatively high success rate observed to date.

CARD11 DN: CADINS

CARD11-associated atopy with dominant interference of NF-κB Signaling (CADINS) patients have germline heterozygous LOF, DN mutations in CARD11 that manifest in severe atopic disease and CID [74–76]. To date, over 70 DN LOF CARD11 mutations have been uncovered in a rapidly expanding patient cohort. Roughly 90% present with allergic inflammation, most commonly atopic dermatitis followed by asthma, rhinitis, food allergies, or eosinophilic esophagitis, aligning with increased IgE and eosinophilia in most patients [15]. About two-thirds of patients also present with serious cutaneous viral infections (e.g. HSV-1, molluscum) and respiratory tract infections. Smaller subsets of patients also present with autoimmunity and malignancy, further classifying CADINS as a true PIRD. Upon TCR stimulation, patient T cells show decreased activation of NF-κB and mTORC1, reduced proliferation, and Th2-skewed cytokine production (elevated IL-4/IL-13, decreased IFN-γ) [74]. Most mutations are localized to the CARD and CC domains, impeding CBM complex assembly by disrupting BCL10-MALT1 binding and oligomerization post-AgR engagement. Although attenuated TCR-induced NF-κB activity helps to confirm diagnosis, effects on other downstream transcription factors (e.g. AP-1) activation remain an open question.

Before the discovery of CADINS disease, Goodnow et al. described ‘unmodulated’ mice harboring a homozygous CC Card11 mutation that developed dermatitis and high IgE levels with age [77]. More recently, a new mouse model expressing a homologous Card11 R30W mutation identified in five separate CADINS patients demonstrated similar T and B cell defects in heterozygosity [78]. Mouse CD4⁺ T cells showed diminished IL-2 and IFN-γ production and decreased CD25 upregulation after AgR stimulation, consistent with impaired NF-κB signaling (decreased IKKα/β phosphorylation and IκBα degradation). Milder B cell activation defects were also apparent (e.g. reduced CD86 upregulation) without an overt IκBα degradation defect, suggesting differential signaling impacts in T and B cells. Most interestingly, aged R30W mice exhibited elevated serum IgE without any signs of Th2 skewing or atopic disease, suggesting other genetic and environmental triggers in CADINS pathogenesis. Although Treg frequency and function are compromised in both mouse models [78, 79], this does not appear to translate to CADINS patients [76]. Recently, Bedsaul et al. proposed a ‘CARD11 oligomer poisoning’ model to describe how LOF CARD11 mutants can dominantly interfere with wild-type (WT) CARD11 in mixed oligomers during two distinct steps of the signaling cascade; either when CARD11 converts to an open conformation and/or interacts with cofactors (BCL10, HOIL-1) necessary for downstream signaling [10].

Too much NF-κB signaling

IKBKB GOF

V203I is the only known IKBKB GOF mutation in humans, manifesting as CID in two unrelated families. All affected patients presented with lymphopenia, recurrent respiratory infections, and hypogammaglobulinemia, with some exhibiting signs of inflammation as well. This mutation in the kinase domain results in increased IκBα and p65 phosphorylation in lymphocytes at baseline and post-AgR stimulation. Whereas both CD4 and CD8 T cells were hyperactivated, B cells also showed decreased proliferation and antibody production following stimulation. Although disease is less severe than the complete LOF of IKBKB, the fact that both LOF and GOF mutations confer immune deficiency underscores the importance of properly tuned NF-κB responses for effective adaptive immunity [80].

CARD11 GOF (BENTA)

B-cell Expansion with NF-κB and T cell Anergy (BENTA) disease is a PIRD caused by inborn heterozygous, GOF mutations in CARD11 and generally results in a mild immunodeficiency [81]. To date, 11 validated GOF mutations have been documented in human patients, largely confined to the LATCH and CC domains [15, 82–84]. BENTA patients present with selective, polyclonal B cell lymphocytosis and splenomegaly early in life, commonly combined lymphadenopathy, and recurrent sinopulmonary infections. Lab findings include increased immature/transitional (CD10⁺), naïve mature (IgM⁺IgD⁺) polyclonal B cells, and decreased class-switched and memory B cells [15]. Autoantibodies have been observed in some BENTA patients, which may relate to enhanced survival of self-reactive B cells [85]. Although autoimmunity is not observed in most BENTA patients, autoimmune cytopenias, atopic inflammation, and severe immune dysregulation (e.g. hemophagocytic lymphohistiocytosis) have been observed in recent case reports [83, 86, 87]. Surprisingly, patient T cells are hyporesponsive in culture with decreased proliferation and IL-2 secretion in response to TCR stimulation, appearing mildly anergic [81]. This phenomenon may help to explain more viral infections in some patients, including disseminated molluscum and moderate EBV viremia.

GOF mutations disrupt complex repressive elements in the inhibitory linker domain, forcing CARD11 into an open conformation [16]. This results in the spontaneous formation of cytoplasmic aggregates called mutant CARD11-dependent shells (mCADS) that include MALT1 and active IKKα/β that drive constitutive NF-κB signaling independent of AgR-engagement, although these aggregates are more dynamic in primary patient T/B cells [88]. Interestingly, one recent mouse study highlighted a role for Card11 in non-canonical regulation of the AKT-FOXO1 signaling axis, independent of NF-κB activation, utilizing the GOF Card11 mutants K215M and E134G [89]. Moreover, the contribution of dysregulated JNK or mTORC1 signaling to clinical pathogenesis remains largely unexplored. Collectively, these threads emphasize a need to investigate how CARD11 mutants affect other crucial non-NF-κB signaling pathways in lymphocytes, keeping in mind that T- and B-cell signaling is not necessarily symmetrical.

BENTA patient B cells have an inherent survival advantage in vitro relative to healthy controls, providing a plausible explanation for B cell accumulation in vivo [90]. This survival advantage appears to be dependent on MALT1 protease activity (unpublished data). As in B cell lymphomas harboring identical somatic CARD11 GOF mutations [91], constitutive NF-κB activation is thought to drive the expansion of the B cell compartment via enhanced survival and resistance to apoptosis. Currently, the role of negative regulators of NF-κB is unknown in BENTA patient lymphocytes. Based on our preliminary data, we hypothesize that the survival advantage in BENTA B cells is linked to a loss of A20-dependent negative regulation, resulting from constitutive cleavage of A20 by active MALT1 paracaspase (unpublished data). Indeed, genetic deletion of A20 is frequently found in B cell lymphomas with elevated NF-κB activity, and ectopic restoration of A20 expression induces apoptosis [92, 93]. CARD11 mutations highlight the importance of elucidating the impact of a novel variants on the function of the encoded proteins and thus the mechanism of pathogenicity.

TNFAIP3 LOF: A20 haploinsufficiency

Protein ubiquitination is a key post-translational modification necessary for regulating signal transduction in diverse inflammatory pathways. Ubiquitin (Ub) chains can form through any of ubiquitin’s seven lysines, allowing for polyubiquitin linkages needed for signal amplification (K63- and M1-linked ubiquitin) or proteasomal degradation of proteins for signal termination (K48-linked ubiquitin). TNFAIP3 encodes A20, a dual function enzyme built from an N-terminal OTU (ovarian tumor) domain with DUB activity, and seven C-terminal zinc finger (ZnF) domains which possess both E3 ligase activity (ZnF4) and Ub-binding capacity (ZnF7) [30]. ZnF4 has a monoubiquitin-binding domain (UBD) and E3 ligase activity which can add K48-linked ubiquitin to the direct destruction of NF-κB signaling intermediates [94]. ZnF7 was shown to inhibit LUBAC-induced NF-κB activation by binding to M1-linked (linear) ubiquitin [95, 96]. However, these experiments were conducted primarily in TNF-stimulated HEK293 cell and did not address MALT1-dependent cleavage of A20 in lymphocytes. Therefore, it is unknown if these mechanisms are involved in regulating AgR-induced NF-κB activity in lymphocytes.

A20 haploinsufficiency (HA20) is observed in patients with heterozygous LOF mutations in TNFAIP3 [97–100]. Patient PBMCs show markedly reduced A20 protein expression and increased NF-κB activation at baseline, with sustained signaling post-TNF stimulation. HA20 patients suffer early in life from retinal scarring and fibrosis, oral ulcers, and dermal lesions/abscesses; periodic fevers and other gastrointestinal and musculoskeletal autoinflammatory manifestations are also reported with remarkable variability. These diverse sequelae are largely explained by the loss of A20-dependent negative regulation of proinflammatory innate signaling pathways. Interestingly, intracellular cytokine staining of patient PBMCs stimulated with LPS or staphylococcal enterotoxin B (SEB) showed increased polarization toward CD4⁺ Th9 and Th17 effector cell lineages [97]. Similarly, when A20 was silenced in IBD and Vogt-Koyanagi-Harada (VKH) patients, CD4⁺ T cells shifted toward a Th1 and Th17 phenotype [101]. These data highlight A20 as a crucial mediator of inflammation and NF-κB signaling in both innate and adaptive immunity.

A20 is constitutively expressed in lymphocytes, with pleiotropic effects in controlling B and T cell function [30]. A20 is the most frequently inactivated gene in B-cell lymphomas, establishing A20 as a critical tumor suppressor gene [92, 93]. Compagno et al. documented biallelic inactivation of TNFAIP3 in ~30% of diffuse large B-cell lymphomas (DLBCL). Moreover, exogenous A20 expression in lymphoma-derived DLBCL lines lacking A20 resulted in suppression of cell growth, induction of apoptosis, and NF-κB downregulation. In addition, germline and somatic genetic TNFAIP3 variations were observed in 77% of patients with mucosa-associated lymphoma associated with primary Sjogren’s syndrome [102, 103]. Congruently, mice lacking A20 expression in B cells exhibited spontaneous B cell activation, expansion of germinal center B cells, and production of autoantibodies [104–106]. Loss of A20 in aged murine B cells was shown to lower their activation threshold, enhance proliferation and survival, and increase NF-κB signaling [104]. A20-deficient B cells also exhibit resistance to Fas-mediated apoptosis, likely related to enhanced expression of anti-apoptotic proteins [106]. These data collectively suggest that A20 acts to restrict B cell growth, survival, and oncogenesis by facilitating cell death, including in autoreactive B cells.

The role of A20 in regulating T cell development and activation is hazy and still under investigation. In mice, A20-deleted CD8⁺ T cells show heightened sensitivity to AgR stimulation, with increased NF-κB and production of IL-2 and IFN-γ [107]. Although these mice exhibit some lymphadenopathy and organ infiltration, there was no detectable pathology. Increased NF-κB activity in adoptively transferred A20-deficient CD8⁺ T cells confers enhanced tumor suppressive activity, with improved control of melanoma growth, enhanced production of IFN-γ and TNFα, and reduced expression of programmed cell death 1 (PD-1), making A20 a potential candidate for T cell-directed immunotherapy [108]. Moreover, A20 expression supported secondary, but not primary, T cell responses to Listeria monocytogenes infection [109]. In contrast, A20-deficient CD4⁺ T cells are susceptible to necroptosis and exhibit decreased autophagy and mTOR activity post-stimulation [110]. A20 also restrains intrathymic differentiation of Tregs and NKT cells [111, 112]. Whereas A20 functions to restrict B cell proliferation and survival, A20 appears to foster proper T cell development and survival, tuning of TCR signaling, and production of memory cells. Considering patients harboring LOF mutations in either CARD11 or A20 display abnormal T cell differentiation and immune dysregulation resulting in clinical disease, further work on these patients should shed new light on the importance of A20 in tuning signals downstream of the CBM complex to maintain immune homeostasis.

The precise mechanism by which A20 regulates AgR-induced NF-κB activity remains nebulous. Previous work suggested that MALT1 initially cleaves A20 in the local CBM complex environment shortly after TCR engagement, allowing signal propagation [37, 113]. NF-κB then induces A20 synthesis to dampen subsequent CBM signaling by cleaving K63-linked ubiquitin chains attached to MALT1 via its DUB activity, ostensibly breaking MALT1-IKK interactions to downregulate further NF-κB signaling [37]. Subsequent work suggests DUB activity is dispensable for A20 function in T cells [114]. In a recent study, Yin et al. demonstrated that A20 and A20-binding inhibitors of NF-κB-1 (ABIN-1) work in concert to modulate T cell activation [115]. A20 utilizes ABIN-1 as an adaptor to engage with the CBM complex and tune both initial and sustained bursts of TCR-induced NF-κB activity. This suggests that A20 and ABIN-1 are recruited to the CBM complex early on to create a threshold that TCR activation has to overcome to allow signal transduction to occur. In the final phases of sustained CBM signaling, the authors conclude that resynthesized A20 is cleaved by MALT1 to allow for signal transduction to proceed. Despite this new insight, it remains unclear which domains of A20 are essential to its localization and regulatory function, including crosstalk with other signaling proteins post-TCR engagement.

Conclusion

The discovery of several IEIs linked to monogenic variants affecting key signaling nodes in AgR-induced NF-κB activation and resolution have yielded fascinating insights into how this pathway is finely tuned to maintain immune homeostasis, effecting multiple aspects of lymphocyte biology. While too little NF-κB activity is clearly associated with impaired lymphocyte responses and PID, too much NF-κB activity often gives rise to immune dysregulation, sometimes accompanied by infectious susceptibilities noted in several PIRDs [4, 38]. Even though pathogenesis often encompasses innate immune dysregulation and non-hematopoietic phenotypes, lymphocytes remain a dominant context for understanding variations in clinical expressivity that result from mistuned NF-κB signaling. Laboratory-based screening of primary patient lymphocytes can often yield valuable diagnostic insights to decipher the impact of novel gene variants. For example, acute aberrations in mitogen or AgR-induced canonical NF-κB signaling can be captured in intracellular flow cytometric assays for differential phosphorylation and degradation of IκB, or phosphorylation/nuclear translocation of p65 [76, 116]. Comparable defects in alternative NF-κB signaling pathways (p100 processing, RELB nuclear translocation) may also be detected with appropriate stimuli (e.g. CD40L). Given the limited or oscillatory nature of NF-κB signaling enforced by negative feedback loops (e.g. IκB/A20 upregulation), more subtle defects may be difficult to ascertain without optimizing the potency and timing of AgR stimulation. Hence definitive diagnoses may require empirical testing of ectopically-expressed mutant gene constructs in relevant in vitro assays that encompass both acute signal transduction events and downstream reporters of NF-κB-dependent transcriptional activity, as we and others have employed [10, 47, 75, 81].

Effective therapeutic strategies for treating these disorders can be complicated, arguably requiring the identification of targets that restore NF-κB activity to equilibrium without overshooting in either direction (Fig. 1B). For example, MALT1 protease inhibitors offer a strategy for turning down AgR-dependent NF-κB signaling in relevant PIRDs (e.g. BENTA) without compromising activity entirely, and yet data from mice and recent human trials revealed unexpected impacts on Treg function that contribute to autoimmune manifestations [15, 117]. Hopefully the accelerating discovery of new IEIs connected to altered NF-κB activity will implicate novel regulatory mechanisms and unveil new therapeutic avenues for restoring balance in this essential pathway.

Glossary

Abbreviations

AD

autosomal dominant

AgR

antigen receptor

AR

autosomal recessive

BCL10

B cell lymphoma 10

BCR

B cell receptor

BENTA

B cell expansion with NF-κB and T cell anergy

CADINS

CARD11-associated atopy with dominant interference of NF-κB signaling;

CARD11

caspase activation and recruitment domain 11

CVID

common variable immunodeficiency

DLBCL

diffuse large B cell lymphoma

DN

dominant negative

DUB

deubiquitinase

EDA-ID

ectodermal dysplasia with immunodeficiency

GOF

gain-of-function

GWAS

genome wide association study

HSCT

hematopoietic stem cell transplantation

IBD

inflammatory bowel disease

IEI

inborn errors of immunity

IKK

IκB kinase

JNK

c-Jun N-terminal kinase

LOF

loss-of-function

LUBAC

linear ubiquitin chain assembly complex

MALT1

mucosa-associated lymphoid tissue lymphoma translocation protein 1

mTOR

mechanistic target of rapamycin

NDAS

NEMO deleted exon 5 autoinflammatory syndrome

NF-κB

nuclear factor of kappa B

NGS

next generation sequencing

NLS

nuclear localization sequence

PBMC

peripheral blood mononuclear cells

PID

primary immune deficiency

PIRD

primary immune regulatory disorder

PRR

pattern recognition receptor

RHD

Rel homology domain

RLR

RIG-I-like receptor

SCID

severe combined immunodeficiency

SNP

single nucleotide polymorphism

TA

transactivation

TCR

T cell receptor

TLR

Toll-like receptor

TNF

tumor necrosis factor

ZnF

zinc finger

Funding

This work was supported by a Specific Defect Research Program grant from the Jeffrey Modell Foundation (A.L.S.).

Conflict of interest

The authors declare no conflicts of interest.Author Contributions

Conception: G.D. and A.L.S. Figure preparation: G.D. Writing and literature review were conducted by both authors. Critical revisions were made by A.L.S. Both authors read, edited, and approved the final version of the article.

Animal Research

ARRIVE guidelines: Not applicable.

Data Availability

Not applicable—no new data were created or analyzed.

Permission to reproduce

Government license—A.L.S. is an employee of the U.S. Government, and this article was written as part of his employment.

Clinical trial registration

Not applicable.