Introduction
Human serine/threonine kinase 4 (STK4) deficiency is a rare autosomal recessive (AR) genetic disorder leading to combined immunodeficiency with severe T cell lymphopenia. This condition is characterized by a predisposition to a wide range of bacterial and viral infectious diseases, mucocutaneous candidiasis, lymphomas, and congenital heart disease [
1]. To date, STK4 deficiency has been reported in relatively few patients; therefore, the extent to which immune signaling and host defense mechanisms are impaired or dysregulated in affected individuals remains incompletely understood. However, the spectrum of clinical manifestations associated with STK4 deficiency has been steadily increasing with each new case report.
STK4 deficiency was first reported by Nehme et al. in two patients from unrelated Turkish families harboring a homozygous nonsense mutation in the
STK4 gene [
2]. The patients experienced complications due to recurrent bacterial and viral infections, most notably persistent Epstein–Barr virus (EBV) viremia, which ultimately resulted in Hodgkin B cell lymphoma. Due to weak expression of the homing receptors CCR7 and CD62L, the authors attributed the underlying mechanism of STK4 deficiency to increased death of naïve and proliferating T cells, and impaired homing of CD8
+ T cells to secondary lymphoid organs [
2]. Abdollahpour et al. reported the cases of three siblings of Iranian descent with a homozygous premature stop codon in the
STK4 gene [
3]. These patients suffered from T and B cell lymphopenia, intermittent neutropenia, and atrial septal defects, as well as recurrent bacterial and viral infections, mucocutaneous candidiasis, cutaneous warts, and skin abscesses. Interestingly, Schipp et al. reported a Turkish patient with STK4 deficiency who developed a highly malignant B cell lymphoma at the age of 10 years and a second, independent Hodgkin lymphoma 5 years later. However, no detectable EBV or other common virus infection was detected in this patient. The authors speculated that the lymphoma may have developed due to the lack of the tumor suppressive function of STK4 or perturbed immune surveillance due to the diminished CD4
+ T cell compartment [
4]. In contrast, most malignancies reported in patients with STK4 deficiency are associated with prolonged EBV viremia, ultimately leading to the development of B cell lymphomas [
2,
5‐
7]. More specifically, patients present with Hodgkin B cell lymphoma [
2], extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue [
8], Burkitt’s lymphoma [
7], or maxillary sinus diffuse large B cell lymphoma [
9]. Additional clinical features in patients with STK4 deficiency include salt-losing tubulopathy, suggestive of an acquired Gitelman syndrome, immune complex glomerulonephritis, and Castleman-like disease [
10], juvenile idiopathic arthritis [
11], human beta-papillomavirus-associated epidermodysplasia verruciformis [
11,
12], primary cardiac T cell lymphoma [
6], and short stature [
13].
Studies in mice and humans have shown that STK4 plays a pivotal role in lymphocyte function by regulating integrin-dependent T lymphocyte trafficking, proliferation, and differentiation [
14,
15]. Of note, the STK4 protein is broadly expressed in various human hemopoietic cells, most notably monocytes, and is not restricted to lymphocytes (
https://www.proteinatlas.org/ENSG00000101109-STK4). However, its role in T cell-independent functions is less well understood. Recently, Jørgensen et al. studied innate immune signaling in the context of STK4 deficiency by in vitro stimulation or infection of PBMCs obtained from an 11-year-old female STK4
−/− patient of a consanguineous Syrian family. These studies revealed defective type I/II and III interferon (IFN) responses to a variety of purified Toll-like receptor (TLR) agonists, live viruses and bacterial lysates due to impaired phosphorylation of the kinase TBK1 and the transcription factor IRF3 [
13]. The results also revealed increased apoptosis in STK4-deficient T cells and neutrophil granulocytes, possibly linked to defective FOXO signaling in STK4-deficient T cells as shown in earlier studies [
2,
3], further supporting the important role of STK4 in T cell survival.
In this study, we identified an AR STK4 deficiency in a child from consanguineous parents, which was due to a novel homozygous stop-gain mutation in a region encoding a coiled-coil domain located downstream of the kinase domain. We investigated the functional consequences of the new variant on innate and adaptive cell-mediated and humoral immune responses.
Discussion
In this study, we identified a novel stop-gain mutation in a patient with AR complete STK4 deficiency. This mutation was found to be located in a genomic region that encodes the coiled-coil domain of STK4, downstream of its protein kinase domain. We were unable to detect even a truncated STK4 protein in the patient using a monoclonal antibody to the N-terminal region of STK4, suggesting that protein expression of the mutated allele is completely abrogated due to nonsense-mediated decay.
In accordance with earlier case reports [
5,
8,
20], we found that the PBMCs isolated from our patient had reduced fractions of CD4
+ naïve, but increased effector memory, T helper cell subsets compared with those in the
STK4wt/mut family members and an unrelated
STK4wt/wt control. Furthermore, flow cytometric analysis showed a considerable proportion of the remaining T helper cell subset in the patient expressed higher levels of PD-1, and our RNA-Seq analyses revealed dysregulation of several pathways in the patient, suggesting elevated T cell exhaustion and impaired effector functions of the residual T cells. Whether this is a consequence of persistent EBV viremia [
21‐
23] or an intrinsic feature of STK4 deficiency, or perhaps both, remains to be established. Previous studies have shown that EBV reactivation correlates with the expression of PD-1/PD-L1 antigens in patients with proliferative glomerulonephritis [
24]. On the other hand, CD4
+ T cell lymphopenia has also been reported in STK4-deficient patients in the absence of detectable EBV infection [
4]. In addition, the patient presented with episodes of intermittent neutropenia, which is consistent with previous observations [
2,
3,
6,
9,
13].
Our results also highlight that STK4 deficiency can lead to the impairment of a variety of T cell-independent and innate immune responses. Indeed, we detected a considerable proportion of CD56
bright NK cells in the PBMCs isolated from the patient. While these cells constitute only a small fraction of NK cells in the peripheral blood of healthy individuals, they represent the majority of NK cells in secondary lymphoid tissues. CD56
bright NK cells are thought to be NK cell precursors [
25] and may have immunoregulatory properties [
26]. We also observed a decreased fraction of pDCs in the patient’s peripheral blood. Whether this is an indirect consequence of active EBV infection, as shown in mouse studies [
27], or whether low levels of pDCs contribute directly to a lack of EBV control, remains unclear. As reported by Jørgensen et al. [
13], we also observed dysregulated type I and II IFN signaling in the patient’s cells. Interestingly, transcriptomic analysis of the patient’s PBMCs in response to IFNAR activation in vitro revealed a marked dysregulation of IFN-regulated gene expression, affecting interferon-stimulated genes (ISGs). Our enrichment analyses of either IFN-a/IFN-b- or PMA/ionomycin-responsive genes that showed differential expression between the patient and controls revealed several gene networks reminiscent of dysregulated cytokine-stimulated cell adhesion, leukocyte chemotaxis, and impaired T cell activation, likely resulting in T cell exhaustion and enhanced immune cell death. Dysregulation of these proinflammatory cytokines and chemokines has also been implicated in cancer pathogenesis [
28]. Moreover, in accordance with our findings, Dang et al. demonstrated that leukocytes of patients with AR STK4 deficiency exhibited impaired chemotaxis after stimulation with CXCL11 and did not bind to ICAM-1 [
5].
It cannot be ruled out that the dysregulated type I IFN-induced gene expression signature in the patient’s PBMCs is, in part, also a consequence of abnormal proportions of some leukocyte subsets. Indeed, we observed decreased fractions of pDCs, which are potent producers of type I IFN under in vitro and in vivo conditions [
29]. Nonetheless, the short duration of the in vitro stimulation and gene expression experiments (2 h) and the low proportions of pDCs, CD56
bright NK cells, and effector memory T cell subsets relative to the total PBMC population make it less likely that abnormal proportions of some leukocytes subsets in the patient have a major effect on their PBMC transcriptome. Overall, the suboptimal IFN signaling may contribute to the T cell immunodeficiency and the vulnerability of STK4-deficient patients to viral infection and cancer development. However, overall fractions of CD19
+ B cells (Fig.
2A) and IgG antibody responses to childhood vaccination (Supplementary Table
S3) or common microbial infection (Fig.
3) did not appear to be diminished in our patient, apart from our observation that the antibodies were predominantly specific for HHV-4 and -5 antigens. Of note, a recent study in STK4
−/− mice and nine patients from five unrelated families with STK4 deficiency suggested that STK4 is required for normal humoral immunity since knockout mice and patients had reduced marginal zone B (MZB) cells as well as reduced numbers of innate-like B-1b cell subsets, while the overall fractions of circulating CD19
+ B cells were normal, as in our patient [
30]. This raises the possibility that patients with STK4 deficiency may also have a selective impairment in the ability to mount robust T cell-independent, polysaccharide-specific antibody responses to control natural infection with encapsulated bacteria, such as
H. influenzae,
K. pneumoniae and
S. pneumoniae, which is consistent with the clinical history of our patient. Polysaccharide-specific antibody responses (or the lack of) are undetectable using the PhIP-Seq assay as it exclusively detects antibodies that target protein antigens and is limited in its capacity to detect conformational and post-translationally modified epitopes [
31]. High efficacy of plain polysaccharide-based vaccines also depends on the maturation of MZB cells, which usually does not occur until the second year of life [
32]. In our patient, the specific antibody responses were at the lower end of our laboratory reference range (Supplementary Table
S3). However, anti-pneumococcal polysaccharide antibodies cannot be used as definitive markers of MZB cell-mediated immunity due to the introduction of the conjugate pneumococcal vaccine into the local routine immunization schedule. The literature shows variability in the specific antibody responses in STK4-deficient patients, ranging from normal to absent [
30]. The history of infection with
H. influenzae,
K. pneumonia, and
S. pneumoniae in our patient could have also interfered with the utility and interpretation of tests of responses to plain polysaccharide vaccines. Therefore, humoral immunity of patients with STK4 deficiency toward encapsulated bacteria requires further investigation.
We also demonstrated a profound impairment of
IL-23 gene expression in the patient’s PBMCs, both at baseline and following in vitro stimulation. IL-23 is produced by innate lymphoid cells, gamma-delta T cells, DCs, and macrophages, and it has been shown that
IL-23-dependent IFN- immunity plays a pivotal role in controlling
Mycobacterium tuberculosis (Mtb) infection [
33]. It is therefore tempting to speculate that impaired
IL-23 gene expression contributed to patient’s susceptibility to pulmonary TB. Despite the clinical evidence of pulmonary TB, the patient’s QuantiFERON test result was indeterminate, which is likely to reflect a combination of cellular dysfunction and profound lymphopenia. Of note, Radwan et al. [
7] also speculated that complications in a 9-year-old Egyptian boy with STK4 deficiency were associated with mycobacterial infection, although tuberculin skin-test results were negative, and the results from QuantiFERON tests were inconclusive.
It remains unclear whether malignancies in STK4-deficient patients are a secondary consequence of persistent EBV viremia, or whether such patients are inherently prone to malignancies due to dysregulation of oncogenes, even in the absence of EBV infection [
4]. Interestingly, our RNA-Seq experiments revealed upregulation of mitogen-induced B cell-activating factor (BAFF) receptor gene (
TNFRSF13C) expression in the patient, suggesting activation of BAFF signaling, in contrast to the controls where this pathway was inhibited following PMA/ionomycin stimulation (Fig.
5 and Supplementary Table
S4). Studies in vitro and in mice have shown that EBV drives autonomous B cell proliferation [
34], which also depends on T cell-independent survival signals provided by the BAFF receptor. Excessive BAFF levels have been implicated in several B-lineage malignancies [
35‐
38], which have also been reported in the context of STK4 deficiency, with or without EBV viremia [
2,
5‐
8,
10]. Our observations provide further mechanistic insights into the susceptibility of STK4-deficient patients to malignancies, although they do not allow firm conclusions to be drawn about the role of EBV in this process. Nonetheless, it is tempting to speculate that STK4-deficient patients, particularly those with persistent EBV viremia, may benefit from treatment with immune checkpoint inhibitors. Using a humanized mouse model, Ma et al. [
39] demonstrated a direct beneficial effect of PD-1/CTLA-4 blockade mediated by monoclonal antibodies against PD-1 or CTLA-4 alone, or in combination, on EBV-associated B cell lymphomas, thereby providing further evidence in support of this hypothesis. However, TB reactivation or primary Mtb infections have also been reported in cancer patients who received checkpoint inhibitors [
40‐
42]. Therefore, the potential therapeutic benefits of checkpoint inhibitors in patients with STK4 deficiency require further investigation.
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