Understanding immunoactinopathies: A decade of research on WAS gene defects

Immunoactinopathies caused by mutations in actin‐related proteins are a growing group of inborn errors of immunity (IEI). Immunoactinopathies are caused by a dysregulated actin cytoskeleton and affect hematopoietic cells especially because of their unique capacity to survey the body for invading pathogens and altered self, such as cancer cells. These cell motility and cell‐to‐cell interaction properties depend on the dynamic nature of the actin cytoskeleton. Wiskott‐Aldrich syndrome (WAS) is the archetypical immunoactinopathy and the first described. WAS is caused by loss‐of‐function and gain‐of‐function mutations in the actin regulator WASp, uniquely expressed in hematopoietic cells. Mutations in WAS cause a profound disturbance of actin cytoskeleton regulation of hematopoietic cells. Studies during the last 10 years have shed light on the specific effects on different hematopoietic cells, revealing that they are not affected equally by mutations in the WAS gene. Moreover, the mechanistic understanding of how WASp controls nuclear and cytoplasmatic activities may help to find therapeutic alternatives according to the site of the mutation and clinical phenotypes. In this review, we summarize recent findings that have added to the complexity and increased our understanding of WAS‐related diseases and immunoactinopathies.

genotype-proteotype-phenotype correlation exists. 42 This makes it possible to predict if a given WAS mutation will result in reduced or completely absent WASp expression. Flow cytometry analysis is needed to confirm the level of WASp expression and may predict the severity of the clinical outcome, especially in the absence of WASp expression and therefore guide clinical decisions for the treatment of the patient. 39,41 However, it is worth highlighting that environmental factors, such as gut microbiota composition and pathogens/antigen exposure during infancy can result in a different clinical course for patients classified with the same score. 44,45 In fact, a multicenter study that enrolled 173 XLT patients from twelve countries has shown that 13.9% had serious bleeding episodes, 6.9% had life-threatening infections, 12.1% developed autoimmunity, and 5.2% malignancy. Fatal bleeding and infection were also reported. 43 Therefore, there is an unmet need to better identify XLT patients (Ochs score 0.5-2) at risk to develop severe WAS disease (Ochs score 3-5).
The third clinical outcome associated with WASp deficiency is the gain-of-function XLN, that, on the Ochs scale, receives score 0, since the XLN patient symptoms do not resemble those of classical WAS and XLT patients. 3,36,46 For XLN patients, severe congenital neutropenia is observed as the main clinical phenotype together with recurrent major bacterial infections and monocytopenia. 47,48 Already in the first description of the disease, structural prediction, and biochemical experiments suggested that WASp harboring the XLN mutation Leucine-270 to Proline (L270P) resides in a constitutively open conformation. Experimental data confirm this prediction by showing increased degradation of XLN-WASp, increased phosphorylation of Tyrosine-291, and increased polymerized actin. [47][48][49][50][51][52] Six unique XLN mutations have been identified to date with amino acid mutations WAS-L270P, I294T, S272P, I290T, R268W, and F271S. 53 All patients have congenital neutropenia and treatment with G-CSF restores a number of blood neutrophils. Because of the possible genetic instability of many hematopoietic cells, 50,52,53 G-CSF should be given with some caution to the development of myelodysplastic syndrome and leukemia as seen upon long-term usage of G-CSF to treat neutropenia patients. 54

| Clinicalaspects,revertantmutations,and unmet needs
The first-line treatment for WAS is allogeneic hematopoietic stem cell transplantation (HSCT) with a human leukocyte antigen (HLA) compatible donor. For HSCT of WAS patients, the overall survival rate was 94% for those treated before the age of 5 years. [55][56][57][58] The outcome after HSCT is influenced by the level of donor engraftment where mixed donor chimerism is associated with an increased risk of poor immune reconstitution. This is a particularly important F I G U R E 1 Summary of the immunoactinopathies group of IEI. Two layers of inborn errors can affect actin cytoskeleton dynamics, one that affects proteins that directly interact with the actin cytoskeleton (marked in red) and the other involving proteins in cell signaling (marked in blue) that modulates the nucleating promoting factors proteins. ARP2/3, actin-related proteins-2/3; CDC42, cell division control protein 42 homologue; DOCK, dedicator of cytokinesis protein; F-actin, filamentous actin; GDP, guanosine diphosphate; GEF, guanine nucleotide-exchange factor; GTP, guanosine triphosphate; LSP-1, leukocyte-specific protein I; MKL1, megakaryoblastic leukemia 1; MSN, moesin; RAC2, Ras-related C3 botulinum toxin substrate 2; SRF, serum response factor; WASp, Wiskott-Aldrich syndrome protein; WRC, WASp family verprolin homologue protein regulatory complex; WDR1, WD repeat-containing protein 1; WIP, WASp-interacting protein. The black arrows indicate the protein-protein interaction/activation. Complete list of immunoactinopathies in Table 1.
aspect for the correction of thrombocytopenia. Other treatment options include haploidentical HSCT from a close relative 59,60 and gene therapy to correct HSCT by lentiviral transfer of a WAS gene (cDNA). Gene therapy is especially suited for primary immunodeficiencies as it targets the HSCs that give rise to all hematopoietic lineage cells. 61 Success rate is directly related to the selective advantage of gene-corrected cells. The gene therapy approach provides less toxicity compared with the full conditioning regimen for allogenic HSCT and the long-term follow-up during an average of 7 years (range, 4-9 years) indicates sustained multilineage engraftment of WASp-expressing cells and clinical resolution of eczema and recurrent infections. [62][63][64] While gene therapy alleviates platelet counts, platelet number is still suboptimal possibly due to the low expression of WASp in platelets. [62][63][64] Somatic revertant mutation mosaicism is a naturally occurring phenomenon in patients affected by immunoactinopathies. It may be of clinical relevance because of the selective growth advantage of the corrected cells and the improvement of disease symptoms.
The WAS gene is located on the X chromosome and only males are affected by the disease whereas carrier mothers are asymptomatic.
Carrier females have random X-inactivation in hematopoietic stem cells, but the strong selective advantage of WASp-expressing cells over WASp null cells leads to mostly WASp-expressing cells in peripheral tissues and especially notable in B-and T-cell lineages. [65][66][67] Somatic revertant mosaicism has been detected in several WAS patients and especially in the T-cell lineage of cells. Interestingly, these revertant mutations in the WAS gene lead to normal WASp expression and functionality. 68

| ThenuclearroleofWASpfamilymembers
WASp has a nuclear localization signal (NLS)-like motif and a nuclear export signal (NES) encoded by exon 7 and exon 2-3, respectively ( Figure 2). WASp lacks a direct DNA binding motif, 97 nevertheless WASp interacts proximal to DNA and chromatin immunoprecipitation has been used to study WASp binding DNA elements. 97,98 Several WASp family members are present in the nucleus, and it is feasible to assume, based on their activity in the cytoplasm, that  Despite the increased propensity to be degraded, WASp-L270P and WASp-I294T, murine WASp-L272P and WASp-I296T, induce increased actin dynamics that contribute to increased cellular stiffness but also increased cellular dynamics. 51,52,110 This is at least partly due to increased phosphorylation of the Tyr291 (murine Tyr293-WASp) and localization to cellular membranes. 53

| Hematopoiesis
In WAS, there is no strong evidence of impaired hematopoiesis,

| Megakaryocytesandplatelets
Thrombocytopenia and small platelet size, known as microthrom- be underreported. The platelets number is normal or low to normal, but thrombocytopenia was also observed with large-sized platelets (macrothrombocytopenia). [47][48][49]54,118 In this context, it was recently observed that neutrophils interact with MK inside the BM and potentially contribute to platelet production, whereas neutropenia blunts thrombopoiesis. 127

| Neutrophils
Neutrophils can rapidly change cell shape and move into tissue and this capacity is important during inflammatory processes. WASp is essential for neutrophil migration by controlling the balance be-

| Monocytesandmacrophages
Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses and this is evidenced by how WAS mutations affect the physiology of monocytes/macrophages. Early studies showed that patient-derived monocytes

F I G U R E 4 WAS mutations cause defects in B, T, and NK cells. Dysfunction of WAS and XLN B cells, T cells, and NK cells. (Upper left panel) TCR signaling defects and decreased cytokine production of WAS T cells. (Upper right panel) Impaired antibody production but increased auto-antibodies caused by cell-intrinsic and -extrinsic defects of WAS B cells. Increased genomic instability with increased γH2AX and R-loops of WAS B and T cells. Increased apoptosis, decreased IgA class switching, and increased plasma cell formation of XLN B cells. (Lower panel) Poor effector function of WAS NK cells and increased cytotoxic activity by XLN NK cells. DSB, double-strand break; GZB, granzyme B.
have decreased migratory response. 134 WASp is important in cell polarity during migration and as part of the podosome initiation complex, being stably recruited by PI3K enriched regions in these structures, together with WIP ( Figure 3). [135][136][137] WASp-Tyr291 phosphorylation is important for macrophage migration 137 by regulating the rate of podosome nucleation, actin filament stability, and matrixdegrading capacity. 138 Whereas WAS and XLT patient macrophages have an impaired podosome formation, XLN patient macrophages have enhanced actin polymerizing activity and an increased number of podosomes ( Figure 3). Interestingly, the XLN podosomes are extremely dynamic with a high rate of turnover. 48

WAS patients
have impaired phagocytosis cup formation contributing to the defective bacterial clearance followed by inflammasome hyperactivity ( Figure 3). 139 Moreover, WASp deficiency was associated with impaired autophagosome formation and trafficking to lysosomes in primary macrophages. 140 The generation and function of antiinflammatory macrophages are defective in both humans and mice in the absence of WASp and therefore associated with inflammatory bowel disease in WAS patients. 141 WASp has a multifaceted function in macrophages and macrophages provide an important example to understand how WASp activity needs to be fine-tuned for the correct cellular response.

| Naturalkiller(NK)cells
A higher tumor susceptibility is observed in WAS patients, and part of the mechanism involved points to a defective immunosurveillance in WASp null NK cells. This is caused by lower motility 142,143 and poor cytotoxicity capacity since WASp promotes granule delivery ( Figure 4). 144 In a tumor-prone mouse model, WASp deficiency in NK cells affects their ability to suppress metastasis. 145 WASp null NK cells have decreased capacity to degranulate and produce IFNγ upon via NKp46 (Figure 4). 146 Interestingly, pretreatment of NK cells with IL-2 ex vivo restored degranulation, IFNγ production, and killing capacity and also, tumor rejection in vivo. 146 The rescue of

| InvariantnaturalkillerT(iNKT)cells
The iNKT cells are involved in the control of pathogen infections and cancer immunosurveillance. iNKT cells are reduced in WAS patients. 149 This deficiency was reproduced in WASp null mice and have defined that WASp is important for iNKT peripheral homeostasis since their number was normal in the thymus and bone marrow. 149 Moreover, it was shown that WASp is important for both maturation, thymic egress, and activation of iNKT cells. 150 These data show that iNKT dysfunction is an additional factor likely to contribute to the clinical features observed in WAS patients.

| Dendriticcells
DCs are active in the interplay between the innate and adaptative immune response, playing a fundamental role in immunosurveillance. In WAS deficiency, DC dysfunction represents an essential component underlying the disease pathogenesis. WAS null DCs have impaired migration, homing, localization in lymph nodes (LNs), and CD8 T-cell priming due to reduced ability to form and stabilize the immune synapse and to provide normal cytokine support to CD4 T-cell activation. [153][154][155][156][157] We recently identified an intricate balance of WASp expression in DCs and CD8 T activation. To understand skin pathology in WAS patients, we used skin infection models in WASp null mice, and we observe an accumulation of DCs and CD8 T cells in the skin and increased IFNγ producing CD8 T cells in the draining lymph node and spleen. 158 Moreover, WASp null DCs induced increased cross-presentation to CD8 T cells due to decreased phagosome acidification of DC in a process dependent on Rac2, upstream of WAVE2 (Figure 3). 158 These data together with the data from NK cells suggest that WASp deficiency induces alternative pathways for Arp2/3 actin polymerization via Rac2-WAVE2 signaling. 159 Among the migratory impact of WASp deficiency in DCs, is reduced to the absent formation of podosomes, dynamic adhesive structures that forms upon Phosphoinositide 3-kinases (PI3K) dependent on WASp clustering. [160][161][162] Other structures related to DC migration were recently described as WASp dependent, such as in providing orthogonal actin patches polymerization in response to mechanical load in a confined and 3D model. 163 WASp deficiency in DCs can also be linked to the autoimmune manifestations observed in WAS and XLT patients. Impaired endosomal maturation and reduced digestion of DNA immunocomplexes lead to a dysregulated activation of the cGAS/STING pathway resulting in pathogenic production of IFN-I, 164 a similar high IFN-I production was also observed in plasmacytoid DCs, the major cell source of this cytokine ( Figure 5). 165 For XLN DCs, we showed that the constitutively active WASp in DCs leads to impaired localization in the draining LN, and the cells also presented a reduced cell surface area and formed larger podosomes structures when compared to wild-type DCs ( Figure 3). 166

WAS patients have a reduced number of T and B cells early in life. 167
When the diversity of T cells was examined, T cells from young WAS patients (less than 15 years old) had a diverse TCR repertoire whereas older WAS patients had an oligoclonal TCR repertoire indicating less diverse T cells. 168 Both CD4 and CD8 T cells are decreased in numbers, especially naïve T cells. In vitro survival of naive CD4 T cells was normal, indicating the capacity to proliferate. 167 WASp null T cells show reduced capacity to transduce signals through the TCR complex, specifically upon CD3 engagement, resulting in defective actin cytoskeleton rearrangement. 169 WASp-deficient CD4 and CD8 T cells have reduced secretion of IL-2, IFNγ, and TNFα and cell proliferation upon TCR/CD28 activation ( Figure 4). 170 This impaired activation leads to reduced recruitment of nuclear factor of activated T cells (NFAT)1 and NFAT2 to the nucleus in CD4 T cells (Figure 4). 171 WASp serves an important role in the formation of the immune synapse, which is the contact area between the antigen-presenting cell and T cell and is composed of supramolecular activation clusters in distal, peripheral, and proximal distance to the TCR. 3 Upon activation of the TCR, WASp localizes to lipid rafts, higher ordered membrane structures are involved in immune synapse formation, and WASp is required for lipid raft clustering to the immune synapse. 172 The requirement for WASp to form the T-cell immune synapse has been debated and the results differ depending on the experimental system used; beads coated with antibodies to CD3 and CD28, lipid bilayer to dynamically visualize synapse formation, superantigen stimulation, and time of synapse formation. By studying the T-cell immune synapse formation using a lipid bilayer, 173 WASp null T cells formed the initial immune synapse. 173 However, the breaking of the immune synapse and reformation of a new synapse is dependent on WASp. 173 It was later shown that WASp takes part in the formation of the immunological synapse by forming distinct actin foci induced by early TCR signaling. 174 The actin foci are associated with actomyosin that generates cytoskeletal tension that actively restrains synapse breaking. 175 These foci elevate cytoplasmic calcium and downstream signals required for an optimal T-cell response. 174,175 For CD8 T cells, WASp is needed to form the cytotoxic synapse with target cells and WASp null cells have a reduced capacity to kill target cells (Figure 4). [176][177][178][179] WASp deficiency leads to a specific loss of protrusions/foci in the central cytotoxic synapse that mediates the release of cytotoxic granules. 180 Figure 5) (52, Liu, 2013# 3520, Liu, 2013. Studies of BCR repertoire to understand the B-cell diversity have revealed alteration and signs of oligoclonality. 190,193,197 These studies show a cell-intrinsic propensity of WASp null B cells to become autoreactive (Figures 4 and 5). Interestingly, deletion of N-WASp in WASp null B cells ameliorate the auto-antibody production, 192,193 suggesting that deletion of WASp may alter B-cell receptor signaling thereby influencing deletion of autoreactive B cells. Specific deletion of WASp in platelets leads to increased cell surface CD40L that triggers autoreactive WASp null B cells ( Figure 5). 124  knowledge has defined gaps that need more attention in the field, as we will discuss in the next sections. Many adult XLT patients, whose clinical phenotype has evolved during time from less to more severe, are also indicated to these curative approaches. Especially, gene therapy can provide better overall survival given the advantage of using autologous HSCs, thus avoiding GvHD disease. [205][206][207] However, the treatment approach for XLT is more conservative, managing the problems as they arise. 40 Since the group of patients is considered to develop the milder disease, probably due to a remaining WASp expression in the hematopoietic compartment rendering a certain level of cell function.

| EMERG INGTHER APIE S
The marked clinical phenotype is microthrombocytopenia and additional clinical manifestations of WAS can evolve through life. 43 Splenectomy is sometimes performed to increase the platelet count, but this procedure elevates the risk of subsequent sepsis and patients are subjected to lifelong antibiotic prophylaxis. 43 The use of thrombopoietic analogs, Eltrombopag and Romiplostin, can provide positive effects in ameliorating thrombocytopenia, but not all patients respond to the treatment, and the activation phenotype of the platelets is not corrected even in the responder groups. 208,209 Some studies have considered to directly target WASp at the cellular level, either aiming to stabilize the residual expression of WASp in XLT patients who have missense mutations in the WIP-binding domain, 108 or in the case of WASp GOF of XLN patients, aiming at degrading the protein. 112 However, no reported studies using these approaches were conducted in XLT or XLN mouse models to verify the in vivo effects and collect data to progress to clinical trials.
Finding new treatment options will benefit WAS/XLT/XLN patients who are not recommended the curative treatments or those from developing countries where HSCT is not always available. Finally, understanding more about actin regulators in the nucleus and how patient mutations affect their nuclear function will add to the possibilities of finding new treatments from drug repurposing.
The next decade will reveal similarities and differences among immunoactinopathies to define the best precision treatment options for patients with immunoactinopathies. This should be based on continued close collaboration between caring physicians, and clinical, translational, and experimental researchers.

CO N FLI C TO FI NTE R E S TS TATE M E NT
The authors declare no conflicts of interest.

PEER R E V I E W
The peer review history for this article is available at https:// www.webof scien ce.com/api/gatew ay/wos/peer-revie w/10.1111/ pai.13951.

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