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Autoimmunity in Wiskott–Aldrich Syndrome: Updated Perspectives

Abstract Wiskott–Aldrich syndrome (WAS) is an uncommon X-linked combined-immunodeficiency disorder characterized by a triad of thrombocytopenia, eczema, and immunodeficiency. Patients with WAS are also predisposed to autoimmunity and malignancy. Autoimmune manifestations have been reported in 26%–72% of patients with WAS. Autoimmunity is an independent predictor of poor prognosis and predisposes to malignancy. Development of autoimmunity is also an early pointer of the need for hematopoietic stem–cell transplantation. In this manuscript, we have collated the published data and present a narrative review on autoimmune manifestations in WAS. A summary of currently proposed immunopathogenic mechanisms and genetic variants associated with development of autoimmunity in WAS is also included.


Introduction
Wiskott-Aldrich syndrome (WAS) is an uncommon X-linked combined immunodeficiency disorder that has a heterogeneous clinical spectrum. [1][2][3] Manifestations vary from a relatively milder form of the disease (intermittent X-linked thrombocytopenia [XLT]) characterized by thrombocytopenia with little or no immunodeficiency to severe WAS, with profound immunodeficiency, bleeding episodes, autoimmunity, and increased risk of malignancy. [3][4][5] Many patients with WAS have intermediate grades of severity. It is this heterogeneity in the clinical spectrum that makes the initial diagnosis of WAS so challenging.
In a US study, WAS incidence was reported to be 3.3-5.2 per million live male births. 6 National registry data on primary immunodeficiency diseases from Sweden and Switzerland estimated WAS incidence to be 3.7 and 4.1 per million live births, respectively. 7,8 In 1994, almost six decades after the initial description of the condition, Derry et al identified the gene responsible for the defect: 9 WAS, which comprises 12 exons and is located on the short arm of the X chromosome (Xp11.23). 10 It encodes for WAS protein (WASp), a 502-amino acid cytosolic protein and a key molecule for actin-cytoskeleton polymerization. [11][12][13][14] WASp is ubiquitously expressed in all nonerythroid hematopoietic cells. 15 It consists of a pleckstrin homology (PH) domain and an enabledvasodilator-stimulated phosphoprotein homology (EVH1, also known as WH1) domain at the amino terminal, a short basic domain (B), a Cdc42-and Rac-interactive binding (CRIB) domain, a large proline-rich region (PRR), and a verprolin/central/acidic (VCA) domain at the carboxyl terminal. (Figure 1) WAS mutations affect actin cytoskeleton-dependent cellular processes, immunological synapse formation, [16][17][18][19][20] cell migration, and signaling, 21,22 and result in impaired functioning of WASp, causing XLT/WAS. Gain-of-function mutations WAS gene manifest as severe congenital Xlinked neutropenia, which is characterized by recurrent bacterial infections, neutropenia, and monocytopenia without thrombocytopenia. 23,24 The clinical course of WAS is complicated by frequent bleeding episodes, eczema, and recurrent infections. Patients with WAS are also predisposed to autoimmune manifestations and malignancies. 3,4 Autoimmunity is an independent independent predictor of poor prognosis and predisposes to malignancy. 25 As such, development of autoimmunity is an early indicator of the need for hematopoietic stem-cell transplantation (HSCT) in patients with XLT/WAS. Though HSCT is curative, it does not completely ameliorate the risk of later development of autoimmunity in WAS. Recent reports on the emergence of post-HSCT autoimmunity in these patients have rekindled interest in this field, and suggest the need for a better understanding of autoimmunity. [26][27][28][29][30] Despite being one of the earliest immunodeficiency syndromes described in the literature, pathophysiological mechanisms of underlying autoimmunity in WAS are still unclear. 31 This review focuses on putative pathogenetic mechanisms, genetic predisposition, and clinical manifestations of XLT/WAS with autoimmunity.

Methods
We carried out a literature search on PubMed, Scopus, Web of Knowledge, Google, and Google Scholar using the keywords "Wiskott Aldrich syndrome," "autoimmunity," "eczema," "anemia," "nephritis," "arthritis," "neutropenia," "vasculitis," "Wiskott Aldrich syndrome protein," and "malignancy." A supplementary manual search to identify additional primary studies was conducted and papers published up to December 2020 collated. Data on autoimmune manifestations were extracted from all single/multicenter cohorts and clinical case reports.
Demographic details, clinical and genetic profiles, WAS clinical scores, WASp expression, and outcomes were tabulated.

Incidence of Autoimmune Manifestations
One of the earliest reports of autoimmunity in WAS was published in 1976 by Gershwin et al, who evaluated patients with primary immunodeficiency diseases for the presence of autoantibodies. 32 Three of eleven children with WAS had autoantibodies against nucleic acids. However, a US multicentre study of a cohort of patients with WAS made no mention of autoimmunity. 6 In 1994, Sullivan et al reported autoimmune manifestations in 40% of patients with WAS. 25 The study highlighted autoimmunity to be a risk factor of the development of malignancy. Since then, autoimmunity has been a well-recognized entity in patients with WAS and an important indicator in predicting clinical outcomes and prognoses. Subsequently, several studies from different centers have reported variable figures (26%-72%) for the occurrence of autoimmune manifestations in patients with WAS (Table 1). 25,[28][29][30][33][34][35][36][37][38][39][40]

Proposed Mechanisms of Autoimmunity
WASp is involved in cytoskeleton remodeling, and absence of or residual WASp-expression defects cause-functional defects in all immune-system cells. Formation of immunological synapses in T cells and T-cell receptor (TCR)-dependent activation is impaired in WAS. [41][42][43] Reduced cytotoxic activity of T cells, natural killer cells, and naturally occurring regulatory T (nT reg ) cells contributes to poor pathogen clearance. [41][42][43][44][45] Motility, adhesion, and migration of B cells is also defective. 45 However, underlying mechanisms for occurrence of autoimmune manifestations are still not completely understood. Some hypotheses currently proposed for development of autoimmunity in WAS are summarized in the following sections.

Role of T Cells in Autoimmunity in WAS
Autoimmunity is caused by failure in self-tolerance mechanisms. T reg cells play a pivotal role in immunotolerance and prevent autoimmunity. They prevent autoimmunity by maintaining tolerance to self-antigens and suppression of excessive immunoresponse. Development and function of T reg cells requires effective TCR signaling with involvement of the CD28 costimulator, expression of master regulator FOXP3, and growth maintenance by IL2. 46 Although peripheral blood T reg -cell numbers have been found to be comparable in patients with WAS and healthy controls, WASp-deficient T reg cells demonstrated impaired ability to suppress proliferation of activated T-effector cells. 47,48 Distribution and phenotype of nT reg cells have also been found to be normal in the thymi and spleens of WASp-deficient mice. 49 nT reg cells, however, have been found to be reduced in inflamed peripheral tissue and lymph nodes. Reduction in nT reg cells correlates with lack of tissue-homing markers like integrin a 4 β 7 , chemokine receptor CCR4, and P-and E-selectin ligands. 44 A mouse model of autoimmunity has failed to control aberrant T-cell activation by WASp-deficient nT reg cells. 45 WASp-deficient nT reg cells fail to suppress B-cell activation and proliferation. Defective granzyme-mediated B-cell killing by nT reg cells has been demonstrated in some studies. 45,47

Role of B Cells in Autoimmunity in WAS
The role of B cells and autoantibodies in the pathogenesis of autoimmune diseases like lupus is well recognized. Bcell dysfunction in patients with WAS is evident from the variable distribution of serum immunoglobulins and demonstration of autoantibodies. Classically, WAS is associated with low serum IgM, normal IgG, and elevated IgE and IgA levels. Patients have impaired response to polysaccharides and other T cell-independent antigens. 3,50 Elevated IgM levels correlate with development of autoimmunity. Around 90% of patients with elevated IgM levels have been found to have developed AIHA in comparison to none with low IgM. 33 WASp deficiency affects adhesion, motility, and homing of B cells. 45,51 Defective B-cell function results in insufficient pathogen clearance, chronic immunoactivation, and failure of peripheral B-cell tolerance. In addition, reduced surface expression of complement receptors CD21 (CR2) and CD35 (CR1) results in impaired opsonization and negative selection of self-reactive B cells, thereby breaking peripheral tolerance and helping in autoantibody production. 52 Murine WASp-deficient B cells demonstrate increased proliferation with autoantibody production and differentiation into plasmablasts. 53 Enhanced proliferation of transitional B cells in response to stimulation by antigen or MYD88 has been seen in both humans and mice. [54][55][56] B reg cells influence the balance and recruitment of T reg cells and T H 17 cells during inflammation. Recent studies have suggested that WASp is required for normal B reg -cell numbers and functions. 57 Murine studies have also revealed reduced levels of IL10 secreting B reg cells (B10) in patients with WAS. 44 Bouma et al found reduced numbers of IL10-producing B reg cells, reduced T reg cells, and increased T H 17 cells in arthritic WAS-knockout mice. Adoptive transfer of wildtype B reg cells ameliorated arthritis and restored the balance between T reg and T H 17 cells. 57  autoreactive B-cell production. 58,59 Reduced numbers, defective invariant NKT cells, and increased IFNγ production have been reported in WASp-deficient mice. 60,61 Role of IFN1 in Autoimmunity in WAS Plasmacytoid dendritic cells (pDCs) are specific subsets that produce IFN1 in response to foreign nucleic acids. Viral infections and certain self-nucleic acids serve as stimulants of TLR7 and TLR9 and keep them persistently activated. Susceptibility to viral infections, impaired clearance, and exposure of self-antigens following cell death activate the IFN1 pathway. 62 This pathogenic mechanism is associated with several autoimmune diseases, such as lupus, Sjögren's syndrome, and psoriasis. 62 WASp deficiency leads to exaggerated activation of TLR9 by its ligand and subsequent increased IFN1 signature. 63 WASp-deficient mice show persistent activation of pDCs and elevated IFN1 levels. Ablation of IFN1 in WASp-deficient mice results in blockade of chronic activation of pDCs. This is accompanied by remission of colitis and reduction in spleen size. 63

Defects in Apoptosis Pathway and Development of Autoimmunity in WAS
Restimulation-induced cell death is a process that augments Fas-mediated apoptosis of activated T cells. It helps eliminate T cells that are activated against chronically expressed antigens like autoantigens. Nikolov et al demonstrated defective FasL in WASp-deficient mice leading to defective elimination of activated T cells and predisposition to autoimmunity. 64 Defective Fas has also been hypothesized to be a pathophysiological mechanism for lymphoproliferative lupus-like syndrome in Fas/FasLnegative mice. 65,66 Role of Inflammasomes in Autoimmunity in WAS Excessive inflammasome activation has been shown in human monocytes and mouse DCs. 67 Some autoimmune manifestations (especially rash and arthralgias) are related to excessive NLRP3-inflammasome activation.
Deletions are spread across the gene, while most missense variants are seen in the PH and EVH1 domains. Missense variants (p.T45M and p.T45K) at position 45 in PH and EVH1 have been found to be associated with autoimmunity. Normal/absent WASp expression was found such patients. 34,36,75,76 Twelve patients with missense variants at position 86 (R86G, R86C, R86H and R86A) developed autoimmunity. Both p.E31K and p. E133K were reported in five patients each that developed autoimmunity.
Patients with XLT can progress to autoimmune WAS. The XLT phenotype with missense variants in PH and EVH1 is most associated with development of autoimmune WAS. Albert et al identified 13 positions associated with XLT to autoimmune WAS progressions, 9 of which lie in PH and EVH1 domain.
Patients with XLT can also develop autoimmunity. Albert et al identified 13 amino acid positions that were prone to progress to autoimmune WAS. 36 We identified 18 variants in 25 patients reported to demonstrate progression of XLT to autoimmune WAS. Of these, 13 (all missense) were located in PH and EVH1.

Clinical Manifestations Autoimmune Hemolytic Anemia (AIHA)
AIHA is the commonest autoimmune manifestation seen in patients with WAS, accounting for 30%-85% of all autoimmune manifestations. The reported frequency of AIHA is 8%-36%. 25,29,40 The mean age of development of AIHA was 13.7-17.5 (1-58) months in two studies (Table 3). 29,33 All patients had AIHA onset aged <5 years. 33 The commonest clinical presentations of AIHA include anemia, jaundice, and hepatosplenomegaly. 29,40 Positive antinuclear antibodies have been demonstrated in 5%-9% of patients with AIHA, while Coombs tests were positive in some patients. 29

Autoimmune Thrombocytopenia (AIT)
Microthrombocytopenia is a cardinal feature of WAS. Abnormality in WASp results in megakaryocyte dysfunction, leading to formation of small platelets. These abnormal microplatelets are recognized by self-antibodies and are prematurely cleared by the spleen. [91][92][93] Immunomediated thrombocytopenia also plays a contributing role in 15%-32% of patients (Tables 1 and  2). 29,30,33,36 Antiplatelet antibodies have been found in 40% of WASp-deficient mice. 94 Assays for antiplatelet antibodies are difficult to standardize and may not be easily available, especially in developing countries. Moreover, these antibodies may have a poor correlation with AIT. 95 AIT may evolve over and exacerbate the underlying thrombocytopenia that is characteristic of WAS. A sudden drop in baseline platelet counts (usually <10×10 9 /L) with or without overt clinical bleeding is an important clinical clue to emergence of AIT. Failure to demonstrate a significant rise or fall in platelet count after platelet transfusion may herald the development of AIT. 95 Most autoimmune manifestations evolve over time, but immunothrombocytopenia may have an early age of onset. In a cohort of patients with WAS aged <2 years with a clinical severity score of 5, ten of 26 (38.4%) had antiplatelet antibody-positive severe refractory thrombocytopenia. 96 Intracranial hemorrhage is a major cause of mortality in patients with WAS. 25 Differentiating AIT from baseline thrombocytopenia in WAS is crucial, as management strategies vary. Institution of appropriate immunosuppressive therapy is needed to maintain platelet counts.

Autoimmune Neutropenia
Autoimmune neutropenia is found in 2%-25% of patients with WAS. 25,29,30,33,35 Vasculitides Vasculitis is the second-commonest autoimmune manifestation, and has been found in 1.5%-29% of patients with WAS. It accounts for 6%-45% of all autoimmune manifestations. 25,30,33,34,36,39,40 (Tables 1 and 2) Two patterns of vasculitic abnormality have been reported in WAS: medium-sized and small-vessel vasculitis of skin, renal, coronary, cerebral, or hepatic arteries, and large-vessel vasculitis involving the aorta and its major branches. 40,5097-101 Involvement of small vessels, especially those of the skin, 25,33,39,102,103 is the commonest vasculitic abnormality (75%). 33 IgA vasculitis (previously termed Henoch-Schönlein purpura) has been reported in 28.5% of patients with vasculitis. 25 Kawakami et al described Kawasaki disease in a patient with WAS. 104 Involvement of small-and medium-sized arteries of the gastrointestinal tract, 97,105,106 heart, 104,105 liver, 98,104 gallbladder, 105 kidneys, 98,106 stomach, 102 and cerebral blood vessels, 98,107 has also been reported. In a singlecenter study of 55 patients of WAS, Dupuis-Girod et al noted cutaneous vasculitis at a mean age of 52.5  months. 33 Lao et al reported large-vessel vasculitis involving the aorta and renal artery in a 5-year-old boy with WAS. 100 Pellier et al described five children with WAS who developed aortic aneurysms predominantly involving the thoracic and abdominal aortae. 101 Four of these five patients with vasculitis were asymptomatic, and aneurysms were discovered only on screening.
Predisposition to developing vasculitis has been ascribed to immunodysregulation in WAS. Patients with WAS typically have depressed levels of IgM and elevated levels of IgA and IgE. It has been suggested that immunodeposition within the vessel wall can lead to necrotizing vasculitis. 98 Alternatively, vasculitis could result from an infectious insult due to the underlying immunodeficiency.

Arthritis
Arthritis has been reported in 1%-29% of patients with WAS, and accounts for 3%-52% of all autoimmune manifestations (Table 1). 25

Renal Disease
Onset of renal disease in XLT/WAS occurs at a relatively later age: 7-20 years. 36,37 Clinical manifestations include transient proteinuria, 33 hematuria, azotemia, and nephritic-nephrotic syndrome. 25,29,100 Reported histopathological patterns include IgA nephropathy, membranoproliferative glomerulonephritis, mesangial proliferation, and interstitial nephritis. [108][109][110][111] IgA nephropathy is the commonest renal disease, described in 27%-41% of patients in various long-term cohorts. 25,35,36 Prevalence appears to be higher in patients with residual WASp expression. 35,36 Screening for renal involvement has been recommended in all patients with XLT/WAS. Aberrant glycosylation of IgA is attributed as a cause for renal disease associated with WAS. Elevated levels of β1,6-N-acetyl-glucosaminyl transferases have been documented in patients with WAS. This leads to aberrant O-glycosylation of sialophorin in patients with WAS. 101,112 Inflammatory Bowel Disease (IBD) Bleeding from the gastrointestinal tract is a common symptom in WAS and often secondary to thrombocytopenia. In our cohort, 49.4% of children presented with bloodstained stools. 40 Autoimmune colitis has also been reported in 1%-9% of patients. 25,29,30,35,36 Both ulcerative colitis and Crohn's disease have been observed. WASassociated colitis is challenging to treat and often refractory to immunosuppressants. 113 In our experience, IBD can be a presenting feature of WAS. Ohya et al reported that 16.6% of patients with IBD had a WAS mutation. 113 Similarly, Cannioto et al showed that 25% of patients aged <2 years with IBD were later shown to have WAS. 114 WASp-deficient mice have been observed to have chronic colitis with mucosal thickening and lymphocytic and neutrophilic infiltrates in the lamina propria. 115 Suggested pathogenic mechanisms for IBD in WAS include WASp deficiency-mediated dysfunction of T reg cells and anti-inflammatory macrophages, increased selfreactive B cells, and altered gut microbiota. 113,116 Other Rheumatic Manifestations Monteferrante et al described lupus nephritis in a patient with WAS. 117 Similarly, other connective-tissue disorders like dermatomyositis, 27 uveitis, 27 autoimmune hepatitis, 27 primary sclerosing cholangitis, 40 amyloidosis, 39 and relapsing polychondritis 118 have been reported in the context of WAS. 27 Crestani et al reported positive antineutrophil cytoplasmic antibodies, and positive antiphospholipid antibodies in patients with WAS. 119

Autoimmune Skin Diseases
Eczematous dermatitis is a cardinal clinical manifestation of WAS. Eosinophilia and elevated levels of serum IgE are associated findings. DC dysfunction and skewed T H 2 immunity may play a role in the development of eczema in this condition. 120 Apart from atopy, other autoimmune skin manifestations have been reported in WAS. These include recurrent angioedema, pyoderma gangrenosum, and erythema nodosum. 25 Alopecia has been reported in 1%-3% of patients with WAS. 25,30,34,36

Posttransplant Autoimmune Manifestations
Development of autoimmunity is considered a predictor of severe disease, and often warrants the need for early HSCT in patients with XLT/WAS. HSCT is curative and is associated with 5-year survival of 91%. 30 However, occurrence of autoimmunity in the posttransplant period has been observed in 13%-20% of patients (Table 4). [26][27][28][29][30] Autoimmune cytopenia is the commonest autoimmune manifestation seen after HSCT. 30 Burroughs et al reported that 75% of patients with autoimmune manifestations responded to immunosuppressive therapy and attained remission within a year of transplant. 30 Mixed or split donor chimerism is an important predictor of development of post-HSCT autoimmunity. 26,27,30 Autoimmunity is most frequently encountered in patients who undergo matched unrelated donor transplants. However, it is also seen in matched related-donor and matched sibling-donor transplants. 26

Is Autoimmunity in WAS a Poor Prognostic Marker? Age at Onset of Autoimmunity
Mahalaoui et al evaluated a subgroup of 26 patients that had onset of autoimmunity before the age of 2 years in their cohort of 160 patients with WAS. 96 The authors concluded that development of early autoimmunity predicted a severe refractory disease course and required early HSCT for survival.

Autoimmunity and Risk of Malignancy
Presence of autoimmunity also increases the risk for development of malignancy in patients with WAS. Sullivan et al observed that 25% of patients with a history of autoimmune disease developed malignancy compared to 5% of patients without autoimmunity. 25 Sallah et al demonstrated that 18% of patients with AIHA developed lymphoreticular malignancy. 121

Treatment of Autoimmunity
Currently, HSCT is the best curative therapy available for WAS. Early studies on HSCT in WAS reported effective

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reconstitution of lymphoid cells, but impaired platelet engraftment. [122][123][124] Long-term overall survival in recipients of unrelated bone-marrow grafts is 70%-78%. 26,125 However, recent studies have reported event-free survival with HLA-identical sibling bone-marrow grafts to be 88%, with overall survival of 90%-95%. 27,30,126 . Results of unrelated-and alternative-donor HSCT have also greatly improved to >90%. 30,37 HSCT with TCRαβ/CD19 depletion or posttransplant cyclophosphamide therapy for haploidentical donors has shown promising results and new opportunities for successful curative therapy in WAS/XLT. 127 Gene therapy is an evolving and promising alternative approach when a transplant is not feasible due to unavailability of matched donors. Gene therapy was attempted with a gibbon ape leukemia virus γ-retroviral vector in 2006. 77,127 Though successful engraftment was reported, it was limited by development of leukemia due to insertional leukemogenesis. Subsequently, therapy with a lentiviral vector was attempted in 31 patients with WAS. This resulted in successful engraftment, discontinuation of intravenous immunoglobulin (IVIg), improvement in platelet counts, and reduction in infections. Resolution of autoimmune manifestations was also demonstrated with gene therapy; however, the rate of de novo autoimmunity posttransplant was comparable with HSCT. 127 Chemoprophylaxis with antimicrobials and monthly VIg are often used for prevention of infections. Management is usually individualized based on disease severity. Treatment of autoimmune manifestations is challenging, and may require require immunosuppressive therapies. These agents can further increase the risk of infections. Corticosteroids remain the first-line therapy for all autoimmune manifestations. Corticosteroids can induce variable rates of remission in patients with AIHA (Table 2). Additional agents are needed in patients who do not respond or attain partial remission. Cyclophosphamide, azathioprine, IVIg, rituximab, cyclosporine, plasma exchange, and tacrolimus have been used with variable results.
High-dose IVIg and oral or parenteral corticosteroids are used as first-line agents in patients with AIT who have significant bleeds. Rituximab is used for refractory cases. Splenectomy significantly increases and often normalizes platelet counts in refractory thrombocytopenia. 77,128,129 However, splenectomy is associated with increased risk of sepsis and necessitates lifelong antimicrobial prophylaxis. 129 Moreover, thrombocytopenia may recur after splenectomy in patients with WAS. 33 Splenectomy is reserved for very severe cases with no prospects from other curative interventions. Severe AIT after splenectomy is usually treated with IVIg, high-dose steroids, azathioprine, and cyclophosphamide. A majority of patients with skin vasculitis, arthritis, IBD, and renal disease associated with WAS respond to standard immunosuppressive regimens containing steroids and cyclosporine. 33

Conclusion
Autoimmune manifestations are well-recognized complications in WAS. Varied clinical manifestations have been associated with the syndrome. Autoimmune cytopenia is the commonest. Development of autoimmunity is a poor prognostic marker and a predictor of development of malignancy. Pathogenic mechanisms for autoimmunity are not clearly defined. Corticosteroids with or without additional immunosuppressive agents are needed for treatment of autoimmune manifestations. HSCT is curative, but there is a risk of development of posttransplant autoimmunity.

Disclosure
The authors report no conflicts of interest in this work.