Discovering the Cause of Wiskott–Aldrich Syndrome and Laying the Foundation for Understanding Immune Cell Structuring

The discovery of the Wiskott–Aldrich syndrome (WAS) protein gene (WASP, now known as WAS) by Derry et al. (1) in 1994 accelerated the field of immune cell biology and studies of the molecular basis of inborn human immune defects, otherwise known as primary immunodeficiencies (PIDs). It was the “novel gene” described in this issue’s Pillars of Immunology article that allowed for the discovery of an entire family of proteins that would help define how all cells gain their structural integrity and led to exciting and groundbreaking findings as to the cell biological requirements for human immunity. Cited almost 1000 times, this article remains a foundation for our understanding of the intracellular mechanics that underpin human immune function.

WAS is a rare X-linked PID that was defined by a classic triad of thrombocytopenia, eczema, and recurrent otitis; later, WAS was known to be associated with increased incidence of many infections, autoimmunity, and malignancy (2). This was historically considered a fatal disease with a short life expectancy for affected patients, almost exclusively boys. The first patient cohort was reported in 1937 by Wiskott (3), when he described three brothers with low platelet counts, bloody diarrhea, skin rash, and recurrent ear infections. The brothers died as a result of their illness; however, it was noted that their sisters were unaffected, leading Wiskott to suggest that the syndrome was hereditary. In 1954, Aldrich (4) painstakingly studied a large Dutch family, describing similar symptoms to those first identified by Wiskott, and documented the X-linked inheritance pattern of the disease, which was subsequently named WAS.

Despite its early description and the devastating effect of this disease on affected families, the molecular etiology of WAS remained unknown for over 50 years. During this time, ongoing studies of these patients and the nature of their disease laid the groundwork for molecular studies that would follow the discovery, in 1994, of the gene encoding WASP. Currently, there are more than 440 known mutations causative of WAS (5). These include mutations that lead to classical WAS, characterized by loss of protein expression, as well as an additional spectrum of mutations that include milder forms of WAS, such as X-linked thrombocytopenia (XLT) (6), intermittent XLT (7), and X-linked neutropenia, a disease that results from constitutively activated WASP (8).

At the time of the discovery of the WASP gene, there was insight into the biology of WAS. Previous linkage analysis had localized the causative defect to the 1-Mbp region of Xp11.22–Xp11.23 (9–14). XLT had been described and mapped to the same region, and it was speculated to arise from defects in the same gene (15, 16). Diverse investigations uncovered important characteristics of T cells from WAS patients, including electron micrographs showing absent or deformed microvillus projections from the cell surface (17, 18) accompanied by decreased or aberrant expression of CD43 (19, 20). This led to the brief speculation that CD43 was the causative gene of WAS, speculation that was ended by the mapping of the CD43 gene to chromosome 16 (21). Other clues as to the function of the protein that would subsequently be identified as WASP included impaired transmembrane signaling (22), decreased chemotaxis, aberrant O-linked glycosylation patterns, and impaired T cell proliferation (23–28). It was recognized that expression of the gene was restricted to the lymphocytic and megakaryocytic lineages, an observation that was critical for the proof of concept that mutations in WASP caused WAS. Despite these useful insights, some of which would be direct demonstrations of WASP function, the disease and its unifying biology remained a great mystery until the gene was identified.

To identify the WASP gene, yeast artificial chromosomes were assembled into a one-Mbp contig spanning the Xp11.22–Xp11.23 region of interest. Affinity capture was used to identify novel cDNA transcripts in combination with Northern hybridization to RNA from cultured lymphoblasts. This identified seven putative genes, including WASP. Notably, protein expression was restricted to lymphocytes and megakaryocytes, and RNA was not detected by Northern hybridization of lymphoblastoid-derived cell lines from two patients with classical WAS. Together, these data linked the putative WASP gene with WAS. The entire WASP cDNA was then isolated from a T cell–derived cDNA library and sequenced; 10 introns and 11 exons were identified. Finally, WASP mutations as the cause of WAS were confirmed by PCR-based sequencing of patient DNA, which identified three independent mutations (T211 deletion and G291A and G291T transversions) in affected patients and also confirmed heterozygosity in female carriers (1).

This article proposed “a direct interaction of WASP with the cytoskeleton” and led to the mechanistic discovery that WASP and its homologous family members are critical regulators of actin cytoskeletal remodeling and are required for hematopoiesis and immune cell function. At the time of writing, Derry et al. (1) suggested the function of the newly discovered WASP gene in cytoskeletal rearrangement based upon previous cell biological studies. It is difficult, however, to overstate the importance of understanding the contribution of WASP to immune cell function and cell biology in general. WASP was the first identified actin nucleation–promoting factor now known to be part of the molecular complex that adds an actin monomer onto an existing actin filament so that a new filament can grow at a 70° angle to the original. This represents nature’s construction work of assembling the structural framework of living cells, akin to joining the girders in a building, providing integrity and form.

In addition to driving countless discoveries about the cell biology of immune cells, it became clear that the critical function of WASP family members also reaches beyond immunity through N-WASP and SCAR/WAVE1–3, which have more ubiquitous tissue expression, and WASP-like proteins WASH and WHAMM, which have functions similar to WASP in immune cell actin dynamics (29–33). Rapid characterization of the WASP protein, following the discovery by Derry et al., led to understanding of its structure and function (reviewed in Ref. 34). Containing a verprolin homology domain–cofilin homology domain–acidic region domain, GTPase binding domain, and proline-rich domain, WASP is held in an autoinhibited conformation through interactions between the verprolin homology domain–cofilin homology domain–acidic region domain and GTPase binding domains and stabilized by WASP-interacting protein. Binding of GTPases, primarily Cdc42, relieves this autoinhibition and enables binding to the Arp2/3 complex to promote actin nucleation upon an existing actin filament. The polyproline region of WASP was noted in the 1994 article and predicted to bind SH3 domain–containing proteins; we now know that Src and Tec family kinases bind this region and can activate WASP through tyrosine phosphorylation. Actin remodeling mediated by WASP is required for processes including directed secretion, cell migration, phagocytic cup formation, and neutrophil effector functions. Although less well understood, the role of WASP in platelet and megakaryocyte formation and function is strikingly underscored by the defects in these cells in WAS patients.

The identification of the WASP gene coincided with technological advances that led to the generation and rapid advance of the field of immune cell biology. Namely, the development of microscopes and imaging systems that enabled the high-resolution study of immune processes launched an explosion of studies into the immunological synapse (IS), with the importance of WASP-mediated actin remodeling at the forefront. Although the T cell IS was first conceptually proposed in 1984 (35), it wasn’t until 1999 that the T cell–APC IS was visualized and functionally dissected, featuring synaptic actin accumulation (36, 37). This was rapidly followed by description of other ISs, including CTL synapses (38), NK cell activating and inhibitory synapses (39), B cell synapses (40), NK cell–dendritic cell synapses (41), and so forth. Beginning with these studies, and continuing to this day, immunologists have risen to the challenge of studying small, highly motile, and diverse cells by being early adopters of sophisticated imaging technologies, including superresolution microscopy, intravital imaging, and microfabrication.

The study of lymphocytes from patients with WAS led to early insight into the importance of actin branching in immune cell function and provided important understanding into the origin of disease in these patients (42, 43). This led the way for further studies that combined high-resolution imaging with analyses of IS structure and function to make seminal discoveries about the cytoskeleton and human immunity (44–46). Immune defects in WAS result from decreased T and B cell numbers and function and impaired NK cell function. In healthy cells, WASP is recruited to the IS, where it acts as a scaffold to promote localized assembly of branched actin filaments that are required for cell adhesion, receptor recruitment, and cell polarization. The loss of WASP function leads not only to impaired lytic effector T cell function but also to impaired proliferation and IL-2 production, increased apoptosis, and decreased numbers of circulating T cells in WAS patients (23, 47–50). The impairment of NK cell effector function is a significant contributor to the devastating infections experienced by WAS patients, including those resulting from abnormal susceptibility to herpesviruses (46, 51). These defects can be partially alleviated by the administration of IL-2, which accesses the function of WAVE2 to mediate actin branching in NK cells in the absence of WASP function (52). Although WAVE2 has baseline function in T cells, in NK cells this function is induced following cytokine stimulation. As such, IL-2 therapy can alleviate low NK cell function in WAS patients, with promising results in a clinical trial (53). Finally, although WAS remains the most common PID as a result of mutations in this family of proteins, a patient with features of WAS has subsequently been identified with deleterious homozygous mutations in WASP-interacting protein (WIPF1) (54).

In addition to acting as the foundation for the study of immune cell biology, this article was among the first to define the molecular basis of a PID. Today, when whole-exome sequencing is de rigueur, the identification of mutation-causing genes includes a significant density of genetic information that is generated by the study of a patient and his or her family members. The challenge lies in first identifying rational candidate genes and then proving causation of disease. However, the same tenets still apply for linking a causative gene to human disease that were demonstrated in this Pillars of Immunology article. The meticulous sequencing efforts, supported by the demonstration of mutations in affected family members that are present in heterozygous female carriers, but not 100 healthy donors, set the high standard that the emerging field of PID research would follow (55). In addition, this article set the stage for an early molecular test for PID, the flow cytometry–based assay for WASP expression (56), and predicted the prospect of gene therapy for WAS. Although hematopoietic stem cell transplant is still the most common therapy for WAS, this prospect became a reality in 2010 with the treatment of 10 WAS patients by retroviral gene therapy (57). The risk of oncogenic transformation has led to the redesign of gene therapy vectors, and success of lentiviral gene therapy has been reported recently in these patients (58). The efficacy of gene therapy was in part monitored by understanding the cell biological impact of WAS gene replacement by studying the NK cell IS in these patients.

In summary, the work of Derry et al. holds the unique distinction of both closing one chapter of a longstanding medical mystery, namely the identification of WASP as the disease-causing gene for WAS, and launching new fields of discovery into the role of actin branching in immune cell function and immunity.