J Clin Immunol (2016) 36:117–122 DOI 10.1007/s10875-016-0232-2 ASTUTE CLINICIAN REPORT Defective Leukocyte Adhesion and Chemotaxis Contributes to Combined Immunodeficiency in Humans with Autosomal Recessive MST1 Deficiency Tarana Singh Dang 1 & Joseph DP Willet1 & Helen R Griffin2 & Neil V Morgan3 & Graeme O’Boyle1 & Peter D Arkwright4 & Stephen M Hughes4 & Mario Abinun1,5 & Louise J Tee3 & Dawn Barge6 & Karin R Engelhardt1 & Michael Jackson5 & Andrew J Cant1,5 & Eamonn R Maher3,7 & Mauro Santibanez Koref2 & Louise N Reynard1 & Simi Ali1 & Sophie Hambleton1,5 Received: 20 March 2015 /Accepted: 4 January 2016 /Published online: 22 January 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract measured, and transwell assays were used to assess chemo- Purpose To investigate the clinical and functional aspects of taxis. Chemokine receptor expression was examined by flow MST1 (STK4) deficiency in a profoundly CD4-lymphopenic cytometry and receptor signalling by immunoblotting. kindred with a novel homozygous nonsense mutation in Results A homozygous nonsense mutation in STK4 STK4. Although recent studies have described the cellular (c.442C > T, p.Arg148Stop) was found in the patients, leading effects of murine Mst1 deficiency, the phenotype of MST1- to a lack of MST1 protein expression. Patient leukocytes ex- deficient human lymphocytes has yet to be fully explored. hibited deficient chemotaxis after stimulation with CXCL11, Patient lymphocytes were therefore investigated in the context despite preserved expression of CXCR3. Patient lymphocytes of current knowledge of murine Mst1 deficiency. were also unable to bind effectively to immobilised ICAM-1 Methods Genetic etiology was identified by whole exome se- under flow conditions, in keeping with a failure to develop quencing of genomic DNA from two siblings, combined with high affinity binding. linkage analysis in the wider family. MST1 protein expression Conclusion The observed abnormalities of adhesion and mi- was assessed by immunoblotting. The ability of patient lym- gration imply a profound trafficking defect among human phocytes to adhere to ICAM-1 under flow conditions was MST1-deficient lymphocytes. By analogy with murine Mst1 deficiency and other defects of leucocyte trafficking, this is likely to contribute to immunodeficiency by impairing key * Sophie Hambleton aspects of T-cell development and function such as positive [email protected] selection in the thymus, thymic egress and immune synapse formation in the periphery. 1 Institute for Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK Keywords Combined immunodeficiency . MST1 . STK4 . 2 Institute of Genetic Medicine, Newcastle University, Newcastle upon lymphocyte adhesion . chemotaxis . CD4 lymphopenia Tyne, UK 3 School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK 4 Department of Paediatric Allergy & Immunology, Royal Manchester Introduction Children’s Hospital, University of Manchester, Manchester, UK 5 Great North Children’s Hospital, Newcastle upon Tyne Hospitals Primary immunodeficiencies (PIDs) are rare, inherited dis- NHS Foundation Trust, Newcastle upon Tyne, UK eases characterised by the altered function or absence of im- 6 Blood Sciences Flow Cytometry Laboratory, Newcastle upon Tyne mune cells. Recently, whole exome sequencing (WES) has Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK emerged as a powerful tool for revealing the genetic etiology 7 Department of Medical Genetics, University of Cambridge, of certain PIDs. The benefits of this approach include the Cambridge, UK potential discovery of novel disease-associated genes, and 118 J Clin Immunol (2016) 36:117–122 the relatively efficient identification of novel variants within ongoing EBV-viremia when assessed in middle childhood. genes already known to be associated with PIDs. This work Both sisters also had documented cryptosporidiosis on more describes an example of the latter, specifically the discovery of than one occasion and both ultimately developed EBV- a novel variant in the serine threonine kinase 4 (STK4) gene. associated lymphoproliferative disease, treated with STK4 encodes a 63kD protein known as MST1, a mamma- Rituximab and steroids before stem cell transplantation. lian orthologue of the Drosophila Hpo protein, and a member Their sister P3 was transplanted at a younger age because of of a family of kinases related to the yeast protein Ste20 (sterile a similar infection history (molluscum, history of severe pri- 20) [1]. Soon after its discovery, MST1 was identified as hav- mary HSV, recurrent herpes zoster) and the availability of a ing roles in regulation of apoptosis and proliferation [2, 3], matched family donor. She developed florid cryptosporidial with other Ste20-like kinases found to have roles in cytoskel- diarrhea on day 0 of transplant, most likely representing the etal rearrangement [4]. The role of MST1 in cell death is recrudescence of chronic infection. After a stormy peri- controversial, with both proapoptotic [5] and antiapoptotic transplant period with veno-occlusive disease, capillary leak [6] functions claimed, although MST1 deficiency has been and GVHD, she engrafted but had not immune reconstituted associated with increased apoptosis in both mouse [7, 8]and when she acquired primary CMVand went on to develop fatal human [9, 10]. MST1 is also a signalling intermediate in the immune dysregulation. P1 also succumbed to transplant- process of ‘inside-out’ signalling in murine lymphocytes, related complications in the form of a cerebrovascular acci- converting signals initiated by chemokine stimulation into dent shortly after conditioning, while P2 has had a good out- subsequent LFA-1 integrin activation and polarization [11, come from HSCT. 12]. MST1 carries out this function by acting downstream of Laboratory work-up in all three children (Table 1) was Rap1, a small GTPase that becomes active after ligation of remarkable for CD4 lymphopenia, absence of naïve T cells chemokine receptors. Rap1 in turn binds RAPL, which binds and hypergammaglobulinemia (yet low IgG2 and poor pneu- to MST1. This complex then mediates the activation and po- mococcal responses) without evidence of autoimmunity. larization of the LFA-1 integrin, and localises with LFA-1 at Thymic size was normal on computer tomographic imaging the leading edge of the cell [12]. The surface redistribution of (data not shown). Given this clinical picture and a consanguin- active LFA-1 is vital for transendothelial migration of lympho- eous background (Fig. 1a), a leaky form of autosomal reces- cytes, as well as for the formation of the immune synapse [13, sive severe combined immunodeficiency was suspected but 14]. known defects were ruled out by a negative genetic screen. Recently, two studies have identified MST1 deficiency as Homozygosity mapping was therefore undertaken, followed the cause of combined immunodeficiency (CID) in three dif- by WES of two of the three patients. This led to the identi- ferent families with homozygous nonsense mutations in the fication of a novel homozygous nonsense variant (c. STK4 gene [9, 10]. This invariably resulted in profound CD4 442C > T, p. Arg148Stop) in STK4, encoding MST1, that lymphopenia and an accompanying phenotype of multiple was confirmed to segregate with disease in the family by bacterial and viral infections, and mucocutaneous candidiasis Sanger sequencing (Fig. 1b). Western blot analysis of pa- [15]. Here, we describe a further family with a biallelic STK4 tient fibroblast and lymphoblastoid B cell lysates con- mutation and similar clinical features. Further analysis of pa- firmed loss of MST1 protein expression and hence the tient cells revealed a defect in LFA-1-mediated adhesion and diagnosis of MST1 deficiency (Fig. 1c). chemotaxis. Confidence in the disease-causing nature of this variant was increased by the publication of two independent reports linking loss-of-function STK4 mutations to CID [9, 10]. Case Study MST1 deficiency was suggested to cause progressive lympho- penia due to the anti-apoptotic role of the enzyme, but the We studied three siblings with early childhood onset of CID reduced adhesive and chemotactic potential found in murine characterized by CD4 lymphopenia and marked susceptibility Mst1−/− lymphocytes [11, 13, 14, 16] was not fully explored in to opportunistic and bacterial infection. The index patient (P2) their human equivalents. was evaluated immunologically at 3 years of age following an Prior to the investigation of the adhesive and chemotactic episode of lobar pneumonia from which she had failed to properties of patient peripheral blood leukocytes (PBLs), the recover fully. Her history was remarkable for a prior episode chemokine receptor expression of these cells was investigated of viral laryngotracheobronchitis requiring intubation and using flow cytometry. Expression of CXCR3 – areceptorfor ventilation, recurrent suppurative otitis media and eczema; CXCL11 – was observed in patient CD4+ T lymphocytes she experienced severe primary herpetic gingivostomatitis, (Fig. 2a), along with expression of CCR1, CCR2, CCR3, had ongoing chronic molluscum contagiosum and proved to CCR5, CCR6, CCR7, CXCR1, and CXCR5, and slightly re- be consistently EBV-viremic. Her elder sister P1 had a similar duced expression of CCR9 and CXCR4 (data not shown). history of recurrent respiratory infection, molluscum and Due to the robust expression of CXCR3 by patient cells,