PMC4769310_01
a Pedigree of affected family, demonstrating consanguinity (double horizontal lines). Males and females are indicated by squares and circles respectively, with open symbols indicating healthy individuals and shaded symbols indicating affected individuals. Triangles indicate unaffected individuals with censored gender, and diagonal lines indicate deceased individuals. b Sanger sequencing of STK4 gene confirmed WES findings - a homozygous C to T substitution at position 442 in patients that was heterozygous in both parents. c Western blotting of P3-derived EBV-transformed B cell lysate and P1 and P3 fibroblast lysates, demonstrating MST1 deficiency. Control lysates were derived from random healthy individuals. Representative of a minimum of two separate experiments
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PMC4769310_01_10875_2016_232_Fig1_HTML.jpg
Fig1
Given this clinical picture and a consanguineous background (Fig. 1a), a leaky form of autosomal recessive severe combined immunodeficiency was suspected but known defects were ruled out by a negative genetic screen. Arg148Stop) in STK4, encoding MST1, that was confirmed to segregate with disease in the family by Sanger sequencing (Fig. 1b). Western blot analysis of patient fibroblast and lymphoblastoid B cell lysates confirmed loss of MST1 protein expression and hence the diagnosis of MST1 deficiency (Fig. 1c).
a Patient CD4+ T lymphocytes exhibit expression of the CXCL11 receptor CXCR3 (red histogram = unstained control, green histogram = anti-CXCR3-PE antibody) (n = 1). b EBV-transformed B cells were treated with 12 nM CXCL11 to stimulate CXCR3 before whole cell lysis, Western transfer analysis and probing for phospho-ERK, total ERK, phospho-AKT and total AKT, with beta-tubulin as a loading control. Numbers indicate time (mins) of treatment with CXCL11. Representative of three separate experiments. c CXCL11 treated patient EBV-transformed B cells (left panel) show reduced binding to ICAM-1-coated chips under flow conditions when compared to control cells (* indicates P = 0.0313). Patient PBLs also show reduced binding when compared to control cells (right panel). Each shape represents one of six separate experiments, with 5 replicates for each. Significance was determined using Wilcoxon signed rank tests. d CXCL11-treated patient PBLs show unchanged capacity for migration across transwell filters when compared to control cells (n = 1)
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PMC4769310_01_10875_2016_232_Fig2_HTML.jpg
Fig2
Expression of CXCR3 - a receptor for CXCL11 - was observed in patient CD4+ T lymphocytes (Fig. 2a), along with expression of CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CXCR1, and CXCR5, and slightly reduced expression of CCR9 and CXCR4 (data not shown). Assessment of ERK1/2 and AKT phosphorylation by Western blot of patient EBV-transformed B-cell lysates demonstrated an intact cellular response to CXCL11 stimulation when compared to control EBV-transformed B-cell lysates (Fig. 2b). Control EBV-transformed B cells exhibited a significant positive change in adhesiveness upon stimulation with CXCL11, with the mean cell count increasing from 24.3 to 45.5 (Fig. 2c left panel; n = 6, P = 0.0313). Conversely, patient EBV-transformed B cells underwent no significant change in adhesiveness in response to the same chemokine, with the mean cell count decreasing from 20.2 to 19.0 (n = 6). In view of this impaired adhesive response to chemokine, the chemotactic ability of primary patient cells was assessed by performing transwell chemotaxis assays (Fig. 2d).