A kindred with mutant IKAROS and autoimmunity

Abstract

IKAROS (encoded by IKZF1) is an important hematopoietic transcription factor critical for early B cell differentiation, with major defects known to lead to low B cell numbers and hypogammaglobulinemia. More perplexing is the link between IKZF1 variants and autoimmunity, including polymorphisms associated with susceptibility to SLE, and recently, rare variants driving monogenic autoimmunity. We identified a novel p.L188V mutation in IKZF1 in the index patient and her father and found this mutation to lead to loss of DNA binding. Peripheral B cells lacking a full complement of IKAROS function show upregulation of molecules accentuating B cell activation, while CD22, a key negative feedback circuit, is suppressed. The resulting hyperresponsiveness of peripheral B cells, in combination with elevated follicular helper T cell (Tfh) numbers, provides a putative mechanistic explanation for the association of IKZF1 variants with the emergence of autoimmune manifestations in this kindred.

Fig. 1.. Heterozygous mutation in IKZF1 is a strong driver of autoimmunity through loss of DNA binding capacity

(A) Segregation of the p.L188V mutant allele and the clinical and immunological phenotype. P1 and P2 are indicated. (B) Schematic representation of the IKAROS protein. Zinc-fingers (ZF) are shown as dark rectangles. The p.L188V mutation is marked by a black arrow. (C) EMSAs were performed using two different probes: IK-bs4 and ΥSat8 as indicated. Quantification data were normalized using the binding of the extracts from WT transfected cells as 100% (6 independent experiments). (D) NIH3T3 cells transfected with Flag-mutant, Flag-WT and/or HA-WT as indicated were stained with anti-Flag antibody +/− an anti-HA antibody followed by fluorochrome-conjugated secondary antibodies. Representative of 4 independent confocal experiments. Scale bar 20μm. (E) IK-bs4 and YSat8 EMSAs, where an increasing amount of L188V mutant was transfected with an equal amount of WT IKAROS. Quantified data were normalized using the binding of the extracts from WT transfected cells set as 100% (4 independent experiments). Statistics were performed using one-way ANOVA. Mean±SEM. **** = p<0.0001, ** = p<0.01, * = p<0.05.

Fig. 2.. Hyperresponsive phenotype of mutant IKAROS B cells is rescued by CD22 overexpression.

(A) CD19+ MFI on B cells from the CFSE assay in Fig. E6. (B) PBMCs stimulated with 2.5μg/ml or 10μg/ml anti-IgM-IgA-IgG were stained for pErk. Shaded area marks one standard deviation above and below the mean of the HC (2 repeats). (C) Baseline MFI of pErk in primary B cells for patients versus HC normalized to the average of HC. (D) PBMCs stimulated with anti-IgM-IgA-IgG 10μg/ml alone or or anti-IgM-IgA-IgG 10μg/ml + CD40L 1μg/ml for 24hrs were stained for CD69 (1 repeat) and (E) CD22 (2 repeat). (F) CD22 mRNA expression normalized to GAPDH and Actin for P and seven HC for 3 repeats. (G) Relative quantification of DNA binding on CD22 specific EMSA probes, normalized to WT binding (3 repeats). (H) ChIP-qPCR of IKAROS in EBV transformed B cells of P2, HC and GM12878 human B lymphocytes for CD22 and ZNF180 (not bound by IKAROS) (3 repeats). (I) CD22 MFI on CD19+ primary B cells after transduction with hCD22 for P2 and HC. (J) pErk MFI on hCD22 transduced CD19+ primary B cells after stimulation with 10μg/ml anti-IgM-IgA-IgG (1 repeat). Mean±SEM. **** = p<0.0001, *** = p<0.001 ** = p<0.01, * = p<0.05.