Supplementary code repository for: Germline biallelic mutation affecting the transcription factor Helios causes pleiotropic defects of immunity

Authors: Tala Shahin,^1,2,3,4 Hye Sun Kuehn,⁵ Mohamed R. Shoeb,¹ Lisa M. Gawriyski,⁶ Sarah Giuliani,^1,2 Peter Repiscak,¹ Birgit Hoeger,^1,2 Özlem Yüce Petronczki,^1,2 Sevgi Köstel Bal,^1,2 Samaneh Zoghi,^1,2 Jasmin Dmytrus,^1,2 Davide Seruggia,¹ Irinka Castanon,¹ Nima Rezaei,^7,8,9 Markku Varjosalo,⁶ Florian Halbritter,¹ Sergio Rosenzweig,⁵ Kaan Boztug^1,2,3

Affiliations:

¹St. Anna Children’s Cancer Research Institute (CCRI), Vienna, Austria

²Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria

³CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

⁴Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria

⁵Immunology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20814, USA

⁶Institute of Biotechnology, Helsinki Institute of Life Science, Proteomics Unit, University of Helsinki, Helsinki, Finland

⁷Research Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran

⁸Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

⁹Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran

Abstract:

Helios, a member of the Ikaros family of transcription factors, is predominantly expressed within the T-cell lineage, including developing thymocytes, activated T cells and regulatory T cells (Tregs). Studies in mice have emphasized its role in maintenance of Treg immunosuppressive functions through stabilizing Foxp3 expression and silencing the IL-2 locus. Yet, its contribution to human immune homeostasis and the precise mechanisms by which Helios regulates other T-cell subsets remain unresolved. Here, we studied a patient with recurrent respiratory infections and hypogammaglobulinemia and identified a germline homozygous missense mutation in IKZF2 encoding Helios (p.Ile325Val). We show that HeliosI325V retains DNA-binding and dimerization properties, but loses interaction with several partners, including epigenetic remodelers HDAC1, HDAC3 and the ATAC complex. Single-cell RNA-sequencing of peripheral blood mononuclear cells revealed gene expression signatures indicative of a shift towards pro-inflammatory, effector-like status in patient T cells, while analysis of patient Tregs has shown a dysregulated transcriptome. Intriguingly, patient T cells were shown to be terminally differentiated, with a pronounced defect in proliferation and a marked reduction in IL-2 production upon stimulation, that was rescued by reverting the mutation in Helios back to wild type in patient-derived T cells. Collectively, we identify a novel germline-encoded inborn error of immunity and highlight a role for Helios in conventional T cells, whereby interactions with specific binding partners is necessary to mediate the transcriptional programs that enable T-cell homeostasis in health and disease.

Repository structure:

• src/RNA-seq - data analysis code for bulk- and single-cell RNA-seq

Analysis workflow:

• RNA-seq: Separate scripts are dedicated to each analysis steps to faciliate understanding and maintaining the workflow. The analysis pipeline is defined in src/RNA-seq/initialize.Rmd where scripts are loaded sequentially. For optimal reproducibility, we used a Docker container mo-ikzf2-v1.Dockerfile, which contains R and all dependent libraries preinstalled.

Data preparation and loading

1. Download and, if needed, preprocess the EGA data into a defined input directory. Metadata table can be found in the Supplementary table S4 Excel worksheet accompanying the manuscript.
2. For RNA-seq analysis, set the path to the input directory at the top of src/RNA-seq/initialize.Rmd and use mo-ikzf2-v1.Dockerfile to run the analysis workflow.

Input data can be obtained from EGA (link below).

Links:

• European Genome-Phenome Archive (EGA): https://ega-archive.org/studies/EGAS00001005675
• Paper: https://doi.org/10.1126/sciimmunol.abe3981