Identification and Characterisation of Novel Thymic Stromal Cell Defects

Background:
Athymia, congenital absence of the thymus, is usually fatal in the first two years of life. Most commonly this problem is associated with complete DiGeorge Syndrome (cDGS), but it is also found in patients with Severe Combined Immunodeficiency (SCID) due to FOXN1- and PAX1-deficiency2,3. The only definitive treatment for athymic patients is allogeneic thymus transplantation1,2. The thymus transplantation programme at UCL’s Institute of Child Health (ICH) and Great Ormond Street Hospital (GOSH) is one of only two programmes worldwide to offer this treatment.

Aims/Objectives:
As demonstrated in cDGS and FOXN1 Nude SCID, complete athymia can be treated with allogeneic thymus transplantation. The general aim of this translational research project is to identify and characterise undefined thymic stromal cell defects, including novel aetiologies for congenital athymia, affecting patients who may undergo haematopoietic stem cell transplantation (HSCT) in the mistaken belief that they have a haematopoietic lineage defect. This is particularly relevant now that universal newborn screening programmes for SCID are about to be introduced in the UK and elsewhere.

Objective 1: An ex vivo T cell differentiation assay, co-culturing bone marrow with a stromal cell line4, will be established to help distinguish genetically undefined T-B+NK+ SCID patients with thymic stromal cell defects from those with haematopoietic defects. This will allow the rapid identification of patients who might benefit from thymus transplantation instead of HSCT.
Objective 2: Patient-derived induced pluripotent stem cells (iPSCs) from patients with suspected thymic defects will be generated and differentiated into thymic epithelial progenitor (TEP) cells3. Analysis of the transcriptional profile of genes involved in thymus development5 will allow confirmation of impaired thymic stromal cell biology. Transcriptomics will also facilitate the identification of new candidate genes.
Objective 3: Using gene editing tools, functional studies will be conducted to validate novel genes as disease-causing, thus increasing our knowledge on thymus development and biology.

Methods:
Patient recruitment and material: This project will have access to a unique cohort of historic and prospective patients with defined and undefined, but suspected thymic stromal cell defects, who are referred to  GOSH’s thymus transplantation programme. Stored patient materiel, including PBMCs, bone marrow (BM) and fibroblasts, will be used. Ex vivo T cell differentiation assay: CD34+ haematopoietic stem and progenitor cells from BM aspirate samples will be co-cultured with a stromal murine cell line (OP9) expressing the Notch ligand Delta-like-ligand 1 (OP9/DL1). The achieved stage of T cell differentiation will be analysed by flow cytometry. Targeted differentiation of iPSCs: Primary skin fibroblasts will be differentiated into iPSCs and subsequently into definitive endoderm and TEP cells adapting existing protocols. scRNA sequencing and gene editing: TEP transcriptome profiling will be done using single-cell transcriptomics. CRISPR/Cas technology will allow the functional assessment of disease-causing mutations and novel candidate genes.

References:
1. Davies EG et al. Thymus transplantation for complete DiGeorge syndrome: European experience. J Allergy Clin Immunol 2017; 140:1660-70.
2. Markert ML et al. First use of thymus transplantation therapy for FOXN1 deficiency (nude/SCID): a report of 2 cases. Blood 2011; 117(2):688-96.
3. Yamazaki Y et al. PAX1 is essential for development and function of the human thymus. Sci Immunol 2020; 5(44).
4. Schmitt TM, Zuniga-Pflucker JC. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 2002; 17(6):749-756.
5. Park JE et al. A cell atlas of human thymic development defines T cell repertoire formation. Science 2020; 367(6480).