Abstract
SummaryRAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7−CD5− to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7−CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.
Highlights
Severe combined immune deficiency (SCID) is a life-threatening disorder caused by a defective acquired immune system due to the absence of functional T cells (Fischer, 2000)
Generation of RAG2-SCID induced pluripotent stem cells (iPSCs) and Isogenic Control iPSCs We generated iPSCs from a female RAG2-SCID patient with a homozygous nonsense mutation (p.R148X) in RAG2 (Figure 1A) by transduction of the patient’s dermal fibroblasts with a lentiviral vector expressing codon-optimized OCT4 and KLF4, SOX2, and MYC (Warlich et al, 2011)
The percentage of CD31+CD34+ DP cells ranged from 0.4% to 1.7%, which was lower than with control H1 embryonic stem cells (ESCs) (3.9%) but similar to skinderived iPSCs from a healthy donor (1.5%)
Summary
Severe combined immune deficiency (SCID) is a life-threatening disorder caused by a defective acquired immune system due to the absence of functional T cells (Fischer, 2000). In addition to T cells, SCID patients sometimes lack B cells and/or natural killer (NK) cells depending on the underlying genetic mutations. Milder forms of SCID can occur as a consequence of hypomorphic mutations. One subgroup of SCID patients, accounting for approximately 30% of the SCID cases, lacks both T and B cells (Gaspar et al, 2013). Mutations causing TnegBneg SCID most often affect genes that play a role in the rearrangement of the B cell receptor (BCR) and T cell receptor (TCR) loci, such as recombination activating genes (RAG) RAG1 and RAG2, DCLRE1C, and PRKDC (Dvorak and Cowan, 2010). RAG1 and RAG2 proteins form a tetrameric complex of two RAG1/RAG2 heterodimers, of which one is bound to a 12-recombination signal sequence (RSS) and one to a 23-RSS in the V-D-J regions of the BCR and TCR loci. The tetrameric RAG complex catalyzes the pairwise cleavage of the RSSs, which are connected through non-homologous end joining, creating the huge V-D-J diversity that underlies the enormously diverse immune repertoire (Notarangelo et al, 2016)
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