Hypomorphic Rag1 mutations alter the preimmune repertoire at early stages of lymphoid development

Abstract

Hypomorphic mutations allowing residual T- and B-cell development have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (CID-G/AI) and abnormalities of the peripheral T- and B-cell repertoire. To examine how hypomorphic mutations affect the earliest stages of lymphocyte development, we used CRISPR/Cas9 to generate mouse models with mutations equivalent to those found in patients with CID-G/AI. Immunological characterization showed partial development of T and B lymphocytes, with persistence of naïve cells and preserved serum immunoglobulin but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with CID-G/AI. By using high-throughput sequencing, we identified marked skewing of and gene usage in early progenitors, with a bias for productive and rearrangements after selection occurred and increased apoptosis of B-cell progenitors. Rearrangement at the locus was impaired, and polyreactive immunoglobulin M antibodies were detected. This study provides novel insights into how hypomorphic mutations alter the primary repertoire of T and B cells, setting the stage for immune dysregulation frequently seen in patients.

RAG gene defects at the verge of immunodeficiency and immune dysregulation

“…T reg abnormalities are also an important factor that may contribute to autoimmune manifestations in RAG deficiency. Reduced number of thymic T reg and a restricted repertoire of peripheral T reg cells have been reported both in patients and in Rag mutant mice . Moreover, we have shown that T reg from patients with OS, AS, and CID‐G/AI have reduced suppressive activity .…”

“…High‐throughput sequencing of the Trb repertoire in DN4 thymocytes from Rag1 R972Q and Rag1 F971L mice has revealed skewed usage of V and J genes, with a pattern that was consistent between the two models . Similarly, these mutant mice presented skewed usage of IghV and IghJ genes in pro‐B cells.…”

“…Moreover, we have shown that T reg from patients with OS, AS, and CID‐G/AI have reduced suppressive activity . Although T reg function was apparently preserved in Rag1 R972Q and Rag1 F971L mice, the restricted repertoire of these cells may translate in decreased suppressive function in response to specific antigens.…”

“…In order to generate such a model, we used CRISPR‐based gene editing to introduce in the coding flank‐sensitive region of RAG1 CTD mutations that are equivalent to those identified in patients with CID‐G/AI. In particular, we generated three mouse models, homozygous for F971L, R972Q and R972W amino acid changes . One of these models (the Rag1 R972Q mouse) carries the same mutation as the MM mouse model reported above.…”

“…Furthermore, the number of both cDC and plasmacytoid DC (pDC) and generation of thymic T reg cells were severely reduced in both Rag1 S723C and Rag2 R229Q mice . The thymic medulla was also very small in Rag1 R972Q , Rag1 F971L , and Rag1 R972W mice, although CMD was partially preserved . As ultimate proof of the importance of lymphostromal cross talk for TEC maturation, we tested in vivo the effect of pre‐TCR signaling on the epithelial compartment.…”