Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans

Abstract

f Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal V␣ and J␣ segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice.T he immune system is the site of intense genome dynamics, in particular during the development and maturation of B and T lymphocytes in bone marrow and the thymus, when antigen receptor genes are rearranged through V(D)J recombination prior to their expression. DNA damages are also likely to occur during the several phases of intense proliferation which accompany the development of B and T cells.V(D)J recombination is the prototypical example of the generation of a programmed DNA double-strand break (DNA-dsb) during lymphoid development through the activity of recombination activating genes 1 and 2 (Rag1/2) on immunoglobulin (Ig) and T cell receptor (TCR) genes (see the work of Helmink and Sleckman [1] for a recent review). The resulting DNA-dsb is resolved by the nonhomologous end-joining (NHEJ) DNA repair pathway, composed of seven core components (see the work of Lieber [2] for a recent review). The Cernunnos-Xrcc4-DNA ligase IV complex ultimately reseals the DNA-dsb. Cernunnos, also known as Xrcc4-like factor (XLF), was the last NHEJ factor that was independently identified, through a survey of RS-SCID patients (3) and a yeast two-hybrid screen with Xrcc4 as a bait (4). Cernunnos and Xrcc4 adopt the same overall three-dimensional crystal structure (5, 6) and, together with DNA ligase IV, are parts of the same complex (4, 7). Cernunnos stimulates the DNA-joining activity of the Xrcc4-DNA ligase IV complex (8, 9). V(D)J recombination constitutes a central checkpoint in the development of the immune system, as its defect leads to abortive B and T cell maturation in vivo, resulting in severe combined immune deficiency (SCID) (10), but its first recognized function was the generation of a diverse antigenic repertoire through the combinatorial association of variable, diversity, and joining segments that encode the variable domains of both Ig and TCRs (11). Numerous examples show that a reduced V(D)J recombinase activity affects the extent of antigenic diversity of immune receptors in mice and humans. The resulting immune deregulation may then lead to autoimmunity, increased susceptibility to infections, or the development of vario...

XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination

“…Vera et al have shown previously that both Xlf knockout mice and an XLFdeficient patient had a skewing of the TRA repertoire toward the more 39 (proximal) V and J genes. 20 They hypothesized that the reduced thymocyte lifespan does not allow the T cells to undergo multiple waves of VaJa rearrangements, which can be necessary for positive selection of T cells. At the IgH and TRB loci, these subsequent rearrangements do not occur, so we wondered whether the combinational diversity was also affected in these rearrangements.…”

“…34 Junction analysis in Xlf-deficient mouse lymphocytes Li et al and Vera et al published nucleotide sequences of DQ52-Jh, Vb18DJb1, and pMX-RSS12/23 junctions obtained from Xlf-deficient lymphocytes. 19,20 We reanalyzed the junctions and determined the deletions, N-nucleotides, and P-nucleotides.…”

“…6,7,18 Studies in XLF-deficient mouse models have confirmed the severe sensitivity to IR and chromosomal instability, but the V(D)J recombination defect seemed relatively mild. 19,20 So far, the most important action of XLF seems to be the stimulation of LIG4 activity, 21,22 probably by stabilizing the XRCC4-LIG4 complex on DNA and promotion of DNA strand alignment. 23 However, recently, Lescale et al have shown that the RAG postcleavage complex (RAG-PCC) and XLF have overlapping functions and have suggested that both the RAG-PCC and XLF ensure stabilization of DNA ends after DNA cleavage by RAG.…”

Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

“…In addition, CSR is reduced in XLF-deficient mouse B cells (29). However, chromosomal V(D)J recombination is largely normal in developing lymphocytes and pro-B-cell lines from XLF-deficient mice (29,30) because of XLF functional redundancy with ATM and downstream substrates, including H2AX and 53BP1 in C-NHEJ (31)(32)(33). Thus, XLF/ATM and XLF/53BP1 double-deficient mice are smaller than single-deficient littermates, have a SCID phenotype associated with severely deficient V(D)J CE and SE joining, and have a greater reduction in CSR than single-deficient mice (31)(32)(33).…”

Paralogie et redondance : maintenir l’intégrité du génome au cours de la recombinaison V(D)J

“…Chez l'homme, comme chez la souris, une déficience de la protéine ATM conduit au syndrome ataxia telangiectasia 1 anomalies du développement plus ou moins sévères et (d) une prédisposition aux cancers [6]. Chez la souris, une déficience de la voie NHEJ (par exemple une absence de XRCC4 ou LIG4) associée à un défaut du point de contrôle du combined immunodeficiency), due à un défaut de réparation des DSB induites par RAG et, par conséquent, une absence totale de lymphocyte mature (excepté dans le cas d'une déficience en XLF, voir ci-dessous), mais également (c) des héréditaire de la voie NHEJ, souffrent d'une grande variété de dysfonctionnements, notamment : (a) une sensibilité accrue aux agents génotoxiques, (b) une immunodéficience combinée sévère, communément appelée SCID (severe [7,8]. En effet, les lymphocytes déficients pour XLF sont capables de réparer les DSB induites par RAG, malgré une incapacité à réparer des DSB induites par des agents géno-toxiques [8].…”