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

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. MATERIALS AND METHODSGeneration of Cernu ؊/؊ mice.Cernu Ϫ/Ϫ mice were developed at the Institut Clinique de la Souris (ICS; Illkirch, France) by flanking exon 4 with LoxP sites (Fig. 1).Mice were genotyped by standard PCR on tail DNA by use of the following primers: Cernu4R (5=-GTCCCCAGCTGTT AAGAGTTTC-3=) for KO and flox mice, CernuExo4F (5=-GGATGAAG GACCTTGAGATCC-3=) for flox mice, and Cernu3F (5=-CTATGGAAG CCAGGAGAGAATG-3=) for KO mice.All animals were maintained in a specific-pathogen-free environment.Analyses were performed on Cernu KO and littermate control animals on a mixed B6/129 background.P53 KO mice were on a mixed B6/129 background as well.All experiments and procedures were performed in compliance with the French Ministry of Agriculture's regulations for animal experiments (act 87847, 19 October 1987; modified in May 2001). Analysis of lymphocyte populations.Cell phenotyping was performed on blood, thymus, bone marrow, lymph nodes, and splenic lymphoid populations by four-color fluorescence analysis as previously described (14).Invariant natural killer T cell (iNKT cell) determination was performed on splenocytes from 4-to 8-week-old mice by staining with allophycocyanin (APC)-mouse CD1d tetramer, loaded or not loaded with