Severe immunodeficiency in mice lacking DNA repair factors XLF and Mri

Department of Clinical and Molecular Medicine (IKOM), Norwegian University of Science and technology, 7491 Trondheim, Norway; St. Olavs Hospital, Trondheim University Hospital, Clinic of Medicine, Postboks 3250 Sluppen, 7006 Trondheim; Behavioural Neurobiology MS program, Theodor-Boveri-Institute, Biocenter, University of Würzburg, 97070 Würzburg, Germany. Department of Biosciences and Nutrition (BioNut), Karolinska Institutet, 14183 Huddinge, Sweden Institute of Clinical Medicine, UiT The Arctic University of Norway, Tromso, Norway These authors contributed equally


Introduction
Non-homologous end-joining (NHEJ) is a DNA repair pathway that recognizes, processes and ligates DNA double-stranded breaks (DSB) throughout the cell cycle. NHEJ is required for lymphocyte development, in particular, to repair DSBs induced by the recombination activating genes (RAG) 1 and 2 in developing B and T lymphocytes, and by activation-induced cytidine deaminase (AID) in mature B cells [1]. NHEJ is initiated when core subunits Ku70 and Ku80 (Ku) are recruited to the DSB sites. Ku, together with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), form the DNA-PK holoenzyme [2]. Subsequently, the nuclease Artemis is recruited to the DSB sites to process DNA hairpins and overhangs [3]. Finally, DNA ligase IV (Lig4), X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF) mediate DNA end ligation. The NHEJ complex is stabilized by a paralogue of XRCC4 and XLF (PAXX) and a modulator of retroviral infection (Mri) [4,5].
In this study, we rescue synthetic lethality from Xlf and Mri by inactivating one or two alleles of Trp53.
We show that resulting Xlf -/-Mri -/-Trp53 +/mice possess a leaky SCID phenotype with severely reduced mature B and T lymphocyte counts in the spleen, low mature T cell counts in the thymus, and accumulated progenitor B cells in bone marrow. Finally, we demonstrate that Mri functions in B and T lymphocyte development in vivo, and its roles are compensated by XLF.

Mice
All experiments involving mice were performed according to the protocols approved by the Comparative Medicine Core Facility (CoMed) at the Norwegian University of Science and Technology (NTNU, Trondheim, Norway). Xlf +/- [11] and Dna-pkcs +/- [2] mice were imported from the laboratory of Professor Frederick W. Alt at Harvard Medical School. Trp53 +/mice [32] were imported from Jackson Laboratories. Paxx +/- [16] and Mri +/-[18] mice were generated by the Oksenych group and described previously.

Lymphocyte development
Lymphocyte populations were analyzed by flow cytometry as described previously [16,18,19,22]. In summary, cells were isolated from the spleen, thymus, and femur of 5-7-week-old mice and treated with red blood cell lysis buffer Hybri-Max TM (Sigma Aldrich, St. Louis, MO, USA; #R7757). The cells were resuspended in PBS (Thermo Scientific, Basingstoke, UK; #BR0014G) containing 5% Fetal bovine serum, FCS (Sigma Life Science, St. Louis, Missouri, United States; #F7524), and counted using a Countess™ II Automated Cell Counter (Invitrogen, Carlsbad, CA, United States; #A27977). Then, the cell suspension was diluted with PBS to get a final cell concentration of 2.5 x 10 7 cells per mL. Finally, surface markers were labeled with fluorochrome-conjugated antibodies and the cell population was analyzed using flow cytometry.

Antibodies
The following antibodies were used for flow cytometry analysis: rat anti-CD4-PE-Cy7 (BD

Leaky SCID in mice lacking XLF and PAXX
Combined inactivation of XLF and PAXX has been shown to result in embryonic lethality in mice [4,14,15,20]. To determine the impact of XLF and PAXX on B and T cell development in vivo, we rescued the synthetic lethality by inactivating one allele of Trp53, as described previously [20]. The  Therefore, we concluded that B cell development is blocked at the pro-B cell stage of Xlf -/-Mri -/-Trp53 +/and Xlf -/-Paxx -/-Trp53 +/mice.

Normal development of Paxx -/-Mri -/mice
Both PAXX and Mri are NHEJ factors that are functionally redundant with XLF in mice. Specifically, we observed that Paxx -/-Mri -/mice had normal thymocyte and splenocyte counts, which encompass CD4+, CD8+, and CD4+CD8+ T cells and CD19+ B cells (Fig. 4C). Paxx inactivation did not affect Ig switch to IgG1 in Mri-deficient B cells (Fig. 4D,E). The quantity of IgG1+ cells after CSR stimulation was similar between Paxx -/-Mri -/and Mri -/naïve B cells (p>0.48), although both were lower than that of WT control, at 72 h and 96 h (p<0.05). From this, we can conclude that there is no genetic interaction between Paxx and Mri in vivo.

Synthetic lethality between Mri and Dna-pkcs in mice
Both Mri and DNA-PKcs are functionally redundant with XLF in mouse development [5,24].
We also found that mice with combined inactivation of Paxx and Mri (Paxx -/-Mri -/-) are liveborn, fertile, and undergo nearly normal B and T cell development (Fig. 4). Moreover, inactivation of Paxx did not affect the CSR efficiency in in vitro stimulated Mri-deficient B cells (Fig. 4) Paxx and Mri both interact genetically with Xlf, we conclude that they do not interact with each other.
Both Mri an Dna-pkcs genetically interact with Xlf. Strikingly, we found that combined inactivation of Mri and Dna-pkcs (Mri -/-Dna-pkcs -/-) leads to embryonic lethality, and that E14.5 Mri -/-Dna-pkcs -/murine embryos were about 40% smaller than single-deficient siblings. DNA-PKcs is associated with the N-terminus of the Mri and Ku heterodimer in the process of recognizing DSBs [5], which may account for genetic interaction between Mri and Dna-pkcs. Thus, inactivation of Trp53, Ku70 or Ku80 may be a viable method to rescue synthetic lethality from Mri -/-Dna-pkcs -/mice.
In conclusion, we have developed several complex genetic models (Fig. 6). All the authors contributed to writing of the final manuscript.

Conflict of interest statement
The authors declare no conflict of interest.