Cutting Edge: Check Your Mice-A Point Mutation in the Ncr1 Locus Identified in CD45.1 Congenic Mice with Consequences in Mouse Susceptibility to Infection

Abstract

B6.SJL-Ptprc^a Pepc^b /Boy (CD45.1) mice have been used in hundreds of congenic competitive transplants, with the presumption that they differ from C57BL/6 mice only at the CD45 locus. In this study, we describe a point mutation in the natural cytotoxicity receptor 1 (Ncr1) locus fortuitously identified in the CD45.1 strain. This point mutation was mapped at the 40th nucleotide of the Ncr1 locus causing a single amino acid mutation from cysteine to arginine at position 14 from the start codon, resulting in loss of NCR1 expression. We found that these mice were more resistant to CMV due to a hyper innate IFN-γ response in the absence of NCR1. In contrast, loss of NCR1 increased susceptibility to influenza virus, a result that is consistent with the role of NCR1 in the recognition of influenza Ag, hemagglutinin. This work sheds light on potential confounding experimental interpretation when this congenic strain is used as a tool for tracking lymphocyte development.

Figure 1. CD45.2 and CD45.1 strains exhibit different susceptibility to infection

(A) History of in-house breeding of CD45.2 and CD45.1 mouse strains. Mouse survival after (B) MCMV, (C) influenza virus, or (D) Citrobacter rodentium infection. Data are from two independent experiments with n = 10 mice per group. Statistics were analyzed using Log-rank Mantel-Cox test.

Figure 2. CD45.1 strain bear a point mutation in the Ncr1 locus causing loss of NCR1 expression

NCR1 expression on (A) spleen NK cells (CD3⁻NK1.1⁺), (B) NK cells from the indicated organs, and (C) intestinal ILCs. (D) Surface and intracellular expression of NCR1 using monoclonal (mAb) or polyclonal (pAb) antibodies. (E) NCR1 expression on NK cells from CD45.1 recipients 3 weeks after gut content transfer. (F) NCR1 expression on donor-derived NK cells from CD45.2⁺ or CD45.1⁺ cells. (G) Genomic DNA and mRNA transcripts of Ncr1 gene. (H) mRNA and protein sequence of Ncr1 gene. (I) Amounts of NCR1 proteins from sorted NK cells. (J, K) NCR1 expression in mutant (NCR1^C14R) and wild-type (NCR1^WT) transfected cells. Data are representative of three independent experiments with n = 4 per group. Statistics were analyzed using t-test, ****p < 0.0001.

Figure 3. Ncr1^C14R mutant mice mount a stronger innate IFNγ response

Frequency of GzmB⁺ cells among NK cells (A) from PolyI:C-treated mice, or (B) after 24 hr. simulation with IL-15 (20ng/ml). (C) Percent of NK specific lysis of YAC-1 cells in the absence or absence of 10 μg/ml of anti-NCR1 antibody. Frequency of IFNγ⁺ cells among NK cells stimulated with (D) IL-12 (1ng/ml) plus IL-18 (2ng/ml), or (E) plate-bound anti-NK1.1 (1μg/ml) antibody for 5 hrs. (F) Amounts of IFNγ secreted by NK cells stimulated with anti-Ly49H antibody for 6 hrs. (G) MCMV titers on day 3. Frequency of IFNγ⁺ cells among NK cells on days 1.5 (H, I, J) and 5 (K, L, M) post-infection. Results are from in vitro re-stimulation with 0.5ng/ml (L) or 1ng/ml (H, I, K, M) of IL-12 supplemented with 2ng/ml of IL-18, or PMA (20ng/ml) plus Ionomycin (1 μg/ml) (J). (N) NCR1 expression in F1 and F2 progeny and parental CD45.1Ncr1^C14R and CD45.2 mice. Mutant and wild-type mice from F2 progeny were infected with MCMV and spleen NK cells were analyzed on d1.5 post-infection for proliferation (O) and GzmB expression (P). IFNγ expression is shown after 5 hr. stimulation with (Q) IL-12 (1ng/ml) plus 2ng/ml of IL-18 (2ng/ml) or (R) PMA/Ionomycin. (S) Dok-1 and p38 phosphorylation from NK cells stimulated with anti-Ly49H antibody for the indicated time points (min). Data are representative of three to four independent experiments with n = 4 per group. Data are mean ± SEM or shown from individual mice. Statistics were analyzed using t-test or two-way ANOVA with Sidak correction, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001.

Figure 4. Tracing the Ncr1^C14R mutation in CD45.1 “congenic” mice

(A) NCR1 expression from CD45.1 mice purchased from commercial vendors between 2015-2017. Data are representative of three independent experiments with n = 4. (B) NCR1 expression from CD45.1 mice purchased from The Jackson Laboratories before 2009 versus after 2015. (C) The colony generation of Jax B6 CD45.1 strain over time. (D) Mice from N22 colony generation could be the source of the Ncr1^C14R mutation.