A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets

Abstract

NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCRB6C14R strain. Ly5.1C14R NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46C14R in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1C14R mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a+ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function.SignificanceInnate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells in vivo. This Ncr1 C14R mutation impairs NKp46 surface expression resulting in destabilization of Ncr1 and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46+ ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively.

Deconstructing deployment of the innate immune lymphocyte army for barrier homeostasis and protection

“…These lines, all derived from Jackson Laboratories, exhibited a single amino acid mutation from cysteine to arginine at amino acid 14 (C14R) which is localized in the region of the signal peptide of Ncr1 . This mutation did not alter the overall expression of Ncr1 mRNA but significantly impaired the surface expression of NKp46 through failed trafficking within the cell . These findings have major implications for previous studies of NK cells and ILC1 where these mice have been used, even further complicating our interpretation of earlier data (Table ).…”

“…The Ncr1 gfp/gfp mice have a complete loss of NKp46 expression, while Ncr1 Noé/Noé mice exhibit a mutation at W32R which affects surface expression of NKp46 . A recent study also suggests that ILC1 development is intrinsically dependent on NKp46 but this work contrasts with other studies that did not observe such effect . Thus, the exact consequences of Ncr1 loss requires deeper investigation to determine precisely how it affects ILC1 and NK cell function.…”

“…ILC1 also preferentially express other surface molecules including IL‐7Rα, TRAIL (Tumor necrosis factor apoptosis‐inducing ligand), and CD49a (also known as integrin alpha‐1) and their survival is strongly regulated by the transcription factor RUNX3 . TRAIL expression is dictated by signaling though the NKp46 receptor . In certain inflammatory conditions, NK cells and ILC3 are converted into ILC1‐like cells (sometimes referred to as ex‐NK cells and ex‐ILC3 respectively) and downregulate EOMES or RORγt expression in favor of T‐BET and TRAIL expression and exhibit enhanced IFN‐γ production .…”

“…Further complicating the interpretation of the data attributable to individual subsets was the emergence of plasticity between a number of ILC subsets implying that alternate flexible programs that did not neatly fit into the subset classification could be identified. In an unexpected twist, recently three groups identified a point mutation in the Ncr1 gene in the congenic CD45.1 + mice used for many studies of lymphocyte tracking and function analysis (Figure A, Table ) . These lines, all derived from Jackson Laboratories, exhibited a single amino acid mutation from cysteine to arginine at amino acid 14 (C14R) which is localized in the region of the signal peptide of Ncr1 .…”

“…The mutant mice, referred to as Ly5.1 C14R , showed normal numbers of NK cells and ILC1 but a modest alteration to the immature and mature NK cell subsets . To date, several models affecting Ncr1 expression have been generated.…”