An ENU mutagenesis approach to dissect "self"-induced immune responses: Unraveling the genetic footprint of immunosurveillance

Abstract

The immune system exerts a critical function as it recognizes and eliminates transformed or neoplastic cells, a process also referred to as immunosurveillance. NK cells play a particularly important role in that they are able to recognize tumor cells via "missing-self"-i.e., the absence of major histocompatibility complex Class I on target cells. Moreover, recent studies suggest that NK cells also participate in the onset and regulation of adaptive immune responses. The exact molecular pathways by which this occurs, however, remain poorly understood. To obtain further insight into the genes that are required for self-induced immune responses via NK cell-mediated cell death, our laboratory initiated a forward genetic approach using N-ethyl-N-nitrosourea (ENU) as a mutagen. Specifically, we tested the ability of NK cells from G3 ENU germline mice to recognize missing-self target cells and induce CD8+ T-cell responses following immunization with irradiated tumor cells. Here we present two ENU germline mutants, designated Ace and Chip, that are defective in the recognition of β-2 microglobulin-deficient target cells, yet exhibit improved clearance of B16 melanoma cells in vivo. Coarse mapping and whole genome sequencing of the Chip mutation revealed a missense mutation causing a T'A amino acid substitution in the highly conserved third immuno-receptor tyrosine-based switch motif of CD244 (2B4). The forward genetic approach described here promises to reveal important insight into critical genes that are required for host responses involved in anticancer immunity.

Figure 1. Identification of ENU germline mutants with impaired NK- and/or CD8⁺ T cell-mediated target cell eradication in vivo. C57BL/6J and G3 ENU germline mice were left untreated (naïve) or were immunized with 5 × 10⁶ irradiated 5E1.TAKO i.p.. Eight days after immunization, mice were injected i.v. with 3 × 10⁶ high CFSE E1B_192–200 peptide loaded C57BL/6J splenocytes (CD8⁺ T cell targets), 3 × 10⁶ medium CFSE β2M^−/− splenocytes (NK cell targets) and 3 × 10⁶ low CFSE C57BL/6J splenocytes (control non-target cells). After 48 h, ocular blood samples were collected and analyzed for the number of fluorescent cells in each CFSE population. Both Chip and Ace mutants exhibited impaired NK yet normal CD8⁺ T cell-mediated killing of target cells compared with C57BL/6J controls.

Figure 2. Improved B16 melanoma cell clearance in Ace and Chip mutants. (A) Reduced clearance of CFSE labeled β2m-deficient splenocytes in Ace and Chip mutants compared with C57BL/6J control mice in vivo. Twenty-four hours after transfer, blood samples were collected and analyzed for the presence of wildtype splenocytes (low-CFSE) and Kb-deficient splenocytes (medium-CFSE). The percentage killing is calculated from the ratio between β2m-deficient and C57BL/6J cells administered to β2m-deficient and control C57BL/6J recipients. Numbers in graph represent the mean % killing ± SD (n = 4). (B,C) The percentage of NKp46⁺ cells in C57BL/6J and mutant mice and their maturation (C) as measured by CD27 and CD11b surface expression (NKp46-gated) (n ≥ 3). (D) C57BL/6J control, anti-asialo treated C57BL/6J and homozygote Ace and Chip mutants were injected 1x10⁵ B16 melanoma cells i.v. Only male mice were used in this expt. After 3 weeks, mice were sacrificed and the number of lung tumor nodules were determined. * = p < 0.05; ** = p < 0.01; *** = p < 0.001

Figure 3. Impaired missing self-recognition in Chip mutants is the result of a missense mutation in the third ITSM motif of CD244. (A) Coarse mapping of the Chip mutation based on 26 mice and a panel of 150 SNPs covering the entire genome. The Chip phenotype was linked to the distal site of chromosome 1. (B), the G→A missense mutation at nucleotide 173,510919 bps on chromosome 1 (C) causes a single amino acid substitution (T→A) in the third ITSM motif of CD244. (D) CD244 surface protein expression is unaffected on NK cells (NKp46⁺) and dendritic cells (CD11c⁺) of homozygote and heterozygote CD244 mice, as determined by flow cytometry.