Molecular Signatures of Natural Killer Cells in CMV-Associated Anterior Uveitis, A New Type of CMV-Induced Disease in Immunocompetent Individuals

Abstract

Cytomegalovirus (CMV) causes clinical issues primarily in immune-suppressed conditions. CMV-associated anterior uveitis (CMV-AU) is a notable new disease entity manifesting recurrent ocular inflammation in immunocompetent individuals. As patient demographics indicated contributions from genetic background and immunosenescence as possible underlying pathological mechanisms, we analyzed the immunogenetics of the cohort in conjunction with cell phenotypes to identify molecular signatures of CMV-AU. Among the immune cell types, natural killer (NK) cells are main responders against CMV. Therefore, we first characterized variants of polymorphic genes that encode differences in CMV-related human NK cell responses (Killer cell Immunoglobulin-like Receptors (KIR) and HLA class I) in 122 CMV-AU patients. The cases were then stratified according to their genetic features and NK cells were analyzed for human CMV-related markers (CD57, KLRG1, NKG2C) by flow cytometry. KIR3DL1 and HLA class I combinations encoding strong receptor-ligand interactions were present at substantially higher frequencies in CMV-AU. In these cases, NK cell profiling revealed expansion of the subset co-expressing CD57 and KLRG1, and together with KIR3DL1 and the CMV-recognizing NKG2C receptor. The findings imply that a mechanism of CMV-AU pathogenesis likely involves CMV-responding NK cells co-expressing CD57/KLRG1/NKG2C that develop on a genetic background of KIR3DL1/HLA-B allotypes encoding strong receptor-ligand interactions.

Keywords: CD57; HLA class I; KLRG1; NKG2C; cytomegalovirus; cytomegalovirus-associated anterior uveitis; killer cell immunoglobulin-like receptors; natural killer cells.

Figure 1

KIR genotypes in CMV-associated anterior uveitis (CMV-AU) patients and healthy controls. KIR genotypes (a) and gene frequencies (b) were determined in 122 individuals with CMV-AU. As a control, KIR genotypes in 208 healthy individuals of the same ethnicity (Chinese Singaporean) are shown from a previous study [25]. CMV-seropositivity was 87% based on a previous study in this population [26]. (a) Shaded boxes indicate presence of KIR genes. The centromeric/telomeric position of KIR loci indicated in columns 4 and 6 are their physical location within the KIR complex on chromosome 19 [27,28]. KIR2DL5, KIR2DS3, and KIR2DS5 genes can locate on either centromeric/telomeric sides of the KIR complex and are thus indicated in this table as such (column 5). KIR B haplotype-specific KIR genes are shown in red. KIR framework genes and pseudogenes are excluded. (b) Black and gray boxes indicate each KIR genotype frequency in CMV-AU and healthy controls, respectively.

Figure 2

Expansion of the CD57⁺KLRG1⁻ or the CD57⁺KLRG1⁺ subsets among the four natural killer (NK) cell subsets distinguished by the differential expression of CD57 and KLRG1. (a) Identification of four NK cell subsets as determined by differential expression of CD57 and KLRG1. Representative profiles of two CMV-seropositive healthy individuals with expansion of either the CD57⁺KLRG1⁺ or CD57⁺KLRG1⁻NK subset. Notably, NKG2A and NKG2C are expressed at different levels on each subset. (b) Frequencies of the four NK subsets are shown for each individual. ANOVA: CMV-AU, p = 0.0025; CMV-seropositive controls, p = 0.002. * p < 0.05, ** p < 0.01, *** p < 0.001. (c) The proportions of NK cell subsets most expanded in the CMV-AU and CMV-seropositive healthy individuals. CMV-AU: n = 17, CMV-seropositive controls: n = 15.

Figure 3

Expansion of CD57/KLRG1 DP NK cells in CMV-AU patients carrying HLA-B Bw4 80Ile and KIR3DL1H. (a) The proportion of expanded NK cell subsets among CMV-AU patients and CMV-seropositive healthy individuals in the presence or absence of HLA-B Bw4 80Ile and KIR3DL1H (CMV-AU, n = 17; CMV-seropositive controls, n = 15). (b) Distributions of KIR3DL1H⁺NK cells among the CD57/KLRG1 subsets. CD57/KLRG1 DP subset frequencies among KIR3DL1H⁺NK cells were significantly elevated in CMV-AU patients carrying HLA-B Bw4 80Ile as compared to CMV-seropositive controls (CMV-AU, n = 12; CMV-seropositive controls, n = 13; ANOVA p = 0.0097). Data represent the mean+ standard deviation. * p < 0.05, ** p < 0.01.

Figure 4

The four NK cell subsets defined by differential expression of CD57 and KLRG1 express disparate levels of NKG2C and iKIR. (a) Proportion of cells expressing NKG2C and iKIR on the four NK cell subsets. (CMV-AU, n = 15; CMV seropositive controls, n = 17. ANOVA: NKG2C, p = 0.047; self-iKIR, p = 0.0001; iKIR, p = 0.0006. Linear trend test: self-iKIR & iKIR, p < 0.0001). * p < 0.05, ** p < 0.01, *** p < 0.001. (b) Proportion of CD57⁺NKG2C⁺, NKG2C⁺, CD57⁺NK cells in CMV-AU and CMV-seropositive healthy individuals (CD57⁺NKG2C⁺, CD57⁺: CMV-AU, n = 17; CMV-seropositive controls, n = 15; NKG2C⁺: CMV-AU, n = 20; CMV-seropositive controls, n = 18). Data represent the mean ± standard deviation.