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. 2007 Apr 1;178(7):4473-81.
doi: 10.4049/jimmunol.178.7.4473.

The human cytomegalovirus MHC class I homolog UL18 inhibits LIR-1+ but activates LIR-1- NK cells

Virginie Prod'homme  1 , Cora Griffin, Rebecca J Aicheler, Eddie C Y Wang, Brian P McSharry, Carole R Rickards, Richard J Stanton, Leszek K Borysiewicz, Miguel López-Botet, Gavin W G Wilkinson, Peter Tomasec
Affiliations

The human cytomegalovirus MHC class I homolog UL18 inhibits LIR-1+ but activates LIR-1- NK cells

Virginie Prod'homme et al. J Immunol. .

Abstract

The inhibitory leukocyte Ig-like receptor 1 (LIR-1, also known as ILT2, CD85j, or LILRB1) was identified by its high affinity for the human CMV (HCMV) MHC class I homolog gpUL18. The role of this LIR-1-gpUL18 interaction in modulating NK recognition during HCMV infection has previously not been clearly defined. In this study, LIR-1(+) NKL cell-mediated cytotoxicity was shown to be inhibited by transduction of targets with a replication-deficient adenovirus vector encoding UL18 (RAd-UL18). Fibroblasts infected with an HCMV UL18 mutant (DeltaUL18) also exhibited enhanced susceptibility to NKL killing relative to cells infected with the parental virus. In additional cytolysis assays, UL18-mediated protection was also evident in the context of adenovirus vector transduction and HCMV infection of autologous fibroblast targets using IFN-alpha-activated NK bulk cultures derived from a donor with a high frequency of LIR-1(+) NK cells. A single LIR-1(high) NK clone derived from this donor was inhibited by UL18, while 3 of 24 clones were activated. CD107 mobilization assays revealed that LIR-1(+) NK cells were consistently inhibited by UL18 in all tested donors, but this effect was often masked in the global response by UL18-mediated activation of a subset of LIR-1(-) NK cells VSports手机版. Although Ab-blocking experiments support UL18 inhibition being induced by a direct interaction with LIR-1, the UL18-mediated activation is LIR-1 independent. .

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FIGURE 1
FIGURE 1
gpUL18 expression inhibits NKL cytotoxicity. HFFF were mock infected or were infected for 96 h with HCMV strains AD169 or AD169 UL18 mutant (ΔUL18) (10 PFU/cell). a, LIR-1 surface expression on NKL was analyzed by flow cytometry with anti-LIR-1 and control isotype mAbs. b, NKL cells were used as effectors in allogeneic chromium release assays. Results are means ± SEM of triplicate samples and are representative of three independent experiments (two-way ANOVA analysis: ΔUL18 vs AD169, p = 0.0005). c, gpUL18 surface expression on HFFF was detected by flow cytometry with anti-gpUL18 and control isotype mAbs.
FIGURE 2
FIGURE 2
gpUL18 expression inhibits NKL cytotoxicity. HFFF were infected for 72 h with a RAd vector containing the complete AD169 UL18 ORF (RAd-UL18) or with the empty control adenovirus (RAd-Ctrl) (100 PFU/cell). a, gpUL18 surface expression on HFFF was detected by flow cytometry with anti-gpUL18 and control isotype mAbs. b, NKL cells were used as effectors in allogeneic chromium release assays (two-way ANOVA analysis: RAd-UL18 vs RAd-Ctrl, p = 0.0014). c, A saturating amount (10 μg/ml) of anti-LIR-1 or control anti-CD56 mAbs was added on NKL for 15 min at 37°C before contact with targets and throughout the allogeneic chromium release assays (two-way ANOVA analysis: RAd-UL18 + anti-CD56 vs RAd-UL18 + anti-LIR-1, p < 0.0001). Results are means ± SEM of triplicate samples and are representative of three independent experiments.
FIGURE 3
FIGURE 3
gpUL18 protection from polyclonal NK cell killing differs between donors. Autologous NK cytolysis assays were set up with T cell-depleted IFN-α-activated PBMC from three HCMV+ healthy donors and primary skin fibroblasts from the same volunteers. a, Fibroblasts were mock infected or infected for 96 h with HCMV strains AD169 and AD169 UL18 mutant (ΔUL18) (10 PFU/cell). ΔUL18 vs AD169 recognition was analyzed with a two-way ANOVA test assuming not-repeated measures for donor 1 (NS), donor 2 (NS), and donor 3 (p < 0.0001). b, Fibroblasts were infected for 72 h with a RAd vector containing the complete AD169 UL18 ORF (RAd-UL18) or with the empty control adenovirus (RAd-Ctrl) (500 PFU/cell). RAd-UL18 vs RAd-Ctrl recognition was analyzed with a two-way ANOVA test assuming not-repeated measures for donor 1 (NS), donor 2 (NS), and donor 3 (p < 0.0001). Results are means ± SEM of triplicate or quadruplicate samples and are representative of three independent experiments.
FIGURE 4
FIGURE 4
gpUL18 expression inhibits LIR-1+ NK cell activation from donor 3. Autologous CD107a mobilization assays were set up with IFN-α-activated PBMC against primary skin fibroblasts from donor 3. Dot plots were gated on CD3−CD56+ NK cells. a, PBMC incubated without any target or with K562 cells are shown as controls. b, Fibroblasts were infected for 72 h with RAd vectors containing the UL18 ORF (RAd-UL18) or the empty control adenovirus (RAd-Ctrl) (500 PFU/cell). c, Fibroblasts were infected for 72 h with RAd vectors containing the UL142 ORF (RAd-UL142) or with the empty control adenovirus (RAd-Ctrl) (500 PFU/cell). d, The dot plot quadrants were adjusted on the LIR-1+ NK cell MFI to allow a comparison of the LIR-1high NK cell response on the upper quadrants with the LIR-1low NK cell degranulation on the lower quadrants. Results are representative of duplicate samples in three independent experiments.
FIGURE 5
FIGURE 5
gpUL18 NK inhibition is mediated via the LIR-1 receptor. Autologous CD107a mobilization assays were set up with IFN-α-activated PBMC against primary skin fibroblasts from donor 3. Fibroblasts were infected for 72 h with RAd vectors containing the UL18 ORF (RAd-UL18) or with the empty control adenovirus (RAd-Ctrl) (500 PFU/cell). A saturating amount (20 μg/ml) of anti-LIR-1 F(ab’)2 or control anti-CD56 mAb was added on PBMC for 15 min at 37°C before contact with targets and throughout the assays (***, p < 0.001 with two-way ANOVA analysis assuming not-repeated measures followed by Bonferroni post tests). Results are means ± SEM of duplicate samples and are representative of three independent experiments.
FIGURE 6
FIGURE 6
gpUL18 expression inhibits LIR-1+ NK cell activation from six tested donors. Allogeneic CD107a mobilization assays were set up with IFN-α-activated PBMC against HFFF infected for 72 h with a RAd vector containing the UL18 ORF (RAd-UL18) or with the empty control adenovirus (RAd-Ctrl) (100 PFU/cell). Results are means ± SEM of duplicate samples and are representative of three independent experiments.
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