Inhibition of NKp46-induced NK cell activation by antibody-mediated triggering of CD161.

<p>PBMC were isolated and stimulated for three hours with the anti-NKp46 mAb 9E2 combined with the anti-CD161 mAb 191B8 or an isotype matched control antibody. The antibodies had been coupled to Fc-receptors on P815 cells. Activation of NK cells was assessed by monitoring CD107a expression on gated CD3^-CD56⁺ cells. (A) Representative dot-plots showing weaker down-regulation of NK cell activation by anti-CD161 triggering in NK cells from a CC carrier compared to cells from a TT individual. (B) Frequency of activated NK cells after stimulation with anti-NKp46 plus isotype control and anti-NKp46 plus anti-CD161. PBMC from nine individuals with CD161 TT and nine carriers of CC were studied. (C) Diminished capacity of CD161 from CC carriers to inhibit NKp46-induced NK cell activation. % Inhibition was calculated as follows: 100 - [(%CD107a⁺ NK cells after stimulation with NKp46-CD161 / % CD107a⁺ NK cells after stimulation with NKp46-isotype) x 100]; *p<0.05 (Unpaired t-test).</p

Inhibition of NK cell activation by LLT1-mediated triggering of CD161.

<p>PBMC were isolated and co-cultivated for three hours with 293-mock or 293-LLT1 transfectants. Activation of NK cells was assessed by monitoring CD107a expression on gated CD3^-CD56⁺ and CD3^-CD56⁺CD161⁺/CD161^- cells. (A) Abrogation of inhibition by antibody-mediated blocking of CD161/LLT1 interactions. Co-cultivation of PBMC with 293 transfectants was performed in the presence of the anti-CD161 mAb 191B8 or an isotype-matched control antibody. (B) Frequency of activated NK cells after cultivation with 293-mock or 293-LLT1 cells. PBMC from nine individuals with CD161 TT and four carriers of CC were studied. (C) Diminished capacity of CD161 from CC carriers to inhibit NK cell activation. % Inhibition was calculated as follows: 100 - [(%CD107a⁺ NK cells in 293-LLT1 co-cultures / %CD107a⁺ NK cells in 293-mock co-cultures) x 100];*p<0.05 (Mann-Whitney). (D) LLT1 induced CD161 down-regulation on IL-12 activated NK cells. PBMC were co-cultured for three hours with either mock transfected 293 cells or cells expressing LLT1. CD161 expression was monitored on gated CD3^-CD56⁺ NK cells. Depicted histograms show CD161 expression in untreated NK cells (open) and after treatment with 293-LLT1 transfectants (filled histograms). The broken vertical line indicates the peak of CD161 observed in LLT1-treated NK cells from a TT individual. The graph summarizes data obtained in experiments using cells from five TT and three CC individuals (mean ± SEM).</p

Assessment of lymphocyte subsets in individuals carrying different CD161 genotypes.

<p>PBMC were isolated and stained with the antibody combinations CD161/CD3/CD4 or CD161/CD3/CD56. Analyses were performed in gated viable lymphocytes as defined by forward- and side-scatter characteristics. (A) CD161 expression patterns on CD3^- NK cells. Representative dot-plots obtained after staining of cells from one TT, TC, and CC individual are shown. Boxes indicate the CD56^brightCD161⁺CD3^- NK cell subset and the frequency. The numbers indicate % positive cells in each quadrant. (B) Decreased frequency of CD3^-CD56⁺ NK cells in CC carriers. PBMC from 27 TT, 51 TC, and 16 carriers of CC were analyzed. Results are expressed as % NK cells among gated lymphocytes; the mean value calculated for each cohort is indicated by the horizontal line; *p< 0.05 (ANOVA). (C) Ratios of CD56^dim to CD56^bright NK cells. The frequencies of CD56^dimCD161⁺CD3^- and CD56^brightCD161⁺CD3^- NK cells were determined in gated lymphocytes from 22 TT, 46 TC, and 15 carriers of CC. The horizontal broken line is an arbitrary cut-off value. (D) Level of CD161 expression. Data are expressed as mean fluorescence intensity/MFI on gated CD3^-CD56⁺ NK cells. (E) Proportion of NK cells co-expressing CD161. Data are expressed as % CD161⁺ cells in gated CD3^-CD56⁺ NK cells. (F) CD161 expression patterns on CD3⁺ T cells. The numbers indicate % positive cells in each quadrant. (G) Proportion of CD4⁺ T cells co-expressing CD161. (H) Proportion of CD8⁺ T cells co-expressing CD161^dim or CD161^bright.</p

The c.503T>C Polymorphism in the Human <i>KLRB1</i> Gene Alters Ligand Binding and Inhibitory Potential of CD161 Molecules

<div><p>Studying genetic diversity of immunologically relevant molecules can improve our knowledge on their functional spectrum in normal immune responses and may also uncover a possible role of different variants in diseases. We characterized the c.503T>C polymorphism in the human <i>KLRB1</i> gene (Killer cell lectin-like receptor, subfamily B, member 1) coding for the cell surface receptor CD161. CD161 is expressed by subsets of CD4⁺ and CD8⁺ T cells and the great majority of CD56⁺ natural killer (NK) cells, acting as inhibitory receptor in the latter population. Genotyping a cohort of 118 healthy individuals revealed 40% TT homozygotes, 46% TC heterozygotes, and 14% carriers of CC. There was no difference in the frequency of CD161 expressing CD4⁺ and CD8⁺ T cells between the different genotypes. However, the frequency of CD161⁺ NK cells was significantly decreased in CC carriers as compared to TT homozygotes. c.503T>C causes an amino acid exchange (p.Ile168Thr) in an extracellular loop of the CD161 receptor, which is regarded to be involved in binding of its ligand Lectin-like transcript 1 (LLT1). Binding studies using soluble LLT1-Fc on 293 transfectants over-expressing CD161 receptors from TT or CC carriers suggested diminished binding to the CC variant. Furthermore, triggering of CD161 either by LLT1 or anti-CD161 antibodies inhibited NK cell activation less effectively in cells from CC individuals than cells from TT carriers. These data suggest that the c.503T>C polymorphism is associated with structural alterations of the CD161 receptor. The regulation of NK cell homeostasis and activation apparently differs between carriers of the CC and TT variant of CD161.</p></div

Characterization of 293 transfectants. 293 cells were “mock”-transfected with the empty pIRES2-AcGFP1 vector or vectors containing CD161 from a homozygous TT or CC individual.

<p>Analyses were performed after selection of stable transfectants. (A) Flow cytometry analysis of 293-mock, 293-CD161/TT, and 293-CD161/CC cells after staining with the PE-conjugated anti-CD161 mAb B199.2. Grey histograms represent staining with an isotype control mAb. The numbers represent mean fluorescence intensity obtained by anti-CD161 staining. (B) Detection of CD161 by Western-blotting. 293 transfectants were lysed, and protein lysates were separated by 9% SDS-PAGE. After transferring to PVDF membranes, samples were probed with the anti-CD161 mAb (B199.2). Binding of B199.2 was visualized by HRP-conjugated goat anti-mouse-IgG and ECL reagents. Protein loading was controlled by staining with a mAb against α-Actin. Representative results of at least two independent experiments are shown. Lysates of PBMC activated for five days with IL-12 were used as a further control to estimate the molecular weight of CD161 molecules.</p