Chopra, A.

Gaudieri, S.

Mpunga, T.

English

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Differential expression levels of activating and inhibitory receptors on NK cell subsets: relevance to NK cell functionIntroductionNatural killer (NK) cells areessential innate immune cells.NK cell cytotoxicity is regulated bythe interplay between activatingand inhibitory membranereceptors, mainly the Killerimmunoglobulin-like receptors(KIRs) and their ligands.CD56 expression can be used todefine two major subsets of NKcells (CD56 Dim and CD56 Bright)that appear to differ in their mainfunction and tissue distribution.CD56 Dim cells are associatedwith cytotoxicity and CD56 Brightcells are associated with immune-regulation[1].HypothesisKIR gene expression is differentbetween NK cell subsets andreflects their main function in bloodand other tissues.Materials and methods PBMC sorting by flow cytometry(isolation of NK cell subsets usingthe cell surface markers CD3 – Tcell, CD56 – NK cell, CD57 –maturation [2]) DNA genotyping of samples usingreal-time PCR (screening for thepresence or absence of KIR genesusing SYBRGreen) RNA expression using Nanostringtechnology (allows absolutequantification withoutamplification)Results1. NK cell subset isolation fromPBMCs by flow cytometry. Usinganti-CD56, anti-CD3 and anti-CD57 antibodies, we were able tosuccessfully isolate NK cellsubpopulations of interest (Fig.1.).Conclusions There are differences in KIRexpression between the NK cellsubsets. The results support thehypothesis that CD56Dim NKcells are likely to have increasedexpression of activating KIRgenes that reflects theircytotoxic function whencompared to CD56Bright NKcells which are less cytotoxic andmainly cytokine producing NKcells.References1. Lopez-Verges, S., et al., CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset. Blood, 2010. 116(19): p.3865-74.2. De Maria, A., et al., Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma onactivation. Proc Natl Acad Sci U S A, 2011. 108(2): p. 728-32.Teddy Mpunga1, Abha Chopra1 and Silvana Gaudieri1, 21Institute for Immunology & Infectious Diseases, Murdoch University, Murdoch, Western Australia, 2School of Anatomy, Physiology and Human Biology, University of Western Australia, Crawley, Western Australia2. DNA genotyping by real-timePCR DNA from PBMC pool wasextracted and genotyped for thepresence or absence of KIRs(Fig.2.).3. KIR Expression results usingNanostring Technology Greaterinhibitory KIR (subgroup_1, ingreen, and subgroup_2 in blue)expression is observed within CD56Bright compared to CD56DimCD57+ and CD56DimCD57- cells,while activating KIRs (in red) aregreatly expressed within CD56DimCD57+ cells compared toCD56DimCD57- cells while CD56BrightCD57- cells showed thelowest activating KIRs expression(Fig.3.).0100200300400500600700Absolute quantificationFig.2. Real-time PCR results obtained from PBMC poolDNA, screening for the presence (+) or absence (-) of the16 KIR genes.Fig.1. NK cell subsets flow cytometric sorting from PBMCspool using: anti-CD3 antibody to differentiate NK cellsfrom T-cells (CD3+), anti-CD56 antibody for NK cellsisolation (CD56+) and anti-CD57 antibody for maturationdistinction within NK cells.Fig.3. Differences in KIR expression between the threedifferent NK subsets (CD56Bright, CD56DimCD57+ andCD56DimCD57-) and controls (NKT, CD3-CD56- andCD3+CD56-).CD57+ CD57-NKTCD3- CD3+Contact: teddympunga@yahoo.fr

Differential expression levels of activating and inhibitory receptors on NK cell subsets: Relevance to NK cell function

https://researchrepository.murdoch.edu.au/id/eprint/36774/1/expressions%20poster.pdf

Differential expression levels of activating and inhibitory receptors on NK cell subsets: Relevance to NK cell function

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