Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69−CD56dim cells Nicole Marquardt, PhD, Eliisa.

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Presentation transcript:

Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69−CD56dim cells Nicole Marquardt, PhD, Eliisa Kekäläinen, MD, PhD, Puran Chen, MD, Egle Kvedaraite, MD, Jennifer N. Wilson, BSc, Martin A. Ivarsson, PhD, Jenny Mjösberg, PhD, Lena Berglin, PhD, Jesper Säfholm, PhD, Martijn L. Manson, PharmD, PhD, Mikael Adner, PhD, Mamdoh Al-Ameri, MD, Per Bergman, MD, PhD, Ann-Charlotte Orre, MD, Mattias Svensson, PhD, Barbro Dahlén, MD, PhD, Sven-Erik Dahlén, MD, PhD, Hans-Gustaf Ljunggren, MD, PhD, Jakob Michaëlsson, PhD Journal of Allergy and Clinical Immunology Volume 139, Issue 4, Pages 1321-1330.e4 (April 2017) DOI: 10.1016/j.jaci.2016.07.043 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Journal of Allergy and Clinical Immunology 2017 139, 1321-1330 Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 CD56dimCD16+ NK cells are the major population of NK cells in human lung. A, Gating strategy to identify NK cells in human lung. B, Frequency of NK cells among live CD14−CD19−CD45+ cells in lung parenchyma and matched peripheral blood (n = 84). Box and whiskers, 5th-95th percentiles. +, Mean. C, Frequency of CD56dimCD16+ and CD56brightCD16− cells among total NK cells (n = 79). Box and whiskers, 5th-95th percentiles. +, Mean. D, NK cell frequencies of live CD14−CD19−CD45+ cells stratified by smoking status. Frequency of NK cells in peripheral blood from age-matched healthy control subjects (Ctrl) is shown for comparison (n = 9-25). Bars indicate means. E, Confocal microscopy analysis of snap-frozen lung tissue from 1 of 4 representative donors. Left panel, Nonparenchymal areas are identified through transmitted light (dashed lines) to reveal NK cell localization. Middle and right panels, Maximum intensity projections of immunofluorescence staining with 4′,6-diamidino-2-phenylindole (DAPI; blue), Eomes (white), and CD3 (red) are presented (higher magnification in right panel). White arrows indicate NK cells (Eomes+CD3−), and red arrows indicate T cells (Eomes−CD3+ or Eomes+CD3+). *P < .05, **P < .01, ***P < .001, and ****P < .0001. Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Human lung NK cells are hypofunctional, even after priming with IFN-α. A, Representative flow plots of degranulation by NK cells freshly isolated from PBMCs (left panels) and lung tissue (right panels). Samples were incubated in medium alone (unstimulated) or stimulated with K562 cells. B and C, Degranulation by CD56dim NK cells after stimulation with K562 cells (Fig 2, B) or 721.221 cells and anti-CD20 mAb (rituximab; Fig 2, C). Background degranulation by unstimulated CD56dim NK cells is subtracted. D, Degranulation by NK cells from current smokers (+) and ex-smokers (ex) in peripheral blood (left panel) and lung tissue (right panel). Background degranulation by unstimulated NK cells is subtracted. E, Degranulation (left panel) and TNF production (right panel) by cryopreserved NK cells after stimulation with K562 cells, with or without overnight priming with IFN-α. Background degranulation and TNF production by unstimulated NK cells are subtracted. F and G, Overlay of representative histograms (Fig 2, F) and summary of data showing degranulation, TNF, and GM-CSF production (Fig 2, G) in CD56bright NK cells with or without phorbol 12-myristate 13-acetate/ionomycin stimulation. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Human lung NK cells are highly differentiated and express self-KIRs more frequently than NK cells in peripheral blood. A, Gating strategy to identify expression of CD57 and NKG2A for analysis of NK cell differentiation. B, Frequencies of CD57−NKG2A+, CD57−NKG2A−, CD57+NKG2A+, and CD57+NKG2A− cells among CD56dimCD16+ NK cells in peripheral blood (open circles) and matched lung tissue (solid circles; n = 58). C, Gating strategy to identify NK cells expressing KIR2DL1, KIR2DL2/S2, KIR2DL3, or KIR3DL1. D, Frequency of KIR+ cells among CD56dimCD16+ NK cells in peripheral blood (open circles) and lung tissue (solid circles) stratified by NK cell differentiation stage (n = 50). E, Frequency of CD56dimCD16+ NK cells lacking expression of KIRs (KIR−), expressing non-self HLA class I–binding KIR+ (non-self), and expressing self-HLA class I–binding KIR (self) in matched peripheral blood (open circles) and lung (solid circles). NK cells were defined as KIR−, non–self-KIR+, and self-KIR+ by using a Boolean function of KIR2DL1, KIR2DL3, and KIR3DL1 expression and KIR-HLA ligand genotype. *P < .05, **P < .01, ***P < .001, and ****P < .0001. Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 NK cells in human lung are predominantly CD69− cells. A, Representative dot plots showing expression of CD69 on NK cells, T cells, and CD127+ ILCs in lung (upper panels) and peripheral blood (lower panels). CD127+ ILCs were defined as CD127+NKG2A−CD3−CD14−CD16−CD19−CD45+ live cells. B, Frequency of CD69+ cells among NK cells, T cells, and CD127+ ILCs in peripheral blood (left panel) and lung tissue (right panel). C, CD69+ cells of CD56dimCD16+ NK cells (left panel) and CD56brightCD16− NK cells (right panel) in peripheral blood (open circles) and lung tissue (solid circles). D, Frequency of cells expressing CD57 (left panel), NKG2A (middle panel), and KIR (right panel) among CD69− and CD69+ CD56dimCD16+ NK cells in the lung. E, Mean fluorescence intensity (MFI) of perforin among CD69− and CD69+ CD56dimCD16+ NK cells. F, Frequency of Ki67+ cells among CD69− and CD69+ CD56dimCD16+ NK cells. G, MFI of NKG2D among CD69− and CD69+ CD56dimCD16+ NK cells. Fig 4, E-G, Open circles, CD69−CD56dimCD16+ NK cells in peripheral blood; gray circles, CD69−CD16+ NK cells in lung tissue; solid circles, CD69+CD16+ NK cells in lung tissue. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Identification of NK cells in lung tissue by means of flow cytometry. A, Detailed gating strategy for NK cells from peripheral blood (upper panels) or lung tissue (lower panels). B, Discrimination of CD56dimCD16+ and CD56brightCD16− NK cells. C, Discrimination of CD56dim and CD56bright NK cells after incubation without (left) or with (right) phorbol 12-myristate 13-acetate/ionomycin. FSC, Forward scatter; SSC, side scatter. Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Frequency of T cells is increased in current smokers and ex-smokers. The frequency of CD3+ T cells in peripheral blood and lung of nonsmokers (−), current smokers (+), and ex-smokers (ex), respectively, is depicted. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Decreased target cell–mediated NK cell responsiveness in lung tissue is independent from CD56bright NK cells. CD56dimCD16+ NK cells from peripheral blood (open circles) and matched lung tissue (solid circles) were sorted and stimulated for 2 hours with K562 or 721.221 cells with or without rituximab, respectively. Degranulation (left) and TNF production (right) in matched experiments are depicted. Background degranulation and TNF production by unstimulated NK cells are subtracted. Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 CD69+ and CD69− CD56dimCD16+ NK cells in peripheral blood have similar expression of CD57, NKG2A, and KIR. Frequency of CD69+ and CD69− CD56dimCD16+ NK cells isolated from peripheral blood expressing CD57 (A), NKG2A (B), and KIRs (C). Journal of Allergy and Clinical Immunology 2017 139, 1321-1330.e4DOI: (10.1016/j.jaci.2016.07.043) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions