Abstract
The activating natural killer (NK)-cell receptor KIR3DS1 has been linked to the outcome of various human diseases, including delayed progression of disease caused by human immunodeficiency virus type 1 (HIV-1), yet a ligand that would account for its biological effects has remained unknown. We screened 100 HLA class I proteins and found that KIR3DS1 bound to HLA-F, a result we confirmed biochemically and functionally. Primary human KIR3DS1+ NK cells degranulated and produced antiviral cytokines after encountering HLA-F and inhibited HIV-1 replication in vitro. Activation of CD4+ T cells triggered the transcription and surface expression of HLA-F mRNA and HLA-F protein, respectively, and induced binding of KIR3DS1. HIV-1 infection further increased the transcription of HLA-F mRNA but decreased the binding of KIR3DS1, indicative of a mechanism for evading recognition by KIR3DS1+ NK cells. Thus, we have established HLA-F as a ligand of KIR3DS1 and have demonstrated cell-context-dependent expression of HLA-F that might explain the widespread influence of KIR3DS1 in human disease.
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Acknowledgements
We thank C. Körner and A. Crespo for advice and discussions; D. Campana (St. Jude Children's Research Hospital) for K562 cells expressing mbIL-15 and CD137L; C. Brander (Ragon Institute of MGH, MIT, and Harvard) for 721.221 cells transduced to express HLA; J. Strominger (Harvard University) for 721.221âHLA-G cells; F. Zhang (Broad Institute of MIT and Harvard) for the lentiCas9-Blast plasmid; L. Ferreira (Harvard University) and T. Meissner (Harvard University) for β2m-targeting single-guide RNA plasmid; and the Ragon Institute Flow Cytometry Core and Virology Core for support and assistance. Supported by the US National Institutes of Health (R01-AI067031-08 and P01-AI104715; F31AI116366 to W.F.G.-B.; the Intramural Research Program, Frederick National Lab, Center for Cancer Research), the National Institute of General Medical Sciences (T32GM007753), the Ragon Institute of MGH, MIT and Harvard, the Frederick National Laboratory for Cancer Research (HHSN261200800001E), the Heinrich-Pette InstituteâLeibniz Institute for Experimental Virology (Program Area Antiviral Targets and Strategies) and the German Center for Infection Research. The content is solely the responsibility of the authors and does not necessarily represent the official views or policies of the National Institute of General Medical Sciences, the National Institutes of Health, or the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
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W.F.G.-B., A.H., G.M., A.W.C., Y.P., C.R.S., T.-E.K., and H.D. performed the experiments; M.R. performed mass spectrometry (not presented here); P.A.L.-M. and T.P. provided resources and reagents; J.D.-M. designed the Jurkat reporter cell assay; S.J. developed NK-cell cloning protocol; G.A., S.J., M.C. and M.A. supervised the research; and W.F.G.-B. analyzed the data and wrote the manuscript.
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Supplementary Figure 1 KIR2DS4-Fc and antibody staining of beads coated with HLA class I.
(a, b) Binding of KIR2DS4-Fc to untreated (complex) versus acid-pulsed (open conformer; OC) beads coated with classical HLA class I allotypes (in a); HLA-E, -F, or -G (in b); or nothing (negative control beads; red star) was measured and reported as median fluorescence intensity (MdFI). Each dot indicates a single HLA class I allotype, and color coding is used in a to indicate HLA class I allotype grouping (see legend and On-line Methods for further details). (c) HLA-E, -F-, and -G-coated beads and negative control beads (NC) were untreated (complex) or acid pulsed (OC) and stained with antiâpan-HLA class I complex antibody (clone: W6/32) (left panel) to assess for complex conformation on beads, anti-β2m antibody (middle panel) to quantify β2m content, and an antibody to HLA class I OCs (clone: HCA2) (right panel) to assess HLA-F reactivity; median fluorescence intensity (MdFI) is reported.
Supplementary Figure 2 Generation and testing of KIRζ+ Jurkat reporter cell lines and assessment of HLA class I OCs on target cells.
(a) Staining of antiâpan-HLA class I complex was assessed in Jurkat cells that underwent CRISPR/Cas9-mediated β2m knockout (KO). (b) KIRζ chimeric receptors for inhibitory and activating KIRs were designed by fusing the extracellular domain (ECD) and transmembrane domain (TMD) of the indicated KIRs with the triple immunoreceptor tyrosine-based activating motif (ITAM)-containing cytoplasmic tail (CYT) of CD3ζ. (c) KIR expression was assessed in Jurkat-β2m-KO cells stably transduced with the indicated KIRζ chimeric receptors via staining with antiâKIR2DL2-KIR2DL3, antiâKIR3DS1-KIR3DL1, and antiâKIR3DL1-KIR3DL2-KIR3DS1; UT signifies untransduced. (d) Reporter activity of untransduced (UT), KIR3DL1ζ+ and KIR3DS1hiζ+ Jurkat cells co-incubated with cell-sized beads covalently conjugated to irrelevant mouse IgG (mIgG) or antiâKIR3DS1-KIR3DL1 antibody (Z27) was measured as percentage of CD69hi Jurkat cells. (e) Untransduced (UT), KIR2DL3ζ+, KIR3DL1ζ+, KIR3DS1hiζ+ and KIR3DL2ζ+ Jurkat cells were co-incubated with streptavidin beads coated with free biotin (negative control; NC) or biotinylated HLA-F monomers (HLA-F) and the percentage of bead-binding cells (left panel) as well as reporter activity as a percentage of CD69hi cells (right panel) was measured. (f) Staining of antiâpan-HLA class I complex (clone: W6/32) and an antibody to HLA class I OCs (clone: HC10) antibodies was measured on untreated and acid-pulsed cell lines to confirm OC generation; 2° only signifies secondary only staining. Data in d is representative of three experiments and shows three technical replicates; a, c, e, and f show one experiment; d shows mean + s.d.
Supplementary Figure 3 Triggering of KIRζ+ Jurkat reporter cells by various cell lines.
(a) Reporter activity of untransduced (UT), KIR3DL1ζ+ and KIR3DS1hiζ+ Jurkat cells co-incubated with untreated (â) or acid pulsed (+) 721.221 cells expressing the indicated HLA class I allotypes was measured as percentage of CD69hi Jurkat cells. (b) Reporter activity of untransduced (UT), KIR3DL1ζ+, KIR3DS1hiζ+ and KIR3DL2ζ+ Jurkat cells co-incubated with untreated (black bars) or acid-pulsed (gray bars) cell lines was measured as percentage of CD69hi Jurkat cells. Cell lines used were EL-4 cells (mouse T-lymphoblastoid cell line), K562 cells (human chronic myelogenous leukemia line), and THP-1 cells (human acute monocytic leukemia line), all of which do not express HLA-F; NT signifies no target cells. (c) Reporter activity of KIR3DS1hiζ Jurkat cells co-incubated with acid-pulsed HLA-Bw4â BCL in the presence of the indicated antibodies or KIR-Fc constructs (50 μg/mL each) was measured as percentage of CD69hi Jurkat cells. The labels indicate the following: NT, no target cells; mIgG1 iso, mouse IgG1 isotype control; α-KIR3DS1, antiâKIR3DS1-KIR3DL1; α-KIR2DL3, antiâKIR2DL2-KIR2DL3; hIgG1 iso, human IgG1 isotype control antibody; KIR3DS1-Fc, KIR3DS1-Fc fusion construct with Fc region of hIgG1; KIR2DL3-Fc, KIR2DL3-Fc fusion construct with Fc region of hIgG1. (d) Reporter activity of untransduced (UT), KIR3DL1ζ+ and KIR3DS1hiζ+ Jurkat cells co-incubated with untreated HLA-Bw4+ BCLs in the presence of the indicated antibodies to KIR or HLA class I (25 μg/mL each) was measured as percentage of CD69hi Jurkat cells. Blocking antibodies used (clone and antigen) are the following: Z27, antiâKIR3DS1-KIR3DL1 antibody; DX9, anti-KIR3DL1; HC10, anti-HLA class I OC; HCA2, anti-HLA class I OC; W6/32, antiâpan-HLA class I complex. Data in a, c, and d show three technical replicates from one experiment; b shows pool data from two independent experiments; a, b, c, and d show mean + s.d. In c, a one-way ANOVA with Tukey multiple comparisons test comparing all columns was performed; only some statistics are shown; *P < 0.01, **P < 0.001 and ***P < 0.0001.
Supplementary Figure 4 NKCL phenotypes and functional responses.
(a, b) NK-cell lines used for plate-bound ligand assays (in a) and clones used for HIV-1 replication inhibition assays (in b) were stained for various NK-cell receptors, as indicated; KIR2Ds in a was measured by staining with antiâKIR2DL2-KIR2DL3 and antiâKIR2DL1-KIR2DS1 having the same fluorophore. In b, â+â denotes >90% expression of the indicated receptor, âââ denotes a frequency of <5%, and â+/ââ denotes a frequency >5% (in both cases for clone #0, ~10%). (c) Representative flow plots of degranulation (i.e. CD107a expression) and production of IFN-γ, TNF-α, and MIP-1β by one KIR3DS1+ NK-cell line seeded onto well plates coated with irrelevant protein (negative control; NC), antiâKIR3DS1-KIR3DL1 (plate-bound α-KIR3DS1), or HLA-F monomers (plate-bound HLA-F) are shown. (d) Degranulation and production of IFN-γ, TNF-α, and MIP-1β by KIR3DS1+ (red dots) and KIR3DS1â (black dots) NKCLs seeded onto well plates coated with irrelevant protein (negative control; NC), human IgG (hIgG), or anti-NKp46 and anti-CD2 (α-NKp46 + α-CD2) were measured. The percentages of CD107a+ (top left panel), IFN-γ+ (top right panel), TNF+ (bottom left panel), and MIP-1β+ (bottom right panel) NKCLs are presented. For d, which shows mean, one-way ANOVA with Sidak multiple comparisons test comparing select columns was performed, and all statistically significant differences are presented; *P < 0.05, **P < 0.01 and ***P < 0.001.
Supplementary Figure 5 Quality-control testing of the antibody to HLA-F used for immunoblot analysis and HLA-F mRNA probes for fluorescence in situ hybridization.
(a) Cell lysates (non-nuclear) from cell lines were run on reducing/denaturing SDS-PAGE, followed by immunoblotting (IB) for HLA-F and β-actin (loading control). Left image shows Jurkat cells (HLA-Fâ) and 721.221 cells (HLA-F+). Middle image shows THP-1 cells (HLA-F-deficient) and THP-1 cells transduced with N-terminally FLAG-tagged HLA-F. Right image shows Jurkat cells and Jurkat cells transduced with N-terminally FLAG-tagged HLA-F. (b) Fluorescent in situ hybridization and flow cytometry was done on an HLA-F+ cell line (BCL) and an HLA-Fâ cell line (Jurkat cells) with probes for HLA-F mRNA and also RPL13a mRNA (a housekeeping gene). Dotted histograms indicate staining without probes (ââ probeâ; negative control) and solid-line, filled histograms are staining with probe (â+ probeâ).
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Garcia-Beltran, W., Hölzemer, A., Martrus, G. et al. Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1. Nat Immunol 17, 1067â1074 (2016). https://doi.org/10.1038/ni.3513
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DOI: https://doi.org/10.1038/ni.3513
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