Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1

Nature Immunology volume 17, pages 1067–1074 (2016)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Binding of KIR-Fc to beads coated with classical and non-classical HLA class I proteins.
Figure 2: Binding of KIRs to HLA-F OCs and functional triggering of KIR3DS1 on reporter cell lines by HLA-F OCs.
Figure 3: HLA-F OCs trigger degranulation and antiviral cytokine production in primary NK cells via KIR3DS1.
Figure 4: Activated CD4+ T cells express HLA-F on their surface and bind KIR3DS1-Fc.
Figure 5: Effect of HIV-1 infection on HLA-F expression, KIR3DS1-Fc binding, and suppression of HIV-1 replication in infected CD4+ T cells by KIR3DS1+ NK cells.

Similar content being viewed by others

References

  1. Parham, P., Norman, P.J., Abi-Rached, L. & Guethlein, L.A. Variable NK cell receptors exemplified by human KIR3DL1/S1. J. Immunol. 187, 11–19 (2011).

    CAS  PubMed  Google Scholar 

  2. Vivier, E. & Ugolini, S. Natural killer cells: from basic research to treatments. Front. Immunol. 2, 18 (2011).

    PubMed  PubMed Central  Google Scholar 

  3. Rajalingam, R. Human diversity of killer cell immunoglobulin-like receptors and disease. Korean J. Hematol. 46, 216–228 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Min-Oo, G., Kamimura, Y., Hendricks, D.W., Nabekura, T. & Lanier, L.L. Natural killer cells: walking three paths down memory lane. Trends Immunol. 34, 251–258 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Waggoner, S.N., Cornberg, M., Selin, L.K. & Welsh, R.M. Natural killer cells act as rheostats modulating antiviral T cells. Nature 481, 394–398 (2011).

    PubMed  PubMed Central  Google Scholar 

  6. Martin, M.P. et al. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat. Genet. 31, 429–434 (2002).

    CAS  PubMed  Google Scholar 

  7. Jiang, Y. et al. KIR3DS1/L1 and HLA-Bw4-80I are associated with HIV disease progression among HIV typical progressors and long-term nonprogressors. BMC Infect. Dis. 13, 405 (2013).

    PubMed  PubMed Central  Google Scholar 

  8. Rivero-Juarez, A. et al. Natural killer KIR3DS1 is closely associated with HCV viral clearance and sustained virological response in HIV/HCV patients. PLoS One 8, e61992 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Trydzenskaya, H. et al. The genetic predisposition of natural killer cell to BK virus-associated nephropathy in renal transplant patients. Kidney Int. 84, 359–365 (2013).

    CAS  PubMed  Google Scholar 

  10. Díaz-Peña, R. et al. Association of the KIR3DS1*013 and KIR3DL1*004 alleles with susceptibility to ankylosing spondylitis. Arthritis Rheum. 62, 1000–1006 (2010).

    PubMed  Google Scholar 

  11. López-Vázquez, A. et al. Protective effect of the HLA-Bw4I80 epitope and the killer cell immunoglobulin-like receptor 3DS1 gene against the development of hepatocellular carcinoma in patients with hepatitis C virus infection. J. Infect. Dis. 192, 162–165 (2005).

    PubMed  Google Scholar 

  12. Besson, C. et al. Association of killer cell immunoglobulin-like receptor genes with Hodgkin's lymphoma in a familial study. PLoS One 2, e406 (2007).

    PubMed  PubMed Central  Google Scholar 

  13. Carrington, M. et al. Hierarchy of resistance to cervical neoplasia mediated by combinations of killer immunoglobulin-like receptor and human leukocyte antigen loci. J. Exp. Med. 201, 1069–1075 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Gagne, K. et al. Donor KIR3DL1/3DS1 gene and recipient Bw4 KIR ligand as prognostic markers for outcome in unrelated hematopoietic stem cell transplantation. Biol. Blood Marrow Transplant. 15, 1366–1375 (2009).

    PubMed  Google Scholar 

  15. Venstrom, J.M. et al. Donor activating KIR3DS1 is associated with decreased acute GVHD in unrelated allogeneic hematopoietic stem cell transplantation. Blood 115, 3162–3165 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Carr, W.H. et al. Cutting edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation. J. Immunol. 178, 647–651 (2007).

    CAS  PubMed  Google Scholar 

  17. Cella, M., Longo, A., Ferrara, G.B., Strominger, J.L. & Colonna, M. NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J. Exp. Med. 180, 1235–1242 (1994).

    CAS  PubMed  Google Scholar 

  18. O'Connor, G.M. et al. Functional polymorphism of the KIR3DL1/S1 receptor on human NK cells. J. Immunol. 178, 235–241 (2007).

    CAS  PubMed  Google Scholar 

  19. Gillespie, G.M. et al. Lack of KIR3DS1 binding to MHC class I Bw4 tetramers in complex with CD8+ T cell epitopes. AIDS Res. Hum. Retroviruses 23, 451–455 (2007).

    CAS  PubMed  Google Scholar 

  20. O'Connor, G.M. et al. Peptide-dependent recognition of HLA-B*57:01 by KIR3DS1. J. Virol. 89, 5213–5221 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Martin, M.P. et al. Innate partnership of HLA-B and KIR3DL1 subtypes against HIV-1. Nat. Genet. 39, 733–740 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Barbour, J.D. et al. Synergy or independence? Deciphering the interaction of HLA class I and NK cell KIR alleles in early HIV-1 disease progression. PLoS Pathog. 3, e43 (2007).

    PubMed  PubMed Central  Google Scholar 

  23. Long, B.R. et al. Conferral of enhanced natural killer cell function by KIR3DS1 in early human immunodeficiency virus type 1 infection. J. Virol. 82, 4785–4792 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Gardiner, C.M. et al. Different NK cell surface phenotypes defined by the DX9 antibody are due to KIR3DL1 gene polymorphism. J. Immunol. 166, 2992–3001 (2001).

    CAS  PubMed  Google Scholar 

  25. Thananchai, H. et al. Cutting edge: allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B. J. Immunol. 178, 33–37 (2007).

    CAS  PubMed  Google Scholar 

  26. Sharma, D. et al. Dimorphic motifs in D0 and D1+D2 domains of killer cell Ig-like receptor 3DL1 combine to form receptors with high, moderate, and no avidity for the complex of a peptide derived from HIV and HLA-A*2402. J. Immunol. 183, 4569–4582 (2009).

    CAS  PubMed  Google Scholar 

  27. Arosa, F.A., Santos, S.G. & Powis, S.J. Open conformers: the hidden face of MHC-I molecules. Trends Immunol. 28, 115–123 (2007).

    CAS  PubMed  Google Scholar 

  28. Wainwright, S.D., Biro, P.A. & Holmes, C.H. HLA-F is a predominantly empty, intracellular, TAP-associated MHC class Ib protein with a restricted expression pattern. J. Immunol. 164, 319–328 (2000).

    CAS  PubMed  Google Scholar 

  29. Goodridge, J.P., Burian, A., Lee, N. & Geraghty, D.E. HLA-F complex without peptide binds to MHC class I protein in the open conformer form. J. Immunol. 184, 6199–6208 (2010).

    CAS  PubMed  Google Scholar 

  30. Trundley, A., Frebel, H., Jones, D., Chang, C. & Trowsdale, J. Allelic expression patterns of KIR3DS1 and 3DL1 using the Z27 and DX9 antibodies. Eur. J. Immunol. 37, 780–787 (2007).

    CAS  PubMed  Google Scholar 

  31. Lee, N., Ishitani, A. & Geraghty, D.E. HLA-F is a surface marker on activated lymphocytes. Eur. J. Immunol. 40, 2308–2318 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Alter, G. et al. Differential natural killer cell-mediated inhibition of HIV-1 replication based on distinct KIR/HLA subtypes. J. Exp. Med. 204, 3027–3036 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Allen, R.L., Raine, T., Haude, A., Trowsdale, J. & Wilson, M.J. Leukocyte receptor complex-encoded immunomodulatory receptors show differing specificity for alternative HLA-B27 structures. J. Immunol. 167, 5543–5547 (2001).

    CAS  PubMed  Google Scholar 

  34. Jones, D.C. et al. HLA class I allelic sequence and conformation regulate leukocyte Ig-like receptor binding. J. Immunol. 186, 2990–2997 (2011).

    CAS  PubMed  Google Scholar 

  35. Lepin, E.J.M. et al. Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors. Eur. J. Immunol. 30, 3552–3561 (2000).

    CAS  PubMed  Google Scholar 

  36. Goodridge, J.P., Burian, A., Lee, N. & Geraghty, D.E. HLA-F and MHC class I open conformers are ligands for NK cell Ig-like receptors. J. Immunol. 191, 3553–3562 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Vivian, J.P. et al. Killer cell immunoglobulin-like receptor 3DL1-mediated recognition of human leukocyte antigen B. Nature 479, 401–405 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Pan, F.H., Liu, X.X. & Tian, W. Characterization of HLA-F polymorphism in four distinct populations in Mainland China. Int. J. Immunogenet. 40, 369–376 (2013).

    CAS  PubMed  Google Scholar 

  39. Shiina, T. et al. Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938-bp HLA class I region. Proc. Natl. Acad. Sci. USA 96, 13282–13287 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Lee, N. & Geraghty, D.E. HLA-F surface expression on B cell and monocyte cell lines is partially independent from tapasin and completely independent from TAP. J. Immunol. 171, 5264–5271 (2003).

    CAS  PubMed  Google Scholar 

  41. Noguchi, K. et al. Detection of anti-HLA-F antibodies in sera from cancer patients. Anticancer Res. 24 5C, 3387–3392 (2004).

    CAS  PubMed  Google Scholar 

  42. Le Gall, S. et al. Nef interacts with the μ subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules. Immunity 8, 483–495 (1998).

    CAS  PubMed  Google Scholar 

  43. Collins, K.L., Chen, B.K., Kalams, S.A., Walker, B.D. & Baltimore, D. HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391, 397–401 (1998).

    CAS  PubMed  Google Scholar 

  44. Cohen, G.B. et al. The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10, 661–671 (1999).

    CAS  PubMed  Google Scholar 

  45. Cerboni, C. et al. Human immunodeficiency virus 1 Nef protein downmodulates the ligands of the activating receptor NKG2D and inhibits natural killer cell-mediated cytotoxicity. J. Gen. Virol. 88, 242–250 (2007).

    CAS  PubMed  Google Scholar 

  46. Derrien, M. et al. Human immunodeficiency virus 1 downregulates cell surface expression of the non-classical major histocompatibility class I molecule HLA-G1. J. Gen. Virol. 85, 1945–1954 (2004).

    CAS  PubMed  Google Scholar 

  47. Boyle, L.H., Gillingham, A.K., Munro, S. & Trowsdale, J. Selective export of HLA-F by its cytoplasmic tail. J. Immunol. 176, 6464–6472 (2006).

    CAS  PubMed  Google Scholar 

  48. Jinushi, M., Hodi, F.S. & Dranoff, G. Therapy-induced antibodies to MHC class I chain-related protein A antagonize immune suppression and stimulate antitumor cytotoxicity. Proc. Natl. Acad. Sci. USA 103, 9190–9195 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Jinushi, M. et al. MHC class I chain-related protein A antibodies and shedding are associated with the progression of multiple myeloma. Proc. Natl. Acad. Sci. USA 105, 1285–1290 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Pelak, K. et al. Copy number variation of KIR genes influences HIV-1 control. PLoS Biol. 9, e1001208 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Pertel, T. et al. TRIM5 is an innate immune sensor for the retrovirus capsid lattice. Nature 472, 361–365 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Mandal, P.K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Cella, M. & Colonna, M. in Methods in Molecular Biology: Natural Killer Cell Protocols: Cellular and Molecular Methods Vol. 121 (eds. Campbell, K. S. & Colonna, M.) 1–4 (Humana Press, 1999).

  55. Imai, C., Iwamoto, S. & Campana, D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 106, 376–383 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

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 721.221–HLA-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 Institute–Leibniz 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.

Author information

Authors and Affiliations

  1. Ragon Institute of MGH, MIT, and Harvard, Cambridge, Massachusetts, USA

    Wilfredo F Garcia-Beltran, Angelique Hölzemer, Amy W Chung, Yovana Pacheco, Camille R Simoneau, Marijana Rucevic, Pedro A Lamothe-Molina, Tae-Eun Kim, Haley Dugan, Galit Alter, Stephanie Jost, Mary Carrington & Marcus Altfeld

  2. Heinrich-Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany

    Angelique Hölzemer, Gloria Martrus & Marcus Altfeld

  3. First Department of Internal Medicine, University Medical Centre Eppendorf, Hamburg, Germany

    Angelique Hölzemer

  4. Departamento de Matemáticas, Facultad de Ciencias, Universidad Nuestra Señora del Rosario, Bogotá, Colombia

    Yovana Pacheco

  5. Center for Neurologic Diseases, Brigham and Women′s Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Thomas Pertel

  6. CNRS, UMR 5164, Université de Bordeaux, Bordeaux, France

    Julie Dechanet-Merville

  7. Cancer and Inflammation Program, Laboratory of Experimental Immunology, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

    Mary Carrington

Authors
  1. Wilfredo F Garcia-Beltran
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angelique Hölzemer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gloria Martrus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amy W Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yovana Pacheco
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Camille R Simoneau
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marijana Rucevic
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pedro A Lamothe-Molina
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Thomas Pertel
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tae-Eun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Haley Dugan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Galit Alter
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Julie Dechanet-Merville
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Stephanie Jost
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Mary Carrington
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Marcus Altfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Marcus Altfeld.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

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 anti–pan-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 anti–pan-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 anti–KIR2DL2-KIR2DL3, anti–KIR3DS1-KIR3DL1, and anti–KIR3DL1-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 anti–KIR3DS1-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 anti–pan-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 (–) 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-Bw4− 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, anti–KIR3DS1-KIR3DL1; α-KIR2DL3, anti–KIR2DL2-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, anti–KIR3DS1-KIR3DL1 antibody; DX9, anti-KIR3DL1; HC10, anti-HLA class I OC; HCA2, anti-HLA class I OC; W6/32, anti–pan-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 anti–KIR2DL2-KIR2DL3 and anti–KIR2DL1-KIR2DS1 having the same fluorophore. In b, ‘+’ denotes >90% expression of the indicated receptor, ‘–’ denotes a frequency of <5%, and ‘+/–’ 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), anti–KIR3DS1-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 KIR3DS1− (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-F−) 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-F− cell line (Jurkat cells) with probes for HLA-F mRNA and also RPL13a mRNA (a housekeeping gene). Dotted histograms indicate staining without probes (‘– probe’; negative control) and solid-line, filled histograms are staining with probe (‘+ probe’).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 1301 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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, 1067–1074 (2016). https://doi.org/10.1038/ni.3513

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3513

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing