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:

RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics

Nature Immunology volume 17, pages 1352–1360 (2016)Cite this article

Subjects

Abstract

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.

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: Identification of human RASGRP1 deficiency.
Figure 2: RASGRP1deficiency causes defective TCR signaling and an aberrant immunophenotype.
Figure 3: RASGRP1 deficiency results in a B cell proliferation and activation defect.
Figure 4: Aberrant cytoskeletal dynamics in NK cells.
Figure 5: RASGRP1 deficiency leads to cell-migration defects that are reversed following treatment with lenalidomide.

Similar content being viewed by others

References

  1. Roose, J. & Weiss, A. T cells: getting a GRP on Ras. Nat. Immunol. 1, 275–276 (2000).

    CAS  PubMed  Google Scholar 

  2. Stone, J.C. Regulation and function of the RasGRP family of Ras activators in blood cells. Genes Cancer 2, 320–334 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Downward, J., Graves, J.D., Warne, P.H., Rayter, S. & Cantrell, D.A. Stimulation of p21ras upon T-cell activation. Nature 346, 719–723 (1990).

    CAS  PubMed  Google Scholar 

  4. Kremer, K.N., Kumar, A. & Hedin, K.E. G alpha i2 and ZAP-70 mediate RasGRP1 membrane localization and activation of SDF-1-induced T cell functions. J. Immunol. 187, 3177–3185 (2011).

    CAS  PubMed  Google Scholar 

  5. Dower, N.A. et al. RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat. Immunol. 1, 317–321 (2000).

    CAS  PubMed  Google Scholar 

  6. Khanna, R. & Burrows, S.R. Human immunology: a case for the ascent of non-furry immunology. Immunol. Cell Biol. 89, 330–331 (2011).

    PubMed  Google Scholar 

  7. Mestas, J. & Hughes, C.C. Of mice and not men: differences between mouse and human immunology. J. Immunol. 172, 2731–2738 (2004).

    CAS  PubMed  Google Scholar 

  8. Waterston, R.H. et al. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002).

    CAS  PubMed  Google Scholar 

  9. Notarangelo, L.D. Functional T cell immunodeficiencies (with T cells present). Annu. Rev. Immunol. 31, 195–225 (2013).

    CAS  PubMed  Google Scholar 

  10. Notarangelo, L.D. Partial defects of T-cell development associated with poor T-cell function. J. Allergy Clin. Immunol. 131, 1297–1305 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Picard, C. et al. Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015. J. Clin. Immunol. 35, 696–726 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Bousfiha, A. et al. The 2015 IUIS phenotypic classification for primary immunodeficiencies. J. Clin. Immunol. 35, 727–738 (2015).

    PubMed  PubMed Central  Google Scholar 

  13. Espinós, C. et al. Mutations in the urocanase gene UROC1 are associated with urocanic aciduria. J. Med. Genet. 46, 407–411 (2009).

    PubMed  Google Scholar 

  14. Chen, Y. et al. Differential requirement of RasGRP1 for γδ T cell development and activation. J. Immunol. 189, 61–71 (2012).

    CAS  PubMed  Google Scholar 

  15. Warnecke, N. et al. TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells. EMBO Rep. 13, 386–391 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Jun, J.E., Rubio, I. & Roose, J.P. Regulation of ras exchange factors and cellular localization of ras activation by lipid messengers in T cells. Front. Immunol. 4, 239 (2013).

    PubMed  PubMed Central  Google Scholar 

  17. Depeille, P. et al. RasGRP1 opposes proliferative EGFR-SOS1-Ras signals and restricts intestinal epithelial cell growth. Nat. Cell Biol. 17, 804–815 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Shen, S. et al. Critical roles of RasGRP1 for invariant NKT cell development. J. Immunol. 187, 4467–4473 (2011).

    CAS  PubMed  Google Scholar 

  19. Montoya, C.J. et al. Characterization of human invariant natural killer T subsets in health and disease using a novel invariant natural killer T cell-clonotypic monoclonal antibody, 6B11. Immunology 122, 1–14 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Priatel, J.J. et al. Chronic immunodeficiency in mice lacking RasGRP1 results in CD4 T cell immune activation and exhaustion. J. Immunol. 179, 2143–2152 (2007).

    CAS  PubMed  Google Scholar 

  21. Ma, C.S. & Deenick, E.K. Human T follicular helper (Tfh) cells and disease. Immunol. Cell Biol. 92, 64–71 (2014).

    CAS  PubMed  Google Scholar 

  22. Bartlett, A., Buhlmann, J.E., Stone, J., Lim, B. & Barrington, R.A. Multiple checkpoint breach of B cell tolerance in Rasgrp1-deficient mice. J. Immunol. 191, 3605–3613 (2013).

    CAS  PubMed  Google Scholar 

  23. Coughlin, J.J., Stang, S.L., Dower, N.A. & Stone, J.C. RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling. J. Immunol. 175, 7179–7184 (2005).

    CAS  PubMed  Google Scholar 

  24. Sun, C. et al. High-density genotyping of immune-related loci identifies new SLE risk variants in individuals with Asian ancestry. Nat. Genet. 48, 323–330 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Tangye, S.G., Ferguson, A., Avery, D.T., Ma, C.S. & Hodgkin, P.D. Isotype switching by human B cells is division-associated and regulated by cytokines. J. Immunol. 169, 4298–4306 (2002).

    CAS  PubMed  Google Scholar 

  26. Thien, M. et al. Excess BAFF rescues self-reactive B cells from peripheral deletion and allows them to enter forbidden follicular and marginal zone niches. Immunity 20, 785–798 (2004).

    CAS  PubMed  Google Scholar 

  27. Perussia, B., Chen, Y. & Loza, M.J. Peripheral NK cell phenotypes: multiple changing of faces of an adapting, developing cell. Mol. Immunol. 42, 385–395 (2005).

    CAS  PubMed  Google Scholar 

  28. Navarro, M.N. & Cantrell, D.A. Serine-threonine kinases in TCR signaling. Nat. Immunol. 15, 808–814 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Okamura, S.M., Oki-Idouchi, C.E. & Lorenzo, P.S. The exchange factor and diacylglycerol receptor RasGRP3 interacts with dynein light chain 1 through its C-terminal domain. J. Biol. Chem. 281, 36132–36139 (2006).

    CAS  PubMed  Google Scholar 

  30. Rodríguez-Crespo, I. et al. Identification of novel cellular proteins that bind to the LC8 dynein light chain using a pepscan technique. FEBS Lett. 503, 135–141 (2001).

    PubMed  Google Scholar 

  31. Huse, M. Microtubule-organizing center polarity and the immunological synapse: protein kinase C and beyond. Front. Immunol. 3, 235 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Mentlik, A.N., Sanborn, K.B., Holzbaur, E.L. & Orange, J.S. Rapid lytic granule convergence to the MTOC in natural killer cells is dependent on dynein but not cytolytic commitment. Mol. Biol. Cell 21, 2241–2256 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Roose, J.P., Mollenauer, M., Gupta, V.A., Stone, J. & Weiss, A. A diacylglycerol-protein kinase C-RasGRP1 pathway directs Ras activation upon antigen receptor stimulation of T cells. Mol. Cell. Biol. 25, 4426–4441 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Li, R. & Gundersen, G.G. Beyond polymer polarity: how the cytoskeleton builds a polarized cell. Nat. Rev. Mol. Cell Biol. 9, 860–873 (2008).

    CAS  PubMed  Google Scholar 

  35. Liu, Y., Zhu, M., Nishida, K., Hirano, T. & Zhang, W. An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response. J. Exp. Med. 204, 93–103 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Ramsay, A.G. et al. Chronic lymphocytic leukemia cells induce defective LFA-1-directed T-cell motility by altering Rho GTPase signaling that is reversible with lenalidomide. Blood 121, 2704–2714 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Richardson, P.G. et al. Lenalidomide, bortezomib, and dexamethasone combination therapy in patients with newly diagnosed multiple myeloma. Blood 116, 679–686 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

    PubMed  Google Scholar 

  39. Gandhi, A.K. et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br. J. Haematol. 164, 811–821 (2014).

    CAS  PubMed  Google Scholar 

  40. Casanova, J.L., Conley, M.E., Seligman, S.J., Abel, L. & Notarangelo, L.D. Guidelines for genetic studies in single patients: lessons from primary immunodeficiencies. J. Exp. Med. 211, 2137–2149 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Fuller, D.M. et al. Regulation of RasGRP1 function in T cell development and activation by its unique tail domain. PLoS One 7, e38796 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Vivier, E., Tomasello, E., Baratin, M., Walzer, T. & Ugolini, S. Functions of natural killer cells. Nat. Immunol. 9, 503–510 (2008).

    CAS  PubMed  Google Scholar 

  43. Thrasher, A.J. & Burns, S.O. WASP: a key immunological multitasker. Nat. Rev. Immunol. 10, 182–192 (2010).

    CAS  PubMed  Google Scholar 

  44. Lanzi, G. et al. A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP. J. Exp. Med. 209, 29–34 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. McGhee, S.A. & Chatila, T.A. DOCK8 immune deficiency as a model for primary cytoskeletal dysfunction. Dis. Markers 29, 151–156 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Liu, X., Kapoor, T.M., Chen, J.K. & Huse, M. Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II. Proc. Natl. Acad. Sci. USA 110, 11976–11981 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Schroeder, C.M., Ostrem, J.M., Hertz, N.T. & Vale, R.D. A Ras-like domain in the light intermediate chain bridges the dynein motor to a cargo-binding region. eLife 3, e03351 (2014).

    PubMed  PubMed Central  Google Scholar 

  48. Jayappa, K.D. et al. Human immunodeficiency virus type 1 employs the cellular dynein light chain 1 protein for reverse transcription through interaction with its integrase protein. J. Virol. 89, 3497–3511 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Zenz, T. Exhausting T cells in CLL. Blood 121, 1485–1486 (2013).

    CAS  PubMed  Google Scholar 

  50. Salzer, E. et al. Combined immunodeficiency with life-threatening EBV-associated lymphoproliferative disorder in patients lacking functional CD27. Haematologica 98, 473–478 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Willmann, K.L. et al. Biallelic loss-of-function mutation in NIK causes a primary immunodeficiency with multifaceted aberrant lymphoid immunity. Nat. Commun. 5, 5360 (2014).

    PubMed  Google Scholar 

  52. Kircher, M. et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet. 46, 310–315 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Dupré, L. et al. Efficacy of gene therapy for Wiskott-Aldrich syndrome using a WAS promoter/cDNA-containing lentiviral vector and nonlethal irradiation. Hum. Gene Ther. 17, 303–313 (2006).

    PubMed  Google Scholar 

  54. Dupré, L. et al. Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction. Mol. Ther. 10, 903–915 (2004).

    PubMed  Google Scholar 

  55. Pichlmair, A. et al. Viral immune modulators perturb the human molecular network by common and unique strategies. Nature 487, 486–490 (2012).

    CAS  PubMed  Google Scholar 

  56. Glatter, T., Wepf, A., Aebersold, R. & Gstaiger, M. An integrated workflow for charting the human interaction proteome: insights into the PP2A system. Mol. Syst. Biol. 5, 237 (2009).

    PubMed  PubMed Central  Google Scholar 

  57. Dow, L.E. et al. A pipeline for the generation of shRNA transgenic mice. Nat. Protoc. 7, 374–393 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Zuber, J. et al. An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance. Genes Dev. 25, 1628–1640 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 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 

  61. Brinkman, E.K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    PubMed  PubMed Central  Google Scholar 

  62. Boztug, K. et al. JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia. Nat. Genet. 46, 1021–1027 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Mellacheruvu, D. et al. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat. Methods 10, 730–736 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Vasconcelos, Z. et al. Individual human cytotoxic T lymphocytes exhibit intraclonal heterogeneity during sustained killing. Cell Rep. 11, 1474–1485 (2015).

    CAS  PubMed  Google Scholar 

  65. Huppa, J.B. et al. TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Fairhead, M., Krndija, D., Lowe, E.D. & Howarth, M. Plug-and-play pairing via defined divalent streptavidins. J. Mol. Biol. 426, 199–214 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Howarth, M. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat. Methods 3, 267–273 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Banerjee, P.P. & Orange, J.S. Quantitative measurement of F-actin accumulation at the NK cell immunological synapse. J. Immunol. Methods 355, 1–13 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Trifari, S. et al. Defective Th1 cytokine gene transcription in CD4+ and CD8+ T cells from Wiskott-Aldrich syndrome patients. J. Immunol. 177, 7451–7461 (2006).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the patient and his family for participating in this study; B. Fleckenstein and M. Schmidt for generating and providing patient-derived T cell lines; and G. Superti-Furga, J. Bigenzahn for providing the inducible protein expression system used for Jurkat T cells and together with N. Serwas, C.D. Conde, A. Kalinichenko, K. Ackerman and R. Martins for critically reviewing the manuscript and providing comments. The research leading to these results was funded by the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement 310857 (K.B.), the Vienna Science and Technology Fund (WWTF) through project LS14-031 (J.B.H. and K.B.), an unrestricted research grant from Celgene Austria (U.J.), the National Institutes of Health (R01AI067946 to J.S.O.), Boehringer Ingelheim Fonds (R.P.), the Austrian Science Fund (FWF): Project M1809-B19 (K.L.W.), and the French Agence Nationale de la Recherche (ANR-13-BSV1-0031 to L.D.).

Author information

Author notes

  1. Deniz Cagdas, Miroslav Hons, Emily M Mace, Michael Sixt, Ilhan Tezcan and Jordan S Orange: These authors contributed equally to this work.

Authors and Affiliations

  1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

    Elisabeth Salzer, Wojciech Garncarz, Özlem Yüce Petronczki, Laurène Pfajfer, Ivan Bilic, Sol A Ban, Katharina L Willmann, Keiryn L Bennett, Loïc Dupré & Kaan Boztug

  2. Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria

    Elisabeth Salzer, Wojciech Garncarz, Özlem Yüce Petronczki, Laurène Pfajfer, Loïc Dupré & Kaan Boztug

  3. Section of Pediatric Immunology, Hacettepe University, Ihsan Dogramaci Children's Hospital, Ankara, Turkey

    Deniz Cagdas, Özden Sanal & Ilhan Tezcan

  4. Institute of Science and Technology Austria, Klosterneuburg, Austria

    Miroslav Hons & Michael Sixt

  5. Center for Human Immunobiology, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas, USA

    Emily M Mace, Malini Mukherjee, Hsiang Ting Hsu, Pinaki P Banerjee, Papiya Sinha & Jordan S Orange

  6. Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

    René Platzer, Verena Supper, Hannes Stockinger & Johannes B Huppa

  7. Centre for Haemato-Oncology, Barts Cancer Institute - a CR-UK Centre of Excellence, Queen Mary University of London, London, UK

    Fabienne McClanahan & John G Gribben

  8. Institute of Immunology, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

    Gerhard J Zlabinger

  9. Christian Doppler Laboratory for Immunomodulation and Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

    Winfried F Pickl

  10. Centre de Physiopathologie de Toulouse Purpan (CPTP), INSERM, UMR1043, Toulouse Purpan University Hospital, Toulouse, France

    Loïc Dupré

  11. Department of Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria

    Ulrich Jäger

  12. Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria

    Kaan Boztug

  13. Department of Pediatrics, St. Anna Kinderspital and Children's Cancer Research Institute, Medical University of Vienna, Vienna, Austria

    Kaan Boztug

Authors
  1. Elisabeth Salzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deniz Cagdas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miroslav Hons
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Emily M Mace
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wojciech Garncarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Özlem Yüce Petronczki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. René Platzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Laurène Pfajfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ivan Bilic
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sol A Ban
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Katharina L Willmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Malini Mukherjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Verena Supper
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Hsiang Ting Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Pinaki P Banerjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Papiya Sinha
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Fabienne McClanahan
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Gerhard J Zlabinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Winfried F Pickl
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. John G Gribben
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Hannes Stockinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Keiryn L Bennett
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Johannes B Huppa
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Loïc Dupré
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Özden Sanal
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Ulrich Jäger
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Michael Sixt
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Ilhan Tezcan
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Jordan S Orange
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Kaan Boztug
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.S. performed most of the experiments, analyzed data, interpreted results, and, together with K.B., wrote the initial draft and revised version of the manuscript. D.C., I.T. and Ö.S. cared for the patient, and provided and interpreted clinical and immunological data. M.H. and E.S. performed migration and Lifeact experiments. M.S. provided critical input to the content of the manuscript. E.M.M., P.S., M.M., P.P.B., H.T.H. and J.S.O. performed and interpreted NK-cell immunological synapse experiments and detailed flow-cytometry-based NK-cell immunophenotyping. S.A.B. and E.S. identified the RASGRP1 mutation and performed initial experiments. R.P. and J.B.H. performed lipid bilayer calcium-flux experiments, and analyzed and interpreted data. L.P. generated an untransformed CD8+ T cell line from the patient and performed the CD8+ T cell cytotoxic assays. H.S. and V.S. performed proliferation analyses together with E.S. and provided critical input. Ö.Y.P. and L.D. performed the immunofluorescence experiments to quantify RhoA activation. K.L.W., W.G. and I.B. performed experiments and provided critical input. F.M., J.G.G. and U.J. helped with migration analyses. K.L.B. performed mass spectrometry analyses. W.F.P. and G.J.Z. performed thymidine incorporation assays, chromium release assays and analyses of autoantibody titers. K.B. conceived of and coordinated the study, provided laboratory resources, interpreted data, supervised E.S., W.G., Ö.Y.P., I.B., S.B. and K.W., wrote the manuscript together with E.S. and took overall responsibility for the study. All of the authors provided critical input and agreed to this publication.

Corresponding author

Correspondence to Kaan Boztug.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Histology of lymphoma and segregation of the mutation with the disease phenotype.

(a) Histology of adenoid biopsy showing low grade B-cell lymphoma compatible with marginal zone lymphoma. Top left to right: Hematoxilin/Eosin staining, CD20 staining, CD3 staining. Bottom left to right: EBER, CD23, Ki67. The described findings suggest a low grade EBV-related B-cell lymphoma developed likely associated with the underlying immunodeficiency. (b) Segregation of the detected mutation among the core family (circle - female; square - male; line - dead; black filling - affected index patient).

Supplementary Figure 2 Functional characterization of T cells.

(a) Invariant natural killer T (iNKT) cells. A prominent TCRvα24-expressing cell population was detectable in patient peripheral blood, however these cells expressed CD8 and CD45RA surface markers, suggesting that they belong to oligoclonally expanded exhausted memory CD8 T cells. HD – healthy donor; SC – Shipment control. (b) Proliferative response of T cells determined by [3H]-thymidine incorporation assay after stimulation with various stimuli after 3 days (OKT3, anti-CD3 antibody (clone OKT3)); PMA, phorbol 12-myristate 13-acetate; SEA: Staphylococcal enterotoxin A; SEB: Staphylococcal enterotoxin B; PHA: phytohaemagglutinin). (c) Proliferation of Jurkat T cells upon shRNA-mediated knockdown of RASGRP1. shRNA against Renilla luciferase was used as negative control. Cells were labeled with violet proliferation dye and analyzed by flow cytometry over the period of 5 days. (d) TCRVb spectratyping of patient and control T cells indicating oligoclonality of the TCR repertoire of the patient. (e) Calcium flux of Jurkat T cells upon inducible shRNA-mediated knockdown of RASGRP-1. shRNA against Renilla luciferase was used as negative control. (f) Fluorescence microscopy of Ca2+-flux of one representative cell is displayed below the graph. Scale bar represents 10 μm. HD–healthy donor; DIC–differential interference contrast. (g) ICAM Ring formation and cSMAC exclusion of hTERT immortalized patient and control T cell lines on a lipid bilayer following OKT3 stimulation. (h) Cropped immunoblot showing downstream TCR signaling in expanded patient and shipment T cells upon CD3/CD28 stimulation. Cells were starved and restimulated with anti-CD3/anti-CD28 antibodies and subsequently analyzed for ERK1/2 phosphorylation. Data is representative of three (h), two (a,c,d,g) or one (b,f,e) independent experiment.

Supplementary Figure 3 Gating Strategy.

Gating strategy for Figures 2 and 3 is presented in this figure.

Supplementary Figure 4 NK-cell studies.

(a) Flow cytometric analysis of intracellular accumulation of IL-5, IL13 and IFNγ in patient and control NK cells. (b) Immune synapse formation in primary patient NK cells (c) Co-immunoprecipitation of Strep-HA tagged RASGRP1 with endogenous dynein light chain 1 (DYNLL1) (RASGRP1wt) wildtype Strep-HA-tagged RASGRP1 and (RASGRP1mut) mutant Strep-HA-tagged RASGRP1. Strep-HA-tagged GFP was used as negative control. (d) Video microscopy of granule convergence in CRISPR-edited NK-92 cells lines HD, healthy donor. (d) Video microscopy of control or sgRASGRP1 NK-92 cells for granule convergence (Granules-red, Microtubuli-green). Data are representative of two biological replicates (a-d). (e) Gating strategy of FACS plots in Figure 4.

Supplementary Figure 5 T-cell activation using CXCL12.

(a-c) Immunofluorescence (a) and quantification (b,c) of patient and healthy donor expanded CD8 T cells following CXCL12 stimulation either stained for total RhoA (green) or active RhoA (pink) and DAPI (blue). Data is representative of two (a-c) independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–8 (PDF 2516 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Salzer, E., Cagdas, D., Hons, M. et al. RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics. Nat Immunol 17, 1352–1360 (2016). https://doi.org/10.1038/ni.3575

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3575

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