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RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics

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.

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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.

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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.).

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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.

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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.

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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

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