The Wiskott-Aldrich syndrome: An Immunodeficiency due to a defective cytoskeleton

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1 The Wiskott-Aldrich syndrome: An Immunodeficiency due to a defective cytoskeleton Raif Geha Children s Hospital, Boston Harvard Medical School Clinical features Wiskott-Aldrich syndrome An X-linked PID that affects to per million individuals. thrombocytopenia with small-sized platelets eczema immunodeficiency - infections (encapsulated bacteria, HSV) - autoimmunity: vasculitis colitis - tumors (leukemia, lymphoma) Immunology IgM, IgA and IgE Impaired response to polysaccharide antigens Progressive decline of lymphocyte count Defective T cells structure, interactions, and activation BasicGBD EVH Pro rich A C V Btk Fyn Toca- Nck Grb? Cortactin CIP4 filament PIP Cdc4 SH3 Basic 9 EVH Y Pro-rich V CA GBD is normally in an inactive state because of intramolecular interactions. Binding of Cdc4 to the GBD or of SH3 domains (Nck, Grb, cortactin) to the proline-rich region of results in breaking the intramolecular bonds. This allows the acidic VCA domain of to interact with the Arp/3 complex and initiate actin polymerization. Siseases associated with mutations Wiskott-Aldrich syndrome (WAS) X-linked thrombocytopenia (XLT) Intermittent X-linked thrombocytopenia (ixlt) X-linked neutropenia (XLN) X-linked myelodysplasia (XLM) mutations PIP Cdc4 SH3 G- Arp /3 EVH Basic GBD Pro-rich V C A Exon Diseases caused by mutations in WAS is due to mutations in the gene that codes for the Wiskott-Aldrich Syndrome protein () resulting in a wide range of clinical phenotypes that depend on the position and nature of the mutation >9% of missense mutations are in EVH domain. >7% of them are XLT patients. missense nonsense insertion, frameshift insertion, inframe deletion, frameshift deletion, inframe splice site complex Disease Mutation Phenotype X-linked thrombocytopenia (XLT) Missense mutations, in frame deletion or insertion in exons -3 Thrombocytopenia and small platelets WAS Nonsense mutations, frame shift caused by deletions or insertions Thrombocytopenia with small platelets, eczema, recurrent infections, increase incidence of autoimmunity and malignancies X-linked neutropenia (XLN) Missense mutations, inframe deletion or insertion in the GBD domain Neutropenia

2 CD8+ (cells/ml) CD4+ (cells/ml) CD9+ (cells/ml) CD3+ (cells/ml) ANALYSIS OF PROTEIN EXPRESSION BY FLOW CYTOMETRY 8 I-XLT XLT WAS T and B cell lymphopenia in WAS/XLT WASp-PE years XLT WAS WAS Revertant CD4 and CD8 Tcell lymphopenia in WAS/XLT 5 Factors that influence the phenotype age. family history (susceptibility genes for allergy/atoimmunity) environment (microbes, hygiene) treatment/prevention years XLT WAS WAS revertant Patients younger than years often have a score 3, even if some of them develop severe symptoms later in life T cell activation defects in WAS - Impaired T cell proliferation, and IL- secretion after stimulation with immobilized, but - Normal response to alloantigens, antigens and soluble presented by APCs - Defective regulatory T cell function

3 Impaired Treg activity in deficient mice KO WAS IPEX: Foxp3 Ratio Resp:T regs CD4 + CD5 - responder T cells (Resp) from mice were co-cultured for 3 days with CD4 + CD5 + T regs cells either from, KO, or KO mice at different ratios. Significance of Treg cell defect Lack of T regs results in autoimmune disease with colitis T cell cytoskeletal defects in WAS Ulcerative colitis occurs in 3-4 % of patients. they also develop vasculitis and anti red cells and antiplts Abs KO mice develop chronic colitis with leukocytic infiltration in the intestinal mucosa. This is reversed by normal T regs KO -Impaired F-actin polymerization after TCR ligation. -Impaired capping of the TCR. -Impaired spreading over coated plates -Impaired homing to lymphoid tissues. Scanning EM of lymphocytes in Wiskott Aldrich syndrome F-actin polymerization in primary T-cells following stimulation is deficient in WAS Normal WAS Control WAS Patient 3

4 L39P NL D77G µm % spread cell Aberrant Capping in -/- Tcells medium Defective spreading of WAS patient primary T cells stimulated with Polylysine 8 p=.6 p= KO NL L39P D77G Immunity. 998 Jul;9():8-9. In vivo homing of -/- T cells Severely impaired in vivo homing of -/- T cells Mesenteric LN Spleen Alexa T cells : + TRITC T cells from, WKO, or DKO hour Spleen Mesenteric LN FACS :.3: -/-.7%.3%.7%.78% -/-.35%.4%.36%.99% :.4: Defects in other immune cells -NK cells : -defective cytolytic function -reduced F-actin at the immunological synapse -B cells: - Defects in MZ B cells (TI Ab. responses to polyscaccharides) and memory B cells. -Macrophages : -defective formation of the phagocytic actin cup -Chemotactic and Migratory defects : - Neutrophils, Macrophages, Dendritic cells and B cells. NK Formation of the cytolytic immunological synapse requires y WASp - NK* DIC F-actin CDa Perforin Merge x z x y x *same with cytochalasin-d DIC F-actin CDa Perforin Merge 4

5 IL- corrects NK cell defect in vitro IMMUNOPHENOTYPE OF IgM MEMORY B cells :CD9+ CD+ IgD+ IgM+ CD7+ IgM memory B cells are markedly decreased in XLT and WAS patients IMMUNOPHENOTYPE OF SWITCHED MEMORY B cells :CD9+ CD+ IgD- IgM- CD7+ % 4 peripheral blood "IgM" memory B lymphocytes HD -y HD >y XLT -y XLT >y WAS -y WAS >y 3 Switched memory B cells are decreased in XLT and WAS patients. Splenectomy, IVIG TREATMENT HD -y HD >y XLT -y XLT >y WAS -y WAS >y % 3 peripheral blood switched memory B lymphocytes. BMT Complete correction of the Wiskott-Aldrich syndrome by allogeneic bone-marrow transplantation. Parkman et al. N Engl J Med. 978 Apr 7;98(7): Gene therapy: ongoing 4. Peptide therapy to stabilize 5

6 The European experience with HSCT in PID: disorders other than SCID Wiskott n=66 Phagocytic n=89 Haemophago n=54 T cell def 4 years Survival rate Wiskott : 7% Phagocytic : 63% Haemophagocytic : 58% T cell def : 47% p<. The European experience with HSCT in WAS: impact of donor type -year survival MRD: 88% MUD: 77% MMRD: 45% mutations PIP Cdc4 SH3 G- Arp /3 EVH Basic GBD Pro-rich V C A Exon >9% of missense mutations are in EVH domain. >7% of them are XLT patients. 5 missense nonsense insertion, frameshift insertion, inframe deletion, frameshift deletion, inframe splice site complex ( Interacting Protein) Verprolin homology domain SH3 binding Proline rich sequence aa Profilin Profilin Nck binding Profilin / Cortactin N- stabilizes : Rationale for stabilization of by overexpression of -/- GAPDH -/- NL NFATp protein mrna WAS * * * * * * n WAS+n n * * n n n n n * n n * n n n * n n * n n 6

7 % % p<. p<. % % p<. p<. p<.5 p<.5 % % p<. p<. % % Relative mrna levels % p<. p<. % NL % spread cell µm L39P D77G µm µm % Interaction of -n with levels are increased by expression of the -binding domain of in -/- T cells B) MPVPPPPAPPPPPTFALANTEKPTLNKTEQAGRNALLSDISKGKKLKKTVTNDRSAPILDKPKGAGASAGGYGGGGGGGGGGGG GGGGSGGNFGGGGPPGLGGLFQAGMPKLRSTANRDNDSGGSRPPILPPGGRATSAKPFSPPSGPGRFPAPSPGHRSGPPEPPRN RMPPPRPDVGSKPDSLPPPVPNTPRPVPSSLHNRGSPAGLGAPRPPFPGNRGAAFGAGSARQNPSGSSSPFPRPPLPPTPSRALD DKPPPPPPPVGNRPSMHREAVPPPPSQTSKPPVPSTPRPGLGSQAPPPPPPPSRPGPPPLPPASNDEIPRLPQRNLSLTSSAPPLP SPGRSGPLPPPPSERPPPPVRDPPGRSGPLPPPPPINRNGSTARALPATPQLPSRSGMDSPRSGPRPPLPPDRPGAGAPPPPPPST SVRNGFQDSSCEDEWESRFYFHPISDLPPPEPYVPTTKTYPSKLARNESRSGSNRRERGAPPLPPIPR A) /- + -/- +- -/- +-n / SCEDEWESRFYFHPISDLPPPEPYVPTTKTYPSKLARNESR Lysate IP: anti / / / /- +-n p<. p<. WB: anti- 4 -n WB: anti- WB: anti protein and mrna levels in WAS patient EBVtransformed B cell lines. A) B) Stabilization of in WAS patient EBV-B cell lines by D77G+ D77G+- D77G+-n N4fs+ N4fs+- N4fs+-n * ** ** ** * EVH domain EVH domain Color key: - -n Normal control Stabilization of in WAS patient EBV-B cell lines by Expression of or n increases spreading of WAS patient primary T cells stimulated with EVH domain Polylysine Polylysine P58R D77G D77H R86H L5P A34T - -n - -n N-terminal to the EVH domain C-terminal to the EVH domain E3K L39P A36G N4fs 8 ** ** * * Polylysine - -n Color key : - -n NL L39P D77G 7

8 Ctl Father Mother Patient Ctl Patient stabilizes Conclusion A 34 a.a long binding peptide fragment of restores levels in EBV B cells and corrects the T cell cytoskeletal defect in T cells from WAS patients with mutations in that destroy binding. This may provide a novel approach to therapy of WAS patients. Phenotype of -/- mice T and B cell phenotype is normal Severe colitis develops in in % of mice -/- T cells Fail to extend membrane protrusions in response to stimulation. Impaired in their ability to form an IS. Fail to increase their F-actin after TCR ligation. Have severely defective T-reg cell number and function Have a disrupted actin network Have drastically low levels of Fail to proliferate to both immobilized and soluble anti- CD3 and to antigens, and fail to develop DTH Fail to respond to IL- by STAT5 phosphorylation, c-myc expression and proliferation, Anton et al. Immunity Electron micrographs of T cells -/- Presentation Family History: Moroccan consanguineous healthy parents (II grade cousins) 4 m/o sister dead of sepsis in Morocco Failure to thrive and feeding difficulties Bullous lesions on the abdomen and impetiginized vesicular lesions RSV penumoioa Colitis - network is disrupted Platelet Count: 59 x ³/µL CRP.3 mg/l Low CD3 CD4 and CD8 Absent protein Intracellular staining T cells Fibrobalsts actin merge Ctl a (3D) a (3D) a (5A5) aactin a (5A5) atubulin aactin Patient 8

9 # Cells SSC Intracellular staining for and isotype control Healthy control patient Mother Father patient parents EX 6 C3G S434X control transduction rescues levels in patient T cells PT Mock PT h CTRL h FSC Sample CTRL h PT h PT h +- PT mock + MFI Conclusion deficient mice have a similar but more severe phenotype than deficient mice and % develop colitis They have severely diminished levels in T cells, which can be corrected by overexpressing deficient patient presents with T cell lymphopenia, poor T cell proliferation to mitogens and severe colitis She had severely diminished levels in T cells, which can be corrected by overexpressing Severine Suresh Michel Ramesh Silvia Giliani 9