Wiskott-Aldrich Syndrome: a cytoskeleton disease Elie Haddad, MD, PhD Pediatric Immunology and Rheumatology CHU Ste-Justine Université de Montréal, Montreal, QC, Canada
Wiskott-Aldrich Syndrome A disease of the hematopietic and immune system X-linked Involving platelets and lymphocytes Responsible of the classical clinical triad : Thrombopenia Immune deficiency Eczema
Platelet abnormalities Constant (100%) and very early finding Thrombopenia Small volume platelets (3.8 to 5 fl) half that of healthy controls From non-life threatening (epistaxis, petechiae, purpura, oral bleeding) to severe manifestations such as intestinal and intracranial bleeding. Death of WAS patients is caused, in 21% of the cases, by haemorrhages
Immune deficiency (Clinical aspects) Progressive Infections Mainly pyogenic Also viral, fungal and opportunistic Auto-immunity Up to 72% of patients Autoimmune hemolytic anemia Vasculitis Neutropenia IBD Renal disease (IgA or HSP like nephropathy) Malignancy (mainly B-cell lymphoma, poor prognosis)
Immune deficiency (biological aspects) Hypo IgM, Hyper IgA-E, normal IgG Impaired antibody response to T-independent antigen (bacterial polysaccharidic Ag), and sometimes to T-dependent protein antigens Progressive T-cell lymphopenia Reduced in vitro T proliferation in response to mitogens, allogenic stimulation and immobilized anti-cd3 mab Difference between XLT et WAS
WAS-XLT scoring system XLT WAS Clinical scores 0.5 1 2 3 4 5A 5B Thrombocytopenia +/- + + + + + + Eczema - - +/- + ++ ++/- ++/- Immuodeficiency - - +/- + ++ ++/- ++/- Autoimmunity - - - - - + - Malignancy - - - - - - + The phenotype may evolve over time and is often incomplete in < 2 years old patients. Progression of the disease is also possible at later age Scoring system according to Zhu et al Blood 1997 and Imai et al Curr Opin Allergy Clin Immunol 2003
The gene and the protein Gene codes for the WASP protein, 502 AA ARNm : exclusively in hematopoïetic cells WASP is involved in the cytoskeleton regulation WAS and XLT result from «loss-of-function» mutations of WAS gene I will not speak of X-linked neuropenia due to gain of function WASP mutation
Structure of WASP SH3 Cdc42-GTP Rac-GTP Adapter proteins: Nck, Grb2, Tyrosine kinases: Fyn, Btk, VCA Cofilin homology domain N WH1/PH B CRIB Y Poly-Pro WH2/VH CH P Fyn WASP-homology domain 2 or Verproline Homology domain C Phosphatase Actine Arp2/3 Functions of WASp dowstream of TCR, including actin polymerisation and IS formation in vitro Actin polymerization
STIMULATION Toca-1 Cdc42 GDP N Cdc42 GTP C CH VH B CRIB Inactive auto-inhibited WASP Cdc42 GTP N B CRIB Active WASP Polymerization VH CH C Arp2/3 Actin
Structure of WASP PiP2 SH3 Cdc42-GTP Rac-GTP Adapter proteins: Nck, Grb2, Tyrosine kinases: Fyn, Btk, VCA N WH1/PH WIP Recruitment of WASP to the Immune Synapse Protects WASP from degradation? Role in the stabilization of auto-inhibited conformation? B CRIB Y Poly-Pro WH2/VH CH P Phosphatase Fyn Filopodia, Cell polarization, Chemotaxis WASP-homology domain 2 or Verproline Homology domain Actine Actin polymerization Cofilin homology domain Arp2/3 C
Role of WASP Transduction of signals from the surface to the actin cytoskeleton Role in actin polymerization, cell polarization Essential functions: Cell locomotion, cell trafficking Intracellular signaling Immune synapse formation and organization Cell-cell interaction Cytotoxicity All functions important for adaptive and innate immunity and immune surveillance
Minor role of WASP in early hematopoietic cell development Significant growth and/or survival disadvantage of mature WASP deficient cells Thrasher et al, Nat Rev Immunol 2010
T-cell dysfunction Progressive T-cell lymphopenia by decreased thymic output and decreased peripheral T cell survival Abnormal T cell morphology Role in the formation of the IS between APC and T-cell lower levels of lipid raft Downstream of T-cell receptor ligation at the IS: Defective actin polymerization Failure to recruit proteins Abnormal proliferative response, defective calcium influx Decreased production of IL-2 and TH1 cytokine (then unbalanced Th2 response) Failure to polarize cytokine secretion toward antigen-specific target cells
T-reg dysfunction WASP-deficient T-reg cells fail to proliferate normally in response to TCR stimulation and their suppressive activity is impaired in vitro Role of T-reg cells in the autoimmunity of mice WAS models is clear Less clear in humans: Number of peripheral T-reg cells are normal Maybe an intrinsic cellular dysfunction
NK and inkt cell dysfunction NK cells: Normal or increased NK cell numbers Impaired NK-cell cytotoxicity, and NK cell migration inkt cells inkt absent in WAS and normal/decreased in XLT WASP is important for inkt homeostasis and function, and also for late-stage thymus development and egress Suggested role of inkt cells in clearance of microorganisms, tumour surveillance and protection from autoimmunity
DC and macrophage/pn dysfunction Human WASP-deficient myeloid lineage cells exhibit impaired phagocytosis monocytes, macrophages, DCs and osteoclasts show almost completely abrogated assembly of podosomes Impaired migration and homing of Macrophages and DC Mobility defect of DC confirmed in vivo Defects in cell-to-cell interactions: abnormal DC-mediated induction of immune synapse formation in T cells
B-cell dysfunction Clinically, clear defect in humoral immunity but until recently, role of WASP in B cells not deeply studied Impaired adhesion, migration and homing Abnormal B cell homeostasis with selective depletion of circulating mature B cells, splenic marginal zone precursors and marginal zone B cells Reduced expression of CD21 and CD35 Intrinsic B-cell defect, not corrected by normal T cells in chimerism situation, likely responsible for humoral immune deficiency and some autoimmunity
Mechanism of thrombopenia? Profound microthrombocypenia is the hallmark of the disease Risk of life-threatening hemorraghea without clear correlation with the intensity of thrombopenia: risk difficult to manage Mechanism still uncompletely understood, likely central and peripheral mechanisms Both an immune-mediated mechanism and intrinsic platelet abnormalities There is also defective function of platelets responsible for bleeding risk
Mechanism of autoimmunity? Role of Treg: abnormal homeostasis and function Role of B cells that intrinsicallly secrete auto-antibodies High frequency of autoimmunity post transplant, associated with mixed chimerism Chimeric mouse model of WASP-/- B cells with WASP + other lineages: increased autoimmunity
Thrasher et al, Nat Rev Immunol 2010
Diagnosis: key words Male Microthrombocytopenia Progressive immune deficiency WASP sequencing
Patient L.N. Uncle, same mutation L.N. s brother, ITP, no mutation ITP in a girl
Management of WAS patients Supportive therapy: Treatment of thrombopenia : somewhat similar to ITP, but more difficult IVIG/ScIG, Bactrim prophylaxis Immunosuppression for autoimmune manifestations Targeted anti-microorganism therapy Bone marrow transplantation HLA id sibling, MUD and matched CB: overall good results Better to obtain a good stable chimerism > 5 years: poorer outcome for MUD but still 73% survival Splenectomy associated with a poorer outcome WAS = indication of HSCT (Moratto et al Blood 2011, Oszahin et al Blood 2008, Filipovich et al Blood 2001 )
Management of WAS patients (2) Gene therapy Surely a good option in next future for patients without a «good» donor To be confirmed: maybe larger indications or maybe not a so good strategy
Conclusions Mutations of WASP are associated with a remarkably wide and variable clinical spectrum Studying the mechanisms of WAS led to a better understanding of the role of the cytoskeleton in the function and homeostasis of the immune system Clinical retrospective studies on XLT and HSCT for WASP have led to a better clinical strategy Some decisions are still very difficult and controversial I recommand: Albert et al Curr Op Hematol 2011, Thrasher et al Nat rev Immunol 2010, Albert et al Blood 2010, Moratto et al Blood 2011