Gerben Bouma

Nonintegrating Lentivector Vaccines Stimulate Prolonged T-Cell and Antibody Responses and Are Effective in Tumor Therapy

ABSTRACT Lentiviral vectors (lentivectors) are effective for stimulation of cell-mediated and humoral immunity following subcutaneous and intramuscular immunization. However, lentivector genome integration carries a risk of perturbation of host gene expression. Here, we demonstrate that lentivectors with multiple mutations that prevent integration are also effective immunogens. First, systemic CD8+ T-cell responses to the model antigen ovalbumin were detected following subcutaneous injection of nonintegrating lentivectors. Transfer of transgenic OT1 T cells demonstrated that antigen presentation persisted for at least 30 days. Furthermore, an enhanced CD8+ T-cell response, peaking at 7 days, was stimulated by coexpression of p38 MAP kinase or an NF-κB activator from the same vector. Second, we demonstrated systemic CD8+ T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.

Wiskott–Aldrich Syndrome: Immunodeficiency resulting from defective cell migration and impaired immunostimulatory activation

Regulation of the actin cytoskeleton is crucial for many aspects of correct and cooperative functioning of immune cells, such as migration, antigen uptake and cell activation. The Wiskott–Aldrich Syndrome protein (WASp) is an important regulator of actin cytoskeletal rearrangements and lack of this protein results in impaired immune function. This review discusses recent new insights of the role of WASp at molecular and cellular level and evaluates how WASp deficiency affects important immunological features and how defective immune cell function contributes to compromised host defence.

B cell-intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.

Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis

To more precisely identify the B-cell phenotype in Wiskott-Aldrich syndrome (WAS), we used 3 distinct murine in vivo models to define the cell intrinsic requirements for WAS protein (WASp) in central versus peripheral B-cell development. Whereas WASp is dispensable for early bone marrow B-cell development, WASp deficiency results in a marked reduction in each of the major mature peripheral B-cell subsets, exerting the greatest impact on marginal zone and B1a B cells. Using in vivo bromodeoxyuridine labeling and in vitro functional assays, we show that these deficits reflect altered peripheral homeostasis, partially resulting from an impairment in integrin function, rather than a developmental defect. Consistent with these observations, we also show that: (1) WASp expression levels increase with cell maturity, peaking in those subsets exhibiting the greatest sensitivity to WASp deficiency; (2) WASp+ murine B cells exhibit a marked selective advantage beginning at the late transitional B-cell stage; and (3) a similar in vivo selective advantage is manifest by mature WASp+ human B cells. Together, our data provide a better understanding of the clinical phenotype of WAS and suggest that gene therapy might be a useful approach to rescue altered B-cell homeostasis in this disease.

Cytoskeletal remodeling mediated by WASp in dendritic cells is necessary for normal immune synapse formation and T-cell priming

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

Phosphorylation of WASp is a key regulator of activity and stability in vivo

The Wiskott-Aldrich syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. Covalent modification of a conserved tyrosine by phosphorylation has emerged as an important potential determinant of activity, although the physiological significance remains uncertain. In a murine knockin model, mutation resulting in inability to phosphorylate Y293 (Y293F) mimicked many features of complete WASp-deficiency. Although a phosphomimicking mutant Y293E conferred enhanced actin-polymerization, the cellular phenotype was similar due to functional dysregulation. Furthermore, steady-state levels of Y293E-WASp were markedly reduced compared to wild-type WASp and Y293F-WASp, although partially recoverable by treatment of cells with proteasome inhibitors. Consequently, tyrosine phosphorylation plays a critical role in normal activation of WASp in vivo, and is indispensible for multiple tasks including proliferation, phagocytosis, chemotaxis, and assembly of adhesion structures. Furthermore, it may target WASp for proteasome-mediated degradation, thereby providing a default mechanism for self-limiting stimulation of the Arp2/3 complex.

The Wiskott-Aldrich syndrome: The actin cytoskeleton and immune cell function

Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive primary immunodeficiency characterised by immune dysregulation, microthrombocytopaenia, eczema and lymphoid malignancies. Mutations in the WAS gene can lead to distinct syndrome variations which largely, although not exclusively, depend upon the mutation. Premature termination and deletions abrogate Wiskott-Aldrich syndrome protein (WASp) expression and lead to severe disease (WAS). Missense mutations usually result in reduced protein expression and the phenotypically milder X-linked thrombocytopenia (XLT) or attenuated WAS [1-3]. More recently however novel activating mutations have been described that give rise to X-linked neutropenia (XLN), a third syndrome defined by neutropenia with variable myelodysplasia [4-6]. WASP is key in transducing signals from the cell surface to the actin cytoskeleton, and a lack of WASp results in cytoskeletal defects that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration.

Functional Gap Junctions Accumulate at the Immunological Synapse and Contribute to T Cell Activation

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca2+ responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell–T cell signaling machinery that regulates T cell activation.

Improvement of Migratory Defects in a Murine Model of Wiskott–Aldrich Syndrome Gene Therapy

Wiskott-Aldrich syndrome (WAS) is an X-linked hematological disease characterized by immunodeficiency, eczema, and thrombocytopaenia, and shows promise for treatment with hematopoietic stem cell gene therapy. The immunopathology of WAS is attributable at least in part to defects of cell migration and localization as a result of chemotactic, adhesive, and chemokinetic defects. Whereas previous studies using either gammaretroviral or lentiviral vectors have demonstrated variable correction of T-cell proliferation and dendritic cell (DC) cytoarchitecture, we have used a lentiviral vector expressing an eGFP-WASp fusion protein to test the potential for restoration of cell migratory defects. Multilineage expression of the fusion transgene was present for up to 10 months after primary engraftment, and also in secondary recipients analyzed after a further 9 months. Transduced bone marrow-derived dendritic cells (BMDCs) demonstrated recovery of podosome numbers and turnover, while B cells, BMDCs, and Langerhans cells (LCs) exhibited enhanced chemotactic responses to specific stimuli. As an indication of functionality in vivo, splenic marginal zone B cells and a cutaneous contact hypersensitivity (CHS) response to dinitrofluorobenzene (DNFB) were both partially restored. These proof of principle experiments demonstrate that WAS protein (WASp) transgene expression can be successfully maintained long term in primary and secondary recipients, and that it is associated with a significant repair of migratory defects.

Recent advances in the understanding of genetic defects of neutrophil number and function

Neutrophils are amongst the first immune cells to arrive at sites of infection and play an important role as the host’s first line of defence against invading pathogens. Defects of neutrophil number or function are usually recognized clinically by recurrent infections that often are life‐threatening. Over the last few years, a number of genetic mutations have been discovered to be the basis for congenital neutropenia, adding to our understanding of the molecular basis of these diseases. While many genetic mutations that cause severe congenital neutropenia result in a differentiation block at the promyelocyte stage, defects of neutrophil function are more heterogeneous on clinical, genetic and mechanistic levels. In this review we discuss recent advances in our understanding of the genetic and molecular basis of human neutrophil disorders.