The impact of the cytoskeleton dynamics on immune responses

Sammanfattning: Immune cells are susceptible to a variety of external stimuli that triggers cell surface receptors, leading to activation of intracellular proteins and changes in the actin cytoskeleton. The Rho-GTPases are a family of proteins that regulate several aspects of actin polymerization. They take part in an intricate network of other proteins and can therefore have a direct or indirect role in several cell functions. In this study I aimed to define the role of the Rho-GTPases Cdc42 and Rac2, as well as the effector proteins WASp and N-WASp in the adaptive immune system. In paper I the aim was to dissect the particular contribution of WASp-deficiency to the induction of skin pathology in Wiskott-Aldrich syndrome patients, using mouse models. Our study showed that WASp-deficient dendritic cells accumulate in the skin; were able to induce an increase in cross-presentation of exogenous antigen and therefore increased CD8+ T cell proliferation and activation in vivo and in vitro. This was caused by increased expression of Rac2 in the cytoplasm, which regulates the phagosomal pH and promotes loading of antigen on MHC class I molecules. In paper II we aimed to understand the role of Cdc42 for B cell activation using a mouse where we deleted Cdc42 conditionally in mature B cells. Mice with Cdc42-deficient B cells showed reduced marginal zone and follicular B cell numbers. Immunized mice had reduced germinal center B cells associated with reduced specific antibody titers in serum. In vitro assays revealed less spreading on antibody-coated surfaces and a skewed CD4+ T cell activation towards more production of IFN? and less IL-2. In paper III we studied the combined contribution of WASp and N-WASp deficiency in B cells. A WASp KO mouse where N-WASp was conditionally deleted only in B cells (cDKO) was used. Compared to WT and WASp KO mice, the cDKO mouse had a large reduction in marginal zone B cells as well as decreased marginal zone B cell precursors and follicular B cells. B cells in cDKO mice also failed to mount a specific antibody response to both T- dependent and T-independent antigens with reduced specific antibody titers, together with decreased antigen uptake by marginal zone B cells and transportation to the follicle. In paper IV we investigated the effect of the hyperactive mutated WASp, found in X-linked neutropenia patients, on murine B and T cells. XLN-WASp mutations induced increased polymerized actin in both T and B cells. We detected reduced spreading but normal chemotaxis toward specific chemokines by both cell types. We also observed reduced proliferation of B cells but not T cells and increased apoptosis of both cell types in vitro. The latter is probably due to increased genomic instability in B and T cells as observed by a FISH-telomere assay. Altogether, this study shows that Rho GTPases and their effector proteins are crucial for correct cell function and the cellular and humoral immune responses. Notably, the phenotypic severity can be increased by depletion of closely related intracellular proteins suggesting the existence of compensatory mechanisms and overlapping roles. The characterization of the Rho GTPases function may contribute to more precise interventions and therapeutic success.