The role of Annexin A6 in T cell activation

Abstract

The activation of T cells by an antigen presenting cell is one of the essential first steps for an effective immune response. Activation of the T cell receptor (TCR) leads to fundamental changes to T cell morphology, cytoskeletal rearrangement, membrane order and the formation of TCR microclusters which then propagate downstream signalling. TCR signalling eventually induces T cell proliferation as well as the production and secretion of interleukin 2 (IL-2) which binds to the IL-2 receptor and further stimulates T cell proliferation in an autocrine and paracrine fashion. Both signalling processes are dependent on specialised membrane domains enriched in cholesterol also termed lipid rafts and were found to sustain transient interactions with the cortical actin meshwork. Annexin A6 (AnxA6) is a calcium-activated cytosolic phospholipid membrane binding protein which has been suspected to play a role in T cell development and is upregulated in a wide range of immune cells including T cells. AnxA6 is involved in receptor endocytosis, vesicle budding and cholesterol homeostasis. It has been found to preferentially bind cholesterol-rich phospholipid membranes and to interact with the cortical cytoskeleton to allow endocytosis and vesicle budding. The aim of this study was to show that AnxA6 is crucial for an effective T cell mediated immune response. It was hypothesised that AnxA6 influences receptor signalling directly by mediating a link between the cortical cytoskeleton and cholesterol-rich membrane domains and thus AnxA6 might be part of a mechanism to target receptors to a specific membrane environment. Alternatively, AnxA6 influences signalling processes indirectly through maintaining the optimal composition of the plasma membrane due to its involvement in cholesterol homeostasis – as AnxA6 plays a role in cholesterol transport from intracellular membranes to the plasma membrane as well as low-density lipoprotein endocytosis. In this study a delayed-type contact hypersensitivity was elicited in AnxA6 knock-out mice to generate a T cell mediated immune response in vivo. The results showed that AnxA6 mice did mount a T cell mediated immune response but the levels of proliferating CD4+ T cells were significantly lower than in wild type mice. Investigating the source of this proliferation defect, western blot, fluorescence microscopy, qPCR and flow cytometry approaches were used to measure the response of primary murine T cells to T cell receptor as well as IL-2 receptor stimulation. Neither the early nor the late response of TCR signalling was affected in AnxA6-/- T cells. Instead, activated AnxA6-/- T cells secreted more IL-2 and the response to IL-2 stimulation was found to be impaired in AnxA6-/- T cells. In parallel, it was investigated if and how the membrane composition of primary AnxA6-/- T cells was different from wild type T cells. The membrane order of TCR stimulated as well as naïve cells was measured with polarity-sensitive dye Laurdan. To characterise the cholesterol content and phospho- and sphingolipid composition the lipid phase was extracted from whole T cell lysates and analysed by mass spectrometry. AnxA6-/- T cells were found to have a lower degree of plasma membrane order which implies that these membranes are more fluid. In agreement with this, AnxA6-/- T cells were found to have an altered membrane lipid composition. Some of these changes potentially affect the fluidity of the T cell plasma membrane: Cholesterol, which decreases plasma membrane fluidity, is less abundant in the T cells of AnxA6-/- mice. Levels of phosphatidylethanolamine with arachidonic acid, a polyunsaturated fatty acid which increases membrane fluidity, were more abundant in AnxA6-/- T cells. In conclusion, it could be shown that AnxA6 does play an important role in T cells and is required for efficient proliferation in a T cell mediated immune response. It is likely that the lower degree of IL-2 signalling efficiency in vitro and proliferation defect of CD4+ T cells observed in AnxA6-/- mice in vivo were a result of the changes in membrane composition.