Regulation of Mucosal Immune Responses by the IgA Cytoplasmic Tail

IgA is the dominant antibody at mucosal sites. IgA protects us from pathogens and maintains mucosal homeostasis. IgA also forms a B cell receptor (BCR) which is highly expressed on the surface of IgA+ plasma cells (PC), suggesting it may have a role in the development and function of these cells. The exact function of membrane IgA and its unique cytoplasmic tail remains unknown.Therefore, we adapted the SWHEL model to oral immunisations with Hen Egg Lysozyme (HEL) and cholera toxin subunit B in H2k congenic mice to enable tracking of SWHEL B cells that have undergone IgA class switching. This response is dominated by IgG1 but also generated IgE in addition to IgA anti-HEL antibodies, making the model potentially useful in future studies of allergic sensitisation. We next truncated the IgA cytoplasmic domain to generate the IgAicΔ mice using CRISP/Cas9. This resulted in marked decrease in cell surface IgA expression on PCs. We next used retroviral transduction to graft the terminal FKVK amino acid residues from the IgM cytoplasmic tail and this rescued surface IgA expression. This was also restored on PCs when the terminal Arginine (R) of the truncated IgA was converted into the Lysine (K) found in IgM (IgAicΔK2).We next retrovirally generated a series of mutant BCR: WT; truncated IgA tail (IgAΔ-TTVR); IgAΔ-FKVK; and IgA-FKVK (+tail). In vitro cultures confirmed that these modifications did not impact on IgA surface expression. Intravenous immunisation with HEL2X-SRBC revealed that IgAΔ-FKVK B cells had reduced Germinal Centre responses compared to IgA-FKVK (+tail) B cells. We therefore generated a second CRISPR mouse line in which the IgA cytoplasmic tail was directly grafted onto the SWHEL IgM receptor.Comprehensive analysis of mammalian IgA evolution showed that placental mammals, but not monotremes or marsupials, have 2 additional amino acids in their IgA transmembrane domain. This misalignment in the literature led us to unintentionally truncate an extra 2 amino acids, thereby affecting stability of the truncated IgA cytoplasmic tail. Future work in which the IgA cytoplasmic tail is truncated to the correct length to preserve the transmembrane domain will be of great interest

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.publishe

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation

This file contains raw data of publication by E.B. Compeer et al. Nature Communications 2018. (link will be updated when paper is published online)