The innate and adaptive immune systems protect the body from harmful environmental sub-stances by sampling, presenting, recognizing and subsequently combating pathogens. Traveling through the blood stream from primary and secondary lymphoid tissues to their tar-get destination, all immune cells interact with and migrate through the endothelium of the blood vessels. Specialized antigen-presenting cells (APC) such as dendritic cells (DC) and macrophages penetrate the epithelial cell barrier to sample antigens on the apical side using their dendritic protrusions. First responding immune cells, granulocytes, including neutrophils and eosino-phils, even transmigrate through the epithelial paracellular space to fight pathogens in the lu-men of e.g. the gut or the lung. When migrating through the paracellular space, immune cells inevitably come into contact with the tight junctions (TJ), a tight meshwork of transmembrane proteins that tighten this space and allow for the passage of ions, water, and macromolecules. There are two different types of TJs: (i) The bicellular TJ (bTJ) constitutes the barrier between two adjacent cells and is predominantly defined by proteins of the claudin family and by occludin and marvelD3. (ii) The tricellular TJ (tTJ) seals the paracellular space at the corners where three or more cells meet and is made up of proteins of the angulin family (LSR, ILDR1 or ILDR2) and tricellulin. Although there has been evidence that certain APCs express different claudins, immune cells transmigrate predominantly trough the tTJ. Therefore, the hypothesis was postulated that im-mune cells express (t)TJ proteins to interact with the tTJ to transmigrate through the paracel-lular space. To investigate this, immune cells were screened for TJ protein expression on mRNA and pro-tein level. RT-qPCR data revealed varying mRNA expression patterns according to activation status. High expression levels of claudin-8 and -12 on mRNA, and LSR on both mRNA and protein level were detected. To clarify protein location, binding experiments with the c-terminal end of Clostridium perfringens enterotoxin (cCPE) and high-resolution microscopy (stimulated emission and depletion, STED) were conducted. cCPE experiments revealed claudin surface expression was not high enough for binding, whereas STED-imaging of claudin-8 and overexpression of claudin-4 in T cells showed that translocation of the protein to the membrane was possible. Also, LSR was detected in the membranes of neutrophils and T cells. So far, subsequent co-immunoprecipitation (Co-IP) assays, however, could not definitively clarify whether immune cell-derived LSR would interact with the tTJ of epithelial monolayers. This indicates that some other not yet identified proteins might regulate the paracellular pas-sage of immune cells through the tTJs. LSR might have different, regulatory functions that could go beyond the already revealed functions in cancer or pathogen interaction. Further-more, it was shown that immune cells express a wide variety of TJ proteins on mRNA and protein level. The expression levels varied according to the state of activation suggesting more non-canonical functions of claudins than so far identified.
