RE: Cell-Cell Communication Prize 2022 Title Trogocytosis – an enigmatic form of cell-cell communication in the immune system. Background Intercellular communication is an inherent property of all metazoans and critically important for many biological processes. In the immune system, most cells communicate via interactions between plasma membrane receptors, or via ligands secreted by one cell and recognised by a receptor on another cell. The interaction of the TCR on CD4+ T cells with peptide-loaded MHC II complexes on antigen presenting cells is a well-studied example of the former. Cytokines secreted from one cell and recognized by a receptor on a distant cell exert major regulatory functions, an example of the latter. Our studies investigate a third form of intercellular communication described decades ago and that is thought to play major roles but remains poorly understood, namely trogocytosis. This activity entails the transfer of plasma membrane from one cell to another1). It is thought to be involved in embryonic development2), pathogenesis3), and immunology4) but, despite being described since the 1980s, its mechanisms and functional implications remain poorly characterized4). Rationale We hypothesize that trogocytosis is a fundamentally important mechanism of intercellular communication in the multi-layered and complex network of the immune system. It allows immune cells to exchange membrane fractions and their embedded proteins. This in turn enables the cells to “borrow” the functional properties of proteins picked from other cells even if the recipient cells do not produce the proteins themselves. Our studies demonstrated for the first time trogocytosis between a critical type of innate immune cells, marginal zone (MZ) B cells, and a fundamental component of the adaptive immune system called dendritic cells (DCs). We showed that MZ B cells of mice and humans acquire from DCs membrane proteins that the B cells cannot produce on their own. We hypothesize that this phenomenon enables MZ B cells to enhance their production of polyreactive antibodies that protect early in life from multiple pathogens by recruiting “help” from T cells5). The anatomical location and molecular mechanisms that enable MZ B cells to trogocytose DC membrane and proteins remains unknown, leaving a major gap in our understanding of this novel form of cooperation between the innate and adaptive immune systems. Preliminary Results and Future Directions We have discovered and recently published in Science6) that MZ B cells, constitutively trogocytose plasma membrane from DCs. The MZ B cells thereby acquire membrane receptors, including peptide-loaded MHC II (pMHC II) complexes generated by DCs. Upon trogocytosis MZ B cells can present pMHC II complexes to T cells they do not generate themselves but acquire from DCs, allowing them to augment or regulate T cell activation in ways that are distinct from DCs. Importantly, this trogocytic protein exchange between MZ B cells and DCs is strictly dependent on a specific receptor-ligand interaction, consisting of complement receptor 2 and complement component 3 (C3). It relies on the formation of a newly-discovered complex comprised of two fundamental molecular components of the innate and adaptive immune systems, namely C3 and MHC II. The formed MHC II-C3 complexes deposited on the DC surface serve as a ligand in the trogocytic cell-cell communication and are recognised by the complement receptor 2 on MZ B cells enabling the trogocytic transfer of DC membrane. The trogocytic MZ B cells thus display pMHC II complexes generated by DCs but also acquire other DC-specific membrane proteins and/or ligands captured by the DCs enabling them to expand the range of functions they can play (summarising chart in Figure1). Our study so far has discovered and characterised three novel findings: (1) the constitutive binding of complement C3 to MHC II on the surface of DCs, (2) the interaction of MZ B cells and DCs in vitro and in vivo via the recognition of the MHC II-C3 complexes by MZ B cell complement receptor 2, and (3) the trogocytic transfer of DCs membrane, protein and antigen presentation functions to MZ B cells through such interaction6) (Figure1). Future studies will further characterise the functional properties acquired by MZ B cells through DC trogocytosis and identify in detail the mechanisms of this trogocytic cell-cell interaction. All specific aims are underpinned by stablished state-of-the-art technology, solid forerunning data and recent publications6, 7) that ensure feasibility. In particular, we aim to deeper understand the role of complement receptor 2 signalling, actin polymerisation in the plasma membrane of the involved cells as well as the role of extracellular vesicles. Furthermore, the visualisation of trogocytosis through (static, live, super-resolution, 3D) microscopy will be pivotal to further understand the process of trogocytosis. The chief investigator (Jose Villadangos) and their laboratories for this project at the University of Melbourne have the expertise and are uniquely equipped for this endeavour to ensure feasibility and the generation of significant new knowledge. Funding has already been secured (Australian Research Council Discovery Project DP220102288#) to expand already existing technologies and collaborations. Figure1 (Schriek et al. 2022 6) Significance of this study This study has already generated new knowledge and discovered hitherto unknown interactions between fundamental cellular and molecular components of the innate and adaptive immune system6). First, the newly discovered MHC II-C3 complex through the covalent binding complement C3 to MHC II at the DC cell surface is itself a highly significant finding because formation of MHC II-C3 complexes may be a novel mechanism for capturing and eliminating potentially harmful C3 constitutively activated by the so-called “Tick-Over” pathway. As expected, given the high degree of conservation of the MHC and complement throughout evolution, we observed MHC II-C3 complexes in both mice and humans6). Second, our discovery of C3-mediated DC trogocytosis and the acquisition of antigen presentation by MZ B cells6) has already been acknowledged for its novelty in the research of cell-cell communication and immunology. While the significance of trogocytosis is clear, its mechanisms remain obscure because it is difficult to measure it under controlled conditions. We will characterise the molecular mechanisms underpinning trogocytosis and our experimental systems in place are ideal to do so as we have established an easy-to-manipulate and tightly controlled setting in vitro to generate data and hypothesis that then can be validated in vivo. In addition, visualisation of trogocytosis and finding physical DC-MZ B cell interactions is novel and of immunological interest, as it underpins forms of cooperation between the two cell types that have not been described yet. Over all this project already has and will further generate fundamental biology that will be published in highly-cited scholarly articles, the primary performance output for biomedical scientists. It will also generate a panel of excellent tools: new genetically manipulated cell lines, mouse models, and techniques for high-end proteomics, all of which will serve as significant resources of knowledge transfer to the research community. This project will generate opportunities for future knowledge generation. Outcomes will form the basis of intellectual property development for new products by biotechnology companies. These future products will improve veterinary and human health services, leading to increased productivity. New knowledge generated by the project and the high-level training of students and researchers will increase the competitiveness of the strategic biotechnology sector. References 1) Joly E, Hudrisier D. What is trogocytosis and what is its purpose? Nat Immunol. 2003 Sep;4(9):815. doi: 10.1038/ni0903-815. PMID: 12942076. 2) Perry JS, Ravichandran KS. Embryonic Trogocytosis: Neighborly Nibbling during Development. Curr Biol. 2017 Jan 23;27(2):R68-R70. doi: 10.1016/j.cub.2016.11.043. PMID: 28118592. 3) Nakada-Tsukui K, Nozaki T. Trogocytosis in Unicellular Eukaryotes. Cells. 2021 Nov 1;10(11):2975. doi: 10.3390/cells10112975. PMID: 34831198; PMCID: PMC8616307. 4) Davis DM. Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol. 2007 Mar;7(3):238-43. doi: 10.1038/nri2020. Epub 2007 Feb 9. PMID: 17290299. 5) Attanavanich K, Kearney JF. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J Immunol. 2004 Jan 15;172(2):803-11. doi: 10.4049/jimmunol.172.2.803. PMID: 14707050. 6) Schriek P, Ching AC, Moily NS, Moffat J, Beattie L, Steiner TM, Hosking LM, Thurman JM, Holers VM, Ishido S, Lahoud MH, Caminschi I, Heath WR, Mintern JD, Villadangos JA. Marginal zone B cells acquire dendritic cell functions by trogocytosis. Science. 2022 Feb 11;375(6581):eabf7470. doi: 10.1126/science.abf7470. Epub 2022 Feb 11. PMID: 35143312. 7) Schriek P, Liu H, Ching AC, Huang P, Gupta N, Wilson KR, Tsai M, Yan Y, Macri CF, Dagley LF, Infusini G, Webb AI, McWilliam HEG, Ishido S, Mintern JD, Villadangos JA. Physiological substrates and ontogeny-specific expression of the ubiquitin ligases MARCH1 and MARCH8. Curr Res Immunol. 2021 Oct 15;2:218-228. doi: 10.1016/j.crimmu.2021.10.004. PMID: 35492398; PMCID: PMC9040089.