ALFA-PRF: a novel approach to detect murine perforin release from CTLs into the immune synapse
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2022-12-22
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Frontiers in Immunology
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FRONTIERS MEDIA SA
Melbourne Authors
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Sir Peter MacCallum Department of Oncology
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Journal Article
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Rudd-Schmidt, J. A., Laine, R. F., Noori, T., Brennan, A. J. & Voskoboinik, I. (2022). ALFA-PRF: a novel approach to detect murine perforin release from CTLs into the immune synapse. Frontiers in Immunology, 13, https://doi.org/10.3389/fimmu.2022.931820.
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Abstract
When killing through the granule exocytosis pathway, cytotoxic lymphocytes release key effector molecules into the immune synapse, perforin and granzymes, to initiate target cell killing. The pore-forming perforin is essential for the function of cytotoxic lymphocytes, as its pores disrupt the target cell membrane and allow diffusion of pro-apoptotic serine proteases, granzyme, into the target cell, where they initiate various cell death cascades. Unlike human perforin, the detection of its murine counterpart in a live cell system has been problematic due its relatively low expression level and the lack of sensitive antibodies. The lack of a suitable methodology to visualise murine perforin secretion into the synapse hinders the study of the cytotoxic lymphocyte secretory machinery in murine models of human disease. Here, we describe a novel recombinant technology, whereby a short ALFA-tag sequence has been fused with the amino-terminus of a mature murine perforin, and this allowed its detection by the highly specific FluoTag®-X2 anti-ALFA nanobodies using both Total Internal Reflection Fluorescence (TIRF) microscopy of an artificial synapse, and confocal microscopy of the physiological immune synapse with a target cell. This methodology can have broad application in the field of cytotoxic lymphocyte biology and for the many models of human disease.