Visualising Cytotoxic Lymphocytes to Understand Human Disease

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Visualising Cytotoxic Lymphocytes to Understand Human Disease

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Author(s): Jesse A. Rudd-Schmidt ¹ ^, , Adrian W. Hodel ¹ , Daniela Castiblanco ¹ , Tahereh Noori ¹ , Joseph A. Trapani ² ^, ³ , Ilia Voskoboinik ¹ ^, ²

Publication date (Electronic): 21 January 2025

Conference name: 13th Asia Pacific Microscopy Congress 2025 (APMC13)

Conference date: 2-7 Febuary 2025

Keywords: Pathobiology, Cytotoxic Lymphocytes, Confocal Microscopy, Immunology, Disease

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Abstract

Cytotoxic T Lymphocytes (CTL) form a critical part of the immune system. They predominantly kill their infected or transformed targets through the granule exocytosis pathway. In this process, CTLs release key effector molecules, perforin (PRF) and granzymes, into the immune synapse to initiate target cell death [ 1]. The pore-forming PRF is essential for the function of CTLs: 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 [ 2]. Using a variety of imaging techniques, our laboratory has deciphered many aspects of the immune synapse, both in health and disease.

In a recent study, we resolved one of the most enigmatic questions in the field of cytotoxic lymphocyte biology: how host lymphocytes are protected against their own toxic cargo [ 3]. Using Atomic Force Microscopy (AFM) we demonstrated that PRF binding and pore formation were abrogated by high lipid packing (which is observed in the plasma membrane of CTLs within the immune synapse). Validating these findings in whole cells through live-cell imaging with the membrane-phase sensitive probe Laurdan, we confirmed that primary CTLs with lower lipid packing showed increased sensitivity to PRF, which we then visualized using a fluorescent fusion protein (GFP-PRF). Additionally, AFM identified negatively charged lipids, most prominently phosphatidylserine (PS), as inactivators of perforin. Our live cell confocal microscopy further confirmed the externalisation of PS on the pre-synaptic membrane of CTLs. Overall we discovered that the condensation of high membrane order (lipid rafts) at the immune synapse, together with PS externalisation, provides two impenetrable layers of protection against PRF, preventing “self-inflicted” damage to CTLs ( Figure 1).

Figure 1

(Reproduced from [3]): Schematic showing CTL (left) protected from the PRF (blue) it releases by condensed, high membrane order lipid rafts (red) and exposed PS (green). Granzymes are shown as black dots and can be seen only entering the target cell (right).

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Publication date (Electronic): 21 January 2025

Electronic Location Identifier: e325

Affiliations

[1 ]Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

[2 ]Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia

[3 ]Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

Author notes

*Corresponding author: Jesse.Rudd-Schmidt@ 123456petermac.org

Author information

Jesse A. Rudd-Schmidt https://orcid.org/0000-0002-3033-2642

Adrian W. Hodel https://orcid.org/0000-0003-0282-1200

Daniela Castiblanco https://orcid.org/0000-0003-1254-4200

Tahereh Noori https://orcid.org/0000-0002-8746-5841

Joseph A. Trapani https://orcid.org/0000-0003-0983-1532

Ilia Voskoboinik https://orcid.org/0000-0002-6410-009X

Article

DOI: 10.14293/APMC13-2025-0325

SO-VID: 5903b05d-20ec-4500-abb2-793e78aa0a37

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Published under Creative Commons Attribution 4.0 International ( CC BY 4.0). Users are allowed to share (copy and redistribute the material in any medium or format) and adapt (remix, transform, and build upon the material for any purpose, even commercially), as long as the authors and the publisher are explicitly identified and properly acknowledged as the original source.

Conference name: 13th Asia Pacific Microscopy Congress 2025

Conference acronym: APMC13

Conference number: 13

Conference location: Brisbane, Australia

Conference date: 2-7 Febuary 2025

History

References

Barry and Bleackley, Nature Reviews Immunology 2 (2002) p. 401.
Lopez et al., The Journal of Immunology 191 (2013) p.2328.
Rudd-Schmidt et al., Nature Communications 10, (2019) 5396
Noori et al., Blood 141 (19) (2023) 2330-2342
Rudd-Schmidt et al., Front. Immunol. 13 (2022) doi: 10.3389/fimmu.2022.931820
Castiblanco et al., Blood 139 (12) (2022) 1833–1849.

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[1] Barry and Bleackley, Nature Reviews Immunology 2 (2002) p. 401.

[2] Lopez et al., The Journal of Immunology 191 (2013) p.2328.

[3] Rudd-Schmidt et al., Nature Communications 10, (2019) 5396

[4] Noori et al., Blood 141 (19) (2023) 2330-2342

[5] Rudd-Schmidt et al., Front. Immunol. 13 (2022) doi: 10.3389/fimmu.2022.931820

[6] Castiblanco et al., Blood 139 (12) (2022) 1833–1849.

Visualising Cytotoxic Lymphocytes to Understand Human Disease

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