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Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

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Abstract

Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein, we aimed to determine how new perforin transits to the synapse if not via lytic granules. We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response.

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Acknowledgements

The authors would like to thank Dr. Pinaki Banerjee and Dr. Emily Mace (Center for Human Immunobiology, Texas Children’s Hospital, Houston, TX) for providing technical advice.

Funding for these studies was provided by the National Institutes of Health (NIH) T32 grant AI053831 (KEL) and R01 grant AI067946 (JSO). The funding institution had no role in collection, analysis, or interpretation of data.

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Authors and Affiliations

  1. Center for Human Immunobiology, Texas Children’s Hospital & Baylor College of Medicine, Houston, TX, USA

    Kelsey Lesteberg, Jordan Orange & George Makedonas

  2. Graduate Program in Immunology, Baylor College of Medicine, Houston, TX, USA

    Kelsey Lesteberg, Jordan Orange & George Makedonas

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  1. Kelsey Lesteberg
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  2. Jordan Orange
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All authors contributed to the research design. KEL preformed experiments and analyzed data, KEL and GM wrote and edited the manuscript, and JSO edited the manuscript.

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Correspondence to George Makedonas.

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Normal human peripheral blood mononuclear cells (PBMCs) were isolated from 1) source leukocyte preparations received from Gulf Coast Regional Blood Center, Houston, TX or 2) blood specimens collected from healthy adult donors after informed consent was obtained in accordance with protocol #H33095, reviewed and approved by the Internal Review Board of Baylor College of Medicine and Texas Children’s Hospital. All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments. Informed consent was obtained from all individual participants included in the study.

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The authors declare that they have no conflict of interests.

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Lesteberg, K., Orange, J. & Makedonas, G. Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin. Immunol Res 65, 1031–1045 (2017). https://doi.org/10.1007/s12026-017-8945-8

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