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A Rapid Ex Vivo Clinical Diagnostic Assay for Fas Receptor-Induced T Lymphocyte Apoptosis

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Abstract

Deleterious mutations in genes involved in the Fas apoptosis pathway lead to Autoimmune Lymphoproliferative Syndrome (ALPS). Demonstration of an apoptosis defect is critical for the diagnosis and study of ALPS. The traditional in vitro apoptosis assay, however, requires a week of experimental procedures. Here, we show that defects in Fas-induced apoptosis in PBMCs can be evaluated directly ex vivo using multicolor flow cytometry to analyze the apoptosis of effector memory T cells, a Fas-sensitive subset of PBMCs. This method allowed us to sensitively quantify defective apoptosis in ALPS patients within a few hours. Some ALPS patients (ALPS-sFAS) without germline mutations have somatic mutations in Fas specifically in double-negative αβ T cells (DNTs), an unusual lymphocyte population that is characteristically expanded in ALPS. Since DNTs have been notoriously difficult to culture, defective apoptosis has not been previously demonstrated for ALPS-sFAS patients. Using our novel ex vivo apoptosis assay, we measured Fas-induced apoptosis of DNTs for the first time and found that ALPS-sFAS patients had significant apoptosis defects in these cells compared to healthy controls. Hence, this rapid apoptosis assay can expedite the diagnosis of new ALPS patients, including those with somatic mutations, and facilitate clinical and molecular investigation of these diseases.

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Acknowledgements

We thank the patients and healthy blood donors. We also thank Josh Milner for use of his LSRFortessa. We thank Julie Niemela for assistance in mutation nomenclature. We also thank Claire Liu for assay name recommendations, and we thank Helen Su and Chryssa Kanellopoulou for critical reading of the manuscript. This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA

    Bernice Lo & Michael J. Lenardo

  2. Immunoregulation Section, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, 20892, USA

    Madhu Ramaswamy & Richard M. Siegel

  3. ALPS Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA

    Joie Davis, Susan Price & V. Koneti Rao

  4. Building 10, Room 11N311, 10 Center Drive, MSC 1892, Bethesda, MD, 20892-1892, USA

    Michael J. Lenardo

Authors
  1. Bernice Lo
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  2. Madhu Ramaswamy
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  3. Joie Davis
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  4. Susan Price
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  5. V. Koneti Rao
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  6. Richard M. Siegel
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  7. Michael J. Lenardo
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Correspondence to Michael J. Lenardo.

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Lo, B., Ramaswamy, M., Davis, J. et al. A Rapid Ex Vivo Clinical Diagnostic Assay for Fas Receptor-Induced T Lymphocyte Apoptosis. J Clin Immunol 33, 479–488 (2013). https://doi.org/10.1007/s10875-012-9811-z

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  • DOI: https://doi.org/10.1007/s10875-012-9811-z

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