Functional assays for the diagnosis of primary defects in lymphocyte cytotoxicity
Author: Chiang, Samuel C C
Date: 2015-11-20
Location: Föreläsningssal 9Q (Månen), Alfred Nobels Allé 8, Karolinska University Hospital, Huddinge.
Time: 09.30
Department: Inst för medicin, Huddinge / Dept of Medicine, Huddinge
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Thesis (2.652Mb)
Abstract
Cytotoxic lymphocytes encompass natural killer (NK) cells and cytotoxic T lymphocytes (CTL). These cells detect and kill virus-infected as well as malignant cells. Primary defects in lymphocyte cytotoxicity are associated with development of a hyperinflammatory syndrome termed hemophagocytic lymphohistiocytosis (HLH). This thesis investigates cytotoxic lymphocyte function in HLH cases as well as the creation of new assays to improve diagnostics.
An NK cell degranulation assay has been developed to quantify NK cell responses as an alternative to the radioactive chromium release assay. CD107a is a transmembrane protein that in unstimulated cells is contained within the inner membrane of perforin-containing cytotoxic granules but is exposed on the cell surface upon cytotoxic granule exocytosis. In a pan-European effort, we established and validated a consensus protocol for the diagnosis of primary HLH patients with defective degranulation (Paper II). NK cell degranulation below 5% predicted a primary defect in exocytosis leading to defective lymphocyte cytotoxicity with 96% sensitivity and 88% specificity. We also provided further optimized protocols for NK cell phenotyping and degranulation (Paper I).
Highlighting the importance of reliable functional assays in primary immunodeficiency discovery, we described novel non-coding aberrations in UNC13D as a cause of HLH and defective degranulation (Paper III, IV). Point mutations were found in a highly conserved intronic region, while a 253kb inversion was identified as the most frequent cause of HLH in Swedish infants.
ORAI1 and STIM1 mediate store-operated Ca2+ entry in T cells, which is required for induction of cytokine expression. However, the role of store-operated Ca2+ entry was not clear. In Paper V, we studied NK cell cytotoxicity using ORAI1 and STIM1-deficient patients, confirming abrogated Ca2+ entry upon target cell stimulation. Importantly, ORAI1 and STIM1-deficient NK cells did not degranulate nor produced proinflammatory cytokines, demonstrating that storeoperated Ca2+ entry is required for overall cytotoxic lymphocyte effector functions.
Primary HLH diagnostics have relied on NK cell functional assays but little had been done to evaluate T cell responses in these patients. In Paper VI, we described how the surface marker CD57 can be used to identify bone fide cytotoxic T lymphocytes, readily allowing identification of a T cell subset that expresses intracellular perforin and can efficiently kill target cells upon TCR engagement. The CD8+CD57+ T cell subset, similar to CD56dim NK cells, was found to display defective degranulation in FHL types 3-5, suggesting CTL and NK cell exocytosis has similar molecular requirements. In paper VII, we compared the established K562 cell-induced NK cell degranulation assay (Paper II) against the newly developed T cell degranulation assay (Paper VI) prospectively evaluating all primary immunodeficiency patients sent to our laboratory during a 3 year period. The T cell assay excelled with respect to sensitivity and specificity (97% and 95%, respectively) for predicting a primary defect in degranulation. Combining NK cell and T cell assays further increased assay specificity.
This thesis demonstrates the power of functional assays in evaluating cytotoxic lymphocyte activity and how this expands our understanding of their biological role and the primary HLH syndrome. These simple, rapid and accurate assays give us the power to quickly diagnose a lifethreatening disease and initiate treatment, thus saving lives.
An NK cell degranulation assay has been developed to quantify NK cell responses as an alternative to the radioactive chromium release assay. CD107a is a transmembrane protein that in unstimulated cells is contained within the inner membrane of perforin-containing cytotoxic granules but is exposed on the cell surface upon cytotoxic granule exocytosis. In a pan-European effort, we established and validated a consensus protocol for the diagnosis of primary HLH patients with defective degranulation (Paper II). NK cell degranulation below 5% predicted a primary defect in exocytosis leading to defective lymphocyte cytotoxicity with 96% sensitivity and 88% specificity. We also provided further optimized protocols for NK cell phenotyping and degranulation (Paper I).
Highlighting the importance of reliable functional assays in primary immunodeficiency discovery, we described novel non-coding aberrations in UNC13D as a cause of HLH and defective degranulation (Paper III, IV). Point mutations were found in a highly conserved intronic region, while a 253kb inversion was identified as the most frequent cause of HLH in Swedish infants.
ORAI1 and STIM1 mediate store-operated Ca2+ entry in T cells, which is required for induction of cytokine expression. However, the role of store-operated Ca2+ entry was not clear. In Paper V, we studied NK cell cytotoxicity using ORAI1 and STIM1-deficient patients, confirming abrogated Ca2+ entry upon target cell stimulation. Importantly, ORAI1 and STIM1-deficient NK cells did not degranulate nor produced proinflammatory cytokines, demonstrating that storeoperated Ca2+ entry is required for overall cytotoxic lymphocyte effector functions.
Primary HLH diagnostics have relied on NK cell functional assays but little had been done to evaluate T cell responses in these patients. In Paper VI, we described how the surface marker CD57 can be used to identify bone fide cytotoxic T lymphocytes, readily allowing identification of a T cell subset that expresses intracellular perforin and can efficiently kill target cells upon TCR engagement. The CD8+CD57+ T cell subset, similar to CD56dim NK cells, was found to display defective degranulation in FHL types 3-5, suggesting CTL and NK cell exocytosis has similar molecular requirements. In paper VII, we compared the established K562 cell-induced NK cell degranulation assay (Paper II) against the newly developed T cell degranulation assay (Paper VI) prospectively evaluating all primary immunodeficiency patients sent to our laboratory during a 3 year period. The T cell assay excelled with respect to sensitivity and specificity (97% and 95%, respectively) for predicting a primary defect in degranulation. Combining NK cell and T cell assays further increased assay specificity.
This thesis demonstrates the power of functional assays in evaluating cytotoxic lymphocyte activity and how this expands our understanding of their biological role and the primary HLH syndrome. These simple, rapid and accurate assays give us the power to quickly diagnose a lifethreatening disease and initiate treatment, thus saving lives.
List of papers:
I. Measurement of NK cell phenotype and activity in humans. Chiang SCC and Bryceson YT. [In] Manual of Molecular and Clinical Laboratory Immunology, 8th ed. Detrick B, Hamilton RG, Schmitz J (ed), ASM Press, Washington, DC. [Accepted]
II. A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes. Bryceson YT, Pende D, Maul-Pavicic A, Gilmour KC, Ufheil H, Vraetz T, Chiang SCC, Marcenaro S, Meazza R, Bondzio I, Walshe D, Janka G, Lehmberg K, Beutel K, zur Stadt U, Binder N, Arico M, Moretta L, Henter JI, and Ehl S. Blood (2012). 119(12):2754-2763.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D. Meeths M, Chiang SCC, Wood SM, Entesarian M, Schlums H, Bang B, Nordenskjöld E, Björklund C, Jakovljevic G, Jazbec J, Hasle H, Holmqvist BM, Rajic L, Pfeifer S, Rosthøj S, Sabel M, Salmi TT, Stokland T, Winiarski J, Ljunggren HG, Fadeel B, Nordenskjöld M, Henter J-I, and Bryceson YT. Blood (2011). 118(22):5783-5793.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Novel deep intronic and missense UNC13D mutations in familial haemophagocytic lymphohistiocytosis type 3. Entesarian M, Chiang SCC, Schlums H, Meeths M, Chan MY, Mya SN, Soh SY, Nordenskjöld M, Henter J-I, and Bryceson YT. British Journal of Haematology (2013). 162(3):415-418.
Fulltext (DOI)
Pubmed
View record in Web of Science®
V. ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis. Maul-Pavicic A, Chiang SCC, Rensing-Ehl A, Jessen B, Fauriat C, Wood SM, Sjöqvist S, Hufnagel M, Schulze I, Bass T, Schamel WW, Fuchs S, Pircher H, McCarl CA, Mikoshiba K, Schwarz K, Feske S, Bryceson YT, and Ehl S. Proceedings of the National Academy of Science U S A (2011). 108(8):3324-3329.
Fulltext (DOI)
Pubmed
View record in Web of Science®
VI. Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production. Chiang SCC, Theorell J, Entesarian M, Meeths M, Mastafa M, Al-Herz W, Frisk P, Gilmour KC, Ifversen M, Langenskiöld C, Machaczka M, Naqvi A, Payne J, Perez-Martinez A, Sabel M, Unal E, Unal S, Winiarski J, Nordenskjöld M, Ljunggren HG, Henter J-I, and Bryceson YT. Blood (2013). 121(8):1345-1356.
Fulltext (DOI)
Pubmed
View record in Web of Science®
VII. Efficacy of the T cell degranulation assay for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis. Chiang SCC, Tesi B, Schulms H, Meeths M, Nejati-Zendegani J, Mördrup K, Horne AC, Ljunggren H-G, Nordenskjöld M, Henter J-I, Bryceson YT. [Manuscript]
I. Measurement of NK cell phenotype and activity in humans. Chiang SCC and Bryceson YT. [In] Manual of Molecular and Clinical Laboratory Immunology, 8th ed. Detrick B, Hamilton RG, Schmitz J (ed), ASM Press, Washington, DC. [Accepted]
II. A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes. Bryceson YT, Pende D, Maul-Pavicic A, Gilmour KC, Ufheil H, Vraetz T, Chiang SCC, Marcenaro S, Meazza R, Bondzio I, Walshe D, Janka G, Lehmberg K, Beutel K, zur Stadt U, Binder N, Arico M, Moretta L, Henter JI, and Ehl S. Blood (2012). 119(12):2754-2763.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D. Meeths M, Chiang SCC, Wood SM, Entesarian M, Schlums H, Bang B, Nordenskjöld E, Björklund C, Jakovljevic G, Jazbec J, Hasle H, Holmqvist BM, Rajic L, Pfeifer S, Rosthøj S, Sabel M, Salmi TT, Stokland T, Winiarski J, Ljunggren HG, Fadeel B, Nordenskjöld M, Henter J-I, and Bryceson YT. Blood (2011). 118(22):5783-5793.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Novel deep intronic and missense UNC13D mutations in familial haemophagocytic lymphohistiocytosis type 3. Entesarian M, Chiang SCC, Schlums H, Meeths M, Chan MY, Mya SN, Soh SY, Nordenskjöld M, Henter J-I, and Bryceson YT. British Journal of Haematology (2013). 162(3):415-418.
Fulltext (DOI)
Pubmed
View record in Web of Science®
V. ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis. Maul-Pavicic A, Chiang SCC, Rensing-Ehl A, Jessen B, Fauriat C, Wood SM, Sjöqvist S, Hufnagel M, Schulze I, Bass T, Schamel WW, Fuchs S, Pircher H, McCarl CA, Mikoshiba K, Schwarz K, Feske S, Bryceson YT, and Ehl S. Proceedings of the National Academy of Science U S A (2011). 108(8):3324-3329.
Fulltext (DOI)
Pubmed
View record in Web of Science®
VI. Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production. Chiang SCC, Theorell J, Entesarian M, Meeths M, Mastafa M, Al-Herz W, Frisk P, Gilmour KC, Ifversen M, Langenskiöld C, Machaczka M, Naqvi A, Payne J, Perez-Martinez A, Sabel M, Unal E, Unal S, Winiarski J, Nordenskjöld M, Ljunggren HG, Henter J-I, and Bryceson YT. Blood (2013). 121(8):1345-1356.
Fulltext (DOI)
Pubmed
View record in Web of Science®
VII. Efficacy of the T cell degranulation assay for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis. Chiang SCC, Tesi B, Schulms H, Meeths M, Nejati-Zendegani J, Mördrup K, Horne AC, Ljunggren H-G, Nordenskjöld M, Henter J-I, Bryceson YT. [Manuscript]
Institution: Karolinska Institutet
Supervisor: Bryceson, Yenan T
Issue date: 2015-11-02
Rights:
Publication year: 2015
ISBN: 978-91-7676-123-6
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