Functional assays for the diagnosis of primary defects in lymphocyte cytotoxicity

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.