T cell exocytosis testing provides superior sensitivity for the diagnosis of familial HLH

Abstract

The rapid diagnosis of primary immunodeficiencies (PID) affecting cytotoxic lymphocyte granule exocytosis is commonly performed by evaluating NK cell surface CD107a expression after K562 challenge. NK cell exocytosis can also be induced by stimulating NK cells with P815 and anti-CD16 antibody. We have previously described how these NK tests can be adapted to study cytotoxic T cell CD107a exocytosis by gating CD3+CD8 +CD57+ cells after TCR engagement with P815 and anti-CD3 antibody. Here we present data comparing 92 patients with variants in genes affecting exocytosis function against 58 other PID patients diagnosed over 10 years from one center. NK and cytotoxic T cell exocytosis was tested on all patient samples. The anti-CD3T cell test returns superior sensitivity and specificity of 97.8% and 94.7% while the K562 NK test a sensitivity of 86.7% and specificity of 96.1%. The anti-CD 16 NK test was found comparable to the T cell test with values of 97.8% and 93.4%. The data was additionally juxtaposed against 63 patients with secondary HLH, 198 healthy adult donors, and 84 transport controls, all giving similar results of high accuracy. We further stratified the data by time taken between venipuncture and arrival at the testing laboratory and found that control samples up to 36 hours old showed comparable function to < 24 hours old samples. This is important as transporting samples from remote primary care centers often cannot be accomplished within 24 hours.While all three exocytosis tests return superior diagnostic sensitivity, we find the anti-CD3 antibody induced exocytosis of T cells and the anti-CD16 antibody induced exocytosis of NK cells more accurate than the current standard K562 cell stimulation for the diagnosis of PIDs affecting cytotoxic lymphocyte exocytosis. Evaluating T cells alongside NK cells acts as a confirmatory test and buffers against technical failures and the lack of NK cells which can be seen in lymphopenic patients. These assays with improved sensitivity may help us better understand PIDs that partially impair exocytosis.