Page 58 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

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Chapter 3
One of the limitations in our study is that we have not been able to distinguish between NT1 patients and healthy controls that have actually encountered the H1N1 influenza virus and those who have not. We could therefore not be sure whether different exposure to the virus explains the lack of differences in H1N1- HA peptide-specific T cell responses between NT1 patients and healthy controls. However, T cell mediated cross-protective immunity generated by previous H1N1 infections was found to be common in the population (Miller et al., 2010). Another limitation in our study is that we are performing our experiments in blood, while the actual destruction of Hcrt-producing neurons is taking place at the other side of the blood-brain barrier. It was recently described that T cell composition in cerebrospinal fluid differed between NT1 patients and healthy controls (Moresco et al., 2018). Repeating our experiments in cerebrospinal fluid would therefore be a better environment to test for cross-reactive T cells. Additionally, detecting proliferation of a small immune subset that could have driven the destruction of the approximately 80,000 Hcrt-producing neurons (Thannickal et al., 2000) in a vast number of non-proliferating immune cells is technically difficult. The procedure used for peptide-specific TCC generation was also used in previous studies for the isolation of HLA-DQ-restricted gluten-specific T cell clones from patients with celiac disease in which we have determined the affinity of such T cells for the cognate HLA-DQ-gluten complexes. We observed that in cell-free assays the affinity values for some of these HLA-DQ-gluten specific TCRs were comparable with affinity values observed for microbial or non-self TCR–pMHC-II interactions, while others exhibited affinity values in line with those of low-affinity autoreactive TCR– pMHC complexes (Petersen et al., 2014). In cellular assays similar substantial differences were observed (Broughton et al., 2012). Therefore, this indicates that our method allows the identification of both high- and low-affinity T cell clones. However, we cannot exclude the possibility that with our technique we missed the detection of very low frequency T cells. The T cell library method that was performed in a recent publication on T cell reactivity to Hcrt (Latorre et al., 2018) would be an interesting technique to screen for these low frequency clones. Other novel approaches, such as mass cytometry, to address this rare immune subset problem are emerging with techniques that can isolate disease- specific immune subsets with unprecedented detail (van Unen et al., 2017). Repeating our experiments with only those subsets that are specific for NT1, would significantly increase the odds of identifying rare cross-reactive immune cells in NT1 patients should they exist.