Page 68 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

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Chapter 4
T cells, CD8+ T cells (CD3+CD7+CD8+), γδ T cells (CD3+CD7+TCRγδdim/+), B cells (CD20+), CD3-CD7+ cells innate lymphoid cells (ILC; CD3-CD7+) and myeloid cell lineages (CD3-CD7-CD11c+) (Figure 4.1 and Figure 4.2; Supplementary figure 4.2). The data of each major immune cell lineage was further explored hierarchically by HSNE, as is exemplified in Figure 4.3 for the memory CD4+ T cell compartment, rendering unique immune cell clusters for each major immune cell lineage. Eventually, exploration of all major immune cell lineages identified a total of 185 immune cell clusters, delineated using unsupervised GMS clustering. For merging of highly similar immune cell clusters Spearman’s rank correlation was performed, as described in more detail in Materials and methods. 3 clusters containing less than 100 cells were excluded from further analysis.
NT1 with recent disease onset vs HLA-DQB1*06:02-matched controls
Subsequently, the composition of the immune system in the peripheral blood of NT1 patients shortly after disease onset was compared to that of HLA- DQB1*06:02-matched healthy controls. Percentages of CD45+ cells did not differ significantly between NT1 patients with recent disease onset and HLA- DQB1*06:02-matched controls for all major immune cell lineages (Figure 4.2). More detailed analysis of the immune cell clusters revealed that in the memory CD4+ T cell compartment differences between both participant groups were observed, namely higher frequencies of CD45RO+CD4+ T cell populations expressing CD27+, CD127+ and/or CD161+ in NT1 patients with recent onset (Figure 4.4 and Supplementary table 4.2). Similarly, also in the CD8+ T cell compartment an immune cell cluster expressing CD27 and CD127 was found more frequently in NT1 patients with recent disease onset (Supplementary figure 4.2).
Regulatory CD4+ T cells, as characterized by the combination of CD4 and CD25 in the absence of CD127, were found more frequently in NT1 patients compared to healthy controls. No differences in effector memory (CD45RO+CCR7-) or central memory (CD45RO+CCR7+) CD4+ T cells were found. CD27+CD8+ T cells were more frequent in NT1 patients compared to healthy controls, albeit not significant, while CD27-CD8+ T cells were significantly less frequent in the NT1 patient population. In contrast to a recent study, we did not observe an increased frequency of CD3-CD56+ natural killer cells in NT1 patients (Figure 4.5).