Tissue-resident lymphocytes play a key role in immune surveillance, but it remains unclear how these inherently stable cell populations respond to chronic inflammation. In the setting of celiac... Show moreTissue-resident lymphocytes play a key role in immune surveillance, but it remains unclear how these inherently stable cell populations respond to chronic inflammation. In the setting of celiac disease (CeD), where exposure to dietary antigen can be controlled, gluten-induced inflammation triggered a profound depletion of naturally occurring Vg4+ /Vd1+ intraepithelial lymphocytes (IELs) with innate cytolytic properties and specificity for the butyrophilin-like (BTNL) molecules BTNL3/ BTNL8. Creation of a new niche with reduced expression of BTNL8 and loss of Vg4+ /Vd1+ IELs was accompanied by the expansion of gluten-sensitive, interferon-g-producing Vd1+ IELs bearing T cell receptors (TCRs) with a shared non-germline-encoded motif that failed to recognize BTNL3/BTNL8. Exclusion of dietary gluten restored BTNL8 expression but was insufficient to reconstitute the physiological Vg4+ /Vd1+ subset among TCRgd+ IELs. Collectively, these data show that chronic inflammation permanently reconfigures the tissue-resident TCRgd+ IEL compartment in CeD. Show less
Fergusson, J.R.; Morgan, M.D.; Bruchard, M.; Huitema, L.; Heesters, B.A.; Unen, V. van; ... ; Spits, H. 2019
It is increasingly recognized that in order to gain further insights into human disorders and develop new therapeutic strategies and diagnostic tools, it is critical to have a comprehensive... Show moreIt is increasingly recognized that in order to gain further insights into human disorders and develop new therapeutic strategies and diagnostic tools, it is critical to have a comprehensive overview of immune cell subsets resident in tissues under physiological and pathological conditions. Flow cytometry has been the golden standard for analyzing immune cell subsets, and with a typical experiment, dozens of immune subsets can be discriminated. Due to the lack of spectrally-resolvable fluorochromes, mass cytometry utilizing metal-conjugated antibodies has shown to be a powerful tool for dissecting the immune landscape even further. Inflammatory intestinal diseases can only be understood by studying specialized cell types within the tissue niche itself. In this thesis, we applied mass cytometry and data-driven, automated analysis approaches to investigate the complex compositions of heterogeneous cell subsets, such as those encountered in intestinal biopsies. These types of data have the potential to greatly improve our understanding of human disease. In concert with clinical data, mass cytometry could enable a finer classification of patients and might aid in the development of improved diagnostics, prognostics and personalized therapeutic regimens. Show less
Laban, S.; Suwandi, J.S.; Unen, V. van; Pool, J.; Wesselius, J.; Hollt, T.; ... ; Roep, B.O. 2018
Auto-reactive CD8 T-cells play an important role in the destruction of pancreatic β-cells resulting in type 1 diabetes (T1D). However, the phenotype of these auto-reactive cytolytic CD8 T-cells... Show moreAuto-reactive CD8 T-cells play an important role in the destruction of pancreatic β-cells resulting in type 1 diabetes (T1D). However, the phenotype of these auto-reactive cytolytic CD8 T-cells has not yet been extensively described. We used high-dimensional mass cytometry to phenotype autoantigen- (pre-proinsulin), neoantigen- (insulin-DRIP) and virus- (cytomegalovirus) reactive CD8 T-cells in peripheral blood mononuclear cells (PBMCs) of T1D patients. A panel of 33 monoclonal antibodies was designed to further characterise these cells at the single-cell level. HLA-A2 class I tetramers were used for the detection of antigen-specific CD8 T-cells. Using a novel Hierarchical Stochastic Neighbor Embedding (HSNE) tool (implemented in Cytosplore), we identified 42 clusters within the CD8 T-cell compartment of three T1D patients and revealed profound heterogeneity between individuals, as each patient displayed a distinct cluster distribution. Single-cell analysis of pre-proinsulin, insulin-DRIP and cytomegalovirus-specific CD8 T-cells showed that the detected specificities were heterogeneous between and within patients. These findings emphasize the challenge to define the obscure nature of auto-reactive CD8 T-cells. Show less
Li, N.; Unen, V. van; Hollt, T.; Thompson, A.; Bergen, J. van; Pezzotti, N.; ... ; Koning, F. 2018
Innate lymphoid cells (ILCs) are abundant in mucosal tissues and involved in tissue homeostasis and barrier function. Although several ILC subsets have been identified, it is unknown if additional... Show moreInnate lymphoid cells (ILCs) are abundant in mucosal tissues and involved in tissue homeostasis and barrier function. Although several ILC subsets have been identified, it is unknown if additional heterogeneity exists, and their differentiation pathways remain largely unclear. We applied mass cytometry to analyze ILCs in the human fetal intestine and distinguished 34 distinct clusters through a t-SNE-based analysis. A lineage (Lin)-CD7+CD127-CD45RO+CD56+ population clustered between the CD127+ ILC and natural killer (NK) cell subsets, and expressed diverse levels of Eomes, T-bet, GATA3, and RORγt. By visualizing the dynamics of the t-SNE computation, we identified smooth phenotypic transitions from cells within the Lin-CD7+CD127-CD45RO+CD56+ cluster to both the NK cells and CD127+ ILCs, revealing potential differentiation trajectories. In functional differentiation assays, the Lin-CD7+CD127-CD45RO+CD56+CD8a- cells could develop into CD45RA+NK cells and CD127+RORγt+ ILC3-like cells. Thus, we identified a previously unknown intermediate innate subset that can differentiate into ILC3 and NK cells. Show less
Buckle, T.; Wal, S. van der; Malderen, S.J.M. van; Muller, L.; Kuil, J.; Unen, V. van; ... ; Leeuwen, F.W.B. van 2017
