Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting... Show moreIdiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting of a course of anti-thymocyte globulin (ATG) followed by long-term use of ciclosporin. However, the immune response that underlies the pathogenesis of AA remains poorly understood. In this study, we applied high-dimensional mass cytometry on bone marrow aspirates of AA patients pre-ATG, AA patients post-ATG and healthy donors to decipher which immune cells may be implicated in the pathogenesis of AA. We show that the bone marrow of AA patients features an immune cell composition distinct from healthy donors, with significant differences in the myeloid, B-cell, CD4+ and CD8+ T-cells lineages. Specifically, we discovered that AA pre-ATG is characterized by a disease-specific immune cell network with high frequencies of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells, CD45RA+CCR7+CD38+ CD8+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST strongly reduced the levels of CD16+ myeloid cells and showed a trend toward normalization of the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+EMRA CD8+ T-cells. Altogether, our study provides a unique overview of the immune landscape in bone marrow in AA at a single-cell level and proposes CCR6 as a potential new therapeutic target in AA. Show less
Unen, V. van; Ouboter, L.F.; Li, N.; Schreurs, M.; Abdelaal, T.; Kooy-Winkelaar, Y.; ... ; Koning, F. 2022
Chronic intestinal inflammation underlies inflammatory bowel disease (IBD). Previous studies indicated alterations in the cellular immune system; however, it has been challenging to interrogate the... Show moreChronic intestinal inflammation underlies inflammatory bowel disease (IBD). Previous studies indicated alterations in the cellular immune system; however, it has been challenging to interrogate the role of all immune cell subsets simultaneously. Therefore, we aimed to identify immune cell types associated with inflammation in IBD using high-dimensional mass cytometry. We analyzed 188 intestinal biopsies and paired blood samples of newly-diagnosed, treatment-naive patients (n=42) and controls (n=26) in two independent cohorts. We applied mass cytometry (36-antibody panel) to resolve single cells and analyzed the data with unbiased Hierarchical-SNE. In addition, imaging-mass cytometry (IMC) was performed to reveal the spatial distribution of the immune subsets in the tissue. We identified 44 distinct immune subsets. Correlation network analysis identified a network of inflammation-associated subsets, including HLA-DR(+)CD38(+) EM CD4(+) T cells, T regulatory-like cells, PD1(+) EM CD8(+) T cells, neutrophils, CD27(+) TCR gamma delta cells and NK cells. All disease-associated subsets were validated in a second cohort. This network was abundant in a subset of patients, independent of IBD subtype, severity or intestinal location. Putative disease-associated CD4(+) T cells were detectable in blood. Finally, imaging-mass cytometry revealed the spatial colocalization of neutrophils, memory CD4(+) T cells and myeloid cells in the inflamed intestine. Our study indicates that a cellular network of both innate and adaptive immune cells colocalizes in inflamed biopsies from a subset of patients. These results contribute to dissecting disease heterogeneity and may guide the development of targeted therapeutics in IBD. Show less
Celiac Disease (CeD) is a complex immune disorder involving villous atrophy in the small intestine that is triggered by gluten intake. Current CeD diagnosis is based on late-stage... Show moreCeliac Disease (CeD) is a complex immune disorder involving villous atrophy in the small intestine that is triggered by gluten intake. Current CeD diagnosis is based on late-stage pathophysiological parameters such as detection of specific antibodies in blood and histochemical detection of villus atrophy and lymphocyte infiltration in intestinal biopsies. To date, no early onset biomarkers are available that would help prevent widespread villous atrophy and severe symptoms and co-morbidities. To search for novel CeD biomarkers, we used single-cell RNA sequencing (scRNAseq) to investigate PBMC samples from 11 children before and after seroconversion for CeD and 10 control individuals matched for age, sex and HLA-genotype. We generated scRNAseq profiles of 9559 cells and identified the expected major cellular lineages. Cell proportions remained stable across the different timepoints and health conditions, but we observed differences in gene expression profiles in specific cell types when comparing patient samples before and after disease development and comparing patients with controls. Based on the time when transcripts were differentially expressed, we could classify the deregulated genes as biomarkers for active CeD or as potential pre-diagnostic markers. Pathway analysis showed that active CeD biomarkers display a transcriptional profile associated with antigen activation in CD4+ T cells, whereas NK cells express a subset of biomarker genes even before CeD diagnosis. Intersection of biomarker genes with CeD-associated genetic risk loci pinpointed genetic factors that might play a role in CeD onset. Investigation of potential cellular interaction pathways of PBMC cell subpopulations highlighted the importance of TNF pathways in CeD. Altogether, our results pinpoint genes and pathways that are altered prior to and during CeD onset, thereby identifying novel potential biomarkers for CeD diagnosis in blood. Show less
Dieckman, T.; Schreurs, M.; Mahfouz, A.; Kooy-Winkelaar, Y.; Neefjes-Borst, A.; Bouma, G.; Koning, F. 2022
BACKGROUND & AIMS: Refractory celiac disease type II (RCDII) is a rare indolent lymphoma in the small intestine characterized by a clonally expanded intraepithelial intracellular CD3 thorn... Show moreBACKGROUND & AIMS: Refractory celiac disease type II (RCDII) is a rare indolent lymphoma in the small intestine characterized by a clonally expanded intraepithelial intracellular CD3 thorn surfa- ceCD3-CD7 thorn CD56- aberrantcell population. However, RCDII pathogenesis is ill-defined. Here, we aimed at single-cell charac-terization of the innate and adaptive immune system in RCDII. METHODS: Paired small intestinal and blood samples from 12 RCDII patients and 6 healthy controls were assessed by single-cell mass cytometry with a 39-cell surface marker antibody panel, designed to capture heterogeneity of the innate and adaptive immune system. A second single-cell mass cytometry panel that included transcription factors and immune checkpoints was used for analysis of paired samples from 5 RCDII patients. Single-cell RNA sequencing analysis was performed on duodenal samples from 2 RCDII patients. Finally, we developed a 40-marker imaging mass cytometry antibody panel to evaluate cell-cell interactions in duodenal biopsy specimens of RCDII patients. RESULTS: We provide evidence for intertumoral and intra-tumoral cell heterogeneity within the duodenal and peripheral aberrant cell population present in RCDII. Phenotypic discrep-ancy was observed between peripheral and duodenal aberrant cells. In addition, we observed that part of the aberrant cell population proliferated and observed co-localization of aber-rant cells with CD163 thorn antigen-presenting cells (APCs) in situ. In addition, we observed phenotypic discrepancy between pe-ripheral and duodenal aberrant cells. CONCLUSIONS: Novel high-dimensional single-cell technolo-gies show substantial intertumoral and intratumoral hetero-geneity in the aberrant cell population in RCDII. This may underlie variability in refractory disease status between pa-tients and responsiveness to therapy, pointing to the need for personalized therapy in RCDII based on patient-specific im-mune profiles. Show less
Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and... Show moreCeliac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of gluten-specific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease. Show less
Graaf, A. van der; Zorro, M.M.; Claringbould, A.; Vosa, U.; Aguirre-Gamboa, R.; Li, C.; ... ; BIOS Consortium 2021
Celiac disease (CeD) is a complex T cell-mediated enteropathy induced by gluten. Although genome-wide association studies have identified numerous genomic regions associated with CeD, it is... Show moreCeliac disease (CeD) is a complex T cell-mediated enteropathy induced by gluten. Although genome-wide association studies have identified numerous genomic regions associated with CeD, it is difficult to accurately pinpoint which genes in these loci are most likely to cause CeD. We used four different in silico approaches-Mendelian randomization inverse variance weighting, COLOC, LD overlap, and DEPICT-to integrate information gathered from a large transcriptomics dataset. This identified 118 prioritized genes across 50 CeD-associated regions. Co-expression and pathway analysis of these genes indicated an association with adaptive and innate cytokine signaling and T cell activation pathways. Fifty-one of these genes are targets of known drug compounds or likely druggable genes, suggesting that our methods can be used to pinpoint potential therapeutic targets. In addition, we detected 172 gene combinations that were affected by our CeD-prioritized genes in trans. Notably, 41 of these trans-mediated genes appear to be under control of one master regulator, TRAF-type zinc finger domain containing 1 (TRAFD1), and were found to be involved in interferon (IFN)gamma signaling and MHC I antigen processing/presentation. Finally, we performed in vitro experiments in a human monocytic cell line that validated the role of TRAFD1 as an immune regulator acting in trans. Our strategy confirmed the role of adaptive immunity in CeD and revealed a genetic link between CeD and IFN gamma signaling as well as with MHC I antigen processing, both major players of immune activation and CeD pathogenesis. Show less
Structural, biochemical and cellular analyses show that bacterial antigens can mimic gliadin epitopes involved in celiac disease being presented by HLA-DQ2.5 and recognized by T cells derived from... Show moreStructural, biochemical and cellular analyses show that bacterial antigens can mimic gliadin epitopes involved in celiac disease being presented by HLA-DQ2.5 and recognized by T cells derived from patients.The human leukocyte antigen (HLA) locus is strongly associated with T cell-mediated autoimmune disorders. HLA-DQ2.5-mediated celiac disease (CeD) is triggered by the ingestion of gluten, although the relative roles of genetic and environmental risk factors in CeD is unclear. Here we identify microbially derived mimics of gliadin epitopes and a parental bacterial protein that is naturally processed by antigen-presenting cells and activated gliadin reactive HLA-DQ2.5-restricted T cells derived from CeD patients. Crystal structures of T cell receptors in complex with HLA-DQ2.5 bound to two distinct bacterial peptides demonstrate that molecular mimicry underpins cross-reactivity toward the gliadin epitopes. Accordingly, gliadin reactive T cells involved in CeD pathogenesis cross-react with ubiquitous bacterial peptides, thereby suggesting microbial exposure as a potential environmental factor in CeD. Show less
Schinkelshoek, M.; Fronczek, R.; Heide, A. van der; Kooy-Winkelaar, Y.; Koning, F.; Lammers, G.J. 2017
OBJECTIVE: Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative... Show moreOBJECTIVE: Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown function: CD56-CD127-, CD56-CD127+, CD56+CD127- and CD56+CD127+. Here we aimed to gain insight into the potential function of these innate IELs in health and disease. DESIGN: We determined the phenotypes, relative abundance and differentiation potential of these innate IEL subsets in duodenal biopsies from controls and patients with CD or patients with refractory CD type II (RCDII). RESULTS: Hierarchical clustering analysis of the expression of 15 natural killer and T cell surface markers showed that innate IELs differed markedly from innate peripheral blood lymphocytes and divided innate IEL subsets into two main branches: a CD127- branch expressing high levels of interleukin (IL) 2/15Rβ but no IL-21R, and a CD127+ branch with the opposite phenotype. While CD was characterised by the contraction of all four innate IEL subsets, a selective expansion of CD56-CD127- and CD56-CD127+ innate IEL was detected in RCDII. In vitro, in the presence of IL-15, CD56-CD127- IEL from controls and patients with CD, but not from patients with RCDII, differentiated into functional natural killer and T cells, the latter largely dependent on notch-signalling. Furthermore, compared with non-coeliac controls, CD56-CD127- IEL from patients with CD expressed more intracellular CD3ε and CD3γ and gave more pronounced T cell differentiation. CONCLUSIONS: Thus, we demonstrate previously unappreciated diversity and plasticity of the innate IEL compartment and its loss of differentiation potential in patients with RCDII. Show less
Petersen, J.; Montserrat, V.; Mujico, J.R.; Loh, K.L.; Beringer, D.X.; Lummel, M. van; ... ; Rossjohn, J. 2014
OBJECTIVE: Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease (CD) characterised by aberrant intraepithelial lymphocytes (IELs) of unknown origin that display an... Show moreOBJECTIVE: Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease (CD) characterised by aberrant intraepithelial lymphocytes (IELs) of unknown origin that display an atypical CD3(-)CD7(+)icCD3(+) phenotype. In approximately 40% of patients with RCDII these lymphocytes develop into an invasive lymphoma. In the current study we aimed to identify the physiological counterpart of these cells. DESIGN: RCDII cell lines were compared with T-cell receptor positive (TCR(+)) IEL (T-IEL) lines by microarray analysis, real-time quantitative PCR and flow cytometry. This information was used to identify cells with an RCDII-associated phenotype in duodenal biopsies from non-refractory individuals by multicolour flow cytometry. RESULTS: RCDII lines were transcriptionally distinct from T-IEL lines and expressed higher levels of multiple natural killer (NK) cell receptors. In addition to the CD3(-)CD7(+)icCD3(+) phenotype, the RCDII lines were distinguishable from other lymphocyte subsets by the absence of CD56, CD127 and CD34. Cells matching this surface lineage-negative (Lin(-)) CD7(+)CD127(-)CD34(-) phenotype expressed a functional interleukin-15 (IL-15) receptor and constituted a significant proportion of IELs in duodenal specimens of patients without CD, particularly children, and were also found in the thymus. In patients without CD, the Lin(-)CD7(+)CD127(-)CD34(-) subset was one of four subsets within the CD3(-)CD7(+)icCD3(+) population that could be distinguished on the basis of differential expression of CD56 and/or CD127. CONCLUSION: Our studies indicate that the CD3(-)CD7(+)icCD3(+) population is heterogeneous and reveal the existence of a Lin(-) subset that is distinct from T, B, NK and lymphoid tissue inducer cells. We speculate that this IL-15 responsive population represents the physiological counterpart of aberrant cells expanded in RCDII and transformed in RCDII-associated lymphoma. Show less
Mujico, J.R.; Dekking, L.; Kooy-Winkelaar, Y.; Verheijen, R.; Wichen, P. van; Streppel, L.; ... ; Koning, F. 2012
