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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 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+ TCRγδ 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.

Celiac disease (CD) is caused by uncontrolled immune responses to the gluten proteins in wheat and related cereals. Gluten is a complex mixture of gliadin and glutenin proteins and most modern wheat varieties contain up to 100 highly related, but distinct gluten proteins. Invariably, these gliadin and glutenin proteins contain immunogenic peptides, particularly so after the peptides have been modified by the enzyme tissue transglutaminase (TG2). This modification results in the conversion of glutamine residues in the gluten peptides into the negatively charged glutamic acid. This generates peptides that bind strongly to the disease predisposing HLA-DQ2.5 or -DQ8 molecules and this facilitates the induction of disease-inducing CD4 T cell responses, a hallmark of CD. It is well-known that the HLA-DQ genotype determines the risk of disease development. Moreover, the abundance of immunogenic peptides in the gluten proteins is likely linked to the observation that polyclonal T cell responses to multiple gluten peptides are usually found in patients with CD. However, not all patients respond to the same set of peptides. Here, I propose a model that integrates these observations and links them to the highly variable clinical spectrum of symptoms that are associated with CD. Moreover, I discuss whether it is feasible to alter wheat and/or gluten to make it suitable for consumption by CD patients.

Coeliac disease (CD) is a T cell mediated inflammatory disorder of the small intestine that affects approximately 1% of the population (1, 2). CD is triggered by gluten ingestion, proteins found in wheat, barley and rye. CD4(+) T cells specific for post-translationally modified gluten peptides bound to the disease-predisposing HLA-DQ2 or HLADQ8 molecules are typically found in patients with CD, explaining the strong association between these HLA-alleles and the occurrence of CD (1, 2). In addition, antibodies specific for modified gluten are also present in patients with CD, implying a role for B to T cell presentation (1). Recent studies have determined the T cell repertoire (TCR) that is utilised by gluten-specific T cells in HLA-DQ2(+) and HLA-DQ8(+) patients and structures between such TCR and HLA-DQ-gluten peptide complexes have been solved (3-7). In HLA-DQ2 patients the TCR repertoire specific for an immunodominant gluten peptide is dominated by the expression of TRAV26 and TRBV7-2 (3-5) while in HLA-DQ8(+) patients such T cells frequently express TRAV26 and TRBV9 (6, 7). Moreover, in the TCR-HLADQ-gluten complexes a non-germline encoded arginine is positioned in the interface between the TCR and HLADQ-gluten which makes critical interactions with both HLA-DQ and the bound peptide (5-7). Collectively, these observations point to stringent selection of a high affinity TCR repertoire in patients with CD. Similar mechanisms are likely to play a role in rheumatoid arthritis (RA).

Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.

Celiac disease (CD) is caused by gluten ingestion in susceptible individuals. Tissue transglutaminase (tTG)-specific Abs are characteristic of CD, and increased tTG activity has been observed in the jejunal biopsies of patients. Here we demonstrate that tTG selectively deamidates gluten peptides, which results in strongly enhanced T cell-stimulatory activity. To our knowledge, this is the first example of an enzymatic modification of a food protein that affects T cell recognition. Moreover, these modifications may lead to the amplification of gluten-specific T cell responses in the gut and consequently may be important for the development of CD.

Celiac disease (CD) is caused by gluten ingestion in susceptible individuals. Tissue transglutaminase (tTG)-specific Abs are characteristic of CD, and increased tTG activity has been observed in the jejunal biopsies of patients. Here we demonstrate that tTG selectively deamidates gluten peptides, which results in strongly enhanced T cell-stimulatory activity. To our knowledge, this is the first example of an enzymatic modification of a food protein that affects T cell recognition. Moreover, these modifications may lead to the amplification of gluten-specific T cell responses in the gut and consequently may be important for the development of CD.

Costs of reforestation projects determine their competitiveness with alternative measures to mitigate rising atmospheric CO2 concentrations. We quantify carbon sequestration in above-ground biomass and soils of plantation forests and secondary forests in two countries in South America-Ecuador and Argentina-and calculate costs of temporary carbon sequestration. Costs per temporary certified emission reduction unit vary between 0.1 and 2.7 USD Mg(-1) CO2 and mainly depend on opportunity costs, site suitability, discount rates, and certification costs. In Ecuador, secondary forests are a feasible and cost-efficient alternative, whereas in Argentina reforestation on highly suitable land is relatively cheap. Our results can be used to design cost-effective sink projects and to negotiate fair carbon prices for landowners.

Celiac disease is an enteropathy caused by intolerance to dietary gluten. The disorder is strongly associated with DQA1*0501/DQB1*0201 (HLA-DQ2) as approximately 95% of celiac patients express this molecule. HLA-DQ2 has unique Ag-binding properties that allow it to present a diverse set of gluten peptides to gluten-reactive CD4+ T cells so instigating an inflammatory reaction. Previous work has indicated that the presence of negatively charged amino acids within gluten peptides is required for specific binding. This, however, only partly explains the scale of the interaction. We have now characterized 432 natural ligands of HLA-DQ2 representing length variants of 155 distinct sequences. The sequences were aligned and the binding cores were inferred. Analysis of the amino acid distribution of these cores demonstrated that negatively charged residues in HLA-DQ2-bound peptides are favored at virtually all positions. This contrasts with a more restricted presence of such amino acids in T cell epitopes from gluten. Yet, HLA-DQ2 was also found to display a strong preference for proline at several anchor and nonanchor positions that largely match the position of proline in gluten T cell epitopes. Consequently, the bias for proline at p6 and p8 facilitates the enzymatic conversion of glutamine into glutamic acid in gluten peptides at p4 and p6, two important anchor sites. These observations provide new insights in the unique ability of HLA-DQ2 to bind a large repertoire of glutamine- and proline-rich gluten peptides. This knowledge may be an important asset in the development of future treatment strategies. J Immunol

Immunotherapy is a highly promising treatment option for cancer. At present, only a small proportion of cancer patients benefits from immunotherapeutic interventions. There is an unmet need to understand which factors determine a patient’s response to immunotherapy as well as to develop novel immunotherapeutic approaches that address shortcomings of current immunotherapies.In this thesis, we have performed an unprecedented characterization of immune cell subsets participating in anti-tumor responses in colorectal cancer and pancreatic ductal adenocarcinoma with different single-cell technologies. Most cancer immunology research studied the role of cytotoxic T cells in both cancer types, while a comprehensive analysis of both innate and adaptive components of cancer immunity was largely lacking. With such an approach, we demonstrated an important involvement of understudied unconventional (γδ T cells) and innate (innate lymphoid cells (ILCs)) immune effector cells in anti-tumor immunity. These immune subsets displayed cytotoxic activity and showed potential for therapeutic exploitation. Further studies will focus on their functional characterization and potential reconversion into a therapeutic agent.This thesis resulted from the collaboration between research groups led by Noel de Miranda (Pathology, LUMC) and Frits Koning (Immunology, LUMC), and underscores the relevance of applying single-cell technologies for the study of complex biological systems.