The inverse electron demand Diels-Alder (IEDDA) pyridazine elimination emerged in 2013 as a new bioorthogonal reaction and constitutes a prime example of what is now known as dissociative... Show moreThe inverse electron demand Diels-Alder (IEDDA) pyridazine elimination emerged in 2013 as a new bioorthogonal reaction and constitutes a prime example of what is now known as dissociative bioorthogonal chemistry. The research described in this Thesis aims to develop synthetic strategies which enable the IEDDA pyridazine elimination to be applied as a versatile toolbox in chemical biology studies. More specifically, it entails modification of antigenic (MHC-I) peptides and (CD1d) glycolipids with a trans-cyclooctene (TCO) moiety to allow chemical control over the recognition of these biomolecules by immune cells. Synthetic advances which encompass the entire scope of the IEDDA pyridazine elimination are additionally described. Show less
Maturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to... Show moreMaturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to identify sialylated proteins at DC surface and comprehend their role and modulation. Mass spectrometry analysis of DC's proteins, pulled down by a sialic acid binding lectin, identified molecules of the major human histocompatibility complex class I (MHC-I), known as human leucocyte antigen (HLA). After desialylation, DCs showed significantly higher reactivity with antibodies specific for properly folded MHC-I-beta 2-microglobulin complex and for beta 2-microglobulin but showed significant lower reactivity with an antibody specific for free MHC-I heavy chain. Similar results for antibody reactivities were observed for TAP2-deficient lymphoblastoid T2 cells, which express HLA-A*02:01. Using fluorescent peptide specifically fitting the groove of HLA-A*02:01, instead of antibody staining, also showed higher peptide binding on desialylated cells, confirming higher surface expression of MHC-I complex. A decay assay showed that desialylation doubled the half-life of MHC-I molecules at cell surface in both DCs and T2 cells. The biological impact of DC ' s desialylation was evaluated in co-cultures with autologous T cells, showing higher number and earlier immunological synapses, and consequent significantly increased production of IFN-gamma by T cells. In summary, sialic acid content modulates the expression and stability of complex MHC-I, which may account for the improved DC-T synapses. Show less
Tumors can evade recognition by the immune system through downregulation of the peptide-presenting MHC-I molecules. In this thesis, we describe that these tumors can still be targeted by a... Show moreTumors can evade recognition by the immune system through downregulation of the peptide-presenting MHC-I molecules. In this thesis, we describe that these tumors can still be targeted by a novel category of CD8 T cells, recognizing a specific peptide presented on those immune-escaped tumors. We describe the selection and characteristics of these specific T cells and show they can protect against a challenge with these MHC-I low tumors. This could be a novel type of therapy for tumors which have become resistant against conventional (immune)therapies. Show less
