Cell-biological mechanisms regulating antigen presentation : Signaling towards adaptive immunity

We, humans, are exposed daily to millions of potential pathogens, through contact, inhalation, or ingestion. Our ability to avoid infection depends on our immune system, which consists of two distinct, yet interrelated and interacting subsystems: the innate and adaptive immune system. The adaptive immune system remembers previous encounters with specific pathogens and destroys them when they attack again. Elicitation of an adaptive response depends on special white blood cells from the innate immune system. This thesis focuses on the central role that the intracelllular endosomal transport network in these specialized white blood cells, e.g. B-cells and Dendritic cells (DCs), plays in elicitating an adaptive immune response. Using live cell fluorescent microscopy on human monocyte-derived DCs, we show that TLR-triggering induced tubulation of late endosomes. However, TLR triggering was insufficient to stimulate tubulation of endosomal recycling compartments (ERCs) in human monocyte-derived DCs. Tubulation of ERCs within human DCs required additional antigen-specific CD8+ T-cell interaction. This tubular remodeling occurs within 30 min of T-cell contact and involved ligation of HLA-A2 and ICAM-1 by T-cell-expressed T-cell receptor and LFA-1, respectively. In addition, wedemonstrated that MICAL-L1 is necessary for tubulation of ERCs in human DCs. Disintegration of microtubules or inhibition of endosomal recycling abolished tubular ERCs, which coincided with reduced antigen-dependent CD8+ T-cell activation. Another mechanism that is involved in endosomal trafficking and underlies antigen presentation, is receptor-mediated uptake of antigens. We targeted exogenous antigen to Fc gamma receptors (FcγRs), which increased CD8+ T-cell activation following antigen cross-presentation by human monocyte-derived and BDCA-3+ DCs. Using several pharmaceutical inhibitors, we show that FcγR-targeted antigens enter both cytosolic and vacuolar pathways of cross-presentation in human DCs. Additionally, we report on two related Common Variable Immunodeficiency (CVID) patients carrying a Leucine to Proline replacement at position 3 in the Src kinase B-lymphoid tyrosine kinase (BLK). Unlike in mice, where functional redundancy exists between Src kinases, we show that L3P-BLK abrogates early B-cell Receptor (BCR) signalling, e.g. Syk phosphorylation, required for B-cell proliferation. Moreover, expression of L3P-BLK accelerates late endosomal/lysosomal BCR-antigen complex delivery, causing destruction of the antigen. Together these events reduce antigen presentation as shown by decreased BCR-mediated HLA-DR-restricted antigen-specific activation of CD4+ T-cells. Thus, defective early BCR signalling may affect endosomal trafficking and diminish interdependent antigen presentation. Whether more CVID patients harbour defective endosomal trafficking and antigen presentation is unknown to date, but we showed disturbed early BCR signaling in a significant proportion of pediatric CVID(-like) patients. In these young CVID-patients, BCR or CD20 crosslinking induces less BCR internalization and antibody-mediated CD20 triggering elicited decreased BCR downstream signaling as measured by secondary Ca2+ fluxes. In conclusion, the work described in this thesis describes the pivotal role of the endosomal network in initiation of adaptive immune responses. Regulating key endosomal processes by interfering a.o. with endosomal tubulation, antigen (Fcγ) receptor-targeting, or Src kinase (BLK) function may contribute to an improvement in future therapeutic intervention strategies contributing to patient survival

MICAL-L1-related and unrelated mechanisms underlying elongated tubular endosomal network (ETEN) in human dendritic cells

The endosomal pathway constitutes a highly dynamic intracellular transport system, which is composed of vesicular and tubular compartments. Endosomal tubules enable geometry-based discrimination between membrane and luminal content. Extended tubular endosomes were suggested to deliver a steady stream of membrane proteins to one location more reliable and effective than vesicular endosomes. Recently, we demonstrated that human dendritic cells (DCs) form a large elongated tubular endosomal network, e.g. ETEN, upon distinct triggers. LPS-stimulation triggered late endosomal tubulation. Additional clustering of class I MHC and ICAM-1 by a cognate interaction between antigen-laden DC and antigen-specific CD8+ T-cells induces formation of transferrin-positive tubules emanating from the endosomal recycling compartment (ERC). We here discuss cell-biological mechanisms that are involved in membrane bending and possibly underlie initiation, elongation, and stabilization of ETEN in human DCs. Using a knock-down approach we demonstrate that MICAL-L1 is necessary for ETEN remodeling originating from ERC in human DCs

Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help

Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4+ T-cell help. We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID. BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4+ T-cells. In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4+ T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients

Recommendations for empowering early career researchers to improve research culture and practice

Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice; and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and examples of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian.Clinical epidemiolog