Classing it up to get noticed : MHC class 1 antigen display in dendritic cells and neuroblastoma

In this thesis I have explored the process of MHC-1-mediated antigen presentation in two distinctive cell types: dendritic cells and neuroblastoma tumor cells. Dendritic cells (DCs) are pivotal players that bridge innate and adaptive immunity. DCs are able to engulf tumor-derived material and cross-present tumor-derived antigen fragments to CD8+ T-cells. In Chapter 2, I give an overview of recent literature about cross-presenting tumor cell material by dendritic cells. Tipping the scale towards antigen preservation causes a complete stop of antigenic peptide production and subsequent loss of antigen cross-presentation, as described in Chapter 3. Here, cowpox virus-derived protein CPXV012 delivered into the endosomal pathway of dendritic cells as a soluble protein. In this endosomal environment, soluble CPXV012 colocalizes with endocytosed antigen. By blocking endosomal acidification, it prevents the degradation of antigen, a process that is required to liberate antigenic peptide to be cross-presented by MHC-1. Peptides that are produced by endosomal processing of antigen do not only reach MHC-1 for presentation, but also MHC 2. MHC 2 presents antigen to helper CD4+ T-cells that aid in the priming of CD8+ T-cells. The benefit of reaching both CD4+ and CD8+ T-cells when initiating an immune response is investigated in Chapter 4. Given the pivotal role of dendritic cells in eliciting cellular immunity, they may be useful for therapeutic purposes. In Chapter 5, I explore this option using a vector derived from Rift Valley Fever virus (bunyaviridae) that targets dendritic cells in vitro and in vivo. In prophylactic and therapeutic settings of vaccination this viral vector was able to confer protection against a lymphoma tumor challenge. Neuroblastoma is the most deadly pediatric solid tumor and currently lacks a cellular immunotherapeutic treatment strategy. Tumor-specific CD8+ T-cells may be effective to destroy neuroblastoma tumor cells. However, neuroblastoma tumors were shown not to be immunogenic due to low MHC-1 expression levels and lack of broadly- expressed antigens. How to increase neuroblastoma immunogenicity is discussed in Chapter 6. I show that the preferred antigen in melanoma (PRAME) is expressed in >90% of high-risk neuroblastoma tumors and PRAME-specific T-cells are able to recognize neuroblastoma cells, but only when MHC-1 levels are upregulated e.g. by previous NK cell attack. The regulation of MHC-1 expression in neuroblastoma is further investigated in Chapter 7. MHC-1 expression, and thus T-cell recognition of neuroblastoma, appears to depend on the activation of the transcription factor NFκB. Through systematic gene deletions, I have identified factors that suppress NFκB activity in neuroblastoma cells. Tumor expression of these NFκB supressors are correlated with worse survival of neuroblastoma patients, suggesting they could be relevant targets for the development of future (immuno)therapies against neuroblastoma. Finally, the role of NFκB suppression in neuroblastoma development, immunogenicity and immunotherapy is reviewed and discussed in Chapter 8

Upregulation of Class I MHC on Neuroblastoma Cells By NK Cell Exposure for Enhanced CTL Reactivity

Neuroblastoma is the most common solid tumor in pediatric patients, with a clear unmet need as survival rates are <20% for stage IV disease. As adjuvant immunotherapy is considered to have additional anti-tumor activity we aim to increase cytotoxic T lymphocyte (CTL)-mediated immune surveillance in neuroblastoma patients by administration of a dendritic cell-based vaccine. Neuroblastoma has developed mechanisms to circumvent CTL recognition; therefore it is our aim to find clinical strategies to make neuroblastoma susceptible for CTL killin

Upregulation of Class I MHC on Neuroblastoma Cells By NK Cell Exposure for Enhanced CTL Reactivity

Neuroblastoma is the most common solid tumor in pediatric patients, with a clear unmet need as survival rates are <20% for stage IV disease. As adjuvant immunotherapy is considered to have additional anti-tumor activity we aim to increase cytotoxic T lymphocyte (CTL)-mediated immune surveillance in neuroblastoma patients by administration of a dendritic cell-based vaccine. Neuroblastoma has developed mechanisms to circumvent CTL recognition; therefore it is our aim to find clinical strategies to make neuroblastoma susceptible for CTL killin

CXCL4 exposure potentiates TLR-driven polarization of human monocyte-derived dendritic cells and increases stimulation of T cells

Chemokines have been shown to play immune-modulatory functions unrelated to steering cell migration. CXCL4 is a chemokine abundantly produced by activated platelets and immune cells. Increased levels of circulating CXCL4 are associated with immunemediated conditions, including systemic sclerosis. Considering the central role of dendritic cells (DCs) in immune activation, in this article we addressed the effect of CXCL4 on the phenotype and function of monocyte-derived DCs (moDCs). To this end, we compared innate and adaptive immune responses of moDCs with those that were differentiated in the presence of CXCL4. Already prior to TLR- or Ag-specific stimulation, CXCL4-moDCs displayed a more matured phenotype. We found that CXCL4 exposure can sensitize moDCs for TLR-ligand responsiveness, as illustrated by a dramatic upregulation of CD83, CD86, and MHC class I in response to TLR3 and TLR7/8-agonists. Also, we observed a markedly increased secretion of IL-12 and TNF-α by CXCL4- moDCs exclusively upon stimulation with polyinosinic-polycytidylic acid, R848, and CL075 ligands. Next, we analyzed the effect of CXCL4 in modulating DC-mediated T cell activation. CXCL4-moDCs strongly potentiated proliferation of autologous CD4+ T cells and CD8+ T cells and production of IFN-γ and IL-4, in an Ag-independent manner. Although the internalization of Ag was comparable to that of moDCs, Ag processing by CXCL4-moDCs was impaired. Yet, these cells were more potent at stimulating Ag-specific CD8+ T cell responses. Together our data support that increased levels of circulating CXCL4 may contribute to immune dysregulation through the modulation of DC differentiation

Nedd4 Binding Protein 1 (N4BP1) and TNFAIP3 Interacting Protein 1 (TNIP1) control MHC-1 display in neuroblastoma

Neuroblastoma is the second most common tumor in children. The cause of neuroblastoma is thought to lie in aberrant development of embryonic neural crest cells and is accompanied by low MHC-1 expression and suppression of the NF-κB transcription factor, thereby gearing cells towards escape from immunosurveillance. Here we assess regulation of the MHC-1 gene in neuroblastoma to enhance its immunogenic potential for therapeutic T-cell targeting. A genome-wide CRISPR screen identified N4BP1 and TNIP1 as inhibitory factors of NF-κB-mediated MHC-1 expression in neuroblastoma. Advanced stage neuroblastoma patients who expressed high levels of TNIP1 and N4BP1 exhibited worse overall survival. Depletion of N4BP1 or TNIP1 increased NF-κB and MHC-1 expression and stimulated recognition by antigen-specific CD8+ T cells. We confirmed that TNIP1 inhibited canonical NF-κB member RelA by preventing activation of the RelA/p50 NF-κB dimer. Furthermore, N4BP1 inhibited both canonical and non-canonical NF-κB through binding of deubiquitinating enzyme CEZANNE, resulting in stabilization of TRAF3 and degradation of NF-κB-inducing kinase NIK. These data suggest N4BP1/CEZANNE or TNIP1 may be candidate targets for immunotherapy in neuroblastoma tumors and should lift NF-κB suppression, thereby triggering increased peptide/MHC1-mediated tumor reactivity to enhance therapeutic T cell targeting

Effect of NSR infection on intracellular and extracellular CD83 levels.

<p>(A) The levels of soluble CD83 in supernatants from cells harvested 24 h after stimulation with LPS, infection with NSR, or from cells mock infected with NSRmock were determined by ELISA. Bars represent average CD83 concentrations ±SD. Results from one of two independently performed experiments with similar results are shown. (B) Detection of CD83 in cell lysates by Western blot at 24 hpi. The different treatments are shown above the top panel and the probed proteins are depicted at the right. The positions of molecular weight standard proteins are shown at the left. The top blot was stripped and re-probed with antibodies against GAPDH and GFP, which served as loading control and control to confirm NSR infection, respectively. Results from one of two independent experiments with cells from two donors are shown.</p