PD-L1 partially protects renal tubular epithelial cells from the attack of CD8+cytotoxic T cells

Background. Activated infiltrating T cells play a crucial role in nephritic inflammation via the direct interaction with proximal tubular epithelial cells (TEC). Under inflammatory conditions, major histocompatibility complex class I and II molecules are upregulated on the surface of renal TEC, enabling them to function as ‘non-professional' antigen-presenting cells (APC) to activate T cells, and, in turn to be targeted by cytotoxic T lymphocytes (CTL) to cause tissue damage. It is known that co-stimulatory (e.g. B7/CD28) and co-inhibitory (e.g. PD-L1/PD-1) signals regulate and determine the magnitude of T cell responses. In this study, we examined the expression of co-stimulatory molecule PD-L1 by renal TEC and the functional role of renal PD-L1/PD-1 pathway in regulating CD8+ T cell responses induced by antigen-presenting renal TEC. Methods. Renal TEC were treated with type I and type II interferons (IFN-α, IFN-β or IFN-γ). PD-L1 expression was then determined with flow cytometry and RT-PCR. To investigate the functional role of renal epithelial PD-L1 on CD8+ CTL responses, H-2Kb-restricted, OVA257-264 peptide-specific CD8+ T cells isolated from OT-1 T cell receptor transgenic mice were co-incubated with IFN-stimulated, OVA257-264 peptide-pulsed congeneic TEC. The activation of OT-1 CD8+ CTL was estimated either by IFN-γ production in the supernatants of co-cultures or by CTL activity. Results. TECs do not constitutively express PD-L1 on their surface. However, a strong and dose-dependent upregulation of PD-L1 was observed on TEC after stimulation with IFN-β or IFN-γ, but not with IFN-α. OVA257-264 peptide pulsed-TEC were able to activate OT-1 CD8+ T cells, indicated by the high amount of IFN-γ production and cytolysis of TEC. Blockade of epithelial PD-L1 with specific mAb significantly increased OT-1 CD8+ T cell activity, indicating that the PD-L1 pathway has a negative effect on CD8+ T cell responses. Moreover, IFN- β- or IFN-γ-stimulated TEC with high surface PD-L1 expression were more resistant to the cytolysis by OT-1 CTL. Conclusion. Together our data reveal that the renal PD-L1/PD-1 pathway has a negative effect on CD8+ CTL activation. PD-L1 might, therefore, act as a protective molecule on TEC, downregulating the cytotoxic renal parenchymal immune respons

PLGA microspheres for improved antigen delivery to dendritic cells as cellular vaccines

Dendritic cells (DC) are currently employed as cellular vaccines in clinical trials of tumor immunotherapy. In most trials, peptide epitopes derived from tumor antigens are being exogenously loaded onto human DC for binding to MHC class I molecules. While this is a convenient method, it suffers from the drawback that the persistence of class I/peptide complexes on the cell surface is in the order of a few hours. This drawback limits the success of vaccination. We have investigated biodegradable poly(D,L-lactide-co-glycolide) microspheres (PLGA-MS) as delivery tools for antigen loading of human monocyte-derived DC (hMoDC). Immature hMoDC readily take up PLGA-MS and present epitopes from encapsulated proteins or peptides both on MHC class I and class II. Interestingly, antigen presentation by hMoDC was markedly prolonged when hMoDC were charged with PLGA-MS-encapsulated as opposed to soluble antigens. The properties of hMoDC with respect to migration, cytokine secretion, survival and allostimulation were not adversely affected by the uptake of PLGA-MS. In this article, we will review the properties of PLGA-MS as an adjuvant and summarize recent data on their potential for antigen delivery to dendritic cells

Photosensitizer and light pave the way for cytosolic targeting and generation of cytosolic CD8 T cells using PLGA vaccine particles

The generation of CTLs is crucial in the immunological fight against cancer and many infectious diseases. To achieve this, vaccine Ags need to be targeted to the cytosol of dendritic cells, which can activate CD8 T cells via MHC class I (MHCI). Therefore, such targeting has become one of the major objectives of vaccine research. In this study, we aimed to bypass the unwanted and default MHC class II Ag presentation and trigger MHCI presentation by using a photosensitizer that, upon light activation, would facilitate cytosolic targeting of codelivered Ag. Poly(lactide-co-glycolide) microparticles ∼1 μm size were loaded with OVA and the photosensitizer tetraphenyl chlorine disulphonate (TPCS2a) and administered intradermally in mice, which were illuminated 1 d later for activation of the photosensitizer. Immunization in the presence of TPCS2a significantly increased activation of CD8 T cells compared with immunization without TPCS2a and as measured by CD8 T cell proliferation, production of proinflammatory IFN-γ, TNF-α, and IL-2, and prevention of tumor growth. Cytotoxicity was demonstrated by granzyme B production in vitro and by in vivo killing of CFSE-labeled targets. CD4-dependent Ab responses were abrogated in mice immunized with TPCS2a-containing particles, suggesting that photosensitization facilitated a shift from default MHC class II toward MHCI Ag presentation. Hence, vaccine particles with Ag and photosensitizers proved an effective vehicle or adjuvant for stimulation of CTLs, and they may find potential application in therapeutic cancer vaccination and in prophylactic and therapeutic vaccination against intracellular infections

Intradermal photosensitisation facilitates stimulation of MHC class-I restricted CD8 T-cell responses of co-administered antigen

The protection or treatment of several immunological disorders is dependent on the antigen-specific and cytotoxic CD8 T cells. However, vaccines aimed at stimulating CD8 T-cell responses are typically ineffective because vaccine antigens are primarily processed by the MHC class-II and not the MHC class-I pathway of antigen presentation: the latter requires cytosolic delivery of antigen. In order to facilitate targeting of antigen to cytosol, the antigen was combined with the photosensitiser TPCS2a (disulfonated tetraphenyl chlorin) and administered intradermally to mice. The photosensitiser was activated by illumination of the injection site. This photochemical internalization (PCI) strongly increased the stimulation of CD8 T-cell responses as measured by antigen-specific proliferation and secretion of pro-inflammatory cytokines. Fluorescence microscopy showed that delivery to cytosol was TPCS2a dependent and occurred by light-induced disruption of TPCS2a- and antigen-containing endosomes. PCI-based vaccination prevented growth of malignant B16 cells as compared with vaccination without PCI. In conclusion, PCI represents a potent tool for delivery of antigens to cytosol for stimulation of cytotoxic CD8 T-cell responses. This study demonstrated a first proof-of-principle for PCI-mediated immunisation with potential application in cancer immunotherapy

Combined Photosensitization and Vaccination Enable CD8 T-Cell Immunity and Tumor Suppression Independent of CD4 T-Cell Help

Cytotoxic T lymphocytes (CTLs) are key players in fighting cancer, and their induction is a major focus in the design of therapeutic vaccines. Yet, therapeutic vaccine efficacy is limited, in part due to the suboptimal vaccine processing by antigen-presenting cells (APCs). Such processing typically takes place via the MHC class II pathway for CD4 T-cell activation and MHC class I pathway for activation of CD8 CTLs. We show that a combination of skin photochemical treatment and immunization, so-called photochemical internalization (PCI) facilitated CTL activation due to the photochemical adjuvant effect induced by photosensitizer, oxygen, and light. Mice were immunized intradermally with antigen and photosensitizer, followed by controlled light exposure. PCI-treated mice showed strong activation of CD8 T cells, with improved IFN-γ production and cytotoxicity, as compared to mice immunized without parallel PCI treatment. Surprisingly, the CD8 T-cell effector functions were not impaired in MHC class II- or CD4 T-cell-deficient mice. Moreover, PCI-based vaccination caused tumor regression independent of MHC class II or CD4 T cells presence in melanoma bearing mice. Together, the data demonstrate that PCI can act as a powerful adjuvant in cancer vaccines, even in hosts with impaired T-helper functions

The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles

Biodegradable microparticles (MP) represent a promising and efficient delivery system for parenteral vaccination. Recently, MP have also been explored as tool for the ex vivo antigen loading of professional antigen presenting cells such as dendritic cells (DC) to be used as cellular vaccines. The purpose of this study was to investigate various polycationic coatings on poly(lactide-co-glycolide) (PLGA) MP, with regard to their effect on phenotypic and functional maturation of monocyte-derived DC (MoDC) that had previously been loaded with the MP in vitro. The preparation and concomitant coating of the PLGA was performed by means of a solvent extraction/evaporation method using a recently developed microextrusion-based technique. The polyelectrolytes tested for MP coating encompassed aminodextran, chitosan, poly(ethylene imine) (PEI), poly(L-lysine) and protamine. Uncoated and differently coated PLGA MP were fed to immature MoDC, which ingested efficiently the different MP types irrespective of their surface coating. The MPloaded immature MoDC were then matured with the help of a cytokine/PGE-2 maturation cocktail. Here, the presence of the ingested MP did not affect the MoDC maturation in terms of expression of the surface markers CD80, CD83, CD86, HLA-DR and MMR, irrespective of the MP surface coating. Importantly, none of the PLGA MP types alone induced significant maturation of MoDC in the absence of the maturation cocktail. MP-loaded and subsequently matured MoDC expressed high levels of the chemokine receptor CCR7, whose functional activity was evidenced by the migration of MoDC towards CCL21, irrespective of the presence of ingested MP. Further, MP-loaded and subsequently matured MoDC also secreted comparable amounts of IL-10 and IL-12p70, irrespective of the presence of ingested MP except for PEI-coated PLGA MP, which enhanced significantly the secretion of IL-12p70 in mature MoDC. In conclusion, phenotypic and functional maturation of MoDC by means of a maturation cocktail remained unchanged irrespective of the presence of previously ingested differently coated PLGA MP. This offers interesting perspectives for using these particulate systems together with entrapped antigens for ex vivo loading of MoDC in view of cellular immunotherapy

Photochemical targeting of antigens to the cytosol for stimulation of MHC class-I-restricted T-cell responses

Tumour chemotherapy with drugs is typically associated with severe systemic and local side effects for which reason immunotherapy represents a safer alternative. However, vaccination often fails to generate the required cytotoxic CD8 T-cell responses due to insufficient access of antigens to the cytosol and the MHC class I pathway of antigen presentation. One important issue of tumour research is therefore to develop strategies that allow cytosolic targeting or endosomal escape of tumour antigens. The objective of the current study was to test whether endocytosed antigen could be delivered to MHC class I by means of photochemical internalisation (PCI). Briefly, the antigen and the photosensitiser Amphinex were loaded in vitro onto bone-marrow-derived murine dendritic cells (DCs). After light activation, which is supposed to cause disruption of OVA- and Amphinex-containing endosomes, the DCs were cultured with OVA-specific CD8 T cells or used for immunisation of mice. PCI facilitated CD8 T-cell responses as measured by IFN-γ secretion in vitro and CD8 T-cell proliferation in vivo. In conclusion, the current proof-of-concept study is the first to describe PCI-mediated immunisation and the results revealed the feasibility of this novel technology in autologous vaccination for stimulation of CD8 T-cell responses

Cell-Specific Delivery Using an Engineered Protein Nanocage

Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and biodistribution. In this study, we examine the utility of a cage-forming variant of the protein lumazine synthase, which was previously designed and evolved to encapsulate biomacromolecular cargo. Linking antibody-binding domains to the exterior of the cage enabled binding of targeting immunoglobulins and cell-specific uptake of encapsulated cargo. Protein nanocages displaying antibody-binding domains appear to be less immunogenic than their unmodified counterparts, but they also recruit serum antibodies that can mask the efficacy of the targeting antibody. Our study highlights the strengths and limitations of a common targeting strategy for practical nanoparticle-based delivery applications.ISSN:1554-8929ISSN:1554-893

Cell-Specific Delivery Using an Engineered Protein Nanocage

Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and biodistribution. In this study, we examine the utility of a cage-forming variant of the protein lumazine synthase, which was previously designed and evolved to encapsulate biomacromolecular cargo. Linking antibody-binding domains to the exterior of the cage enabled binding of targeting immunoglobulins and cell-specific uptake of encapsulated cargo. Protein nanocages displaying antibody-binding domains appear to be less immunogenic than their unmodified counterparts, but they also recruit serum antibodies that can mask the efficacy of the targeting antibody. Our study highlights the strengths and limitations of a common targeting strategy for practical nanoparticle-based delivery applications.ISSN:1554-8929ISSN:1554-893