Role In Cross-presentation Research Articles

Wdfy4-deficiency reveals a critical role for cross-presentation in anti-viral and anti-tumor responses

Abstract Cross-presentation allows for the presentation of exogenous viral and tumor antigens to CD8+ T cells by XCR1+ classical dendritic cells (cDC1s). Previously, the role of cross-presentation in cytotoxic T cell responses in vivo was unclear due to the lack of mouse models specifically deficient in cross-presentation. We developed a CRISPR-Cas9 screen to find novel regulators of cross-presentation and found that the protein WDFY4 is essential for cross-presentation of cell-associated antigens but not for direct presentation or presentation on MHCII. WDFY4-deficient mice had normal cDC1 development and were able to survive infections with Toxoplasma gondii through production of IL-12. However, WDFY4-deficient mice failed to prime CD8+ T cells to viral antigens and were completely unable to control immunogenic tumors. WDFY4 interacts with proteins related to vesicular formation and trafficking such as clathrin and HSP90ab1 and localizes near the cell surface, suggesting its involvement in vesicular targeting after antigen uptake during cross-presentation.

Abstract 2778: Discovery and preclinical characterization of BAY 1905254 a novel immune checkpoint inhibitor for cancer immunotherapy targeting the immunoglobulin-like domain containing receptor 2 (ILDR2)

Abstract ILDR2 is a type I transmembrane protein belonging to the B7 family of immunomodulatory receptors with 98 % sequence identity between the extracellular domains of human and murine orthologues. The exact physiological role of ILDR2 is still unclear, but it has been implicated in the development of type 2 diabetes and the formation of tri-cellular junctions. Our detailed analyses reveal that ILDR2 is expressed in kidney, testis, liver, and lymph node fibroblastic reticular cells (FRC). The latter is particularly interesting since FRC belong to the CD45- stromal population, a specialized cell subset located in the T cell zone. These cells are essential for recruitment of naïve T cells and activated dendritic cells to the lymph node and have been reported to exhibit immuno-regulatory properties. Due to its structural similarity to members of the B7 family and due to its expression in FRCs, we speculated that ILDR2 might play a role e.g. in T cell priming and the initiation of antigen-specific T cell responses. In line with this, we and our collaborators from Compugen were able to show that an ILDR2 -Fc fusion protein is able to a) bind to activated (but not naïve) T cells, b) suppress TCR-stimulated cytokine secretion and c) exhibit immunomodulatory effects in several models of autoimmune diseases, i.e. multiple sclerosis, rheumatoid arthritis, and type 1 diabetes. As a consequence, we set out to generate antibodies against this novel immuno-oncology target and describe here for the first time the characterization of BAY 1905254, a human/mouse cross-reactive IgG2 antibody blocking the immunosuppressive activity of ILDR2. BAY 1905254 specifically binds to ILDR2 but not to the closely related family members ILDR1 and ILDR3/LSR. It enhances antigen-specific T cell proliferation in vivo in an ovalbumin vaccination model with OT-I transgenic T cells. BAY 1905254 exhibits anti-tumor activity in different syngeneic mouse models with efficacy correlating with increasing mutational load. Furthermore, additive/synergistic anti-tumor effects can be observed in combination with either an anti-PD-L1 Ab, an immunogenic cell death inducing chemotherapeutic (Docetaxel) or tumor antigen immunization. Ex vivo analysis reveal that BAY 1905254 treatment results in increased intra-tumoral IFN-y levels and enhanced infiltration of CD45+ cells, e.g. of CD8α+ dendritic cells (DCs). Interestingly, this DC population is known to play a crucial role in cross-presentation, thus, in the initiation of CD8+ T cell responses. In summary, BAY 1905254 is a function-blocking Ab able to counteract the immuno-suppressive function of the newly discovered immuno-oncology target ILDR2. The Ab exhibits in vivo efficacy both in monotherapy as well as in combination with various other approaches, and it is planned to advance BAY 1905254 into FiM trials in 2018. Citation Format: Julia Huetter, Uwe Gritzan, Ilona Gutcher, Sven Golfier, Wolf-Dietrich Doecke, Merlin Verena Luetke-Eversloh, Helge Roider, John Hunter, Andrew Pow, Spencer Liang, Zurit Levine, Ofer Levy, Ilan Vaaknin, Bertolt Kreft, Lars Roese. Discovery and preclinical characterization of BAY 1905254 a novel immune checkpoint inhibitor for cancer immunotherapy targeting the immunoglobulin-like domain containing receptor 2 (ILDR2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2778.

Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins

Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c+/CD8+ dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel.

Serpinb9 is a marker of antigen cross-presenting dendritic cells.

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (grB) that protects cytotoxic lymphocytes from grB-mediated death. In addition, Sb9 is also expressed in accessory immune cells, including dendritic cells (DCs), although its role is debated. Recently, we have demonstrated that Sb9 plays a grB-independent role in cross-presentation of antigens by CD8+ DCs. Here, using a mouse line expressing green fluorescent protein knocked in under the control of the Sb9 promoter, we demonstrate that Sb9 expression is highest in those tissue-resident and migratory DC subsets capable of cross-presentation. Further, we show that CD8+ DCs can be divided into two subsets based on Sb9 expression, and that only the subset expressing higher levels of Sb9 is capable of cross-presentation. These findings add support for role for Sb9 cross-presentation, and indicate that high Sb9 expression is a novel marker of cross-presentation capable DCs.

Heat-Shock Proteins in Autoimmunity

Heat-Shock Proteins in Autoimmunity

Tumor-Derived Autophagosome Vaccine: Mechanism of Cross-Presentation and Therapeutic Efficacy

We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8(+) T cells. Here, we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), which is derived from tumor cells after inhibition of protein degradation, and to provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy. DRibbles were characterized by Western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole-tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed, and the antitumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma. The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiP), and damage-associated molecular pattern molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional and plasmacytoid dendritic cells (DC), and cross-presentation was partially CLEC9A dependent. Furthermore, this autophagy-assisted antigen cross-presentation pathway involved both caveolae- and clathrin-mediated endocytosis and endoplasmic reticulum-associated degradation machinery. It depends on proteasome and TAP1, but not lysosome functions of antigen-presenting cells. Importantly, DCs loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma. These data documented the unique characteristics and potent antitumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials.

CD8α+ Dendritic Cells Are the Critical Source of Interleukin-12 that Controls Acute Infection by Toxoplasma gondii Tachyzoites

CD8α(+) dendritic cells (DCs) are important invivo for cross-presentation of antigens derived from intracellular pathogens and tumors. Additionally, secretion of interleukin-12 (IL-12) by CD8α(+) DCs suggests a role for these cells in response to Toxoplasma gondii antigens, although it remains unclear whether these cells are required for protection against T.gondii infection. Toward this goal, we examined T.gondii infection of Batf3(-/-) mice, which selectively lack only lymphoid-resident CD8α(+) DCs and related peripheral CD103(+) DCs. Batf3(-/-) mice were extremely susceptible to T.gondii infection, with decreased production of IL-12 and interferon-γ. IL-12 administration restored resistance in Batf3(-/-) mice, and mice in which IL-12 production was ablated only from CD8α(+) DCs failed to control infection. These results reveal that the function of CD8α(+) DCs extends beyond a role in cross-presentation and includes a critical role for activation of innate immunity through IL-12 production during T.gondii infection.

Efficient scavenger receptor-mediated uptake and cross-presentation of negatively charged soluble antigens by dendritic cells.

Exogenous antigens endocytosed in large amounts by antigen-presenting cells (APC) are presented on major histocompatibility complex (MHC) class I molecules as well as on class II molecules, a process called cross-presentation. Among APC, dendritic cells (DC) play a key role in cross-presentation by transporting internalized antigen to the cytosol. The present study shows that ovalbumin (OVA) introduced with negative charges by succinylation (Suc-OVA), maleylation (Mal-OVA) or cis-aconitylation (Aco-OVA) was efficiently taken up by DC via scavenger receptors (SR). Mal-OVA and Aco-OVA were efficiently cross-presented by DC, while cross-presentation of Suc-OVA was hardly observed. MHC class I presentation of acylated OVA introduced directly into the cytosol was inefficient and presentation of exogenous native OVA but not of Aco-OVA was markedly augmented by chloroquine, an inhibitor of endosomal acidification, suggesting that deacylation in endosomes or lysosomes is necessary for cross-presentation of acylated OVA. MHC class I presentation of exogenous native OVA and Aco-OVA by DC was blocked by lactacystin and brefeldin A, demonstrating that exogenous antigens taken up by DC are cross-presented through the conventional cytosolic pathway. Therefore, SR-mediated delivery of antigen to DC leads to efficient cross-presentation, although the pathway of chemical modification should be considered.