Cross-presentation Of Exogenous Antigens Research Articles

Machine Learning-Directed Conversion of Glioblastoma Cells to Dendritic Cell-Like Antigen-Presenting Cells as Cancer Immunotherapy.

Immunotherapy has limited efficacy in glioblastoma (GBM) due to the blood-brain barrier and the immunosuppressed or "cold" tumor microenvironment (TME) of GBM, which is dominated by immune-inhibitory cells and depleted of CTL and dendritic cells (DC). Here, we report the development and application of a machine learning precision method to identify cell fate determinants (CFD) that specifically reprogram GBM cells into induced antigen-presenting cells with DC-like functions (iDC-APC). In murine GBM models, iDC-APCs acquired DC-like morphology, regulatory gene expression profile, and functions comparable to natural DCs. Among these acquired functions were phagocytosis, direct presentation of endogenous antigens, and cross-presentation of exogenous antigens. The latter endowed the iDC-APCs with the ability to prime naïve CD8+ CTLs, a hallmark DC function critical for antitumor immunity. Intratumor iDC-APCs reduced tumor growth and improved survival only in immunocompetent animals, which coincided with extensive infiltration of CD4+ T cells and activated CD8+ CTLs in the TME. The reactivated TME synergized with an intratumor soluble PD1 decoy immunotherapy and a DC-based GBM vaccine, resulting in robust killing of highly resistant GBM cells by tumor-specific CD8+ CTLs and significantly extended survival. Lastly, we defined a unique CFD combination specifically for the human GBM to iDC-APC conversion of both glioma stem-like cells and non-stem-like cell GBM cells, confirming the clinical utility of a computationally directed, tumor-specific conversion immunotherapy for GBM and potentially other solid tumors.

STEM-04. FATE-BASED CONVERSION FOR IN SITU VACCINATION IN GLIOBLASTOMA

Abstract BACKGROUND Although immune checkpoint inhibitors have shown high benefit for other solid tumors, their efficacy is limited in GBM due in large part to the “cold” tumor microenvironment (TME) of the GBM tumor. New approaches are necessary to overcome these challenges. We propose a shift in the scientific and treatment paradigm of cancer immunotherapy, calling for tumor cells to take on a central, active role in initiating their own immunity right in the TME. METHODS Using AI, we identified cell fate determinants (CFDs) to convert GBM cells directly into induced dendritic cells (iDC) inside the TME. CFDs were delivered using a viral vector and conversion assessed by immunophenotyping, scRNA-seq, and functional assays for classical DC properties. RESULTS A four-CFD subnetwork anchored by PU.1 was sufficient to convert mouse GBM cells to iDCs (CD45+MHCII+CD80/86+MHCI+). iDCs are growth arrested, exhibit 3-fold higher phagocytic activity and upregulate the canonical antigen processing and presenting machineries by 10-40-fold, resulting in 100-fold greater efficiency at processing ovalbumin and cross-presenting SIINFEKL to naive OTI-CD8+ cytotoxic T lymphocytes (CTL). In addition, iDCs efficiently cross-present exogenous antigens to naïve CD8+ CTLs and elicit >30-fold higher activation and cytotoxicity in tumor-specific T cells compared to native GBM cells, confirming their DC-like properties. Lastly, intratumoral GBM-DC conversion in a syngeneic orthotopic GBM model resulted in significant reduction in tumor burden and increase in survival compared to controls (Log-rank, p = 0.01) with synergistic efficacy with ICIs and classical DC-based tumor vaccination. We have also successfully converted human GBM cells to iDCs with similar properties using a 2-CFD combination also anchored by PU.1. CONCLUSIONS GBM-derived iDCs acquired DC-like functions and were capable of eliciting robust intratumor immune activation, thereby overcoming many current limitations of immunotherapy in GBM. Our goal is to develop a low cost, off-the-shelf gene therapy-based fate conversion tumor immunotherapy.

Perforin-2 is a pore-forming effector of endocytic escape in cross-presenting dendritic cells.

During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1-/- mice failed to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.

Abstract NG01: Dendritic cell intrinsic androgen receptor signaling reduces dendritic cell function and anti-tumor immunity

Abstract NG01: Dendritic cell intrinsic androgen receptor signaling reduces dendritic cell function and anti-tumor immunity

Bioinspired vaccines to enhance MHC class-I antigen cross-presentation.

Cross-presentation of exogenous antigen on MHC class-I is a crucial process for generating a CD8+ T cell response, and is therefore an important design consideration in the development of T-cell-engaging vaccines against viruses, intracellular bacteria, and cancers. Here, we briefly summarize known cross-presentation pathways and highlight how synthetic vaccines can be engineered to enhance MHC-I presentation of exogenous peptide and protein antigens by professional antigen-presenting cells (APCs). In particular, we summarize how molecular engineering and nanotechnology are being harnessed to enhance antigen delivery to lymph nodes and to cross-presenting dendritic cells, to bypass endosomal trafficking of exogenous antigen to promote delivery of antigen to the cytosol of APCs, and to coordinate the delivery of antigen withimmune-stimulating adjuvants that can act synergistically to augment antigen cross-presentation.

The In Vitro Differentiation of Human CD141+CLEC9A+ Dendritic Cells from Mobilized Peripheral Blood CD34+ Hematopoietic Stem Cells.

As shown in various preclinical studies, conventional type-1 dendritic cells, or cDC1s, play a critical role in the immunological rejection of tumors and in the defense against pathogens. This indispensability stems from their potent capacity to activate cytotoxic T cells, especially via the cross-presentation of exogenous antigens. For this reason, cDC1s have become an attractive target for immunotherapy. Here we report a simplified method for generating large numbers of cDC1-like cells in vitro from mobilized human peripheral blood CD34+ hematopoietic stem cells using FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF). An important aspect of this Protocol is the growth of cells on a non-tissue culture-treated surface rather than on a tissue culture-treated surface since the latter suppresses cDC1-marker expression. The resulting CD11c+ DCs express high levels of cDC1-specific markers such as CD141, CLEC9A, TLR3, and several DC maturation markers. Compared to alternative differentiation methods, this method generates large numbers of cDC1-like cells without the need for immortalized feeder cells and should prove useful for studying cDC1 immunobiology and clinical applications of this DC subset. © 2022 Wiley Periodicals LLC. Basic Protocol: Generation of human CD141+CLEC9A+ dendritic cells from mobilized peripheral blood CD34+ hematopoietic stem cells Support Protocol: Flow cytometric immunophenotyping of CD141+ dendritic cells.

Enhanced antigen cross-presentation in human colorectal cancer-associated fibroblasts through upregulation of the lysosomal protease cathepsin S

BackgroundCross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including...

Generation of cDC-like cells from human induced pluripotent stem cells via Notch signaling

BackgroundDendritic cells (DCs) play critical roles in regulating the innate and adaptive immune responses, and have long been a major focus of cancer immunotherapy. Accumulating evidence suggests that conventional type...

Investigating the role of Quaking in antigen presentation by Dendritic Cells

Abstract Dendritic Cells (DCs) are considered the most potent antigen-presenting cells due to their superior capacity to cross-present exogenous antigens to CD8 T cells for effective adaptive immunity. They internalize foreign antigens by phagocytosis, receptor-mediated endocytosis or macropinocytosis, which are then processed in endo/lysosomal compartments and loaded on MHC class I. However, the molecular mechanisms regulating exogenous antigen uptake and cross-presentation by DCs remain unclear. We revealed a potential regulatory role of an RNA-binding protein, Quaking (QKI), in antigen presentation. Our previous studies in neural stem cells and microglia identified QKI as a novel regulator of phagosome maturation and endosome signaling via interaction with the transcription factor PPARβ/δ. Therefore, we hypothesize that QKI, together with PPARβ/δ enhances DC antigen uptake and cross-presentation via promoting phagosome and endolysosome signaling. We found QKI and PPARβ/δ to be significantly unregulated in human monocyte-derived DCs, and demonstrated that activating the QKI-PPARβ/δ complex in DCs upregulates QKI targets and significantly promotes DC phagocytosis activity, suggesting QKI cooperates with PPARβ/δ to enhance antigen uptake. The biological role of QKI in antigen cross-presentation and the effect of QKI enhancement on induction of CD8 T cell response by DC are currently under investigation. Understanding the role of QKI in DC may reveal how antigen presentation is regulated and provide new strategies to improve DC-based immunotherapy.

Cross-presentation of exogenous antigens on MHC I molecules.

In order to get recognized by CD8 T cells, most cells present peptides from endogenously expressed self or foreign proteins on MHC class I molecules. However, specialized antigen-presenting cells, such as DCs and macrophages, can present exogenous antigen on MHC-I in a process called cross-presentation. This pathway plays key roles in antimicrobial and antitumor immunity, and also immune tolerance. Recent advances have broadened our understanding of the underlying mechanisms of cross-presentation. Here, we review some of these recent advances, including the distinct pathways that result in the cross-priming of CD8 T cells and the source of the class I molecules presenting exogenous peptides.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Effective cancer immunotherapy by natural mouse conventional type-1 dendritic cells bearing dead tumor antigen

BackgroundThe manipulation of dendritic cells (DCs) for cancer vaccination has not reached its full potential, despite the revolution in cancer immunotherapy. DCs are fundamental for CD8+ T cell activation, which relies on cross-presentation of exogenous antigen on MHC-I and can be fostered by immunogenic cancer cell death. Translational and clinical research has focused on in vitro-generated monocyte-derived DCs, while the vaccination efficacy of natural conventional type 1 DCs (cDC1s), which are associated with improved anti-tumor immunity and specialize on antigen cross-presentation, remains unknown.MethodsWe isolated primary spleen mouse cDC1s and established a protocol for fast ex vivo activation and antigen-loading with lysates of tumor cells that underwent immunogenic cell death by UV irradiation. Natural tumor antigen-loaded cDC1s were transferred and their potential for induction of endogenous CD8+ and CD4+ T cell responses in vivo, cancer prevention and therapy were assessed in three grafted cancer models. Further, we tested the efficacy of natural cDC1 vaccination in combination and comparison with anti-PD-1 treatment in two “wildtype” tumor models not expressing exogenous antigens.ResultsHerein, we reveal that primary mouse cDC1s ex vivo loaded with dead tumor cell-derived antigen are activated and induce strong CD8+ T cell responses from the endogenous repertoire upon adoptive transfer in vivo through tumor antigen cross-presentation. Notably, cDC1-based vaccines enhance tumor infiltration by cancer-reactive CD8+ and CD4+ T cells and halt progression of engrafted cancer models, including tumors that are refractory to anti-PD-1 treatment. Moreover, combined tumor antigen-loaded primary cDC1 and anti-PD-1 therapy had strong synergistic effects in a PD-1 checkpoint inhibition susceptible cancer model.ConclusionsThis preclinical proof-of-principle study is first to support the therapeutic efficacy of cancer immunotherapy with syngeneic dead tumor cell antigen-loaded mouse cDC1s, the equivalents of the human dendritic cell subset that correlates with beneficial prognosis of cancer patients. Our data pave the way for translation of cDC1-based cancer treatments into the clinic when isolation of natural human cDC1s becomes feasible.

Cross-presentation of Exogenous Antigens.

Presentation of exogenous antigens loaded on major histocompatibility complex class I molecules by antigen presenting cells, termed cross-presentation, is essential for the induction of CD8+ T cells and is performed mainly by specialized dendritic cell subsets. Research into this field has described two main mechanisms of cross-presentation, the cytosolic pathway and the vacuolar pathway. As the first step in cross-presentation, surface receptors relating to cross-presentation are required in the recognition and uptake of Ags, which include C-type lectin receptors, immunoglobulin γ Fc region receptor, chemokine receptor, scavenger receptor etc. After uptake by the cells, there are also many molecules that enable Ags to participate in cross-presentation pathways. By this approach, exogenous Ags can induce CD8+ T cells into cytotoxic T lymphocytes, which is of great significance to induce antitumor and antiviral immune responses, and the molecular mechanism would facilitate the development of related adjuvants. However, the detailed mechanisms of cross-presentation still remain unknown. In this paper, some latest researches, including two major pathways, DC surface receptors and application prospects are summarized.

Tumor-Derived Microvesicles Enhance Cross-Processing Ability of Clinical Grade Dendritic Cells.

Tumor cells release extracellular microvesicles (MVs) in the microenvironment to deliver biological signals to neighboring cells as well as to cells in distant tissues. Tumor-derived MVs appear to play contradictory role promoting both immunosuppression and tumor growth and both evoking tumor specific immune response. Recent evidences indicate that tumor-derived MVs can positively impact Dendritic Cells (DCs) immunogenicity by reprogramming DC antigen processing machinery and intracellular signaling pathways, thus promoting anti-tumor response. DCs are considered pivot cells of the immune system due to their exclusive ability to coordinate the innate and acquired immune responses, cross-present exogenous antigens, and prime naïve T cells. DCs are required for the induction and maintenance of long-lasting anti-tumor immunity and their exploitation has been extensively investigated for the design of anti-tumor vaccines. However, the clinical grade culture conditions that are required to generate DCs for therapeutic use can strongly affect their functions. Here, we investigated the immunomodulatory impact of MVs carrying the MUC1 tumor glycoantigen (MVsMUC1) as immunogen formulation on clinical grade DCs grown in X-VIVO 15 (X-DCs). Results indicated that X-DCs displayed reduced performance of the antigen processing machinery in term of diminished phagocytosis and acidification of the phagosomal compartment suggesting an altered immunogenicity of clinical grade DCs. Pulsing DCs with MVsMUC1 restored phagosomal alkalinization, triggering ROS increase. This was not observed when a soluble MUC1 protein was employed (rMUC1). Concurrently, MVsMUC1 internalization by X-DCs allowed MUC1 cross-processing. Most importantly, MVsMUC1 pulsed DCs activated IFNγ response mediated by MUC1 specific CD8+ T cells. These results strongly support the employment of tumor-derived MVs as immunogen platforms for the implementation of DC-based vaccines.

Encapsulation of Poly I:C and the natural phosphodiester CpG ODN enhanced the efficacy of a hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticle vaccine in TC-1-grafted tumors

FDA approval of CpG oligodeoxynucleotide (CpG ODN) adjuvants for a human hepatitis B virus vaccine has been delayed until late 2017 because of concerns regarding the severe side effects, which may be attributed to the high dosage and systemic diffusion of this proinflammatory material. Considering that PLGA could provide shelter to resist nucleases in tissue and that cationic lipids could confine anionic oligonucleotides in the nanoparticles via electrostatic attraction to avoid systemic diffusion, we encapsulated a natural phosphodiester or the expensive phosphorothioate CpG ODNs in our previously reported hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticles and evaluated vaccine efficacy in a TC-1-grafted mouse model. Our results showed that together with Poly I:C, CpG ODN could promote the maturation of bone marrow-derived dendritic cells and the cross-presentation of exogenous antigens in vitro. For the coencapsulation with Poly I:C, in vivo studies showed that adjuvant effects on the vaccine efficacy of tumor depression, immune cell activation, and memory T-cell elevation of phosphodiester CpG ODNs were comparable to those of the phosphorothioate CpG ODNs at a low concentration (5 µg/dose). In conclusion, the combination of oligonucleotide adjuvants and synthetic particulate systems not only potentiated the immunogenicity of these nanoparticles but also made these adjuvants safer and more economical, which may be helpful for their wide application.

The levels of DNGR-1 and its ligand-bearing cells were altered after human and simian immunodeficiency virus infection.

Dendritic cell NK lectin Group Receptor-1 (DNGR-1), also known as C-type lectin domain family 9, member A (CLEC9A), is a member of C-type lectin receptor superfamily expressed primarily on dendritic cells (DC) that excel in cross-presentation of exogenous antigens. To find out whether and how it is affected in human immunodeficiency virus infections or acquired immunodeficiency syndromes (HIV/AIDS), DNGR-1 expression and DNGR-1-binding cells in simian/human immunodeficiency virus (SHIV) and simian immunodeficiency virus (SIV)-infected rhesus macaques and antiretroviral therapy (ART)-treated AIDS patients were examined by real-time RT-PCR, flow cytometry, and confocal microscopy. DNGR-1 expression was observed in both lymphoid and non-lymphoid tissues including gut-associated lymphoid tissues (GALT) of rhesus macaques. DNGR-1 mRNA levels were significantly reduced in the blood while significantly elevated in the GALT of SHIV/SIV-infected rhesus macaques. DNGR-1 transcription levels were also significantly reduced in the blood of ART-treated AIDS patients irrespective of viral status. White blood cells with exposed DNGR-1 ligands were significantly increased in ART-treated AIDS patients, while significantly decreased in SIV-infected rhesus macaques. These data indicate that DNGR-1 expression, and by extension, the function of cross-presentation of antigens associated with dead/damaged cells might be compromised in HIV/SIV infection, which might play a role in HIV/AIDS pathogenesis and should be taken into consideration in therapeutic AIDS vaccine development.

Fast isolation of mouse XCR-1+ dendritic cells

Abstract Cross-presenting CD8-+ conventional dendritic cells (cDCs) are important for the eradication of cancers and viral infections due to their ability to induce cytotoxic T lymphocyte (CTL) responses. Cross presenting DCs are a very rare subset and in the past, studies of these cells have been limited by their scarcity of specific cell surface markers. Therefore, methods for the detection and isolation of these cells were commonly based on a multitude of immunophenotypic criteria, such as the expression of CD11c and CD8- and the absence of CD3, CD4, SIRP-- and CD11b. Recently, it was demonstrated that crosspresenting cDCs in lymphoid and non-lymphoid tissues specifically express XCR1, which correlates with the ability to take up and cross-present exogenous antigens. Combining our recombinant REAfinity™ Anti-XCR1 mouse mAb with MACS® Technology based on UltraPure MicroBeads, we developed a new method for the fast and easy isolation of cross-presenting DCs. Using this method XCR1+ DCs can be routinely enriched with high recovery and purity without the need for time-consuming laborious flow sorting. This will facilitate the study of cross-presenting DC subset, which will ultimately help to develop new therapeutic strategies employing DCs in the future.

Molecular and Cellular Mechanisms of Antitumor Immune Response Activation by Dendritic Cells

Dendritic cells (DCs) play a crucial role in the initiation and regulation of the antitumor immune response. Already , DC-based antitumor vaccines have been thoroughly explored both in animal tumor models and in clinical trials. DC-based vaccines are commonly produced from DC progenitors isolated from peripheral blood or bone marrow by culturing in the presence of cytokines, followed by loading the DCs with tumor-specific antigens, such as DNA, RNA, viral vectors, or a tumor cell lysate. However, the efficacy of DC-based vaccines remains low. Undoubtedly, a deeper understanding of the molecular mechanisms by which DCs function would allow us to enhance the antitumor efficacy of DC-based vaccines in clinical applications. This review describes the origin and major subsets of mouse and human DCs, as well as the differences between them. The cellular mechanisms of presentation and cross-presentation of exogenous antigens by DCs to T cells are described. We discuss intracellular antigen processing in DCs, cross-dressing, and the acquisition of the antigen cross-presentation function. A particular section in the review describes the mechanisms of tumor escape from immune surveillance through the suppression of DCs functions.

Immunotherapy based on dendritic cells pulsed with CTPFoxM1 fusion protein protects against the development of hepatocellular carcinoma.

Application of dendritic cells (DCs) pulsed with tumor-associated antigens is considered attractive in immunotherapy for hepatocellular carcinoma (HCC). In order to efficiently prime tumor-associated antigens specific for cytotoxic T lymphocytes (CTLs), it is important that DCs present tumor-associated antigens on MHC class I. MHC class I generally present endogenous antigens expressed in the cytosol. In this study, we developed a new antigen delivery tool based on cross presentation of exogenous antigens in DCs by using cytoplasmic transduction peptide (CTP). CTP protein could transduce FoxM1 tumor antigen into the cytosol of DCs, and CTP-FoxM1 fusion protein could stimulate activation and maturation of DCs. DCs pulsed with CTP-FoxM1 could induce specific CTLs. More importantly, the immunity induced by DCs loaded with CTP-FoxM1 could significantly inhibit tumor growth and metastasis in HCC-bearing mice, which was more potent than that induced by DCs loaded with FoxM1 or CTP, alone. Our results indicate that DCs pulsed with CTP-FoxM1 might be a promising vaccine candidate for HCC therapy and provide new insight into the design of DC-based immunotherapy.

Langerin-mediated internalization of a modified peptide routes antigens to early endosomes and enhances cross-presentation by human Langerhans cells.

The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8+ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells (LCs) were able to cross-present a synthetic long peptide (SLP) to CD8+ T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1+ early endosomes. The potency of LCs to enhance CD8+ T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic:polycytidylic acid (pI:C). Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses.