A Biomimetic Aggregation-Induced Emission Photosensitizer with Antigen-Presenting and Hitchhiking Function for Lipid Droplet Targeted Photodynamic Immunotherapy.

Photodynamic therapy (PDT) is a promising alternative approach for effective cancer treatment that is associated with an antitumor immune response. However, immunosuppression of the tumor microenvironment limits the immune response induced by PDT. Stimulation and proliferation of T cells is a critical step for generating immune responses and depends on the efficient presentation of tumor antigens and co-stimulatory molecules by antigen-presenting cells (APCs). Here, biomimetic aggregation-induced emission (AIE) photosensitizers with antigen-presenting and hitchhiking abilities (DC@AIEdots) are developed by coating dendritic cell (DC) membranes on the nanoaggregates of the AIEgens. Notably, the inner AIE molecules can selectively accumulate in lipid droplets of tumor cells, and the outer cell membrane can facilitate the hitchhiking of DC@AIEdots onto the endogenous T cells and enhance the tumor delivery efficiency by about 1.6 times. Furthermore, DC@AIEdots can stimulate the in vivo proliferation and activation of T cells and trigger the immune system. The potential applications of therapeutic agents targeting lipid droplets for immunotherapy are indicated and a new hitchhiking approach for drug delivery is provided. Lastly, the study presents a photoactive and artificial antigen-presenting platform for effective T cell stimulation and cancer photodynamic immunotherapy.

Facile Transformation of Murine and Human Primary Dendritic Cells into Robust and Modular Artificial Antigen-Presenting Systems by Intracellular Hydrogelation.

The growing enthusiasm for cancer immunotherapies and adoptive cell therapies has prompted increasing interest in biomaterials development mimicking natural antigen-presenting cells (APCs) for T-cell expansion. In contrast to conventional bottom-up approaches aimed at layering synthetic substrates with T-cell activation cues, transformation of live dendritic cells (DCs) into artificial APCs (aAPCs) is demonstrated herein using a facile and minimally disruptive hydrogelation technique. Through direct intracellular permeation of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel monomer and UV-activated radical polymerization, intracellular hydrogelation is rapidly accomplished on DCs with minimal influence on cellular morphology and surface antigen display, yielding highly robust and modular cell-gel hybrid constructs amenable to peptide antigen exchange, storable by freezing and lyophilization, and functionalizable with cytokine-releasing carriers for T-cell modulation. The DC-derived aAPCs are shown to induce prolonged T-cell expansion and improve anticancer efficacy of adoptive T-cell therapy in mice compared to nonexpanded control T cells, and the gelation technique is further demonstrated to stabilize primary DCs derived from human donors. The work presents a versatile approach for generating a new class of cell-mimicking biomaterials and opens new venues for immunological interrogation and immunoengineering.

E-Cadherin Expression and Blunted Interferon Response in Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive neoplasm derived from plasmacytoid dendritic cells (pDCs). In this study, we investigated by immunohistochemical analysis the expression of E-cadherin (EC) on pDCs in reactive lymph nodes and tonsils, bone marrow, and in BPDCN. We compared the expression of EC in BPDCN to that in leukemia cutis (LC) and cutaneous lupus erythematosus (CLE), the latter typically featuring pDC activation. In BPDCN, we also assessed the immunomodulatory activity of malignant pDCs through the expression of several type I interferon (IFN-I) signaling effectors and downstream targets, PD-L1/CD274, and determined the extent of tumor infiltration by CD8-expressing T cells. In reactive lymph nodes and tonsils, pDCs expressed EC, whereas no reactivity was observed in bone marrow pDCs. BPDCN showed EC expression in the malignant pDCs in the vast majority of cutaneous (31/33 cases, 94%), nodal, and spleen localizations (3/3 cases, 100%), whereas it was more variable in the bone marrow (5/13, 38,5%), where tumor cells expressed EC similarly to the skin counterpart in 4 cases and differently in other 4. Notably, EC was undetectable in LC (n=30) and in juxta-epidermal pDCs in CLE (n=31). Contrary to CLE showing robust expression of IFN-I-induced proteins MX1 and ISG5 in 20/23 cases (87%), and STAT1 phosphorylation, BPDCN biopsies showed inconsistent levels of these proteins in most cases (85%). Expression of IFN-I-induced genes, IFI27, IFIT1, ISG15, RSAD2, and SIGLEC1, was also significantly (P<0.05) lower in BPDCN as compared with CLE. In BPDCN, a significantly blunted IFN-I response correlated with a poor CD8+T-cell infiltration and the lack of PD-L1/CD274 expression by the tumor cells. This study identifies EC as a novel pDC marker of diagnostic relevance in BPDCN. The results propose a scenario whereby malignant pDCs through EC-driven signaling promote the blunting of IFN-I signaling and, thereby, the establishment of a poorly immunogenic tumor microenvironment.

Case Report: In Situ Vaccination by Autologous CD16+ Dendritic Cells and Anti-PD-L 1 Antibody Synergized With Radiotherapy To Boost T Cells-Mediated Antitumor Efficacy In A Psoriatic Patient With Cutaneous Squamous Cell Carcinoma.

The combination of radiotherapy and immunotherapy improves the survival rate of patients with malignancies developed through escape from T-cell-mediated immune surveillance. Immune checkpoint inhibitors, such as anti-programmed cell death protein-ligand 1 (anti-PD-L1) antibody, are used to rescue exhausted T cells. Simultaneously, dendritic cells (DCs) which are antigen-presenting cells that can initiate T-cell activation, are used to induce a tumor-specific immune response. However, the synergistic antitumor efficacy of the aforementioned combinational immunotherapy with intratumoral injection of low-dose DCs has not been reported, and the underlying therapeutic mechanism requires further investigation. Herein, we present the special case of a psoriatic patient with cutaneous squamous cell carcinoma (cSCC) in the right inguinal region, these two diseases characterized by opposing contradiction, further complicating treatments and side-effect management efforts. To treat the intractable SCC without exaggerating psoriasis, we developed the triple-regimen therapy (TRT) with the intratumoral injection of low-dose autologous DCs and anti-PD-L1 combined with radiotherapy. The injected DCs were obtained simply through leukapheresis without prior G-CSF administration for mobilization nor tumor-antigen loading for expansion. The patient received three radiation doses (24, 18, and 18 Gy) combined with three intratumoral injections of anti-PD-L1 antibody (40, 60, and 120 mg) plus autologous DCs (80% of the DC subpopulation being CD16+ myeloid DC with approximate amounts of 7.3 × 104, 2.5 × 106, and 1.7 × 107) within 10 weeks. The efficacy of the TRT was encouraging in shrinking tumor mass with remarkable SUVmax reduction (approximately 42%) on FDG PET-Scan despite relatively low-dose DCs were available. The low-dose intratumoral immunotherapy induced mild cutaneous side effects as expected. The transcriptomes were compared between pre-TRT and post-TRT biopsies to analyze underlying mechanical pathways of the TRT protocol. Over 10 highly significantly enriched T-cell-related pathways (P <0.0001) were identified in post-TRT biopsies. In addition, the activation of both innate and adaptive immunity was significantly enriched in post-TRT peripheral blood samples. We develop the easily accessible TRT which produces both local anti-tumor T-cell responses and systemic antitumor immunity for treating cSCC patients, especially for those with autoimmune disease.

Biomimetic hybrid membrane-based nanoplatforms: synthesis, properties and biomedical applications.

The rapid clearance and capture by the immune system pose a big challenge in targeted drug delivery using nanocarriers. Cell membrane coating endows nanoplatforms with prolonged blood circulation, enhanced immune escape, and improved targeting capability. However, monotypic cell membrane fails to meet the omnifarious needs of biomedical applications. The combination of different types of cell membranes provides a promising solution to provide multifunctional biomimetic nanoplatforms. In this review, we first discuss the feasibility of constructing biomimetic hybrid membranes and summarize current methods of preparing biomimetic hybrid membrane-based nanoplatforms (BHMNs) and their biomedical applications including drug delivery, cancer detection, detoxification, and cancer vaccines. Finally, the prospects and challenges of utilizing BHMNs for personalized medicine are also discussed.

Adoptive cellular immunotherapy of tumors via effective CpG delivery to dendritic cells using dendrimer-entrapped gold nanoparticles as a gene vector.

The major obstacle that hinders current cancer immunotherapies is the development of an effective approach to promote a proper immune response for effective tumor killing through activated T cells. Herein, we report an effective T cell-based tumor immunotherapy approach through nonviral delivery of a cytosine-guanine (CpG) oligonucleotide using dendrimer-entrapped gold nanoparticles (Au DENPs). In our work, Au DENPs partially decorated with methoxy polyethylene glycol (mPEG) were synthesized and characterized to be used as a vector for CpG delivery to bone marrow-derived dendritic cells (BMDCs). The BMDCs matured via CpG delivery were used to activate T cells for adoptive immunotherapy of cancer cells. We show that the developed PEGylated Au DENPs are able to effectively transfect CpG leading to the maturation of BMDCs that can be used to activate T cells for subsequent adoptive immunotherapy of cancer cells in vitro and a xenografted melanoma tumor model in vivo after intravenous injection. Importantly, the developed approach to genetically engineer BMDCs enables a triggered adaptive immune response and memory of T cells, which can be beneficial for effective inhibition of tumor metastasis and recurrence. The developed nonviral gene delivery approach using Au DENPs as a vector for T cell-based immunotherapy can be applied to different cancer types.

Simultaneous determination of eight arginine-related metabolites in cellular extracts using liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and rapid liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of arginine and its pathway-related metabolites (ornithine, proline, citrulline, glutamate, agmatine, spermidine, and spermine) in cellular extracts. Cells were lysed and cellular proteins precipitated by the addition of acetonitrile followed by ultra-sonication. Supernatants were analyzed using a Chromolith High Resolution RP-18 endcapped column (100 × 4.6 mm, 1.15 µm, 150 Å), with mobile phases of 0.1% formic acid solution and 0.1% formic acid in acetonitrile. Detection was carried out in multiple reaction monitoring (MRM) mode. Calibration curves showed linearity (r2 > 0.99) for all metabolites over the calibration ranges used. The intra- and inter-day precision was less than 13.5%, and the accuracy was between 91.3 and 114.7%. The method developed in this study was successfully applied to measure arginine and its pathway-related metabolites, which are related to nitric oxide synthase/arginase pathways in mouse bone marrow-derived dendritic cells (BMDCs). The ability to simultaneously measure arginine and its pathway-related metabolites is valuable for better understanding local and systemic inflammatory processes.

Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas.

Single-cell RNA sequencing (scRNA-seq) enables the systematic identification of cell populations in a tissue, but characterizing their spatial organization remains challenging. We combine a microarray-based spatial transcriptomics method that reveals spatial patterns of gene expression using an array of spots, each capturing the transcriptomes of multiple adjacent cells, with scRNA-Seq generated from the same sample. To annotate the precise cellular composition of distinct tissue regions, we introduce a method for multimodal intersection analysis. Applying multimodal intersection analysis to primary pancreatic tumors, we find that subpopulations of ductal cells, macrophages, dendritic cells and cancer cells have spatially restricted enrichments, as well as distinct coenrichments with other cell types. Furthermore, we identify colocalization of inflammatory fibroblasts and cancer cells expressing a stress-response gene module. Our approach for mapping the architecture of scRNA-seq-defined subpopulations can be applied to reveal the interactions inherent to complex tissues.

scPred: accurate supervised method for cell-type classification from single-cell RNA-seq data.

Single-cell RNA sequencing has enabled the characterization of highly specific cell types in many tissues, as well as both primary and stem cell-derived cell lines. An important facet of these studies is the ability to identify the transcriptional signatures that define a cell type or state. In theory, this information can be used to classify an individual cell based on its transcriptional profile. Here, we present scPred, a new generalizable method that is able to provide highly accurate classification of single cells, using a combination of unbiased feature selection from a reduced-dimension space, and machine-learning probability-based prediction method. We apply scPred to scRNA-seq data from pancreatic tissue, mononuclear cells, colorectal tumor biopsies, and circulating dendritic cells and show that scPred is able to classify individual cells with high accuracy. The generalized method is available at https://github.com/powellgenomicslab/scPred/.

Modular actin nano-architecture enables podosome protrusion and mechanosensing.

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.

9-O-acetyl sialic acid levels identify committed progenitors of plasmacytoid dendritic cells.

The origins of plasmacytoid dendritic cells (pDCs) have long been controversial and progenitors exclusively committed to this lineage have not been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-pDCs were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-pDCs. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the pDC lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

Dual Expression of TCF4 and CD123 Is Highly Sensitive and Specific For Blastic Plasmacytoid Dendritic Cell Neoplasm.

The diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN) has been based on the expression status of multiple markers, including CD123. TCF4 was discovered recently to be an obligatory master regulator of plasmacytoid dendritic cells. We postulated that a tissue-based assay designed to detect dual CD123 and TCF4 expression would provide a highly reliable and practical marker for BPDCN in biopsy material. We designed, optimized, and validated a dual-color TCF4/CD123 immunohistochemistry stain for use in formalin-fixed paraffin-embedded tissue sections. The performance characteristics of the TCF4/CD123 stain were evaluated in 48 confirmed BPDCN cases. TCF4/CD123 coexpression was detected reproducibly in plasmacytoid dendritic cells. In BPDCN, the TCF4/CD123 stain showed coexpression in all (48/48; 100%) cases analyzed. Cases with concurrent samples from different anatomic sites showed comparable staining characteristics. In contrast, of 464 non-BPDCN cases comprising a wide range of hematolymphoid neoplasms and cutaneous lesions that might enter in the differential diagnosis of BPDCN, we identified dual expression of TCF4 and CD123 in only 1 case of B-lymphoblastic leukemia/lymphoma. On the basis of these findings, the TCF4/CD123 dual-color immunohistochemical stain had an analytic sensitivity of 100% and a specificity of 99.8%. Receiver operator characteristic analysis demonstrated an area under the curve of 1.000 (95% confidence interval: 0.999-1.000). In summary, the dual-color TCF4/CD123 immunohistochemistry stain provides a robust standalone and cost-effective assay for the diagnosis of BPDCN.

Immunomodulatory Effects of Dendritic Poly(ethyleneimine) Glycoarchitectures on Human Multiple Myeloma Cell Lines, Mesenchymal Stromal Cells, and in Vitro Differentiated Macrophages for an Ideal Drug Delivery System in the Local Treatment of Multiple Myeloma.

The use of a drug delivery system (DDS) represents a novel therapeutic approach in the treatment of multiple myeloma in bone lesion. We show the immunomodulatory effects of anionic and cationic dendritic poly(ethyleneimine) glycoarchitectures (PEI-DGAs) on human myeloma cell lines and cells in their microenvironment, in vitro differentiated macrophages, and mesenchymal stromal cells (MSCs). PEI-DGAs do not influence the secretion of IL-6, which is a major growth and survival factor in multiple myeloma. Cationic PEI-DGAs in turn have cytostatic properties on multiple myeloma cell lines. Anionic PEI-DGAs induce the secretion of proinflammatory cytokines IL-1ß, TNFα, and IL-6 in macrophages and MSCs, whereas cationic PEI-DGAs do not. Macrophages and MSCs show remarkably high cell viability in the presence of high concentration of PEI-DGAs. RNA sequencing of MSCs exposed to cationic PEI-DGAs supports the hypothesis that smaller cationic PEI-DGAs are less toxic and could improve osteogenic differentiation in an ideal DDS.

In vitro assessment of PD-L1+ microvesicles in the cyst fluid of non-syndromic odontogenic keratocysts.

Odontogenic keratocysts (OKCs) are jaw cystic lesions which are characterized by local invasion and high recurrence rate. The majority of OKCs are exposed to microorganisms and occur along with focal inflammatory infiltrates. Cyst fluids are biological fluids that contain a large content of cytokines and immune globulins. Inhibitory receptor such as programmed death receptor 1 (PD-1) and its ligand programmed death-ligand 1 (PD-L1), which can induce a coinhibitory signal in activated T cells, plays a vital role in the differentiation, exhaustion and apoptosis of T cells. Cell derived microvesicles, carrying a cargo of functional proteins, nucleic acids and lipids, are important communication tools in the development of diseases. However, the expression of PD-L1 in OKCs tissues and whether PD-L1 could be carried by microvesicles are unexplored. Presently, we have isolated cyst fluid microvesicles and identified cell derived PD-L1+ cyst fluid microvesicles. PD-L1 was located in the membrane of the cyst fluid microvesicles. The main cellular origins of PD-L1+ cyst fluid microvesicles were dendritic cells followed by lymphocytes. Elevated PD-L1+ cyst fluid microvesicles were detected in the OKCs compared with dentigerous cysts. Isolated cyst fluid microvesicles could bind to the membrane of activated CD8 T cells and inhibit proliferation of stimulated peripheral blood CD8 T cells. In conclusion, the present study suggests that elevated PD-L1+ cyst fluid microvesicles might be related with the cyst development of OKCs.

Plasmodium falciparum Activates CD16+ Dendritic Cells to Produce Tumor Necrosis Factor and Interleukin-10 in Subpatent Malaria.

Background: The malaria causing parasite Plasmodium subverts host immune responses by several strategies including the modulation of dendritic cells (DCs). Methods: In this study, we show that Plasmodium falciparum skewed CD16+ DC cytokine responses towards interleukin (IL)-10 production in vitro, distinct to the cytokine profile induced by Toll-like receptor ligation. To determine CD16+ DC responsiveness in vivo, we assessed their function after induced P falciparum infection in malaria-naive volunteers. Results: CD16+ DCs underwent distinctive activation, with increased expression of maturation markers human leukocyte antigen (HLA)-DR and CD86, enhanced tumor necrosis factor (TNF) production, and coproduction of TNF/IL-10. In vitro restimulation with P falciparum further increased IL-10 production. In contrast, during naturally acquired malaria episode, CD16+ DCs showed diminished maturation, suggesting increased parasite burden and previous exposure influence DC subset function. Conclusions: These findings identify CD16+ DCs as the only DC subset activated during primary blood-stage human Plasmodium infection. As dual cytokine producers, CD16+ DCs contribute to inflammatory as well as regulatory innate immune processes.

Efficacy of different hemodialysis methods on dendritic cell marker CD40 and CD80 and platelet activation marker CD62P and P10 in patients with chronic renal failure.

BACKGROUND: Chronic renal failure (CRF) has become a major public health concern, which increases the risk of stroke and systemic thromboembolism. Therefore, therapeutic strategies are in urgent requirement. This study was conducted for investigating efficacy of hemodialysis (HD), hemodiafiltration (HDF), and hemoperfusion (HP) in patients with CRF and the correlation with the presence of complications following HD therapy. METHODS: The therapeutic effect, living quality, biochemical indicators, and dry weight were detected before and after the treatment regimens. Flow cytometry was conducted to detect expressions of dendritic cell markers (CD40 and CD80) and platelet activation markers (CD62P and P10), and the relationship between their expression and therapeutic effect as well as the association of these expressions with complications was analyzed. RESULTS: After HD therapy, patients presented with decreased serum creatinine, serum phosphorus, triglyceride, parathyroid hormone, and ß2 -MG expression; increased hemoglobin, plasma albumin expressions, and dry weight; and enhanced therapeutic effect and living quality. CD62P and P10 expressions decreased, while CD40 and CD80 expressions increased following HD therapy. The therapeutic effect improved in patients with low expressions of CD40 and CD80 and high expressions of CD62P and P10 following HP treatment and complications were lower after treatment of HDF and HP. CONCLUSION: The aforementioned results indicated that CRF patients treated with HP exhibited higher expression of CD40 and CD80 and lower expression of CD62P and P10, suggesting that HP is conferred to have better efficacy than HDF and HD. Therefore, HP may be a promising clinical regimen for treatment of CRF patients.

A method for manual and automated multiplex RNAscope in situ hybridization and immunocytochemistry on cytospin samples.

In situ analysis of biomarkers is essential for clinical diagnosis and research purposes. The increasing need to understand the molecular signature of pathologies has led to the blooming of ultrasensitive and multiplexable techniques that combine in situ hybridization (ISH) and immunohistochemistry or immunocytochemistry (IHC or ICC). Most protocols are tailored to formalin-fixed paraffin embedded (FFPE) tissue sections. However, methods to perform such assays on non-adherent cell samples, such as patient blood-derived PBMCs, rare tumor samples, effusions or other body fluids, dissociated or sorted cells, are limited. Typically, a laboratory would need to invest a significant amount of time and resources to establish one such assay. Here, we describe a method that combines ultrasensitive RNAscope-ISH with ICC on cytospin cell preparations. This method allows automated, sensitive, multiplex ISH-ICC on small numbers of non-adherent cells. We provide guidelines for both chromogenic and fluorescent ISH/ICC combinations that can be performed either in fully automated or in manual settings. By using a CD8+ T cells in vitro stimulation paradigm, we demonstrate that this protocol is sensitive enough to detect subtle differences in gene expression and compares well to commonly used methods such as RT-qPCR and flow cytometry with the added benefit of visualization at the cellular level.

Direct loading of CTL epitopes onto MHC class I complexes on dendritic cell surface in vivo.

Dendritic cell (DC)-based cytotoxic T lymphocyte (CTL) epitope vaccines are effective to induce CTL responses but require complex ex vivo DC preparation and epitope-loading. To take advantage of DC-based epitope vaccines without involving the ex vivo procedures, we aimed to develop carriers to directly load CTL epitopes onto DCs in vivo. Here, we first engineered a carrier consisting of a hydrophilic polypeptide, immune-tolerant elastin-like polypeptide (iTEP) and a substrate peptide of matrix metalloproteinases-9 (sMMP). The iTEP was able to solubilize CTL epitopes. CTL epitopes were connected to the carrier, iTEP-sMMP, through sMMP so that the epitopes can be cleaved from the carrier by MMP-9. iTEP-sMMP was found to release its epitope payloads in the DC culture media, which contained MMP-9 released from DCs. iTEP-sMMP allowed for the direct loading of CTL epitopes onto the surface MHC class I complexes of DCs. Importantly, iTEP-sMMP resulted in greater epitope presentation by DCs both in vitro and in vivo than a control carrier that cannot directly load epitopes. iTEP-sMMP also induced 2-fold stronger immune responses than the control carrier. To further enhance the direct epitope-loading strategy, we furnished iTEP-sMMP with an albumin-binding domain (ABD) and found the new carrier, ABD-iTEP-sMMP, had greater lymph node (LN) accumulation than iTEP-sMMP. ABD-iTEP-sMMP also resulted in greater immune responses than iTEP-sMMP by 1.5-fold. Importantly, ABD-iTEP-sMMP-delivered CTL epitope vaccine induced stronger immune responses than free CTL epitope vaccine. Taken together, these carriers utilized two physiological features of DCs to realize direct epitope-loading in vivo: the accumulation of DCs in LNs and MMP-9 released from DCs. These carriers are a potential substitute for DC-based CTL epitope vaccines.