Search

(A) Induction of an MDSC phenotype by AML-derived soluble factors, (B) Induction of IDO-activity by AML-EVs, (C) ZetaView-based analysis of AML-EVs, and (D) Impact of chemotherapy on EV release by AML cells.

(A) Impact of anti-TLR4 antibodies on MDSC induction, (B) Impact of anti-TLR2 antibodies on pCREB activation, (C) Impact of PrK pre-treatment of AML-EV uptake, (D) Impact of chemotherapy on protein palmitoylation in AML-cells, and (E) Impact of 2-BP on EV release by AML-cells.

Changes of mitochondrial (A) membrane potential, (B) ROS production, and (C) mass in monocytes upon AML-EV uptake.

(A) Induction of MDSCs by AML-EVs released by chemotherapy-pretreated AML celllines, (B) Phagocytotic activity of monocytes upon AML-EV uptake, (C) Induction of MDSCs by AML-EVs isolated by gradient ultracentrifugation, (D) Induction of pCREB by AML-EVs.

(A) CD33 expression on AML-EVs, (B) Cytokine-content of AML-EVs, (C) Localization analysis of AML-EVs in monocytes, (D) Control experiment regarding EV-independent uptake of PKH26.

Acute myeloid leukemia (AML) represents the most common acute leukemia among adults. Despite recent progress in diagnosis and treatment, long-term outcome remains unsatisfactory. The success of allogeneic stem cell transplantation underscores the immunoresponsive nature of AML, creating the basis for further exploiting immunotherapies. However, emerging evidence suggests that AML, similar to other malignant entities, employs a variety of mechanisms to evade immunosurveillance. In light of this, T-cell inhibitory myeloid-derived suppressor cells (MDSC) are gaining interest as key facilitators of immunoescape. Accumulation of CD14+HLA-DRlow monocytic MDSCs has been described in newly diagnosed AML patients, and deciphering the underlying mechanisms could help to improve anti-AML immunity. Here, we report that conventional monocytes readily take-up AML-derived extracellular vesicles (EV) and subsequently undergo MDSC differentiation. They acquired an CD14+HLA-DRlow phenotype, expressed the immunomodulatory indoleamine-2,3-dioxygenase, and upregulated expression of genes characteristic for MDSCs, such as S100A8/9 and cEBPβ. The Akt/mTOR pathway played a critical role in the AML-EV–induced phenotypical and functional transition of monocytes. Generated MDSCs displayed a glycolytic switch, which rendered them more susceptible toward glycolytic inhibitors. Furthermore, palmitoylated proteins on the AML-EV surface activated Toll-like receptor 2 as the initiating event of Akt/mTOR-dependent induction of MDSC. Therefore, targeting protein palmitoylation in AML blasts could block MDSC accumulation to improve immune responses.Significance:These findings indicate that targeting protein palmitoylation in AML could interfere with the leukemogenic potential and block MDSC accumulation to improve immunity.

Information regarding patient characteristics, antibodies, and primers.

Extracellular vesicles (EVs) have been proposed to regulate the deposition of Aβ. Multiple publications have shown that APP, amyloid processing enzymes and Aβ peptides are associated with EVs. However, very little Aβ is associated with EVs compared with the total amount Aβ present in human plasma, CSF, or supernatants from cultured neurons. The involvement of EVs has largely been inferred by pharmacological inhibition or whole body deletion of the sphingomyelin hydrolase neutral sphingomyelinase-2 (nSMase2) that is a key regulator for the biogenesis of at-least one population of EVs. Here we used a Cre-Lox system to selectively delete nSMase2 from pyramidal neurons in APP/PS1 mice (APP/PS1-SMPD3-Nex1) and found a ~ 70% reduction in Aβ deposition at 6 months of age and ~ 35% reduction at 12 months of age in both cortex and hippocampus. Brain ceramides were increased in APP/PS1 compared with Wt mice, but were similar to Wt in APP/PS1-SMPD3-Nex1 mice suggesting that elevated brain ceramides in this model involves neuronally expressed nSMase2. Reduced levels of PSD95 and deficits of long-term potentiation in APP/PS1 mice were normalized in APP/PS1-SMPD3-Nex1 mice. In contrast, elevated levels of IL-1β, IL-8 and TNFα in APP/PS1 mice were not normalized in APP/PS1-SMPD3-Nex1 mice compared with APP/PS1 mice. Mechanistic studies showed that the size of liquid ordered membrane microdomains was increased in APP/PS1 mice, as were the amounts of APP and BACE1 localized to these microdomains. Pharmacological inhibition of nSMase2 activity with PDDC reduced the size of the liquid ordered membrane microdomains, reduced the localization of APP with BACE1 and reduced the production of Aβ

The design and development of novel adjuvants, which enhance, accelerate, and prolong the immune responses triggered by antigens, hold great importance for targeting infectious diseases and cancer. Here, we designed a biotinylated peptide amphiphile (Biotin-PA) nanofiber, which serves as a noncovalent binding location for antigens. We showed that presenting the antigens on synthetic Biotin-PA nanofibers generated a higher immune response than the free antigens delivered with a CpG ODN (TLR9 agonist) adjuvant. Antigen attached Biotin-PA nanofibers triggered splenocytes to produce high levels of cytokines (IFN-γ, IL-12, TNF-α, and IL-6) and to exhibit a superior cross-presentation of the antigen. Both Biotin-PA nanofibers and CpG ODN induce a Th-1-biased IgG subclass response; however, delivering the antigen with Biotin-PA nanofibers induced significantly greater production of total IgG and subclasses of IgG compared to delivering the antigen with CpG ODN. Contrary to CpG ODN, Biotin-PA nanofibers also enhanced antigen-specific splenocyte proliferation and increased the proportion of the antigen-specific CD8(+) T cells. Given their biodegradability and biocompatibility, Biotin-PA nanofibers have a significant potential in immunoengineering applications as a biomaterial for the delivery of a diverse set of antigens derived from intracellular pathogens, emerging viral diseases such as COVID-19, or cancer cells to induce humoral and cellular immune responses against the antigens. This article is protected by copyright. All rights reserved.

Acute myeloid leukemia (AML) represents the most common acute leukemia among adults. Despite recent progress in diagnosis and treatment, long-term outcome remains unsatisfactory. The success of allogeneic stem cell transplantation underscores the immunoresponsive nature of AML, creating the basis for further exploiting immunotherapies. However, emerging evidence suggests that AML, similar to other malignant entities, employs a variety of mechanisms to evade immunosurveillance. In light of this, T-cell inhibitory myeloid-derived suppressor cells (MDSC) are gaining interest as key facilitators of immunoescape. Accumulation of CD14+HLA-DRlow monocytic MDSCs has been described in newly diagnosed AML patients, and deciphering the underlying mechanisms could help to improve anti-AML immunity. Here, we report that conventional monocytes readily take-up AML-derived extracellular vesicles (EV) and subsequently undergo MDSC differentiation. They acquired an CD14+HLA-DRlow phenotype, expressed the immunomodulatory indoleamine-2,3-dioxygenase, and upregulated expression of genes characteristic for MDSCs, such as S100A8/9 and cEBPβ. The Akt/mTOR pathway played a critical role in the AML-EV–induced phenotypical and functional transition of monocytes. Generated MDSCs displayed a glycolytic switch, which rendered them more susceptible toward glycolytic inhibitors. Furthermore, palmitoylated proteins on the AML-EV surface activated Toll-like receptor 2 as the initiating event of Akt/mTOR-dependent induction of MDSC. Therefore, targeting protein palmitoylation in AML blasts could block MDSC accumulation to improve immune responses. Significance: These findings indicate that targeting protein palmitoylation in AML could interfere with the leukemogenic potential and block MDSC accumulation to improve immunity.

Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 30%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. Here, we demonstrate increased frequencies of CD14+ monocytic MDSCs in newly diagnosed AML that co-express CD33 but lack HLA-DR (HLA-DRlo). AML-blasts induce HLA-DRlo cells from healthy donor-derived monocytes in vitro that suppress T-cells and express indoleamine-2,3-dioxygenase (IDO). We investigated whether a CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with pre-clinical activity against AML-blasts by redirection of T-cells can eradicate CD33+ MDSCs. In fact, T-cells eliminate IDO+CD33+ MDSCs in the presence of AMG 330. Depletion of total CD14+ cells (including MDSCs) in peripheral blood mononuclear cells from AML patients did not enhance AMG 330-triggered T-cell activation and expansion, but boosted AML-blast lysis. This finding was corroborated in experiments showing that adding MDSCs into co-cultures of T- and AML-cells reduced AML-blast killing, while IDO inhibition promotes AMG 330-mediated clearance of AML-blasts. Taken together, our results suggest that AMG 330 may achieve anti-leukemic efficacy not only through T-cell-mediated cytotoxicity against AML-blasts but also against CD33+ MDSCs, suggesting that it is worth exploring the predictive role of MDSCs for responsiveness towards an AMG 330-based therapy. Electronic supplementary material The online version of this article (10.1186/s40425-018-0432-9) contains supplementary material, which is available to authorized users.

Table S1. Fluorochrome-coupled antibodies and/or chemical dyes for flow cytometry. Table S2. AML-derived PBMCs (n=12) were treated with AMG 330 for three days. The median fluorescence intensity (MFI) of granzyme B (Grz B), CD107, perforin, CD69, CD137, CD25, CD154, IL2, IFNγ, and the cells’ expansion index was assessed by FACS in CD4+/CD8+ CD3+ T-cells as indicated. The association between those variables and the PBMCs’ initial frequency of HLA-DRlo cells among CD14+ cells was calculated using a Pearson correlation analysis. Abbreviations: p, p-value; r, Pearson correlation. Table S3. AML-derived PBMCs (n=12) were treated with AMG 330 for three days. The median fluorescence intensity (MFI) of granzyme B (Grz B), CD107, perforin, CD69, CD137, CD25, CD154, IL2, and IFNγ was assessed by FACS in CD4+/CD8+ CD3+ T-cells. The association between those variables and the PBMCs’ initial frequency of CD3+ T-cells was calculated using a Pearson correlation analysis. Abbreviations: p, p-value; r, Pearson correlation. (DOCX 50 kb)

The design and development of vaccines, which can induce cellular immunity, particularly CD8+ T cells hold great importance since these cells play crucial roles against cancers and viral infections. Covalent conjugation of antigen and adjuvant molecules has been used for successful promotion of immunogenicity in subunit vaccines; however, the stimulation of the CD8+ T-cell responses by this approach has so far been limited. This study demonstrates a modular system based on noncovalent attachment of biotinylated antigen to a hybrid nanofiber system consisting of biotinylated self-assembling peptide and CpG oligodeoxynucleotides (ODN) molecules, via biotin-streptavidin interaction. These peptide/oligonucleotide hybrid nanosystems are capable of bypassing prior limitations related with inactivated or live-attenuated virus vaccines and achieve exceptionally high CD8+ T-cell responses. The nanostructures are found to trigger strong IgG response and effectively modulate cross-presentation of their antigen "cargo" through close proximity between the antigen and peptide/ODN adjuvant system. In addition, the biotinylated peptide nanofiber system is able to enhance antigen uptake and induce the maturation of antigen-presenting cells. Due to its versatility, biocompatibility, and biodegradability with a broad variety of streptavidin-linked antigens, the nanosystem shown here can be utilized as an efficient strategy for new vaccine development.

There is an urgent need for more efficient treatment of chronic wounds in diabetic patients especially with a high risk of leg amputation. Biomaterials capable of presenting extracellular matrix-mimetic signals may assist in the recovery of diabetic wounds by creating a more conducive environment for blood vessel formation and modulating the immune system. In a previous study, we showed that glycosaminoglycan-mimetic peptide nanofibers are able to increase the rate of closure in STZ-induced diabetic rats by induction of angiogenesis. The present study investigates the effect of a heparin-mimetic peptide amphiphile (PA) nanofiber gel on full-thickness excisional wounds in a db/db diabetic mouse model, with emphasis on the ability of the PA nanofiber network to regulate angiogenesis and the expression of pro-inflammatory cytokines. Here, we showed that the heparin-mimetic PA gel can support tissue neovascularization, enhance the deposition of collagen and expression of alpha-smooth muscle actin (α-SMA), and eliminate the sustained presence of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the diabetic wound site. As the absence of neovascularization and overexpression of pro-inflammatory markers are a hallmark of diabetes and interfere with wound recovery by preventing the healing process, the heparin-mimetic PA treatment is a promising candidate for acceleration of diabetic wound healing by modulating angiogenesis and local immune response. © 2017 The Royal Society of Chemistry.

Synthetic vaccines utilize viral signatures to trigger immune responses. Although the immune responses raised against the biochemical signatures of viruses are well characterized, the mechanism of how they affect immune response in the context of physical signatures is not well studied. In this work, we investigated the ability of zero- and one-dimensional self-assembled peptide nanostructures carrying unmethylated CpG motifs (signature of viral DNA) for tuning immune response. These nanostructures represent the two most common viral shapes, spheres and rods. The nanofibrous structures were found to direct immune response towards Th1 phenotype, which is responsible for acting against intracellular pathogens such as viruses, to a greater extent than nanospheres and CpG ODN alone. In addition, nanofibers exhibited enhanced uptake into dendritic cells compared to nanospheres or the ODN itself. The chemical stability of the ODN against nuclease-mediated degradation was also observed to be enhanced when complexed with the peptide nanostructures. In vivo studies showed that nanofibers promoted antigen-specific IgG production over 10-fold better than CpG ODN alone. To the best of our knowledge, this is the first report showing the modulation of the nature of an immune response through the shape of the carrier system.