Induce T-cell Proliferation Research Articles

Evaluating Nanoparticulate Vaccine Formulations for Effective Antigen Presentation and T-Cell Proliferation Using an In Vitro Overlay Assay.

Inducing T lymphocyte (T-cell) activation and proliferation with specificity against a pathogen is crucial in vaccine formulation. Assessing vaccine candidates' ability to induce T-cell proliferation helps optimize formulation for its safety, immunogenicity, and efficacy. Our in-house vaccine candidates use microparticles (MPs) and nanoparticles (NPs) to enhance antigen stability and target delivery to antigen-presenting cells (APCs), providing improved immunogenicity. Typically, vaccine formulations are screened for safety and immunostimulatory effects using in vitro methods, but extensive animal testing is often required to assess immunogenic responses. We identified the need for a rapid, intermediate screening process to select promising candidates before advancing to expensive and time-consuming in vivo evaluations. In this study, an in vitro overlay assay system was demonstrated as an effective high-throughput preclinical testing method to evaluate the immunogenic properties of early-stage vaccine formulations. The overlay assay's effectiveness in testing particulate vaccine candidates for immunogenic responses has been evaluated by optimizing the carboxyfluorescein succinimidyl ester (CFSE) T-cell proliferation assay. DCs were overlaid with T-cells, allowing vaccine-stimulated DCs to present antigens to CFSE-stained T-cells. T-cell proliferation was quantified using flow cytometry on days 0, 1, 2, 4, and 6 upon successful antigen presentation. The assay was tested with nanoparticulate vaccine formulations targeting Neisseria gonorrhoeae (CDC F62, FA19, FA1090), measles, H1N1 flu prototype, canine coronavirus, and Zika, with adjuvants including Alhydrogel® (Alum) and AddaVax™. The assay revealed robust T-cell proliferation in the vaccine treatment groups, with variations between bacterial and viral vaccine candidates. A dose-dependent study indicated immune stimulation varied with antigen dose. These findings highlight the assay's potential to differentiate and quantify effective antigen presentation, providing valuable insights for developing and optimizing vaccine formulations.

The MARIANE-trial: Multicenter phase 1b/2 trial testing safety and efficacy of neoadjuvant intradermal ipilimumab and nivolumab in high-risk stage II melanoma.

TPS9615 Background: Adjuvant anti-PD1 (nivolumab or pembrolizumab) has been shown to improve relapse free survival for patients with high-risk stage II melanoma (IIB-C). However, this therapy probably won’t be reimbursed as standard of care in most European countries due to high costs, and the benefit-risk-ratio. For macroscopic stage III melanoma, neoadjuvant ipilimumab (IPI; anti-CTLA4) plus nivolumab (NIVO; anti-PD1) has been shown to induce high pathologic response rates in the resected lymph nodes, long-term event free survival, and this schedule is currently being compared to adjuvant NIVO in a phase 3 study (NCT04949113). This combination schedule, however, is unsuitable for earlier disease stages, due to high costs and clinical toxicity. In preclinical models, a local tumor draining lymph node directed delivery of anti-PD-1 plus anti-CTLA-4 has been demonstrated to outperform systemic administration on induction of T-cell proliferation and tumor control. In patients, local delivery of anti-PD-1 or anti-CTLA-4 has been suggested to be equally effective in tumor control with a reduction of adverse events. These data have formed the rationale for testing intradermal injections at the excision site of the primary tumor with IPI and NIVO prior to the re-excision and sentinel node procedure in high-risk stage II melanoma patients. Methods: This multicenter, dose-escalating phase Ib/II trial, using a Simon’s two-stage design, is the first trial to test feasibility and efficacy of intradermal IPI plus NIVO in high-risk stage II melanoma patients. In total 21 to 80 patients, diagnosed with a stage T3-4 cutaneous melanoma with ≥ 50% chance of positive sentinel node, naïve for re-excision, sentinel node surgery and immunotherapy, will be included in this trial. Patients will receive 2 cycles of intradermal IPI 0.5 mg + NIVO 1 mg at the excision site of the primary melanoma (every 3 weeks; arm A); 6 cycles intradermal IPI 0.5 mg + NIVO 1 mg (every week; arm B); 2 cycles intradermal IPI 10 mg + NIVO 20 mg (every 3 weeks, arm C); or 2 cycles of intravenous nivolumab 240 mg every 3 weeks combined with the most optimal intradermal regimen of IPI + NIVO (based on the results of arm A, B or C) in arm D. In week 6, patients will undergo sentinel node surgery, whereafter only patients with a non-MPR (>10% vital tumor) will be treated with adjuvant anti-PD1 according to current standard of care. We expect the first patient to be enrolled in March 2024and to be accruing for 1-1.5 years. Clinical trial information: NCT06240143 .

A comprehensive review uncovering the anticancerous potential of genkwanin (plant-derived compound) in several human carcinomas

Abstract Plant-derived bioactive compounds displayed major therapeutic and chemo-preventive roles in the pathogenesis of numerous chronic malignancies such as cancer and enhanced oxidative stress and inflammation. Antioxidants found in food, such as genkwanin, may reduce oxidative stress and the release of cytokines or pathways that promote inflammation. The goal of this work is to summarize the potential for anticancer effects of genkwanin, a methoxyflavone that is present in a variety of plant species. This review examined and analyzed numerous research studies on identifying, isolating, measuring, and analyzing anticancer properties of genkwanin. The mechanisms involved cellular and molecular activities at various levels, including apoptosis induction and cancer cell growth and proliferation inhibition. Preclinical studies have demonstrated genkwanin’s effects and mechanism of action; however, further research is required to investigate its therapeutic potential thoroughly. Additional research is needed to further our understanding of the pharmacodynamic effects of genkwanin. Additional toxicological study is necessary to evaluate the clinical efficacy and safety of genkwanin, which would help scientists to elucidate a potent drug candidate for cancer management.

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity

BackgroundThe paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor...

SiRNA-Mediated B7H7 Knockdown in Gastric Cancer Lysate-Loaded Dendritic Cells Amplifies Expansion and Cytokine Secretion of Autologous T Cells.

Gastric cancer, ranked as the fifth most common cancer worldwide, presents multiple treatment challenges. These obstacles often arise due to cancer stem cells, which are associated with recurrence, metastasis, and drug resistance. While dendritic cell (DC)-based immunotherapy has shown promise as a therapeutic strategy, its efficacy can be limited by the tumor microenvironment and certain inhibitory immune checkpoint molecules, such as B7H7. SiRNA-medicated knockdown of B7H7 in tumor cell lysate-pulsed DCs can increase cytokine secretion and autologous T lymphocyte expansion. This study aimed to evaluate the impact of B7H7 suppression in gastric cancer cell lysate-pulsed DCs on the stimulatory potential of autologous CD3+ T lymphocytes. Peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were obtained; then, they were differentiated to immature DCs (iDCs) by GM-CSF and IL-4. Tumor cell lysates from human gastric cancer cell lines were harvested, and iDCs were transformed into mature DCs (mDCs) by stimulating iDCs with tumor cell lysate and lipopolysaccharide. B7H7-siRNA was delivered into mDCs using electroporation, and gene silencing efficiency was assessed. The phenotypic characteristics of iDCs, mDCs, and B7H7-silenced mDCs were evaluated using specific surface markers, an inverted light microscope, and flow cytometry. CD3+ T cells were isolated via magnetically activated cell sorting. They were labeled with CFSE dye and co-cultured with mDCs and B7H7-silenced mDCs to evaluate their ability to induce T-cell proliferation. T-cell proliferation was assessed using flow cytometry. The concentration of TGF-β, IL-4, and IFN-γ secreted from CD3+ T cells in the co-cultured supernatant was evaluated to investigate the cytokine secretory activity of the cells. Transfection of B7H7 siRNA into mDCs was performed in optimal conditions, and the siRNA transfection effectively reduced B7H7 mRNA expression in a dose-dependent manner. SiRNA-mediated B7H7 knockdown in mDCs enhanced maturation and activation of the DCs, as demonstrated by an increased surface expression of CD11c, CD86, and CD40. Co-culture experiments revealed that B7H7-silenced mDCs had more capacity to induce T cell proliferation compared to non-transfected mDCs. The cytokine production patterns of T cells were also altered. Upon examining the levels of TGF-β, IL-4, and IFN-γ released by CD3+ T cells in the co-culture supernatant, we found that silencing B7H7 in mDCs resulted in a rise in IL-4 secretion and a reduction in TGF-β levels compared to mDCs that were not transfected. The study found that suppressing B7H7 expression in DCs significantly enhances their maturation and stimulatory activity when exposed to gastric cancer cell lysate. These B7H7-silenced DCs can substantially increase cytokine production and promote co-cultured T-cell expansion. Consequently, inhibiting B7H7 in DCs may offer a practical strategy to enhance the ability of DCs to initiate T lymphocyte responses and improve the effectiveness of DC-based cell therapy for cancer patients.

BCMA-Targeted Bortezomib Nanotherapy (BCMA-BTZ-Nps) Targets Tumor, Enhances Therapeutic Efficacy, Triggers Immunogenic Cell Death, Overcomes Drug Resistance, and Reduces Off-Target Toxicity in Multiple Myeloma (MM)

Bortezomib (BTZ) is a standard-of-care treatment for multiple myeloma (MM). In addition to its direct anti-MM effects, we recently reported that BTZ triggers immunogenic cell death associated with improved outcome. However, adverse effects limit its long-term clinical use. To improve its therapeutic index, we encapsulated bortezomib in PEG-PLGA nanoparticles linked with antibodies that target B-cell maturation antigen (BCMA-BTZ-NPs) to specifically target MM cells. BCMA-BTZ-NPs showed significantly increased cellular internalization into wild-type (WT) H929 cells after 6h (99.2±0.7%) than non-targeted NPs (57.7±0.8%) (p<0.0001), with a time-dependent increase in internalization. Importantly, cellular internalization of the BCMA-BTZ-NPs in BCMA-KO H929 cells (15.9±2.5%) was significantly depleted (p<0.0001). BCMA-BTZ-NPs showed target-specific cytotoxicity against MM cell lines and primary tumor cells from MM patients. Furthermore, BCMA-BTZ-NPs induced cell death more efficiently than non-targeted nanoparticles or free BTZ, triggering potent mitochondrial depolarization assessed by JC1 staining in MM.1S (79.06±3.7% vs. 57.4±1.9% for BCMA-BTZ-NPs vs non-targeted NPs, p<0.01); and H929 cells (47.23±1.9% vs. 31.26±0.7% for BCMA-BTZ-NPs vs. non-targeted NPs, p<0.001). This was associated with enhanced activation of caspases 8, 9 and 3, followed by apoptosis. We also found that BCMA-BTZ-NPs were still effective in BTZ-resistant MM cell lines (36.66±1.8% residual chymotryptic activity in RPMI-Dox40 [resistant] and 40.29±1.0% in MM.1S [sensitive] cells), whereas free BTZ showed no inhibitory effect (100.01±4.6% residual chymotryptic activity in RPMI-Dox40), likely due to BCMA-BTZ-NPs entering the cell through receptor-mediated uptake which averts acquired BTZ resistance based on the drug pump P-glycoprotein (PgP). Importantly, BCMA-BTZ-NPs also enhanced immunogenic cell death (ICD) and activated the autophagic pathway more than free BTZ, evidenced by increased calreticulin cell surface exposure (free BTZ: 45.13±3.7% vs BCMA-BTZ-NP: 71.5±3.4%,) and induction of T-cell proliferation in both MM.1S (free BTZ: 36.5±2.6% vs. BCMA-BTZ-NP: 65.2±2.3%, p<0.001) and AMO-1 cells (free BTZ: 37.03±1.3% vs. BCMA-BTZ-NP: 66.33±3.3%, p<0.001). Of note, increased levels of TNFα, Granzyme B, and IFNγ were also present in BCMA-BTZ-NPs treated MM cells with DC (Dendritic cells) and T cells co-culture supernatants. Finally, we evaluated the in vivo target selectivity and therapeutic efficacy of BCMA-BTZ-NPs against human MM cells in a murine xenograft model. BCMA-BTZ-NPs selectively accumulated in the tumor more efficiently than non-targeted BTZ-NPs and were associated with more efficient tumor reduction and host survival. In summary, our study indicates that BCMA-BTZ nanotherapy accumulates at the tumor site, enhances therapeutic efficacy, triggers immunogenic cell death, overcomes drug resistance, and reduces off-target toxicity. These results provide the framework for clinical evaluation of BCMA-BTZ-NPs to increase the therapeutic index of BTZ and improve patient outcome in MM.

Outcomes Analysis of Lenalidomide-Induced Rashes in Myeloma Patients

CONCLUSIONS Lenalidomide is an immunomodulatory drug fundamental in treating and managing patients with multiple myeloma (MM). It works through various pathways to induce T-cell proliferation and activation of natural killer (NK) cells. This mechanism of T-cell activation is hypothesized to be one of the mechanisms that cause lenalidomide-related skin reactions. Skin reactions are a well-known adverse effect of lenalidomide treatment, with some studies showing that 44% of patients developed a rash while receiving treatment. Rash development has been associated with improved outcomes with other chemotherapy agents in patients with solid tumors. Initial investigation demonstrated that lenalidomide-induced rashes in myeloma patients were associated with improved survival. Other immune-mediated adverse reactions, such as lenalidomide-related diarrhea (LRD), have also been associated with improved patient outcomes. Lenalidomide-related diarrhea and rash likely stem from the same pathways involving the activation of Th2 and NK cells. This single-site retrospective study analyzed charts from patients with multiple myeloma whose treatment course included lenalidomide single therapy or lenalidomide plus dexamethasone therapy. Patients who were only treated with lenalidomide in combination with other chemotherapies were excluded from the study. The development of a rash while receiving lenalidomide single therapy was recorded. We also recorded the number of patients who developed severe diarrhea requiring hospitalization or a change in therapy regimen. Other adverse reactions that altered lenalidomide treatment were also recorded. Outcomes were assessed using 5-year survival (5YS), overall survival (OS), and time to next treatment (TTNT). We analyzed the charts of 100 patients who met the criteria for the study and found that 25 (25%) patients developed rashes compared to 75 (75%) that did not. There was not a significant difference in OS or 5YS between the rash group compared to the non-rash groups [(OS: 40.0%, 38.7%, p = 0.90); (5YS: 68%, 52%, p = 0.196) respectively]. There was a statistically significant difference in TTNT between the rash group and the non-rash group (43.0 months, 21.2 months, p = 0.035). We compared 31 (31%) patients who displayed immune-related adverse effects (diarrhea and/or rash) to the 69 (69%) patients who developed no rashes or diarrhea. In these groups, there was a significant difference in TNTT and 5YS ( p = 0.035; p = 0.017 respectively) but not in OS ( p = 0.857). TTNT was measured in this study and is similar to progression-free survival, as patients whose diseases progressed initiated another treatment. We observed similar results to previous studies showing that patients who developed rashes on lenalidomide exhibited longer TTNT. We observed that patients who developed rashes or LRD exhibited longer TTNT and had significantly higher 5-year survival. These reactions may be manifestations of an increased immune response contributing to their improved response to lenalidomide. Many patients with multiple myeloma undergo complex treatment regimens involving multiple therapies. We observed occasions when patients developed drug rashes on bortezomib, lenalidomide, and dexamethasone triple therapy but didn't develop a rash later on lenalidomide maintenance therapy. To isolate the rash to lenalidomide, these rashes were not counted in our study, skewing the number of patients counted as developing rashes.

An Immune-suppressive Triterpenoid from the Stem of Kadsura coccinea

Abstract: Kadsura coccinea has been used as a folk medicine in China, treating various diseases, including chronic gastritis and rheumatoid arthritis (RA). Chemical studies have revealed that lignans and triterpenes are the main constituents of its roots and stems. In this paper, a chemical investigation on the stem of K. coccinea was performed and a previously undescribed 3,4-secolanostane (1) was afforded. The structure of 1 was determined through comprehensive NMR and HRESI-MS data analysis. The stereo-configuration of 1 was established based on a comparison of experimental and calculated ECD spectra. Bioactivity assay carried out in immune cells indicated that 1 could show moderate effects on ConA (5 μg/mL)-induced T-cell proliferation with an IC50 value of 13.28 μM (cyclosporine A as the positive drug, IC50 = 0.13 μM) while the IC50 value was 23.12 μM in LPS (10 μg/mL)-induced B cells (mycophenolate mofetil as the positive drug, IC50 = 16.71 μM).

Inhibitor of Apoptosis Proteins Antagonist Induces T-cell Proliferation after Cross-Presentation by Dendritic Cells.

Cross-presentation of tumor antigens by dendritic cells (DC) is crucial to prime, stimulate and restimulate CD8+ T cells. This process is important in initiating and maintaining an antitumor response. Here, we show that the presence of conventional type 1 DCs (cDC1), a DC subtype that excels in cross-presentation, in the tumor correlated with response to neoadjuvant immune checkpoint blockade (ICB) in melanoma. This led us to hypothesize that patients failing to respond to ICB could benefit from enhanced cross-presentation of tumor antigens. We therefore established a cross-presentation assay to screen over 5,500 compounds for enhancers of DC cross-presentation using induced T-cell proliferation as the readout. We identified 145 enhancers, including AZD5582, an antagonist of inhibitor of apoptosis proteins (IAP) cIAP1, cIAP2, and XIAP. AZD5582 treatment led to DC activation of the noncanonical NF-kB pathway, enhanced antigen import from endolysosomes into the cytosol, and increased expression of genes involved in cross-presentation. Furthermore, it upregulated expression of CD80, CD86, MHC class II, CD70 and secretion of TNF by DCs. This enhanced DC activation and maturation program was observed also in tumor-bearing mice upon AZD5582 treatment, culminating in an increased frequency of systemic tumor antigen-specific CD8+ T cells. Our results merit further exploration of AZD5582 to increase antigen cross-presentation for improving the clinical benefit of ICB in patients who are unlikely to respond to ICB.

Complement protein C3a enhances adaptive immune responses towards FVIII products.

The most serious complication in the treatment of hemophilia A (HA) is the development of factor (F)VIII inhibitors or antidrug antibodies (ADA) occurring in 25-35% of patients with severe HA. The immunological mechanisms underlying the development of ADA against FVIII products have not been completely understood yet. Immunological danger signals associated with events such as infection or surgery have been suggested to play a critical role. In previous studies, we demonstrated that plasma-derived (pd)FVIII but not recombinant (r)FVIII can activate human monocyte-derived dendritic cells (DC) in a danger signal-dependent manner, which subsequently mediate the proliferation of autologous CD4+ T cells. In this study, we investigated the ability of plasma components, naturally present in pdFVIII products, to mediate T-cell responses. In fact, we show that addition of plasma to rFVIII plus lipopolysaccharide (LPS)-stimulated DC induces proliferation of autologous CD4+ T cells. Interestingly, although DC pulsed with LPS plus plasma induce T-cell proliferation upon co-culture, the addition of FVIII significantly increases the number of proliferating as well as FVIII-specific CD4+ T cells. Total proliferating CD4+ T cells and FVIII-specific subsets were identified mainly as central memory T cells. Experiments using blocking antibodies and receptor antagonists revealed that the complement proteins C3a and, to a lesser extent, C5a are critically involved in these LPS-mediated T-cell responses. Collectively, our results indicate that complement proteins are potent drivers of T-cell responses to FVIII. Data presented provide a model how event-related substitution of FVIII in HA patients might contribute to inhibitor development.

ХАРАКТЕРИСТИКА ПАРАМЕТРОВ ИММУНИТЕТА ПРИ ПРИМЕНЕНИИ РЕКОМБИНАНТНОГО ЧЕЛОВЕЧЕСКОГО ИНТЕРЛЕЙКИНА-2

Recombinant human interleukin-2 induces T-cell proliferation and is a central regulator of immune responses. Objective: to study the parameters of immunity when using rIL-2. Materials and Methods. As part of the scientific process, methods were applied to determine circulating immune formations using precipitation reactions with polyethylene glycol, as well as to assess the function of phagocytosis by leukocytes according to the method proposed by Slavskaya and Berman. The SOLAR PV 1251C was used to collect spectral data and morphological observations were made using optical microscopy. The processing and interpretation of the results were carried out using the Statistica 10.0 software. Results. In the main group, a significantly higher concentration of Ig A was observed than in the comparison group No. 2 – Ig G (p ˂ 0.05). The phagocytosis completion index and the absolute phagocytic index were at a level below normal values only in comparison groups No. 1 and No. 2, in which cytokine preparations were not administered, and in the rIL-2 application group (main group), these parameters were at a normal level, which indicates on the effectiveness of the use of this drug in terms of maintaining both absorption and digestion phagocytic functions. Conclusions Recombinant cytokines act exclusively within the wound, indicating not only the effectiveness, but also the safety of this group of drugs in patients with SSI.

Cleistanthins A and B Ameliorate Testosterone-Induced Benign Prostatic Hyperplasia in Castrated Rats by Regulating Apoptosis and Cell Differentiation.

Background The aging male population is at higher risk for benign prostatic hyperplasia (BPH) wherein increased proliferation of stromal and epithelial cells of the prostate is observed. In this study, we investigated the effect of cleistanthins A and B on the inhibition of testosterone-induced BPH in castrated rats. Methodology Male Wistar rats were divided into eight groups (n = 6) and surgical castration was performed. BPH was induced by the administration of testosterone propionate in corn oil at 5 mg/kg for four weeks. The control group received corn oil, and the model group received testosterone propionate. The standard treatment group received finasteride orally along with testosterone. Cleistanthins A and B at 0.3, 1, and 3 mg/kg were administered by oral gavage along with testosterone. After four weeks, rats were sacrificed, and prostates were weighed and assessed for histomorphological, inflammatory, apoptotic, and proliferative markers. Results Cleistanthins A and B decreased prostatic enlargement and histopathological abnormalities. Elevated serum dihydrotestosterone levels were lowered significantly in both the cleistanthin A andcleistanthin B groups compared to the BPH model group. Cleistanthins A and B significantly lowered the serum interleukin (IL)-1β and tumor necrosis factor-alpha inflammatory markers in the test groups. Western blot analysis revealed cleistanthin A downregulated the IL-6, signal transducer and activator of transcription 3/cyclin D1 signaling pathway. Both cleistanthins A and B upregulated the apoptotic markers caspase-3 and cleaved caspase-3, whereas the cell proliferation markers cyclin D1 and proliferating cell nuclear antigen were found to be downregulated. Conclusions Both cleistanthins A and B inhibited BPH in a rat model by apoptotic induction and impeded cell proliferation.

IGFBP3 inhibits tumor growth and invasion of lung cancer cells and is associated with improved survival in lung cancer patients

The insulin-like growth factor (IGF)-pathway is involved in tumor cell proliferation, metastasis, and survival. We aimed to find out what effects IGF binding protein 3 (IGFBP3) exerted on H1299 lung cancer (LC) cells in terms of tumor growth and invasion and whether IGFBP3 was associated with clinical and pathological parameters in a prospective cohort of LC patients. H1299 cells were transfected with an IGFBP3-expressing vector. Its influence on apoptosis induction via flow cytometry annexin V FITC assay, cell proliferation in 2D and 3D cell culture, and invasion were examined. Expression of several matrix metalloproteinases (MMPs) and inhibitors (TIMP-1) were also investigated in IGFBP3-transfected LC cells. Further, data on LC patients (n = 131), tumor characteristics, and survival were prospectively collected and correlated with IGFBP3 plasma levels. IGFBP3 did not influence apoptosis induction and 2D cell proliferation. However, both spheroid growth (3D proliferation) and invasion of IGFBP3-transfected cells planted in an extracellular matrix-based gel were significantly inhibited. IGFBP3 inhibited MMP-1 release, and the total MMP activity. In LC patients, higher IGFBP3 plasma levels correlated with both lower clinical tumor stage, grading, Ki-67 staining, and the absence of necrosis (P < 0.05, respectively). Increased IGFBP3 plasma levels were associated with improved overall survival (hazard ratio 0.37, P = 0.01). In conclusion, overexpressed IGFBP3 in a LC cell line inhibited tumor growth and invasion. Translating from bench to bedside, investigation of clinicopathological parameters confirmed these experimental results showing that higher IGFBP3 plasma levels were associated with less aggressive tumor growth, reduced tumor spread, and improved survival of LC patients.

Inhibition of O-GlcNAcase Inhibits Hematopoietic and Leukemic Stem Cell Self-Renewal and Drives Dendritic Cell Differentiation via STAT3/5 Signaling.

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34+ hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34+ HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T-cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage-agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages.

Establishment of the “Valsa pyri metabolites (VpM)-suspension cell”-based system to study the response of pears to VpM

Valsa canker, caused by Valsa pyri (Vp), is a necrotrophic fungal disease that seriously damaged the pear industry in the world. Investigations on resistant mechanisms are helpful for resistance breeding and effective control methods. The current study obtained suspension cells of a resistant rootstock ‘Duli-G03’ (Pyrus betulifolia bunge) and a susceptible species ‘Yuluxiang’ (Pyrus bretschneideri Rehd.). Furthermore, the study explored the culture methods of callus and suspension cells of both varieties. Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L 6-benzylaminopurine (6-BA) was suitable for callus induction and suspension cell proliferation of ‘Yuluxiang’, while Nitsch & Nitsch Medium (NN69) with 5 mg/L 6-BA and 0.4 mg/L 1-naphthlcetic acid (NAA) was suitable for ‘Duli-G03’. After suspension cells treated with Vp metabolites (VpM), differentially expressed genes (DEGs) were mainly involved in “signal transduction”, “plant-pathogen interaction”, “plant hormone signal transduction”. Among these, disease resistance protein- and tobacco mosaic virus (TMV) resistance protein-related genes, such as GWHGAAYT037865, GWHGAAYT031455, GWHGAAYT037851, were only promptly induced in ‘Duli-G03’. On the contrary, DEGs related to Hypersensitive response (HR) (gene4043), ethylene (ET) (gene26613) and brassinosteroid (BR) (gene33208) were aroused only in ‘Yuluxiang’. The results supplied important implications for understanding the molecular mechanisms of pear resistance to Valsa canker. Some of the screened genes can be further investigated for disease resistance effects and pave the ground for the mining of disease resistance genes.

Breaking Entry-and Species Barriers: LentiBOOST® Plus Polybrene Enhances Transduction Efficacy of Dendritic Cells and Monocytes by Adenovirus 5.

Due to their ability to trigger strong immune responses, adenoviruses (HAdVs) in general and the serotype5 (HAdV-5) in particular are amongst the most popular viral vectors in research and clinical application. However, efficient transduction using HAdV-5 is predominantly achieved in coxsackie and adenovirus receptor (CAR)-positive cells. In the present study, we used the transduction enhancer LentiBOOST® comprising the polycationic Polybrene to overcome these limitations. Using LentiBOOST®/Polybrene, we yielded transduction rates higher than 50% in murine bone marrow-derived dendritic cells (BMDCs), while maintaining their cytokine expression profile and their capability to induce T-cell proliferation. In human dendritic cells (DCs), we increased the transduction rate from 22% in immature (i)DCs or 43% in mature (m)DCs to more than 80%, without inducing cytotoxicity. While expression of specific maturation markers was slightly upregulated using LentiBOOST®/Polybrene on iDCs, no effect on mDC phenotype or function was observed. Moreover, we achieved efficient HAdV5 transduction also in human monocytes and were able to subsequently differentiate them into proper iDCs and functional mDCs. In summary, we introduce LentiBOOST® comprising Polybrene as a highly potent adenoviral transduction agent for new in-vitro applications in a set of different immune cells in both mice and humans.

Effect of topical application of lipopolysaccharide on contact hypersensitivity

Lipopolysaccharide (LPS) is the principal component of the outer membrane of gram-negative bacteria. The prior oral administration of LPS attenuates inflammatory responses, such as intestinal injury and atopic dermatitis, in mouse models; however, the underlying mechanism remains unclear. Here, we examined the effect of topical LPS application on allergic contact dermatitis and its mechanism of action using a murine contact hypersensitivity (CHS) model. Prolonged LPS application to the skin significantly suppressed 2,4-dinitrofluorobenzene (DNFB)-induced CHS. LPS application to the skin also reduced the phagocytosis of fluorescein isothiocyanate (FITC)-dextran by Langerhans and dendritic cells. Cutaneous cell migration into the skin-draining lymph nodes (LNs) induced by FITC painting was reduced by LPS application. During the CHS response, DNFB application induced T-cell proliferation and inflammatory cytokine production in skin-draining LNs, whereas prolonged LPS application inhibited DNFB-induced T-cell growth and interferon gamma production, indicating suppression of DNFB-induced sensitization. These results suggest that prolonged LPS application suppressed DNFB-induced sensitization and subsequently CHS response. Our findings imply that topical application of LPS may prevent allergic dermatitis such as CHS.

Soluble CD127 potentiates IL-7 activity in vivo in healthy mice.

IntroductionSoluble forms of cytokine receptors can be involved in the endogenous regulation of cytokine activity. Soluble interleukin 7 receptor α (sCD127) naturally binds IL‐7, therefore there is interest in its potential application as an immunotherapeutic agent to regulate IL‐7. With the hypothesis that sCD127 enhances IL‐7 activity, thus promoting T‐cell proliferation in vivo, we sought to assess the effect of sCD127, IL‐7 or IL‐7 + sCD127 treatment on CD4+ and CD8+ T‐cells in the blood and spleen of mice.MethodsPeripheral blood mononuclear cells and splenocytes were prepared, and analyzed for T‐cell number, phenotype and proliferation (Ki67+) by flow cytometry.ResultsIL‐7 treatment induced T‐cell proliferation, increased T‐cell number, and triggered T‐cell differentiation each of which was enhanced with the addition of sCD127. IL‐7 + sCD127 treatment significantly increased spleen weight over that seen with IL‐7 treatment alone. More pronounced proliferation and a greater increase in cell number was observed in CD8+ T‐cells relative to the effect on CD4+ T‐cells.ConclusionsThese findings suggest that the addition of sCD127 enhances IL‐7‐mediated T‐cell proliferation and suggests a potential therapeutic use for sCD127.

Pembrolizumab with low-dose carboplatin for recurrent platinum-resistant ovarian, fallopian tube, and primary peritoneal cancer: survival and immune correlates

BackgroundAnti-programmed death 1 (PD1)/programmed cell death ligand 1 (PD-L1) therapies have shown modest activity as monotherapy in recurrent ovarian cancer. Platinum chemotherapies induce T-cell proliferation and enhance tumor recognition. We...

Modulation of Xenogeneic T-cell Proliferation by B7 and mTOR Blockade of T Cells and Porcine Endothelial Cells.

Activation of porcine endothelial cells (PECs) is the mechanistic centerpiece of xenograft rejection. This study sought to characterize the immuno-phenotype of human T cells in response to PECs and to explore the immuno-modulation of B7 and mammalian target of rapamycin blockade of T cells and/or PECs during xeno-responses. Rapid memory T-cell (TM) responses to PECs were assessed by an intracellular cytokine staining. T-cell proliferation to PEC with or without belatacept or rapamycin was evaluated by a mixed lymphocyte-endothelial cell reaction (MLER). Additionally, rapamycin-pretreated PECs were used in MLER. Cell phenotypes were analyzed by flow cytometry. Tumor necrosis factor-α/interferon-γ producers were detected in CD8+ cells stimulated by human endothelium but not PECs. MLER showed proliferation of CD4+ and CD8+ cells with predominantly memory subsets. Purified memory and naive cells proliferated following PEC stimulation with an increased frequency of TM in PEC-stimulated naive cells. Proliferating cells upregulated programmed cell death-1 (PD-1) and CD2 expression. Belatacept partially inhibited T-cell proliferation with reduced CD2 expression and frequency of the CD8+CD2highCD28- subset. Rapamycin dramatically inhibited PEC-induced T-cell proliferation, and rapamycin-preconditioned PECs failed to induce T-cell proliferation. PD-1 blockade did not restore T-cell proliferation to rapamycin-preconditioned PECs. Humans lack rapid TM-mediated responses to PECs but induce T-cell proliferative responses characterized largely as TM with increasing CD2 and PD-1 expression. B7-CD28 and mammalian target of rapamycin blockade of T cells exhibit dramatic inhibitory effects in altering xeno-proliferating cells. Rapamycin alters PEC xeno-immunogenicity leading to inhibition of xeno-specific T-cell proliferation independent of PD-1-PD ligand interaction.