Activation Of Human Lymphocytes Research Articles

Methods for Analysis of Nanoparticle Immunosuppressive Properties.

Adverse drug effects on immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of drug withdrawal from the market is attributed to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is T cell dependent antibody response (TDAR). This method involves a 28-day in vivo study evaluating the animal's antibody titer to a known antigen (Keyhole Limpet Hemocyanin; KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human lymphocyte activation (HuLA) assay has been developed to substitute the traditional TDAR assay during early preclinical development. In this test, leukocytes isolated from healthy donors vaccinated with the current year's flu vaccine are incubated with Fluzone in the presence or absence of nanoparticles. The antigen-specific lymphocyte proliferation is then measured by ELISA analyzing incorporation of BrdU into DNA of the proliferating cells. Here we describe the experimental procedures for investigating immunosuppressive properties of nanoparticles by both TDAR and HuLA assays, discuss the in vitro-in vivo correlation of these methods, and show a case study using the iron oxide nanoparticle formulation, Feraheme.

Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients.

The comet assay-based in vitro DNA repair assay has become a common tool for quantifying base excision repair (BER) activity in human lymphocytes or cultured cells. Here, we optimized the protocol for studying BER in human placental tissue because the placenta is a non-invasive tissue for biomonitoring of early-life exposures, and it can be used to investigate molecular mechanisms associated with prenatal disorders. The optimal protein concentration of placental protein extracts for optimal damage recognition and incision was 2 mg protein/mL. The addition of aphidicolin did not lead to reduced non-specific incisions and was, therefore, not included in the optimized protocol. The interval between sample collection and analysis did not affect BER activity up to 70 min. Finally, this optimized protocol was tested on pre-eclamptic (PE) placental tissues (n = 11) and significantly lower BER activity in PE placentas compared to controls (n = 9) was observed. This was paralleled by a significant reduction in the expression of BER-related genes and increased DNA oxidation in PE placentas. Our study indicates that BER activity can be determined in placentas, and lower activity is present in PE compared with healthy. These findings should be followed up in prospective clinical investigations to examine BER's role in the advancement of PE.

Impact of chemical mixtures from wastewater treatment plant effluents on human immune cell activation: An effect-based analysis

BackgroundHumans are exposed to many different chemicals on a daily basis, mostly as chemical mixtures, usually from food, consumer products and the environment. Wastewater treatment plant effluent contains mixtures of chemicals that have been discarded or excreted by humans and not removed by water treatment. These effluents contribute directly to water pollution, they are used in agriculture and may affect human health. The possible effect of such chemical mixtures on the immune system has not been characterized. ObjectiveThe aim of this study was to investigate the effect of extracts obtained from four European wastewater treatment plant effluents on human primary immune cell activation. MethodsImmune cells were exposed to the effluent extracts and modulation of cell activation was performed by multi-parameter flow cytometry. Messenger-RNA (mRNA) expression of genes related to immune system and hormone receptors was measured by RT-PCR. ResultsThe exposure of immune cells to these extracts, containing 339 detected chemicals, significantly reduced the activation of human lymphocytes, mainly affecting T helper and mucosal-associated invariant T cells. In addition, basophil activation was also altered upon mixture exposure. Concerning mRNA expression, we observed that 12 transcripts were down-regulated by at least one extract while 11 were up-regulated. Correlation analyses between the analyzed immune parameters and the concentration of chemicals in the WWTP extracts, highlighted the most immunomodulatory chemicals. DiscussionOur results suggest that the mixture of chemicals present in the effluents of wastewater treatment plants could be considered as immunosuppressive, due to their ability to interfere with the activation of immune cells, a process of utmost importance for the functionality of the immune system. The combined approach of immune effect-based analysis and chemical content analysis used in our study provides a useful tool for investigating the effect of environmental mixtures on the human immune response.

43001 Phase 1 study of fianlimab, a human lymphocyte activation gene-3 (LAG-3) monoclonal antibody, in combination with cemiplimab in advanced melanoma: Expansion cohort analysis

43001 Phase 1 study of fianlimab, a human lymphocyte activation gene-3 (LAG-3) monoclonal antibody, in combination with cemiplimab in advanced melanoma: Expansion cohort analysis

A study on Amygdalin's Genotoxicological Safety and Modulatory Activity in Human Peripheral Lymphocytes in vitro.

Amygdalin (AMY), a plant secondary metabolite containing nitrile, is a major component of the seeds of Rosaceae family plants. It is known that this compound has many pharmacological activities such as cancer prevention, antipyretic, and cough suppressant. In this study, the genotoxic and modulatory effects of amygdalin were assessed by chromosomal aberration (CA), sister chromatid exchange (SCE), and cytokinesis-block micronucleus assay (CBMN) assays using human peripheral lymphocytes (HPLs) in the absence and presence of metabolic activator (S9 mix). Lymphocytes were exposed to various concentrations of amygdalin (0.86, 1.72, 3.43, 6.86, and 13.75 μg/mL) alone and in combination with mitomycin-C (MMC, 0.20 μg/mL) or cyclophosphamide (CP, 12 μg/mL). The mitotic index (MI), replication index (RI), cytokinesis-block proliferation index (CBPI), and cytostasis were also evaluated to determine cytotoxicity. Amygdalin alone did not exhibit genotoxic and cytotoxic effects at all the tested concentrations both in the absence and presence of the S9 mix. In contrast, amygdalin significantly reduced the frequencies of CA (especially at 48 h treatments), SCE, and MN (except 0.86 μg/mL in pre- and simultaneous treatment) induced by MMC in all the tested concentrations and treatment protocols. It has also considerably decreased CP-induced CA and SCE frequencies at all the concentrations (except 0.86 μg/mL) in simultaneous treatment. This study demonstrated that amygdalin alone was not genotoxic, on the contrary, it has revealed modulatory effects against chemotherapy agents that induced genomic damage in human lymphocytes, suggesting its chemopreventive potential. This article is protected by copyright. All rights reserved.

Influence of Paleobacteria on the Proliferative Activity of Human Lymphocytes In Vitro

When testing the proliferative activity of 14 strains of permafrost microorganisms in the reaction of blast transformation of human lymphocytes in vitro, a strain (Alcaligenes sp.) with mitogen properties was isolated (20-fold increase in the rate of lymphocyte proliferation in comparison with the control). Four strains activated lymphocyte proliferation by 3-9 times in comparison with the control. Three strains produced substances with cytostatic properties and reduced proliferation activity by 33-43% and one strain (Bacillus sp.) almost completely suppressed phytohemagglutinin-induced lymphocyte proliferation. These data indicate that strains with a unique immunobiological potential are concentrated in the population of permafrost microorganisms that have undergone rigorous evolutionary selection.

Phase 1 study of fianlimab, a human lymphocyte activation gene-3 monoclonal antibody, in combination with cemiplimab in advanced melanoma: Expansion cohort analysis

Phase 1 study of fianlimab, a human lymphocyte activation gene-3 monoclonal antibody, in combination with cemiplimab in advanced melanoma: Expansion cohort analysis

PP.42 Phase 1 Study of Fianlimab, a Human Lymphocyte Activation Gene-3 (LAG-3) Monoclonal Antibody, in Combination With Cemiplimab in Advanced NSCLC

PP.42 Phase 1 Study of Fianlimab, a Human Lymphocyte Activation Gene-3 (LAG-3) Monoclonal Antibody, in Combination With Cemiplimab in Advanced NSCLC

150P Phase I study of fianlimab: A human lymphocyte activation gene-3 (LAG-3) monoclonal antibody, in combination with cemiplimab in advanced melanoma (mel) - Subgroup analysis

Concurrent blockade of LAG-3 may enhance efficacy of anti-programmed cell death-1 (PD-1) therapies. We previously presented safety and efficacy data from the phase 1 study in patients (pts) with advanced mel treated with anti-LAG-3 (fianlimab) in combination with anti-PD-1 (cemiplimab). We demonstrated high clinical activity among pts with anti–PD-1/PD-ligand (L)1-naive (expansion cohort [EC] 6 and 15) advanced mel. Among pts in EC6+EC15 (N=80), the objective response rate (ORR) was 63.8%, the disease control rate (DCR) was 80.0%, and the median duration of response (mDOR) was not reached (NR).

127P Phase I study of fianlimab: A human lymphocyte activation gene-3 (LAG-3) monoclonal antibody, in combination with cemiplimab in advanced NSCLC

Concurrent blockade of LAG-3 may enhance efficacy of anti–programmed cell death-1 (anti–PD-1) therapies. We present safety and clinical activity data from a phase 1 study in patients (pts) with NSCLC treated with anti–LAG-3 (fianlimab) + anti–PD-1 (cemiplimab).

400P Phase I study of fianlimab, a human lymphocyte activation gene-3 (LAG-3) monoclonal antibody, in combination with cemiplimab in advanced melanoma (mel)

Concurrent blockade of LAG-3 may enhance efficacy of anti-programmed cell death-1 (PD-1) therapies. We present updated safety and clinical activity data from patients (pts) with advanced mel treated with concurrent anti-LAG-3 (fianlimab) and anti-PD-1 (cemiplimab).

790MO Phase I study of fianlimab, a human lymphocyte activation gene-3 (LAG-3) monoclonal antibody, in combination with cemiplimab in advanced melanoma (mel)

Concurrent blockade of LAG-3 may enhance efficacy of anti-programmed cell death-1 (PD-1) therapies. We present updated safety and clinical activity data from patients (pts) with advanced mel treated with concurrent anti-LAG-3 (fianlimab) and anti-PD-1 (cemiplimab).

Ipecac root extracts and isolated circular peptides differentially suppress inflammatory immune response characterised by proliferation, activation and degranulation capacity of human lymphocytes in vitro.

Circular peptides are attractive lead compounds for drug development; this study investigates the immunomodulatory effects of defined root powder extracts and isolated peptides (called cyclotides) from Carapichea ipecacuanha (Brot.) L. Andersson ('ipecac'). Changes in the viability, proliferation and function of activated human primary T cells were analysed using flow cytometry-based assays. Three distinct peptide-enriched extracts of pulverised ipecac root material were prepared via C18 solid-phase extraction and analysed by reversed-phase HPLC and mass spectrometry. These extracts induced caspase 3/7 dependent apoptosis, thus leading to a suppressed proliferation of activated T cells and a reduction of the number of cells in the G2 phase. Furthermore, the stimulated T cells had a lower activation potential and a reduced degranulation capacity after treatment with ipecac extracts. Six different cyclotides were isolated from C. ipecacuanha and an T cell proliferation inhibiting effect was determined. Furthermore, the degranulation capacity of the T cells was diminished specifically by some cyclotides. In contrast to kalata B1 and its analog T20K, secretion of IL-2 and IFN- γ was not affected by any of the caripe cyclotides. The findings add to our increased understanding of the immunomodulating effects of cyclotides, and may provide a basis for the use of ipecac extracts for immunomodulation in conditions associated with an exessive immune responses.

IL-2 and Zoledronic Acid Therapy Restores the In Vivo Anti-Leukemic Activity of Human Lymphocytes Pre-Exposed to Simulated Microgravity.

We have previously shown that the anti-tumor activity of human lymphocytes is diminished in vitro after 12-hours pre-exposure to simulated microgravity (SMG). Here we used an immunocompromised mouse model to determine if this loss of function would extend in vivo, and to also test the efficacy of IL-2 and zoledronic acid (ZOL) therapy as a potential countermeasure against SMG-induced immune dysfunction. We adoptively transferred human lymphocytes that were exposed to either SMG or 1G-control into NSG-Tg (Hu-IL15) mice 1-week after they were injected with a luciferase-tagged human chronic myeloid leukemia (K562) cell line. Tumor growth was monitored 2x weekly with bioluminescence imaging (BLI) for up to 6-weeks. Mice that received lymphocytes exposed to SMG showed greater tumor burden compared to those receiving lymphocytes exposed to 1G (week 6 BLI: 1.8e10 ± 8.07e9 versus 2.22e8 ± 1.39e8 photons/second; p < 0.0001). Peak BLI was also higher in the SMG group compared to 1G-control (2.34e10 ± 1.23e10 versus 3.75e8 ± 1.56e8 photons/second; p = 0.0062). Exposure to SMG did not affect the ability of human lymphocytes to engraft or evoke xeno-graft-versus-host disease in the mice. Additionally, we injected the mice with IL-2 and zoledronic acid (ZOL) to expand and activate the anti-tumor activity of NK cells and γ δ-T cells, respectively. This treatment was found to revive the loss of anti-leukemic function observed in vivo when lymphocytes were pre-exposed to SMG. Microgravity plays a contributory role in loss of tumor control in vivo. Immuno-stimulating agents like ZOL+IL-2 may offer an important countermeasure for immune dysregulation during prolonged spaceflight.

Targeting of Nrf2 improves antitumoral responses by human NK cells, TIL and CAR T cells during oxidative stress

BackgroundAdoptive cell therapy using cytotoxic lymphocytes is an efficient immunotherapy against solid and hematological cancers. However, elevated levels of reactive oxygen species (ROS) in the hostile tumor microenvironment can impair...

Anti-LAG-3 antibody LBL-007 in combination with toripalimab in patients with unresectable or metastatic melanoma: A phase Ⅰ, open-label, multicenter, dose escalation/expansion study.

9538 Background: LBL007 is a novel, fully human IgG4 monoclonal antibody targeting human Lymphocyte-activation gene3 (LAG-3). Dual inhibition of anti-programmed cell death protein 1 (PD-1) and LAG-3 is anticipated to synergistically increase immune response against tumor growth. Here we report the preliminary safety and efficacy of LBL-007 in combination with Toripalimab (an anti-PD-1 antibody has approved for treatment of melanoma in China) in patients (pts) with unresectable or metastatic melanoma. Methods: Pts with unresectable or metastatic melanoma with or without prior anti-PD-(L)1 therapy were enrolled. This trial comprised 2 parts, namely part 1 (dose escalation), pts received LBL-007 (0.25/1/3/6 mg/kg) /Toripalimab (3 mg/kg) both i.v., Q2W; and part 2 (expansion), pts received LBL-007 (3/6 mg/kg)/Toripalimab (3 mg/kg) both i.v., Q2W. The primary objective was safety, the second objectives included pharmacokinetics, pharmacodynamics and efficacy (per RECIST 1.1). Results: By Jan 2022, 37 pts (15 [40.5%] male, median age 59 [range 31-74] years, 9 pts [24.3%] with baseline LDH elevation, 18 [48.6%] with acral, 12 [32.4%] mucosal, 5 [13.5%] nonacral cutaneous, 2 [5.4%] primary site unknown) were enrolled, with 17 in part 1 and 20 in part 2. No dose-limiting toxicity was observed in part 1, and the MTD was not reached. Of all 37 pts, the most common treatment-emergent adverse events (TEAEs) included anemia (24.3%), creatine phosphokinase elevation (24.3%), hypothyroidism (21.6%), and aspartate aminotransferase elevation (21.6%). For 32 radiologically evaluable pts, ORR was 12.5%, DCR was 53.1%. In a preplanned subtype-specific analysis in anti-PD-(L)1 treatment-naïve pts, ORR was 27.3 % vs. 0%, and DCR was 81.8% vs. 50.0% in acral and mucosal melanoma subtypes, respectively. For anti-PD-(L)1-resistant pts (n = 11), DCR was 18.2%. Conclusions: LBL007/Toripalimab combination is well tolerated and promising efficacy in pts with unresectable or metastatic melanoma, especially in the acral type without prior anti-PD-(L)1 therapy. Clinical trial information: NCT04640545.

Antigenotoxic Effect of Ascorbic Acid and Resveratrol in Erythrocytes of Ambystoma mexicanum, Oreochromis niloticus and Human Lymphocytes Exposed to Glyphosate.

Glyphosate is a controversial herbicide. Its genotoxicity and presence in various ecosystems have been reported. The use of ascorbic acid and resveratrol could protect different organisms from glyphosate-induced genetic damage. In the present study, specific genetic damage induced by glyphosate was evaluated in erythrocytes of Oreochromis niloticus, Ambystoma mexicanum and human lymphocytes. Simultaneously, the antigenotoxic capacity of various concentrations of ascorbic acid and resveratrol was evaluated by means of pretreatment and simultaneous treatment protocols. The 0.03, 0.05 and 0.07 mM concentrations of glyphosate induced significant genotoxic activity (p < 0.05) in human lymphocytes and in erythrocytes of the species studied, and could cause genomic instability in these populations. The reduction in genetic damage observed in human lymphocytes exposed to high concentrations of glyphosate is only apparent: excessive genetic damage was associated with undetectable excessive tail migration length. A significant (p < 0.05) antigenotoxic effect of ascorbic acid and resveratrol was observed in all concentrations, organisms and protocols used. Both ascorbic acid and resveratrol play an important role in maintaining the integrity of DNA. Ascorbic acid in Oreochromis niloticus, Ambystoma mexicanum reduced glyphosate-induced genetic damage to a basal level. Therefore, our data indicate that these antioxidants could help preserve the integrity of the DNA of organisms exposed to glyphosate. The consumption of antioxidants is a useful tool against the genotoxicity of glyphosate.

Extended‐Synaptotagmins Modulate Diacylglycerol Dynamics at the Immunological Synapse in Human T Lymphocytes

Cytotoxic T‐lymphocytes (CTLs) regulate the immune response by recognizing and destroying cancerous or virus‐infected cells through the polarized release of apoptosis‐inducing proteins at the contact area known as the immunological synapse (IS). The synaptic membrane is a heterogenous region where protein complexes rearrange on the cell surface. Within the membrane are phospholipids that assemble to relay information from extracellular receptors to induce global cellular changes. Diacylglycerol (DAG) is a key secondary messenger of T‐cell receptor (TCR) activation which induces microtubule‐organizing center polarization towards the IS and signaling via MAPK and NFkB pathways. Abnormal lipid homeostasis at the synapse is linked to autoimmune disorders. However, our understanding on how cells regulate dynamic transfer of lipids from one cellular compartment to another is still scant. Evidence points to the role of lipid transport proteins (LTPs) in the regulation of lipid homeostasis without the need for membrane fusion. Extended‐Synaptotagmins (E‐Syts) are a group of LTPs tethering the endoplasmic reticulum (ER) to plasma membrane (PM), thus controlling membrane lipid compositions by transporting DAG down its concentration gradient. Herein, we tested the hypothesis that E‐Syts play a role in the modulation of diacylglycerol at the synaptic membrane, ER‐PM morphology and signaling dynamics by performing the following experiments: 1) assessing the role of E‐Syts in regulating the effector functions of human lymphocytes by measuring cellular cytotoxicity, degranulation, TCR signaling via phospho‐protein analysis and intracellular Ca2+ levels; 2) testing the role of E‐Syts in DAG distribution at the IS via stimulated emission depletion (STED) microscopy. Our results indicate that E‐Syts down‐modulate human lymphocyte activation as their knock‐down leads to enhanced TCR signaling, degranulation, and cytokine production measured by flow cytometry. Using GFP‐based bioprobes for DAG and tagged E‐Syt constructs, we observed proximal localization of E‐Syts with signaling protein LAT and CD4 via super‐resolution microscopy. Accordingly, E‐Syt1 knockout Jurkat cells at resting state express localized DAG accumulation similar to activated WT Jurkat cells. Our results provide, for the first time, an understanding of spatio‐temporal distribution as well as the role of lipid regulators at membrane junction sites during IS formation. We believe that E‐Syts play an essential role in maintenance of synaptic lipids via regulation of non‐vesicular lipid transport, whose loss might contribute to enhanced activity of CTLs at resting conditions. Collectively, our results suggest that E‐Syts are critical components to orchestrate lipid distribution during synapse formation. Future experiments will be required to shed light on additional steady state regulators of synaptic membrane lipids, highlighting the importance of membrane crosstalk in the setting of CTLs.

The Sphingolipid Inhibitors Ceranib-2 and SKI-II Reduce Measles Virus Replication in Primary Human Lymphocytes: Effects on mTORC1 Downstream Signaling.

The bioactive sphingolipids ceramide and sphingosine-1-phosphate (S1P) are involved in the regulation of cell homeostasis and activity ranging from apoptosis to proliferation. We recently described that the two compounds ceranib-2 (inhibiting acid ceramidase) and SKI-II [inhibiting the sphingosine kinases 1 and − 2 (SphK1/2)] reduce mTORC1 activity and measles virus (MV) replication in human primary peripheral blood lymphocytes (PBL) by about one log step. We now further investigated whether mTORC1 downstream signaling and viral protein expression may be affected by ceranib-2 and/or SKI-II. Western blot analyses showed that in uninfected cells the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) was reduced by both inhibitors. Interestingly, MV infection led to an increase of rpS6 protein levels and phosphorylation of eIF4E. Treatment with both inhibitors reduced the rpS6 protein expression, and in addition, SKI-II reduced rpS6 phosphorylation. The phosphorylation of eIF4E was slightly reduced by both inhibitors. In addition, SKI-II led to reduced levels of IKK in MV-infected cells. Both inhibitors reduced the expression of viral proteins and the titers of newly synthesized MV by approximately one log step. As expected, SKI-II and rapamycin reduced also the virally encoded GFP expression; however, ceranib-2 astonishingly led to increased levels of GFP fluorescence. Our findings suggest that the inhibitors ceranib-2 and SKI-II act via differential mechanisms on MV replication. The observed effects on mTORC1 downstream signaling, predominantly the reduction of rpS6 levels by both inhibitors, may affect the translational capacity of the cells and contribute to the antiviral effect in human primary PBL.

Antigenotoxic and antimutagenic activities of &lt;i&gt;Psittacanthus calyculatus&lt;/i&gt; (Loranthaceae) leaves water extract

Mistletoe (&lt;i&gt;Psittacanthus calyculatus&lt;/i&gt;) is used for the prevention and treatment of numerous diseases. Samples of leaves from &lt;i&gt;P. calyculatus&lt;/i&gt; were collected in April of 2019, and prepared an aqueous extract. The extract was lyophilized, and its polyphenols, flavonoids, and anthocyanins content were determined. Then, concentrations of lyophilized extract were prepared (5, 50 and 100 ppm) and assessed their antigenotoxic, antimutagenic and genotoxicity activities in human lymphocytes were evaluated using the comet assay system. The dry aqueous extract contained 73.54 mg of polyphenols AGE per g sample, 39.37 mg of flavonoid CE per g, and 0.1 mg of anthocyanins Cy-3-gluc E per g. No significant genotoxic activity was observed, with the exception of the concentration of 100 ppm at 10 hours of exposure (p &lt;0.05). There was also significant (p &lt;0.05) antigenotoxic and antimutagenic activity (p &lt;0.05). Clearly, low concentrations and short-duration exposures to lyophilized &lt;i&gt;P. calyculatus&lt;/i&gt; do not induce genetic damage; however, high concentrations are genotoxic. The antigenotoxic and antimutagenic effects were due to a protective effect not only against induced DNA damage but also against basal genetic damage.