Concentration Of Effector Cells Research Articles

Intratumoral Injection of Large Surface Area Microparticle Taxanes in Carcinomas Increases Immune Effector Cell Concentrations, Checkpoint Expression, and Synergy with Checkpoint Inhibitors: A Review of Preclinical and Clinical Studies.

This review summarizes development of large surface area microparticle paclitaxel (LSAM-PTX) and docetaxel (LSAM-DTX) for local treatment of primary carcinomas with emphasis on immunomodulation. Intratumoral (IT) delivery of LSAM-PTX and LSAM-DTX provides continuous, therapeutic drug levels for several weeks. Preclinical studies and clinical trials reported a reduction in tumor volume (TV) and immunomodulation in primary tumor and peripheral blood with increases in innate and adaptive immune cells and decreases in suppressor cells. Increased levels of checkpoint expression of immune cells occurred in clinical trials of high-risk non-muscle-invasive bladder cancer (LSAM-DTX) and unresectable localized pancreatic cancer (LSAM-PTX). TV reduction and increases in immune effector cells occurred following IT LSAM-DTX and IT LSAM-PTX together with anti-mCTLA-4 and anti-mPD-1, respectively. Synergistic benefits from combinatorial therapy in a 4T1-Luc breast cancer model included reduction of metastasis with IT LSAM-DTX + anti-mCTLA-4. IT LSAM-PTX and LSAM-DTX are tumoricidal, immune enhancing, and may improve solid tumor response to immune checkpoint inhibitors without additional systemic toxicity.

Superoxide dismutase inhibits cytotoxic killing of Fasciola gigantica newly excysted juveniles expressed by sheep invitro

Fasciola gigantica faces a series of threats from various free radicals produced by the host immune system during its invasion through the abdominal cavity and establishment in the bile duct of ruminants, limiting the fluke viability. The role of the superoxide radical produced by Muzaffarnagari sheep immune effector cells against F. gigantica newly excysted juveniles (NEJs) is highlighted in this study, as is the critical role of superoxide dismutase enzyme (SOD) in dismutation of superoxide radicals derived from host immune effector cells in vitro. Three concentrations of the ovine immune effector cells viz. 2.5, 5, and 10 × 106 cells were tested for their ability to induced cytotoxic killing of the parasite. All the three cell concentrations caused significant (p < 0.01) cytotoxic killing of NEJs in comparison to the control groups. Also, reduction of the immune effector cell concentration directly correlates with the NEJs killing. Attachment of immune effector cells to the parasite tegument in the presence of anti-F. gigantica antibodies was found to be critical in inducing NEJs killing via antibody-dependent cell-mediated cytotoxicity (ADCC). However, the addition of SOD greatly inhibits cytotoxic killing of NEJs, demonstrating the importance of SOD enzyme in fluke survival and parasite evasion of the host immunity. Thus, F. gigantica SOD warrants a promising candidate for immunoprophylactic studies in ruminants against the tropical liver fluke.

Chemokines’ role in immunopathogenesis of atherosclerosis

The results obtained and conflicting data of immunopathogenesis of inflammation and atherosclerosis are analyzed. The role of macrophages and T-lymphocytes in the endothelium damage and formation of an atherosclerotic plaque is shown. Chemokine-leading processes initiating the data processes - macrophage chemotactic protein, interleukin 8, fractalkine. An important role in the initiation of intercellular interaction with the subsequent development of the alliance CD40 and their ligands, expressed by various cell types, participants in the immune-inflammatory process - CD154. The role of subpopulations of T-lymphocytes - 1st, 2nd and 3rd, as well as regulatory T-lymphocytes in the atherosclerotic process was analyzed. An experimental data on the role of chemokines in the development of atherosclerotic process is described. The role of the factor inhibiting migration and providing the concentration of effector cells in the area of atherosclerotic plaque, which lead to its destabilization, is of great importance. It has been shown experimentally that the expression of inhibition factor migration correlates with the intensity of atherosclerotic process. The information on the factor contributing to the output of stromal cells is presented. Experimental data show that the development of coronary heart disease is associated with it. Its deficiency contributes to the development of the disease, and an increase - improves the course of the disease and has a stabilizing effect on atherosclerotic plaques. The role of hemokine (CXC motif) ligand 10 and 11 in the process of atherosclerosis has been experimentally proved. They have a controlled effect on chemotaxis and T-lymphocyte function in the process of atherosclerotic lesion formation.

In silico tumor control induced via alternating immunostimulating and immunosuppressive phases

Despite recent advances in the field of Oncoimmunology, the success potential of immunomodulatory therapies against cancer remains to be elucidated. One of the reasons is the lack of understanding on the complex interplay between tumor growth dynamics and the associated immune system responses. Toward this goal, we consider a mathematical model of vascularized tumor growth and the corresponding effector cell recruitment dynamics. Bifurcation analysis allows for the exploration of model's dynamic behavior and the determination of these parameter regimes that result in immune-mediated tumor control. In this work, we focus on a particular tumor evasion regime that involves tumor and effector cell concentration oscillations of slowly increasing and decreasing amplitude, respectively. Considering a temporal multiscale analysis, we derive an analytically tractable mapping of model solutions onto a weakly negatively damped harmonic oscillator. Based on our analysis, we propose a theory-driven intervention strategy involving immunostimulating and immunosuppressive phases to induce long-term tumor control.

Cancer therapeutic potential of combinatorial immuno- and vasomodulatory interventions.

Currently, most of the basic mechanisms governing tumour-immune system interactions, in combination with modulations of tumour-associated vasculature, are far from being completely understood. Here, we propose a mathematical model of vascularized tumour growth, where the main novelty is the modelling of the interplay between functional tumour vasculature and effector cell recruitment dynamics. Parameters are calibrated on the basis of different in vivo immunocompromised Rag1(-/-) and wild-type (WT) BALB/c murine tumour growth experiments. The model analysis supports that tumour vasculature normalization can be a plausible and effective strategy to treat cancer when combined with appropriate immunostimulations. We find that improved levels of functional tumour vasculature, potentially mediated by normalization or stress alleviation strategies, can provide beneficial outcomes in terms of tumour burden reduction and growth control. Normalization of tumour blood vessels opens a therapeutic window of opportunity to augment the antitumour immune responses, as well as to reduce intratumoral immunosuppression and induced hypoxia due to vascular abnormalities. The potential success of normalizing tumour-associated vasculature closely depends on the effector cell recruitment dynamics and tumour sizes. Furthermore, an arbitrary increase in the initial effector cell concentration does not necessarily imply better tumour control. We evidence the existence of an optimal concentration range of effector cells for tumour shrinkage. Based on these findings, we suggest a theory-driven therapeutic proposal that optimally combines immuno- and vasomodulatory interventions.

Targeting AML Using an Fc-Engineered BST1/CD157 Monoclonal Antibody

Antibody-based immunotherapy represents a promising strategy to target and eliminate acute myeloid leukemia (AML) tumor cells. We evaluated BST1/CD157, a novel, slow internalizing surface antigen, for its suitability as immunotherapeutic target in AML. An Fc-optimized antibody against BST1/CD157 was developed (MEN1112/OBT357NF), which binds to the Fc-receptor on natural killer (NK) cells with enhanced affinity. The target antigen expression profile of BST1/CD157 on AML bulk cells as well as on leukemic stem cells (LSC) was analyzed with flow cytometry using a commercially available antibody. BST1/CD157 expression was detected in > 97% of AML patients at primary diagnosis (n=81) and at time of relapse (n=20) using specific fluorescence intensity (SFI, positivity: SFI > 1. 5). Importantly, BST1/CD157 was also expressed on CD34+/CD38-cells (n=20), the compartment with a high percentage of LSCs.Functional titration experiments of MEN1112/OBT357NF showed antibody-mediated lysis of U937 cells in the low picomolar range (EC50 = 30–140 pM) and demonstrated superior ADCC activity compared to its non Fc-engineered parental analogue. To evaluate the additive effect of the complement cascade, healthy donor (HD) monocytes were co-incubated with autologous NK cells and MEN1112/OBT357NF in the presence or absence of 5% donor serum. After 20 hours of incubation, a significantly enhanced lysis efficacy by MEN1112/OBT357NF through the addition of serum compared to control cultures without serum (p=0.02) was demonstrated.For further functional characterization of MEN1112/OBT357NF standard chromium release assays using AML cell lines and NK cells from HDs were performed. In this setting, efficient target cell lysis (30–50%) at various effector cell:target (E:T) ratios (50:1–6:1) was observed. Compared to Rituximab-mediated lysis of RAJI cells, MEN1112/OBT357NF triggered more efficient target cell lysis at much lower effector cell concentrations (33% lysis of OCI-AML3 cells vs. 11% lysis of RAJI cells at E:T 6:1).Furthermore, the cytotoxic potential of NK cells from AML patients after intense double induction chemotherapy on AML cell lines was tested. The results were highly variable with efficient lysis in about one third of samples tested (n=10). As a positive control NK cells from HDs were used which showed superior lysis as compared to patient-derived NK cells. In an autologous set-up using NK cells from either primary diagnosis or at time of remission, we could show efficient lysis of primary AML cells in one of two patient samples (patient in complete remission) in the presence of MEN1112/OBT357NF.In summary, our analysis demonstrates that BST1/CD157 is a promising target antigen for antibody-based therapy in AML. The effective in-vitro ADCC activity supports further development of MEN1112/OBT35NF as an immunotherapy for patients with AML. DisclosuresKrupka:Oxford BioTherapeutics Ltd: Research Funding. Jansen:Oxford BioTherapeutics Ltd: Research Funding. Aud:Oxford BioTherapeutics Ltd: Employment. Dusek:Oxford BioTherapeutics Ltd: Employment. Bisht:Oxford BioTherapeutics Ltd: Employment. Pombo-Villar:Oxford BioTherapeutics Ltd: Employment, Equity Ownership. Rohlff:Oxford BioTherapeutics Ltd: Employment. Subklewe:Oxford BioTherapeutics Ltd: Research Funding.

Bi-specific Aptamers Mediating Tumor Cell Lysis

Antibody-dependent cellular cytotoxicity plays a pivotal role in antibody-based tumor therapies and is based on the recruitment of natural killer cells to antibody-bound tumor cells via binding of the Fcγ receptor III (CD16). Here we describe the generation of chimeric DNA aptamers that simultaneously bind to CD16α and c-Met, a receptor that is overexpressed in many tumors. By application of the systematic evolution of ligands by exponential enrichment (SELEX) method, CD16α specific DNA aptamers were isolated that bound with high specificity and affinity (91 pm-195 nm) to their respective recombinant and cellularly expressed target proteins. Two optimized CD16α specific aptamers were coupled to each of two c-Met specific aptamers using different linkers. Bi-specific aptamers retained suitable binding properties and displayed simultaneous binding to both antigens. Moreover, they mediated cellular cytotoxicity dependent on aptamer and effector cell concentration. Displacement of a bi-specific aptamer from CD16α by competing antibody 3G8 reduced cytotoxicity and confirmed the proposed mode of action. These results represent the first gain of a tumor-effective function of two distinct oligonucleotides by linkage into a bi-specific aptamer mediating cellular cytotoxicity.

Evaluating ELISPOT summary measures with criteria for obtaining reliable estimates

The ELISPOT assay is a commonly used technique for quantifying the occurrence of T lymphocyte cells secreting a cytokine after stimulation with an antigen or peptide. The assay endpoint, the number of spot-forming cells (SFC) at a specific concentration of effector cells, is typically estimated using either a simple arithmetic mean or the predicted value from a linear regression model. We compare statistical modeling approaches for summarizing these assays using data from the Pediatric AIDS Clinical Trial Group (PACTG) study 299. A simulation study was conducted to compare methods under controlled conditions. Assuming the optimal effector cell concentration is known, we demonstrate that the simple mean is appropriate if assays are conducted at the same concentration for all samples. Normalizing simple means to a summary concentration using results from a range of concentrations is not valid. A random effects or mixed model is superior to the simple mean when a large within-assay (subject) variance relative to between-subject variance exists. When assays are conducted over a range of effector cell concentrations for each individual, the theoretical linearity assumption of the regression model is often violated and can result in biased estimates. In this case, nonlinear models provide more accurate estimation. Collecting data over a range of concentrations allows reassessment of the optimal cell concentration after the data are generated.

Simplified quantitation of cytotoxicity by integration of specific lysis against effector cell concentration at a constant target cell concentration and measuring the area under the curve

Experience with the lytic unit (LU) as a measure of cytolytic efficiency has indicated that its accuracy is limited, even if expressed in a logarithmic format. A new method of quantifying cytotoxicity from effector dilution assays is proposed: the area under the curve (AUC) of the Ig ( E/ T) ratio vs. percentage cytotoxicity plot, gives an overall measure of lytic efficiency. The AUC derived from the Briggs-Haldane kinetic model is dependent on both the kinetic parameters that determine the efficiency of effector cells (the Michaelis constant K M and the catalytic constant k cat). AUC provides an index of inhibition or stimulation of lysis, independent of whether the modulation is kinetically competitive, uncompetitive or mixed. The relationship between the AUC and the LU suggests that a broad range of LU data could have the same AUC value. In practice the method may be applied to interpret simple cytotoxicity assay data, where effector cells are being used in standardised screening for modifiers of the cytolytic response. Illustrative data of LAK cytotoxicity influenced by dose of the LAK response modifiers IL-2, TGFβ, TDSF and 5-FU, show different relationships between lytic units, K M and AUC. These data also shows a wide range in the Hill coefficient and would be consistent with a cooperative effect dependent on the effector cell efficacy. This confirms that using LU as a simple measure of cytolytic efficiency could be erroneous and suggest that cytolytic response modifiers can produce a variety of kinetic changes. the AUC method, however, provides a comparative measure of efficiency in these situations, independent of mechanism.

Kinetic assay of cytotoxic T lymphocyte clones: I. Methodology and data analysis

A kinetic assay for cytotoxic T lymphocyte (CTL) clones expanded briefly in culture is described. The Hill equation for allosteric kinetics is used to fit curves to dose-response data. In this assay, K 0 - the target cell concentration required for half-maximal velocity of killing — is determined for clones generated from a population's most frequent CTL precursors. Because each clone's K 0 is a constant under given conditions and a description of relative cytolytic efficiency, K 0 values can be used to compare the clonal composition of disparate or dynamic populations. The parameter n is the target concentration exponent in the Hill equation and the slope of the line in the Hill plot. This parameter further specifies the shape of a clone's dose response curve, describing a clone's responsiveness to changes in target cell concentration. Because the assay is designed for small, undetermined effector cell concentrations, maximum velocities are not compared. The method described herein requires minimal supplies, equipment, and biological materials.

Natural cytotoxicity of tilapia leucocytes

Natural cytotoxicity of tilapia leucocytes

Kinetic analysis of cloned cytotoxic T lymphocyte reactivity against normal and leukaemic target cells.

The kinetics of unrestricted killing of normal and leukaemic lymphocyte target cells by a Qa-1b-specific murine cytotoxic T lymphocyte (CTL) clone were evaluated in a manner analogous to enzyme kinetic assays in which the effector and target cells corresponded to the enzyme and substrate, respectively. In order to apply the enzyme-substrate analogy to clonal cytolytic reactions, it was first established that the lytic reactions exhibited initial steady-state velocity of lysis at the effector and target cell concentrations used. The lytic reaction maintained linearity for velocity of lysis during the first 90 min of incubation, then plateaued. Vmax (the maximal rate of target cell lysis achieved by a given effector population) and Km (the target cell number resulting in 1/2Vmax) values were determined over a wide range of target and effector cell concentrations. Both parameters were found to be directly proportional to the number of effector cells. At a given concentration of cloned CTL, the lytic parameters of Vmax and Km were not significantly different for normal or leukaemic target cells that express Qa-1b. Additional kinetic parameters for lysis of normal and leukaemic target cells by a cloned CTL were also compared. The lytic efficiency of the CTL clone (i.e. maximal killing rate with an infinite number of targets) and the intrinsic affinity between effector and target cells were the same with either normal or leukaemic targets. However, the maximal lysis of target cells at an infinite number of effectors was significantly less for normal compared with leukaemic targets. This suggests that the normal target cells were more heterogeneous in their expression of the target (Qa-1b) antigen. Enzyme-like kinetic analysis of cell-mediated lysis reactions can be useful for comparing the relative affinities of effector and target cells obtained from various sources.

Antibody-dependent cytotoxicity of human and mouse mononuclear cells against Trypanosoma cruzi epimastigotes

Human peripheral mononuclear cells were cytotoxic to antibody-sensitized Trypanosoma cruzi epimastigotes. The cytotoxic effect depended on the concentration of effector cells and antiserum, and was progressive until 17 hr of incubation at 28 °C. After 3 hr of incubation the highest specific activity was achieved at a 50:1 effector to target cell ratio. A nonspecific cytotoxic effect in the absence of antiserum was observed at a 100:1 parasite to cell ratio or after 17 hr of incubation. When the human mononuclear cell population was depleted of adherent cells by Sephadex G-10 filtration or adsorption to glass, the cytotoxic effect was greatly reduced. Similar results were obtained using mouse spleen cells, indicating that only the adherent cells were cytotoxic to sensitized T. cruzi in both systems. When human mononuclear cells were incubated with amobarbital, cyanide, azide, or aminotriazole, an inhibition of cytotoxicity against sensitized T. cruzi was observed, suggesting that oxygen reduction products and myeloperoxidase were involved in the destruction of sensitized T. cruzi epimastigotes by normal human mononuclear cells.

CELL-MEDIATED CYTOTOXICITY TOWARD THE DONOR IN PATIENTS WITH WELL-FUNCTIONING KIDNEY GRAFTS

Patients with well-functioning kidney allografts from one-HLA-haplotype--mismatched related donors have strongly reduced donor-specific cell-mediated cytotoxicity 2-10 years after having received their grafts. This could also be demonstrated in secondary in vitro cell-mediated lympholysis (CML). Even though secondary donor-specific CML did not exceed 10% at an effector-to-target cell ratio of 50:1, specific lysis of donor target cells increased significantly with increasing effector cell concentrations. The weak cytotoxicity toward the donor could be enhanced neither through pool-cell stimulation nor by adding exogenous Interleukin 2 (IL-2) during the induction phase. Furthermore, growth factors were produced in the cultures during the induction of cytotoxic cells in concentrations comparable to those of the sibling control. Thus no evidence could be obtained that lack of IL-2 was causing the decreased cytotoxicity in these patients. Our studies indicate that in vivo depletion of cytotoxic cells with high lytic efficiency is probably the reason for the strongly reduced donor-specific cytotoxicity.

Quantitation and estimation of cooperation between target-binding sites in natural and antibody-dependent cell-mediated cytotoxicity by use of a mathematical transformation. Influence of interferon.

We have studied the use of a mathematical transformation, the Hill transformation, in natural and antibody-dependent cell-mediated cytotoxicity as a function of effector cell concentration. It can be concluded that when adherent cells are removed from the effector cell suspension, cooperation between binding sites for effector cells on the target cell changes from negative to zero. After stimulation of nonadherent cells with alpha-interferon, the binding sites still remain noninteracting. This is the case both in natural and in antibody-dependent cell-mediated cytotoxicity experiments. The Hill transformation seems to be an easy and easily reproducible way of quantitating cytotoxicity.

Inhibition of whole blood antibody dependent cellular cytotoxicity by heat aggregated human IgG

Inhibition of the whole blood antibody dependent cellular cytotoxicity (ADCC) of a lymphoblastoid cell line by heat aggregated human IgG (HAI) is described. Optimal sensitising antibody and effector cell concentrations were established to permit the detection of 0.1 μg/ml HAI. Conditions which avoid the ADCC inhibitory effects of normal human sera were defined. Twelve paired normal human sera were stored at −70°C for prolonged (> 3 years) and shorter (<6 months) periods of time. Sensitised sheep red cells were used to determine complement depletion in serum dilutions heated at 40°C, 45°C, 50°C and 56°C. Concentrations of human IgG (1 000–0.1 μg/ml) were prepared in foetal calf serum before heating at 63°C for 30 min to aggregate the IgG. ADCC inhibitory activity, frequently characteristic of normal human serum was minimised (<10%) when sera were stored at −70°C and heated to 50°C for 30 min to inactivate serum complement. This assay provides an economical, reproducible and sensitive test for the detection of circulating IgG complexes.

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

Effector cells in avian spontaneous and antibody-dependent cell-mediated cytotoxicity.

Using effector cells from chickens and quails, antibody-dependent and spontaneous cell-mediated cytotoxicity (ADCC and SCMC) were studied in a 51chromium-release microcytotoxicity assay against erythrocytes and several tumor cells as targets. The effector cells mediating both ADCC and SCMC against tumor targets, as well as those active in ADCC against erythrocytes, exhibited similar characteristics: they were adherent cells of high density, which could be removed by treatment with carbonyl iron and magnet. The effector cell concentration was highest in the peripheral blood and lower in the spleen, whereas no activity was found in thymus and bursa. Lysis was detectable after 15 min of incubation, and the reaction was completed after a few hours. A rabbit antiserum was found to be able to induce ADCC against sheep erythrocytes, but it did so less effectively than a chicken antiserum. Studies on the target specificity of SCMC revealed a preferential destruction of xenogeneic lymphoma targets. These findings are discussed with respect to differences between the avian effector cells and the mammalian K and NK cells.

Antibody-dependent cell-mediated antibacterial activity of human mononuclear cells. I. K lymphocytes and monocytes are effective against meningococi in cooperation with human imune sera.

In cooperation with human heat-inactivated antisera from adults immunized with group C meningococcal polysaccharide, normal human peripheral blood mononuclear cells significantly decreased the viability of group C meningococci (Mgc) in vitro. K lymphocytes (Null cells) and monocytes, (but not T or B lymphocytes) were capable of effecting antibody-dependent cell-mediated (ADC) antibacterial activity in this system. The degree to which meningococcal viability was decreased was a function of the length of the test incubation, the concentration of effector cells, and the amount of antiserum used in the assay. When specific antibodies directed against Mgc were adsorbed from the antiserum, cell-mediated antibacterial activity was abolished. ADC antibacterial activity was also abrogated by performing the assay at 4 degrees C or by heating effector cells to 46 degrees C for 15 min before the assay, Similarities between the ADC antibacterial system and previously described ADCC assays are discussed. The data suggest the K cells (as well as monocytes) may play a role in host immune defense against pathogenic bacteria.

Antibody-dependent cytolysis of Trypanosoma cruzi by human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes were cytotoxic to Trypanosoma cruzi epimastigotes in the presence of specific antibodies from Chagas' disease patients and sera from rabbits immunized with homogenates of T. cruzi epimastigotes. Cytotoxicity was evaluated by the in vitro release of [ 3H]uridine-labeled RNA from culture forms. Under the same conditions, mononuclear cells were not cytotoxic. Pretreatment of target cells with antibodies was as effective as the continued presence of antisera during the reaction, while sera-treated effector cells did not mediate cytotoxicity. The cytotoxic effect depended on the concentration of effector cells and antibody and was progressive until 4 hr of incubation at 28 °C.