Antibody Binding to Membrane of Cultured Melanoma Cells by Sera of Melanoma Patients

1979 ◽  
Vol 65 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Silvana Canevari ◽  
Giuseppe Fossati ◽  
Paolo Vezzoni ◽  
Sergio Biguzzi ◽  
Jose Garcia-Puche ◽  
...  
Keyword(s):  
Binding Activity ◽  
Absorption Capacity ◽  
Target Cells ◽  
Similar Amount ◽  
Experimental Conditions ◽  
Healthy Donors ◽  
Fetal Bovine ◽  
Melanoma Patients ◽  
Quantitative Absorption ◽  
Sepharose 4B

One hundred and nine sera from 75 patients with malignant melanoma and 69 sera from as many healthy donors were assayed by isotopic antiglobulin technique (IAT) on 2 melanoma cell lines. The same picture of reactivity was observed with patients’ and healthy donors’ sera, and in both groups 35% of the cases were high responders on 1 line and 21% on the other one. The specificity of the reactions was analyzed by absorption experiments using 12 melanoma sera selected for their high binding activity. Pools of human erythrocytes or leukocytes did not remove, except in 1 case respectively, the activity of the sera, suggesting that it was not directed against alloantigens. Quantitative absorption experiments were done with the 2 melanoma lines and with 1 colon carcinoma line. The results, evaluated on the basis of absorption capacity per cell, indicate that the 2 melanoma lines had a similar amount of shared antigens, whereas the colon line was also effective in absorbing out the serum activity, but less frequently and less efficiently. Further experiments performed to analyze the influence of culturing the target cells in presence of fetal bovine serum (FBS), showed that the activity of sera was removed, at various degrees for different sera, by absorption with free FBS, with FBS coupled to Sepharose 4B, and with normal leukocytes cultured overnight with 10% FBS. The same positive melanoma sera became negative when assayed on the same melanoma line cultured in γ-globulin-depleted human AB serum. In conclusion, in our experimental conditions, the activity of melanoma sera seems mostly directed against components of FBS absorbed on cell membrane during culturing.

Tim-3 expression and function in natural killer cells from advanced melanoma patients.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8571-8571
Author(s):  
Ines Esteves Domingues Pires Da Silva ◽  
Sonia Jimenez-Baranda ◽  
Anne Gallois ◽  
Vijay Kuchroo ◽  
Iman Osman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Advanced Melanoma ◽  
Target Cells ◽  
Cell Cytotoxicity ◽  
Healthy Donors ◽  
Melanoma Patients ◽  
Ifn Γ ◽  
Nk Cell Cytotoxicity ◽  
And Function

8571 Background: The concept of CD8+ T cell exhaustion in the context of metastatic cancer has been reinforced by the recent success of immunotherapies targeting the exhaustion markers CTLA-4 and PD-1 in advanced melanoma. T-cell immunoglobulin 3 (Tim-3), another exhaustion marker, is also expressed in natural killer (NK) cells, however its role is still unknown. Recent reports have shown that NK cells, innate immune cells that eliminate tumors through cytotoxicity and IFN-g production, are functionally impaired in advanced melanoma patients, although no receptor has been linked with that phenotype so far. In this study, we characterize the role of Tim-3 in NK cells, particularly in the presence of its natural ligand, Galectin-9 (Gal-9), that is known to be expressed/secreted by some tumor cells including melanoma. Methods: We compared 20 advanced melanoma donors NK cells with 40 healthy donors NK cells as it relates to Tim-3 expression (by flow cytometry) and function (cytotoxicity, IFN-γ production and proliferation). NK cells cytotoxicity was measured by lamp-1 expression, and two different target cells were used: i) K562 cells (Gal-9-) and ii) Gmel Gal-9+ and Gmel Gal-9- sorted melanoma cells. Proliferation was quantified by CFSE after 6 days in the presence of rhIL-2. Recombinant rhGal9 effect was tested in cytotoxicity and IFN-γ production. Results: Melanoma patients NK cells express higher levels of Tim-3 compared to healthy donors NK cells (p<0.05). Melanoma patients NK cells have a defect in cytotoxicity, proliferation and IFN-γ production. Tim-3 expression by itself (without engagement of specific ligands) does not negatively affect NK cell functions (p<0.05). However, when rhGal9 is added to the system, a decrease in NK cell cytotoxicity and IFN-γ production (p<0.05) was observed. Finally, the expression of Gal-9 by the target cells induces a defect in NK cell cytotoxicity (Gmel Gal-9+ vs Gmel Gal-9-). Conclusions: These data suggest that advanced melanoma patients NK cells are exhausted, although it still remains unclear if Tim-3 is involved in this phenotype. In addition,the expression/secretion of Galectin-9, immunosuppressive for NK cells, may be a possible mechanism for tumors to evade immune surveillance.

scholarly journals 151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A159-A159
Author(s):  
Michael Whang ◽  
Ming-Hong Xie ◽  
Kate Jamboretz ◽  
Hadia Lemar ◽  
Chao Guo ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Adoptive Cell Therapy ◽  
Therapeutic Window ◽  
Target Cells ◽  
Healthy Donors ◽  
Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

scholarly journals The Fatty Acid and Protein Profiles of Circulating CD81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4157
Author(s):  
Giovanni Paolino ◽  
Veronica Huber ◽  
Serena Camerini ◽  
Marialuisa Casella ◽  
Alberto Macone ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Extracellular Vesicles ◽  
Ad Hoc ◽  
Early Stage ◽  
Disease Onset ◽  
Protein Composition ◽  
Disease Stage ◽  
Biological Macromolecules ◽  
Healthy Donors ◽  
Melanoma Patients

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II–IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0–I, II and III–IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0–I) from late (III–IV) stages’ CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III–IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV’ FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.

Cellular basis for burn-mediated cardiac dysfunction in adult rabbits

1996 ◽  
Vol 271 (6) ◽  
pp. H2615-H2621 ◽  
Author(s):  
J. W. Horton
Keyword(s):  
Cardiac Myocyte ◽  
Burn Injury ◽  
Total Body Surface Area ◽  
Ringer Solution ◽  
Polymorphonuclear Neutrophils ◽  
Experimental Conditions ◽  
Cardiac Contractile ◽  
Fetal Bovine ◽  
Total Body ◽  
And Control

We have shown that cutaneous burn injury impairs cardiac contractile performance; however, the mechanisms remain unclear. In this study, New Zealand White rabbits were anesthetized with isoflurane, given a full-thickness scald burn over 30% of total body surface area, and resuscitated with lactated Ringer solution (4 ml.kg-1.%burn-1 for 24 h); rabbits handled in an identical fashion were given a sham burn. Serum obtained from burned and control (sham-burned) rabbits was aliquoted and frozen at -70 degrees C until assay. Polymorphonuclear neutrophils (PMN) were isolated 24 h postburn from both sham and burned rabbits to yield preparations with > 95% PMN with > 95% viability. Cardiac myocytes were isolated by retrograde perfusion of hearts with Ca(2+)-free collagenase-Tyrode buffer, suspended in Krebs-Henseleit buffer containing 10% fetal bovine serum and 1.8 mM Ca2+, and incubated (1 x 10(5) cells/well) in a CO2 incubator under several experimental conditions, including buffer alone, buffer plus 10% burn serum, buffer plus 10% sham serum, or buffer plus either burn or sham PMN (25 x 10(5) cells/well). Myocyte viability (%) and creatine kinase (CK; units.ml-1.10(5) cells-1) were unchanged after incubation with sham plasma or sham PMN. Incubation of sham myocytes with burn plasma caused viability to fall (from 79 +/- 3 to 54 +/- 4%, P < 0.002), whereas CK rose (from 1,639 +/- 115 to 2,803 +/- 132 units.ml-1.10(5) cells-1, P < 0.01). Similarly, incubation of sham myocytes with burn PMN reduced viability (from 83 +/- 2 to 50 +/- 3%, P < 0.01), whereas CK remained unchanged (1,880 +/- 168 units.ml-1.10(5) cells-1). Our data indicate that circulating myocardial depressant factors after burn injury contribute to cardiac myocyte injury.

Specificity of afferent and efferent regeneration in the cockroach: establishment of a reflex pathway between contralaterally homologous target cells

1978 ◽  
Vol 41 (4) ◽  
pp. 885-895 ◽  
Author(s):  
C. R. Fourtner ◽  
C. D. Drewes ◽  
T. W. Holzmann
Keyword(s):  
Motor Neuron ◽  
Temporal Analysis ◽  
Normal Activity ◽  
Monosynaptic Reflex ◽  
Target Cells ◽  
Experimental Conditions ◽  
Stimulus Interval ◽  
Experimental Procedure ◽  
Metathoracic Ganglion ◽  
The Right

1. In 132 cockroaches the main leg nerve on one side (right), of the metathoracic segment was crossed to the opposite (left) side and allowed to regenerate. In 3-8 wk, 59% of the animals displayed reflex activity in the left leg (behaviorally demonstrated by leg withdrawal following tarsal stimulation). 2. EMGs from the femoral extensor revealed potentials characteristic of normal activity in the extensor, which is innervated by an identified motor neuron, Ds. 3. Intracellular recordings from processes within the right hemiganglion of the metathoracic ganglion (CNS) demonstrated 1:1 activity between a unit in the CNS recording and the EMG of the left extensor. Subsequent intracellular staining revealed that the unit was on the right side of the CNS and was identified as motor neuron Ds by the location of its soma and dendrites. This finding indicated that specific, contralateral, efferent reinnervation occurs in the cockroach. 4. In normal cockroaches a monosynaptic reflex exists between hair plate afferents and Ds. A temporal analysis (stimulus-interval histogram) indicated that the reflex is also established in the crossed-regenerated animals. These data suggested that specific contralateral afferent reinnervation also occurs in the cockroach and that the monosynaptic nature of the normal reflex was reestablished. 5. Therefore, cell-to-cell specificity in neuron-to-neuron or neuron-to-muscle interactions not only occurs in normally developing or regenerating animals but also occurs between contralaterally homologous target cells, given the proper experimental conditions. It is also suggested that this experimental procedure of redesigning pathways may be a useful tool for further studies of behavior.

scholarly journals The Contribution of IgG Glycosylation to Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) in Hashimoto’s Thyroiditis: An in Vitro Model of Thyroid Autoimmunity

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 171 ◽  
Author(s):  
Marta Ząbczyńska ◽  
Katarzyna Polak ◽  
Kamila Kozłowska ◽  
Grzegorz Sokołowski ◽  
Ewa Pocheć
Keyword(s):  
Cell Line ◽  
Hashimoto’S Thyroiditis ◽  
Leukemia Cell Line ◽  
Hashimoto's Thyroiditis ◽  
Target Cells ◽  
Healthy Donors ◽  
Complement Dependent Cytotoxicity ◽  
Dependent Cell ◽  
Igg Glycosylation

Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are involved in destruction of thyroid tissue in Hashimoto’s thyroiditis (HT). N-glycosylation of the Fc fragment affects the effector functions of IgG by enhancing or suppressing the cytotoxicity effect. The aim of the present study was to assess the impact of HT-specific IgG glycosylation in ADCC and CDC, using in vitro models. The normal thyroid Nthy-ori 3-1 cell line and thyroid carcinoma FTC-133 cells were used as the target cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors and the HL-60 human promyelotic leukemia cell line served as the effector cells. IgG was isolated from sera of HT and healthy donors and then treated with α2-3,6,8-neuraminidase to cut off sialic acids (SA) from N-glycans. We observed more intensive cytotoxicity in the presence of IgG from HT patients than in the presence of IgG from healthy donors. Removal of SA from IgG N-glycans increased ADCC intensity and reduced CDC. We conclude that the enhanced thyrocyte lysis resulted from the higher anti-TPO content in the whole IgG pool of HT donors and from altered IgG glycosylation in HT autoimmunity.

The shared tumor-associated antigen cytochrome P450 1B1 is recognized by specific cytotoxic T cells

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3287-3294 ◽  
Author(s):  
Britta Maecker ◽  
David H. Sherr ◽  
Robert H. Vonderheide ◽  
Michael S. von Bergwelt-Baildon ◽  
Naoto Hirano ◽  
...  
Keyword(s):  
T Cells ◽  
Cytochrome P450 ◽  
Cytotoxic T Cells ◽  
Phase 1 ◽  
Cell Epitope ◽  
Target Cells ◽  
Cytochrome P450 1B1 ◽  
Tumor Associated Antigen ◽  
Healthy Donors ◽  
Binding Peptides

AbstractCytochrome P450 1B1 (CYP1B1), a drug-metabolizing extrahepatic enzyme, was recently shown to be overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for anticancer therapy, particularly cancer immunotherapeutics. We identified HLA-A*0201–binding peptides and a naturally processed and presented T-cell epitope capable of inducing CYP1B1-specific cytotoxic T lymphocytes (CTLs) in HLA-A2 transgenic mice. Furthermore, the induction of CYP1B1-specific T cells was demonstrated in healthy donors and cancer patients. These T cells efficiently lysed target cells pulsed with the cognate peptide. More important, HLA-A2–matched tumor cell lines and primary malignant cells were also recognized by CYP1B1-specific CTLs. These findings form the basis of a phase 1 clinical trial exploring a DNA-based vector encoding CYP1B1 for widely applicable cancer immunotherapy conducted at the Dana-Farber Cancer Institute.

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