PBMC-07- Stimulation of human peripheral blood mononuclear cells with immobilized anti-CD3 and soluble anti-CD28 antibodies. v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Massimilano Legnaro ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
Emanuela Rasini ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Functional Responses ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

This protocol was designed to activate the lymphocytes T of a population of peripheral blood mononuclear cells (PBMCs), simulating their physiological response to antigen/MHC complex acting on T Cell Receptors-TCR , in order to test their functional responses including cell proliferation and cytokine production. The co-stimulation protocol include: i)anti-CD3 antibody a polyclonal activator specific for invariant framework epitopes on TCR complex (in particular, we use UCHT1 clone an anti-human CD3 antibody that recognizes the ε-chain of CD3 which is used for immobilized option of activation) (http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf) ii) anti-CD28 antibody used to cooperate with TCR signals promoting activation of T cells The procedure has been reproduced following the indications contained in the protocol of "EBiooscience" (https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf). Pilot experiments on PBMC were carried out to determine the best concentrations of anti-CD3 and anti-CD28 to induce optimal proliferation of PBMC and production of cytokines TNF-α and IFN-γ. We found a dose dependent correlation between immobilized anti-CD3 and cells functional responses. The selected amount was 2 µg/mL for both anti-CD3 and anti-CD28 that was the concentration below the maximum response which allows also to test possible modulations by therapeutic agents. References http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf https://www.bdbiosciences.com/ds/pm/tds/555330.pdf https://www.bdbiosciences.com/ds/pm/tds/555726.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using,sterile culture mediumand sterile plastic disposable as well.

scholarly journals Impaired Induction of the CD28-Responsive Complex in Granulocyte Colony-Stimulating Factor Mobilized CD4 T Cells

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 347-352 ◽  
Author(s):  
Junji Tanaka ◽  
Marco Mielcarek ◽  
Beverly Torok-Storb
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Stimulating Factor

Abstract Use of the CD28/B7 costimulatory signal for T-cell activation was analyzed in granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood mononuclear cells (G-PBMCs) and in peripheral blood mononuclear cells obtained before administration of G-CSF (preG-PBMCs). CTLA4Ig inhibition of OKT3-stimulated proliferation was significantly lower in G-PBMCs compared with preG-PBMCs (39.9% ± 5.6% and 72.2% ± 5.4%, respectively; P < .001). Furthermore, as shown in electrophoretic mobility-shift assays, the inducible level of the T-cell transcription factor CD28 responsive complex (CD28RC) was suppressed in CD4 cells derived from G-PBMC. However, depletion of CD14 cells from G-PBMCs restored CD28RC induction to normal levels. Taken together, these findings suggest that the large number of CD14 monocytes in G-PBMCs may limit T-cell responsiveness by suppressing the induction of the CD28RC.

scholarly journals Human erythroid burst-promoting activity produced by phytohemagglutinin- stimulated, radioresistant peripheral blood mononuclear cells

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1050-1057 ◽  
Author(s):  
D Meytes ◽  
JA Ma Ortega ◽  
NA Shore ◽  
PP Dukes
Keyword(s):  
T Cell ◽  
Red Blood Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract The regulation of erythroid burst-colony formation was studied in cultures of human peripheral blood mononuclear cells. Numbers of erythropoietin-stimulated colonies obtainable from the cells in response to various treatments were compared. One-day preincubation of the cells with phytohemagglutinin (PHA) doubled the yield of colonies. Irradiation of the cells with 3000 rad eliminated their ability to form erythroid bursts, but did not impair the ability of PHA-treated cells to enhance burst formation when added to a fresh batch of cells. This was due to a humoral factor, since media conditioned by PHA-treated washed cells were as effective as the cells themselves. When cells were separated into subpopulations by an adherence procedure and according to their ability to form rosettes with sheep red blood cells, it was found that the PHA-dependent burst-promoting activity released into the medium originated in a nonadherent, nonrosetting (T-cell depleted) cell population.

Effect of the antirheumatic agent Tenidap on CD3, CD4, and CD8 expression and interleukin-1 and leukotriene B4 secretion in human peripheral blood mononuclear cells

1994 ◽  
Vol 72 (9-10) ◽  
pp. 397-402 ◽  
Author(s):  
Pio Conti ◽  
Marcella Reale ◽  
Renato C. Barbacane ◽  
Stephano Stuard ◽  
Fernanda Placido
Keyword(s):  
T Cell ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Interleukin 1 ◽  
Inhibitory Effect ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Antirheumatic Agent

Thymocytes that express the complete CD3−T-cell receptor (TCR) complex are CD4− and CD8−. The CD4+ T-cell population can be subdivided into at least two quite distinct subsets, TH1 and TH2 cells, based upon cytokine expression. Interleukin-1 (IL-1) appears to be required for optimal proliferation of T cells in response to antigen and it seems that in the absence of IL-1, TH2 clones proliferate less in response to antigen. Tenidap is an antirheumatic agent that has an inhibitory effect on IL-1 production. In these studies, we show that isolated human peripheral blood mononuclear cells (PBMCs) treated in vitro with Tenidap (15 μg/mL) for 48-h incubations significantly (p < 0.05) enhanced the present of CD4+ expression compared with untreated cells (control), as determined by cytofluorimetric analysis. Lipopolysaccharide and Bacillus Calmette-Guérin were used as positive controls. When the cells were tested for CD3 or CD8 receptor expression, no differences were found between the untreated PBMCs and the treated (15 μg/mL Tenidap) cells. No change was found when cells were incubated for 72 h. Moreover, our data show a strong dose-dependent inhibitory effect of Tenidap (15 μg/mL) on IL-1α, IL-1β, and leukotriene B4 secretion in PBMCs treated overnight. The increased CD4+ expression by Tenidap in PBMCs may suggest an important role for this new antirheumatic agent in immunity and may hold future therapeutic promise for diseases involving IL-1 and leukotriene B4 as mediators.Key words: Tenidap, lymphocyte receptors, leukotriene B4, interleukin-1, lipopolysaccharide.

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