Hello,
I would like to analyse naive CD4+ T-cell activation and intracellular processes with a machine that does not allow CD3-coated plates. I thought of activating my cells with plate-bound CD3 and soluble CD28 for 24h, and then analyzing them.
The issue is that I do not know how long activated naive CD4+ T- cells remain completely activated (both metabolically and in terms of cytokine production) after removal from plate-bound CD3/soluble CD28. Does anybody have experience with this? Do the cells remain completely activated for several hours after removal of CD3/28, or does that drop rapidly?
Alternatively, would culturing the cells on plate-bound anti-CD3/soluble CD28 followed by harvesting the cells and adding soluble CD3/CD28 also work for a short experiment (few hours) or are CD3/28-linked beads necessary here?
Thank you in advance!
Hello, Liselotte!
You look at these protocols, please:
https://clincancerres.aacrjournals.org/content/5/2/461
T-Cell Activation and Expansion.
Twenty-four-well cell culture plates (Costar, Cambridge, MA) were coated with antihuman CD3 mAb (OKT3; Ortho Pharmaceutical Corp., Raritan, NJ) at a concentration of 2 μg/ml in PBS or coated with the combination of antihuman CD3 plus antihuman CD28 mAb (PharMingen, San Diego, CA) at a ratio of 1:1. The concentration of each antibody in the combination was 2 μg/ml in PBS. Each well of a 24-well plate was coated with 600 μl of anti-CD3 or the anti-CD3/anti-CD28 antibody combination at 4°C overnight or at room temperature for 5–6 h. The coated plates were then washed twice with PBS. Purified VPLN T cells were placed on the precoated and washed plates at 1 × 106–2 × 106 cells per ml in 2 ml of X-Vivo-15 medium and cultured for 4 days in 5% humidified CO2. After exposure to the immobilized antibodies, the cells were harvested and counted. The cells were then expanded in IL-2 (Chiron, Emeryville, CA) containing X-Vivo-15 medium starting at a concentration of 3 × 105 cells/ml in six-well culture plates (Costar) for 7 days. The concentration of IL-2 was 50 units/ml. During the 7-day cell expansion period, additional IL-2-containing medium was added and cells were split as they grew to keep the cell density below 3 × 106 cells/ml.
Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: Differing impact on CD8 T cell phenotype and responsiveness to restimulation
Results Time course for T cell response to stimulation The CFSE-labeled CD4 and CD8 T cells in MNCs began dividing 40-50 hours after stimulation with anti-CD3/ CD28 beads or soluble anti-CD3. CD8 cells divided slightly more rapidly than CD4 cells, and there were no consistent differences in early response to the two stimuli (Figure 1A and 1C). The number of viable cells at hour 40 (just before proliferation began) was similar in unstimulated, bead-stimulated, and anti-CD3-stimulated wells indicating neither stimulus caused extensive early activation-induced cell death (AICD) (Figure 1B and 1D). Consistent with the timing of cell division, expansion in cell number in response to either stimulus was delayed until 50-60 hours after stimulation.