GSE135936
Human plasma-like medium improves T lymphocyte activation
T lymphocytes are critical effectors of the immune system and the ability to study their behavior in synthetic media in vitro has facilitated major discoveries in their development, function, and fate. Recently, it has been shown that the constituents in human plasma differ markedly from commonly used synthetic media and these differences have important effects on cell physiology. We therefore compared T lymphocyte activation in human plasma-like medium (HPLM) to RPMI conventional medium both with and without serum. We found that HPLM and RPMI induced vastly different transcriptional programs, especially for transcripts encoding proteins involved in activation, proliferation and metabolism, in human peripheral blood T cells after T cell receptor (TCR) stimulation. We also found that in dialyzed fetal bovine serum (dFBS), HPLM supports a stronger response to TCR engagement compared to RPMI. In reconstruction experiments, we demonstrated that this activation difference is due to RPMI being hypocalcemic relative to the in vivo milieu and that addition of calcium chloride to RPMI can improve human T lymphocyte activation. Thus, we conclude that investigators should be cognizant of differences between commonly used media formulations and the in vivo environment since this could profoundly affect their experimental results and that physiologic media is a valuable new way to study immune cells in culture Overall design: T cells from healthy human volunteers were isolated from PBMCs via magnetic cell sorting (Miltenyi) and cultured in either RPMI (US biologicals) or HPLM (Cantor et al. 2017). Both cultures were supplemented with dialyzed FBS (Thermo Fisher) at 10% by volume. Maxisorp 96 well plates (Thermo Fisher) were coated with 1 mg/ml of anti-CD3 and anti-CD28 antibodies (clone Hit3a, Biolegend) overnight at 4°C. T cells were then stimulated using these plates at a concentration of 0.5 x 10^6 cells/ml for 48 or 120 hours. For the 120 hour cultures, media was changed and cells were re-normalized to a concentration of 0.5 x 10^6 cells/ml at 48 and 96 hours post activation.100 ng – 1 ug of total RNA as the input to an mRNA capture with oligo-dT coated magnetic beads using Illumina TruSeq protocol. The mRNA is fragmented, and then a random-primed cDNA synthesis is performed. The resulting double-strand cDNA is used as the input to a standard Illumina library prep with end-repair, adapter ligation and PCR amplification and then quantitated by qPCR followed by cluster generation and sequencing.