TCR-mediated functions are enhanced in activated peripheral blood T cells isolated from leucocyte reduction systems

Abstract

Buffy coats are the most common method for the acquisition of activated primary human T cells for research or clinical applications, but recently leukocyte reduction system (LRS) cones have emerged as a viable source for these cells. In this study, we determined if activated human T cells derived from buffy coats or LRS cones had different functionality. No changes in the expression of surface receptors were observed except for a significant increase in CD44 expression on T cells isolated from LRS cones. LRS cone-derived T cells trended towards higher receptor-mediated cytokine production and had significantly increased donor-to-donor variability in IFN-γ production. TCR-induced ERK1/ERK2 and AKT phosphorylation was also increased in T cells isolated from LRS cones. In conclusion, LRS cones are an excellent source of T cells for clinical and research applications, but these cells have subtle functional differences from T cells isolated using standard buffy coats.

Keywords: Buffy coat; Leukocyte reduction systems; T cell receptor; T cells.

Figure 1

Activated T cells from standard buffy coat isolation and LRS cone isolation have similar surface receptor expression. (A) The expression of surface markers on expanded T cells from the two isolation methods was assessed by flow cytometry. (B) The percentage of cells expressing surface markers was determined by flow cytometry after setting the isotype control stained cells to 4–5% positive. The bar is the average ± the 95% confidence interval for six separate donors for each isolation methods with the outliers removed as described in the Materials and Methods. The p value for significant changes in expression of CD44 is shown. (C) The median fluorescence intensity of live cells was determined by flow cytometry. The results are an average ± 95% confidence intervals for six donors for each isolation method with the outliers removed as described in the Materials and Methods. No significant changes were observed. (D) The co-expression of surface markers on expanded T cells from the LRS cones was assessed by flow cytometry. (E) The median fluorescence intensity of CD44 in live cells that were positive or negative for the indicated surface protein was determined by flow cytometry. The results are an average ± 95% confidence intervals for four donors. The p value for differences in expression of CD44 is shown.

Figure 2

Cytokine production and donor-to-donor variability is increased in the LRS cone isolated cells. T cells isolated from buffy coats or LRS cones were unstimulated (US) or stimulated with plate-bound anti-human CD3 (2 μg/ml) in the absence or presence of anti-CD28 (1 μg/ml), Fibronectin (1 μg/ml) or anti-CD44 (1 μg/ml) for 48 hours at 37 degrees. Supernatants were collected and assayed by ELISA for (A) IL-2 production and (B) IFN-γ production. The averages ± SEM of six different donors for each isolation method are shown with the outliers removed as described in the Materials and Methods.

Figure 3

Early TCR-mediated signaling is enhanced in T cells isolated from LRS cones. (A) T cells isolated using standard buffy coats or LRS cones were stimulated with anti-CD3 (10 μg/mL) and anti-CD4 (2 μg/mL) for various times. The phosphorylation of ERK1/ERK2 and AKT and the expression of ERK1/ERK2, AKT and actin were examined by immunoblotting. The results are a representative of six donors for each isolation method. The level phosphorylation of ERK1/ERK2 and AKT at each timepoint was normalized as described in the materials and methods. The relative phosphorylation levels for (B) ERK1/ERK2 and (C) AKT or the fold activation from the 0 timepoint for (D) ERK1/ERK2 and (E) AKT are shown as the average ± SEM of six donors for each isolation method with the outliers removed as described in the Materials and Methods. The p values for the statistical analysis of each point are shown on the graphs.