A dual neutrophil-T cell purification procedure and methodological considerations in studying the effects of estrogen on human Th17 cell differentiation

Abstract

New procedures are required to optimize the use of blood samples to study different cell types. The purification of neutrophils and T cells from the same blood sample is not commonly described. We have previously used PolymorphPrep™ (P) or LymphoPrep™ (L) for purifying neutrophils or T cells, respectively. In this study, we describe a new method for purifying both of these cells using P and L from the same sample, and methodological considerations required to obtain consistent Th17 differentiation results. For T cell studies, we first isolated mononuclear cells from peripheral blood of healthy humans using either P alone, L alone or sequential isolation with P and then L (P + L). CD3+ lymphocytes comprise up to 73% of peripheral blood mononuclear cells (PBMCs) obtained by sequential isolation, with 29% and 36% for P and L, respectively. T lymphocyte subsets, Th1, Th17 or double-positive (Th17/1), were then amplified. Four days of amplification culture after isolation by P alone led to over-expression of Th17/1 cells and of Th17 cells in comparison to cells isolated by L or by sequential P + L. Th17/1 cells comprised 11.0 ± 6.8% (P alone) vs 1.2 ± 0.28% (L alone) vs 0.45 ± 0.11% (P + L) and Th17 cells comprised 2.8 ± 0.4% (P alone) 0.88 ± 0.15% (L alone) vs 0.86 ± 0.14% (P + L). As the second step, we examined T cell purification and differentiation. A higher purity of 97.1 ± 0.44% naïve CD4+ T cell was reached after P + L followed by immunomagnetic bead sorting in comparison to 70 ± 9.3% (L) vs 21.0 ± 8.5% (P). These cells grew well in the density range of 25, 000 to 100, 000 cells per well in 96-well plates during Th17 cell differentiation; higher or lower cell density did not support Th17 cell differentiation. Lastly, to investigate the effect of estrogen on Th17 cell differentiation, serum-free AIM V medium without phenol red was chosen to minimize the hormonal effects of the medium. We found that exogenous estrogen (1 nM) inhibited Th17 cell differentiation in this medium. Taken together, we devised a method to isolate both neutrophils and T cells from the same blood sample and show that high PBMC purity, selected culture medium and an optimal cell density of the initial cell culture produced the most robust and consistent results for Th17 differentiation.