Nrp-1 expression defines a highly activated CD8⁺ T cell phenotype.

Abstract

Splenic CD8+ T cells from naïve C57BL/6 mice were stimulated with αCD3 and αCD28 in vitro. (A) Nrp-1, CD69 and PD-1 expression of CD8+ T cells was analyzed by flow cytometry after 0, 24, 48, 72 or 96 hours of in vitro stimulation with 1 μg/ml αCD3/αCD28. (B) Nrp-1 expression on gated CD8+ T cells (left) and the expression of activation-associated molecules was analyzed by flow cytometry on Nrp-1+ (black bars) and Nrp-1- CD8+ T cells (white bars) 48 hours after in vitro stimulation with 1 μg/ml, 0.5 μg/ml or 0.1 μg/ml αCD3/αCD28 and is summarized as mean ± SD. Experiments have been performed as technical duplicates of stimulated T cells from one mouse. Mean values of duplicates have been included as one data point that represents one biological replicate (cells from one mouse). (C) The stability of Nrp-1 expression after in vitro induction was analyzed by isolating Nrp-1+CD8+ T cells using FACS after 48 hours of stimulation. The cells were re-cultured with IL-2 and Nrp-1 expression was analyzed at 0, 24, 48, 72 and 96 hours after re-cultivation by flow cytometry as shown by exemplary contour plots. Data from one to two independent experiments with (A) n = 8 mice in total and (B) n = 3–8 mice in total and (C) one experiment with n = 3 mice in total are presented as mean values with SD. (A, B) Ordinary one-way ANOVA with Holm-Sidak’s or Tukey’s multiple comparisons test or (C) Kruskal-Wallis test with Dunn’s multiple comparisons test were used to test significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p