Re-stimulated Nrp-1⁺CD8⁺ T cells maintained their high level of T cell activation.

Abstract

CD8+ T cells from Nrp-1tdTomato reporter mice or from C57BL/6 mice were separated into Nrp-1tdtomato+ or Nrp-1+ fluorochrome-labeled antibody-stained (represented as black bars) and Nrp-1- not endogenously expressing tdTomato or negative for Nrp-1 fluorochrome-labeled antibody staining (white bars) by FACS sorting 48 hours after in vitro stimulation. Cells were then stimulated again with αCD3 and αCD28 for 48 hours and analyzed by flow cytometry. (A) Nrp-1 expression of Nrp-1+ or Nrp-1- CD8+ T cells after sorting (post-sort) and re-stimulation with indicated concentrations of αCD3 and αCD28 was measured by flow cytometry. (B and C) The activation state (CD44, PD-1), effector function (GzmB) and proliferation (Ki-67) of CD8+ T cells that were either Nrp-1+ or Nrp-1- prior to re-stimulation was analyzed 48 hours after re-stimulation with (B) 1 μg/ml, (C) 0.5 μg/ml or 0.1 μg/ml αCD3/αCD28 on gated CD8+ T cells by flow cytometry. (D) The supernatants of this re-cultivation (with 1 ug/ml αCD3/αCD28) were collected and the concentrations of cytokines were determined using Luminex technology. Data from two to three independent experiments with n = 5–8 mice in total are shown as mean ± SD. Depending on the sorted cell number of Nrp-1+ or Nrp-1- CD8+ T cells, some of the experiments have been performed as technical duplicates. In this case, the mean was calculated and plotted as one dot. (A, C) ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test, (B) Student’s t-test or (D) Mann-Whitney test were used to test significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p