Neutrophils can activate T cells following RBC phagocytosis. (A) Schematic overview of the autologous TT-specific T-cell assay. On day 0, (CFSE-labeled) T cells were incubated with neutrophils ± RBCs or RBC-ops TT. On day 4, T cells were restimulated with neutrophils ± RBCs or RBC-ops and ± TT. Flow cytometry was used as readout on days (1, 4, and) 8. (B) T-cell activation was measured by flow cytometry by determining the percentage of CD25⁺, CD38, or HLA-DR⁺ CD4⁺ T cells after 1, 4, and 8 days. Results are shown for T cells incubated with neutrophils (Neutro) ± RBCs or RBC-ops. All conditions are plus TT. T cells plus RBCs served as a negative control and T cells plus PBMCs as positive control (mean ± SEM; n = 3). (C) Histograms showing CD25, CD38, and HLA-DR staining on CD4⁺ T cells on days 1, 4, and 8 of a representative donor. As a positive control for CD25, CD38 and HLA-DR staining T cells stimulated with anti-CD3 and anti-CD28 were used. An isotype control was used as negative control. (D) CD4⁺ T-cell proliferation was measured by flow cytometry by determining the percentage of CFSE low T cells after 8 days. Percentage proliferation is shown for T cells incubated with neutrophils ± RBCs or RBC-ops and ± TT. T cells ± RBCs served as a negative control and T cells plus PBMCs as positive control (mean ± SEM; n = 8). (E) The experiment was repeated with the addition of an MHC-II blocking antibody to show that neutrophil-induced T-cell proliferation is MHC-II restricted (mean ± SEM; n = 6). (F) Graph showing IFN-γ levels in the medium at day 8 (n = 6). Asterisks above the straight-line bars represent significant differences compared with T cells plus RBCs day 1; asterisks above the inverted U–shaped spanner bars represent significant differences between the indicated bars (****P < .0001; ***P < .001; **P < .01; *P < .05). Nonsignificant results have not been indicated.