Ovalbumin-pulsed neutrophils injected via subcutaneous route induce naive CD8⁺ T-cell differentiation into effector cells. (A) CFDA-SE–labeled or unlabeled OT1 CD8⁺ T cells were adoptively transferred into β2-microglobulin^−/− mice. One day later, mice were injected subcutaneously with CFA plus PBS, Ova, Ova-pulsed LB27.4, Ova-pulsed BMNs, or Ova-pulsed DCs and OT1 activation was measured at day 5. (B) OT1 CD8⁺ T-cell proliferation was expressed as the percentage of CD8⁺ T cells in each cycle (mean ± SD of 3 separate experiments). ANOVA test was performed. The data were statistically significant for DCs and PMNs (*P < .001) when compared with controls. (C) At day 5, IFN-γ and IL-2 production was determined by ELISA 24 hours after in vitro restimulation. Results are expressed in ng/mL as mean (± SD) of 3 separate experiments. Mann-Whitney test was performed. The data were statistically significant for BMNs, PENs, and DCs (*P < .03) when compared with controls. (D) In vivo cytolysis assays were performed by injecting 3 × 10⁶ unpulsed CFDA-SE^low–labeled splenocytes and 3 × 10⁶ CFDA-SE^high–labeled SIINFEKL-pulsed splenocytes. Three hours later, CDFA-SE expression was analyzed by FACS in spleen and lymph nodes. Percentage of lysis was calculated as described in “In vivo cytolysis assay.” Data are presented as a percentage of lysis of peptide-pulsed targets of 3 separate experiments (mean ± SD). The data were statistically significant for DCs and PMNs when compared with controls (ANOVA test, *P < .001).