A speculative explanation for this observation is that after vaccination, the stimulated NK cells may migrate to the infected cells and/or to secondary lymphoid organs where the antigen-bearing NK cells could settle in the T-cell area where they may stimulate T cells. live attenuated PRV vaccine strain triggers manifestation of MHC class II on porcine NK 5(6)-Carboxyfluorescein cells, that porcine NK cells can internalize debris from PRV-infected target cells, and that NK cells can stimulate proliferation of CD8+ and CD4+CD8+ PRV-experienced T cells. These results focus on the potential of focusing on these NK cell features in future vaccination strategies. comparisons between different conditions were performed using Tukey’s range test. Results Porcine NK Cells Internalize Debris Derived From Killed PRV-Infected Target Cells Recently, using the NK-susceptible cell collection K562, we showed that porcine NK cells are able to perform actin polymerization-dependent internalization of cell debris derived from their killed target cells (14). Here, we investigated whether porcine NK cells may also internalize debris from killed PRV-infected target cells, which is an important prerequisite for potential antigen showing properties of porcine NK cells in the context 5(6)-Carboxyfluorescein of an alphaherpesvirus infection. 5(6)-Carboxyfluorescein To test this, main porcine NK cells of healthy blood donors were used in cytolytic assays using CFSE-labeled mock-infected and crazy type (WT) PRV-infected swine kidney (SK) target cells. SK cells were infected at a MOI of 10 which we showed earlier to result in a 100% illness rate (22). Illness rate was confirmed for each assay by cell surface staining of viral protein gD and 5(6)-Carboxyfluorescein circulation cytometric analysis and was constantly 100% (data not demonstrated). Earlier, we also have demonstrated that co-incubation of NK cells KSHV ORF26 antibody with PRV-infected or mock-infected SK cells prospects to preferential killing of PRV-infected SK cells compared to mock-infected cells (23, 24). At different time points post co-incubation of NK and target cells, NK cells were analyzed by circulation cytometry for CFSE fluorescence as an indication for internalization of target cell debris, as described earlier for killed K562 target cells (14). To ensure that NK cells do not take up free CFSE from lysed target cells which has not covalently bound to cellular proteins, a control experiment was performed where NK cells were incubated for 2 h with either CFSE-labeled K562 cells or with supernatant of CFSE-labeled K562 cells that had been incubated before for 2 h with NK cells to result in K562 cell killing. NK cells incubated with supernatant of killed CFSE-labeled K562 cells did not become CFSE positive (Supplemental Number 1). After 2 h of co-incubation of NK cells with CFSE-labeled PRV-infected or mock-infected SK cells, a statistically significant higher amount (imply SD) (8.1 2.1%) of CFSE-positive NK cells were detected upon co-incubation with PRV-infected target cells compared to co-incubation with mock-infected cells (2.4 0.7%), indicative for internalization of debris derived from PRV-infected target cells from the NK cells (Number ?(Figure1).1). This increase in the number of CFSE-positive NK cells was followed by a progressive decrease (from 7.2 3.0% at 4 h to 4.7 1.9% at 8 h) (Number ?(Figure1),1), most in line with earlier results in K562 cells (14), suggesting that NK cells are able internalize debris and further process the internalized debris of PRV-infected target cells. Open in a separate window Number 1 Porcine NK cells internalize fragments of killed PRV-infected target cells. (A) Histograms display the CFSE transmission of IL-2-primed NK cells that were incubated for the indicated instances with PRV WT-infected SK cells (NK:target ratio 25:1) that had been labeled with CFSE (reddish open histogram), CFSE-labeled mock-infected SK cells (black open histogram) or not incubated with target cells (gray shaded histogram) of one representative pig (out of three). The amount of CFSE-positive cells (%) is definitely indicated in the histograms. (B) Graph shows the amount of CFSE-positive IL-2-primed NK cells that were co-incubated for the indicated instances with CFSE-labeled mock-infected SK cells or PRV crazy type-infected SK cells (effector target percentage of 25:1). Dot.
