III. ABSTRACTS AND POSTERS / ORAL PRESENTATIONS
6. Mice deficient in interferon regulatory factor 4 (IRF4) are more susceptible to infection with mouse-adapted influenza A/Aichi/2/68
2.5.4. Cytotoxic T-lymphocytes are activated following SARS-CoV DNA immunization
A standard 4-h 51Cr release assay was performed to characterize the cytotoxicity of the in vitro restimulated splenocytes. RMA/S cells (4 x 103) pulsed with each of the five specific high-binding epitopes (S436, S497, S525, S884 and S1116) were used as targets. Assays with an effector-to-target ratio of 100:1 to 11:1 were performed in triplicate.
The negative controls, i.e. RMA/S cells pulsed with an irrelevant epitope (Ova257-264) as well as non-epitope-presenting RMA/S cells, showed no cytotoxicity.
Restimulated splenocytes of mice immunized with PBS (Fig. 2.12) as well as vector alone (Fig. 2.13) did not show any cytotoxicity. The highest cytotoxicity levels were observed for restimulated splenocytes of mice immunized with S-RGD/His and S-His against S1116-pulsed RMA/S cells. The cytotoxicity of S-His cultures (Fig. 2.14) was slightly higher than that of S-RGD/His (Fig. 2.15).
53 Figure 2.9. 4 hour-51Cr Cytotoxicity assay of OTI splenocytes effector cells in vitro stimulated with ConA against various target cells (EL4 pulsed with 20àM Ova257-264
peptide, RMA/S cells pulsed with 20àM Ova257-264 peptide and E.G7-Ova transfected cells) labeled with 100μCi 51Cr (in the form of Na2[51Cr]O4). Effector cells, from splenocytes harvested from OT1 mouse (stimulated with ConA for three days before being subjected to the cytotoxicity assay) were added at a specific number to a constant number (4 x 103 cells/well) of the three target cell lines (EL4 pulsed with Ova257-264 peptide, RMA/S cells pulsed with Ova257-264 peptide and E.G7-Ova transfected cells). Con-A-in vitro-stimulated OTI splenocytes were able to effectively lyse and kill all three target cell lines.
% specific lysis = [(experimental release-spontaneous release) / (maximum release-spontaneous release)].
Each cytotoxicity assay well was performed in triplicates and representative data is shown.
54 Figure 2.10. 4 hour-51Cr Cytotoxicity assay of purified-CD8+ cytotoxic T cells from C57BL/6 spleen, previously primed with irradiated RMA/S-Ova257-264-peptide-pulsed cells, then in vitro-stimulated with Ova257-264-peptide-pulsed RMA/S cells, against various target cells (Unpulsed-EL4 cells, E.G7-Ova transfected cells, Unpulsed/Unloaded-RMA/S cells and H2-Kb-restricted-OVA257-264-pulsed RMA/S cells) labeled with 100μCi 51Cr (in the form of Na2[51Cr]O4). Splenocytes were from C57BL/6 mice immunized intra-peritoneally twice with 5 x 105 irradiated peptide- pulsed RMA/S cells at two-week intervals. These cells were in vitro-stimulated with 7800 Rads-irradiated Ova257-264-peptide-pulsed-RMA/S cells for three to five days before being purified using a EasySep™ Mouse CD8a Positive Selection Kit (StemCell Technologies, Canada). Purified CD8+ T cells were then subjected to cytotoxicity assay. Prepared targets cells labeled with 51Cr were dispensed to 96-well round bottom plate at 4 x 103 cells/well. A graded increase in effector cells numbers were then mixed to specific wells containing a constant number of the target cells.
Minimum or no cytotoxicity was observed against unpulsed EL4 and unpulsed/unloaded RMA/S target cells.
% specific lysis = [(experimental release-spontaneous release) / (maximum release-spontaneous release)].
Each cytotoxicity assay well was performed in triplicates and representative data is shown.
55 Figure 2.11. 4 hour-51Cr Cytotoxicity assay of crude/unpurified C57BL/6 splenocytes, previously primed with irradiated RMA/S-Ova257-264-peptide-pulsed cells, then in vitro-stimulated with Ova257-264-peptide-pulsed RMA/S cells, against various target cells (Unpulsed-EL4 cells, E.G7-Ova transfected cells, Unpulsed/Unloaded-RMA/S cells and H2-Kb-restricted-OVA257-264-pulsed RMA/S cells). Splenocytes were harvested from C57BL/6 mice immunized intra-peritoneally twice with 5 x 105 irradiated peptide-pulsed RMA/S cells at two-week intervals. These cells were in vitro-stimulated with 7800 Rads-irradiated Ova257-264-peptide-pulsed-RMA/S cells for three to five days before being subjected to cytotoxicity assay. Prepared targets cells labeled with 51Cr were dispensed to 96-well round bottom plate at 4 x 103 cells/well.
A graded increase in effector cells numbers were then mixed to specific wells containing a constant number of the target cells. Splenocytes showed cytotoxicity against Ova257-264-peptide pulsed RMA/S. Only a mild cytotoxicity was observed against E.G7-Ova transfected cells. Minimum or no cytotoxicity was observed against unpulsed EL4 and unpulsed/unloaded RMA/S target cells.
% specific lysis = [(experimental release-spontaneous release) / (maximum release-spontaneous release)].
Each cytotoxicity assay well was performed in triplicates and representative data is shown.
56 Figure 2.12. Cytotoxicity of PBS control immunised mice. 51Cr cytotoxicty assay of in vitro stimulated splenocytes of mice immunized with PBS, No cytotoxicity was observed for in vitro stimulated splenocytes of mice mock-immunized with PBS or vector only. RMA/S cells were cultured overnight, and pulsed with 20μM of each respective peptide for 1h. Each target line was pulsed with 50μCi of 51Cr at 37ºC for 1h. Assays were performed in triplicate, and cytotoxicity was calculated as (sample release – spontaneous release) divided by (maximum release – spontaneous release).
Data are expressed as the means of triplicate wells.
Figure 2.13. Cytotoxicity of splenocytes from mice immunized with DNA vector alone. 51Cr cytotoxicty assay of in vitro stimulated splenocytes of mice immunized with pcDNA3.1 Vector only. No cytotoxicity was observed for in vitro stimulated splenocytes of mice mock-immunized with vector only. RMA/S cells were cultured overnight, and pulsed with 20μM of each respective peptide for 1h. Each target line was pulsed with 50μCi of 51Cr at 37ºC for 1h. Assays were performed in triplicate, and cytotoxicity was calculated as (sample release – spontaneous release) divided by (maximum release – spontaneous release). Data are expressed as the means of triplicate wells.
57 Figure 2.14. Cytotoxcity of splenocytes from mice immunized with S-His DNA vaccine. 51Cr cytotoxicty assay of in vitro stimulated splenocytes of mice immunized with S-His. RMA/S cells were cultured overnight, and pulsed with 20μM of each respective peptide for 1h. Each target line was pulsed with 50μCi of 51Cr at 37ºC for 1h. Assays were performed in triplicate, and cytotoxicity was calculated as (sample release – spontaneous release) divided by (maximum release – spontaneous release).
Data are expressed as the means of triplicate wells.
Figure 2.15. Cytotoxcity of splenocytes from mice immunized with S-RGD/His DNA vaccine. 51Cr cytotoxicty assay of in vitro stimulated splenocytes of mice immunized with S-RGD/His. RMA/S cells were cultured overnight, and pulsed with 20μM of each respective peptide for 1h. Each target line was pulsed with 50μCi of 51Cr at 37ºC for 1h. Assays were performed in triplicate, and cytotoxicity was calculated as (sample release – spontaneous release) divided by (maximum release – spontaneous release). Data are expressed as the means of triplicate wells.