Fig. 8.

pDNA induces CD4 T cell proliferative responses early after intramuscular immunisation. To readout Ag presentation to CD4⁺ T cells in vivo, we transferred CFSE-labelled Eα-specific TEa T cells into B6 recipients and analysed the kinetics of T cell activation and cell division following injection with Eα-expressing plasmids. Mice that had received CFSE-labelled TEa Tg T cells 1 day previously, were immunised with pCI-EαRFP, pCI-EαGFP or the control plasmid pCIneo. Draining lymph nodes (popliteal and inguinal), distal peripheral lymph nodes (cervical, brachial and axial) and the spleen were collected at different times after immunisation (12 h–10 days) and analysed by flow cytometry for blastogenesis, clonal expansion and division of Tg lymphocytes. (A) At d3 post-immunisation we observed an increase in both the percentage and blastogenesis of Eα-specific Tg T cells in draining LNs (pooled popliteal and inguinal), distal peripheral LNs and the spleens of pCI-EαRFP- and pCI-EαGFP-immunised mice. No such increases were observed in the pCIneo group. (B and C) We observed division of TEa T cells in draining and distal lymph nodes and spleen at d5, d7 and d10 post-immunisation following injection with pCI-EαRFP but not the control plasmid pCIneo. Kinetic analysis of cell division (C) revealed that by day 5 post-pCI-EαRFP immunisation Eα-specific lymphocytes in the popLN and spleen had started to divide, and this number had increased by days 7 and 10. In contrast Eα-specific lymphocytes in distal lymph nodes has increased in number by day 7, but no further increased was observed at day 10. Cells from pCIneo-immunised mice showed only background staining at all timepoints. Error bars show standards errors and asterisk (*) indicates statistical significance compared to pCIneo-immunised group.