Induction of c-fos is additive during sequential in vivo antigen-specific stimulations.

<p>A. Experimental design: CD45.1 Marilyn TCR transgenic mice were injected with 5 µg Dby peptide and splenocytes and LN cells were harvested 30 minutes post-injection and left unlabeled. In parallel, control Marilyn mice were injected with PBS and splenocytes and LN cells were harvested 30 minutes post-injection and labeled with Cell-Tracker Blue. CD4+ T cells from both sources were purified by negative selection and mixed at a 1∶1 ratio before adoptive transfer into wildtype CD45.2 C57BL/6 congenic mice. Host mice received an injection of 5 µg Dby peptide or PBS, and splenocytes were harvested 30 minutes later for intracellular staining of c-fos followed by FACS analysis. B. Left panel: Upon adoptive transfer, Marilyn T cells can be identified in the host spleen based on CD45.1 expression. Furthermore, Marilyn T cells purified from mice that received peptide (orange box, +Dby) can be distinguished from those purified from mice that did not receive peptide (blue box, +PBS) based on Cell-Tracker Blue labeling (left panel). Right panel: A single stimulation with Dby induces upregulation of c-fos (blue line) but two sequential stimulations result in a further increase in c-fos levels (orange line). Shaded gray histograms show c-fos levels in endogenous CD4 T cells as a baseline comparison. C. Change in of c-fos protein levels in Marilyn T cells at the end of the second in vivo stimulation for the indicated combination of stimulation. Results are compiled from 2 independent experiments and shown as the percentage (mean±sem) of the maximum c-fos fluorescence observed in each experiments.