Figure 2. CD27 is proteolytically cleaved from the surface of T cells in a CD70 dependent manner.

A. CD27^−/− BMDCs were matured in vitro with either LPS or αCD40/PolyIC and pulsed with the SIINFEKL peptide and then co-cultured with WT OT1 T cells (white histogram), CD27^−/− OT1 T cells (gray histogram), WT OT1 T cells plus the blocking antibody RM27 (dashed line), or without T cells (black histogram). BMDCs were stained for the acquisition of CD27 and shown are the flow cytometry histograms. B. Quantitation of groups shown in A. C. WT and CD70^−/− BMDCs were matured with LPS and pulsed with SIINFEKL as in A and B. BMDCs were then labeled with CFSE (green) and OT1 T cells were labeled with CMTMR (blue). 2–6 hours after co-culturing labeled OT1s with BMDCs cells were stained with an APC conjugated CD27 antibody (red) and paraformaldehyde fixed before visualizing on a spinning disk microscope. White scale bar is 10μm in length. The image was captured with a 40x objective. D. Microscope slides were divided into sections and imaged. Fold increase in CD27 staining over BMDCs cultured without T cells was calculated using based on all images for both WT and CD70−/− BMDCs. E. As above, matured BMDCs were co-cultured with T cells in the presence or absence of an MMP8 inhibitor or a CD70 blocking antibody. 18–24 hours later the T cells were stained with αCD27 antibody and the MFI compared to the αCD27 MFI on T cells before incubation with BMDCs (pre). Each experiment was repeated three or more times with at least three replicates per group with similar results. Data shown are representative of these experiments. Data shown is the mean +/−SEM.One asterisk represents differences with a p-value less than 0.05, three asterisks represent a p-value less than 0.001 by student’s t test. Only values that were significant across experiments are shown with an asterisk.