Inhibition of T cell activation by VARV B22.

<p><b>A)</b> Sequence comparison of MPXV 197 and its orthologs by Geneious v5.6.3. Black, green, and red bars show consensus sequence, conserved, and hydrophobic residues, respectively. <b>B)</b> CHO cells were transduced with Ad-B22R, Ad-197 and Ad-tTA for 24 h followed by immunoblotting with αFLAG. Right panel: Overexposure reveals a >250 kDa band (asterisk). <b>C)</b> CHO cells were transduced as in B). After 24 h, cell surface proteins were biotinylated followed by immunoprecipitation with NeutrAvidin, electrophoretic separation and immunoblotting with αFLAG. <b>D)</b> CHO cells were transfected with pCDNA3.1-B22-CFlag (24 h), fixed, and either permeabilized (‘intracellular’) or left unpermeabilized (‘cell-surface’). The samples were stained with αFLAG and analyzed by LSCM. The scale bar is 20 µm. <b>E)</b> BEAS-2b cells, uninfected (UN) or transduced with Ad-197/Ad-tTA (‘Ad-197’) or Ad-tTA only (‘Ad-control’) were used to stimulate human <i>Mtb</i>-specific T cell clone D466 D6 with CFP10_2-12 peptide. <b>F)</b> CM9-specific T-cells were incubated (18 h) with CHO cells either uninfected (UN) or transduced with Ad-B22R/Ad-tTA (‘Ad-B22R’) or Ad-tTA only (‘Ad-control’) followed by stimulation with CM9-peptide pulsed BLCLs.