HLA class II tetramers were produced as previously described. Briefly, recombinant α- and β-chains (HLA-DRA1 and DRB5*01:01) were folded in the presence of either the wild-type IE1211-225 peptide, P3 variant, or P7 variant of the wild-type peptide. The resulting monomers were tetramerized with PE- or APC-conjugated streptavidin.
PBMC's from a donor previously determined to recognize the DRB5*01:01 restricted CMV IE1211–225 epitope (Braendstrup et al. 2014 (link)) was expanded on this epitope. The cells were double-stained with PE-labeled wild-type IE1211–225 -DRB5*01:01 tetramer and APC-labeled mutant peptide-DRB5*01:01 tetramer as previously described (Braendstrup et al. 2013 (link)). The cells were washed and subsequently stained with anti-CD3-Pacific blue and anti-CD4-PerCP antibody (Biolegend, San Diego, USA) for 30 min. The stained cells were analyzed by flow cytometry on a Fortessa (BD Biosciences).
PBMC's from a donor previously determined to recognize the DRB5*01:01 restricted CMV IE1211–225 epitope (Braendstrup et al. 2014 (link)) was expanded on this epitope. The cells were double-stained with PE-labeled wild-type IE1211–225 -DRB5*01:01 tetramer and APC-labeled mutant peptide-DRB5*01:01 tetramer as previously described (Braendstrup et al. 2013 (link)). The cells were washed and subsequently stained with anti-CD3-Pacific blue and anti-CD4-PerCP antibody (Biolegend, San Diego, USA) for 30 min. The stained cells were analyzed by flow cytometry on a Fortessa (BD Biosciences).
