Ex vivo MHC class II tetramer staining and sorting.

Two x 10⁷ PBMCs were incubated with PE- and APC-conjugated HLA-DRB5*01∶01-IE1_{211–225(H6)} tetramers for 2 h at 37°C. Approximately 1000 tetramer-positive cells were isolated on FACS-ARIAII and cultured for 14 days with irradiated autologous dendritic cells pulsed with IE1_211–225. One thousand tetramer-negative, CD4+ T cells were isolated as control and cultured under identical conditions. Following in vitro culture, tetramer-positive cells expanded to approximately 6×10⁵ cells whereas tetramer-negative cells expanded to 10⁴ cells. A) Gated tetramer-positive CD4+ T cells (encircled) shown on FACS-ARIAII following ex vivo staining. B) Dot plots showing the percentage of cells that stain with tetramer following in vitro culture of tetramer-positive and tetramer-negative sorted cells. The cells were stained with PE-conjugated HLA-DRB5*01∶01-IE1_{211–225(H6)} (y-axis) and APC-conjugated HLA-DRB5*01∶01-CLIP(H6) (x-axis). Ninety-seven point nine % of ex vivo tetramer-positive sorted in vitro cultured cells stain with HLA-DRB5*01∶01-IE1_{211–225(H6)} (left dot plot) compared to 2.6% among ex vivo tetramer-negative in vitro cultured cells (right dot plot). No staining with irrelevant tetramer can be observed in either population. C) ICS of in vitro cultured tetramer-positive cells stimulated with IE1_211–225–pulsed irradiated autologous dendritic cells. Ninety-four % produce cytokine (IFN-γ and TNF-α) and up regulate CD69.