Effective MHC class I tetramer–mediated depletion of antigen-specific CD8⁺ T cells. (A) Splenocytes of mice transgenic for the NP_366-374-specific F5 TCR (top), or HY_738-746-specific TCR (bottom) were mixed with wild-type splenocytes and subjected to 2 rounds of MHC tetramer–based depletion with D^b-NP or D^b-HY tetramers. Prior to depletion (Before), and after depletion (After), the percentage of NP- or HY-specific CD8⁺ T cells (top right corner) was determined by combined staining with α-CD8 antibody and D^b-NP or D^b-HY tetramers. Depletion was performed by staining with PE-labeled antigen-specific D^b tetramer, followed by negative selection with α-PE microbeads (Miltenyi Biotec). (B-C) Depletion of naive, polyclonal HY-specific T cells. Splenocytes from female B6 mice were depleted with D^b-HY tetramers and α-PE microbeads, or mock-depleted with α-PE microbeads alone. Cells (40 × 10⁶) were transferred into female TCRα-deficient mice, and 4 days after transfer, mice received an HY₇₃₈ peptide vaccination in combination with α-CD40 antibody treatment. At day 10 after vaccination, spleens were harvested and spleen cells were restimulated in vitro for 14 days. The percentage of HY₇₃₈-specific CD8⁺ T cells was assessed by D^b-HY tetramer staining (B), and by intracellular IFNγ staining (C), upon a 5-hour in vitro restimulation with the HY_738-746 peptide (P = .002), or with the irrelevant NP_366-374 peptide. Each dot plot in panel B represents a single mouse, and bars in panel C represent mean responses of 3 mice ± SD (representative of at least 2 independent experiments).