Figure 3.

Validation of specific binding and killing of CD8⁺ T cells by SAP-coupled K^d tetramers in vitro and in vivo. (a) K^d tetramers prepared with SA-SAP retain binding specificity, and free SAP does not bind CD8⁺ T cells. Peripheral blood lymphocytes from NOD 8.3 and NOD CL4 mice were incubated with tetramers at 4°C for 1 h, washed extensively, and probed for surface SAP binding with polyclonal anti-SAP Abs. (b) T cells are killed by cognate SAP-coupled tetramers in 72 h. Cultured T cells were harvested at 1, 24, 48 or 72 h. (c) T cells are killed by cognate SAP-coupled tetramers in dose-dependent fashion. T cells were harvested at 72 h; results are normalized to percent survival with non-toxic tetramer treatment alone. The EC50 values for killing of NOD 8.3 T cells were 0.254 and 0.564 nM in two independent experiments. In (b) and (c), T cells were incubated with tetramers at 37°C for 1 h, then washed and cultured in medium alone for the indicated times; harvested cells were analyzed by flow cytometry after staining with anti-CD8 mAb and 7-AAD. (d) The K^d-NRP-V7-SAP tetramer can eliminate naïve cognate CD8⁺ T cells in vivo. Purified Thy1.2⁺ NOD 8.3 and NOD CL4 T cells (1 × 106 each) were mixed and transferred i.v. into NOD Thy1.1⁺ hosts. To verify equivalent transfer, PBLs were collected from recipient mice 1 day later and analyzed for Thy1.2⁺ tetramer⁺ CD8⁺ T cells (upper panel); mice were then injected i.v. with either K^d-NRP-V7 or K^d-NRP-V7-SAP tetramers. Three days after treatment, splenocytes were stained with K^d-HA-PE, K^d-NRP-V7-AlexaFluor647, 7-AAD, and an anti-CD8 mAb, and analyzed by flow cytometry, showing the loss of NOD 8.3, but not NOD CL4 control, T cells following treatment with the K^d-NRP-V7-SAP tetramer (lower panel).