A D^b-SAP tetramer retains toxicity and TCR specificity, and is endocytosed by target T cells in vitro. (A) Coupling of SA-SAP to pMHC monomers does not diminish SAP potency. The gpC9M-SAP tetramer and SA-SAP demonstrate similar ribosome inhibition in a rabbit reticulocyte lysate translation assay. Nonlinear regression was used to calculate EC₅₀ values (free SAP, 6.4 pM; SA-SAP, 190 pM; and gpC9M-SAP, 43.8 pM). (B) Coupling of SAP to pMHC monomers does not alter tetramer-binding specificity, and free SAP does not bind CD8⁺ T cells. FITC-labeled anti-SAP Abs were only detected on P14 T cells following incubation with the cognate gpC9M-SAP tetramer. (C) A SAP-coupled tetramer does not bind to B cells, CD4⁺ cells, or NK T cells, or to antigen-presenting cells. The CD8⁺ T-cell population alone was labeled with anti-SAP Abs after incubation of bulk P14 splenocytes with gpC9M-SAP tetramer. CD4⁺ T cells constituted approximately 7% of splenocytes in these transgenic mice. (D) A SAP-coupled tetramer is internalized by metabolically active cognate T cells. P14 T cells incubated at 37°C with gpC9M-SAP, but not hyC2A-SAP, exhibit internal PE fluorescence that corresponds to endocytosed SAP. In all experiments, the gpC9M tetramer served as a negative control. Results represent 2 (A, C) or 3 (B, D) separate experiments.