Fig. 5.

Self-peptide-MHC contributes to class I-restricted thymocyte signaling in a peptide-specific manner. (A) Surface Plasmon Resonance (SPR) measurement of binding of soluble dimeric complexes of the indicated self-peptide-K^bs to immobilized OT-I TCR. The Mapk1 and Stat3 self-peptide-K^bs bound OT-I with a measurable affinity, while the engineered “null” SIAAFASL-K^b did not. (B) Dimeric but not monomeric Ova-K^b drives calcium signaling by OT-I DP thymocytes. Thymocytes were taken from OT-I H-2K^−/− H-2D^−/− mice, loaded with the ratiometric calcium dye Fura2am, and exposed to high concentrations of soluble monomeric or dimeric Ova-K^b. Fura2 ratios (340/380 nm) are shown in a false-color scale. (C) Homodimeric or heterodimeric Ova-K^b, but not monomeric Ova-K^b, drives negative selection in whole thymi. E16 FTOC were established from OT-I H-2K^−/− H-2D^−/− mice, and the indicated soluble monomeric or dimeric H-2K^bs were added at varying concentrations. After 3–4 days, FTOC were disrupted, and the resulting thymocyte populations were assayed by FACS: the percentage of surviving DP thymocytes, relative to untreated control FTOCs, are shown. Ova-K^b monomers had no effect on thymocyte selection, even when added at concentrations above the K_D of the Ova-K^b/OT-I interaction, where more than 50% of the thymocytes’ TCRs are expected to be bound by the Ova-K^b. Ova-K^b homodimers, or Ova-K^b–self-peptide-K^b heterodimers, could drive negative selection of the OT-I thymocytes to a similar extent, and at an equally low dose. (D) The ability of Ova-K^b–self-peptide-K^b to negatively select depends on the self-peptide-K^b’s affinity for the OT-I TCR. FTOC were established and assayed as in part (C). Negative selection did not result from even very high doses of the Ova-K^b–SIAAFASL-K^b (in which the peptide’s OT-I-contacting residues are mutated), or the Ova-K^b–SIINFEK^(BIO)L-K^b (in which a primary predicted OT-I contacting residue in the Ova peptide, the p7 K, is modified with a biotin at the lysine’s free amine group). Data are originally from Ref. 38.