ANALYSIS OF THE EXPRESSION OF PEPTIDE-MAJOR HISTOCOMPATIBILITY COMPLEXES USING HIGH-AFFINITY SOLUBLE DIVALENT T-CELL RECEPTORS

Understanding the regulation of cell surface expression of specific pe ptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC compl exes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR-Ig) that h ave high affinity for their cognate peptide-MHC ligands. The generalit y of this approach was demonstrated by specific staining of peptide-pu lsed cells with two different TCR-Ig complexes: one specific for the m urine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by human histocompatibility leukocyte a ntigens-A2. Further, using 2C TCR-Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several intere sting findings. Soluble divalent 2C TCR-Ig detected significant change s in the level of specific antigenic-peptide MHC cell surface expressi on in cells treated with gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complex es recognized by 2C TCR-Ig were distinct from its effects on total H-2 L-d expression; thus, lower doses of gamma-IFN were required to incre ase expression of cell surface class I MHC complexes than were require d for upregulation of expression of specific peptide-MHC complexes. An alysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands un expectedly revealed that the affinity of the 2C TCR-IS for the natural ly occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 K-bm3, is very low, weaker than 71 mu M The affinity of the 2C TCR for the other naturally occurring, negatively selecting, allore active complex, p2Ca-H-2 L-d, is similar to 1000-fold higher. Thus, ne gatively selecting peptide-MHC complexes do not necessarily have intri nsically high affinity for copnate TCR. These results, uniquely reveal ed by this analysis, indicate the importance of using high affinity bi ologically relevant cognates, such as soluble divalent TCR, in further ing our understanding of immune responses.