J. Exp. Med.
© The Rockefeller University Press
0022-1007/97/10/1333/13 $2.00
Volume 186, Number 8, October 20, 1997 1333-1345

Analysis of the Expression of Peptide-Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors

By Sean M. O'Herrin,^* Michael S. Lebowitz,^* Joan G. Bieler,^* Basel K. al-Ramadi, Ursula Utz,^§ Alfred L.M. Bothwell, and Jonathan P. Schneck^*

From the ^* Johns Hopkins University, Department of Pathology and Medicine, Division of Immunopathology, Baltimore, Maryland 21205;  Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520; and ^§ Laboratoire d'Immunologic Institut de recherches cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada

Understanding the regulation of cell surface expression of specific peptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR-Ig) that have high affinity for their cognate peptide-MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR-Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by human histocompatibility leukocyte antigens-A2. Further, using 2C TCR- Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several interesting findings. Soluble divalent 2C TCR-Ig detected significant changes in the level of specific antigenic-peptide MHC cell surface expression in cells treated with -interferon (-IFN). Interestingly, the effects of -IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 L^d expression; thus, lower doses of -IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide-MHC complexes. Analysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands unexpectedly revealed that the affinity of the 2C TCR-Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 K^bm3, is very low, weaker than 71 µM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 L^d, is ~1000-fold higher. Thus, negatively selecting peptide-MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.

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