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Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors.

O'Herrin SM, Lebowitz MS, Bieler JG, al-Ramadi BK, Utz U, Bothwell AL, Schneck JP - J. Exp. Med. (1997)

Bottom Line: Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-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.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.

View Article: PubMed Central - PubMed

Affiliation: Johns Hopkins University, Department of Pathology and Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
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 gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-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 Kbm3, is very low, weaker than 71 microM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 Ld, is approximately 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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Proposed structure and biochemical characterization of soluble divalent TCR–Ig chimeras. A schematic of the chimeric protein  showing the TCR-α polypeptide (heavily shaded) linked to IgG1 heavy  chain and TCR-β polypeptide (lightly shaded) linked to Ig kappa light  chain is shown in A. The linkers between the chimeric chains consist of  glycine/serine spacers. Presumptive binding sites of mAb 1B2, an anticlonotypic 2C TCR-specific mAb, and anti–human Vβ13.1-specific mAb  on the putative TCR–Ig structures are also noted. Detection of chimeras in  baculovirus supernatants (Fig. 1, B and C). Plates were coated with goat  anti–mouse Fc. For detection of TCR–Ig, the secondary antibody was biotinylated and consisted of either the anti 2C mAb, 1B2 (B) or the anti– human Vβ13.1 TCR-specific mAb H131.21Y (C).
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Figure 1: Proposed structure and biochemical characterization of soluble divalent TCR–Ig chimeras. A schematic of the chimeric protein showing the TCR-α polypeptide (heavily shaded) linked to IgG1 heavy chain and TCR-β polypeptide (lightly shaded) linked to Ig kappa light chain is shown in A. The linkers between the chimeric chains consist of glycine/serine spacers. Presumptive binding sites of mAb 1B2, an anticlonotypic 2C TCR-specific mAb, and anti–human Vβ13.1-specific mAb on the putative TCR–Ig structures are also noted. Detection of chimeras in baculovirus supernatants (Fig. 1, B and C). Plates were coated with goat anti–mouse Fc. For detection of TCR–Ig, the secondary antibody was biotinylated and consisted of either the anti 2C mAb, 1B2 (B) or the anti– human Vβ13.1 TCR-specific mAb H131.21Y (C).

Mentions: The details of construction, expression, purification, and characterization of soluble divalent 2C TCR–Ig and A6 TCR–Ig were carried out as described elsewhere (O'Herrin, S.M., M.S. Lebowitz, and J.P. Schneck, manuscript in preparation). In brief, to generate the soluble divalent TCR, cDNAs encoding the TCR α and β chains were genetically linked via glycine/serine spacer to cDNAs encoding IgG1 heavy chains and kappa light chains, respectively (see Fig. 1 for protein schematic). Soluble monovalent 2C TCR was made and purified as previously described (12).


Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors.

O'Herrin SM, Lebowitz MS, Bieler JG, al-Ramadi BK, Utz U, Bothwell AL, Schneck JP - J. Exp. Med. (1997)

Proposed structure and biochemical characterization of soluble divalent TCR–Ig chimeras. A schematic of the chimeric protein  showing the TCR-α polypeptide (heavily shaded) linked to IgG1 heavy  chain and TCR-β polypeptide (lightly shaded) linked to Ig kappa light  chain is shown in A. The linkers between the chimeric chains consist of  glycine/serine spacers. Presumptive binding sites of mAb 1B2, an anticlonotypic 2C TCR-specific mAb, and anti–human Vβ13.1-specific mAb  on the putative TCR–Ig structures are also noted. Detection of chimeras in  baculovirus supernatants (Fig. 1, B and C). Plates were coated with goat  anti–mouse Fc. For detection of TCR–Ig, the secondary antibody was biotinylated and consisted of either the anti 2C mAb, 1B2 (B) or the anti– human Vβ13.1 TCR-specific mAb H131.21Y (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211717&req=5

Figure 1: Proposed structure and biochemical characterization of soluble divalent TCR–Ig chimeras. A schematic of the chimeric protein showing the TCR-α polypeptide (heavily shaded) linked to IgG1 heavy chain and TCR-β polypeptide (lightly shaded) linked to Ig kappa light chain is shown in A. The linkers between the chimeric chains consist of glycine/serine spacers. Presumptive binding sites of mAb 1B2, an anticlonotypic 2C TCR-specific mAb, and anti–human Vβ13.1-specific mAb on the putative TCR–Ig structures are also noted. Detection of chimeras in baculovirus supernatants (Fig. 1, B and C). Plates were coated with goat anti–mouse Fc. For detection of TCR–Ig, the secondary antibody was biotinylated and consisted of either the anti 2C mAb, 1B2 (B) or the anti– human Vβ13.1 TCR-specific mAb H131.21Y (C).
Mentions: The details of construction, expression, purification, and characterization of soluble divalent 2C TCR–Ig and A6 TCR–Ig were carried out as described elsewhere (O'Herrin, S.M., M.S. Lebowitz, and J.P. Schneck, manuscript in preparation). In brief, to generate the soluble divalent TCR, cDNAs encoding the TCR α and β chains were genetically linked via glycine/serine spacer to cDNAs encoding IgG1 heavy chains and kappa light chains, respectively (see Fig. 1 for protein schematic). Soluble monovalent 2C TCR was made and purified as previously described (12).

Bottom Line: Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-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.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.

View Article: PubMed Central - PubMed

Affiliation: Johns Hopkins University, Department of Pathology and Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
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 gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-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 Kbm3, is very low, weaker than 71 microM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 Ld, is approximately 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.

Show MeSH
Related in: MedlinePlus