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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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The affinity of soluble divalent 2C TCR–Ig for peptide–H-2  Ld complexes is higher than that of soluble monovalent 2C TCR. RMA  S-Ld cells were loaded with peptides (QL9, □, and  ; p2Ca, ⋄; or pMCMV, ▵) and subsequently incubated with a constant amount of FITC-labeled 30.5.7 Fab and varying concentrations of either 2C TCR–Ig (solid  lines) or soluble monovalent 2C TCR (sm2C TCR; dashed line). Binding  of FITC–30.5.7 Fab was determined by flow cytometry. Plotted as the  percent maximal (no 2C TCR analogue) 30.5.7 binding versus the concentration of 2C TCR analogue. Apparent affinities were determined  from a replot of 1/(percent maximal 30.5.7 binding) versus [TCR analogue] (see text and Table 2 for further discussion). Data shown are from  one representative experiment that has been repeated at least three times.  Each data point is the average of duplicates.
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Figure 5: The affinity of soluble divalent 2C TCR–Ig for peptide–H-2 Ld complexes is higher than that of soluble monovalent 2C TCR. RMA S-Ld cells were loaded with peptides (QL9, □, and ; p2Ca, ⋄; or pMCMV, ▵) and subsequently incubated with a constant amount of FITC-labeled 30.5.7 Fab and varying concentrations of either 2C TCR–Ig (solid lines) or soluble monovalent 2C TCR (sm2C TCR; dashed line). Binding of FITC–30.5.7 Fab was determined by flow cytometry. Plotted as the percent maximal (no 2C TCR analogue) 30.5.7 binding versus the concentration of 2C TCR analogue. Apparent affinities were determined from a replot of 1/(percent maximal 30.5.7 binding) versus [TCR analogue] (see text and Table 2 for further discussion). Data shown are from one representative experiment that has been repeated at least three times. Each data point is the average of duplicates.

Mentions: 2C TCR–Ig inhibited binding of 30.5.7 Fab to H-2 Ld molecules loaded with either QL9 or p2Ca peptides, but did not inhibit 30.5.7 Fab binding to pMCMV-loaded H-2 Ld molecules (Fig. 5). The affinity of soluble divalent 2C TCR–Ig for QL9 loaded molecules is 13.3 nM (Fig. 5 and Table 2). As expected, the affinity of 2C TCR–Ig for p2Ca-loaded molecules, 90 nM, is lower than that for QL9-loaded H-2 Ld. Although a small amount of competitive inhibition was seen with SL9 loaded cells, the affinity of the soluble divalent 2C TCR–Ig chimeras for SL9-loaded molecules is too low to be accurately measured under the conditions tested (data not shown).


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)

The affinity of soluble divalent 2C TCR–Ig for peptide–H-2  Ld complexes is higher than that of soluble monovalent 2C TCR. RMA  S-Ld cells were loaded with peptides (QL9, □, and  ; p2Ca, ⋄; or pMCMV, ▵) and subsequently incubated with a constant amount of FITC-labeled 30.5.7 Fab and varying concentrations of either 2C TCR–Ig (solid  lines) or soluble monovalent 2C TCR (sm2C TCR; dashed line). Binding  of FITC–30.5.7 Fab was determined by flow cytometry. Plotted as the  percent maximal (no 2C TCR analogue) 30.5.7 binding versus the concentration of 2C TCR analogue. Apparent affinities were determined  from a replot of 1/(percent maximal 30.5.7 binding) versus [TCR analogue] (see text and Table 2 for further discussion). Data shown are from  one representative experiment that has been repeated at least three times.  Each data point is the average of duplicates.
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Related In: Results  -  Collection

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Figure 5: The affinity of soluble divalent 2C TCR–Ig for peptide–H-2 Ld complexes is higher than that of soluble monovalent 2C TCR. RMA S-Ld cells were loaded with peptides (QL9, □, and ; p2Ca, ⋄; or pMCMV, ▵) and subsequently incubated with a constant amount of FITC-labeled 30.5.7 Fab and varying concentrations of either 2C TCR–Ig (solid lines) or soluble monovalent 2C TCR (sm2C TCR; dashed line). Binding of FITC–30.5.7 Fab was determined by flow cytometry. Plotted as the percent maximal (no 2C TCR analogue) 30.5.7 binding versus the concentration of 2C TCR analogue. Apparent affinities were determined from a replot of 1/(percent maximal 30.5.7 binding) versus [TCR analogue] (see text and Table 2 for further discussion). Data shown are from one representative experiment that has been repeated at least three times. Each data point is the average of duplicates.
Mentions: 2C TCR–Ig inhibited binding of 30.5.7 Fab to H-2 Ld molecules loaded with either QL9 or p2Ca peptides, but did not inhibit 30.5.7 Fab binding to pMCMV-loaded H-2 Ld molecules (Fig. 5). The affinity of soluble divalent 2C TCR–Ig for QL9 loaded molecules is 13.3 nM (Fig. 5 and Table 2). As expected, the affinity of 2C TCR–Ig for p2Ca-loaded molecules, 90 nM, is lower than that for QL9-loaded H-2 Ld. Although a small amount of competitive inhibition was seen with SL9 loaded cells, the affinity of the soluble divalent 2C TCR–Ig chimeras for SL9-loaded molecules is too low to be accurately measured under the conditions tested (data not shown).

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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