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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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Soluble divalent 2C TCR–Ig retains the relative reactivity of  2C T cells towards specific peptide-stabilized H-2 Ld molecules. RMA-S  Ld cells were incubated under various conditions as described in Fig. 2.  Peptide stabilized H-2 Ld molecules were analyzed by flow cytometry as  described above. To facilitate comparison of peptide-stabilized H-2 Ld  molecules, data are presented as mean channel fluorescence.
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Figure 3: Soluble divalent 2C TCR–Ig retains the relative reactivity of 2C T cells towards specific peptide-stabilized H-2 Ld molecules. RMA-S Ld cells were incubated under various conditions as described in Fig. 2. Peptide stabilized H-2 Ld molecules were analyzed by flow cytometry as described above. To facilitate comparison of peptide-stabilized H-2 Ld molecules, data are presented as mean channel fluorescence.

Mentions: 2C TCR–Ig reactivity showed exquisite peptide specificity. Peptides (see Table 1 for sequences) were loaded into empty H-2 Ld molecules on RMA-S Ld cells. As expected, all H-2 Ld–binding peptides stabilized expression of the epitope recognized by mAb 30.5.7 (Fig. 2, B–D and Fig. 3). Only H-2 Ld molecules loaded with 2C-reactive peptides, peptides p2Ca, QL9, and SL9, expressed peptide/H-2Ld epitopes that reacted with 2C TCR–Ig (Fig. 2, F–H and Fig. 3). MCF increased ∼10–200-fold, from an MCF of 10 for either unloaded cells or cells loaded with an irrelevant H-2 Ld–binding peptide, to as high as 2,200 for RMA-S Ld cells loaded with peptide QL9 (Fig. 3). The pattern of reactivity mimicked the known affinities of monovalent 2C TCR for peptide–H-2 Ld complexes (see Table 1 for affinities). RMA-S Ld cells loaded with peptide QL9, p2Ca, or SL9 had MCF values of 2,200, 550, and 100, respectively, when stained with 2C TCR–Ig. Thus, soluble divalent 2C TCR–Ig chimeras reacted strongly with QL9–H-2 Ld complexes, modestly with p2Ca–H-2 Ld complexes, and weakly with SL9–H-2 Ld complexes. The fact that 2C TCR–Ig bound to SL9-loaded H-2 Ld molecules indicates that even in a direct flow cytometry assay, soluble divalent 2C TCR–Ig chimeras could be used to detect specific peptide–MHC complexes that have affinities as weak as 71 μM for monovalent 2C TCR.


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)

Soluble divalent 2C TCR–Ig retains the relative reactivity of  2C T cells towards specific peptide-stabilized H-2 Ld molecules. RMA-S  Ld cells were incubated under various conditions as described in Fig. 2.  Peptide stabilized H-2 Ld molecules were analyzed by flow cytometry as  described above. To facilitate comparison of peptide-stabilized H-2 Ld  molecules, data are presented as mean channel fluorescence.
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Related In: Results  -  Collection

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Figure 3: Soluble divalent 2C TCR–Ig retains the relative reactivity of 2C T cells towards specific peptide-stabilized H-2 Ld molecules. RMA-S Ld cells were incubated under various conditions as described in Fig. 2. Peptide stabilized H-2 Ld molecules were analyzed by flow cytometry as described above. To facilitate comparison of peptide-stabilized H-2 Ld molecules, data are presented as mean channel fluorescence.
Mentions: 2C TCR–Ig reactivity showed exquisite peptide specificity. Peptides (see Table 1 for sequences) were loaded into empty H-2 Ld molecules on RMA-S Ld cells. As expected, all H-2 Ld–binding peptides stabilized expression of the epitope recognized by mAb 30.5.7 (Fig. 2, B–D and Fig. 3). Only H-2 Ld molecules loaded with 2C-reactive peptides, peptides p2Ca, QL9, and SL9, expressed peptide/H-2Ld epitopes that reacted with 2C TCR–Ig (Fig. 2, F–H and Fig. 3). MCF increased ∼10–200-fold, from an MCF of 10 for either unloaded cells or cells loaded with an irrelevant H-2 Ld–binding peptide, to as high as 2,200 for RMA-S Ld cells loaded with peptide QL9 (Fig. 3). The pattern of reactivity mimicked the known affinities of monovalent 2C TCR for peptide–H-2 Ld complexes (see Table 1 for affinities). RMA-S Ld cells loaded with peptide QL9, p2Ca, or SL9 had MCF values of 2,200, 550, and 100, respectively, when stained with 2C TCR–Ig. Thus, soluble divalent 2C TCR–Ig chimeras reacted strongly with QL9–H-2 Ld complexes, modestly with p2Ca–H-2 Ld complexes, and weakly with SL9–H-2 Ld complexes. The fact that 2C TCR–Ig bound to SL9-loaded H-2 Ld molecules indicates that even in a direct flow cytometry assay, soluble divalent 2C TCR–Ig chimeras could be used to detect specific peptide–MHC complexes that have affinities as weak as 71 μM for monovalent 2C TCR.

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