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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 selectively reacts with  H-2 Ld molecules loaded with  peptides known to interact with  2C T cells. After overnight incubation of RMA-S Ld cells at  27°C, cells were cultured in the  presence or absence of various  H-2 Ld binding peptides: no  peptide cells maintained at 27°C  (A and E), tum− (B and F), p2Ca  (C and G), and QL9 (D and H)  were added to the cultures and  incubated as described in Materials and Methods. Cells were then  harvested and processed for flow  cytometry analysis. Cells were  stained with either purified mAb  30.5.7 (A–D), or 2C TCR–Ig  culture supernatants (E–H) diluted to 20–40 μg/ml final concentration. In each panel, the  histogram of treated cells (solid  line) is contrasted with that of cells not treated with any peptide and cultured for 1 h at 37°C (broken line). Histograms shown are from one representative experiment that has been repeated at least three times.
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Figure 2: Soluble divalent 2C TCR–Ig selectively reacts with H-2 Ld molecules loaded with peptides known to interact with 2C T cells. After overnight incubation of RMA-S Ld cells at 27°C, cells were cultured in the presence or absence of various H-2 Ld binding peptides: no peptide cells maintained at 27°C (A and E), tum− (B and F), p2Ca (C and G), and QL9 (D and H) were added to the cultures and incubated as described in Materials and Methods. Cells were then harvested and processed for flow cytometry analysis. Cells were stained with either purified mAb 30.5.7 (A–D), or 2C TCR–Ig culture supernatants (E–H) diluted to 20–40 μg/ml final concentration. In each panel, the histogram of treated cells (solid line) is contrasted with that of cells not treated with any peptide and cultured for 1 h at 37°C (broken line). Histograms shown are from one representative experiment that has been repeated at least three times.

Mentions: The temperature-dependent reactivity of RMA-S Ld with 2C TCR–Ig was significantly different than the reactivity of RMA-S Ld with mAb 30.5.7. As expected (44, 45), RMA-S Ld cells, cells that express empty MHC molecules, expressed more serologically reactive H-2 Ld molecules recognized by mAb 30.5.7 on cells cultured at 27°C than when cells were cultured at 37°C (Fig. 2 A); mean channel fluorescence (MCF)1 increased approximately fivefold. Thus, the epitope on H-2 Ld molecules recognized by mAb 30.5.7 can be stabilized by incubating cells at low temperatures. In contrast, RMA-S Ld cells expressed very low amounts of H-2 Ld molecules recognized by 2C TCR–Ig on cells cultured at either 27 or at 37°C (Fig. 2, E). This finding is consistent with the expected peptide-dependent reactivity of 2C TCR–Ig which should not recognize MHCs that have not been pulsed with peptides even when conformationally stabilized by decreased temperature.


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 selectively reacts with  H-2 Ld molecules loaded with  peptides known to interact with  2C T cells. After overnight incubation of RMA-S Ld cells at  27°C, cells were cultured in the  presence or absence of various  H-2 Ld binding peptides: no  peptide cells maintained at 27°C  (A and E), tum− (B and F), p2Ca  (C and G), and QL9 (D and H)  were added to the cultures and  incubated as described in Materials and Methods. Cells were then  harvested and processed for flow  cytometry analysis. Cells were  stained with either purified mAb  30.5.7 (A–D), or 2C TCR–Ig  culture supernatants (E–H) diluted to 20–40 μg/ml final concentration. In each panel, the  histogram of treated cells (solid  line) is contrasted with that of cells not treated with any peptide and cultured for 1 h at 37°C (broken line). Histograms shown are from one representative experiment that has been repeated at least three times.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Soluble divalent 2C TCR–Ig selectively reacts with H-2 Ld molecules loaded with peptides known to interact with 2C T cells. After overnight incubation of RMA-S Ld cells at 27°C, cells were cultured in the presence or absence of various H-2 Ld binding peptides: no peptide cells maintained at 27°C (A and E), tum− (B and F), p2Ca (C and G), and QL9 (D and H) were added to the cultures and incubated as described in Materials and Methods. Cells were then harvested and processed for flow cytometry analysis. Cells were stained with either purified mAb 30.5.7 (A–D), or 2C TCR–Ig culture supernatants (E–H) diluted to 20–40 μg/ml final concentration. In each panel, the histogram of treated cells (solid line) is contrasted with that of cells not treated with any peptide and cultured for 1 h at 37°C (broken line). Histograms shown are from one representative experiment that has been repeated at least three times.
Mentions: The temperature-dependent reactivity of RMA-S Ld with 2C TCR–Ig was significantly different than the reactivity of RMA-S Ld with mAb 30.5.7. As expected (44, 45), RMA-S Ld cells, cells that express empty MHC molecules, expressed more serologically reactive H-2 Ld molecules recognized by mAb 30.5.7 on cells cultured at 27°C than when cells were cultured at 37°C (Fig. 2 A); mean channel fluorescence (MCF)1 increased approximately fivefold. Thus, the epitope on H-2 Ld molecules recognized by mAb 30.5.7 can be stabilized by incubating cells at low temperatures. In contrast, RMA-S Ld cells expressed very low amounts of H-2 Ld molecules recognized by 2C TCR–Ig on cells cultured at either 27 or at 37°C (Fig. 2, E). This finding is consistent with the expected peptide-dependent reactivity of 2C TCR–Ig which should not recognize MHCs that have not been pulsed with peptides even when conformationally stabilized by decreased temperature.

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