Limits...
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.

Show MeSH
Soluble divalent 2C  TCR–Ig detects SIY–MHC complexes, but not dEV-8–MHC  complexes. T2 cells transfected  with either H-2 Kb, H-2 Kbm3,  or H-2 Kbm11 were incubated  overnight at 27°C and loaded  with peptides dEV-8 (broken  line), SIY (solid line), or pVSV  (dotted line) as described in Materials and Methods. Cells were  stained with purified 2C TCR– Ig (∼50 μg/ml) and GAM-IgG-RPE as described in Materials  and Methods, and analyzed by  FACS®. Resultant histograms are  shown; (A), T2-Kb cells, (B) T2-Kbm3, (C), T2-Kbm11. In the histograms presented 2C TCR–Ig  reactivity with either dEV-8 (broken line) or pVSV (dotted line) was  virtually identical leading to difficulty in discriminating between  these two histograms.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211717&req=5

Figure 6: Soluble divalent 2C TCR–Ig detects SIY–MHC complexes, but not dEV-8–MHC complexes. T2 cells transfected with either H-2 Kb, H-2 Kbm3, or H-2 Kbm11 were incubated overnight at 27°C and loaded with peptides dEV-8 (broken line), SIY (solid line), or pVSV (dotted line) as described in Materials and Methods. Cells were stained with purified 2C TCR– Ig (∼50 μg/ml) and GAM-IgG-RPE as described in Materials and Methods, and analyzed by FACS®. Resultant histograms are shown; (A), T2-Kb cells, (B) T2-Kbm3, (C), T2-Kbm11. In the histograms presented 2C TCR–Ig reactivity with either dEV-8 (broken line) or pVSV (dotted line) was virtually identical leading to difficulty in discriminating between these two histograms.

Mentions: Peptide SIY-loaded T2 Kb, T2 Kbm3, or T2 Kbm11 cells all expressed epitopes recognized by 2C TCR–Ig (Fig. 6, A–C). MCF of cells incubated with 2C TCR–Ig increased ∼20-fold, from 14 for pVSV (H-2 Kb binding peptide isolated from vesicular stomatitis virus NP residues) -loaded to 276 for SIY-loaded T2 Kb and from 16 for pVSV-loaded to 250 for SIY-loaded T2 Kbm11. SIY-loaded T2 Kbm3 cells showed a much weaker but still significant interaction with 2C TCR–Ig (Fig. 6 B); compare SIY-loaded (solid lines; MCF of 36) to pVSV-loaded (dotted lines; MCF of 12) T2 Kbm3 cells. The 2C TCR–Ig-binding data to SIY–MHC complexes was consistent with 2C CTL-mediated lysis on various SIY–MHC targets (Fig. 7). 2C CTL-mediated efficient lysis of SIY-loaded T2 Kb and T2 Kbm11 cells (Fig. 7 B and data not shown, 50% lethal dose (LD50) of ∼10 ng/ml for SIY T2 Kb). 2C CTL-mediated lysis of SIY-loaded T2 Kbm3 cells was significantly less efficient (Fig. 7 C, LD50 ∼100 ng/ml).


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 detects SIY–MHC complexes, but not dEV-8–MHC  complexes. T2 cells transfected  with either H-2 Kb, H-2 Kbm3,  or H-2 Kbm11 were incubated  overnight at 27°C and loaded  with peptides dEV-8 (broken  line), SIY (solid line), or pVSV  (dotted line) as described in Materials and Methods. Cells were  stained with purified 2C TCR– Ig (∼50 μg/ml) and GAM-IgG-RPE as described in Materials  and Methods, and analyzed by  FACS®. Resultant histograms are  shown; (A), T2-Kb cells, (B) T2-Kbm3, (C), T2-Kbm11. In the histograms presented 2C TCR–Ig  reactivity with either dEV-8 (broken line) or pVSV (dotted line) was  virtually identical leading to difficulty in discriminating between  these two histograms.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Soluble divalent 2C TCR–Ig detects SIY–MHC complexes, but not dEV-8–MHC complexes. T2 cells transfected with either H-2 Kb, H-2 Kbm3, or H-2 Kbm11 were incubated overnight at 27°C and loaded with peptides dEV-8 (broken line), SIY (solid line), or pVSV (dotted line) as described in Materials and Methods. Cells were stained with purified 2C TCR– Ig (∼50 μg/ml) and GAM-IgG-RPE as described in Materials and Methods, and analyzed by FACS®. Resultant histograms are shown; (A), T2-Kb cells, (B) T2-Kbm3, (C), T2-Kbm11. In the histograms presented 2C TCR–Ig reactivity with either dEV-8 (broken line) or pVSV (dotted line) was virtually identical leading to difficulty in discriminating between these two histograms.
Mentions: Peptide SIY-loaded T2 Kb, T2 Kbm3, or T2 Kbm11 cells all expressed epitopes recognized by 2C TCR–Ig (Fig. 6, A–C). MCF of cells incubated with 2C TCR–Ig increased ∼20-fold, from 14 for pVSV (H-2 Kb binding peptide isolated from vesicular stomatitis virus NP residues) -loaded to 276 for SIY-loaded T2 Kb and from 16 for pVSV-loaded to 250 for SIY-loaded T2 Kbm11. SIY-loaded T2 Kbm3 cells showed a much weaker but still significant interaction with 2C TCR–Ig (Fig. 6 B); compare SIY-loaded (solid lines; MCF of 36) to pVSV-loaded (dotted lines; MCF of 12) T2 Kbm3 cells. The 2C TCR–Ig-binding data to SIY–MHC complexes was consistent with 2C CTL-mediated lysis on various SIY–MHC targets (Fig. 7). 2C CTL-mediated efficient lysis of SIY-loaded T2 Kb and T2 Kbm11 cells (Fig. 7 B and data not shown, 50% lethal dose (LD50) of ∼10 ng/ml for SIY T2 Kb). 2C CTL-mediated lysis of SIY-loaded T2 Kbm3 cells was significantly less efficient (Fig. 7 C, LD50 ∼100 ng/ml).

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