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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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Modulation of endogenous 2C-specific peptide–H-2 Ld  complexes on the surface of RENCA cells by γ-IFN. RENCA cells were  cultured for 48 h with 0 (A and E), 5 (B and F), 10 (C and G), or 50 (D  and H), U/ml γ-IFN. As described in Results, γ-IFN is known to have a  direct effect on class I expression, making it necessary to establish background binding of 2C TCR–Ig to γ-IFN–treated cells. This was accomplished by incubating RENCA cells with saturating amounts of the H-2  Ld–binding peptide, MCMV, which efficiently displaced the endogenous  H-2 Ld–bound peptides, including any 2C-reactive peptides. Cells were  harvested, stained with 2C TCR–Ig (75 μg/ml; A–D) or the mAb 30.5.7  (45 μg/ml; E–H) as described in Materials and Methods. Cells were subsequently stained with GAM-IgG-RPE and analyzed by FACS®. Resultant histograms are shown. Solid lines represent histograms of cultures  with no added peptide, whereas dotted lines represent histograms from  cultures incubated with pMCMV. All experiments were done in duplicate and repeated at least three times. Note the differences in the extents  of fluorescence (see the scales on the histograms) upon staining with 2C-TCR–Ig versus staining with 30.5.7.
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Figure 8: Modulation of endogenous 2C-specific peptide–H-2 Ld complexes on the surface of RENCA cells by γ-IFN. RENCA cells were cultured for 48 h with 0 (A and E), 5 (B and F), 10 (C and G), or 50 (D and H), U/ml γ-IFN. As described in Results, γ-IFN is known to have a direct effect on class I expression, making it necessary to establish background binding of 2C TCR–Ig to γ-IFN–treated cells. This was accomplished by incubating RENCA cells with saturating amounts of the H-2 Ld–binding peptide, MCMV, which efficiently displaced the endogenous H-2 Ld–bound peptides, including any 2C-reactive peptides. Cells were harvested, stained with 2C TCR–Ig (75 μg/ml; A–D) or the mAb 30.5.7 (45 μg/ml; E–H) as described in Materials and Methods. Cells were subsequently stained with GAM-IgG-RPE and analyzed by FACS®. Resultant histograms are shown. Solid lines represent histograms of cultures with no added peptide, whereas dotted lines represent histograms from cultures incubated with pMCMV. All experiments were done in duplicate and repeated at least three times. Note the differences in the extents of fluorescence (see the scales on the histograms) upon staining with 2C-TCR–Ig versus staining with 30.5.7.

Mentions: The specificity and affinity of 2C TCR–Ig for peptide–MHC complexes suggested that one might be able to use this reagent to probe the influence of lymphokines on endogenous, cell surface, peptide–MHC complexes. To analyze this possibility and follow the expression of endogenous 2C-reactive peptide– H-2 Ld complexes within a heterogeneous peptide–MHC environment, the influence of γ-IFN on the H-2 Ld–expressing murine cell line RENCA was studied. RENCA cells were cultured in the presence of variable amounts of γ-IFN to induce upregulation of naturally loaded peptide–MHC complexes. 2C TCR–Ig binding to RENCA cells increased as a function of γ-IFN induction (Fig. 8, A–D, solid lines). The effect of γ-IFN was dose dependent with a maximal two- to threefold increase seen on cells treated with 10 U/ml of γ-IFN. Since γ-IFN is known to have a direct effect on class I expression (Fig. 8, E–H and reference 23), it is necessary to normalize for any nonspecific 2C TCR–Ig binding secondary to increased expression of H-2 Ld. This was accomplished by incubating RENCA cells with a control irrelevant H-2 Ld–binding peptide from pp89 of murine cytomegalovirus, pMCMV. Since p2Ca is known to have a weak affinity for H-2 Ld (14, 15) exchange with a higher affinity H-2 Ld–binding peptide like pMCMV (14) should be very efficient. Therefore, background reactivity of 2C TCR–Ig could be determined by the efficient displacement of endogenous p2Ca or p2Ca-like peptides by incubating the cells with saturating amounts of the control pMCMV peptide. In all cases, 2C TCR–Ig binding could be blocked by earlier incubation of cells with the control H-2 Ld–binding pMCMV (Fig. 8, A–D, dotted lines). Earlier incubation of RENCA cells with a 2C-specific peptide, QL9, induced a dramatic increase in 2C TCR–Ig binding (data not shown). The results of these experiments indicate that 2C TCR–Ig could be used to analyze the impact of cytokines on cell surface expression of endogenous 2C-reactive peptide–MHC complexes.


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)

Modulation of endogenous 2C-specific peptide–H-2 Ld  complexes on the surface of RENCA cells by γ-IFN. RENCA cells were  cultured for 48 h with 0 (A and E), 5 (B and F), 10 (C and G), or 50 (D  and H), U/ml γ-IFN. As described in Results, γ-IFN is known to have a  direct effect on class I expression, making it necessary to establish background binding of 2C TCR–Ig to γ-IFN–treated cells. This was accomplished by incubating RENCA cells with saturating amounts of the H-2  Ld–binding peptide, MCMV, which efficiently displaced the endogenous  H-2 Ld–bound peptides, including any 2C-reactive peptides. Cells were  harvested, stained with 2C TCR–Ig (75 μg/ml; A–D) or the mAb 30.5.7  (45 μg/ml; E–H) as described in Materials and Methods. Cells were subsequently stained with GAM-IgG-RPE and analyzed by FACS®. Resultant histograms are shown. Solid lines represent histograms of cultures  with no added peptide, whereas dotted lines represent histograms from  cultures incubated with pMCMV. All experiments were done in duplicate and repeated at least three times. Note the differences in the extents  of fluorescence (see the scales on the histograms) upon staining with 2C-TCR–Ig versus staining with 30.5.7.
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Related In: Results  -  Collection

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Figure 8: Modulation of endogenous 2C-specific peptide–H-2 Ld complexes on the surface of RENCA cells by γ-IFN. RENCA cells were cultured for 48 h with 0 (A and E), 5 (B and F), 10 (C and G), or 50 (D and H), U/ml γ-IFN. As described in Results, γ-IFN is known to have a direct effect on class I expression, making it necessary to establish background binding of 2C TCR–Ig to γ-IFN–treated cells. This was accomplished by incubating RENCA cells with saturating amounts of the H-2 Ld–binding peptide, MCMV, which efficiently displaced the endogenous H-2 Ld–bound peptides, including any 2C-reactive peptides. Cells were harvested, stained with 2C TCR–Ig (75 μg/ml; A–D) or the mAb 30.5.7 (45 μg/ml; E–H) as described in Materials and Methods. Cells were subsequently stained with GAM-IgG-RPE and analyzed by FACS®. Resultant histograms are shown. Solid lines represent histograms of cultures with no added peptide, whereas dotted lines represent histograms from cultures incubated with pMCMV. All experiments were done in duplicate and repeated at least three times. Note the differences in the extents of fluorescence (see the scales on the histograms) upon staining with 2C-TCR–Ig versus staining with 30.5.7.
Mentions: The specificity and affinity of 2C TCR–Ig for peptide–MHC complexes suggested that one might be able to use this reagent to probe the influence of lymphokines on endogenous, cell surface, peptide–MHC complexes. To analyze this possibility and follow the expression of endogenous 2C-reactive peptide– H-2 Ld complexes within a heterogeneous peptide–MHC environment, the influence of γ-IFN on the H-2 Ld–expressing murine cell line RENCA was studied. RENCA cells were cultured in the presence of variable amounts of γ-IFN to induce upregulation of naturally loaded peptide–MHC complexes. 2C TCR–Ig binding to RENCA cells increased as a function of γ-IFN induction (Fig. 8, A–D, solid lines). The effect of γ-IFN was dose dependent with a maximal two- to threefold increase seen on cells treated with 10 U/ml of γ-IFN. Since γ-IFN is known to have a direct effect on class I expression (Fig. 8, E–H and reference 23), it is necessary to normalize for any nonspecific 2C TCR–Ig binding secondary to increased expression of H-2 Ld. This was accomplished by incubating RENCA cells with a control irrelevant H-2 Ld–binding peptide from pp89 of murine cytomegalovirus, pMCMV. Since p2Ca is known to have a weak affinity for H-2 Ld (14, 15) exchange with a higher affinity H-2 Ld–binding peptide like pMCMV (14) should be very efficient. Therefore, background reactivity of 2C TCR–Ig could be determined by the efficient displacement of endogenous p2Ca or p2Ca-like peptides by incubating the cells with saturating amounts of the control pMCMV peptide. In all cases, 2C TCR–Ig binding could be blocked by earlier incubation of cells with the control H-2 Ld–binding pMCMV (Fig. 8, A–D, dotted lines). Earlier incubation of RENCA cells with a 2C-specific peptide, QL9, induced a dramatic increase in 2C TCR–Ig binding (data not shown). The results of these experiments indicate that 2C TCR–Ig could be used to analyze the impact of cytokines on cell surface expression of endogenous 2C-reactive peptide–MHC complexes.

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