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Molecular requirements for T cell recognition by a major histocompatibility complex class II-restricted T cell receptor: the involvement of the fourth hypervariable loop of the Valpha domain.

Thatte J, Qadri A, Radu C, Ward ES - J. Exp. Med. (1999)

Bottom Line: In contrast, the effect of mutating E69 to alanine is less marked.CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade.The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology and Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-8576, USA.

ABSTRACT
The role of two central residues (K68, E69) of the fourth hypervariable loop of the Valpha domain (HV4alpha) in antigen recognition by an MHC class II-restricted T cell receptor (TCR) has been analyzed. The TCR recognizes the NH2-terminal peptide of myelin basic protein (Ac1-11, acetylated at NH2 terminus) associated with the class II MHC molecule I-Au. Lysine 68 (K68) and glutamic acid 69 (E69) of HV4alpha have been mutated both individually and simultaneously to alanine (K68A, E69A). The responsiveness of transfectants bearing wild-type and mutated TCRs to Ac1-11-I-Au complexes has been analyzed in the presence and absence of expression of the coreceptor CD4. The data demonstrate that in the absence of CD4 expression, K68 plays a central role in antigen responsiveness. In contrast, the effect of mutating E69 to alanine is less marked. CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade. The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.

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Surface expression of TCR on WT and mutant transfectants and responsiveness  to antibody-mediated cross-linking or PMA stimulation. (A) Cells were stained with the  anti-Vβ8 mAb F23.1 (10 μg/ml), followed by anti–mouse Ig-FITC. Controls (shaded  curves) were incubated with the secondary antibody only. For analyses of responsiveness,  transfectants (1 × 105) were stimulated with (B) 10 ng/ml PMA + 500 ng/ml ionomycin, or  (C) 10 μg/ml plate-bound anti-CD3ε mAb 145-2C11, or (D) anti-Vβ8 mAb F23.1. IL-2  production was quantitated using the IL-2–dependent cell line, CTLL-2. Background cpm  were <4000 cpm for all transfectants. The stimulation data are representative of three independent experiments.
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Figure 2: Surface expression of TCR on WT and mutant transfectants and responsiveness to antibody-mediated cross-linking or PMA stimulation. (A) Cells were stained with the anti-Vβ8 mAb F23.1 (10 μg/ml), followed by anti–mouse Ig-FITC. Controls (shaded curves) were incubated with the secondary antibody only. For analyses of responsiveness, transfectants (1 × 105) were stimulated with (B) 10 ng/ml PMA + 500 ng/ml ionomycin, or (C) 10 μg/ml plate-bound anti-CD3ε mAb 145-2C11, or (D) anti-Vβ8 mAb F23.1. IL-2 production was quantitated using the IL-2–dependent cell line, CTLL-2. Background cpm were <4000 cpm for all transfectants. The stimulation data are representative of three independent experiments.

Mentions: Mycophenolic acid–resistant transfectants were analyzed for expression levels of surface TCR by flow cytometry using a Vβ8-specific mAb, F23.1. Since the expression levels varied between transfectants, transfectants with comparable levels of surface TCR were chosen for further analysis (Fig. 2). These transfectants were analyzed for responsiveness (assessed by quantitating IL-2 production) to PMA in addition to anti-Vβ8 (F23.1) and anti-CD3ε (145-2C11) antibody-mediated cross-linking. Responses to all three types of stimulation did not differ markedly, indicating that the signaling machinery of the transfectants was intact and that the Vα mutations do not affect signaling via antibody-mediated β chain cross-linking (Fig. 2 D). Differences observed upon 145-2C11 (Fig. 2 C) stimulation probably reflect minor differences in the surface TCR levels observed in Fig. 2 A. To control for variability, responses to cognate pMHC (below) have been normalized with respect to those obtained from stimulation with 145-2C11 and expressed as percentages of 145-2C11 responses. A similar approach was taken by Patten and colleagues for the analysis of anti-cytochrome c–I-Ek responses by transfectants expressing the WT 2B4 TCR and its mutated derivatives (43).


Molecular requirements for T cell recognition by a major histocompatibility complex class II-restricted T cell receptor: the involvement of the fourth hypervariable loop of the Valpha domain.

Thatte J, Qadri A, Radu C, Ward ES - J. Exp. Med. (1999)

Surface expression of TCR on WT and mutant transfectants and responsiveness  to antibody-mediated cross-linking or PMA stimulation. (A) Cells were stained with the  anti-Vβ8 mAb F23.1 (10 μg/ml), followed by anti–mouse Ig-FITC. Controls (shaded  curves) were incubated with the secondary antibody only. For analyses of responsiveness,  transfectants (1 × 105) were stimulated with (B) 10 ng/ml PMA + 500 ng/ml ionomycin, or  (C) 10 μg/ml plate-bound anti-CD3ε mAb 145-2C11, or (D) anti-Vβ8 mAb F23.1. IL-2  production was quantitated using the IL-2–dependent cell line, CTLL-2. Background cpm  were <4000 cpm for all transfectants. The stimulation data are representative of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192911&req=5

Figure 2: Surface expression of TCR on WT and mutant transfectants and responsiveness to antibody-mediated cross-linking or PMA stimulation. (A) Cells were stained with the anti-Vβ8 mAb F23.1 (10 μg/ml), followed by anti–mouse Ig-FITC. Controls (shaded curves) were incubated with the secondary antibody only. For analyses of responsiveness, transfectants (1 × 105) were stimulated with (B) 10 ng/ml PMA + 500 ng/ml ionomycin, or (C) 10 μg/ml plate-bound anti-CD3ε mAb 145-2C11, or (D) anti-Vβ8 mAb F23.1. IL-2 production was quantitated using the IL-2–dependent cell line, CTLL-2. Background cpm were <4000 cpm for all transfectants. The stimulation data are representative of three independent experiments.
Mentions: Mycophenolic acid–resistant transfectants were analyzed for expression levels of surface TCR by flow cytometry using a Vβ8-specific mAb, F23.1. Since the expression levels varied between transfectants, transfectants with comparable levels of surface TCR were chosen for further analysis (Fig. 2). These transfectants were analyzed for responsiveness (assessed by quantitating IL-2 production) to PMA in addition to anti-Vβ8 (F23.1) and anti-CD3ε (145-2C11) antibody-mediated cross-linking. Responses to all three types of stimulation did not differ markedly, indicating that the signaling machinery of the transfectants was intact and that the Vα mutations do not affect signaling via antibody-mediated β chain cross-linking (Fig. 2 D). Differences observed upon 145-2C11 (Fig. 2 C) stimulation probably reflect minor differences in the surface TCR levels observed in Fig. 2 A. To control for variability, responses to cognate pMHC (below) have been normalized with respect to those obtained from stimulation with 145-2C11 and expressed as percentages of 145-2C11 responses. A similar approach was taken by Patten and colleagues for the analysis of anti-cytochrome c–I-Ek responses by transfectants expressing the WT 2B4 TCR and its mutated derivatives (43).

Bottom Line: In contrast, the effect of mutating E69 to alanine is less marked.CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade.The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology and Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-8576, USA.

ABSTRACT
The role of two central residues (K68, E69) of the fourth hypervariable loop of the Valpha domain (HV4alpha) in antigen recognition by an MHC class II-restricted T cell receptor (TCR) has been analyzed. The TCR recognizes the NH2-terminal peptide of myelin basic protein (Ac1-11, acetylated at NH2 terminus) associated with the class II MHC molecule I-Au. Lysine 68 (K68) and glutamic acid 69 (E69) of HV4alpha have been mutated both individually and simultaneously to alanine (K68A, E69A). The responsiveness of transfectants bearing wild-type and mutated TCRs to Ac1-11-I-Au complexes has been analyzed in the presence and absence of expression of the coreceptor CD4. The data demonstrate that in the absence of CD4 expression, K68 plays a central role in antigen responsiveness. In contrast, the effect of mutating E69 to alanine is less marked. CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade. The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.

Show MeSH
Related in: MedlinePlus