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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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Cell surface expression of TCR and CD4 by transfectants and responsiveness to  antibody-mediated cross-linking. (A) Surface TCR expression (left-hand histograms) was  analyzed by staining cells with the anti-Vβ8 mAb F23.1 (10 μg/ml; thick lines), followed by  anti–mouse Ig-FITC. Controls (shaded) were incubated with the secondary antibody only.  For CD4 staining (right-hand histograms) the anti-CD4 mAb, GK1.5 (10 μg/ml; thick  lines), followed by anti–rat Ig-FITC antibody were used. Controls (shaded) were treated  similarly as for TCR staining. Data from 104 cells was collected on FACScan® flow cytometer (Becton-Dickinson) and analyzed using the Cellquest program. For antibody-mediated  cross-linking, transfectants (5 × 104) were stimulated with (B) plate bound anti-CD3ε mAb  145-2C11, or (C) plate bound anti-Vβ8 mAb F23.1. To coat wells of 96-well plates, antibodies were used at 10 μg/ml. IL-2 activity in the culture supernatants was analyzed using  the IL-2–dependent cell line, CTLL-2. Background cpm were <3000 cpm for all transfectants. The stimulation data are representative of three separate experiments. All transfectants  gave similar but lower responses with antibodies coated at 3 and 5 μg/ml, indicating overlapping dose response curves (data not shown).
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Figure 4: Cell surface expression of TCR and CD4 by transfectants and responsiveness to antibody-mediated cross-linking. (A) Surface TCR expression (left-hand histograms) was analyzed by staining cells with the anti-Vβ8 mAb F23.1 (10 μg/ml; thick lines), followed by anti–mouse Ig-FITC. Controls (shaded) were incubated with the secondary antibody only. For CD4 staining (right-hand histograms) the anti-CD4 mAb, GK1.5 (10 μg/ml; thick lines), followed by anti–rat Ig-FITC antibody were used. Controls (shaded) were treated similarly as for TCR staining. Data from 104 cells was collected on FACScan® flow cytometer (Becton-Dickinson) and analyzed using the Cellquest program. For antibody-mediated cross-linking, transfectants (5 × 104) were stimulated with (B) plate bound anti-CD3ε mAb 145-2C11, or (C) plate bound anti-Vβ8 mAb F23.1. To coat wells of 96-well plates, antibodies were used at 10 μg/ml. IL-2 activity in the culture supernatants was analyzed using the IL-2–dependent cell line, CTLL-2. Background cpm were <3000 cpm for all transfectants. The stimulation data are representative of three separate experiments. All transfectants gave similar but lower responses with antibodies coated at 3 and 5 μg/ml, indicating overlapping dose response curves (data not shown).

Mentions: Since the differences in response to peptide were most striking between the WT and the K68A mutant, transfectants expressing these TCRs were analyzed further. The T cell coreceptor, CD4, has been shown to enhance the capability of thymocytes and mature T cells to recognize antigen (reviewed in references 19 and 51). To analyze whether CD4 can compensate for the effect of the K68A mutation, cotransfections of the 1934.4 WT or K68A mutant α chain together with WT β chain and CD4 expression constructs using the 58α−β− thymoma cell line as recipient were carried out. The surface TCR and CD4 levels are comparable between the WT and K68A transfectants (Fig. 4 A), although the TCR levels for the transfectant K68A/CD4-32 are slightly lower. These CD4+ transfectants show similar responses to 145-2C11 and F23.1 stimulation (Fig. 4, B and C). In addition to shifting the dose response curve of the WT transfectants, cotransfection of CD4 almost completely (K68A/CD4-12 mutant) or partially (K68A/CD4-32 mutant) restores the K68A mutant responses to cognate pMHC to the levels seen with the WT transfectant, WT/CD4-43 (Fig. 5). The WT transfectant WT/CD4-33 is, however, still more responsive than the two mutant transfectants and this is particularly so for the Ac1-11 peptide (Fig. 5 A). This significant gain of function for the K68A mutants suggests that either the increased avidity and/or enhancing the efficiency of TCR signaling by CD4 coexpression can compensate, in part at least, for the suboptimal nature of the K68A TCR–pMHC interaction. In addition, the enhancing effect of CD4 is much greater for the K68A mutants than for the WT transfectants (Figs. 3 and 5).


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)

Cell surface expression of TCR and CD4 by transfectants and responsiveness to  antibody-mediated cross-linking. (A) Surface TCR expression (left-hand histograms) was  analyzed by staining cells with the anti-Vβ8 mAb F23.1 (10 μg/ml; thick lines), followed by  anti–mouse Ig-FITC. Controls (shaded) were incubated with the secondary antibody only.  For CD4 staining (right-hand histograms) the anti-CD4 mAb, GK1.5 (10 μg/ml; thick  lines), followed by anti–rat Ig-FITC antibody were used. Controls (shaded) were treated  similarly as for TCR staining. Data from 104 cells was collected on FACScan® flow cytometer (Becton-Dickinson) and analyzed using the Cellquest program. For antibody-mediated  cross-linking, transfectants (5 × 104) were stimulated with (B) plate bound anti-CD3ε mAb  145-2C11, or (C) plate bound anti-Vβ8 mAb F23.1. To coat wells of 96-well plates, antibodies were used at 10 μg/ml. IL-2 activity in the culture supernatants was analyzed using  the IL-2–dependent cell line, CTLL-2. Background cpm were <3000 cpm for all transfectants. The stimulation data are representative of three separate experiments. All transfectants  gave similar but lower responses with antibodies coated at 3 and 5 μg/ml, indicating overlapping dose response curves (data not shown).
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Figure 4: Cell surface expression of TCR and CD4 by transfectants and responsiveness to antibody-mediated cross-linking. (A) Surface TCR expression (left-hand histograms) was analyzed by staining cells with the anti-Vβ8 mAb F23.1 (10 μg/ml; thick lines), followed by anti–mouse Ig-FITC. Controls (shaded) were incubated with the secondary antibody only. For CD4 staining (right-hand histograms) the anti-CD4 mAb, GK1.5 (10 μg/ml; thick lines), followed by anti–rat Ig-FITC antibody were used. Controls (shaded) were treated similarly as for TCR staining. Data from 104 cells was collected on FACScan® flow cytometer (Becton-Dickinson) and analyzed using the Cellquest program. For antibody-mediated cross-linking, transfectants (5 × 104) were stimulated with (B) plate bound anti-CD3ε mAb 145-2C11, or (C) plate bound anti-Vβ8 mAb F23.1. To coat wells of 96-well plates, antibodies were used at 10 μg/ml. IL-2 activity in the culture supernatants was analyzed using the IL-2–dependent cell line, CTLL-2. Background cpm were <3000 cpm for all transfectants. The stimulation data are representative of three separate experiments. All transfectants gave similar but lower responses with antibodies coated at 3 and 5 μg/ml, indicating overlapping dose response curves (data not shown).
Mentions: Since the differences in response to peptide were most striking between the WT and the K68A mutant, transfectants expressing these TCRs were analyzed further. The T cell coreceptor, CD4, has been shown to enhance the capability of thymocytes and mature T cells to recognize antigen (reviewed in references 19 and 51). To analyze whether CD4 can compensate for the effect of the K68A mutation, cotransfections of the 1934.4 WT or K68A mutant α chain together with WT β chain and CD4 expression constructs using the 58α−β− thymoma cell line as recipient were carried out. The surface TCR and CD4 levels are comparable between the WT and K68A transfectants (Fig. 4 A), although the TCR levels for the transfectant K68A/CD4-32 are slightly lower. These CD4+ transfectants show similar responses to 145-2C11 and F23.1 stimulation (Fig. 4, B and C). In addition to shifting the dose response curve of the WT transfectants, cotransfection of CD4 almost completely (K68A/CD4-12 mutant) or partially (K68A/CD4-32 mutant) restores the K68A mutant responses to cognate pMHC to the levels seen with the WT transfectant, WT/CD4-43 (Fig. 5). The WT transfectant WT/CD4-33 is, however, still more responsive than the two mutant transfectants and this is particularly so for the Ac1-11 peptide (Fig. 5 A). This significant gain of function for the K68A mutants suggests that either the increased avidity and/or enhancing the efficiency of TCR signaling by CD4 coexpression can compensate, in part at least, for the suboptimal nature of the K68A TCR–pMHC interaction. In addition, the enhancing effect of CD4 is much greater for the K68A mutants than for the WT transfectants (Figs. 3 and 5).

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