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Dependence of T cell antigen recognition on the dimensions of an accessory receptor-ligand complex.

Wild MK, Cambiaggi A, Brown MH, Davies EA, Ohno H, Saito T, van der Merwe PA - J. Exp. Med. (1999)

Bottom Line: The T cell antigen receptor (TCR) and its ligand peptide-major histocompatibility complex (MHC) are small (approximately 7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23-50 nm).Immunol.Today. 17:177-187).

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, United Kingdom.

ABSTRACT
The T cell antigen receptor (TCR) and its ligand peptide-major histocompatibility complex (MHC) are small (approximately 7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23-50 nm). We have proposed that molecules at the T cell/antigen-presenting cell (APC) interface segregate according to size, with small "accessory" molecules (e.g., CD2, CD4, CD8, CD28, and CD154) contributing to the formation of a close-contact zone, within which the TCR engages peptide-MHC, and from which large molecules are excluded (Davis, S.J., and P.A. van der Merwe. 1996. Immunol. Today. 17:177-187). One prediction of this model is that increasing the size of these small accessory molecules will disrupt their function. Here, we test this prediction by varying the dimensions of the CD2 ligand, CD48, and examining how this affects T cell antigen recognition. Although the interaction of CD2 on T cells with wild-type or shortened forms of CD48 on APCs enhances T cell antigen recognition, the interaction of CD2 with elongated forms of CD48 is strongly inhibitory. Further experiments indicated that elongation of the CD2/CD48 complex inhibited TCR engagement of peptide-MHC, presumably by preventing the formation of sufficiently intimate contacts at the T cell/APC interface. These findings demonstrate the importance of small size in CD2/CD48 function, and support the hypothesis that T cell antigen recognition requires segregation of cell surface molecules according to size.

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The CD2/CD48 interaction enhances T cell antigen recognition. (A) Antigen recognition by 2B4.CD2 cells using as APCs untransfected I-Ek+ CHO cells (CD48 neg CHO), I-Ek+ CHO cells stably transfected with CD48 (CD48 CHO), or CD48− revertant cells derived from the latter clone (CD48 CHO revertant). In each well, 5 × 104 2B4.CD2 cells were mixed with the same number of APCs and the indicated concentrations of peptide. After 18 h of culture, supernatants were analyzed for IL-2. Error bars represent the SE of triplicate cultures. (B) Comparison of antigen recognition by 2B4 cells expressing high levels (2B4.CD2) or very low levels (2B4) of CD2 using CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. The results were normalized to aid comparison between the different 2B4 cells, letting 100% equal maximal peptide-stimulated IL-2 secretion in the presence of CD48 neg CHOs. (C) Comparison of antigen recognition by 2B4 cells expressing full-length (2B2.CD2) or truncated (2B4.CD2trunc) forms of CD2 cells with CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. (D) Binding of CD48-coated beads to 2B4.CD2 cells is blocked by a CD2 mAb. 2B4.CD2 T cells were incubated with a mouse CD2 (RM2-1) or control (OX11) mAb before incubation with CD5- (control) or CD48-coated fluorescent beads, followed by flow cytometry. Identical results were obtained with 2B4.CD2trunc T cells.
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Figure 3: The CD2/CD48 interaction enhances T cell antigen recognition. (A) Antigen recognition by 2B4.CD2 cells using as APCs untransfected I-Ek+ CHO cells (CD48 neg CHO), I-Ek+ CHO cells stably transfected with CD48 (CD48 CHO), or CD48− revertant cells derived from the latter clone (CD48 CHO revertant). In each well, 5 × 104 2B4.CD2 cells were mixed with the same number of APCs and the indicated concentrations of peptide. After 18 h of culture, supernatants were analyzed for IL-2. Error bars represent the SE of triplicate cultures. (B) Comparison of antigen recognition by 2B4 cells expressing high levels (2B4.CD2) or very low levels (2B4) of CD2 using CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. The results were normalized to aid comparison between the different 2B4 cells, letting 100% equal maximal peptide-stimulated IL-2 secretion in the presence of CD48 neg CHOs. (C) Comparison of antigen recognition by 2B4 cells expressing full-length (2B2.CD2) or truncated (2B4.CD2trunc) forms of CD2 cells with CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. (D) Binding of CD48-coated beads to 2B4.CD2 cells is blocked by a CD2 mAb. 2B4.CD2 T cells were incubated with a mouse CD2 (RM2-1) or control (OX11) mAb before incubation with CD5- (control) or CD48-coated fluorescent beads, followed by flow cytometry. Identical results were obtained with 2B4.CD2trunc T cells.

Mentions: Although the interaction of human CD2 with its ligand, CD58, has been shown to enhance T cell antigen recognition, such an effect has not been formally demonstrated for the equivalent murine interaction between CD2 and CD48. This is an important point because CD2-deficient mice have surprisingly mild alterations in T cell function 2729, and the mouse and rat CD2/CD48 interactions have an ∼6-fold lower solution affinity 4653 and ∼40-fold lower membrane affinity 54 than the human CD2/CD58 interaction 41. Therefore, we examined the effect that CD48 expression on I-Ek+ CHO cells had on antigen recognition by 2B4 T cells. Because the original 2B4 hybridoma expressed only very low levels of CD2 (Fig. 2 C), we used cells that had been stably transfected with full-length mouse CD2 (2B4.CD2; reference 26). T cell antigen recognition by 2B4.CD2 cells, as measured by IL-2 secretion, was substantially enhanced by the expression of CD48 on I-Ek+ CHO APCs (Fig. 3 A). This enhancement was inhibited by incubating the APCs with a blocking CD48 mAb (Fig. 4 C), and was lost in CD48− revertants of these APCs (Fig. 3 A). In contrast, the CD48 mAb had no effect on the 2B4 cell response to CD48− I-Ek+ CHO cells (Fig. 4 C) nor on the CTLL line used in the IL-2 assay (data not shown), indicating that the inhibitory effect was not the result of binding to 2B4 or CTLL-2 cells (which also express CD48). The CD48 mAb consistently inhibited IL-2 secretion to below the levels seen with CD48− I-Ek+ CHO cells (Fig. 4 C). This may be because mAb treatment killed up to 30% of CD48+ I-Ek+ CHO cells (data not shown).


Dependence of T cell antigen recognition on the dimensions of an accessory receptor-ligand complex.

Wild MK, Cambiaggi A, Brown MH, Davies EA, Ohno H, Saito T, van der Merwe PA - J. Exp. Med. (1999)

The CD2/CD48 interaction enhances T cell antigen recognition. (A) Antigen recognition by 2B4.CD2 cells using as APCs untransfected I-Ek+ CHO cells (CD48 neg CHO), I-Ek+ CHO cells stably transfected with CD48 (CD48 CHO), or CD48− revertant cells derived from the latter clone (CD48 CHO revertant). In each well, 5 × 104 2B4.CD2 cells were mixed with the same number of APCs and the indicated concentrations of peptide. After 18 h of culture, supernatants were analyzed for IL-2. Error bars represent the SE of triplicate cultures. (B) Comparison of antigen recognition by 2B4 cells expressing high levels (2B4.CD2) or very low levels (2B4) of CD2 using CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. The results were normalized to aid comparison between the different 2B4 cells, letting 100% equal maximal peptide-stimulated IL-2 secretion in the presence of CD48 neg CHOs. (C) Comparison of antigen recognition by 2B4 cells expressing full-length (2B2.CD2) or truncated (2B4.CD2trunc) forms of CD2 cells with CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. (D) Binding of CD48-coated beads to 2B4.CD2 cells is blocked by a CD2 mAb. 2B4.CD2 T cells were incubated with a mouse CD2 (RM2-1) or control (OX11) mAb before incubation with CD5- (control) or CD48-coated fluorescent beads, followed by flow cytometry. Identical results were obtained with 2B4.CD2trunc T cells.
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Figure 3: The CD2/CD48 interaction enhances T cell antigen recognition. (A) Antigen recognition by 2B4.CD2 cells using as APCs untransfected I-Ek+ CHO cells (CD48 neg CHO), I-Ek+ CHO cells stably transfected with CD48 (CD48 CHO), or CD48− revertant cells derived from the latter clone (CD48 CHO revertant). In each well, 5 × 104 2B4.CD2 cells were mixed with the same number of APCs and the indicated concentrations of peptide. After 18 h of culture, supernatants were analyzed for IL-2. Error bars represent the SE of triplicate cultures. (B) Comparison of antigen recognition by 2B4 cells expressing high levels (2B4.CD2) or very low levels (2B4) of CD2 using CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. The results were normalized to aid comparison between the different 2B4 cells, letting 100% equal maximal peptide-stimulated IL-2 secretion in the presence of CD48 neg CHOs. (C) Comparison of antigen recognition by 2B4 cells expressing full-length (2B2.CD2) or truncated (2B4.CD2trunc) forms of CD2 cells with CD48+ or CD48− I-Ek+ CHO cells as APCs. Experiment performed as in A. (D) Binding of CD48-coated beads to 2B4.CD2 cells is blocked by a CD2 mAb. 2B4.CD2 T cells were incubated with a mouse CD2 (RM2-1) or control (OX11) mAb before incubation with CD5- (control) or CD48-coated fluorescent beads, followed by flow cytometry. Identical results were obtained with 2B4.CD2trunc T cells.
Mentions: Although the interaction of human CD2 with its ligand, CD58, has been shown to enhance T cell antigen recognition, such an effect has not been formally demonstrated for the equivalent murine interaction between CD2 and CD48. This is an important point because CD2-deficient mice have surprisingly mild alterations in T cell function 2729, and the mouse and rat CD2/CD48 interactions have an ∼6-fold lower solution affinity 4653 and ∼40-fold lower membrane affinity 54 than the human CD2/CD58 interaction 41. Therefore, we examined the effect that CD48 expression on I-Ek+ CHO cells had on antigen recognition by 2B4 T cells. Because the original 2B4 hybridoma expressed only very low levels of CD2 (Fig. 2 C), we used cells that had been stably transfected with full-length mouse CD2 (2B4.CD2; reference 26). T cell antigen recognition by 2B4.CD2 cells, as measured by IL-2 secretion, was substantially enhanced by the expression of CD48 on I-Ek+ CHO APCs (Fig. 3 A). This enhancement was inhibited by incubating the APCs with a blocking CD48 mAb (Fig. 4 C), and was lost in CD48− revertants of these APCs (Fig. 3 A). In contrast, the CD48 mAb had no effect on the 2B4 cell response to CD48− I-Ek+ CHO cells (Fig. 4 C) nor on the CTLL line used in the IL-2 assay (data not shown), indicating that the inhibitory effect was not the result of binding to 2B4 or CTLL-2 cells (which also express CD48). The CD48 mAb consistently inhibited IL-2 secretion to below the levels seen with CD48− I-Ek+ CHO cells (Fig. 4 C). This may be because mAb treatment killed up to 30% of CD48+ I-Ek+ CHO cells (data not shown).

Bottom Line: The T cell antigen receptor (TCR) and its ligand peptide-major histocompatibility complex (MHC) are small (approximately 7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23-50 nm).Immunol.Today. 17:177-187).

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, United Kingdom.

ABSTRACT
The T cell antigen receptor (TCR) and its ligand peptide-major histocompatibility complex (MHC) are small (approximately 7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23-50 nm). We have proposed that molecules at the T cell/antigen-presenting cell (APC) interface segregate according to size, with small "accessory" molecules (e.g., CD2, CD4, CD8, CD28, and CD154) contributing to the formation of a close-contact zone, within which the TCR engages peptide-MHC, and from which large molecules are excluded (Davis, S.J., and P.A. van der Merwe. 1996. Immunol. Today. 17:177-187). One prediction of this model is that increasing the size of these small accessory molecules will disrupt their function. Here, we test this prediction by varying the dimensions of the CD2 ligand, CD48, and examining how this affects T cell antigen recognition. Although the interaction of CD2 on T cells with wild-type or shortened forms of CD48 on APCs enhances T cell antigen recognition, the interaction of CD2 with elongated forms of CD48 is strongly inhibitory. Further experiments indicated that elongation of the CD2/CD48 complex inhibited TCR engagement of peptide-MHC, presumably by preventing the formation of sufficiently intimate contacts at the T cell/APC interface. These findings demonstrate the importance of small size in CD2/CD48 function, and support the hypothesis that T cell antigen recognition requires segregation of cell surface molecules according to size.

Show MeSH
Related in: MedlinePlus