Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of elongated and shortened forms of CD48 in I-Ek+ CHO cells. (A) Schematic representation of the various forms of CD48 used in this study. Segments derived from mouse CD48 and segments inserted from human CD2 or mouse CD22 are depicted as heavy and light lines, respectively. The asterisk represents the CD22 mutation, R130A. (B) Western blot of the CD48 constructs expressed on I-Ek+ CHO cells. Triton X-100 lysates of 105 I-Ek+ CHO cells expressing no CD48 (CD48 neg) or the indicated form of CD48 were run under reducing conditions and blotted with the mouse CD48 mAb, OX78. Soluble recombinant mouse CD48 with an oligohistidine tag (sCD48his, 50 ng) was included for comparison. Parallel blots of the same samples with an isotype-matched control mAb (OX11) gave no staining (not shown).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195552&req=5

Figure 1: Expression of elongated and shortened forms of CD48 in I-Ek+ CHO cells. (A) Schematic representation of the various forms of CD48 used in this study. Segments derived from mouse CD48 and segments inserted from human CD2 or mouse CD22 are depicted as heavy and light lines, respectively. The asterisk represents the CD22 mutation, R130A. (B) Western blot of the CD48 constructs expressed on I-Ek+ CHO cells. Triton X-100 lysates of 105 I-Ek+ CHO cells expressing no CD48 (CD48 neg) or the indicated form of CD48 were run under reducing conditions and blotted with the mouse CD48 mAb, OX78. Soluble recombinant mouse CD48 with an oligohistidine tag (sCD48his, 50 ng) was included for comparison. Parallel blots of the same samples with an isotype-matched control mAb (OX11) gave no staining (not shown).

Mentions: Four different mouse CD48 constructs were made: wild-type CD48 with a silent BssHII site NH2-terminal to the glycosyl phosphatidylinositol (GPI) signal sequence for the insertion of additional domains; elongated molecules with two-domain human CD2 (CD48-CD2) or three-domain mouse CD22 (CD48-CD22) inserts; and a shortened CD48 construct (CD48d1) in which the membrane-proximal domain (d2) had been deleted (see Fig. 1 A). The wild-type CD48 construct was made by PCR in two steps using as template the vector pCD.MBCM-1, which contained the sequence for full-length mouse CD48 23. In the first PCR, the 5′ primer 1130 (tagtagtctagaccccatccgctcaagcaggccaccATGTGCTTCATAAAACAGGGATGGTG) encoded an XbaI site (restriction sites are underlined in all primers), the rat CD4 5′-untranslated region (in lower case), and the first 26 nucleotides of the CD48 sequence beginning with the start codon (upper case; compare with Wong et al. [23]). The 3′ primer GTTGTGACCACTAGCCAAGTTGCAGTCCAACATACTCCAGAAGAgCgcGC-TAGATCACAAGGTAG encoded for the 3′ end of the second domain of CD48, a silent restriction site (BssHII), and part of the GPI anchor signal sequence. The resulting product was elongated in a second PCR using the same 5′ primer (1130) with the 3′ primer ctactagGaTCcTCAGGTTAACAaGATCCTGTGAATGATGA-GTGTTGTGACCACTAGCCAAGTTGC. This primer overlaps the 3′ primer used in the first PCR and encodes for the remainder of the CD48 sequence followed by a BamHI site. In addition, a silent mutation (lower case “a”) was introduced to eliminate an internal BamHI site. The PCR product was digested with XbaI and BamHI and ligated into vector pEF-BOS-XB 40.


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)

Expression of elongated and shortened forms of CD48 in I-Ek+ CHO cells. (A) Schematic representation of the various forms of CD48 used in this study. Segments derived from mouse CD48 and segments inserted from human CD2 or mouse CD22 are depicted as heavy and light lines, respectively. The asterisk represents the CD22 mutation, R130A. (B) Western blot of the CD48 constructs expressed on I-Ek+ CHO cells. Triton X-100 lysates of 105 I-Ek+ CHO cells expressing no CD48 (CD48 neg) or the indicated form of CD48 were run under reducing conditions and blotted with the mouse CD48 mAb, OX78. Soluble recombinant mouse CD48 with an oligohistidine tag (sCD48his, 50 ng) was included for comparison. Parallel blots of the same samples with an isotype-matched control mAb (OX11) gave no staining (not shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195552&req=5

Figure 1: Expression of elongated and shortened forms of CD48 in I-Ek+ CHO cells. (A) Schematic representation of the various forms of CD48 used in this study. Segments derived from mouse CD48 and segments inserted from human CD2 or mouse CD22 are depicted as heavy and light lines, respectively. The asterisk represents the CD22 mutation, R130A. (B) Western blot of the CD48 constructs expressed on I-Ek+ CHO cells. Triton X-100 lysates of 105 I-Ek+ CHO cells expressing no CD48 (CD48 neg) or the indicated form of CD48 were run under reducing conditions and blotted with the mouse CD48 mAb, OX78. Soluble recombinant mouse CD48 with an oligohistidine tag (sCD48his, 50 ng) was included for comparison. Parallel blots of the same samples with an isotype-matched control mAb (OX11) gave no staining (not shown).
Mentions: Four different mouse CD48 constructs were made: wild-type CD48 with a silent BssHII site NH2-terminal to the glycosyl phosphatidylinositol (GPI) signal sequence for the insertion of additional domains; elongated molecules with two-domain human CD2 (CD48-CD2) or three-domain mouse CD22 (CD48-CD22) inserts; and a shortened CD48 construct (CD48d1) in which the membrane-proximal domain (d2) had been deleted (see Fig. 1 A). The wild-type CD48 construct was made by PCR in two steps using as template the vector pCD.MBCM-1, which contained the sequence for full-length mouse CD48 23. In the first PCR, the 5′ primer 1130 (tagtagtctagaccccatccgctcaagcaggccaccATGTGCTTCATAAAACAGGGATGGTG) encoded an XbaI site (restriction sites are underlined in all primers), the rat CD4 5′-untranslated region (in lower case), and the first 26 nucleotides of the CD48 sequence beginning with the start codon (upper case; compare with Wong et al. [23]). The 3′ primer GTTGTGACCACTAGCCAAGTTGCAGTCCAACATACTCCAGAAGAgCgcGC-TAGATCACAAGGTAG encoded for the 3′ end of the second domain of CD48, a silent restriction site (BssHII), and part of the GPI anchor signal sequence. The resulting product was elongated in a second PCR using the same 5′ primer (1130) with the 3′ primer ctactagGaTCcTCAGGTTAACAaGATCCTGTGAATGATGA-GTGTTGTGACCACTAGCCAAGTTGC. This primer overlaps the 3′ primer used in the first PCR and encodes for the remainder of the CD48 sequence followed by a BamHI site. In addition, a silent mutation (lower case “a”) was introduced to eliminate an internal BamHI site. The PCR product was digested with XbaI and BamHI and ligated into vector pEF-BOS-XB 40.

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