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Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway.

Reinhold MI, Lindberg FP, Kersh GJ, Allen PM, Brown EJ - J. Exp. Med. (1997)

Bottom Line: Here, we show that mAbs recognizing IAP can enhance proliferation of primary human T cells in the presence of low levels of anti-CD3, but have no effect on T cell proliferation on their own.IAP costimulation does not require CD28.We hypothesize that anti-IAP generates the costimulatory signal because it modulates interactions of the IgV domain with other plasma membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain of IAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The integrin-associated protein (IAP, CD47) is a 50-kD plasma membrane protein with a single extracellular immunoglobulin variable (IgV)-like domain, a multiply membrane-spanning segment, and alternatively spliced short cytoplasmic tails. On neutrophils, IAP has been shown to function in a signaling complex with beta 3 integrins. However, the function of IAP on T cells, which express little or no beta 3 integrin, is not yet defined. Here, we show that mAbs recognizing IAP can enhance proliferation of primary human T cells in the presence of low levels of anti-CD3, but have no effect on T cell proliferation on their own. Together with suboptimal concentrations of anti-CD3, engagement of IAP also enhances IL-2 production in Jurkat cells, an apparently integrin-independent function of IAP. Nonetheless, costimulation by IAP ligation requires cell adhesion. IAP costimulation does not require CD28. Furthermore, anti-IAP, but not anti-CD28, synergizes with suboptimal anti-CD3 to enhance tyrosine phosphorylation of the CD3 zeta chain and the T cell-specific tyrosine kinase Zap70. Ligation of human IAP transfected into the hemoglobin-specific 3.L2 murine T cell hybridoma costimulates activation for IL-2 secretion both with anti-CD3 and with antigenic peptides on antigen-presenting cells (APCs). Moreover, ligation of IAP but not CD28 can convert antagonist peptides into agonists in 3.L2 cells. Using costimulation by IAP ligation as an assay to analyze the structure-function relationships in IAP signaling, we find that both the extracellular and multiply membrane-spanning domains of IAP are necessary for synergy with the antigen receptor, but the alternatively spliced cytoplasmic tails are not. These data demonstrate that IAP ligation initiates an adhesion-dependent costimulatory pathway distinct from CD28. We hypothesize that anti-IAP generates the costimulatory signal because it modulates interactions of the IgV domain with other plasma membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain of IAP. This model may have general significance for how IAP functions in cell activation.

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IAP/CD7 cannot costimulate IL-2 production. (A) 3.L2  clones, transfected with IAP/CD7, were plated on plates coated with  CD3 at the indicated concentration in the presence of either anti-CD28  (37.51), anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1). (B) IAP/ CD7-transfected 3.L2 clones (open symbol) or hIAP form 2 (closed symbol)  were activated with the indicated concentration of Hb(64–76) peptide in  the presence of anti-IAP mAbs 2E11, 2D3, or B6H12, or a control mAb  (IB4) and T cell activation was analyzed. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.
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Figure 8: IAP/CD7 cannot costimulate IL-2 production. (A) 3.L2 clones, transfected with IAP/CD7, were plated on plates coated with CD3 at the indicated concentration in the presence of either anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1). (B) IAP/ CD7-transfected 3.L2 clones (open symbol) or hIAP form 2 (closed symbol) were activated with the indicated concentration of Hb(64–76) peptide in the presence of anti-IAP mAbs 2E11, 2D3, or B6H12, or a control mAb (IB4) and T cell activation was analyzed. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.

Mentions: To determine whether the IAP Ig domain alone is able to enhance IL-2 production, we replaced the multiply membrane-spanning domain and cytoplasmic tail of IAP with the CD7 transmembrane domain (see Fig. 1). All anti-IAP mAbs recognized this chimeric protein when expressed in the 3.L2 clones (see Fig. 4). Moreover, this construct restored vitronectin bead binding when transfected into an IAP-deficient cell expressing αvβ3 and αvβ5 integrins (31). Thus, both mAb and functional data suggest that the Ig domain conformation was unaltered. Nonetheless, ligation of IAP/CD7 did not costimulate in either 3.L2 clone (Fig. 8 A). These results show that the extracellular domain of IAP is not sufficient to synergize with anti-CD3.


Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway.

Reinhold MI, Lindberg FP, Kersh GJ, Allen PM, Brown EJ - J. Exp. Med. (1997)

IAP/CD7 cannot costimulate IL-2 production. (A) 3.L2  clones, transfected with IAP/CD7, were plated on plates coated with  CD3 at the indicated concentration in the presence of either anti-CD28  (37.51), anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1). (B) IAP/ CD7-transfected 3.L2 clones (open symbol) or hIAP form 2 (closed symbol)  were activated with the indicated concentration of Hb(64–76) peptide in  the presence of anti-IAP mAbs 2E11, 2D3, or B6H12, or a control mAb  (IB4) and T cell activation was analyzed. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.
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Related In: Results  -  Collection

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Figure 8: IAP/CD7 cannot costimulate IL-2 production. (A) 3.L2 clones, transfected with IAP/CD7, were plated on plates coated with CD3 at the indicated concentration in the presence of either anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1). (B) IAP/ CD7-transfected 3.L2 clones (open symbol) or hIAP form 2 (closed symbol) were activated with the indicated concentration of Hb(64–76) peptide in the presence of anti-IAP mAbs 2E11, 2D3, or B6H12, or a control mAb (IB4) and T cell activation was analyzed. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.
Mentions: To determine whether the IAP Ig domain alone is able to enhance IL-2 production, we replaced the multiply membrane-spanning domain and cytoplasmic tail of IAP with the CD7 transmembrane domain (see Fig. 1). All anti-IAP mAbs recognized this chimeric protein when expressed in the 3.L2 clones (see Fig. 4). Moreover, this construct restored vitronectin bead binding when transfected into an IAP-deficient cell expressing αvβ3 and αvβ5 integrins (31). Thus, both mAb and functional data suggest that the Ig domain conformation was unaltered. Nonetheless, ligation of IAP/CD7 did not costimulate in either 3.L2 clone (Fig. 8 A). These results show that the extracellular domain of IAP is not sufficient to synergize with anti-CD3.

Bottom Line: Here, we show that mAbs recognizing IAP can enhance proliferation of primary human T cells in the presence of low levels of anti-CD3, but have no effect on T cell proliferation on their own.IAP costimulation does not require CD28.We hypothesize that anti-IAP generates the costimulatory signal because it modulates interactions of the IgV domain with other plasma membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain of IAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The integrin-associated protein (IAP, CD47) is a 50-kD plasma membrane protein with a single extracellular immunoglobulin variable (IgV)-like domain, a multiply membrane-spanning segment, and alternatively spliced short cytoplasmic tails. On neutrophils, IAP has been shown to function in a signaling complex with beta 3 integrins. However, the function of IAP on T cells, which express little or no beta 3 integrin, is not yet defined. Here, we show that mAbs recognizing IAP can enhance proliferation of primary human T cells in the presence of low levels of anti-CD3, but have no effect on T cell proliferation on their own. Together with suboptimal concentrations of anti-CD3, engagement of IAP also enhances IL-2 production in Jurkat cells, an apparently integrin-independent function of IAP. Nonetheless, costimulation by IAP ligation requires cell adhesion. IAP costimulation does not require CD28. Furthermore, anti-IAP, but not anti-CD28, synergizes with suboptimal anti-CD3 to enhance tyrosine phosphorylation of the CD3 zeta chain and the T cell-specific tyrosine kinase Zap70. Ligation of human IAP transfected into the hemoglobin-specific 3.L2 murine T cell hybridoma costimulates activation for IL-2 secretion both with anti-CD3 and with antigenic peptides on antigen-presenting cells (APCs). Moreover, ligation of IAP but not CD28 can convert antagonist peptides into agonists in 3.L2 cells. Using costimulation by IAP ligation as an assay to analyze the structure-function relationships in IAP signaling, we find that both the extracellular and multiply membrane-spanning domains of IAP are necessary for synergy with the antigen receptor, but the alternatively spliced cytoplasmic tails are not. These data demonstrate that IAP ligation initiates an adhesion-dependent costimulatory pathway distinct from CD28. We hypothesize that anti-IAP generates the costimulatory signal because it modulates interactions of the IgV domain with other plasma membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain of IAP. This model may have general significance for how IAP functions in cell activation.

Show MeSH
Related in: MedlinePlus