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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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Anti-IAP costimulates IL-2 production in 3.L2 clones transfected with hIAP form 1. (A) 3.L2 clones, transfected with hIAP form 1,  were cultured on surfaces coated with anti-CD3 at the indicated concentration plus anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb  (YTS 213.1) and IL-2 production was measured. (B) 3.L2 clones, transfected with hIAP form 1, 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). T cell activation was analyzed as described in Fig. 2 B. The values shown represent averages of triplicates of 1  experiment of >3 with similar results.
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Figure 7: Anti-IAP costimulates IL-2 production in 3.L2 clones transfected with hIAP form 1. (A) 3.L2 clones, transfected with hIAP form 1, were cultured on surfaces coated with anti-CD3 at the indicated concentration plus anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1) and IL-2 production was measured. (B) 3.L2 clones, transfected with hIAP form 1, 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). T cell activation was analyzed as described in Fig. 2 B. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.

Mentions: To begin to understand the domains of IAP required for costimulation, we tested whether the cytoplasmic tail of IAP is required for costimulatory activity. IAP form 1, which has a cytoplasmic tail of only four amino acids, is a naturally occurring form of IAP expressed in keratinocytes and several transformed cell lines (9). 3.L2 clones transfected with hIAP form 1 (see Fig. 4) were tested for their ability to synergize with anti-CD3. All anti-human IAP mAbs tested (2E11, 2D3, or B6H12) were able to costimulate IL-2 production in these cells (Fig. 7 A). 3.L2 transfectants expressing the tailless form of IAP also were tested with antigenic peptide. In these transfectants, the addition of anti-hIAP mAbs 2E11 or 2D3 resulted in a marked increase in IL-2 production (Fig. 7 B). Thus, in two assays, the IAP cytoplasmic tail was not required for costimulation.


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)

Anti-IAP costimulates IL-2 production in 3.L2 clones transfected with hIAP form 1. (A) 3.L2 clones, transfected with hIAP form 1,  were cultured on surfaces coated with anti-CD3 at the indicated concentration plus anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb  (YTS 213.1) and IL-2 production was measured. (B) 3.L2 clones, transfected with hIAP form 1, 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). T cell activation was analyzed as described in Fig. 2 B. 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 7: Anti-IAP costimulates IL-2 production in 3.L2 clones transfected with hIAP form 1. (A) 3.L2 clones, transfected with hIAP form 1, were cultured on surfaces coated with anti-CD3 at the indicated concentration plus anti-CD28 (37.51), anti-hIAP (2E11, 2D3), or control mAb (YTS 213.1) and IL-2 production was measured. (B) 3.L2 clones, transfected with hIAP form 1, 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). T cell activation was analyzed as described in Fig. 2 B. The values shown represent averages of triplicates of 1 experiment of >3 with similar results.
Mentions: To begin to understand the domains of IAP required for costimulation, we tested whether the cytoplasmic tail of IAP is required for costimulatory activity. IAP form 1, which has a cytoplasmic tail of only four amino acids, is a naturally occurring form of IAP expressed in keratinocytes and several transformed cell lines (9). 3.L2 clones transfected with hIAP form 1 (see Fig. 4) were tested for their ability to synergize with anti-CD3. All anti-human IAP mAbs tested (2E11, 2D3, or B6H12) were able to costimulate IL-2 production in these cells (Fig. 7 A). 3.L2 transfectants expressing the tailless form of IAP also were tested with antigenic peptide. In these transfectants, the addition of anti-hIAP mAbs 2E11 or 2D3 resulted in a marked increase in IL-2 production (Fig. 7 B). Thus, in two assays, the IAP cytoplasmic tail was not required for costimulation.

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