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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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The multiply membrane-spanning domain of IAP is not  sufficient for T cell costimulation. (A) Jurkat clones, transfected with  CD8MC2, were cultured on plates coated with anti-CD3 plus antiCD28 (9.3), anti-IAP (2E11), two different anti-CD8 (YTS, KT15), or  control mAb (IB4). Supernatants were harvested after 24 h and IL-2 concentration measured using CTLL-2 as described in Fig. 2 B. A third antiCD8 mAb (53.67) also failed to costimulate these transfected Jurkat cells.  (B) 3.L2 clones transfected with CD8MC2 (open symbols) or hIAP form 2  (closed symbol) were activated by the indicated amounts of Hb(64-76)  peptide presented by CH27 cells in the presence of anti-IAP (2D3), antiCD8 (53.67; KT15; YTS), or control (anti-KLH) mAb and IL-2 production was measured. The values represent averages of triplicates of 1 experiment of >3 with similar results.
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Figure 9: The multiply membrane-spanning domain of IAP is not sufficient for T cell costimulation. (A) Jurkat clones, transfected with CD8MC2, were cultured on plates coated with anti-CD3 plus antiCD28 (9.3), anti-IAP (2E11), two different anti-CD8 (YTS, KT15), or control mAb (IB4). Supernatants were harvested after 24 h and IL-2 concentration measured using CTLL-2 as described in Fig. 2 B. A third antiCD8 mAb (53.67) also failed to costimulate these transfected Jurkat cells. (B) 3.L2 clones transfected with CD8MC2 (open symbols) or hIAP form 2 (closed symbol) were activated by the indicated amounts of Hb(64-76) peptide presented by CH27 cells in the presence of anti-IAP (2D3), antiCD8 (53.67; KT15; YTS), or control (anti-KLH) mAb and IL-2 production was measured. The values represent averages of triplicates of 1 experiment of >3 with similar results.

Mentions: To determine whether the multiply membrane-spanning domain of IAP was sufficient for IAP function, we replaced the IAP extracellular domain with that of mouse CD8α (CD8MC2). We used IAP form 2 for the chimeric construct because this is the endogenous form of IAP expressed in T cells and in the Jurkat cell line. Control chimeras consisting of the mouse CD8α extracellular and transmembrane domain with (CD8C2; see Fig. 1) and without (CD8*; see Fig. 1) the IAP cytoplasmic tail also were transfected (see Fig. 4; data not shown). Jurkat clones expressing the CD8 constructs did not show costimulatory activity when activated by a low concentration of anti-CD3 and any of three different antiCD8 mAbs tested (Fig. 9 A; data not shown). As for all cDNAs, these chimeras were transfected into two independent Jurkat clones each, with identical results. Expression of the chimeric molecules did not prevent costimulation of Jurkat cells, since activation of the endogenous IAP with 2E11 mAb still resulted in elevated IL-2 levels (Fig. 9 A). To test the CD8MC2 chimera in the antigen-induced activation, it was transfected into two 3.L2 clones (see Fig. 4). Anti-CD8 mAbs failed to enhance IL-2 production over background in this assay as well (Fig. 9 B). To rule out the possibility that low expression of the chimera led to failure to costimulate, a transfectant population was selected stably expressing fivefold more CD8MC2. Although these clones expressed the chimera at a level equivalent to expression of the wild-type human IAP, anti-CD8 still failed to costimulate IL-2 production (data not shown).


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)

The multiply membrane-spanning domain of IAP is not  sufficient for T cell costimulation. (A) Jurkat clones, transfected with  CD8MC2, were cultured on plates coated with anti-CD3 plus antiCD28 (9.3), anti-IAP (2E11), two different anti-CD8 (YTS, KT15), or  control mAb (IB4). Supernatants were harvested after 24 h and IL-2 concentration measured using CTLL-2 as described in Fig. 2 B. A third antiCD8 mAb (53.67) also failed to costimulate these transfected Jurkat cells.  (B) 3.L2 clones transfected with CD8MC2 (open symbols) or hIAP form 2  (closed symbol) were activated by the indicated amounts of Hb(64-76)  peptide presented by CH27 cells in the presence of anti-IAP (2D3), antiCD8 (53.67; KT15; YTS), or control (anti-KLH) mAb and IL-2 production was measured. The values 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 9: The multiply membrane-spanning domain of IAP is not sufficient for T cell costimulation. (A) Jurkat clones, transfected with CD8MC2, were cultured on plates coated with anti-CD3 plus antiCD28 (9.3), anti-IAP (2E11), two different anti-CD8 (YTS, KT15), or control mAb (IB4). Supernatants were harvested after 24 h and IL-2 concentration measured using CTLL-2 as described in Fig. 2 B. A third antiCD8 mAb (53.67) also failed to costimulate these transfected Jurkat cells. (B) 3.L2 clones transfected with CD8MC2 (open symbols) or hIAP form 2 (closed symbol) were activated by the indicated amounts of Hb(64-76) peptide presented by CH27 cells in the presence of anti-IAP (2D3), antiCD8 (53.67; KT15; YTS), or control (anti-KLH) mAb and IL-2 production was measured. The values represent averages of triplicates of 1 experiment of >3 with similar results.
Mentions: To determine whether the multiply membrane-spanning domain of IAP was sufficient for IAP function, we replaced the IAP extracellular domain with that of mouse CD8α (CD8MC2). We used IAP form 2 for the chimeric construct because this is the endogenous form of IAP expressed in T cells and in the Jurkat cell line. Control chimeras consisting of the mouse CD8α extracellular and transmembrane domain with (CD8C2; see Fig. 1) and without (CD8*; see Fig. 1) the IAP cytoplasmic tail also were transfected (see Fig. 4; data not shown). Jurkat clones expressing the CD8 constructs did not show costimulatory activity when activated by a low concentration of anti-CD3 and any of three different antiCD8 mAbs tested (Fig. 9 A; data not shown). As for all cDNAs, these chimeras were transfected into two independent Jurkat clones each, with identical results. Expression of the chimeric molecules did not prevent costimulation of Jurkat cells, since activation of the endogenous IAP with 2E11 mAb still resulted in elevated IL-2 levels (Fig. 9 A). To test the CD8MC2 chimera in the antigen-induced activation, it was transfected into two 3.L2 clones (see Fig. 4). Anti-CD8 mAbs failed to enhance IL-2 production over background in this assay as well (Fig. 9 B). To rule out the possibility that low expression of the chimera led to failure to costimulate, a transfectant population was selected stably expressing fivefold more CD8MC2. Although these clones expressed the chimera at a level equivalent to expression of the wild-type human IAP, anti-CD8 still failed to costimulate IL-2 production (data not shown).

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