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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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Costimulation with anti-IAP and not anti-CD28 results in  enhanced ζ chain and Zap70 tyrosine phosphorylation. (A and C) Jurkat  cells (106cells, A; 5 × 106 cells, C) were stimulated for the indicated timepoints with either an optimal high concentration of anti-CD3 (100) or a  low concentration of anti-CD3 (1) coimmobilized with anti-IAP, antiHLA, or anti-CD28. Cell lysates were immunoprecipitated with anti-ζ  chain (A) or anti-Zap70 (C) polyclonal Abs and analyzed by SDS-PAGE  followed by Western blotting with antiphosphotyrosine. (B and D) ζ  chain and Zap70 tyrosine phosphorylation expressed as fold increase over  control noncostimulatory conditions (low anti-CD3 plus anti-HLA). Bars  represent the mean and SEM of three independent experiments at either  5 min (B) or 15 min (D). Phosphorylation of both ζ chain and Zap70 was  increased by cell adhesion to the costimulatory combination of anti-CD3  and anti-IAP compared to control and compared with adhesion to antiCD3 and anti-CD28 (P <0.05 in all cases). In contrast, adhesion to the  costimulatory combination of anti-CD3 and anti-CD28 did not stimulate  ζ chain or Zap70 phosphorylation compared with control.
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Figure 3: Costimulation with anti-IAP and not anti-CD28 results in enhanced ζ chain and Zap70 tyrosine phosphorylation. (A and C) Jurkat cells (106cells, A; 5 × 106 cells, C) were stimulated for the indicated timepoints with either an optimal high concentration of anti-CD3 (100) or a low concentration of anti-CD3 (1) coimmobilized with anti-IAP, antiHLA, or anti-CD28. Cell lysates were immunoprecipitated with anti-ζ chain (A) or anti-Zap70 (C) polyclonal Abs and analyzed by SDS-PAGE followed by Western blotting with antiphosphotyrosine. (B and D) ζ chain and Zap70 tyrosine phosphorylation expressed as fold increase over control noncostimulatory conditions (low anti-CD3 plus anti-HLA). Bars represent the mean and SEM of three independent experiments at either 5 min (B) or 15 min (D). Phosphorylation of both ζ chain and Zap70 was increased by cell adhesion to the costimulatory combination of anti-CD3 and anti-IAP compared to control and compared with adhesion to antiCD3 and anti-CD28 (P <0.05 in all cases). In contrast, adhesion to the costimulatory combination of anti-CD3 and anti-CD28 did not stimulate ζ chain or Zap70 phosphorylation compared with control.

Mentions: One of the earliest events in T cell activation is the tyrosine phosphorylation of the TCR ζ chain and the syk family tyrosine kinase Zap70. To determine whether costimulation by IAP affects the phosphorylation status of ζ and Zap70, we analyzed ζ chain and Zap70 immunoprecipitates after activation of Jurkat clones under costimulatory (low anti-CD3 plus anti-IAP or anti-CD28) and control conditions (low anti-CD3 plus anti-HLA). We found that ζ chain tyrosine phosphorylation in the presence of anti-IAP mAbs was enhanced over control and was almost equivalent to optimal anti-CD3 (Fig. 3, A and B). In contrast, the costimulatory combination of anti-CD28 with anti-CD3 did not enhance ζ chain tyrosine phosphorylation. Similarly, Zap70 phosphorylation was equivalent for optimal anti-CD3 and the costimulatory combination of CD3 and IAP mAb. In contrast, anti-CD28 did not costimulate Zap70 phosphorylation above control levels (Fig. 3, C and D). These data demonstrate that IAP-mediated costimulation occurs by a signaling pathway distinct from that initiated by CD28.


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)

Costimulation with anti-IAP and not anti-CD28 results in  enhanced ζ chain and Zap70 tyrosine phosphorylation. (A and C) Jurkat  cells (106cells, A; 5 × 106 cells, C) were stimulated for the indicated timepoints with either an optimal high concentration of anti-CD3 (100) or a  low concentration of anti-CD3 (1) coimmobilized with anti-IAP, antiHLA, or anti-CD28. Cell lysates were immunoprecipitated with anti-ζ  chain (A) or anti-Zap70 (C) polyclonal Abs and analyzed by SDS-PAGE  followed by Western blotting with antiphosphotyrosine. (B and D) ζ  chain and Zap70 tyrosine phosphorylation expressed as fold increase over  control noncostimulatory conditions (low anti-CD3 plus anti-HLA). Bars  represent the mean and SEM of three independent experiments at either  5 min (B) or 15 min (D). Phosphorylation of both ζ chain and Zap70 was  increased by cell adhesion to the costimulatory combination of anti-CD3  and anti-IAP compared to control and compared with adhesion to antiCD3 and anti-CD28 (P <0.05 in all cases). In contrast, adhesion to the  costimulatory combination of anti-CD3 and anti-CD28 did not stimulate  ζ chain or Zap70 phosphorylation compared with control.
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Related In: Results  -  Collection

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Figure 3: Costimulation with anti-IAP and not anti-CD28 results in enhanced ζ chain and Zap70 tyrosine phosphorylation. (A and C) Jurkat cells (106cells, A; 5 × 106 cells, C) were stimulated for the indicated timepoints with either an optimal high concentration of anti-CD3 (100) or a low concentration of anti-CD3 (1) coimmobilized with anti-IAP, antiHLA, or anti-CD28. Cell lysates were immunoprecipitated with anti-ζ chain (A) or anti-Zap70 (C) polyclonal Abs and analyzed by SDS-PAGE followed by Western blotting with antiphosphotyrosine. (B and D) ζ chain and Zap70 tyrosine phosphorylation expressed as fold increase over control noncostimulatory conditions (low anti-CD3 plus anti-HLA). Bars represent the mean and SEM of three independent experiments at either 5 min (B) or 15 min (D). Phosphorylation of both ζ chain and Zap70 was increased by cell adhesion to the costimulatory combination of anti-CD3 and anti-IAP compared to control and compared with adhesion to antiCD3 and anti-CD28 (P <0.05 in all cases). In contrast, adhesion to the costimulatory combination of anti-CD3 and anti-CD28 did not stimulate ζ chain or Zap70 phosphorylation compared with control.
Mentions: One of the earliest events in T cell activation is the tyrosine phosphorylation of the TCR ζ chain and the syk family tyrosine kinase Zap70. To determine whether costimulation by IAP affects the phosphorylation status of ζ and Zap70, we analyzed ζ chain and Zap70 immunoprecipitates after activation of Jurkat clones under costimulatory (low anti-CD3 plus anti-IAP or anti-CD28) and control conditions (low anti-CD3 plus anti-HLA). We found that ζ chain tyrosine phosphorylation in the presence of anti-IAP mAbs was enhanced over control and was almost equivalent to optimal anti-CD3 (Fig. 3, A and B). In contrast, the costimulatory combination of anti-CD28 with anti-CD3 did not enhance ζ chain tyrosine phosphorylation. Similarly, Zap70 phosphorylation was equivalent for optimal anti-CD3 and the costimulatory combination of CD3 and IAP mAb. In contrast, anti-CD28 did not costimulate Zap70 phosphorylation above control levels (Fig. 3, C and D). These data demonstrate that IAP-mediated costimulation occurs by a signaling pathway distinct from that initiated by CD28.

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