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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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Related in: MedlinePlus

Schematic representation of the native and chimeric molecules used in this study. Generation of the individual chimeras is described  in Materials and Methods.
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Related In: Results  -  Collection


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Figure 1: Schematic representation of the native and chimeric molecules used in this study. Generation of the individual chimeras is described in Materials and Methods.

Mentions: Standard techniques were used for nucleic acid manipulations. PCR was performed to amplify the human IAP transmembrane domain plus cytoplasmic tail and the cytoplasmic tail alone from human IAP form 2 in pBS (pIAP3) (8). The IAP transmembrane plus cytoplasmic tail segment was obtained using primers containing NheI and XbaI–SacI cloning sites (sense oligonucleotide, 5′-GTTTCATGGGCTAGCCCAAATGAAAATATTCTT-3′; anti-sense oligonucleotide, 5′-ATCGAGCTCATGGTTCTAGAACACAAGTGT-3′). The IAP cytoplasmic tail segment was obtained using primers containing SalI and XbaI–SacI cloning sites (sense oligonucleotide, 5′-TTACTTGGACTAGGTCGACTGAAATTTGTG-3′; anti-sense oligonucleotide as above; cloning sites are underlined). These fragments were digested with NheI and SacI or SalI and SacI, respectively. The cut fragments were ligated into a CD8-expressing plasmid, CD8-8-45 in pBS (18), using the SpeI and SacI or SalI and SacI sites. These chimeric cDNAs were cloned into the expression vector BSRαEN (gift of Dr. A. Shaw, Washington University School of Medicine, St. Louis, MO) using the XhoI and XbaI sites. This generated two cDNAs encoding chimeric proteins. One encodes the extracellular Ig domain of CD8 and the multiply membrane-spanning and cytoplasmic domains of IAP form 2 (CD8MC2, Fig. 1). The other encodes a chimera of the extracellular and transmembrane domain of CD8, and the cytoplasmic tail of IAP form 2 (CD8C2; Fig. 1). The CD8-8-* (Fig. 1) construct was generated using the following primers, 5′-CGATTAATCTAGAGAGCT-3′ and 5′-CTCTAGATTAAT-3′, which generate, upon annealing, a stop codon immediately followed by an XbaI site (underlined) plus ClaI and SacI overhangs (double underlined). Upon annealing, this fragment was ligated into ClaI and SacI cut CD8-8-45 in pBS, and the Xho and Xba fragment was then subcloned into BSRαEN.


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)

Schematic representation of the native and chimeric molecules used in this study. Generation of the individual chimeras is described  in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211576&req=5

Figure 1: Schematic representation of the native and chimeric molecules used in this study. Generation of the individual chimeras is described in Materials and Methods.
Mentions: Standard techniques were used for nucleic acid manipulations. PCR was performed to amplify the human IAP transmembrane domain plus cytoplasmic tail and the cytoplasmic tail alone from human IAP form 2 in pBS (pIAP3) (8). The IAP transmembrane plus cytoplasmic tail segment was obtained using primers containing NheI and XbaI–SacI cloning sites (sense oligonucleotide, 5′-GTTTCATGGGCTAGCCCAAATGAAAATATTCTT-3′; anti-sense oligonucleotide, 5′-ATCGAGCTCATGGTTCTAGAACACAAGTGT-3′). The IAP cytoplasmic tail segment was obtained using primers containing SalI and XbaI–SacI cloning sites (sense oligonucleotide, 5′-TTACTTGGACTAGGTCGACTGAAATTTGTG-3′; anti-sense oligonucleotide as above; cloning sites are underlined). These fragments were digested with NheI and SacI or SalI and SacI, respectively. The cut fragments were ligated into a CD8-expressing plasmid, CD8-8-45 in pBS (18), using the SpeI and SacI or SalI and SacI sites. These chimeric cDNAs were cloned into the expression vector BSRαEN (gift of Dr. A. Shaw, Washington University School of Medicine, St. Louis, MO) using the XhoI and XbaI sites. This generated two cDNAs encoding chimeric proteins. One encodes the extracellular Ig domain of CD8 and the multiply membrane-spanning and cytoplasmic domains of IAP form 2 (CD8MC2, Fig. 1). The other encodes a chimera of the extracellular and transmembrane domain of CD8, and the cytoplasmic tail of IAP form 2 (CD8C2; Fig. 1). The CD8-8-* (Fig. 1) construct was generated using the following primers, 5′-CGATTAATCTAGAGAGCT-3′ and 5′-CTCTAGATTAAT-3′, which generate, upon annealing, a stop codon immediately followed by an XbaI site (underlined) plus ClaI and SacI overhangs (double underlined). Upon annealing, this fragment was ligated into ClaI and SacI cut CD8-8-45 in pBS, and the Xho and Xba fragment was then subcloned into BSRαEN.

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