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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 synergy with CD3. (A) Human peripheral blood T cells  were incubated on plates coated with a low concentration of anti-CD3  together with increasing concentrations of anti-IAP mAb 2E11 or 2D3,  or anti-CR1 (3D9). Cells were pulsed with [3H]thymidine for the last 16 h  of a 90 h incubation. Shown are averages of triplicate wells from 1 experiment of >3 with similar results. Cells plated on 2E11, 2D3, and 3D9  alone had <1,000 CPM. Maximum stimulation by high concentration of  anti-CD3 was 40,000 cpm. (B) Jurkat cells were incubated on plates  coated with increasing concentrations of anti-CD3 and the same concentration of anti-IAP (2D3), anti CD28 (9.3), or anti-HLA (W6/32) mAbs.  Supernatants were harvested after 24 h and IL-2 concentration measured  by assay on CTLL-2 cells. The values shown represent triplicates of  [3H]thymidine incorporation by the CTLL-2 cells in 1 experiment of >3  with similar results. Quantitation of IL-2 concentration showed that stimulation of Jurkat cells by low levels of anti CD3 (1) in the presence of  anti-IAP or anti-CD28 mAbs led to 1 U/ml, compared with 0.1 U/ml  for the negative control mAb. Neither anti-IAP or anti-HLA caused detectable IL-2 secretion in the absence of anti-CD3. Additional mAbs that  do not costimulate with anti-CD3 include anti-CD61 (integrin β3), antiCD18 (integrin β2), and anti-CD29 (integrin β1).
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Figure 2: IAP synergy with CD3. (A) Human peripheral blood T cells were incubated on plates coated with a low concentration of anti-CD3 together with increasing concentrations of anti-IAP mAb 2E11 or 2D3, or anti-CR1 (3D9). Cells were pulsed with [3H]thymidine for the last 16 h of a 90 h incubation. Shown are averages of triplicate wells from 1 experiment of >3 with similar results. Cells plated on 2E11, 2D3, and 3D9 alone had <1,000 CPM. Maximum stimulation by high concentration of anti-CD3 was 40,000 cpm. (B) Jurkat cells were incubated on plates coated with increasing concentrations of anti-CD3 and the same concentration of anti-IAP (2D3), anti CD28 (9.3), or anti-HLA (W6/32) mAbs. Supernatants were harvested after 24 h and IL-2 concentration measured by assay on CTLL-2 cells. The values shown represent triplicates of [3H]thymidine incorporation by the CTLL-2 cells in 1 experiment of >3 with similar results. Quantitation of IL-2 concentration showed that stimulation of Jurkat cells by low levels of anti CD3 (1) in the presence of anti-IAP or anti-CD28 mAbs led to 1 U/ml, compared with 0.1 U/ml for the negative control mAb. Neither anti-IAP or anti-HLA caused detectable IL-2 secretion in the absence of anti-CD3. Additional mAbs that do not costimulate with anti-CD3 include anti-CD61 (integrin β3), antiCD18 (integrin β2), and anti-CD29 (integrin β1).

Mentions: To investigate the function of IAP on lymphocytes, we evaluated its role in T cell activation. When human T lymphocytes were purified from peripheral blood, no concentration of antiIAP alone stimulated proliferation (data not shown). In contrast, when combined with a suboptimal concentration of anti-CD3, three different anti-IAP mAbs enhanced proliferation of purified peripheral blood T cells. Using the same low concentration of anti-CD3, the nonbinding isotype-matched negative control 3D9 (anti-CR1/CD35) (Fig. 2 A) and mAb W6/32 (anti-HLA), which binds to a different cell surface antigen (data not shown), did not significantly enhance proliferation. In contrast with assays in which IAP has been shown to function with β3 integrins, the anti-IAP mAb B6H12 was much less potent than antiIAP 2D3, which recognizes a distinct epitope on the Ig domain (1) (data not shown). This suggested the possibility that the role for IAP is different in synergy with anti-CD3 than in cooperation with β3 integrins. 2E11, an anti-IAP mAb recognizing a third distinct epitope, also was costimulatory. Anti-IAP mAbs 2E11 and 2D3 also synergized with suboptimal concentrations of anti-CD3 to increase IL-2 production in Jurkat cells, while anti-HLA did not (Fig. 2 B; data not shown). This synergy is unlikely to be dependent on IAP cooperation with an integrin, since mAbs against β1, β2, and β3 integrins did not increase T cell proliferation or Jurkat IL-2 production in combination with antiCD3 (data not shown). The αvβ3 ligand vitronectin also was unable to costimulate T cell proliferation or Jurkat IL-2 production with suboptimal anti-CD3 (data not shown). These results imply that IAP enhancement of IL-2 production and T cell proliferation is independent of IAP association with integrins. For costimulation, anti-IAP and anti-CD3 had to be on the same surface. If either or both antibodies were used in solution, there was no costimulation even when the antibodies were cross-linked with a secondary antibody (data not shown). This suggests that the costimulatory signal arises from adhesion to a surface presenting ligands for both IAP and the TCR complex. When IL-2 production was quantitated, IAP-mediated enhancement of IL-2 production with low concentration of anti-CD3 was similar to that seen with the well characterized costimulator CD28 (Fig. 2 B). To determine whether costimulation by IAP required CD28, we tested whether IAP was able to augment IL-2 production in the CD28 deficient human cutaneous T cell lymphoma, HUT 78. Although without effect on their own, anti-IAP Abs enhanced IL-2 production with suboptimal anti-CD3 in HUT 78, equivalent to their effect in Jurkat cells (data not shown). Thus, antiIAP-mediated costimulation does not require expression of 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)

IAP synergy with CD3. (A) Human peripheral blood T cells  were incubated on plates coated with a low concentration of anti-CD3  together with increasing concentrations of anti-IAP mAb 2E11 or 2D3,  or anti-CR1 (3D9). Cells were pulsed with [3H]thymidine for the last 16 h  of a 90 h incubation. Shown are averages of triplicate wells from 1 experiment of >3 with similar results. Cells plated on 2E11, 2D3, and 3D9  alone had <1,000 CPM. Maximum stimulation by high concentration of  anti-CD3 was 40,000 cpm. (B) Jurkat cells were incubated on plates  coated with increasing concentrations of anti-CD3 and the same concentration of anti-IAP (2D3), anti CD28 (9.3), or anti-HLA (W6/32) mAbs.  Supernatants were harvested after 24 h and IL-2 concentration measured  by assay on CTLL-2 cells. The values shown represent triplicates of  [3H]thymidine incorporation by the CTLL-2 cells in 1 experiment of >3  with similar results. Quantitation of IL-2 concentration showed that stimulation of Jurkat cells by low levels of anti CD3 (1) in the presence of  anti-IAP or anti-CD28 mAbs led to 1 U/ml, compared with 0.1 U/ml  for the negative control mAb. Neither anti-IAP or anti-HLA caused detectable IL-2 secretion in the absence of anti-CD3. Additional mAbs that  do not costimulate with anti-CD3 include anti-CD61 (integrin β3), antiCD18 (integrin β2), and anti-CD29 (integrin β1).
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Figure 2: IAP synergy with CD3. (A) Human peripheral blood T cells were incubated on plates coated with a low concentration of anti-CD3 together with increasing concentrations of anti-IAP mAb 2E11 or 2D3, or anti-CR1 (3D9). Cells were pulsed with [3H]thymidine for the last 16 h of a 90 h incubation. Shown are averages of triplicate wells from 1 experiment of >3 with similar results. Cells plated on 2E11, 2D3, and 3D9 alone had <1,000 CPM. Maximum stimulation by high concentration of anti-CD3 was 40,000 cpm. (B) Jurkat cells were incubated on plates coated with increasing concentrations of anti-CD3 and the same concentration of anti-IAP (2D3), anti CD28 (9.3), or anti-HLA (W6/32) mAbs. Supernatants were harvested after 24 h and IL-2 concentration measured by assay on CTLL-2 cells. The values shown represent triplicates of [3H]thymidine incorporation by the CTLL-2 cells in 1 experiment of >3 with similar results. Quantitation of IL-2 concentration showed that stimulation of Jurkat cells by low levels of anti CD3 (1) in the presence of anti-IAP or anti-CD28 mAbs led to 1 U/ml, compared with 0.1 U/ml for the negative control mAb. Neither anti-IAP or anti-HLA caused detectable IL-2 secretion in the absence of anti-CD3. Additional mAbs that do not costimulate with anti-CD3 include anti-CD61 (integrin β3), antiCD18 (integrin β2), and anti-CD29 (integrin β1).
Mentions: To investigate the function of IAP on lymphocytes, we evaluated its role in T cell activation. When human T lymphocytes were purified from peripheral blood, no concentration of antiIAP alone stimulated proliferation (data not shown). In contrast, when combined with a suboptimal concentration of anti-CD3, three different anti-IAP mAbs enhanced proliferation of purified peripheral blood T cells. Using the same low concentration of anti-CD3, the nonbinding isotype-matched negative control 3D9 (anti-CR1/CD35) (Fig. 2 A) and mAb W6/32 (anti-HLA), which binds to a different cell surface antigen (data not shown), did not significantly enhance proliferation. In contrast with assays in which IAP has been shown to function with β3 integrins, the anti-IAP mAb B6H12 was much less potent than antiIAP 2D3, which recognizes a distinct epitope on the Ig domain (1) (data not shown). This suggested the possibility that the role for IAP is different in synergy with anti-CD3 than in cooperation with β3 integrins. 2E11, an anti-IAP mAb recognizing a third distinct epitope, also was costimulatory. Anti-IAP mAbs 2E11 and 2D3 also synergized with suboptimal concentrations of anti-CD3 to increase IL-2 production in Jurkat cells, while anti-HLA did not (Fig. 2 B; data not shown). This synergy is unlikely to be dependent on IAP cooperation with an integrin, since mAbs against β1, β2, and β3 integrins did not increase T cell proliferation or Jurkat IL-2 production in combination with antiCD3 (data not shown). The αvβ3 ligand vitronectin also was unable to costimulate T cell proliferation or Jurkat IL-2 production with suboptimal anti-CD3 (data not shown). These results imply that IAP enhancement of IL-2 production and T cell proliferation is independent of IAP association with integrins. For costimulation, anti-IAP and anti-CD3 had to be on the same surface. If either or both antibodies were used in solution, there was no costimulation even when the antibodies were cross-linked with a secondary antibody (data not shown). This suggests that the costimulatory signal arises from adhesion to a surface presenting ligands for both IAP and the TCR complex. When IL-2 production was quantitated, IAP-mediated enhancement of IL-2 production with low concentration of anti-CD3 was similar to that seen with the well characterized costimulator CD28 (Fig. 2 B). To determine whether costimulation by IAP required CD28, we tested whether IAP was able to augment IL-2 production in the CD28 deficient human cutaneous T cell lymphoma, HUT 78. Although without effect on their own, anti-IAP Abs enhanced IL-2 production with suboptimal anti-CD3 in HUT 78, equivalent to their effect in Jurkat cells (data not shown). Thus, antiIAP-mediated costimulation does not require expression of 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