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Cooperative B7-1/2 (CD80/CD86) and B7-DC costimulation of CD4+ T cells independent of the PD-1 receptor.

Shin T, Kennedy G, Gorski K, Tsuchiya H, Koseki H, Azuma M, Yagita H, Chen L, Powell J, Pardoll D, Housseau F - J. Exp. Med. (2003)

Bottom Line: B7-1/B7-2-deficient DCs, while strongly diminished in their ability to stimulate naive CD4+ T cells, nonetheless retain partial activity.B7-DC costimulates expression of CD40L with faster kinetics than B7-1 and displays potent synergy with B7-1 and B7-2 for T cell proliferation and cytokine production, indicating that these B7 family members work in concert to stimulate T cells.Finally, costimulation with B7-DC alone or in conjunction with B7-1 is PD-1 independent, indicating that B7-DC costimulates T cells via a second receptor.

View Article: PubMed Central - PubMed

Affiliation: Sidneu Kimmel Comprehensive Cancer Centre, Johns Hopkins School of Medicine, Baltimore, MD 21231, USA.

ABSTRACT
B7-DC is a recently discovered member of the B7 family that binds to PD-1 and is selectively expressed by dendritic cells (DCs). It has been shown to either costimulate or inhibit T cell responses. To assess the role of B7-DC in DC-T cell interactions, DCs from B7-DC knockout (KO) mice were generated and compared with DCs from wild-type (WT) and B7-1/B7-2 double KO mice. B7-1/B7-2-deficient DCs, while strongly diminished in their ability to stimulate naive CD4+ T cells, nonetheless retain partial activity. DCs from B7-DC KO mice are diminished in their ability to activate CD4+ T cells, demonstrating that DC-expressed B7-DC serves a predominantly stimulatory rather than inhibitory function in the initiation of T cell responses. B7-DC costimulates expression of CD40L with faster kinetics than B7-1 and displays potent synergy with B7-1 and B7-2 for T cell proliferation and cytokine production, indicating that these B7 family members work in concert to stimulate T cells. Finally, costimulation with B7-DC alone or in conjunction with B7-1 is PD-1 independent, indicating that B7-DC costimulates T cells via a second receptor.

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Immobilized B7-DC strongly costimulates CD4+ T cells and synergizes with B7–1. (a) FACS® analysis of CD40L expression. Purified CD4+ T cells were stimulated with 3 ng/well (left panel) or 30 ng/well (right panel) of precoated anti-CD3 in presence or absence of an equimolar amount of immobilized B7-DC-Ig and B7–1-Ig or the combination of both. Cells were harvested at indicated times, washed, and stained 30 min with PE-conjugated anti-CD40L mAb and FITC-conjugated anti-CD4 mAb. (b) Culture supernatants were collected at indicated times and assayed for cytokine secretion by ELISA when T cells were stimulated with 3 ng/well (left) or 30 ng/well (right) of immobilized anti-CD3 alone, in presence of plate bound B7-1-Ig, B7-DC-Ig, or both. Open bars, no costimulation; gray bars, B7-1-Ig; hatched bars, B7-DC-Ig; black bars, B7-1-Ig + B7-DC-Ig (c) B7-DC-Ig and B7-1-Ig titration. CD4+ T cells were stimulated with a constant amount of anti-CD3 (30 ng/well) in presence of increasing quantities of immobilized B7-DC-Ig and/or increasing amount of B7–1/2-Ig. Symbols: black squares, no B7–1/2-Ig; black circles, 25 ng/ml B7–1-Ig; open circles, 50 ng/ml B7–1-Ig; black triangles, 25 ng/ml B7–2-Ig; open triangles, 50 ng/ml B7–2-Ig. (d) Immobilized B7-DC-Ig costimulates preactivated CD4+ T cells as well as resting T cells. Purified CD4+ T cells freshly isolated from spleens or preactivated 2 d with plate-bound anti-CD3 (30 ng/well), were stimulated with immobilized anti-CD3 (30 ng/well) in the presence or absence of immobilized B7–1-Ig and/or B7-DC-Ig. Open bars, no costimulation; gray bars, B7–1-Ig; hatched bars, B7-DC-Ig; black bars, B7–1-Ig + B7-DC-Ig. Cytokine release was measured by ELISA on 24 h culture supernatants.
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fig2: Immobilized B7-DC strongly costimulates CD4+ T cells and synergizes with B7–1. (a) FACS® analysis of CD40L expression. Purified CD4+ T cells were stimulated with 3 ng/well (left panel) or 30 ng/well (right panel) of precoated anti-CD3 in presence or absence of an equimolar amount of immobilized B7-DC-Ig and B7–1-Ig or the combination of both. Cells were harvested at indicated times, washed, and stained 30 min with PE-conjugated anti-CD40L mAb and FITC-conjugated anti-CD4 mAb. (b) Culture supernatants were collected at indicated times and assayed for cytokine secretion by ELISA when T cells were stimulated with 3 ng/well (left) or 30 ng/well (right) of immobilized anti-CD3 alone, in presence of plate bound B7-1-Ig, B7-DC-Ig, or both. Open bars, no costimulation; gray bars, B7-1-Ig; hatched bars, B7-DC-Ig; black bars, B7-1-Ig + B7-DC-Ig (c) B7-DC-Ig and B7-1-Ig titration. CD4+ T cells were stimulated with a constant amount of anti-CD3 (30 ng/well) in presence of increasing quantities of immobilized B7-DC-Ig and/or increasing amount of B7–1/2-Ig. Symbols: black squares, no B7–1/2-Ig; black circles, 25 ng/ml B7–1-Ig; open circles, 50 ng/ml B7–1-Ig; black triangles, 25 ng/ml B7–2-Ig; open triangles, 50 ng/ml B7–2-Ig. (d) Immobilized B7-DC-Ig costimulates preactivated CD4+ T cells as well as resting T cells. Purified CD4+ T cells freshly isolated from spleens or preactivated 2 d with plate-bound anti-CD3 (30 ng/well), were stimulated with immobilized anti-CD3 (30 ng/well) in the presence or absence of immobilized B7–1-Ig and/or B7-DC-Ig. Open bars, no costimulation; gray bars, B7–1-Ig; hatched bars, B7-DC-Ig; black bars, B7–1-Ig + B7-DC-Ig. Cytokine release was measured by ELISA on 24 h culture supernatants.

Mentions: To further assess the costimulatory capacity of B7-DC, alone and in conjunction with B7–1, a series of in vitro experiments using immobilized B7–1 and/or B7-DC (as Ig dimers) and RENCA cells expressing B7–1 and/or B7-DC, were performed. Initially, stimulation of CD4+ T cells was assessed by analyzing the expression of T cell activation markers such as CD40L, CD25 and CD69. Fig. 2 demonstrates that for both high and low concentrations of anti-CD3 (signal 1), B7-DC-Ig costimulates the expression of CD40L with much faster kinetics than B7–1, inducing significant up-regulation after 6 h. In contrast B7–1 costimulation of CD40L was not observed until 18 h. Importantly, B7-DC does not inhibit the up-regulation of CD40L on CD4+ T cells by B7–1 costimulation. Interestingly, at low concentrations of anti-CD3, B7-DC alone efficiently up-regulated CD40L expression whereas B7–1 did not. The differential kinetics and sensitivity of CD4 T cells to costimulation with B7-DC versus B7–1 was not observed for other T cell surface molecules such as CD25 and CD69 (data not depicted), which are involved in later stages of T cell activation than CD40L. These results suggest that B7-DC costimulation is particularly important in modulating early events in crosstalk between T cells and DC as CD40L induced on T cells is a potent amplifier of DC function (18, 19) whereas B7-DC cross-linking has been shown to stimulate CD40 expression by DCs (12).


Cooperative B7-1/2 (CD80/CD86) and B7-DC costimulation of CD4+ T cells independent of the PD-1 receptor.

Shin T, Kennedy G, Gorski K, Tsuchiya H, Koseki H, Azuma M, Yagita H, Chen L, Powell J, Pardoll D, Housseau F - J. Exp. Med. (2003)

Immobilized B7-DC strongly costimulates CD4+ T cells and synergizes with B7–1. (a) FACS® analysis of CD40L expression. Purified CD4+ T cells were stimulated with 3 ng/well (left panel) or 30 ng/well (right panel) of precoated anti-CD3 in presence or absence of an equimolar amount of immobilized B7-DC-Ig and B7–1-Ig or the combination of both. Cells were harvested at indicated times, washed, and stained 30 min with PE-conjugated anti-CD40L mAb and FITC-conjugated anti-CD4 mAb. (b) Culture supernatants were collected at indicated times and assayed for cytokine secretion by ELISA when T cells were stimulated with 3 ng/well (left) or 30 ng/well (right) of immobilized anti-CD3 alone, in presence of plate bound B7-1-Ig, B7-DC-Ig, or both. Open bars, no costimulation; gray bars, B7-1-Ig; hatched bars, B7-DC-Ig; black bars, B7-1-Ig + B7-DC-Ig (c) B7-DC-Ig and B7-1-Ig titration. CD4+ T cells were stimulated with a constant amount of anti-CD3 (30 ng/well) in presence of increasing quantities of immobilized B7-DC-Ig and/or increasing amount of B7–1/2-Ig. Symbols: black squares, no B7–1/2-Ig; black circles, 25 ng/ml B7–1-Ig; open circles, 50 ng/ml B7–1-Ig; black triangles, 25 ng/ml B7–2-Ig; open triangles, 50 ng/ml B7–2-Ig. (d) Immobilized B7-DC-Ig costimulates preactivated CD4+ T cells as well as resting T cells. Purified CD4+ T cells freshly isolated from spleens or preactivated 2 d with plate-bound anti-CD3 (30 ng/well), were stimulated with immobilized anti-CD3 (30 ng/well) in the presence or absence of immobilized B7–1-Ig and/or B7-DC-Ig. Open bars, no costimulation; gray bars, B7–1-Ig; hatched bars, B7-DC-Ig; black bars, B7–1-Ig + B7-DC-Ig. Cytokine release was measured by ELISA on 24 h culture supernatants.
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fig2: Immobilized B7-DC strongly costimulates CD4+ T cells and synergizes with B7–1. (a) FACS® analysis of CD40L expression. Purified CD4+ T cells were stimulated with 3 ng/well (left panel) or 30 ng/well (right panel) of precoated anti-CD3 in presence or absence of an equimolar amount of immobilized B7-DC-Ig and B7–1-Ig or the combination of both. Cells were harvested at indicated times, washed, and stained 30 min with PE-conjugated anti-CD40L mAb and FITC-conjugated anti-CD4 mAb. (b) Culture supernatants were collected at indicated times and assayed for cytokine secretion by ELISA when T cells were stimulated with 3 ng/well (left) or 30 ng/well (right) of immobilized anti-CD3 alone, in presence of plate bound B7-1-Ig, B7-DC-Ig, or both. Open bars, no costimulation; gray bars, B7-1-Ig; hatched bars, B7-DC-Ig; black bars, B7-1-Ig + B7-DC-Ig (c) B7-DC-Ig and B7-1-Ig titration. CD4+ T cells were stimulated with a constant amount of anti-CD3 (30 ng/well) in presence of increasing quantities of immobilized B7-DC-Ig and/or increasing amount of B7–1/2-Ig. Symbols: black squares, no B7–1/2-Ig; black circles, 25 ng/ml B7–1-Ig; open circles, 50 ng/ml B7–1-Ig; black triangles, 25 ng/ml B7–2-Ig; open triangles, 50 ng/ml B7–2-Ig. (d) Immobilized B7-DC-Ig costimulates preactivated CD4+ T cells as well as resting T cells. Purified CD4+ T cells freshly isolated from spleens or preactivated 2 d with plate-bound anti-CD3 (30 ng/well), were stimulated with immobilized anti-CD3 (30 ng/well) in the presence or absence of immobilized B7–1-Ig and/or B7-DC-Ig. Open bars, no costimulation; gray bars, B7–1-Ig; hatched bars, B7-DC-Ig; black bars, B7–1-Ig + B7-DC-Ig. Cytokine release was measured by ELISA on 24 h culture supernatants.
Mentions: To further assess the costimulatory capacity of B7-DC, alone and in conjunction with B7–1, a series of in vitro experiments using immobilized B7–1 and/or B7-DC (as Ig dimers) and RENCA cells expressing B7–1 and/or B7-DC, were performed. Initially, stimulation of CD4+ T cells was assessed by analyzing the expression of T cell activation markers such as CD40L, CD25 and CD69. Fig. 2 demonstrates that for both high and low concentrations of anti-CD3 (signal 1), B7-DC-Ig costimulates the expression of CD40L with much faster kinetics than B7–1, inducing significant up-regulation after 6 h. In contrast B7–1 costimulation of CD40L was not observed until 18 h. Importantly, B7-DC does not inhibit the up-regulation of CD40L on CD4+ T cells by B7–1 costimulation. Interestingly, at low concentrations of anti-CD3, B7-DC alone efficiently up-regulated CD40L expression whereas B7–1 did not. The differential kinetics and sensitivity of CD4 T cells to costimulation with B7-DC versus B7–1 was not observed for other T cell surface molecules such as CD25 and CD69 (data not depicted), which are involved in later stages of T cell activation than CD40L. These results suggest that B7-DC costimulation is particularly important in modulating early events in crosstalk between T cells and DC as CD40L induced on T cells is a potent amplifier of DC function (18, 19) whereas B7-DC cross-linking has been shown to stimulate CD40 expression by DCs (12).

Bottom Line: B7-1/B7-2-deficient DCs, while strongly diminished in their ability to stimulate naive CD4+ T cells, nonetheless retain partial activity.B7-DC costimulates expression of CD40L with faster kinetics than B7-1 and displays potent synergy with B7-1 and B7-2 for T cell proliferation and cytokine production, indicating that these B7 family members work in concert to stimulate T cells.Finally, costimulation with B7-DC alone or in conjunction with B7-1 is PD-1 independent, indicating that B7-DC costimulates T cells via a second receptor.

View Article: PubMed Central - PubMed

Affiliation: Sidneu Kimmel Comprehensive Cancer Centre, Johns Hopkins School of Medicine, Baltimore, MD 21231, USA.

ABSTRACT
B7-DC is a recently discovered member of the B7 family that binds to PD-1 and is selectively expressed by dendritic cells (DCs). It has been shown to either costimulate or inhibit T cell responses. To assess the role of B7-DC in DC-T cell interactions, DCs from B7-DC knockout (KO) mice were generated and compared with DCs from wild-type (WT) and B7-1/B7-2 double KO mice. B7-1/B7-2-deficient DCs, while strongly diminished in their ability to stimulate naive CD4+ T cells, nonetheless retain partial activity. DCs from B7-DC KO mice are diminished in their ability to activate CD4+ T cells, demonstrating that DC-expressed B7-DC serves a predominantly stimulatory rather than inhibitory function in the initiation of T cell responses. B7-DC costimulates expression of CD40L with faster kinetics than B7-1 and displays potent synergy with B7-1 and B7-2 for T cell proliferation and cytokine production, indicating that these B7 family members work in concert to stimulate T cells. Finally, costimulation with B7-DC alone or in conjunction with B7-1 is PD-1 independent, indicating that B7-DC costimulates T cells via a second receptor.

Show MeSH
Related in: MedlinePlus