B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells

Abstract

Dendritic cells (DCs), unique antigen-presenting cells (APCs) with potent T cell stimulatory capacity, direct the activation and differentiation of T cells by providing costimulatory signals. As such, they are critical regulators of both natural and vaccine-induced immune responses. A new B7 family member, B7-DC, whose expression is highly restricted to DCs, was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC fails to bind the B7.1/2 receptors CD28 and cytotoxic T lymphocyte-associated antigen (CTLA)-4, but does bind PD-1, a receptor for B7-H1/PD-L1. B7-DC costimulates T cell proliferation more efficiently than B7.1 and induces a distinct pattern of lymphokine secretion. In particular, B7-DC strongly costimulates interferon gamma but not interleukin (IL)-4 or IL-10 production from isolated naive T cells. These properties of B7-DC may account for some of the unique activity of DCs, such as their ability to initiate potent T helper cell type 1 responses.

Figure 1

Aa sequence of B7-DC and comparison with other B7 family members. (a) Aa sequence comparison of murine B7-DC to human B7-DC. The mB7-DC putative leader and transmembrane domain are underlined. The alignment was done using Clustawl-Gonnet Pam250 matrix. The red lettering shows identical aa and the blue lettering shows conservative substitutions. The asterisks indicate cysteine residues that may be important in the formation of disulfide bonds inside the IgV or IgC domains. (b) Aa sequence comparison of mB7-DC to mB7-H1/ PD-L1. (c) Both hB7-DC and B7-H1/PD-L1 are localized on human chromosome 9p24 and map to within 150 kb of each other on BAC clone RPCI-11.2. hB7-DC is centromeric to B7-H1.

Figure 1

Aa sequence of B7-DC and comparison with other B7 family members. (a) Aa sequence comparison of murine B7-DC to human B7-DC. The mB7-DC putative leader and transmembrane domain are underlined. The alignment was done using Clustawl-Gonnet Pam250 matrix. The red lettering shows identical aa and the blue lettering shows conservative substitutions. The asterisks indicate cysteine residues that may be important in the formation of disulfide bonds inside the IgV or IgC domains. (b) Aa sequence comparison of mB7-DC to mB7-H1/ PD-L1. (c) Both hB7-DC and B7-H1/PD-L1 are localized on human chromosome 9p24 and map to within 150 kb of each other on BAC clone RPCI-11.2. hB7-DC is centromeric to B7-H1.

Figure 1

Aa sequence of B7-DC and comparison with other B7 family members. (a) Aa sequence comparison of murine B7-DC to human B7-DC. The mB7-DC putative leader and transmembrane domain are underlined. The alignment was done using Clustawl-Gonnet Pam250 matrix. The red lettering shows identical aa and the blue lettering shows conservative substitutions. The asterisks indicate cysteine residues that may be important in the formation of disulfide bonds inside the IgV or IgC domains. (b) Aa sequence comparison of mB7-DC to mB7-H1/ PD-L1. (c) Both hB7-DC and B7-H1/PD-L1 are localized on human chromosome 9p24 and map to within 150 kb of each other on BAC clone RPCI-11.2. hB7-DC is centromeric to B7-H1.

Figure 2

B7-DC is differentially expressed between DCs and macrophages. (a) Distribution of mB7-DC mRNA in murine bone marrow DCs, splenic DCs, macrophages (Mφ), and macrophage lines was assessed by hybridization analysis using 0.5 μg/lane purified cDNA generated by SMART cDNA synthesis and run on a 1% agarose gel. G3PDH is used as control. J774A1, RAW 264.7, Pu5-1.8, and WEHI are macrophage cell lines. act., activated; BM, bone marrow. (b) hB7-DC is expressed in human DCs. Lane 1 shows human placenta and lane 2 shows human DCs cultured with GM-CSF plus IL-4. Oligos from the 5′ and 3′ UTR of human B7-DC were used to make PCR DNA probe for virtual Northern analysis of total RNA of human DCs. β-actin was used as control to ensure the quality of mRNA.

Figure 2

B7-DC is differentially expressed between DCs and macrophages. (a) Distribution of mB7-DC mRNA in murine bone marrow DCs, splenic DCs, macrophages (Mφ), and macrophage lines was assessed by hybridization analysis using 0.5 μg/lane purified cDNA generated by SMART cDNA synthesis and run on a 1% agarose gel. G3PDH is used as control. J774A1, RAW 264.7, Pu5-1.8, and WEHI are macrophage cell lines. act., activated; BM, bone marrow. (b) hB7-DC is expressed in human DCs. Lane 1 shows human placenta and lane 2 shows human DCs cultured with GM-CSF plus IL-4. Oligos from the 5′ and 3′ UTR of human B7-DC were used to make PCR DNA probe for virtual Northern analysis of total RNA of human DCs. β-actin was used as control to ensure the quality of mRNA.

Figure 3

B7-DC binds PD-1 but not CD28 or CTLA-4. 293T cells were transiently transfected with pCAGGS-B7.1 or pCAGGS-B7-DC. Transfectants were stained with PD-1-Ig, CD28-Ig, and CTLA-4-Ig fusion molecules followed by PE-labeled secondary antibody. Staining of pCAGGS (empty vector) transfectants was negative (data not shown).

Figure 4

Costimulation of T cells by B7-DC. (a) Purified T cells (CD4 plus CD8) were cultured in the presence of increasing concentrations of precoated antibody against CD3 (monoclonal antibody 2C11) and a constant amount of immobilized B7.1-Ig (♦), B7-DC-Ig (•), or isotype control (▴). The concentration of the Ig-fusion molecules used was 0.1 μg/ml. Data depict one representative experiment of three. (b) Purified CD8 T cells were cultured in the presence of increasing concentrations of precoated antibody against CD3 and a constant amount of immobilized B7.1-Ig (♦), B7-DC-Ig (•), or isotype control (▴) as in panel a. Data depict one representative experiment of two. Cells were incubated for 72 h and labeled with [³H]thymidine. (c) For analysis of cytokine secretion, purified T cells were cultured in the presence of precoated anti-CD3 (0.12 μg/ml) and 0.1 μg/ml of immobilized B7.1-Ig (♦), B7-DC-Ig (•), or isotype control (▴) as in panel a. Supernatants were collected after 24 and 48 h culture and assayed for the indicated lymphokines using ELISA. Data depict one representative experiment of three. (d) IFN-γ–treated RENCA cells expressing MHC class II were loaded with 12.5 μg/ml HA110–120 peptide and incubated with purified HA plus I-E^d–specific transgenic T cells together with 2 μg/ml of soluble B7.1-Ig (♦), B7-DC-Ig (•), or isotype control (▴). Data depict one representative experiment of two. Supernatants were collected after 24 and 48 h culture and assayed for the indicated lymphokines using ELISA.