The B7 family and other T cell co-stimulatory/co-inhibitory molecules are widely recognized as therapeutic targets for regulating the immune response. Butyrophilins are a novel class of co-stimulatory/co-inhibitory molecules that are structurally related to the B7 family and appear to have similar immunomodulatory functions. These parallels suggest that the butyrophilins or their receptors may also serve as drug targets. Recognizing this potential, R&D Systems exclusively offers bioactive recombinant butyrophilin proteins to further research on these molecules.
Overview of T cell activation, the B7 Family, and Butyrophilins
T cell activation requires two signals: 1) recognition of the antigenic peptide/major histocompatibility complex (MHC) by the T cell receptor (TCR) and 2) antigen-independent co-stimulation induced by interactions between co-signaling molecules expressed on antigen-presenting cells (APCs) and their T cell-expressed receptors. B7 family proteins are co-signaling molecules that interact with T cell-expressed immune receptors belonging to the CD28 family to yield both co-stimulatory and co-inhibitory signals. Integration of these signals contributes to the outcome and magnitude of a T cell response including the enhancement or suppression of T cell proliferation, differentiation, and/or cytokine secretion. Additionally, several other protein families such as the butyrophilins, SLAM and TIM family proteins, and members of the TNF receptor superfamily have also been shown to regulate T cell co-signaling. Like the B7-CD28 protein families, these molecules are of particular interest as multiple studies have shown that blockade of T cell co-inhibitory signaling can improve the anti-tumor immune response.
The butyrophilins are structurally closely related to the B7 family proteins and appear to have similar immunomodulatory functions. To date, thirteen human butyrophilin proteins have been identified including BTN1A1, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, BTN3A3, and the butyrophilin-like proteins, BTNL2, BTNL3, BTNL8, BTNL9, BTNL10, and SKINT-like (SKINTL). With the exception of BTNL2 and BTN3A2, butyrophilins are type I transmembrane proteins that contain one IgV-like and one IgC-like domain in their extracellular regions and a cytoplasmic B30.2 domain, which distinguishes them from the B7 family. Most butyrophilins that have been characterized to date, including human BTN1A1, BTN2A2, BTN3A1, BTNL2, and mouse BTNL1, act through unidentified receptors to inhibit T cell proliferation and cytokine production. The exception is BTNL8 which enhances T cell proliferation and cytokine secretion. Further investigation is necessary to identify the butyrophilin receptors and determine if co-inhibitory signaling by butyrophilins can also be targeted to enhance anti-tumor immunity. R&D Systems exclusively offers Recombinant Human and Mouse BTN1A1, Recombinant Human BTN3A1, and Recombinant Mouse BTNL2.
B7 Family Proteins and Butyrophilins Function as Co-Signaling Molecules to Regulate T Cell Activation. The B7 family consists of ten cell surface glycoproteins including B7-1/CD80, B7-2/CD86, PD-L1/B7-H1, B7-DC/PD-L2, B7-H2/ICOS L, B7-H3, B7-H4, B7-H5/PD-1H, B7-H6, and B7-H7/HHLA2. These proteins primarily interact with T cell-expressed immune receptors belonging to the CD28 family (CD28, CTLA4, PD-1, ICOS, and BTLA) to transduce both co-stimulatory and co-inhibitory signals that regulate T cell activation. Additionally, these interactions can have bidirectional effects (indicated by the two-headed arrows in the graphic). Similar to the B7 family, proteins belonging to the butyrophilin subfamily of the Immunoglobulin superfamily have also been shown to regulate T cell proliferation and cytokine production. With the exception of BTNL8, the buytrophilins that have been characterized to date, including human BTN1A1, BTN2A2, BTN3A1, BTNL2, and mouse BTNL1, act through unidentified receptors to inhibit T cell proliferation and cytokine production.
Recombinant Butyrophilin Proteins Available from R&D Systems
Butyrophilin Antibodies Available from R&D Systems
Assessment of the Bioactivity and Purity of R&D Systems® Recombinant Butyrophilin Proteins
BTN1A1 and BTN3A1 Inhibit Anti-CD3-Induced IL-2 Production by Human T Cells in a Manner Similar to B7-H1/PD-L1. Human T cells were incubated with immobilized Mouse Anti-Human CD3ε Monoclonal Antibody (Catalog # MAB100; 1 ug/mL) and the indicated concentrations of (A) Recombinant Human BTN1A1 (Catalog # 8467-BT), (B) Recombinant Human BTN3A1/CD277 Fc Chimera (Catalog # 8539-BT), or (C) Recombinant Human B7-H1/PD-L1 Fc Chimera (Catalog # 156-B7). IL-2 secretion was measured in cell culture supernatants using the Human IL-2 Quantikine® ELISA Kit (Catalog # D2050). The ED50 for this effect is typically 0.5-2.5 ug/mL for Recombinant Human BTN1A1, 1-5 ug/mL for Recombinant Human BTN3A1/CD277, and 2-10 ug/mL for Recombinant Human B7-H1/PD-L1. The purity of (A) Recombinant Human BTN1A1 (Catalog # 8467-BT; 1 ug/lane), (B) Recombinant Human BTN3A1/CD277 (Catalog # 8539-BT; 1 ug/lane), and (C) Recombinant Human B7-H1/PD-L1 (Catalog # 156-B7; 1 ug/lane) was assessed by SDS-PAGE analysis under reducing (R) and non-reducing (NR) conditions and visualized by silver staining (inset).
|BTN3A1 Inhibits Human T Cell Proliferation in a Dose-Dependent Manner. Human T cells were incubated with immobilized Mouse Anti-Human CD3ε Monoclonal Antibody (1 ug/mL; Catalog # MAB100) and the indicated concentrations of Recombinant Human BTN3A1/CD277 Fc Chimera (Catalog # 8539-BT) or Recombinant Human IgG1-Fc (Catalog # 110-HG) for 72 hours. Cell proliferation was assessed by a fluorometric assay using the redox sensitive dye Resazurin (Catalog # AR002).
||BTN3A1 Binds to Activated Human T Cells. Human CD3+ T cells were isolated from peripheral blood mononuclear cells using the MagCellect™ Human CD3+ T Cell Isolation Kit (Catalog # MAGH101) and stimulated with immobilized Mouse Anti-Human CD3ε Monoclonal Antibody (Catalog # MAB100; 1 ug/mL) for 48 hours. Resting T cells (left) or activated T cells (right) were incubated with Recombinant Human BTN3A1/CD277 Fc Chimera (Catalog # 8539-BT) or Recombinant Human IgG1-Fc (Catalog # 110-HG) for 1 hr. Protein binding to the cell surface was analyzed by flow cytometry using a biotinylated goat anti-human IgG Fc antibody and PE-conjugated Streptavidin (Catalog # F0040).
In addition to the butyrophilins, R&D Systems offers a wide selection of reagents for other molecules involved in T cell co-signaling. For a complete product listing, please visit the T Cell Costimulatory/Co-inhibitory Molecules page of our website.