Products for Butyrophilin-related Research: R&D Systems

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

Molecule	Species	Catalog #	Tag
BTN1A1	Human	8467-BT	His
	Human	AVI8467	Biotin, His
	Mouse	8540-BT	His
BTN2A1	Human	9058-BT	His
BTN2A1	Human	AVI10909	Biotin, Fc
BTN2A2	Human	8918-BT	Fc
BTN2A2	Mouse	8997-BT	His
BTN3A1/CD277	Human	8539-BT	Fc
BTN3A2	Human	9514-BT	His
BTN3A2	Human	AVI9514	Biotin, His
BTN3A3	Human	1350-BT	His
BTNL2	Mouse	8605-BT	His
BTNL3	Human	9658-BT	Fc
BTNL3	Mouse	10222-BT	Fc
BTNL4	Mouse	9590-BT	Fc
	Mouse	9594-BT	His
BTNL6	Mouse	9638-BT	His
BTNL8	Human	9359-BT	Fc
BTNL9	Human	9659-BT	Fc
	Mouse	9555-BT	Fc
BTNL10/Butyrophilin-like 10	Human	10014-BT	Fc
	Mouse	1444-BT	Fc

Butyrophilin Antibodies Available from R&D Systems

Molecule	Species	Catalog #	Applications
BTN1A1	Human	MAB84671	FC, ICC/IF
	Human	FAB84671	FC
	Mouse	AF4765	WB
BTN2A2	Human	AF8645	IHC, WB
BTN2A2	Mouse	AF4917	WB
BTN3A1/CD277	Human	MAB7136	FC
	Human	FAB7136	FC
BTN3A1/2/3	Human	AF7136	WB
BTNL2	Mouse	AF5236	WB
BTNL8	Human	FAB9359 MAB9359	FC FC, WB
BTNL8	Human	MAB113641	IHC, WB

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.