Butyrophilins As Immune Checkpoint Proteins

Butyrophilins Regulate T Cell Functions

Butyrophilins are a more recently identified class of T cell co-inhibitory/co-stimulatory molecules that belong to the immunoglobulin superfamily. These proteins are structurally related to the B7 family and appear to have similar immunomodulatory functions. In humans,the butyrophilin family consists of seven butyrophilins, BTN1A1, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, BTN3A3, five butyrophilin-like proteins, BTNL2, BTNL3, BTNL8, BTNL9, BTNL10, and the SKINT-like (SKINTL) protein. In mice, eleven butyrophilin family members have been described to date, BTN1A1, BTN2A2, BTNL1-2, BTNL4-7, BTNL9-10, and SKINTL, although these typically differ from their human counterparts.

With the exception of BTN3A2, BTNL2, and BTNL10, butyrophilins have one extracellular IgV-like domain and one extracellular IgC-like domain, a transmembrane region, and a cytoplasmic B30.2 domain, which distinguishes them from the B7 family. In contrast to the other butyrophilin family members, BTNL2 has two extracellular IgV-like and two IgC-like domains and lacks the intracellular B30.2 domain, as do BTN3A2 and BTNL10. Most butyrophilins that have been characterized to date inhibit T cell proliferation and cytokine production by acting through currently unidentified receptors.1-4 The exceptions are BTN3A2, BTNL8, BTN2A1, and BTN3A1.BTN3A2 was shown to enhance intraepithelial infiltration of CD4+ and CD8+ T cells in ovarian cancer tissues, while BTNL8 was found to promote anti-CD3-stimulated human T cell proliferation and cytokine secretion.5, 6 Additionally, BTN2A1 and BTN3A1 have recently been shown to be involved in the Vgamma9Vdelta2 TCR-mediated recognition of phosphoantigens on stressed cells, which results in activation of the major peripheral subset of gamma delta T cells.7-10 Although further research is needed to identify the key binding partners of many of the other butryophilin proteins and the pathways by which they regulate T cell functions, structural and functional similarities between these proteins and the B7 family members suggest that the butyrophilins should be investigated as potential targets for cancer immunotherapy.

Bio-Techne exclusively offers a large selection of R&D SystemsTM bioactive recombinant butyrophilin proteins, along with human or mouse antibodies against a number of butyrophilin family members to further researchers’ abilities to investigate the functions of these molecules as immune checkpoint regulators.

Butyrophilins Mediate T Cell Co-stimulatory and Co-inhibitory Interactions

Butyrophilins are structurally similar to the B7 family proteins and function as co-stimulatory or co-inhibitory secondary signals to regulate T cell activation.

Butyrophilins Function as Co-Signaling Molecules to Regulate T Cell Activation. Similar to the B7 family, proteins belonging to the butyrophilin subfamily of the immunoglobulin superfamily regulate T cell proliferation and cytokine production. Most of 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.

Bioactivity Testing of R&D Systems Butyrophilin Proteins

Analysis of the purity of R&D Systems Recombinant Human BTN1A1 and its effect on IL-2 production by activated T cells.

BTN1A1 Inhibits Anti-CD3-induced IL-2 Production by Human T Cells. Human T cells were incubated with an immobilized Mouse Anti-Human CD3 Monoclonal Antibody (R&D Systems, Catalog # MAB100; 1μg/mL) and the indicated concentrations of Recombinant Human BTN1A1 (R&D Systems, Catalog # 8467-BT). IL-2 secretion was measured in cell culture supernatants using the Human IL-2 QuantikineTM ELISA Kit (R&D Systems, Catalog # D2050). The ED50 for this effect is typically 0.5-2.5 μg/mL. The purity of Recombinant Human BTN1A1 (R&D Systems, Catalog # 8467-BT; 1 μg/lane) was assessed by SDS-PAGE analysis under reducing (R) and non-reducing (NR) conditions (inset).

Analysis of the purity of R&D Systems Recombinant Human BTN3A1/CD277 Fc Chimera and its effect on IL-2 production by activated T cells.

BTN3A1 Inhibits Anti-CD3-induced IL-2 Production by Human T Cells. Human T cells were incubated with an immobilized Mouse Anti-Human CD3 Monoclonal Antibody (R&D Systems, Catalog # MAB100; 1μg/mL) and the indicated concentrations of Recombinant Human BTN3A1/CD277 Fc Chimera (R&D Systems, Catalog # 8539-BT). IL-2 secretion was measured in cell culture supernatants using the Human IL-2 Quantikine ELISA Kit (R&D Systems, Catalog # D2050). The ED50 for this effect is typically1-5 μg/mL. The purity of Recombinant Human BTN3A1/CD277 (R&D Systems, Catalog # 8539-BT; 1 μg/lane) was assessed by SDS-PAGE analysis under reducing (R) and non-reducing (NR) conditions (inset).

BTNL9 Inhibits Human CD4+ T Cell Proliferation: Bioactivity assay data showing R&D Systems Recombinant Human BTNL9 inhibits human CD4+ T cell proliferation.

BTNL9 Inhibits Human CD4+ T Cell Proliferation. Human CD3+ T cells were isolated from PBMCs using the MagCellectTM Human CD3+ T Cell Isolation Kit (R&D Systems, Catalog # MAGH101). CFSE-labeled T cells were treated with a combination of plate-bound anti-CD3 and either plate bound Recombinant Human BTNL9 Fc Chimera (R&D Systems, Catalog # 9659-BT;10 μg/mL) or vehicle control for 5 days. Cells were stained with an APC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (R&D Systems, Catalog # FAB3791A) or an APC-conjugated Anti-Human CD8 Monoclonal Antibody (R&D Systems, Catalog # FAB1509A) and gated by flow cytometry.

References

  1. Nguyen, T. et al. (2006) BTNL2, a butryophilin-like molecule that functions to inhibit T cell activation. J. Immunol. 176:7354. PMID: 16751379.

  2. Yamazaki, T. et al. (2010) A butyrophilin family member critically inhibits T cell activation. J. Immunol. 185:5907. PMID: 20944003.

  3. Cubillos-Ruiz, J.R. et al. (2010) CD277 is a negative co-stimulatory molecule universally expressed by ovarian cancer microenvironmental cells. Oncotarget 1:329. PMID: 21113407.

  4. Yamashiro, H. et al. (2010) Stimulation of human butyrophilin 3 molecules results in negative regulation of cellular immunity. J. Leukoc. Biol. 88:757. PMID: 20610803.

  5. LePage, C. et al. (2012) BTN3A2 expression in epithelial ovarian cancer is associated with higher tumor infiltrating T cells and a better prognosis. PLoS One 7:38541. PMID: 22685580.

  6. Chapoval, A.I. et al. (2013) BTNL8, a butyrophilin-like molecule that costimulates the primary immune response. Mol. Immunol. 56:819. PMID: 24036152.

  7. Peigne, C.M. et al. (2017) The juxtamembrane domain of butyrophilin BTN3A1 controls phosphoantigen-mediated activation of human Vgamma9Vdelta2 T cells. J. Immunol. 198:4228. PMID: 28461569.

  8. Gu, S. et al. (2017) Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vgamma9Vdelta2 T cell activation. Proc. Natl. Acad. Sci. USA PMID: 28807997.

  9. Karunakaran, M.M. et al. (2020) Butyrophilin-2A1 directly binds germline-encoded regions of the Vgamma9Vdelta2 TCR and is essential for phosphoantigen sensing. Immunity 52:487. PMID: 32155411.

  10. Rigau, M. et al. (2020) Butyrophilin 2A1 is essential for phosphoantigen reactivity by gammadelta T cells. Science 367:5516. PMID: 31919129.