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B7-1 and B7-2 Provide Secondary Signals that Regulate T Cell Activation

B7-1, B7-2 and T Cell Activation

CD28 and CTLA-4 are T cell-expressed receptors that bind to the B7 family proteins, B7-1/CD80 and B7-2/CD86, which are primarily expressed on antigen-presenting cells (APCs). While CD28 is constitutively expressed on naïve T cells and provides a co-stimulatory signal upon binding to these ligands, CTLA-4 is up-regulated on T cells following T cell activation and delivers a co-inhibitory signal following ligand-binding. CTLA-4 binds to both B7-1/CD80 and B7-2/CD86 with a higher affinity than CD28, and as a result, the T cell response is down-regulated following up-regulation of CTLA-4.1 

CTLA-4 is also constitutively expressed on regulatory T cells and has been shown to remove B7-1/CD80 and B7-2/CD86 from APCs by trans-endocytosis, suggesting another mechanism by which CTLA-4 may down-regulate the T cell response.2, 3 In addition, CTLA-4 induces the production of indoleamine 2,3-dioxygenase (IDO) by dendritic cells (DCs), leading to degradation of the amino acid tryptophan and the production of immunosuppressive metabolites such as kynurenine.4, 5 Both the depletion of tryptophan and the production of kynurenine inhibit T cell activation and proliferation, indicating that CTLA-4 may also indirectly regulate T cell activity by acting on DCs.

Up-regulated and sustained expression of CTLA-4 along with other inhibitory receptors is associated with T cell exhaustion in cancer.6-8 T cell exhaustion is a dysfunctional state that is characterized by reduced T cell proliferation, decreased cytokine production, and reduced cytotoxicity, which leads to a down-regulation of the T cell-mediated anti-tumor immune response. As a result, blockade of CTLA-4 may not only improve anti-tumor immune responses by suppressing CTLA-4-mediated T cell co-inhibitory signaling and inhibiting the suppressive activity of regulatory T cells, but also by partially restoring the functions of exhausted T cells. To date, CTLA-4, along with PD-1, and PD-L1 are the most common proteins being targeted for cancer immunotherapy.8

B7-1/CD80 and B7-2/CD86 Regulate T Cell Activation by Binding to CD28 or CTLA-4

B7-1 B7-2 CD28 Figure

B7-1/CD80 and B7-2/CD86 mediate T cell co-stimulatory and co-inhibitory effects by binding to either CD28 or CTLA-4. Following recognition of the antigenic peptide/major histocompatibility complex (MHC) by the T cell receptor (TCR), binding of B7-1/CD80 or B7-2/CD86 to CD28 provides a co-stimulatory signal to promote T cell activation. Following T cell activation, the co-inhibitory receptor, CTLA-4, is up-regulated and binds with higher affinity to B7-1/CD80 and B7-2/CD86 than CD28, resulting in a down-regulation of T cell activity. In cancer, tumor-associated macrophages express B7-1/CD80 and B7-2/CD86, which allows them to suppress the functions of activated T cells through CTLA-4. Up-regulated expression of CTLA-4 and other inhibitory receptors is also associated with tumor-specific T cell exhaustion. This dysfunctional T cell state is characterized by a decrease in proliferative capacity and reduced T cell effector functions that prevent effective tumor elimination.

Analysis of the Binding Properties of R&D Systems B7-1 and CTLA-4 Proteins by SPR

Surface plasmon resonance data showing the affinity measurements and binding kinetics between the CTLA-4 and B7-1 proteins.

Affinity Measurements and Binding Kinetics of the CTLA-4:B7-1 Interaction by Surface Plasmon Resonance. Sensorgram data of captured Avi-tag Biotinylated Recombinant Human CTLA-4 Fc Chimera (R&D Systems, Catalog # AVI7268) binding to Recombinant Human B7-1 Fc Chimera (R&D Systems, Catalog # 10133-B1). Briefly, Avi-tag Biotinylated Recombinant Human CTLA-4 Fc Chimera was captured at a low coupling density to the active flow cell via the Avi-tag biotin. Recombinant Human B7-1 Fc Chimera at a concentration range between 82 pM and 21 nM was flowed over both active and uncoupled reference flow cells at each concentration. Kinetic sensorgrams were fit to a 1:1 binding model to determine the binding kinetics and affinity, with an interaction affinity of KD=0.2511 nM. The corresponding overlaid kinetic fits with the residual plot shown below. The corresponding steady state affinity fit is shown at the bottom. The experiment was performed on a Biacore T200, GE Healthcare.

Assessment of the Bioactivity of R&D Systems Recombinant Human CTLA-4 Protein

Analysis of the effect of R&D Systems Recombinant Human CTLA-4 Fc Chimera on IL-2 secretion by Jurkat cells.

CTLA-4 Inhibits IL-2 Secretion by Jurkat Cells. Jurkat human acute T cell leukemia cells were stimulated with Recombinant Human B7-1/CD80 Fc Chimera (R&D Systems, Catalog # 140-B1; 1 μg/mL) and then treated with the indicated concentrations of Recombinant Human CTLA-4 Fc Chimera (R&D Systems, Catalog # 7268-CT). 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 0.03-0.15 μg/mL.

Background Information

  1. Sansom, D.M. (2000) CD28, CTLA-4 and their ligands: who does what and to whom? Immunology 101:169. PMID: 11012769.

  2. Qureshi, O.S. et al. (2011) Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4. Science 332:600. PMID: 21474713.

  3. Ovcinnikovs, V. et al. (2019) CTLA-4-mediated transendocytosis of costimulatory molecules primarily targets migratory dendritic cells. Sci. Immunol. PMID: 31152091.

  4. Grohmann, U. et al. (2002) CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3:1097. PMID: 12368911.

  5. Munn, D.H. et al. (2004) Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J. Immunol. 172:4100. PMID: 15034022.

  6. Wherry, E.J. & M. Kurachi (2015) Molecular and cellular insights into T cell exhaustion. Nat. Rev. Immunol. 15:486. PMID: 26205583.

  7. Zhao, Y. et al. (2020) Exhaustion and senescence: two crucial dysfunctional states of T cells in the tumor microenvironment. Cell. Mol. Immunol. 17:27. PMID: 31853000.

  8. Seidel, J.A. et al. (2018) Anti-PD-1 and anti-CTLA-4 therapies in cancer: mechanisms of action, efficacy, and limitations. Front. Oncol. 8:86. PMID: 296444214.