Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses

Abstract

Pathways in the B7:CD28 family of costimulatory molecules regulate T cell activation and tolerance. B7-dependent responses in Cd28(-/-)Ctla4(-/-) T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 molecules (PD-L1 or PD-L2) have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine production were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient versus PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient versus CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. Our findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an additional dimension to immunoregulatory functions of the B7:CD28 family.

Figure 1. Equilibrium binding constants of B7-1 and PD-L1 interactions

A) Equilibrium binding responses were measured using surface plasmon resonance of fusion proteins of B7-1, B7-2, and PD-1 to immobilized PD-L1 and PD-L2 fusion proteins. No binding response was seen for B7-2 to PD-L1 or PD-L2 (not shown). The Kds for the three significant interactions are shown (arrows). B) Scatchard plot shows the dissociation constant for B7-1 to immobilized PD-L1 is 1.6 μM. The linear fit and its 90% confidence interval are shown. C) Summary of dissociation constants obtained from Biacore equilibrium binding experiments.

Figure 2. Localization of the B7-1:PD-L1 binding site

A) B7-1-Ig and PD-L1-Ig and the products of cross-linking with the heterobifunctional crosslinker sulfo-SBED are shown in an SDS-PAGE gel stained with Coomassie Blue. Arrow indicates band of gel that was cut out for mass spectrometric analysis. B) Peptides were categorized into sites on PD-L1 and B7-1 and grouped according to their lysines. Amino acid numbers start from the position after signal cleavage. The number of distinct peptides containing the sites and the cross-linker were counted and the results are shown graphically. C and D) Molecular model of (C) B7-1 and (D) PD-L1 showing sites of interaction identified by cross-linking. On each structure, lysines are colored: site 1 lysines are colored red, site 2 lysine(s) are colored orange, and site 3 lysine(s) are colored green. The binding site for CTLA-4 (Stamper et al., 2001) is shown in yellow on the B7-1 model, and the binding site for PD-1 (Wang et al., 2003) is shown in yellow on the PD-L1 model. Dotted lines are shown to summarize the putative B7-1:PD-L1 binding site. There is partial overlap among the B7-1:CTLA-4, B7-1:PD-L1, and PD-L1:PD-1 interfaces.

Figure 3. Cell adhesion assay shows specific binding of B7-1 to PD-L1

A) Adhesion assay with 300.19-B7-1 cells shows specific binding to PD-L1. The mouse pre-B cell line 300.19 was stably transfected with mB7-1 (300.19-B7-1) and labeled with BCECF. Some cells were pre-incubated with 40 μg/mL of anti-B7-1 mAb or CTLA4-Ig, as indicated. Wells were coated with PD-L1-Ig, PD-L2-Ig, CTLA-4-Ig, or hIgG1-Fc and blocked. Fluorescent 300.19-B7-1 cells were introduced into the wells. Fluorescence was measured before and after washing. Untransfected 300.19 cells showed less than 1% binding to similarly coated wells (not shown). B. Adhesion assay with 300.19 cells stably transfected with mPD-L1 (300.19-PD-L1) demonstrates the differential ability of two anti-PD-L1 mAbs to block the interaction between PD-L1 and PD-1. C. Adhesion assay with 300.19-PD-L1 cells shows specific binding to B7-1. Wells were coated with B7-1-Ig, B7-2-Ig, PD-1-Ig, or hIgG1-Fc and blocked, then some cells were incubated with 20 μg/mL of the indicated mAbs. 300.19-PD-L1 cells were labeled with BCECF, introduced into the wells, and fluorescence was measured before and after washing. Untransfected 300.19 cells showed less than 4% binding (not shown).

Figure 4. Proliferation of CD28/CTLA-4^-/- T cells is reduced by B7-1

CD28/CTLA4^-/- or WT CD4 T cells were stimulated with beads coated with anti-CD3 plus either B7-1-Ig (“B7-1”) or hIgG1Fc (“Ig”) control. A. T cell proliferation was measured by ³H-thymidine incorporation for the last 16 hours of a 64 hour culture. These results are representative of at least 5 independent experiments. B. T cell expansion was measured by flow cytometric analysis of CFSE dilution at 64 hours. These results are representative of 3 independent experiments. C. Cytokines were measured by BD Cytokine Bead Array in supernatants obtained at 48 hrs.

Figure 5. Markers of activation are diminished in CD28/CTLA-4^-/- T cells stimulated with anti-CD3 plus B7-1

CD28/CTLA4^-/- CD4 T cells were cultured with beads coated with anti-CD3 plus either B7-1-Ig (“B7-1”) or hIgG1Fc (“Ig”). T cells were then stained and analyzed by FACS after 24 or 48 hours for A. activation markers and B. costimulatory receptors and ligands. Shaded histogram indicates control cells.

Figure 6. Proliferation of PD-1^-/- T cells is reduced by PD-L1

PD-1^-/- or WT T cells were stimulated with beads coated with anti-CD3 plus either PD-L1-Ig (“PD-L1”) or hIgG1Fc (“Ig”) control. A. T cell proliferation was measured by ³H-thymidine incorporation for the last 16 hours of a 64 hour culture. These results are representative of at least 3 independent experiments. B. T cell expansion was measured by flow cytometric analysis of CFSE dilution at 64 hours. These results are representative of 3 independent experiments. C. Cytokines were measured by BD Cytokine Bead Array in supernatants obtained at 48 hrs.

Figure 7. Specificity of B7-1:PD-L1 interactions

A. WT, CD28/CTLA-4^-/-, or CD28/CTLA-4/PD-L1^-/- CD4 T cells were cultured in wells coated with anti-CD3 antibody (10 μg/mL) plus either PD-L1-Ig (10 μg/mL) or control Ig (10 μg/mL). CFSE dilution was examined by flow cytometry at 48 hours. B. WT, PD-1^-/-, or PD-1/B7-1^-/- CD4 T cells were cultured in wells coated with anti-CD3 antibody (5 μg/mL) plus either PD-L1-Ig (10 μg/mL) or control Ig (10 μg/mL). CFSE dilution was examined by flow cytometry at 48 hours.