Fusobacterium nucleatum CbpF Mediates Inhibition of T Cell Function Through CEACAM1 Activation

Abstract

F. nucleatum is an anaerobic bacterium that is associated with several tumor entities and promotes tumorigenesis. Recent evidence suggests that F. nucleatum binds the inhibitory receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) via the trimeric autotransporter adhesin CbpF. However, whether this binding is functional or whether other fusobacterial trimeric autotransporter adhesins are involved in CEACAM1 activation is unknown. In this study, using F. nucleatum mutants lacking the type 5c trimeric autotransporter adhesins fvcA (CbpF), fvcB, fvcC, and fvcD, we show that F. nucleatum CbpF binds and activates CEACAM1 and also binds carcinoembryonic antigen (CEA), a tumor-associated protein. We further find that CEACAM antibodies directed against the CEACAM N-terminal domain block the CbpF-CEACAM1 interaction. In functional assays, we demonstrate CbpF-dependent inhibition of CD4⁺ T cell response. Thus, we characterize an immune evasion mechanism in which F. nucleatum uses its surface protein CbpF to inhibit T cell function by activating CEACAM1.

Keywords: CEA; CEACAM1; CbpF; F. nucleatum; trimeric autotransporter adhesins.

Figure 1

Fnn specifically binds to the N-terminal domain of human CEACAM1. (A) 721.221 cells coated with FITC-labeled Fnn were stained with 2 μg of human CEACAM1-Ig, ∆N CEACAM1-Ig that lacks the N-terminal domain, CEACAM3-Ig, CEA-Ig, CEACAM6-Ig, CEACAM8-Ig, mouse CEACAM1-Ig (mCEACAM1-Ig) or rhesus macaque CEACAM1-Ig (macCEACAM1-Ig). Filled grey histograms represent staining with secondary antibody only. Representative histograms from three independent repeats are shown. (B) CEACAM1-Ig and (C) CEA-Ig staining quantified and shown as fold increase in MFI relative to secondary only. Bars represent means of three independent experiments ± SD. Statistical significance was assessed using a two-tailed unpaired t test. *p ≤ 0.05.

Figure 2

The Fnn ∆CbpF mutant fails to bind CEACAM1. (A) FITC-labeled Fnn and single mutants of the trimeric autotransporter adhesins fvcA (cbpF), fvcB, fvcC, fvcD, as well as a mutant lacking all four adhesins (Fnn fvcABCD) were stained with 3 μg of human CEACAM1-Ig. 721.221 cells were used as carrier cells. Filled gray histograms represent staining with secondary antibody only. One representative staining out of two is shown. (B) Schematic representation of the BW reporter assay. BW5147 cells stably transfected with human CEACAM1 fused to the transmembrane and cytoplasmic domain of mouse CD3ζ chain (chimeric recptor-CD3ζ) are incubated with above-mentioned Fnn strains. Activation of CEACAM1 by a ligand results in mIL-2 quantified by ELISA. (C) CEACAM1-expressing BW cells were incubated with Fnn strains at a ratio of 1:300. Mouse IL-2 in the supernatants was determined by ELISA 48 hours later. Bars represent means of three independent experiments performed in triplicates ± SD. Fnn bacteria were arbitrarily set to 1 and other values were normalized accordingly. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test. **p ≤ 0.01.

Figure 3

Binding of CEACAM1 to Fnn is blocked by anti-CEACAM1 18/20 and 6G5J. (A) FITC-labeled Fnn were stained with 2 μg of human CEACAM1-Ig preincubated with 1 µg of anti-PVR or different anti-CEACAM1 antibodies. 721.221 cells were used as carrier cells. Filled grey histograms represent staining with secondary antibody only. (B) Quantification of three independent experiments. Bars represent means of three independent experiments ± SD. Values obtained without blocking antibody were arbitrarily set to 1 and all other values were normalized accordingly. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test. *p ≤ 0.05; ***p ≤ 0.001.

Figure 4

Fnn inhibits CD4⁺ T cell IFN-γ secretion by activating CEACAM1. (A) Schematic representation of the redirected cytokine assay. Mouse mastocytoma P815 cells expressing FcγR were coated with anti-CD3 to enable activation of T cells and subsequent IFN-γ secretion. Cells were incubated with Fnn or the Fnn ∆CbpF mutant at a E:T ratio of 1:600 and IFN-γ secretion was quantified by ELISA after 48 hours of incubation. (B) Flow cytometry staining of primary IL-2 activated human CD4⁺ T cells with anti-CEACAM1 and anti-CD4. Filled grey histograms represent staining with secondary antibody only. (C) Quantification of IFN-γ secretion determined by ELISA. Bars represent means of triplicates ± SD. The graphs represent data collected from two independent experiments. Statistical significance was assessed using a two-tailed unpaired t test. ***p ≤ 0.001. NS, not significant.