Evolutionarily divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway

Abstract

The herpesvirus entry mediator (HVEM), a member of the TNF receptor (TNFR) superfamily, can act as a molecular switch that modulates T cell activation by propagating positive signals from the TNF-related ligand LIGHT (TNFR superfamily 14), or inhibitory signals through the Ig superfamily member B and T lymphocyte attenuator (BTLA). Competitive binding analysis and mutagenesis reveals a unique BTLA binding site centered on a critical lysine residue in cysteine-rich domain 1 of HVEM. The BTLA binding site on HVEM overlaps with the binding site for the herpes simplex virus 1 envelope glycoprotein D, but is distinct from where LIGHT binds, yet glycoprotein D inhibits the binding of both ligands, potentially nullifying the pathway. The binding site on HVEM for BTLA is conserved in the orphan TNFR, UL144, present in human CMV. UL144 binds BTLA, but not LIGHT, and inhibits T cell proliferation, selectively mimicking the inhibitory cosignaling function of HVEM. The demonstration that distinct herpesviruses target the HVEM-BTLA cosignaling pathway suggests the importance of this pathway in regulating T cell activation during host defenses.

Fig. 1.

Topography of BTLA, LIGHT, and gD binding to HVEM. (A and B) Dermal fibroblasts (2 × 10⁴) stably expressing hBTLA or mBTLA were incubated with graded amounts of hHVEM (A) or mHVEM-Fc (B)in50 μl of binding buffer for 60 min, washed, and stained with phycoerythrin (PE)-conjugated goat anti-human IgG, and fluorescence was detected by flow cytometry. MFI, mean fluorescence intensity. (C) HEK293 cells transfected with hHVEM or hBTLA expression plasmids were incubated with graded concentrations of either hBTLA-Fc or hHVEM-Fc as described above. (D) HEK293 cells transfected with hHVEM were incubated with graded concentrations of hLIGHTt66 (FLAG epitope), and bound ligand was detected with goat anti-FLAG-PE. (E) For competition binding assay, graded concentrations of LIGHTt66 were incubated with hHVEM-expressing HEK293 cells in the presence of 25 μg/ml of BTLA-Fc. (F) HEK293 cells stably transfected with mHVEM or hLIGHT-EL4 cells were incubated with graded concentrations of hLIGHTt66 in the presence of mBTLA-T (1.4 μg/ml) or mHVEM-Fc (2 μg/ml; detected with goat anti-human IgG-PE). Control for nonspecific staining with mBTLA-T was based on 293T cells. (G) Graded concentrations of soluble gD (gDtΔ90-99) was used to compete for mBTLA-T (1.4 μg/ml) binding to mHVEM-HEK293 cells or mHVEM-Fc (2 μg/ml) to hLIGHT-EL4 cells as in F. (H) Graded concentrations of hBTLA-Fc or mouse anti-LIGHT Omniclone were incubated with hLIGHT-expressing EL4 cells in the presence of 6 μg/ml of biotinylated hHVEM-Fc. The parental EL4 cells were used as negative control (data not shown).

Fig. 2.

Binding analyses of BTLA-Fc, soluble LIGHT, and gD to HVEM mutants. (A) Location of site-directed mutations in the structure of hHVEM (Protein Data Bank ID code 1JMA, Swiss-Pdb Viewer). The α-carbon backbone of hHVEM with side chains of mutated amino acids is shown. (Left) CRD1, gray; CRD2, purple; CRD3, blue; cysteine residues, yellow. (Right) Mutated amino acid residues: Arg-62 (R62), Lys-64 (K64), and Glu-65 (E65), red; Tyr-47 (Y47), Ser-58, (S58), Y61, E76, and R113 (green); some side chains are not shown for clarity. (B) 293T cells transfected with the expression plasmids of WT hHVEM or individual substitution mutants were stained with anti-HVEM antibody, hBTLA-Fc (100 μg/ml), soluble hLIGHT (400 nM), and gD-Fc (0.4 μg/ml). Binding analyses were performed by flow cytometry. Binding profiles of HVEM ligands to HVEM-293T cells (dark lines) and mock-transfected 293T parental cells (light lines) are shown.

Fig. 3.

Sequence alignment of HVEM and UL144 CRD1. hHVEM and mHVEM CRD1 alignment and representative sequences from the five subtypes of UL144 were aligned with hHVEM (clustalw, PAM350 series, macvector 7). The asterisk denotes Lys-64 in hHVEM that is critical for binding BTLA. The GenBank accession nos. for the UL144 sequences are group 1A, DQ100368; 1B, AF085003; 1C, AF179208; 2, DQ100369; and 3, AF084982.

Fig. 4.

Specific binding between UL144 and BTLA. (A) Graded concentrations of hBTLA-Fc were incubated with UL144-transfected 293T cells (groups 1A, 1B, 1C, 2, and 3) and Fiala (type 3). Histograms show transfected cells stained with hBTLA-Fc at 200 μg/ml (dark lines) or mock-transfected 293T cells as a negative control (light lines). The specific fluorescence of cells stained with graded concentrations (25, 50, 100, and 200 μg/ml) of hBTLA-Fc is shown. (B) Competition binding assay for hBTLA-Fc binding to UL144(1C). Graded concentrations of hHVEM-Fc were added to UL144(1C)-transfected 293T cells in the presence of hBTLA-Fc (50 μg/ml). Specific BTLA-Fc binding was detected with phycoerythrin-conjugated goat anti-human IgG after correction for nonspecific HVEM-Fc staining was determined at each concentration of HVEM-Fc in the absence of BTLA-Fc.

Fig. 5.

Inhibition of T cell proliferation by HVEM-Fc and UL144-Fc. Purified CD4⁺ T cells from human peripheral blood were cultured in 96-well plates at 4 × 10⁵ cells per well and stimulated with graded concentrations of plate-bound anti-CD3 and 1 μg/ml soluble anti-CD28 in the presence of 10 μg/ml human IgG, hLTβR-Fc, UL144:Fc (Fiala, group 3), or hHVEM:Fc immobilized with anti-human IgG1Fc antibody adsorbed to plastic. (B) Graded amounts of hIgG, UL144-Fc(Fiala), or HVEM-Fc were incubated with anti-human IgG1Fc antibody adsorbed to plastic. Wells were coated with 10 μg/ml anti-CD3, and anti-CD28 was added to stimulate purified CD4⁺ T cells. Results represent mean values ± SEM of triplicate wells and are representative of three experiments.