Unconventional ligand activation of herpesvirus entry mediator signals cell survival

Abstract

The herpesvirus entry mediator (HVEM; TNFRSF14) activates NF-kappaB through the canonical TNF-related cytokine LIGHT, serving as a costimulatory pathway during activation of T cells. HVEM also functions as a ligand for the Ig superfamily members B and T lymphocyte attenuator (BTLA) and CD160, both of which limit inflammatory responses initiated by T cells. Emerging evidence indicates BTLA also promotes T cell survival, but its structural differences from LIGHT intimate BTLA is unlikely to function as an activator of HVEM. We demonstrate here that BTLA, CD160, and herpes simplex virus envelope glycoprotein D (gD) function as activating ligands for HVEM, promoting NF-kappaB activation and cell survival. Membrane-expressed BTLA and CD160, as well as soluble dimeric receptor surrogates BTLA-Fc and gD-Fc specifically activated HVEM-dependent NF-kappaB. BTLA and CD160 engagement induced recruitment of TNF receptor-associated factor 2 (TRAF2), but not TRAF3, to HVEM that specifically activated the RelA but not the RelB form of NF-kappaB in a mucosal epithelial tumor cell line. Moreover, Btla(-/-) T cells survived poorly following activation but were rescued with BTLA-Fc, indicating HVEM-BTLA bidirectional signaling may serve as a critical cell-survival system for lymphoid and epithelial cells.

Fig. 1.

BTLA activates HVEM-dependent NF-κB. (A) LIGHT activates HVEM signaling. NF-κB-dependent luciferase reporter vector was transfected into 293T-HVEM and 293T-HVEM-BTLA coexpressing cells. LIGHT-expressing EL4 cells (EL4-LIGHT) were cocultured with 239T-HVEM cells for 24 h and then assessed for luciferase activity in cell lysates (Left). Dose–response: EL4-LIGHT cells or soluble LIGHTt66 were incubated at the indicated ratio or concentration with 293T-HVEM, and HVEM signaling was assessed by using a luciferase reporter assay (Right). (B) 293T-BTLA or 293T cells were cocultured with either 293T-HVEM cells (1:1 ratio) transfected with luciferase NF-κB reporter or mock-transfected cells. NF-κB activation was measured as in A. (C) 293T-BTLA cells were cocultured at the indicated ratios with transfected 293T cells expressing HVEM-Y61A mutant or WT HVEM, along with luciferase reporter (Left). EL4-LIGHT cells cocultured with 293T-HVEM-Y61A cells expressing luciferase reporter (Right). (D) Mouse anti-human BTLA mAb (J168 clone) was added to 293T-BTLA cells cocultured with 293T-HVEM cells transfected with NF-κB reporter plasmids, and luciferase activity was measured 24 h later. (E) 293T cells transfected with HVEM and NF-κB reporter plasmids were cocultured with 293T-BTLA cells with LIGHTt66 as indicated. (F) Graded concentrations of BTLA-Fc or LTβR-Fc were incubated with 293T-HVEM cells transfected with NF-κB reporter plasmids (Left). Anti-Fc IgG was added to BTLA-Fc or LTβR-Fc and incubated with 293T-HVEM cells (Right). (G) Assessment of BTLA oligomerization by FRET. 293T cells transfected individually or cotransfected with BTLA-CFP and BTLA-DsRed were detected at the CFP channel (excitation at 405 nm, emission at 425–475 nm), DsRed channel (excitation at 488 nm, emission at 562–588 nm), and FRET channel (excitation at 405 nm, emission at 564–606 nm). Note that at high levels of CFP and DsRed expression, with reference to the HVEM-CFP and BTLA-DsRed coexpressing cells (Upper Left), there was minimal CFP spectral overlap and DsRed coexcitation detected in the FRET channel (Lower Left). (Right) An overlay of the FRET channel.

Fig. 2.

CD160 activates HVEM-dependent NF-κB. (A) CD160-mediated HVEM signaling. CD160-expressing EL4 cells (EL4-CD160) were cocultured with 293T-HVEM cells, and NF-κB reporter luciferase assay was performed after 24 h of incubation. (B) CD160-binding site on HVEM. HVEM-Y61A-Fc fusion protein was incubated with EL4-CD160 cells, and binding was assessed by flow cytometry. (C) Specificity of BTLA-mediated HVEM signaling. EL4-CD160 cells were cocultured with 293T-HVEM or HVEM-Y61A cells transfected with NF-κB reporter plasmids at the indicated ratios. (D) Herpes simplex virus gD-mediated HVEM signaling. Graded concentrations (0.3–20 μg/mL) of gD-Fc were incubated with 293T-HVEM cells transfected with NF-κB reporter plasmids. Rabbit IgG (20 μg/mL) was used as a negative control.

Fig. 3.

Activation of HVEM signaling by BTLA and CD160. (A) HVEM-dependent RelA nuclear translocation in human HT29 cells. HT29 cells were incubated with agonistic antibodies to HVEM or LTβR, lysed, nuclear fractions isolated, and Western blotted for NF-κB RelA (Top) or RelB (Middle), or the cytosolic fraction for the processing of p100 to p52 (Bottom). (B) HVEM-dependent TRAF2 recruitment in HT29 cells. HT29 cells were stimulated with BTLA-Fc (BTLA-Fc and anti-Fc antibody, each at 5 μg/mL, lanes 1 and 2) or cocultured with EL4-CD160 cells (1:1 ratio) for 60 min (lane 4), lysed and immunoprecipitated with anti-HVEM, and Western blotted for TRAF2 or HVEM. LTβR-Fc and EL4 cells (lanes 1 and 3) were used as negative controls. (C) BTLA- and CD160-mediated, HVEM-dependent RelA nuclear translocation in HT29 cells. Immunocytochemical staining for RelA translocation was performed on HT29 cells following incubation with anti-HVEM antibody (5 μg/mL), goat IgG (5 μg/mL), BTLA-Fc (15 μg/mL), mouse LTβR-Fc (15 μg/mL), EL4-CD160 (1:5 ratio), or EL4 cells (1:5 ratio). (Magnification: 400×.)

Fig. 4.

BTLA-mediated HVEM signaling enhances T cell survival. (A) BTLA-mediated T cell proliferation. (Left) WT or Btla^−/− naïve CD4 or CD8 T cells cultured in vitro with plate-bound anti-CD3ε (1 μg/mL) and soluble anti-CD28 mAb (0.5 μg/mL) with IgG control or BTLA-Fc (10 μg/mL). CFSE staining was performed at day 3. (Right) The level of T cell proliferation under each condition was calculated as the ratio of dividing cells to nondividing cells. (B) Effect of BTLA-HVEM transinteractions on CD4 T cell survival. (Left) Btla^−/− naïve CD4 T cells cultured in vitro as in A. CFSE and 7-AAD stainings were performed at day 5. (Right) The level of T cell survival under each condition was calculated as the ratio of viable divided cells to dead divided cells. The experiment shown is representative of 3 similarly conducted experiments. (C) BTLA-mediated, HVEM-dependent NF-κB activation in CD4 T cells. CD4⁺ T cells were isolated from Hvem^−/− or Btla^−/− mice and treated with 20 μg/mL anti-HVEM antibody (clone 14C1.1) or 15 μg/mL mBTLA-Fc for 2 h. Immunohistochemical staining was performed as described above. The percentage of cells with specific RelA nuclear translocation was determined by subtracting nonspecific staining in WT CD4⁺ T cells.