Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus

Abstract

B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.

Fig. 1

Anti-CD3ε T cell proliferation is unaffected by co-immobilized anti-B and T lymphocyte attenuator (BTLA) reagents (a): cross-linked herpesvirus entry mediator (HVEM-Fc) and some anti-mBTLA monoclonal antibodies (mAbs) suppress this proliferation (b). Mouse CD4⁺ T cells were purified from C57BL/6 mouse splenocytes by AutoMACS-negative selection (Miltenyi Biotec). In schematic (a) 100 000 T cells in a U-bottomed 96-well plate were activated in vitro by plate-bound hamster anti-mouse CD3ε (clone 145-2C11 at 0·1 µg per plate) for 72 h and [³H]-labelled tritium was added to the cell culture medium for the last 18 h; the test reagent was co-immobilized with the activating stimulus at the indicated amounts. In the cross-linked plate in schematic (b), 1 µg per well of a polyclonal goat anti-mFc reagent was added just prior to the activating stimulus and the test reagents were added for the last 18 h at the indicated amounts. Cells were harvested onto a filter after 72 h of stimulation and radioactivity was assessed as a measure of cell proliferation. Analysis of secreted cytokines by multi-analyte profiling showed that secreted levels of interferon-γ correlated well with cell proliferation (data not shown). Epitope grouping experiments using BIACore showed that the anti-mBTLA mAbs that inhibited T cell proliferation grouped to the same epitope on the mBTLA molecule (see Fig. S3).

Fig. 2

B cell proliferation is not inhibited by co-immobilized anti-B and T lymphocyte attenuator (BTLA) reagents. Mouse B cells were purified from C57BL/6 mouse splenocytes by AutoMACS-negative selection (Miltenyi Biotec). Purified B cells (10⁵) were incubated in duplicate in 96-well flat-bottomed plates in RPMI-1640 (Invitrogen, Inc.) with 10% heat inactivated fetal bovine serum (Gibco). Cells were stimulated with 2 µg/ml of lipopolysaccaride (List Biological Laboratories, Inc.) or 1 µg/ml of anti-CD40 monoclonal antibody (mAb) (BD Biosciences, Inc.) plus 2 µg/ml of goat F(ab')₂ anti-mouse immunoglobulin M (IgM) (Jackson ImmunoResearch, West Grove, PA, USA) in the presence of various concentrations of anti-BTLA reagent or murine herpesvirus entry mediator (mHVEM-Fc) protein. Cells were incubated for 72 h at 37°C and 5% CO₂ and pulsed with radioactive [³H] thymidine for the last 18 h to assess proliferation.

Fig. 3

Bead-absorbed αCD3ε causes T cell proliferation: only anti-B and T lymphocyte attenuator (BTLA) reagents in the cis, and not the trans, format relative to the activating stimulus inhibit this proliferation. Mouse CD4⁺ T cells were purified from C57BL/6 mouse splenocytes by AutoMACS-negative selection (Miltenyi Biotec). In a U-bottomed 96-well plate, 100 000 T cells were activated in vitro by bead-absorbed anti-mouse CD3ε[clone 145-2C11] at 0·1 µg per 10⁶ cells on tosyl activated 4·5 µM beads (Dynal) for 72 h and [³H]-labelled tritium was added to the cell culture medium for the last 18 h: the test reagent was either co-immobilized with the activating stimulus (cis format) or on a separate bead (trans format) at the indicated amounts. Cells were harvested onto a filter after 72 h of stimulation and radioactivity was assessed as a measure of cell proliferation. Analysis of secreted cytokines by multi-analyte profiling showed that secreted levels of interferon-γ correlated well with cell proliferation (data not shown).

Fig. 4

Anti-B and T lymphocyte attenuator (BTLA) monoclonal antibodies (mAbs) do not affect in vivo activation of DO11.10 T cells transferred to syngeneic mice; 20 × 10⁶ DO11.10 splenocytes were adoptively transferred into BALB/c recipients. The next day mice were treated intraperitoneally with 15 mg/kg of anti-BTLA reagent or control reagent (5 mg/kg for mCTLA4-hFc). Three h after protein treatment animals were administered 10 µg of biotin-labelled rat amIL-2 (clone JES6-5H4) to capture secreted interleukin (IL)-2 (Finkelman et al., Int. Immunol., 11, 1999). Mice were then injected in the footpad with 100 µg of ovalbumin protein to activate the monoclonal population of transferred DO11.10 T cells. The mice were rested for 18 h before exsanguination and then serum IL-2 was detected by enzyme-linled immunosorbent assay. A double asterisk indicates no detectable IL-2.

Fig. 5

Bead-absorbed αCD3ε causes T cell proliferation: Only anti-B and T lymphocyte attenuator (BTLA) reagents in the cis, and not the trans, format relative to the activating stimulus inhibit this proliferation. Shown above is a hypothetical schematic that models a possible explanation for this observation. (a) Bead-absorbed αCD3ε clusters and activates the T cell receptor (TCR) and the cell proliferates. Anti-BTLA reagents on the same bead can localize BTLA to synapse, bringing the BTLA molecule in juxtaposition to the TCR. This allows the activation of BTLA to recruit the SHP-2 phosphatase adjacent to the intracellular domain of the TCR, resulting in dephosphorylation of the TCR complex and countering T cell proliferation. (b) Bead-absorbed αCD3ε clusters and activates the TCR and the cell proliferates. An anti-BTLA reagent on a different bead is dislocated physically from the immunological synapse and is unable to localize BTLA to the synapse. Hence, the SHP-2 phosphatase cannot be recruited adjacent to the intracellular domain of the TCR and T cell proliferation is unaffected.