Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy

Abstract

The 4-1BB receptor (CDw137), a member of the tumor necrosis factor receptor superfamily, has been shown to costimulate the activation of T cells. Here we show that anti-mouse 4-1BB monoclonal antibodies (mAbs) inhibit thymus-dependent antibody production by B cells. Injection of anti-4-1BB mAbs into mice being immunized with cellular or soluble protein antigens induced long-term anergy of antigen-specific T cells. The immune response to the type II T cell-independent antigen trinintrophenol-conjugated Ficoll, however, was not suppressed. Inhibition of humoral immunity occurred only when anti-4-1BB mAb was given within 1 wk after immunization. Anti-4-1BB inhibition was observed in mice lacking functional CD8(+) T cells, indicating that CD8(+) T cells were not required for the induction of anergy. Analysis of the requirements for the anti-4-1BB-mediated inhibition of humoral immunity revealed that suppression could not be adoptively transferred with T cells from anti-4-1BB-treated mice. Transfer of BALB/c splenic T cells from sheep red blood cell (SRBC)-immunized and anti-4-1BB-treated mice together with normal BALB/c B cells into C.B-17 severe combined immunodeficient mice failed to generate an anti-SRBC response. However, B cells from the SRBC-immunized, anti-4-1BB-treated BALB/c mice, together with normal naive T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by anti-4-1BB mAbs.

Figure 1

Pharmacokinetics of 1D8 mAbs. Serum levels of anti–4-1BB mAbs at varying time points were determined by ELISA. Five 8–12-wk-old female BALB/c mice were given four doses of 200 mg of 1D8 mAb intravenously on days 0, 2, 4, and 6. At the indicated times, sera were collected from each mouse, and the concentration of serum 1D8 was determined by measuring its binding to 4-1BB–huIgG1 fusion protein immobilized on ELISA plates and comparing the results to those obtained from a standard curve generated using purified 1D8 mAb. The figure shows the clearance rates of five mice. Each assay was carried out in triplicate. Serum concentrations at various time points were determined as described. The results are shown as the mean ± SD of three mice.

Figure 2

Inhibition of anti-SRBC and anti-huIgG responses by 1D8 mAb but not by 4-1BB–huIg. Groups of five BALB/c mice were immunized with SRBC or huIgG in 0.5 ml of PBS by tail vein injection. On days 0, 2, 4, and 6, mice were injected with 200 mg of 1D8 (•), 6E9 (♦) in PBS, PBS alone (▪), or the 4-1BB–huIg soluble fusion protein (▴). Serum samples were collected as indicated and assayed in triplicate by ELISA for anti-SRBC–reactive antibodies of all IgG isotypes (A). The results shown are the mean ± SD for each group of five mice. Similarly, mice immunized with huIgG (B) and injected with 1D8 (•), 6E9 (♦) or PBS (▪) were likewise assayed for an anti-huIgG response. At week 7, the mice were challenged with a second dose of SRBC or huIgG, and their anti-SRBC/huIgG titers were followed through week 11.

Figure 2

Inhibition of anti-SRBC and anti-huIgG responses by 1D8 mAb but not by 4-1BB–huIg. Groups of five BALB/c mice were immunized with SRBC or huIgG in 0.5 ml of PBS by tail vein injection. On days 0, 2, 4, and 6, mice were injected with 200 mg of 1D8 (•), 6E9 (♦) in PBS, PBS alone (▪), or the 4-1BB–huIg soluble fusion protein (▴). Serum samples were collected as indicated and assayed in triplicate by ELISA for anti-SRBC–reactive antibodies of all IgG isotypes (A). The results shown are the mean ± SD for each group of five mice. Similarly, mice immunized with huIgG (B) and injected with 1D8 (•), 6E9 (♦) or PBS (▪) were likewise assayed for an anti-huIgG response. At week 7, the mice were challenged with a second dose of SRBC or huIgG, and their anti-SRBC/huIgG titers were followed through week 11.

Figure 3

Anti–4-1BB mAbs block humoral immunity only if given during the first week of immunization. Two groups (five mice per group) of BALB/c mice were immunized on day 0 with SRBC as described above. One group of mice received anti–4-1BB mAb 1D8 as described in the Fig. 1 legend on day 0. Injection of anti–4-1BB in the second group was carried out as for the first group, but injection was delayed until day 10. When anti–4-1BB mAbs were injected at the time of immunization, the mice failed to generate either a primary or secondary humoral response to SRBC (♦). However, if the injection of anti–4-1BB mAbs was delayed by 10 d (•), there was no inhibition of either a primary or secondary response to SRBC antigens.

Figure 4

Anti–4-1BB mAbs fail to block type II TI humoral responses. Three groups (five mice per group) of 8–12-wk-old female BALB/c mice were injected intravenously with 50 mg of TNP–Ficoll on days 0 and 14. In addition, one group was injected intravenously with 200 mg of 3E1 (♦) on day 0; a second group was injected intravenously with 200 mg of 6E9 (▴), and the last group was injected with PBS (•).

Figure 5

Adoptively transferred T cells from 3E1-treated, SRBC-immunized mice fail to support normal anti-SRBC responses in C.B-17 SCID mice. BALB/c mice were immunized with SRBC and injected intravenously with 200 mg of 3E1. 2 wk later, the mice were challenged with SRBC, and 3 wk thereafter, the mice were killed and 2–4 × 10⁷ splenic T or B cells were injected intravenously into recipient C.B-17 SCID mice. SCID mice receiving T cells from anti–4-1BB–treated mice were injected with an equal number of B cells from naive BALB/c mice (•). Mice that received B cells from anti–4-1BB–injected mice were injected with an equal number of T cells from naive mice (▪). A third group of SCID mice received T and B cells from naive mice (♦). At weeks 5 and 9 after transfer, SCID mice were challenged with SRBC. At week 9, mice were immunized with an unrelated antigen, keyhole limpet hemocyanin (KLH). Serum samples were collected weekly and assayed for anti-SRBC titer. Results are expressed as the mean ± SD from each treatment group. KLH responses were normal (▴).