4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses

Abstract

The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

Figure 1

Anti–4-1BB mAbs block 4-1BBL binding to activated DO.11.10 T cells. T cells were activated as described above. Cells were then incubated with dilutions of anti–4-1BB mAbs for 30 min before the addition of 4-1BBL–CD8 fusion protein at 1 μg/ml and the cells were incubated for an additional 30 min. Cells were washed twice in PBS, incubated with PE-conjugated anti-CD8 mAb, and assayed by FACS^® for ligand binding.

Figure 2

Anti–4-1BB mAbs costimulate anti-CD3–induced T cell proliferation. BALB/c T cells were cultured for 72 h at 2 × 10⁵/well in 96-well flat-bottomed microtiter plates with the indicated concentrations of 145.2C11 with PBS control (-•-) or with the following: anti–4-1BB mAbs 15B9 (-□-), 1D8 (--○--), 21E5 (--▴--), 3B8 (...•...), 3H3 (.-♦.-), and 3E1 (..-▿-..), each at 10 μg/ ml. Results are expressed as the mean ± SD of triplicate wells.

Figure 3

(A) Anti–4-1BB mAbs differentially costimulate in vitro proliferation of CD4⁺ and CD8⁺ T cell subsets. Purified resting CD4⁺ or CD8⁺ BALB/c splenic T cells were cultured for 72 h in 96-well microtiter plates at 1 × 10⁵/well with 0.5% APC ± anti– 4-1BB mAb at 10 μg/ml and the indicated concentration of anti-CD3 mAb. Cells were pulsed with 0.5 μCi/well [³H]thymidine for the final 12 h of culture, harvested, and counted by liquid scintillation spectroscopy. (B) 4-1BB–Ig fusion protein inhibits anti-CD3– induced T cell proliferation. CD4⁺ and CD8⁺ T cells with APC were stimulated with anti-CD3 mAb at 300 ng/ml in the absence or presence of soluble 4-1BB–Ig fusion protein. Cells were pulsed during the final 12 h of culture with [³H]thymidine, harvested, and counted by liquid scintillation spectroscopy.

Figure 3

(A) Anti–4-1BB mAbs differentially costimulate in vitro proliferation of CD4⁺ and CD8⁺ T cell subsets. Purified resting CD4⁺ or CD8⁺ BALB/c splenic T cells were cultured for 72 h in 96-well microtiter plates at 1 × 10⁵/well with 0.5% APC ± anti– 4-1BB mAb at 10 μg/ml and the indicated concentration of anti-CD3 mAb. Cells were pulsed with 0.5 μCi/well [³H]thymidine for the final 12 h of culture, harvested, and counted by liquid scintillation spectroscopy. (B) 4-1BB–Ig fusion protein inhibits anti-CD3– induced T cell proliferation. CD4⁺ and CD8⁺ T cells with APC were stimulated with anti-CD3 mAb at 300 ng/ml in the absence or presence of soluble 4-1BB–Ig fusion protein. Cells were pulsed during the final 12 h of culture with [³H]thymidine, harvested, and counted by liquid scintillation spectroscopy.

Figure 3

(A) Anti–4-1BB mAbs differentially costimulate in vitro proliferation of CD4⁺ and CD8⁺ T cell subsets. Purified resting CD4⁺ or CD8⁺ BALB/c splenic T cells were cultured for 72 h in 96-well microtiter plates at 1 × 10⁵/well with 0.5% APC ± anti– 4-1BB mAb at 10 μg/ml and the indicated concentration of anti-CD3 mAb. Cells were pulsed with 0.5 μCi/well [³H]thymidine for the final 12 h of culture, harvested, and counted by liquid scintillation spectroscopy. (B) 4-1BB–Ig fusion protein inhibits anti-CD3– induced T cell proliferation. CD4⁺ and CD8⁺ T cells with APC were stimulated with anti-CD3 mAb at 300 ng/ml in the absence or presence of soluble 4-1BB–Ig fusion protein. Cells were pulsed during the final 12 h of culture with [³H]thymidine, harvested, and counted by liquid scintillation spectroscopy.

Figure 4

Anti-CD28 (3N7) and anti-4-1BB (3H3 and 3E1) mAbs costimulate opposite T cell subsets. Purified CD4⁺ and CD8⁺ resting T cells were costimulated with either anti-CD3 and anti-CD28 or anti-CD3 and anti–4-1BB mAbs for 24, 48, or 72 h. Cultures were pulsed with [³H]thymidine for the final 12 h of culture.

Figure 5

Cross-linking 4-1BB initiates distinct patterns and kinetics of protein tyrosine phosphorylation in CD4⁺ and CD8⁺ T cell subsets. BALB/c splenocytes were activated as described earlier. CD4⁺ (A) and CD8⁺ (B) subsets were incubated with 10 ng/ml of anti-CD3, anti–4-1BB (3E1), or both at 10 ng/ml each. After 15-min incubation at room temperature, the cells were equilibrated to 37°C and cross-linked with 50 ng/ml of RG7, a mouse anti–rat IgG that cross-reacts with hamster IgG. Where both anti-CD3 and anti-4-1BB were added together, RG7 was added at 100 ng/ml. Untreated (lane 1), 1 min (lane 2), 3 min (lane 3), 9 min (lane 4).

Figure 5

Cross-linking 4-1BB initiates distinct patterns and kinetics of protein tyrosine phosphorylation in CD4⁺ and CD8⁺ T cell subsets. BALB/c splenocytes were activated as described earlier. CD4⁺ (A) and CD8⁺ (B) subsets were incubated with 10 ng/ml of anti-CD3, anti–4-1BB (3E1), or both at 10 ng/ml each. After 15-min incubation at room temperature, the cells were equilibrated to 37°C and cross-linked with 50 ng/ml of RG7, a mouse anti–rat IgG that cross-reacts with hamster IgG. Where both anti-CD3 and anti-4-1BB were added together, RG7 was added at 100 ng/ml. Untreated (lane 1), 1 min (lane 2), 3 min (lane 3), 9 min (lane 4).

Figure 6

(A) Anti–4-1BB mAbs enhance or inhibit in vivo generation of CTL responses during GVHD responses. GVHD was established by intravenous injection of 5 × 10⁷ C57BL/6 (H-2^b) splenocytes into DBA/2 (H-2^bd) mice as described in the Materials and Methods. The administration of mAbs was carried out as described earlier. On day 10, spleens were collected from GVHD mice and single cell suspensions prepared. CTL activity in this population was measured directly by assaying CTL-mediated killing of ⁵¹Cr-labeled P815 cells. (B) Total splenocyte levels are markedly reduced in mice receiving anti–4-1BB mAbs. The total number of viable splenocytes obtained from groups of three spleens pooled from each treatment group was assessed by microscopy and trypan blue exclusion. (C) Percentages of viable CD8⁺ T cells were determined for each treatment group using FITC-conjugated anti-murine CD8 mAb (PharMingen) and flow cytometry.

Figure 6

(A) Anti–4-1BB mAbs enhance or inhibit in vivo generation of CTL responses during GVHD responses. GVHD was established by intravenous injection of 5 × 10⁷ C57BL/6 (H-2^b) splenocytes into DBA/2 (H-2^bd) mice as described in the Materials and Methods. The administration of mAbs was carried out as described earlier. On day 10, spleens were collected from GVHD mice and single cell suspensions prepared. CTL activity in this population was measured directly by assaying CTL-mediated killing of ⁵¹Cr-labeled P815 cells. (B) Total splenocyte levels are markedly reduced in mice receiving anti–4-1BB mAbs. The total number of viable splenocytes obtained from groups of three spleens pooled from each treatment group was assessed by microscopy and trypan blue exclusion. (C) Percentages of viable CD8⁺ T cells were determined for each treatment group using FITC-conjugated anti-murine CD8 mAb (PharMingen) and flow cytometry.

Figure 6

(A) Anti–4-1BB mAbs enhance or inhibit in vivo generation of CTL responses during GVHD responses. GVHD was established by intravenous injection of 5 × 10⁷ C57BL/6 (H-2^b) splenocytes into DBA/2 (H-2^bd) mice as described in the Materials and Methods. The administration of mAbs was carried out as described earlier. On day 10, spleens were collected from GVHD mice and single cell suspensions prepared. CTL activity in this population was measured directly by assaying CTL-mediated killing of ⁵¹Cr-labeled P815 cells. (B) Total splenocyte levels are markedly reduced in mice receiving anti–4-1BB mAbs. The total number of viable splenocytes obtained from groups of three spleens pooled from each treatment group was assessed by microscopy and trypan blue exclusion. (C) Percentages of viable CD8⁺ T cells were determined for each treatment group using FITC-conjugated anti-murine CD8 mAb (PharMingen) and flow cytometry.

Figure 7

Costimulation of CD8⁺ T cells with anti-4-1BB but not anti-CD28 leads to IFN-γ production. Unseparated and CD4⁺ or CD8⁺ resting T cells were stimulated with anti-CD3 (•) or anti-CD3 and either anti-CD28 (▪) or the anti–4-1BB mAb 3H3 (▴). Activated T cell culture SN were collected at 24, 48, 72, and 96 h and assayed by ELISA for IL-2, IL-4, IL-10, and IFN-γ.