Figure 6.
Treatment with antagonistic anti–TIM-1 mAb does not affect Tcon-cell expansion or proliferation in vivo or in vitro. (A) TIM-1 blockade does not affect murine T-cell proliferation in MLR cell culture. Allogeneic responder Tcon cells were labeled with CellTrace violet and stimulated with irradiated splenocytes as antigen-presenting cells (APC) for 7 days at a ratio of 1:2 in the presence or absence of anti–TIM-1 mAb (3D10). In separate cultures, responder T cells were stimulated with anti-CD3/CD28 Dynabeads at 1:2 ratio in the presence or absence of anti–TIM-1 mAb (3D10; 20 μg/mL). Pooled data from 2 independent experiments are shown (n = 8 per group). (B) TIM-1 blockade does not affect human T cell proliferation in MLR cell culture. Monocyte-derived immature dendritic cells were generated from PBMCs. The dendritic cells were irradiated (30 Gy) and cocultured with allogeneic responder PBMCs for 7 days at a ratio of 1:5 in the presence or absence of antagonistic anti–TIM-1 mAb (1D12; 20 μg/mL). In separate cultures, responder PBMCs were stimulated with anti-CD3/CD28 Dynabeads (ThermoFisher) at a 1:1 ratio in the presence of anti–TIM-1 mAb or isotype control. Pooled data from 3 independent experiments are shown (n = 12 donors per group) for the MLR and (n = 6 donors per group) for anti-CD3/CD28 stimulation with statistical evaluation using the paired Student t test. (C) Representative serial bioluminescence images at days +3, +5, +7, +14, and +23 after transplantation of from Luc+ B6 donor mice to assess the proliferative capacity of T cells in the presence of anti–TIM-1 mAb (3D10). (D) Bioluminescence signal intensity time course. Allogeneic transplanted BALB/c recipient mice after injection of luc+ Tcon cells alone (▼) or Tcon cells and anti–TIM-1 mAb (3D10) (●). Mice that were injected only with BM (☐) were used as controls. Error bars indicate SEM. (E) Cytokine levels in the supernatant of splenocytes stimulated ex vivo with anti-CD3/CD28 Dynabeads beads, as assessed by a multiplex assay (Luminex). Splenocytes were isolated at day 9 after transplantation from spleens of BALB/c recipient mice treated with isotype control (white bars) or anti–TIM-1 mAb (3D10) (black bars). Shown are pooled data from 3 independent experiments. For statistical analysis, the 2-tailed Student t test was used (*P ≤ .05, **P ≤ .01, ***P ≤ .01). Error bars indicate SEM.

Treatment with antagonistic anti–TIM-1 mAb does not affect Tcon-cell expansion or proliferation in vivo or in vitro. (A) TIM-1 blockade does not affect murine T-cell proliferation in MLR cell culture. Allogeneic responder Tcon cells were labeled with CellTrace violet and stimulated with irradiated splenocytes as antigen-presenting cells (APC) for 7 days at a ratio of 1:2 in the presence or absence of anti–TIM-1 mAb (3D10). In separate cultures, responder T cells were stimulated with anti-CD3/CD28 Dynabeads at 1:2 ratio in the presence or absence of anti–TIM-1 mAb (3D10; 20 μg/mL). Pooled data from 2 independent experiments are shown (n = 8 per group). (B) TIM-1 blockade does not affect human T cell proliferation in MLR cell culture. Monocyte-derived immature dendritic cells were generated from PBMCs. The dendritic cells were irradiated (30 Gy) and cocultured with allogeneic responder PBMCs for 7 days at a ratio of 1:5 in the presence or absence of antagonistic anti–TIM-1 mAb (1D12; 20 μg/mL). In separate cultures, responder PBMCs were stimulated with anti-CD3/CD28 Dynabeads (ThermoFisher) at a 1:1 ratio in the presence of anti–TIM-1 mAb or isotype control. Pooled data from 3 independent experiments are shown (n = 12 donors per group) for the MLR and (n = 6 donors per group) for anti-CD3/CD28 stimulation with statistical evaluation using the paired Student t test. (C) Representative serial bioluminescence images at days +3, +5, +7, +14, and +23 after transplantation of from Luc+ B6 donor mice to assess the proliferative capacity of T cells in the presence of anti–TIM-1 mAb (3D10). (D) Bioluminescence signal intensity time course. Allogeneic transplanted BALB/c recipient mice after injection of luc+ Tcon cells alone (▼) or Tcon cells and anti–TIM-1 mAb (3D10) (●). Mice that were injected only with BM (☐) were used as controls. Error bars indicate SEM. (E) Cytokine levels in the supernatant of splenocytes stimulated ex vivo with anti-CD3/CD28 Dynabeads beads, as assessed by a multiplex assay (Luminex). Splenocytes were isolated at day 9 after transplantation from spleens of BALB/c recipient mice treated with isotype control (white bars) or anti–TIM-1 mAb (3D10) (black bars). Shown are pooled data from 3 independent experiments. For statistical analysis, the 2-tailed Student t test was used (*P ≤ .05, **P ≤ .01, ***P ≤ .01). Error bars indicate SEM.

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