Figure 2.
Figure 2. The blockade of CD137-CD137L interactions inhibits proliferation, survival, and in vivo growth of CTCL tumor cells. (A-B) Hut78 and MyLa cells (1.0 × 105 per well) were cultured with anti-CD137 (5 μg/mL) and/or anti-CD137L (5 μg/mL) neutralizing antibodies for 48 hours (A). EL-4 cells (1.0 × 105 per well) were cultured with anti-CD137 antibody (5 or 10 μg/mL) or anti-CD137L–neutralizing antibody (5 or 10 μg/mL) for 48 hours (B). Viable cells were counted at indicated time points by trypan blue exclusion. Statistical difference was compared with isotype-treated cells. (C) PBMCs from 5 SS patients and 4 age-matched healthy controls (1.0 × 105 per well) were cultured with anti-CD137L–neutralizing antibody (5 μg/mL) for 48 hours, and the percentage of CD4+CD7− and CD4+CD7+ T cells in SS patients or CD4+ T cells from healthy controls were determined by flow cytometry. Viable cells were counted at 48-hour time points by trypan blue exclusion. Statistical difference was compared with isotype-treated cells (paired Student t test). (D-E) Hut78, MyLa, and EL-4 cells (1.0 × 105 per well) were cultured with anti-CD137L–neutralizing antibody (5 μg/mL) for 24 hours. Apoptosis (Annexin V+ and 7-AAD−) was evaluated with Annexin V and 7-aminoactinomycin D (7-AAD) staining for flow cytometric analysis. (F) Hut78, MyLa, HH, and SeAx cells (5.0 × 104 per well) were cultured with plate-coated recombinant CD137L (10 μg/mL) for 48 hours. Viable cells were counted. Data are presented as mean plus or minus SD. *P < .05, **P < .01 (A-F). (G-H) Hut78 cells (5.0 × 106) were injected into NSG mice with anti-CD137L–neutralizing antibody (50 μg/mL) or isotype immunoglobulin G (IgG) (G). EL-4 cells (3.0 × 106) were injected into C57BL/6 mice with anti-CD137L–neutralizing antibody (50 μg/mL) or isotype IgG (H). Each reagent was injected on days 4, 7, and 11. The tumor volume was calculated using the equation: V = π (L1 × L22)/6, where V = volume (mm3), L1 = longest diameter (mm), and L2 = shortest diameter (mm). Data are presented as means plus standard error of the mean (SEM; n = 12). *P < .05, ** P < .01 by Mann-Whitney U test compared with control group.

The blockade of CD137-CD137L interactions inhibits proliferation, survival, and in vivo growth of CTCL tumor cells. (A-B) Hut78 and MyLa cells (1.0 × 105 per well) were cultured with anti-CD137 (5 μg/mL) and/or anti-CD137L (5 μg/mL) neutralizing antibodies for 48 hours (A). EL-4 cells (1.0 × 105 per well) were cultured with anti-CD137 antibody (5 or 10 μg/mL) or anti-CD137L–neutralizing antibody (5 or 10 μg/mL) for 48 hours (B). Viable cells were counted at indicated time points by trypan blue exclusion. Statistical difference was compared with isotype-treated cells. (C) PBMCs from 5 SS patients and 4 age-matched healthy controls (1.0 × 105 per well) were cultured with anti-CD137L–neutralizing antibody (5 μg/mL) for 48 hours, and the percentage of CD4+CD7− and CD4+CD7+ T cells in SS patients or CD4+ T cells from healthy controls were determined by flow cytometry. Viable cells were counted at 48-hour time points by trypan blue exclusion. Statistical difference was compared with isotype-treated cells (paired Student t test). (D-E) Hut78, MyLa, and EL-4 cells (1.0 × 105 per well) were cultured with anti-CD137L–neutralizing antibody (5 μg/mL) for 24 hours. Apoptosis (Annexin V+ and 7-AAD−) was evaluated with Annexin V and 7-aminoactinomycin D (7-AAD) staining for flow cytometric analysis. (F) Hut78, MyLa, HH, and SeAx cells (5.0 × 104 per well) were cultured with plate-coated recombinant CD137L (10 μg/mL) for 48 hours. Viable cells were counted. Data are presented as mean plus or minus SD. *P < .05, **P < .01 (A-F). (G-H) Hut78 cells (5.0 × 106) were injected into NSG mice with anti-CD137L–neutralizing antibody (50 μg/mL) or isotype immunoglobulin G (IgG) (G). EL-4 cells (3.0 × 106) were injected into C57BL/6 mice with anti-CD137L–neutralizing antibody (50 μg/mL) or isotype IgG (H). Each reagent was injected on days 4, 7, and 11. The tumor volume was calculated using the equation: V = π (L1 × L22)/6, where V = volume (mm3), L1 = longest diameter (mm), and L2 = shortest diameter (mm). Data are presented as means plus standard error of the mean (SEM; n = 12). *P < .05, ** P < .01 by Mann-Whitney U test compared with control group.

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