An Antitumor Antibody Response to Attenuate Metastatic Melanoma in a Murine Model
Poster Feb 14, 2017
M. Usman Ahmad, Sylvia Rehakova, Louise Van Der Weyden, David Adams, Anneliese Speak, Reza Motallebzadeh, Gavin J. Pettigrew
To establish a murine model of melanoma, 0.5x105 C57BL/6-derived B16 melanoma cells expressing OVA were injected via the tail vein into wild-type C57BL/6 mice. 24h later mice were injected with either 4x106 or 5x105 MHC class II mismatched CD4(+) T-cells from the bm12 mice. The bm12 mice are C57BL/6 that harbor a mutation in the H2-Ab1bm12 allele resulting in a variant MCH class II allele. CD4 T cells were separated from mouse spleens using MACS separation with positive selection (Miltenyi). Controls included no injection and autologous cell transfer. Two independent experiments were conducted consisting of either 5 or 10 mice per each control or treatment arm. Mice were sacrificed on either day 10 or day 20 post cell transfer and the effect on tumor burden was measured by counting pulmonary metastasis. Serum antibody responses were measured using an indirect ELISA specific for OVA (Chondrex Kit #3011). Germinal centers in the spleen were counted using immunohistochemistry with fluorescently labeled antibodies to GL7, a germinal center marker and B220, a B-cell marker.
Two independent experiments showed statistically significant reduction of pulmonary metastasis compared to control (n=10, mean met count; 14.6 vs. 32.2, p=0.02) and (n=20, mean met count; 13.5 vs. 28.8, p=0.01) at day 10 post adoptive cell transfer of either 4x106 or 5x105 cells. There was also statistically significant reduction of pulmonary metastasis compared to autologous cell transfer control (n=20, mean met count; 13.5 vs. 27.9, p=0.008) when 5x105 cells were used. The level of serum anti-OVA antibodies was increased in both experiments. However, only the experiment using 5x105 cells showed the increase to be statistically significant when compared to no cell transfer control (n=20, mean conc. ng/mL, 15.25 vs. 2.78, p=0.0003) and autologous cell transfer control (n=20, mean conc. ng/mL, 15.25 vs. 0.05, p<0.0001). Consistent with this observation was the finding that the frequency of secondary follicles was statistically significantly increased compared to autologous cell transfer control (n=20, mean % frequency of secondary follicles/total follicles; 69% vs. 14%, p<0.0001). In contrast, pulmonary metastasis was identical in all arms when analyzed at day 20 post adoptive cell transfer.
These preliminary results show antitumor activity of adoptive cell transfer of MHC class mismatched CD4(+) T-cells. The finding of reduced numbers of pulmonary metastases correlates with increased serum anti-OVA IgG and increased germinal center development in the spleen. Interestingly, using an 8-fold higher number of MHC class mismatched CD4(+) T-cells did not result in a statistically significant increase of serum OVA specific IgG. Additional research is required to further characterize this anti-tumor antibody response induced by the adoptive cell transfer of MHC class mismatched CD4(+) T-cells. For this purpose experiments are currently underway using a four-fold higher tumor burden.
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