Figure 7. Anti–TIM-1 inhibits the expansion of ABM TCR-Tg T cells, tipping the balance from effector to regulatory T cells.
(A) Following adoptive transfer of 2 × 106 ABM TCR-Tg T cells into nude B6 mice, recipients were transplanted with bm12 skin grafts and received anti–TIM-1 mAb or were left untreated. After 7 days, the dLNs and the spleen were harvested, and the total leukocyte number in the different compartments was determined. Leukocytes were stained for expression of Vα2.1 and Vβ8.1, and the percentage of these cells was determined. The percentage and total leukocyte count were then used to calculate the absolute number of ABM TCR-Tg T cells present in the dLNs and spleen. Anti–TIM-1 mAb significantly decreased the number of ABM TCR-Tg T cells present in the dLNs and spleen compared with untreated controls. (B) Cells harvested from dLNs and spleen were cultured with PMA/ionomycin and brefeldin A for 4 hours and stained with mAbs against IFN-γ. Cytokine production was determined by flow cytometry after gating on Vα2.1+Vβ8.1+ cells. Anti–TIM-1 mAb significantly decreased the frequency of allospecific IFN-γ–producing T cells present in the dLNs and spleen compared with untreated controls. Values represent the mean ± SEM of data obtained from 5 separate experiments. (C) Using the adoptive transfer model on day 7 after engraftment, allospecific Tg T cells were harvested from lymphoid compartments. The percentage of Tg cells expressing the CD4+CD25+FoxP3+ regulatory phenotype was then enumerated using flow cytometry. As demonstrated in C, anti–TIM-1 triggered a significant increase in the percentage of Tg T cells expressing a regulatory phenotype in the dLNs However, given the overall decrease in the size of the Tg cell compartment (A), there was no significant change in the absolute number of Tregs after anti–TIM-1 treatment in this model.