Figure 1.

Accumulation and proliferation of T reg cells in lymphoid organs of tumor-bearing rodents. (A) CD4⁺CD25⁺ cells in DLNs from C57BL6 mice bearing B16F10 tumors. The expression of CD4 and CD25 was analyzed by flow cytometry on gated CD3⁺ T cells isolated from inguinal DLNs of 15-d-old B16F10 TBM or tumor-free controls. These cells were FACS purified and then subjected to intracellular staining for the detection of FOXP3, whereas background levels were determined with an isotype control (bottom). Percent values represent the mean ± SEM (n = 6 distinct animals). (B) Inhibitory effect of T reg cell splenocytes. 10⁵ splenocytes harvested from a TFM were depleted in CD25⁺ cells and stimulated with 2 μg/ml of ConA. CD25⁺ T cells purified from TFM or TBM were added to the culture. Proliferation was determined after 3 d of culture using [³H]thymidine incorporation. Values represent the mean ± SEM of triplicates. (C) Proliferation of T reg cells in TBM. C57BL6 mice bearing B16F10 tumors (as in A) or TFM were injected with BrdU, and the frequency of BrdU⁺ cells was determined among CD3⁺CD4⁺CD25⁺ cells by flow cytometry. Values represent the mean ± SEM (n = 6). (D) Immunophenotyping of axillary DLNs from rats bearing 28-d-old PROb tumors or tumor free control LNs. Percent values represent the mean ± SEM (n = 6). RT-PCR analysis of FOXP3 expression in CD25⁺ and CD25⁻ T cells from TBR and TFR. Data are representative of three experiments. (E) Antiproliferative action of T reg cells from rats. The experiment was analogous to that shown in B, with the difference that all cells were derived from BD-IX rats instead of C57BL/6 mice. (F) Proliferation of T reg cells in vivo in TBR. BD-IX rats bearing PROb tumors (as in D) or TFR were injected with BrdU, and the frequency of BrdU⁺ cells was determined among CD3⁺CD4⁺CD25⁺ cells as in C. Values represent the mean ± SEM (n = 6).