IL-2 Therapy Promotes the Expansion of Human CD4+CD25+ Regulatory T Cells and Selectively Upregulates the Expression of FOXP3 in T Cells In Vivo.

Recombinant IL-2 has been used extensively in clinical trials to enhance a wide range of immune responses. Overall this strategy has had limited efficacy. Recent evidence suggests that IL-2 plays a key role in the generation and maintenance of CD4+CD25+ regulatory T cells (Treg) in vivo. In our study, we investigated the effect of prolonged administration of recombinant IL-2 on Treg in vivo. In a retrospective analysis, we first examined CD4+CD25+ Treg in blood samples collected from 21 cancer patients before and after they started continuous treatment with IL-2 at a dose of 2 X 105 U/m2/day for 3 months. Nine patients received IL-2 beginning 3 months after CD6 T cell depleted allogeneic bone marrow transplantation (BMT) for CML. The remaining 12 patients received IL-2 as treatment for advanced solid tumors. Overall toxicity was minimal and none of the transplant patients developed GVHD following IL-2 administration. Previous reports demonstrated that this prolonged treatment with low-dose IL-2 resulted in the expansion of CD56+CD3− NK cells in peripheral blood. Further analysis showed that 15 patients exhibited an expansion of Treg in peripheral blood 26 to 77 days after beginning IL-2 as demonstrated by an increase in the CD4+CD25+/CD3+ ratio (median fold increase 2.68; range 1.3 to 59). Three patients had no significant change and 3 patients demonstrated a decreased Treg/CD3 ratio. Using RNA from the same samples we also measured the expression of the Treg specific transcription factor FOXP3 by quantitative PCR. Nineteen of 21 patients showed a marked increase in FOXP3 expression following IL-2 treatment (8.38 median fold increase; range 1.4 to 41.5). Only 2 of 21 patients had lower FOXP3 expression after IL-2 administration. Since IL-2 treatment resulted in the expansion of NK cells as well as Treg, we purified CD56+CD3− NK cells and CD4+ T cells from patient samples collected post-IL-2 treatment, and measured FOXP3 gene expression in both subsets. In 4 analyzed cases, FOXP3 was selectively expressed in CD4+ T cells. Further analysis of purified Treg and NK cells incubated with IL-2 in vitro confirmed that FOXP3 expression was selectively induced in Treg, and also suggested that the in vivo increase in FOXP3 expression resulted from both Treg expansion and up-regulation of gene expression at the single cell level. To study the duration of the IL-2 effect, we analyzed additional samples collected 2 to 8 months after IL-2 treatment was completed. Nine of 10 patient samples tested showed a decrease in the CD4+CD25+/CD3+ ratio (1.39 median fold decrease; range 1.13 to 15.02). Using quantitative PCR, expression of FOXP3 decreased for 6 of 8 patients tested (10.76 median fold decrease; range 1.22 to 88.31). These results indicate that prolonged administration of IL-2 promotes the expansion of CD4+CD25+ Treg in vivo and also has a direct effect on FOXP3 expression. Although administration of IL-2 has previously been used to enhance T and NK cell responses, this study demonstrates that IL-2 therapy predominantly reinforces the regulatory component of the immune response, and may provide a means for controlling immune reactions in vivo.