Treatment-enhanced CD4+ Foxp3+ glucocorticoid-induced TNF receptor family RelatedHigh regulatory tumor-infiltrating T cells limit the effectiveness of cytokine-based immunotherapy

Regulatory T cells can suppress activated CD4(+) and CD8(+) T effector cells and may serve as an impediment to spontaneous or therapeutic type 1 antitumor immunity. In a previous study, we observed minimal therapeutic impact, but significantly enhanced T cell cross-priming and lesional infiltration of tumor-reactive CD8(+) T cells into established CMS4 sarcomas after combined treatment of BALB/c mice with rFLt3 ligand (rFL) and recombinant GM-CSF (rGM-CSF). In this study, we show that this cytokine regimen also results in the profound enhancement of CD4(+) tumor-infiltrating lymphocytes (TIL) expressing FoxP3, IL-10, and TGF-beta mRNA, with 50 or 90% of CD4(+) TIL coexpressing the CD25 and glucocorticoid-induced TNFR family related molecules, respectively. Intracellular staining for Foxp3 protein revealed that combined treatment with rFL plus rGM-CSF results in a significant increase in CD4(+)Foxp3(+) T cells in the spleen of both control and tumor-bearing mice, and that nearly half of CD4(+) TIL expressed this marker. In addition, CD4(+) TIL cells were of an activated/memory (ICOS(high)CD62L(low)CD45RB(low)) phenotype and were capable of suppressing allospecific T cell proliferation and IFN-gamma production from (in vivo cross-primed) anti-CMS4 CD8(+) T cells in vitro, via a mechanism at least partially dependent on IL-10 and TGF-beta. Importantly, in vivo depletion of CD4(+) T cells resulted in the ability of previously ineffective, rFL plus rGM-CSF therapy-induced CD8(+) T cells to now mediate tumor regression.