ABSTRACT
Immunological tolerance is maintained by specialized subsets of T cells including CD4(+)CD25(+)FOXP3(+) regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. Moreover, foxp3 gene transfer in murine CD4(+)CD25(-) T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4(+)CD25(-) T lymphocytes leads to STAT3 Tyr(705) phosphorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4(+)CD25(-) T cell conversion into Treg through costimulation with TCR/CD28 and TGF-beta1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-gamma. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differentiated CD4(+)CD25(+) T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4(+)CD25(+) T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4(+) lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance.
Subject(s)
Forkhead Transcription Factors/biosynthesis , Graft vs Host Disease/immunology , Immunotherapy, Adoptive/methods , Neoplasms/immunology , STAT3 Transcription Factor/physiology , T-Lymphocytes, Regulatory/immunology , Animals , CD28 Antigens/immunology , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Disease Models, Animal , Forkhead Transcription Factors/antagonists & inhibitors , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Transplantation , Phenotype , Phosphorylation , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/drug effects , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes, Regulatory/transplantationABSTRACT
OBJECTIVE: CD4(+)CD25(+) regulatory T lymphocytes (Treg) have been initially shown to prevent organ-specific autoimmunity. It is now accepted that Treg homeostasis depends in part on the peripheral conversion of naïve CD4(+)CD25(-) T cells. This conversion implicates acquisition of the Treg-specific markers, forkhead winged helix protein 3 (Foxp3), after CD28 costimulation. Because natural killer cells (NK) are critical for efficient cytotoxic T-cell priming and TH1 polarization, we investigated their role in Foxp3 induction in CD4(+) T lymphocytes. MATERIALS AND METHODS: Human CD4(+)CD25(-) T lymphocytes were activated in vitro by CD28 costimulation in the presence of interleukin-2-activated NK. Three days after initial activation, Foxp3 protein and RNA expression were determined by flow cytometry and reverse transcription polymerase chain reaction. In vivo influence of activated NK on Foxp3 expression was studied in an adoptive transfer model of CD45.2(+) CD4(+)CD25(-) lymphocytes into CD45.1(+) mice. RESULTS: Interleukin-2-activated NK decreased Treg conversion of adoptively transferred murine CD4(+)CD25(-) T cells in vivo. Likewise, human-activated NK, but not resting NK, decreased CD28-driven Foxp3 expression in CD4(+)CD25(-) T lymphocytes, while at the same time increasing proliferation and interferon-gamma (IFN-gamma) production. Neutralization of IFN-gamma partially restored Treg conversion and prevented TH1 polarization after CD28 costimulation. CONCLUSION: The current study suggests that activated NK interfere with CD28-mediated Foxp3 expression in CD4(+)CD25(-) T lymphocytes. Our experiments further underline a molecular interaction between IFN-gamma and Foxp3 downstream of CD28 signaling. Together, these results demonstrate that activated NK play a critical role at the initiation step of immune responses by modulating peripheral Treg differentiation.
