Exposure to inflammatory cytokines selectively limits GM-CSF production by induced T regulatory cells.
Understanding processes that can limit this potentially deleterious effect of Treg cells in a therapeutic setting is therefore important.We show that iTreg cells can produce significant amounts of three proinflammatory cytokines (IFN-γ, GM-CSF and TNF-α) upon secondary TCR stimulation.Furthermore, we show that IL-6 and IL-27 individually, or IL-2 and TGF-β in combination, can mediate the selective loss of GM-CSF production by iTreg cells.
Affiliation: MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK.
- Encephalomyelitis, Autoimmune, Experimental/immunology*
- Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis*
- T-Lymphocytes, Regulatory/immunology*
- Forkhead Transcription Factors/biosynthesis
- Lymphocyte Activation/immunology
- Mice, Inbred C57BL
- Mice, Knockout
- Receptors, Antigen, T-Cell/immunology
- Th1 Cells
- Transforming Growth Factor beta/pharmacology
- Tumor Necrosis Factor-alpha/biosynthesis
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fig04: Autoantigen-responsive iTreg cells are suppressive rather than pathogenic and selectively lose the ability to produce GM-CSF in response to antigen in an inflammatory context. (A) Clinical course of passive EAE in C57BL/6 x B10.PL mice (five per group) that received either CD4+ effector cells (4 × 106/mouse) or iTreg cells (6 × 106/mouse) generated from naïve Tg4.Foxp3LuciDTR-4 T cells was monitored daily. (B) Clinical course of active EAE in C57BL/6 x B10.PL mice (five per group) that received 1 × 106 naïve CD4+ Tg4 cells, either alone, or together with an equal number of Tg4 × Foxp3LuciDTR-4 iTreg cells, one day before immunization with MBP peptide, was monitored daily. Data are shown as mean ± SEM of the indicated number of mice from single experiments representative of two performed. (C–G) C57BL/6 × B10.PL mice (four per group) received 2 × 106 naïve CD4+ Tg4.CD90.1 cells, either alone (–) or together with an equal number of Tg4.Foxp3LuciDTR-4 iTreg cells (+), one day before immunization with MBP peptide. After 7 days, draining lymph nodes were analyzed by flow cytometry for (C and D) the presence of the transferred naïve CD4+ Tg4.CD90.1 cells and (E–G) the ability of those cells to produce (E) IFN-γ, (F) GM-CSF, and (G) TNF-α after overnight culture of lymph node cells with the MBP peptide. Each data point represents an individual mouse and data shown are from single experiments representative of three performed. *p < 0.05 as determined by Mann–Whitney U test; ns = not significant. (H–J) B10.PL mice received 2 × 106 Tg4 × Foxp3LuciDTR-4 iTreg cells alone one day before immunization with the MBP peptide as above. After 7 days, spleens were harvested and cultured and stained for cytokine production as above. Plots are gated on CD45.1+ donor iTreg cells (for gating strategy, see Supporting Information Fig. 2) showing expression of Foxp3 and production of (H) IFN-γ, (I) TNF-α, and (J) GM-CSF. Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. Data shown are from a single experiment representative of three performed.
iTreg cells can therefore produce GM-CSF, IFN-γ, and TNF-α, as well as showing expression of T-bet 16. This phenotype is commonly seen in myelin-responsive CD4+ T cells that can induce passive EAE upon adoptive transfer 19–21. We have previously reported only very poor pathogenic activity with “ex-iTreg” cells (which in that case had received a second stimulation in the presence of IL-12 to further boost T-bet expression and IFN-γ production) 16. However, we reasoned that those cells might have had reduced GM-CSF production, due to the inhibitory effect of IL-12 20. Given that T cell production of GM-CSF (and not IFN-γ) is now believed to be essential for EAE induction 17,18,20,22,23, we revisited this issue, by testing the pathogenic activity of GM-CSF-producing primary iTreg cells (not exposed to IL-12). Using MBP-responsive iTreg cells generated from Tg4.Foxp3LuciDTR-4 mice, we found that transfer of these cells did not provoke clinical signs of EAE (Fig. 4A). Moreover, we confirmed the maintained suppressive activity of these autoreactive iTreg cells, because their cotransfer alongside naive Tg4 T responder cells into C57BL/6 × B10.PL mice prevented EAE upon subsequent immunization with the MBP peptide in CFA (Fig. 4B).