c-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells

Abstract

The development of natural Foxp3+ CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen dependent generation of CD25+GITRhiFoxp3−CD4+ nTreg precursors followed by the cytokine induction of Foxp3. Using mutant mouse models that lack c-Rel, the critical NF-κB transcription factor required for nTreg differentiation, we establish that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2 dependent induction of Foxp3 without a need for c-Rel reactivation.

c-Rel

“…By intercrossing c-rel -/-mice with another mouse strain that expresses a GFP-Foxp3 fusion protein created by inserting GFP in frame within the N-terminal coding region of the foxp3 gene, 17 19 The reduction in Treg precursors in c-rel -/-mice had been these cells to undergo further maturation. The qualitative nature of the intracellular signals generated by the strength and duration (avidity) of these antigen-receptor interactions dictate thymocyte fates, with only a range of TCR avidity being compatible with further thymocyte maturation.…”

“…25 The phenotypic similarities of the nTreg defects in c-rel -/-and cns3 -/-mice have led c-Rel to be designated a 'pioneer transcriptional regulator' for the nTreg lineage, with the proposal that c-Rel binding to CNS3 establishes a permissible state for the subsequent IL-2 dependent induction of foxp3 transcription. 27 Irrespective of whether c-Rel controls foxp3 transcription through CNS2 and/or CNS3, the finding that c-Rel is not in the nucleus in Treg precursors or nTregs, 7,19 and is not activated by IL-2 or IL-15 despite being required for the cytokine induction of Foxp3, can only be reconciled if foxp3 transcription is programmed by c-Rel prior to the generation of nTreg precursors. Presumably c-Rel would remodel the chromatin structure or methylation status ( Fig.…”

“…19 This reduced cytokine responsiveness appears to be due to multiple defects in the intracellular signal transduction pathway downstream of the cytokine receptors, including impaired STAT5 phosphorylation by JAK, an activation step that is required for STAT5 dependent foxp3 transcription. 19 Although c-Rel is necessary for the cytokine dependent induction of Foxp3, neither IL-2 or IL-15 promotes c-Rel nuclear translocation in nTreg precursors, 19 a finding consistent with the inability of these cytokines to activate the NF-κB pathway. Despite this conundrum, an emerging body of data does point to c-Rel regulating foxp3 transcription.…”

“…23,24 The c-Rel binding sites within the foxp3 promoter have been touted as being important for directing the assembly of a transcription complex essential for foxp3 expression. 23 However, the observation that the remaining c-rel -/-thymic nTregs express normal levels of Foxp3 7,19 indicates that c-Rel is required to establish foxp3 transcription rather than control its constitutive expression. This has focused attention on CNS2 and CNS3, the other conserved non-coding regions of the foxp3 gene that bind c-Rel.…”

“…10 Furthermore, while c-Rel is expressed at high levels in nTreg precursors, it exists in an inactive cytoplasmic state in these cells. 19 This indicates that c-Rel is not utilized to maintain the Treg precursor population; rather, it is required in the generation of these cells.…”