The NF-κB transcription factor RelA is required for the tolerogenic function of Foxp3+ regulatory T cells
Introduction
CD4+ regulatory T cells (Tregs) that express the Forkhead box P3 (FOXP3) transcription factor control peripheral immune cell homeostasis by restraining normal immune responses and suppressing auto-reactive T cells [1], [2], [3]. Tregs exert immune modulatory functions through a variety of effector mechanisms that include the production of immune suppressive molecules such as IL-10 and TGF-β, the consumption of IL-2, inhibiting dendritic cell (DC) co-stimulation or the direct killing of antigen-presenting cells and T cells [4], [5]. Like conventional CD4+ T cells, Tregs comprise sub-populations with distinct functions and properties [6], [7]. The majority of Tregs recirculating through peripheral lymphoid organs have a naïve phenotype defined by high levels of the homing receptor CD62L and low to moderate levels of CD44, a marker of T cell activation. By contrast, most Tregs in non-lymphoid organs have an effector-like phenotype. Although effector Treg (eTreg) phenotypic variability is influenced by factors including microbes, inflammation and the surrounding environment, these cells typically display low levels of CD62L, high levels of CD44, ICOS and GITR, plus variable expression of KLRG1, CD69 and CD103 [6], [7], [8].
The diverse phenotypic, migratory and functional properties of Tregs reflect specific transcriptional programs elicited by particular immune signals. FOXP3 is essential for the immune suppressive properties of all Tregs [9], [10], [11], but alone is insufficient to create or maintain these functions [12], [13], [14]. Instead, FOXP3 serves to stabilize transcriptional landscapes established during Treg development or post-thymic Treg differentiation through the combined activity of other ubiquitous and lineage-restricted transcription factors [12], [13], [14], [15], [16]. For example, the transcriptional regulators Tbet, IRF4, GATA-3, RORγt and Bcl6 that selectively dictate the differentiation of specific conventional CD4+ T helper (Th) subsets (Th1, Th2, Th17 and Tfh) are also employed by different types of eTregs [6], [7], [17], [18], [19], [20], [21], [22]. These Th polarizing transcription factors co-operate with FOXP3 to endow eTreg subsets with a capacity to customise functional programs tailored to respond to specific types of immune responses controlled by different Th cells. The roles other transcription factors known to be important in T effector cell biology play in eTreg development and function remains to be determined.
Canonical NF-κB transcription factors comprising homodimers or heterodimers of RelA, c-Rel and NF-κB1 serve a diverse range of functions in T lineage cells, including controlling thymic derived Treg (tTreg) development [23], [24], [25], [26]. c-Rel is the most important of the canonical NF-κB proteins in developing tTregs, with its absence resulting in a loss of ∼85% of the thymic Treg population [27]. RelA appears to serve a minor role in tTreg development, while NF-κB1 is dispensable [27]. Despite a paucity of information regarding the post-developmental function of NF-κB in Tregs, autoimmune disease induced in mice by the conditional inactivation of different upstream activators of the canonical NF-κB pathway in FOXP3+ cells indicates that Tregs require NF-κB activity to maintain peripheral tolerance [28], [29], [30]. Which canonical NF-κB factors are active in Tregs and what functional role these proteins serve, remains unclear. Here we show that murine Tregs exhibit a pattern of NF-κB activity reminiscent of activated conventional CD4+ T cells and that conditional targeting of a single family member Rela in FOXP3+ cells, induces a rapid onset multi-focal autoimmune disease. Our findings that RelA promotes the maintenance of FOXP3 expression and eTreg homeostasis supports a model in which peripheral tolerance depends on RelA controlling the stability and fitness of eTregs.
Section snippets
Mice
Unless otherwise stated, all mouse strains were maintained on an inbred Ly5.2 C57BL6/J background (>10 generation backcross). Rela+/− (heterozygous for the Rela null allele) mice [31], Foxp3gfp mice [10] and Foxp3rpf mice [32] have been described previously. The Foxp3rpf and C57BL6/J Ly5.1 strains were inter-crossed to create Ly5.1Foxp3rfp mice. Relawt/flFoxp3cre mice heterozygous for a loxP targeted Rela allele [33] and homozygous (females) or hemizygous (males) for a version of Foxp3
The canonical NF-κB transcription factor RelA is active in Tregs
The finding that the tolerogenic properties of peripheral Tregs depend on the NF-κB pathway [28], [29], [30] prompted an examination of nuclear NF-κB DNA binding activity in splenic Tregs using electrophoretic mobility gel shift assays (EMSA). NF-κB activity was assessed in nuclear extracts prepared from Tregs (CD4+CD25+GFP+) and conventional T cells (CD4+CD25−GFP−) co-purified from the spleens of Foxp3gfp mice. NF-κB levels in the nucleus of unstimulated conventional CD4+ T cells were
Discussion
In addition to the lineage defining protein FOXP3, increasing numbers of transcription factors are being identified that contribute to the suppressive capacity and function of Tregs [16], [17], [18], [19], [20], [21], [22]. Amongst the many transcription factors involved in controlling immune responses, NF-κB proteins play a central role regulating immune effector functions in a range of cell types. While c-Rel is known to be important for Treg development [27], not until the conditional
Acknowledgments
The authors thank Drs Roland Schmidt, Alexander Rudensky and John Silke for the generous provision of mouse strains used in this study. This work is supported by NHMRC program (#1016701) and project (#1029822) grant funding to SG. The authors declare they have no actual or potential conflicts of interest.
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These authors contributed equally to this study.