Control of Regulatory T Cell Functional Specialization by AP-1 Transcription Factors

Foxp3-expressing regulatory T cells (Tregs) are essential mediators of immunological tolerance in both mice and humans. Although Tregs have traditionally been considered a relatively homogeneous population, they are now thought to comprise numerous cellular subtypes each with distinct roles in maintaining organismal homeostasis. In this regard, Tregs undergo functional specialization leading to diverse phenotypes and effector functions depending on (1) the inflammatory milieu encountered during an immune response, and (2) the specific anatomical location in which they reside. This process of context-dependent adaptation of effector function is facilitated by changes in transcriptional programming resulting from the coordinated action of numerous transcription factors (TFs). However, aside from the demonstrated roles for classical lineage-defining transcription factors – such as RORt, GATA-3, T-bet and Bcl-6 – the transcriptional regulatory circuity underlying Treg specialization has remained largely unexplored.

The activator protein-1 (AP-1) superfamily consists of a large number of TFs which bind similar DNA sequences as either homo- or hetero-dimers to influence transcription. Transcriptomic profiling studies have demonstrated that numerous AP-1 TFs exhibit subset- or tissue-specific expression patterns in Tregs, suggesting that these factors might regulate Treg specialization. Specifically, AP-1 TFs c-Maf and JunB were shown to exhibit preferential expression in specialized Tregs and have established regulatory roles in the differentiation of helper T cells. Therefore, I hypothesized that c-Maf and JunB might play undiscovered roles controlling the process of Treg specialization.

Using mice with Treg-specific ablation of Maf (encoding c-Maf), I evaluated the role of c-Maf in Treg specialization within the intestinal immune compartment that hosts a variety of specialized Treg subsets. Mice with Tregs lacking c-Maf appeared largely normal, with no apparent health problems, indicating that c-Maf was dispensable for general Treg function. However, using multicolor flow cytometry to perform comprehensive immunophenotyping of these mice, I found that c-Maf played a critical role in the generation of RORt+ Tregs, which constitute a major subset of specialized Tregs in the intestine but are rarely found in other organs. Additionally, these analyses showed that c-Maf was important for the generation of a second specialized Treg subset, T follicular regulatory (Tfr) cells, which are found in secondary lymphoid organs and are important for controlling antibody responses. However, in contrast to RORt+ Tregs and Tfr cells, c-Maf was not required for differentiation of other types of specialized Tregs, indicating a novel and specific role for c-Maf in Treg specialization. Mechanistically, in vitro culture experiments showed that upregulation of c-Maf and RORt in Tregs could be driven by exposure to IL-6 signaling in the presence of TGF, suggesting that c-Maf functions to link environmental signals to Treg specialization in the intestine. This work demonstrated that c-Maf is a novel and critical regulator of subset-specific Treg specialization for RORt+ Tregs and Tfr cells.

Separately, I investigated the role of JunB in Treg specialization by employing a similar approach as for c-Maf. Surprisingly, I found that mice with Treg-specific ablation of JunB developed a spontaneous phenotype of immune dysregulation which lead to weight loss and colonic inflammation, indicating that JunB was critical for normal Treg functioning. JunB expression was greatly elevated in intestinal Tregs relative to other populations and inflammation was most pronounced within the colons of animals with JunB-deficient Tregs, suggesting that JunB played a specific role in the function of colon-resident Tregs. Like c-Maf, JunB was absolutely required for the development of Tfr cells; however, JunB was otherwise dispensable for the development of all other specialized Tregs examined. Unexpectedly, I found that the loss of Tfr cells in mice with Tregs lacking JunB stemmed from impaired maintenance of the CD25- Treg population, which may be due to novel JunB-dependent metabolic requirements in these cells. Examining the colon-specific effects of JunB ablation using RNA-sequencing of intestinal Tregs, I found that JunB was required for expression of a tissue-specific set of Treg effector genes, such as granzyme B, which may represent a novel suppressive mechanism employed by multiple subtypes of colonic Tregs. Therefore, JunB plays a novel, organ-specific role in Treg specialization of both Tfr cells and all colonic Tregs.

Taken together, my work has revealed novel functions for two AP-1 family TFs – c-Maf and JunB – in the control of Treg specialization. Although c-Maf and JunB are both important for Treg specialization, their contributions to this process are distinct. c-Maf predominantly plays a subset-specific role in RORt+ Tregs and Tfr cells, whereas JunB plays both subset-specific and organ-specific roles depending on anatomical location. Of note, both c-Maf and JunB are essential for differentiation of Tfr cells, although the underlying molecular mechanisms appear to be different. These findings highlight the importance of AP-1 TFs in the control of Treg diversification and functional specialization and suggest that AP-1-mediated transcriptional regulatory circuits are an important mechanism controlling subset- and tissue-specific gene expression in Tregs.