Browsing by Author "Ciofani, Maria"

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.

Autoimmune diseases are caused by dysregulated immune responses against self. Multiple sclerosis (MS) is one such autoimmune disease in which the central nervous system (CNS) is affected by chronic inflammation, and Th17 cells are critical mediators of disease pathogenesis. While targeting leukocyte trafficking is effective in treating autoimmunity, there are currently no therapeutic interventions that specifically block encephalitogenic Th17 cell migration. Here, we report integrin α3 as a Th17 cell-selective determinant of pathogenicity in experimental autoimmune encephalomyelitis, a mouse model of MS. CNS-infiltrating Th17 cells express high integrin α3, the expression of which is induced by transcription factors that are required for Th17 cell specification. The deletion integrin α3 in CD4+ T cells or IL-17A-fate-mapped cells attenuated disease severity. Mechanistically, integrin α3 promoted the polarization, proliferation, and transmigration of Th17 cells, and integrin α3-deficiency enhanced the retention of CD4+ T cells in the perivascular space of the blood-brain barrier. Notably, differential RNA-seq expression analysis revealed that Th17 cells continuously depend on integrin α3 to maintain Th17 cell identity and effector function. The requirement of integrin α3 in Th17 cell pathogenicity suggests integrin α3 as a therapeutic target for MS treatment.

Lymphocytes take on effector programs coordinated by lineage-defining transcription factors (LDTF), resulting in the production of cytokines that fight specific types of pathogens. Therefore, both adaptive and innate lymphocyte lineages can take on specialized effector programs; the type 1 program mediated by T-bet for killing intracellular pathogens and tumors, the type 2 program controlled by GATA3 for protection against helminths, and the type 3 program mediated by RORγt for fighting extracellular bacteria and fungi. While each program can be defined by a single LDTF, many context-dependent situations arise that lead to more than one LDTF being expressed in a cell at a given time. The dual expression of LDTFs can result in the switching of effector programs within a differentiated cell. Nevertheless, LDTFs work in a cooperative manner with signal-dependent TFs and other TFs that sense environmental cues to ultimately control effector fates.

Environmental signals can be sensed by various classes of cell-surface receptors that modulate the downstream signaling effectors and subsequent transcriptional output of a cell for differentiation, proliferation, maintenance, and effector function. Surface receptors, such as the T cell receptor (TCR), cytokine receptors, and costimulatory receptors, translate the environmental cues into downstream signaling cascades that act in concert to promote the differentiation of lymphocyte subsets. Cytokines fine-tune the activation and repression of lymphocytes through phosphorylation of signal transducer and activator of transcription (STAT) TFs that translocate into the nucleus, bind DNA, and regulate gene expression at key loci. Acting alongside STAT TFs, AP-1 TFs are basic leucine zipper (bZIP) TFs that help translate environmental cues into effector programming through binding to key TF and effector cytokine loci.

The ability of a differentiated cell to switch to an alternative fate is referred to as plasticity. Innate lymphoid cells (ILCs) are remarkably plastic at steady state and fate-mapping studies in the mouse intestine revealed that RORγt+ ILCs (ILC3s) can upregulate T-bet and shut down RORγt expression for full conversion to a type 1 ILC (ILC1). ILC3s help maintain healthy mucosal barriers through the production of IL-22 that promotes the release of antimicrobial peptides from epithelial cells. ILC3 to ILC1 plasticity therefore results in a shift from IL-22 to IFNγ production. While increased IFNγ production can be protective against viruses and intracellular pathogens, it can result in many autoimmune and inflammatory diseases when dysregulated. Notably, ILC3 plasticity is implicated in Crohn's disease.

Although the environmental cues regulating ILC3 plasticity were somewhat known, the molecular mechanisms governing ILC3 plasticity were undefined. Here, we identified the AP-1 TF c-Maf as an essential regulator of ILC3 homeostasis and plasticity that limits physiological ILC1 conversion. Phenotypic analysis of effector status in Maf-deficient CCR6- ILC3s using flow cytometry revealed a skewing towards T-bet and IFNγ production. To determine the molecular mechanisms by which c-Maf supported the type 3 program, we evaluated the global changes in transcriptome (RNA-seq), chromatin accessibility (ATAC-seq), and transcription factor motif enrichment. We found that c-Maf promoted ILC3 accessibility and supported RORγt activity and expression of type 3 effector genes. Conversely, c-Maf restrained T-bet expression and function, thereby antagonizing the type 1 program. We performed ATAC-seq on transitioning subsets in the CCR6- ILC3 compartment all the way through conversion to ILC1s to understand the chromatin landscape changes taking place during ILC3 plasticity. These results solidified c-Maf as a gatekeeper of type 1 regulatory transformation and a controller of ILC3 fate.