Maria Ciofani

/ Summary: A wealth of noncoding regulatory elements has been described across mammalian cell types, yet determining their functional role remains a challenge. Regulatory control of gene expression is critical during active processes such as the adaptive immune response. Upon antigen presentation, a naive CD4+ T cell undergoes major transcriptional and structural reorganization necessary for establishment of subset identity and immune function. In this study, we systematically measure the regulatory potential of candidate regulatory elements associated with open chromatin across five mouse CD4+ T cell subsets. Using ATAC-STARR-seq, we found that approximately one quarter of open chromatin regions demonstrate regulatory activity. Most exhibit shared functional potential across subsets, though we identify enhancers with activity that is restricted to specific cellular contexts. To distinguish regulatory potential from endogenous function, we performed CRISPR-based epigenome editing screens at noncoding regions of Th17 cells and identified a set of core elements essential for subset polarization. Integrating Region Capture Micro-C, we resolved precise 3D chromatin topologies that explain functional regulatory networks via physical contacts. We characterize examples of active regulatory hubs formed through multiple CTCF-independent interactions organized in a hierarchical architecture. Furthermore, we discover a critical Batf enhancer that operates via these contacts. Using targeted perturbations, we disrupt local chromatin topology and gene expression with profound consequence to downstream Th17 cell phenotypes. We confirm the physiological necessity of these functional enhancers in vivo , demonstrating the importance of noncoding elements for Th17 cell identity. Together, this work reveals how DNA sequence and chromatin cooperate to shape the regulatory logic of Th17 cells, with implications for cis-regulatory principles beyond the immune system.

T helper 17 (Th17) cells have been implicated in numerous inflammatory autoimmune diseases. Clinical benefits from targeting Th17 cell-related cytokines, such as IL-17 and IL-23, highlight how knowledge of Th17 cell development and effector function can be translated into treatments for inflammatory disease. Here we discuss the pathogenic roles of Th17 cells in autoimmune diseases such as multiple sclerosis, inflammatory bowel disease and psoriasis, with emphasis on the cytokines, transcriptional regulators and metabolites that influence Th17 cell differentiation and pathogenicity. Moreover, we address how intestinal environments and physiological responses affect Th17 cells in autoimmune diseases. We also examine current and emerging therapeutic strategies aimed at regulating Th17 cell-driven inflammation to mitigate autoimmune diseases.

Interleukin-17 (IL-17)-producing γδ T (Tγδ17) cells are innate-like mediators of intestinal barrier immunity. Although IL-17-producing helper T cell and group 3 innate lymphoid cell plasticity have been extensively studied, the mechanisms governing Tγδ17 cell effector flexibility remain undefined. Here, we combined type 3 fate mapping with single-cell ATAC-sequencing/RNA-sequencing multiome profiling to define the cellular features and regulatory networks underlying Tγδ17 cell plasticity. During homeostasis, Tγδ17 cell effector identity was stable across tissues, including for intestinal T-bet⁺ Tγδ17 cells that restrained interferon-γ production. However, Salmonella enterica subsp. enterica serovar Typhimurium infection induced intestinal V_γ6⁺ Tγδ17 cell conversion into type 1 effectors, with loss of IL-17A production and partial RORγt downregulation. Multiome analysis revealed a trajectory along V_γ6⁺ Tγδ17 cell effector conversion, with TIM-3 marking ex-Tγδ17 cells with enhanced type 1 functionality. Last, we characterized and validated a critical AP-1 regulatory axis centered around JUNB and FOSL2 that controls V_γ6⁺ Tγδ17 cell plasticity by stabilizing type 3 identity and restricting type 1 effector conversion.

Allergic diseases are common and affect a large proportion of the population. Interleukin-13 (IL-13)-expressing follicular helper T (T_FH13) cells are a newly identified population of T_FH cells that have been associated with high-affinity IgE responses. However, the origins, developmental signals, transcriptional programming and precise functions of T_FH13 cells are unknown. Here, we examined the developmental signals for T_FH13 cells and found a direct and progressive differentiation pathway marked by the production of IL-21. These two pathways differed in kinetics and extrinsic requirements. However, both pathways converged, forming transcriptionally similar T_FH13 cells that express the transcription factor JunB as a critical stabilizing factor. Using an intersectional genetics-based T_FH13-diphtheria toxin receptor model to perturb these cells, we found that T_FH13 cells were essential to drive broad germinal center responses and allergen-specific IgG and IgE. Moreover, we found that IL-21 is a broad positive regulator of allergen germinal center B cells and synergizes with IL-13 produced by T_FH13 cells to amplify allergic responses. Thus, T_FH13 cells orchestrate multiple features of allergic inflammation.

IL-17-producing γδ T (Tγδ17) cells are innate-like mediators of intestinal barrier immunity. While Th17 cell and ILC3 plasticity have been extensively studied, the mechanisms governing Tγδ17 cell effector flexibility remain undefined. Here, we combined type 3 fate-mapping with single cell ATAC/RNA-seq multiome profiling to define the cellular features and regulatory networks underlying Tγδ17 cell plasticity. During homeostasis, Tγδ17 cell effector identity was stable across tissues, including for intestinal T-bet ⁺ Tγδ17 cells that restrained IFNγ production. However, S. typhimurium infection induced intestinal Vγ6 ⁺ Tγδ17 cell conversion into type 1 effectors, with loss of IL-17A production and partial RORγt downregulation. Multiome analysis revealed a trajectory along Vγ6 ⁺ Tγδ17 effector conversion, with TIM-3 marking ex-Tγδ17 cells with enhanced type 1 functionality. Lastly, we characterized and validated a critical AP-1 regulatory axis centered around JunB and Fosl2 that controls Vγ6 ⁺ Tγδ17 cell plasticity by stabilizing type 3 identity and restricting type 1 effector conversion.

Invasive fungal infections are a leading cause of death worldwide. Translating molecular insights into clinical benefits is challenging because fungal pathogens and their hosts share similar eukaryotic physiology. Consequently, current antifungal treatments have limited efficacy, may be poorly fungicidal in the host, can exhibit toxicity, and are increasingly compromised by emerging resistance. We have established that the phosphatase calcineurin (CaN) is required for invasive fungal disease and an attractive target for antifungal drug development. CaN is a druggable target, and there is vast clinical experience with the CaN inhibitors FK506 and cyclosporin A (CsA). However, while FK506 and its natural analog FK520 exhibit antifungal activity, they are also immunosuppressive in the host and thus not fungal-selective. We leverage our pathogenic fungal CaN-FK506-FKBP12 complex X-ray structures and biophysical data to support structure-based ligand design as well as structure–activity relationship analyses of broad-spectrum FK506/FK520 derivatives with potent antifungal activity and reduced immunosuppressive activity. Here we apply molecular docking studies to develop antifungal C22- or C32-modified FK520 derivatives with improved therapeutic index scores. Among them, the C32-modified FK520 derivative JH-FK-44 ( 7 ) demonstrates a significantly improved therapeutic index compared to JH-FK-08, our lead compound to date. NMR binding studies with C32-derivatives are consistent with our hypothesis that C32 modifications disrupt the hydrogen bonding network in the human complex while introducing favorable electrostatic and cation–π interactions with the fungal FKBP12 R86 residue. These findings further reinforce calcineurin inhibition as a promising strategy for antifungal therapy. Significance: Invasive fungal infections cause significant mortality worldwide, and current antifungal treatments are often ineffective, toxic, or face growing resistance. This research identifies calcineurin (CaN), a critical protein for fungal survival, as a potential target for developing new antifungal drugs. Although existing CaN inhibitors such as FK506 (tacrolimus) and FK520 (ascomycin) possess antifungal properties, their immunosuppressive effects limit their clinical utility. By studying the structure of human and fungal FKBP12-FK506 or FK520 complexes with CaN, we have designed and synthesized modified FK520 derivatives with strong antifungal activity and reduced immunosuppressive effects. These new derivatives are expected to have significantly improved therapeutic profiles, offering hope for more effective and safer antifungal treatments.

T cell receptor (TCR) signaling regulates important developmental transitions, partly through induction of the E protein antagonist, Id3. Although normal γδ T cell development depends on Id3, Id3 deficiency produces different phenotypes in distinct γδ T cell subsets. Here, we show that Id3 deficiency impairs development of the Vγ3⁺ subset, while markedly enhancing development of NKγδT cells expressing the invariant Vγ1Vδ6.3 TCR. These effects result from Id3 regulating both the generation of the Vγ1Vδ6.3 TCR and its capacity to support development. Indeed, the Trav15 segment, which encodes the Vδ6.3 TCR subunit, is directly bound by E proteins that control its expression. Once expressed, the Vγ1Vδ6.3 TCR specifies the innate-like NKγδT cell fate, even in progenitors beyond the normally permissive perinatal window, and this is enhanced by Id3-deficiency. These data indicate that the paradoxical behavior of NKγδT cells in Id3-deficient mice is determined by its stereotypic Vγ1Vδ6.3 TCR complex.

T cell receptor (TCR) signals regulate important developmental transitions through induction of the E protein antagonist, Id3; however, Id3-deficiency produces paradoxical effects on γδ T cell subsets. Here, we show here that Id3-deficiency attenuates the development of Vγ3-expressing γδ T cells, while markedly enhancing the development of Vγ1Vδ6.3-expressing NKγδT cells. Id3-deficiency does so by regulating both the generation of the stereotypic Vγ1Vδ6.3 TCR expressed by NKγδT cells and its capacity to support development. Indeed, we determined that the Trav15 segment, which encodes the Vδ6.3 TCR subunit, is directly bound by E proteins that control its expression. Moreover, once expressed, the resulting Vγ1Vδ6.3 TCR in capable of specifying the innate-like NKγδT cell fate in a cell-autonomous and developmentally-unrestricted manner that is restrained by the Id3/E axis. Together, these data indicate that the paradoxical behavior of NKγδT cells in the Id3-deficient setting is entirely determined by its stereotypic Vγ1Vδ6.3 TCR complex.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by CNS-infiltrating leukocytes, including T _H 17 cells that are critical mediators of disease pathogenesis. Although targeting leukocyte trafficking is effective in treating autoimmunity, there are currently no therapeutic interventions that specifically block encephalitogenic T _H 17 cell migration. Here, we report integrin α3 as a T _H 17 cell–selective determinant of pathogenicity in experimental autoimmune encephalomyelitis. CNS-infiltrating T _H 17 cells express high integrin α3, and its deletion in CD4 ⁺ T cells or Il17a fate-mapped cells attenuated disease severity. Mechanistically, integrin α3 enhanced the immunological synapse formation to promote the polarization and proliferation of T _H 17 cells. Moreover, the transmigration of T _H 17 cells into the CNS was dependent on integrin α3, and integrin α3 deficiency enhanced the retention of CD4 ⁺ T cells in the perivascular space of the blood-brain barrier. Integrin α3–dependent interactions continuously maintain T _H 17 cell identity and effector function. The requirement of integrin α3 in T _H 17 cell pathogenicity suggests integrin α3 as a therapeutic target for MS treatment.