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Mathis and colleagues show that the transcriptional regulator Aire preferentially localizes in and activates super-enhancers. Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. We found that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized to and activated long chromatin stretches that were overloaded with transcriptional regulators, known as super-enhancers. We also identified topoisomerase 1 as a cardinal Aire partner that colocalized on super-enhancers and was required for the interaction of Aire with all of its other associates. We propose a model that entails looping of super-enhancers to efficiently deliver Aire-containing complexes to local and distal transcriptional start sites.

A combination of transcription factors, enhancers, and epigenetic marks determines the expression of the key transcription factor FoxP3 in regulatory T cells (Tregs). Adding an additional layer of complexity, the long noncoding RNA (lncRNA) Flicr (Foxp3 long intergenic noncoding RNA) is a negative regulator that tunes Foxp3 expression, resulting in a subset of Tregs with twofold- to fivefold-lower levels of FoxP3 protein. The impact of Flicr is particularly marked in conditions of IL-2 deficiency, and, conversely, IL-2 represses Flicr expression. Flicr neighbors Foxp3 in mouse and human genomes, is specifically expressed in mature Tregs, and acts only in cis. It does not affect DNA methylation, but modifies chromatin accessibility in the conserved noncoding sequence 3 (CNS3)/Accessible region 5 (AR5) region of Foxp3. Like many lncRNAs, Flicr’s molecular effects are subtle, but by curtailing Treg activity, Flicr markedly promotes autoimmune diabetes and, conversely, restrains antiviral responses. This mechanism of FoxP3 control may allow escape from dominant Treg control during infection or cancer, at the cost of heightened autoimmunity.

Significance A unique population of Foxp3 ⁺CD4 ⁺ regulatory T (T ᵣₑg) cells resides in visceral adipose tissue of lean mice. VAT T ᵣₑgₛ are important regulators of local and systemic inflammation and metabolism. Here, we show that the VAT T ᵣₑg signature is imposed early in life, well before the typical age-dependent expansion of the adipose-tissue T ᵣₑg population. VAT T ᵣₑgₛ in obese mice lose the signature typical of lean individuals but gain an additional set of over- and underrepresented transcripts. In striking parallel to a pathway recently elucidated in adipocytes, the obese mouse VAT T ᵣₑg signature depends on phosphorylation of a specific residue of PPARγ. These findings are important to consider in designing drugs to target type 2 diabetes and other features of the “metabolic syndrome.” A unique population of Foxp3 ⁺CD4 ⁺ regulatory T (T ᵣₑg) cells resides in visceral adipose tissue (VAT) of lean mice, especially in the epididymal fat depot. VAT T ᵣₑgₛ are unusual in their very high representation within the CD4 ⁺ T-cell compartment, their transcriptome, and their repertoire of antigen-specific T-cell receptors. They are important regulators of local and systemic inflammation and metabolism. The overall goal of this study was to learn how the VAT T ᵣₑg transcriptome adapts to different stimuli; in particular, its response to aging in lean mice, to metabolic perturbations associated with obesity, and to certain signaling events routed through PPARγ, the “master-regulator” of adipocyte differentiation. We show that the VAT T ᵣₑg signature is imposed early in life, well before age-dependent expansion of the adipose-tissue T ᵣₑg population. VAT T ᵣₑgₛ in obese mice lose the signature typical of lean individuals but gain an additional set of over- and underrepresented transcripts. This obese mouse VAT T ᵣₑg signature depends on phosphorylation of the serine residue at position 273 of PPARγ, in striking parallel to a pathway recently elucidated in adipocytes. These findings are important to consider in designing drugs to target type 2 diabetes and other features of the “metabolic syndrome.”

Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity. Differentiating neutrophil functional states is difficult. Here the authors show, using single cell RNA-sequencing and trajectory analyses, that mouse neutrophils can be presented as a transcriptome continuum rather than discrete subsets, but are affected by inflammation to express distinct transcriptional states.

FoxP3⁺ T regulatory (Treg) cells are central elements of immunologic tolerance. They are abundant in many tumors, where they restrict potentially favorable antitumor responses. We used a three-pronged strategy to identify genes related to the presence and function of Tregs in the tumor microenvironment. Gene expression profiles were generated from tumor-infiltrating Tregs (TITRs) of both human and mouse tumors and were compared with those of Tregs of lymphoid organs or normal tissues from the same individuals. A computational deconvolution of whole-tumor datasets from the Cancer Genome Atlas (TCGA) was performed to identify transcripts specifically associated with Tregs across thousands of tumors from different stages and locations. We identified a set of TITR-differential transcripts with striking reproducibility between tumor types in mice, between mice and humans, and between different human patients spanning tumor stages. Many of the TITR-preferential transcripts were shared with “tissue Tregs” residing in nonlymphoid tissues, but a tumor-preferential segment could be identified. Many of these TITR signature transcripts were confirmed by mining of TCGA datasets, which also brought forth transcript modules likely representing the parenchymal attraction of, or response to, tumor Tregs. Importantly, the TITR signature included several genes encoding effective targets of tumor immunotherapy. A number of other targets were validated by CRISPR-based gene inactivation in mouse Tregs. These results confirm the validity of the signature, generating a wealth of leads for understanding the role of Tregs in tumor progression and identifying potential targets for cancer immunotherapy.

Vertebrates typically harbor a rich gastrointestinal microbiota, which has coevolved with the host over millennia and is essential for several host physiological functions, in particular maturation of the immune system. Recent studies have highlighted the importance of a single bacterial species, segmented filamentous bacteria (SFB), in inducing a robust T-helper cell type 17 (Th17) population in the small-intestinal lamina propria (SI-LP) of the mouse gut. Consequently, SFB can promote IL-17-dependent immune and autoimmune responses, gut-associated as well as systemic, including inflammatory arthritis and experimental autoimmune encephalomyelitis. Here, we exploit the incomplete penetrance of SFB colonization of NOD mice in our animal facility to explore its impact on the incidence and course of type 1 diabetes in this prototypical, spontaneous model. There was a strong cosegregation of SFB positivity and diabetes protection in females, but not in males, which remained relatively disease-free regardless of the SFB status. In contrast, insulitis did not depend on SFB colonization. SFB-positive, but not SFB-negative, females had a substantial population of Th17 cells in the SI-LP, which was the only significant, repeatable difference in the examined T-cell compartments of the gut, pancreas, or systemic lymphoid tissues. Th17-signature transcripts dominated the very limited SFB-induced molecular changes detected in SI-LP CD4⁺ T cells. Thus, a single bacterium, and the gut immune system alterations associated with it, can either promote or protect from autoimmunity in predisposed mouse models, probably reflecting their variable dependence on different Th subsets.

Given mounting evidence of the importance of gut-microbiota/immune-cell interactions in immune homeostasis and responsiveness, surprisingly little is known about leukocyte movements to, and especially from, the gut. We address this topic in a minimally perturbant manner using Kaede transgenic mice, which universally express a photoconvertible fluorescent reporter. Transcutaneous exposure of the cervical lymph nodes to violet light permitted punctual tagging of immune cells specifically therein, and subsequent monitoring of their immigration to the intestine; endoscopic flashing of the descending colon allowed specific labeling of intestinal leukocytes and tracking of their emigration. Our data reveal an unexpectedly broad movement of leukocyte subsets to and from the gut at steady state, encompassing all lymphoid and myeloid populations examined. Nonetheless, different subsets showed different trafficking proclivities (e.g., regulatory T cells were more restrained than conventional T cells in their exodus from the cervical lymph nodes). The novel endoscopic approach enabled us to evidence gut-derived Th17 cells in the spleens of K/BxN mice at the onset of their genetically determined arthritis, thereby furnishing a critical mechanistic link between the intestinal microbiota, namely segmented filamentous bacteria, and an extraintestinal autoinflammatory disease.

There is increasing appreciation of the important role of B cells in many autoimmune diseases and consequently, increasing interest in treating these disorders through B cell-depletion therapy with rituximab, an anti-CD20 monoclonal antibody. Yet, precisely how this and related drugs exert their therapeutic effects remains controversial. In particular, it is unclear how, in a number of contexts, rituximab can greatly reduce the titer of serum autoantibodies without substantially altering the overall antibody titer. We have studied the action of this drug in the K/BxN mouse model of inflammatory arthritis after first crossing in a human CD20 transgene. Rituximab treatment of these mice led to a decrease in the titer of serum antibodies targeting glucose-6-phosphate isomerase, the relevant autoantigen, but not in the total antibody titer. Glucose-6-phosphate isomerase-specific plasma cells did not reside primarily in the bone marrow as expected but rather in the spleen and lymph nodes, where they had short lives, expressed CD20, and were rapidly depleted by rituximab. These data support a model whereby autoreactive plasma cells (at least certain specificities thereof) are intrinsically different from protective antimicrobial plasma cells in their differentiation, migration, and survival properties. Rituximab targets the former and spares the latter.

The functionality of these Tregs is retained, and the enhanced production of IL-10 mediates a protective effect in a colitis model using T cell transfer. Conclusions The articles in this Research Topic illustrate that tissue-localized Tregs are far more than static suppressors of immunity: they are dynamic, context-dependent players that integrate transcriptional, genetic, and environmental cues to shape outcomes across health and disease. Conflict of interest Authors BS and AZ are employed by TRex Bio, Inc. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Costimulatory molecules of the CD28 family on T lymphocytes integrate cues from innate immune system sensors and modulate activation responses in conventional CD4 ⁺ T cells (Tconv) and their FoxP3 ⁺ regulatory counterparts (Treg). To better understand how costimulatory and coinhibitory signals might be integrated, we profiled the changes in gene expression elicited in the hours and days after engagement of Treg and Tconv by anti-CD3 and either anti-CD28, -CTLA4, -ICOS, -PD1, -BLA, or -CD80. In Tconv, a shared “main response” was induced by CD28, ICOS, and, surprisingly, BTLA and CD80, with very limited CD28-specific (primarily Il2) or ICOS-specific elements (including Th1 and Th2 but not the follicular T signature). CTLA4 and PD1 had a very subtle impact in this system, similarly inhibiting the response to anti-CD3. Treg responded to the same costimulatory hierarchy and to the same extent as Tconv, but inducing different clusters of genes. In this reductionist system, costimulatory or coinhibitory engagement mainly elicits generic responses, suggesting that the variability of their effects in vivo result from temporal or anatomical differences in their engagement, rather than from inherently different wiring.