The tissue-resident regulatory T cell pool is shaped by transient multi-tissue migration and a conserved residency program.

The tissues are the site of many important immunological reactions, yet how the immune system is controlled at these sites remains opaque. Recent studies have identified Foxp3+ regulatory T (Treg) cells in non-lymphoid tissues with unique characteristics compared with lymphoid Treg cells. However, tissue Treg cells have not been considered holistically across tissues. Here, we performed a systematic analysis of the Treg cell population residing in non-lymphoid organs throughout the body, revealing shared phenotypes, transient residency, and common molecular dependencies. Tissue Treg cells from different non-lymphoid organs shared T cell receptor (TCR) sequences, with functional capacity to drive multi-tissue Treg cell entry and were tissue-agnostic on tissue homing. Together, these results demonstrate that the tissue-resident Treg cell pool in most non-lymphoid organs, other than the gut, is largely constituted by broadly self-reactive Treg cells, characterized by transient multi-tissue migration. This work suggests common regulatory mechanisms may allow pan-tissue Treg cells to safeguard homeostasis across the body.

"IL-2 immunotherapy for targeting regulatory T cells in autoimmunity".

FOXP3+ regulatory T cells (Treg) are indispensable for immune homoeostasis and for the prevention of autoimmune diseases. Interleukin-2 (IL-2) signalling is critical in all aspects of Treg biology. Consequences of defective IL-2 signalling are insufficient numbers or dysfunction of Treg and hence autoimmune disorders in human and mouse. The restoration and maintenance of immune homoeostasis remain central therapeutic aims in the field of autoimmunity. Historically, broadly immunosuppressive drugs with serious side-effects have been used for the treatment of autoimmune diseases or prevention of organ-transplant rejection. More recently, ex vivo expanded or in vivo stimulated Treg have been shown to induce effective tolerance in clinical trials supporting the clinical benefit of targeting natural immunosuppressive mechanisms. Given the central role of exogenous IL-2 in Treg homoeostasis, a new and promising focus in drug development are IL-2-based approaches for in vivo targeted expansion of Treg or for enhancement of their suppressive activity. In this review, we summarise the role of IL-2 in Treg biology and consequences of dysfunctional IL-2 signalling pathways. We then examine evidence of efficacy of IL-2-based biological drugs targeting Treg with specific focus on therapeutic candidates in clinical trials and discuss their limitations.

Regulatory T-cell stability and functional plasticity in health and disease.

FOXP3-expressing regulatory T cells (Treg ) are indispensable for immune homeostasis and tolerance, and in addition tissue-resident Treg have been found to perform noncanonical, tissue-specific functions. For optimal tolerogenic function during inflammatory disease, Treg are equipped with mechanisms that assure lineage stability. Treg lineage stability is closely linked to the installation and maintenance of a lineage-specific epigenetic landscape, specifically a Treg -specific DNA demethylation pattern. At the same time, for local and directed immune regulation Treg must possess a level of functional plasticity that requires them to partially acquire T helper cell (TH ) transcriptional programs-then referred to as TH -like Treg . Unleashing TH programs in Treg , however, is not without risk and may threaten the epigenetic stability of Treg with consequently pathogenic ex-Treg contributing to (auto-) inflammatory conditions. Here, we review how the Treg -stabilizing epigenetic landscape is installed and maintained, and further discuss the development, necessity and lineage instability risks of TH 1-, TH 2-, TH 17-like Treg and follicular Treg .

Unstable regulatory T cells, enriched for naïve and Nrp1neg cells, are purged after fate challenge.

Regulatory T cells (Tregs) are indispensable for the control of immune homeostasis and have clinical potential as a cell therapy for treating autoimmunity. Tregs can lose expression of the lineage-defining Foxp3 transcription factor and acquire effector T cell (Teff) characteristics, a process referred to as Treg plasticity. The extent and reversibility of such plasticity during immune responses remain unknown. Here, using a murine genetic fate-mapping system, we show that Treg stability is maintained even during exposure to a complex microbial/antigenic environment. Furthermore, we demonstrate that the observed plasticity of Tregs after adoptive transfer into a lymphopenic environment is a property limited to only a subset of the Treg population, with the nonconverting majority of Tregs being resistant to plasticity upon secondary stability challenge. The unstable Treg fraction is a complex mixture of phenotypically distinct Tregs, enriched for naïve and neuropilin-1-negative Tregs, and includes peripherally induced Tregs and recent thymic emigrant Tregs These results suggest that a "purging" process can be used to purify stable Tregs that are capable of robust fate retention, with potential implications for improving cell transfer therapy.

A robust pipeline with high replication rate for detection of somatic variants in the adaptive immune system as a source of common genetic variation in autoimmune disease.

The role of somatic variants in diseases beyond cancer is increasingly being recognized, with potential roles in autoinflammatory and autoimmune diseases. However, as mutation rates and allele fractions are lower, studies in these diseases are substantially less tolerant of false positives, and bio-informatics algorithms require high replication rates. We developed a pipeline combining two variant callers, MuTect2 and VarScan2, with technical filtering and prioritization. Our pipeline detects somatic variants with allele fractions as low as 0.5% and achieves a replication rate of >55%. Validation in an independent data set demonstrates excellent performance (sensitivity > 57%, specificity > 98%, replication rate > 80%). We applied this pipeline to the autoimmune disease multiple sclerosis (MS) as a proof-of-principle. We demonstrate that 60% of MS patients carry 2-10 exonic somatic variants in their peripheral blood T and B cells, with the vast majority (80%) occurring in T cells and variants persisting over time. Synonymous variants significantly co-occur with non-synonymous variants. Systematic characterization indicates somatic variants are enriched for being novel or very rare in public databases of germline variants and trend towards being more damaging and conserved, as reflected by higher phred-scaled combined annotation-dependent depletion (CADD) and genomic evolutionary rate profiling (GERP) scores. Our pipeline and proof-of-principle now warrant further investigation of common somatic genetic variation on top of inherited genetic variation in the context of autoimmune disease, where it may offer subtle survival advantages to immune cells and contribute to the capacity of these cells to participate in the autoimmune reaction.

Abnormal differentiation of B cells and megakaryocytes in patients with Roifman syndrome.

BACKGROUND: Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12-type intron splicing, were identified recently as driving Roifman syndrome. OBJECTIVE: We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder. METHODS: We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages. RESULTS: The patients exhibited a dramatic reduction in B-cell numbers, with differentiation halted at the transitional B-cell stage. Despite abundant B-cell activating factor availability, development past this B-cell activating factor-dependent stage was crippled, with disturbed minor splicing of the critical mitogen-activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased α-granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively. CONCLUSION: Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome.

Defective germinal center B-cell response and reduced arthritic pathology in microRNA-29a-deficient mice.

MicroRNA (miR) are short non-coding RNA sequences of 19-24 nucleotides that regulate gene expression by binding to mRNA target sequences. The miR-29 family of miR (miR-29a, b-1, b-2 and c) is a key player in T-cell differentiation and effector function, with deficiency causing thymic involution and a more inflammatory T-cell profile. However, the relative roles of different miR-29 family members in these processes have not been dissected. We studied the immunological role of the individual members of the miR-29 family using mice deficient for miR-29a/b-1 or miR-29b-2/c in homeostasis and during collagen-induced arthritis. We found a definitive hierarchy of immunological function, with the strong phenotype of miR-29a-deficiency in thymic involution and T-cell activation being reduced or absent in miR-29c-deficient mice. Strikingly, despite elevating the Th1 and Th17 responses, loss of miR-29a conferred near-complete protection from collagen-induced arthritis (CIA), with profound defects in B-cell proliferation and antibody production. Our results identify the hierarchical structure of the miR-29 family in T-cell biology, and identify miR-29a in B cells as a potential therapeutic target in arthritis.

Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes.

Type 1 (T1D) and type 2 (T2D) diabetes share pathophysiological characteristics, yet mechanistic links have remained elusive. T1D results from autoimmune destruction of pancreatic beta cells, whereas beta cell failure in T2D is delayed and progressive. Here we find a new genetic component of diabetes susceptibility in T1D non-obese diabetic (NOD) mice, identifying immune-independent beta cell fragility. Genetic variation in Xrcc4 and Glis3 alters the response of NOD beta cells to unfolded protein stress, enhancing the apoptotic and senescent fates. The same transcriptional relationships were observed in human islets, demonstrating the role of beta cell fragility in genetic predisposition to diabetes.

The microRNA-29 Family Dictates the Balance Between Homeostatic and Pathological Glucose Handling in Diabetes and Obesity.

The microRNA-29 (miR-29) family is among the most abundantly expressed microRNA in the pancreas and liver. Here, we investigated the function of miR-29 in glucose regulation using miR-29a/b-1 (miR-29a)-deficient mice and newly generated miR-29b-2/c (miR-29c)-deficient mice. We observed multiple independent functions of the miR-29 family, which can be segregated into a hierarchical physiologic regulation of glucose handling. miR-29a, and not miR-29c, was observed to be a positive regulator of insulin secretion in vivo, with dysregulation of the exocytotic machinery sensitizing ß-cells to overt diabetes after unfolded protein stress. By contrast, in the liver both miR-29a and miR-29c were important negative regulators of insulin signaling via phosphatidylinositol 3-kinase regulation. Global or hepatic insufficiency of miR-29 potently inhibited obesity and prevented the onset of diet-induced insulin resistance. These results demonstrate strong regulatory functions for the miR-29 family in obesity and diabetes, culminating in a hierarchical and dose-dependent effect on premature lethality.

Fundamental properties of unperturbed haematopoiesis from stem cells in vivo.

Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation. Chromosomal marking, viral integration and barcoding of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2(+) HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse's life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated 'short-term' stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease.

Premature thymic involution is independent of structural plasticity of the thymic stroma.

The thymus is the organ devoted to T-cell production. The thymus undergoes multiple rounds of atrophy and redevelopment before degenerating with age in a process known as involution. This process is poorly understood, despite the influence the phenomenon has on peripheral T-cell numbers. Here we have investigated the FVB/N mouse strain, which displays premature thymic involution. We find multiple architectural and cellular features that precede thymic involution, including disruption of the epithelial-endothelial relationship and a progressive loss of pro-T cells. The architectural features, reminiscent of the human thymus, are intrinsic to the nonhematopoietic compartment and are neither necessary nor sufficient for thymic involution. By contrast, the loss of pro-T cells is intrinsic to the hematopoietic compartment, and is sufficient to drive premature involution. These results identify pro-T-cell loss as the main driver of premature thymic involution, and highlight the plasticity of the thymic stroma, capable of maintaining function across diverse interstrain architectures.

Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3⁺ and Foxp3⁻ T cells.

Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.

The thymic microenvironment differentially regulates development and trafficking of invariant NKT cell sublineages.

The regulatory role of the thymic microenvironment during trafficking and differentiation of the invariant NKT (iNKT) cell lineage remains poorly understood. In this study, we show that fractalkine receptor expression marks emigrating subpopulations of the NKT1, NKT2, and NKT17 sublineages in the thymus and peripheral organs of naive mice. Moreover, NKT1 sublineage cells can be subdivided into two subsets, namely NKT1(a) and NKT1(b), which exhibit distinct developmental and tissue-specific distribution profiles. More specifically, development and trafficking of the NKT1(a) subset are selectively dependent upon lymphotoxin (LT)α1ß2-LTß receptor-dependent differentiation of thymic stroma, whereas the NKT1(b), NKT2, and NKT17 sublineages are not. Furthermore, we identify a potential cellular source for LTα1ß2 during thymic organogenesis, marked by expression of IL-7Rα, which promotes differentiation of the NKT1(a) subset in a noncell-autonomous manner. Collectively, we propose a mechanism by which thymic differentiation and retention of the NKT1 sublineage are developmentally coupled to LTα1ß2-LTß receptor-dependent thymic organogenesis.

A novel Zap70 mutation with reduced protein stability demonstrates the rate-limiting threshold for Zap70 in T-cell receptor signalling.

Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3(+) regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70(A243V) variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70(rps) allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.

Mast cell chymase degrades the alarmins heat shock protein 70, biglycan, HMGB1, and interleukin-33 (IL-33) and limits danger-induced inflammation.

During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W(sash))-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3⁺ regulatory T cells.

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Smad3 binding to the foxp3 enhancer is dispensable for the development of regulatory T cells with the exception of the gut.

Regulatory T cells (T reg cells) are essential for the prevention of autoimmunity throughout life. T reg cell development occurs intrathymically but a subset of T reg cells can also differentiate from naive T cells in the periphery. In vitro, Smad signaling facilitates conversion of naive T cells into T reg cells but results in unstable Foxp3 expression. The TGF-ß-Smad response element in the foxp3 locus is located in the CNS1 region in close proximity to binding sites for transcription factors implicated in TCR and retinoic acid signaling. From in vitro experiments it was previously postulated that foxp3 transcription represents a hierarchical process of transcription factor binding in which Smad3 would play a central role in transcription initiation. However, in vitro conditions generate T reg cells that differ from T reg cells encountered in vivo. To address the relevance of Smad3 binding to the CNS1 enhancer in vivo, we generated mice that exclusively lack the Smad binding site (foxp3(CNS1mut)). We show that binding of Smad3 to the foxp3 enhancer is dispensable for T reg cell development in newborn and adult mice with the exception of the gut.

Thymus-autonomous T cell development in the absence of progenitor import.

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis.

Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin,a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation,whereas serglycin −/− MCs completely lacked this ability.Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist,which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex.Moreover, IL-13 degradation was abrogated in MC-CPA −/−MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein.Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.