Thymic regulatory T cells arise via two distinct developmental programs.

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

CD8αα intraepithelial lymphocytes arise from two main thymic precursors.

TCRαß+CD4-CD8α+CD8ß- intestinal intraepithelial lymphocytes (CD8αα IELs) are an abundant population of thymus-derived T cells that protect the gut barrier surface. We sought to better define the thymic IEL precursor (IELp) through analysis of its maturation, localization and emigration. We defined two precursor populations among TCRß+CD4-CD8- thymocytes by dependence on the kinase TAK1 and rigorous lineage-exclusion criteria. Those IELp populations included a nascent PD-1+ population and a T-bet+ population that accumulated with age. Both gave rise to intestinal CD8αα IELs after adoptive transfer. The PD-1+ IELp population included more strongly self-reactive clones and was largely restricted by classical major histocompatibility complex (MHC) molecules. Those cells localized to the cortex and efficiently emigrated in a manner dependent on the receptor S1PR1. The T-bet+ IELp population localized to the medulla, included cells restricted by non-classical MHC molecules and expressed the receptor NK1.1, the integrin CD103 and the chemokine receptor CXCR3. The two IELp populations further differed in their use of the T cell antigen receptor (TCR) α-chain variable region (Vα) and ß-chain variable region (Vß). These data provide a foundation for understanding the biology of CD8αα IELs.

Expansible residence decentralizes immune homeostasis.

In metazoans, specific tasks are relegated to dedicated organs that are established early in development, occupy discrete locations and typically remain fixed in size. The adult immune system arises from a centralized haematopoietic niche that maintains self-renewing potential1,2, and-upon maturation-becomes distributed throughout the body to monitor environmental perturbations, regulate tissue homeostasis and mediate organism-wide defence. Here we examine how immunity is integrated within adult mouse tissues, and address issues of durability, expansibility and contributions to organ cellularity. Focusing on antiviral T cell immunity, we observed durable maintenance of resident memory T cells up to 450 days after infection. Once established, resident T cells did not require the T cell receptor for survival or retention of a poised, effector-like state. Although resident memory indefinitely dominated most mucosal organs, surgical separation of parabiotic mice revealed a tissue-resident provenance for blood-borne effector memory T cells, and circulating memory slowly made substantial contributions to tissue immunity in some organs. After serial immunizations or cohousing with pet-shop mice, we found that in most tissues, tissue pliancy (the capacity of tissues to vary their proportion of immune cells) enables the accretion of tissue-resident memory, without axiomatic erosion of pre-existing antiviral T cell immunity. Extending these findings, we demonstrate that tissue residence and organ pliancy are generalizable aspects that underlie homeostasis of innate and adaptive immunity. The immune system grows commensurate with microbial experience, reaching up to 25% of visceral organ cellularity. Regardless of the location, many populations of white blood cells adopted a tissue-residency program within nonlymphoid organs. Thus, residence-rather than renewal or recirculation-typifies nonlymphoid immune surveillance, and organs serve as pliant storage reservoirs that can accommodate continuous expansion of the cellular immune system throughout life. Although haematopoiesis restores some elements of the immune system, nonlymphoid organs sustain an accrual of durable tissue-autonomous cellular immunity that results in progressive decentralization of organismal immune homeostasis.

Novel antiinflammatory biologics shaped by parasite-host coevolution.

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus.

TCRαß+ CD8α+ CD8ß- intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely ß2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+ . However, selective depletion of ß2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.

Harnessing helminth-driven immunoregulation in the search for novel therapeutic modalities.

Parasitic helminths have coevolved with humans over millennia, intricately refining and developing an array of mechanisms to suppress or skew the host's immune system, thereby promoting their long-term survival. Some helminths, such as hookworms, cause little to no overt pathology when present in modest numbers and may even confer benefits to their human host. To exploit this evolutionary phenomenon, clinical trials of human helminth infection have been established and assessed for safety and efficacy for a range of immune dysfunction diseases and have yielded mixed outcomes. Studies of live helminth therapy in mice and larger animals have convincingly shown that helminths and their excretory/secretory products possess anti-inflammatory drug-like properties and represent an untapped pharmacopeia. These anti-inflammatory moieties include extracellular vesicles, proteins, glycans, post-translational modifications, and various metabolites. Although the concept of helminth-inspired therapies holds promise, it also presents a challenge to the drug development community, which is generally unfamiliar with foreign biologics that do not behave like antibodies. Identification and characterization of helminth molecules and vesicles and the molecular pathways they target in the host present a unique opportunity to develop tailored drugs inspired by nature that are efficacious, safe, and have minimal immunogenicity. Even so, much work remains to mine and assess this out-of-the-box therapeutic modality. Industry-based organizations need to consider long-haul investments aimed at unraveling and exploiting unique and differentiated mechanisms of action as opposed to toe-dipping entries with an eye on rapid and profitable turnarounds.

RelB-Deficient Dendritic Cells Promote the Development of Spontaneous Allergic Airway Inflammation.

RelB is a member of the NF-κB family, which is essential for dendritic cell (DC) function and maturation. However, the contribution of RelB to the development of allergic airway inflammation (AAI) is unknown. Here, we identify a pivotal role for RelB in the development of spontaneous AAI that is independent of exogenous allergen exposure. We assessed AAI in two strains of RelB-deficient (RelB-/-) mice: one with a targeted deletion and one expressing a major histocompatibility complex transgene. To determine the importance of RelB in DCs, RelB-sufficient DCs (RelB+/+ or RelB-/-) were adoptively transferred into RelB-/- mice. Both strains had increased pulmonary inflammation compared with their respective wild-type (RelB+/+) and heterozygous (RelB+/-) controls. RelB-/- mice also had increased inflammatory cell influx into the airways, levels of chemokines (CCL2/3/4/5/11/17 and CXCL9/10/13) and T-helper cell type 2-associated cytokines (IL-4/5) in lung tissues, serum IgE, and airway remodeling (mucus-secreting cell numbers, collagen deposition, and epithelial thickening). Transfer of RelB+/- CD11c+ DCs into RelB-/- mice decreased pulmonary inflammation, with reductions in lung chemokines, T-helper cell type 2-associated cytokines (IL-4/5/13/25/33 and thymic stromal lymphopoietin), serum IgE, type 2 innate lymphoid cells, myeloid DCs, γδ T cells, lung Vß13+ T cells, mucus-secreting cells, airway collagen deposition, and epithelial thickening. These data indicate that RelB deficiency may be a key pathway underlying AAI, and that DC-encoded RelB is sufficient to restore control of this inflammation.

Solution structure of the N-terminal extension domain of a Schistosoma japonicum asparaginyl-tRNA synthetase.

Several secreted proteins from helminths (parasitic worms) have been shown to have immunomodulatory activities. Asparaginyl-tRNA synthetases are abundantly secreted in the filarial nematode Brugia malayi (BmAsnRS) and the parasitic flatworm Schistosoma japonicum (SjAsnRS), indicating a possible immune function. The suggestion is supported by BmAsnRS alleviating disease symptoms in a T-cell transfer mouse model of colitis. This immunomodulatory function is potentially related to an N-terminal extension domain present in eukaryotic AsnRS proteins but few structure/function studies have been done on this domain. Here we have determined the three-dimensional solution structure of the N-terminal extension domain of SjAsnRS. A protein containing the 114 N-terminal amino acids of SjAsnRS was recombinantly expressed with isotopic labelling to allow structure determination using 3D NMR spectroscopy, and analysis of dynamics using NMR relaxation experiments. Structural comparisons of the N-terminal extension domain of SjAsnRS with filarial and human homologues highlight a high degree of variability in the ß-hairpin region of these eukaryotic N-AsnRS proteins, but similarities in the disorder of the C-terminal regions. Limitations in PrDOS-based intrinsically disordered region (IDR) model predictions were also evident in this comparison. Empirical structural data such as that presented in our study for N-SjAsnRS will enhance the prediction of sequence-homology based structure modelling and prediction of IDRs in the future.Communicated by Ramaswamy H. Sarma.

TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αß and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

Antioxidant and Anti-Inflammatory Activities of Endemic Plants of the Australian Wet Tropics.

Plants have been a vital source of natural antioxidants since ancient times. Plants growing under various abiotic stress conditions often produce more defensive secondary metabolites such as phenolics, flavonoids, and terpenoids during adaptation to the environment. Many of these secondary metabolites are known to possess antioxidant and anti-inflammatory properties. This study tested seven plants sourced from the mountaintop areas (above 1000 m elevation) of Mount Lewis National Park (falls under the Wet Tropics of Queensland), Australia, for their antioxidant and anti-inflammatory activities. Of the seven studied plants, hydroethanolic extracts of six plants (Leptospermum wooroonooran, Ceratopetalum hylandii, Linospadix apetiolatus, Garcinia brassii, Litsea granitica, and Polyscias willmottii) showed high 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging activity in a dose-dependent (25-1000 µg/mL) manner. At the highest concentration of 1 mg/mL, the DPPH free radical scavenged percentage varied between 75.4% and 92.3%. Only the species Alyxia orophila was inactive in the DPPH free radical scavenging assay. Pseudo-IC50 values of the extracts' ferric reducing antioxidant power (FRAP) based on dose-response curves showed a significant positive correlation with total phenolic content. Five out of the seven plants, namely G. brassii, C. hylandii, L. apetiolatus, L. wooroonooran, and A. orophila, showed inhibitory effects on the secretion of proinflammatory cytokines, tumour necrosis factor (TNF), and interleukins (IL)-23 in a lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) assay. The results of this study demonstrate the value of tropical mountaintop plants in the biodiscovery of antioxidant and anti-inflammatory lead compounds.

Anti-inflammatory properties of novel galloyl glucosides isolated from the Australian tropical plant Uromyrtus metrosideros.

Two new galloyl glucosides, galloyl-lawsoniaside A (4) and uromyrtoside (6), were isolated from the polar fraction of Uromyrtus metrosideros leaf extract along with another four previously identified phytochemicals (1, 2, 3, and 5). The structures of these six compounds were characterised using low and high-resolution mass spectrometry (L/HRMS) and 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy. These compounds were not toxic to human peripheral blood mononuclear cells (PBMCs) at 10 µg/mL over 24 h, yet showed significant in vitro suppression of proinflammatory cytokines involved in the pathogenesis of inflammatory bowel disease (IBD). Specifically, the release of interferon γ (IFN-γ), interleukin (IL)-17A, and IL-8 from phorbol myristate acetate/ionomycin (P/I) and anti-CD3/anti-CD28-activated cells were significantly suppressed by compounds 4 and 5. Interestingly, no effect on tumour necrosis factor (TNF) release was observed. These results show that the newly characterised compound 4 has promising cytokine suppressive properties, which could be further investigated as a candidate for IBD treatment.

Peptides derived from hookworm anti-inflammatory proteins suppress inducible colitis in mice and inflammatory cytokine production by human cells.

A decline in the prevalence of parasites such as hookworms appears to be correlated with the rise in non-communicable inflammatory conditions in people from high- and middle-income countries. This correlation has led to studies that have identified proteins produced by hookworms that can suppress inflammatory bowel disease (IBD) and asthma in animal models. Hookworms secrete a family of abundant netrin-domain containing proteins referred to as AIPs (Anti-Inflammatory Proteins), but there is no information on the structure-function relationships. Here we have applied a downsizing approach to the hookworm AIPs to derive peptides of 20 residues or less, some of which display anti-inflammatory effects when co-cultured with human peripheral blood mononuclear cells and oral therapeutic activity in a chemically induced mouse model of acute colitis. Our results indicate that a conserved helical region is responsible, at least in part, for the anti-inflammatory effects. This helical region has potential in the design of improved leads for treating IBD and possibly other inflammatory conditions.

A netrin domain-containing protein secreted by the human hookworm Necator americanus protects against CD4 T cell transfer colitis.

The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases.

Revisiting Inflammatory Bowel Disease: Pathology, Treatments, Challenges and Emerging Therapeutics Including Drug Leads from Natural Products.

Inflammatory bowel disease (IBD) is a chronic and life-long disease characterized by gastrointestinal tract inflammation. It is caused by the interplay of the host's genetic predisposition and immune responses, and various environmental factors. Despite many treatment options, there is no cure for IBD. The increasing incidence and prevalence of IBD and lack of effective long-term treatment options have resulted in a substantial economic burden to the healthcare system worldwide. Biologics targeting inflammatory cytokines initiated a shift from symptomatic control towards objective treatment goals such as mucosal healing. There are seven monoclonal antibody therapies excluding their biosimilars approved by the US Food and Drug Administration for induction and maintenance of clinical remission in IBD. Adverse side effects associated with almost all currently available drugs, especially biologics, is the main challenge in IBD management. Natural products have significant potential as therapeutic agents with an increasing role in health care. Given that natural products display great structural diversity and are relatively easy to modify chemically, they represent ideal scaffolds upon which to generate novel therapeutics. This review focuses on the pathology, currently available treatment options for IBD and associated challenges, and the roles played by natural products in health care. It discusses these natural products within the current biodiscovery research agenda, including the applications of drug discovery techniques and the search for next-generation drugs to treat a plethora of inflammatory diseases, with a major focus on IBD.

Intestinal CD8αα IELs derived from two distinct thymic precursors have staggered ontogeny.

CD8αα intraepithelial lymphocytes (IELs) are abundant T cells that protect the gut epithelium. Their thymic precursors (IELps) include PD-1+ type A and Tbet+ type B populations, which differ in their antigen-receptor specificities. To better understand CD8αα IEL ontogeny, we performed "time-stamp" fate mapping experiments and observed that it seeds the intestine predominantly during a narrow time window in early life. Adoptively transferred IELps parked better in the intestines of young mice than in adults. In young mice, both type A and type B IELps had an S1PR1+ and α4ß7+ emigration- and mucosal-homing competent phenotype, while this was restricted to type A IELps in adults. Only CD8αα IELs established in early life were enriched in cells bearing type B IELp TCR usage. Together, our results suggest that the young intestine facilitates CD8αα IEL establishment and that early IELs are distinct from IELs established after this initial wave. These data provide novel insight into the ontogeny of CD8αα IELs.

Development, ontogeny, and maintenance of TCRαß+ CD8αα IEL.

The intestinal epithelium is the outermost cellular layer that separates the body from the gut lumen. The integrity of this protective mucosal barrier is crucial and maintained by specialized cells-intraepithelial lymphocytes (IEL). Much research has been conducted on these cells and our overall understanding of them is increasing rapidly. In this review we focus on the TCRαß+ subset of CD8αα IEL. We discuss recent studies that shed light on the development, ontogeny, maintenance, and functional characteristics of CD8αα IEL, and highlight yet unanswered questions for future studies.

Intravenous Labeling and Analysis of the Content of Thymic Perivascular Spaces.

Following development in the thymus, T cells are thought to exit into the periphery predominantly through perivascular spaces (PVS). This exit route is used by conventional T cells, and likely also applies to unconventional T cell subsets, such as precursors of CD8αα and TCRγδ intraepithelial lymphocytes, regulatory T cells and natural killer T cells. Additional cell types might also be found in the PVS and initiate interactions with exiting T cells. The exact content of the PVS, and the processes within, are not well studied. To distinguish vascular from resident cells within various tissues by flow cytometry, intravenous (i.v.) labeling is becoming a commonly employed method. We recently used anti-CD45.2 antibodies and magnetic enrichment to further evaluate this technique, and compared labeled and unlabeled cells in the thymus and blood. This assay can be used to specifically investigate hematopoietic cell subsets within the PVS of the thymus.

Autoimmune-Mediated Thymic Atrophy Is Accelerated but Reversible in RelB-Deficient Mice.

Polymorphisms impacting thymic function may decrease peripheral tolerance and hasten autoimmune disease. The NF-κB transcription factor subunit, RelB, is essential for the development and differentiation of medullary thymic epithelial cells (mTECs): RelB-deficient mice have reduced thymic cellularity and markedly fewer mTECs, lacking AIRE. The precise mechanism of this mTEC reduction in the absence of RelB is unclear. To address this, we studied mTECs and dendritic cells (DCs), which critically regulate negative selection, and thymic regulatory T-cells (tTreg) in RelB-/- mice, which have spontaneous multiorgan autoimmune disease. RelB-/- thymi were organized, with medullary structures containing AIRE- mTECs, DCs, and CD4+ thymocytes, but fewer tTreg. Granulocytes infiltrated the RelB-/- thymic cortex, capsule, and medulla, producing inflammatory thymic medullary atrophy, which could be treated by granulocyte depletion or RelB+ DC immunotherapy, with concomitant recovery of mTEC and tTreg numbers. These data indicate that central tolerance defects may be accelerated by autoimmune thymic inflammation where impaired RelB signaling impairs the medullary niche, and may be reversible by therapies enhancing peripheral Treg or suppressing inflammation.