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Variation in food quality and abundance requires animals to decide whether to stay on a poor food patch or leave in search of better food. An important question in behavioral ecology asks when is it optimal for an animal to leave a food patch it is depleting. Although optimal foraging is central to evolutionary success, the neural and molecular mechanisms underlying it are poorly understood. Here we investigate the neuronal basis for adaptive food-leaving behavior in response to resource depletion in Caenorhabditis elegans, and identify several of the signaling pathways involved. The ASE neurons, previously implicated in salt chemoattraction, promote food-leaving behavior via a cGMP pathway as food becomes limited. High ambient O2 promotes food-leaving via the O2-sensing neurons AQR, PQR, and URX. Ectopic activation of these neurons using channelrhodopsin is sufficient to induce high food-leaving behavior. In contrast, the neuropeptide receptor NPR-1, which regulates social behavior on food, acts in the ASE neurons, the nociceptive ASH neurons, and in the RMG interneuron to repress food-leaving. Finally, we show that neuroendocrine signaling by TGF-β/DAF-7 and neuronal insulin signaling are necessary for adaptive food-leaving behavior. We suggest that animals integrate information about their nutritional state with ambient oxygen and gustatory stimuli to formulate optimal foraging strategies.

Regulatory T cells (Tregs) hold promise for treating autoimmune disease and transplant rejection, yet generation of autologous products for adoptive transfer can suffer donor variability and slow turnaround, limiting their use in urgent indications. We therefore examine whether allogeneic, pre-manufactured (‘off-the-shelf’) Tregs could overcome these barriers. In a human skin-xenograft model, HLA-mismatched Tregs are swiftly eliminated by recipient CD8 + T cells and fail to protect grafts. Stringent matching of HLA class I and II restores efficacy but is clinically impractical. Using non-viral CRISPR editing we disrupt B2M and CIITA while inserting an HLA-E- B2M fusion, generating hypo-immunogenic Tregs that evade both T and NK cell attack. Engineered cells retain FOXP3 stability and potent in vitro suppression, and after a single low-dose infusion, prolong human skin graft survival in a humanized mouse model comparably to autologous Tregs. Histology and spatial transcriptomics reveal minimal cytotoxic infiltration and enrichment of immunoregulatory and tissue-repair programmes. Multiplex HLA engineering thus enables ready-to-use allogeneic Tregs that withstand host immune attack for adoptive transfer. Adoptive regulatory T cell (Treg) therapy holds promise for the treatment of a range of immunopathological conditions. Here the authors explore the HLA engineering of allogenic Treg products that avoid T cell and NK cell attack and maintain immunomodulatory function in a human skin-xenograft model.

Regulatory T cell (Treg) therapy for immune modulation is a promising therapeutic strategy for the treatment and prevention of autoimmune disease and graft-versus-host disease (GvHD) after bone marrow transplantation. However, Treg are heterogeneous and express a variety of chemokine receptor molecules. The optimal subpopulation of Treg for therapeutic use may vary according to the pathological target. Indeed, clinical trials of Treg for the prevention of GvHD where the skin is a major target of the anti-host response have employed Treg derived from a variety of different sources. We postulated that for the effective treatment of GvHD-related skin pathology, Treg must be able to migrate to skin in order to regulate local alloimmune responses efficiently. To test the hypothesis that different populations of Treg display distinct efficacy in vivo based on their expression of tissue-specific homing molecules, we evaluated the activity of human Treg derived from two disparate sources in a model of human skin transplantation. Treg were derived from adult blood or cord blood and expanded in vitro. While Treg from both sources displayed similar in vitro suppressive efficacy, they exhibited marked differences in the expression of skin homing molecules. Importantly, only adult-derived Treg were able to prevent alloimmune-mediated human skin destruction in vivo, by virtue of their improved migration to skin. The presence of Treg within the skin was sufficient to prevent its alloimmune-mediated destruction. Additionally, Treg expressing the skin homing cutaneous lymphocyte antigen (CLA) were more efficient at preventing skin destruction than their CLA-deficient counterparts. Our findings highlight the importance of the careful selection of an effective subpopulation of Treg for clinical use according to the pathology of interest.

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

Regulatory T cells (Tregs) can control excessive or undesirable immune responses toward autoantigens, alloantigens, and pathogens. In transplantation, host immune responses against the allograft are suppressed through the use of immunosuppressive drugs, however this often results in life-threatening side effects including nephrotoxicity and an increased incidence of cancer and opportunistic infections. Tregs can control graft-vs.-host disease and transplant rejection in experimental models, providing impetus for the use of Tregs as a cellular therapy in clinical transplantation. One of the major barriers to the widespread use of Treg cellular therapy is the requirement to expand cells to large numbers in order to alter the overall balance between regulatory and effector cells. Methods that enhance suppressive capacity thereby reducing the need for expansion are therefore of interest. Here, we have compared the function of freshly-isolated and -manipulated human Tregs in a pre-clinical humanized mouse model of skin transplantation. Sorted human CD127 CD25 CD4 Tregs were assessed in three different conditions: freshly-isolated, following transient activation with antiCD3/antiCD28 beads or after -expansion for 2 weeks in the presence of antiCD3/antiCD28 beads and recombinant human IL2. While -expansion of human Tregs increased their suppressive function moderately, transient -activation of freshly isolated Tregs resulted in a powerful enhancement of Treg activity sufficient to promote long-term graft survival of all transplants . In order to investigate the mechanisms responsible for these effects, we measured the expression of Treg-associated markers and susceptibility to apoptosis in activated Tregs. Transiently activated Tregs displayed enhanced survival and proliferation and . On a molecular level, Treg activation resulted in an increased expression of anti-apoptotic (encoding BCL-XL) which may be at least partially responsible for the observed enhancement in function. Our results suggest that activation of human Tregs arms them with superior proliferative and survival abilities, enabling them to more effectively control alloresponses. Importantly, this transient activation results in a rapid functional enhancement of freshly-isolated Tregs, thereby providing an opportunity to eliminate the need for expansion in select circumstances. A protocol employing this technique would therefore benefit from a reduced requirement for large cell numbers for effective therapy.

In non-small cell lung cancer (NSCLC), stroma-resident and tumour-infiltrating macrophages may facilitate an immunosuppressive tumour microenvironment (TME) and hamper immunotherapeutic responses. Analysis of tumour-associated macrophage (TAM) plasticity in NSCLC is largely lacking. We established a novel, multi-marker, dual analysis approach for assessing monocyte-derived macrophage (Mφ) polarisation and M1/M2 phenotypic plasticity. We developed a flow cytometry-based, two-marker analysis (CD64 and CD206) of CD14+ cells. The phenotype and immune function of in vitro-induced TAMs was studied in a heterotypic spheroid and tumour-derived explant model of NSCLC. Heterotypic spheroids and NSCLC explants skewed Mφs from an M1- (CD206loCD64hi) to M2-like (CD206hiCD64lo) phenotype. Lipopolysaccharide (LPS) and IFNγ treatment reversed M2-like Mφ polarisation, indicating the plasticity of Mφs. Importantly, antigen-specific CD8+ T cell responses were reduced in the presence of tumour explant-conditioned Mφs, but not spheroid-conditioned Mφs, suggesting explants are likely a more relevant model of the immune TME than cell line-derived spheroids. Our data indicates the importance of multi-marker, functional analyses within Mφ subsets and the advantages of the ex vivo NSCLC explant model in immunomodulation studies. We highlight the plasticity of the M1/M2 phenotype using the explant model and provide a tool for studying therapeutic interventions designed to reprogram M2-like Mφ-induced immunosuppression.

This research aimed to explore the trainee perspective on factors affecting recruitment into old age psychiatry higher training in the UK. A qualitative survey was created by the Faculty of Old Age Psychiatry and distributed to current higher trainees in all psychiatric subspecialties. A total of 324 higher trainees responded to the survey, representing a broad demographic range. Thematic analysis was carried out, with sufficient responses to achieve saturation. Key themes included concerns about the future of the specialty, issues with social care, lack of support with patients’ physical health needs, issues with training posts, and workload. The need to improve core trainees’ experience of the specialty was highlighted. Many positive themes arose from the data; however, a number of barriers to recruitment were also identified. The findings have implications for recruitment to the specialty and should be used to inform recruitment strategy moving forward.

Regulatory T cells (Tregs), lymphocytes that suppress immunological reactions, are of great interest for our comprehension of basic immunology and as a therapeutic agent to treat immune-mediated pathologies. Understanding the physiology of these cells will help to inform clinical strategies targeting Tregs. In order to study the homing of human Tregs, we utilised genetic engineering to drive expression of fluorescent protein in human Tregs, permitting in vivo cell tracking. We optimised a protocol for lentivirus-mediated transduction of human Tregs during in vitro expansion, to generate high yields of stably-engineered cells. After infusing labelled cells into a humanised mouse model of skin allotransplantation, we detected human Tregs within a human skin graft by PCR and visualised Tregs moving in the graft, in a live mouse, by two-photon microscopy. Through reverse genetic analyses, we explored molecular mechanisms that allow Tregs to respond adaptively to environmental cues. Neuropilin-1 (NRP1), a transmembrane co-receptor, has been implicated in the function of mouse Tregs. Tregs transduced with shRNA to knock down NRP1 were severely impaired in their capacity to suppress cell proliferation in vitro and to prolong allograft survival in a humanised mouse model. qRT-PCR analysis revealed that transcription the gene encoding the anti-inflammatory cytokine IL-10, and the autophagy-associated genes BECN1, COPS4 and MAP1LC3B, was significantly diminished in NRP1-deficient Tregs. We concluded that in human Tregs, NRP1 is necessary for suppressive function, most likely via regulation of NRP1-dependent regulation of cytokine production and metabolism. Having identified a molecular target via which Treg function might be potentiated, we explored methods to target such molecules for cell therapy applications. Tregs engineered to over-express IL-10, but not NRP1, exerted significantly enhanced suppression of cell proliferation in vitro. Thus, relatively straightforward genetic engineering, compatible with generation of therapeutic cell yields, could be exploited to improve the efficacy of Treg cellular therapy.

The potential to harness regulatory T cells (Tregs) for the treatment of autoimmune diseases and transplant rejection has been restricted by several barriers: donor variability, manufacturing complications, and time-consuming expansion processes. These issues further complicate the use of autologous Tregs during acute disease phases or when Tregs are low in number or dysfunctional. Here we explore the potential of ‘off-the-shelf’ allogeneic Tregs, from healthy donors or universal sources, to provide a more practical solution. We discover that the efficacy of these cells is undermined by the recipient’s immune response, and that that rigorous matching of HLA classes I and II overcomes this barrier. Importantly, genetically manipulating HLA expression enables the use of unmatched allogeneic Tregs with in vivo efficacy. Our findings underscore the transformative potential of HLA-engineered Tregs, offering a novel, ready-to-use therapeutic avenue for treating a wide array of inflammatory diseases. Matching or engineering of HLA-I and HLA-II facilitates allogeneic ‘off-the-shelf’ regulatory T cells for immunoregulation.