Multiscale topology classifies cells in subcellular spatial transcriptomics.

Spatial transcriptomics measures in situ gene expression at millions of locations within a tissue1, hitherto with some trade-off between transcriptome depth, spatial resolution and sample size2. Although integration of image-based segmentation has enabled impactful work in this context, it is limited by imaging quality and tissue heterogeneity. By contrast, recent array-based technologies offer the ability to measure the entire transcriptome at subcellular resolution across large samples3-6. Presently, there exist no approaches for cell type identification that directly leverage this information to annotate individual cells. Here we propose a multiscale approach to automatically classify cell types at this subcellular level, using both transcriptomic information and spatial context. We showcase this on both targeted and whole-transcriptome spatial platforms, improving cell classification and morphology for human kidney tissue and pinpointing individual sparsely distributed renal mouse immune cells without reliance on image data. By integrating these predictions into a topological pipeline based on multiparameter persistent homology7-9, we identify cell spatial relationships characteristic of a mouse model of lupus nephritis, which we validate experimentally by immunofluorescence. The proposed framework readily generalizes to new platforms, providing a comprehensive pipeline bridging different levels of biological organization from genes through to tissues.

Towards regulatory cellular therapies in solid organ transplantation.

Organ transplantation is a modern medical success story. However, since its inception it has been limited by the need for pharmacological immunosuppression. Regulatory cellular therapies offer an attractive solution to these challenges by controlling transplant alloresponses through multiple parallel suppressive mechanisms. A number of cell types have seen an accelerated development into human trials and are now on the threshold of a long-awaited breakthrough in personalized transplant therapeutics. Here we assess recent developments with a focus on the most likely candidates, some of which have already facilitated successful immunosuppression withdrawal in early clinical trials. We propose that this may constitute a promising approach in clinical transplantation but also evaluate outstanding issues in the field, providing cause for cautious optimism.

Banff 2022 Vascularized Composite Allotransplantation Meeting Report: Diagnostic criteria for vascular changes.

As more data become available, the Banff 2007 working classification of skin-containing vascularized composite allograft (VCA) pathology is expected to evolve and develop. This report represents the Banff VCA Working Group's consensus on the first revision of the 2007 scoring system. Prior to the 2022 Banff-CanXadian Society of Transplantation Joint Meeting, 83 clinicians and/or researchers were invited to a virtual meeting to discuss whether the 2007 Banff VCA system called for a revision. Unanimously, it was determined that the vascular changes were to be included in the first revision. Subsequently, 2 international online surveys, each followed by virtual discussions, were launched. The goals were (1) to identify which changes define severe rejection, (2) to grade their importance in the evaluation of severe rejection, and (3) to identify emerging criteria to diagnose rejection. A final hybrid (in-person and virtual) discussion at the Banff/Canadian Society of Transplantation Joint Meeting finalized the terminology, the definition, a scoring system, and a reporting system of the vascular changes. This proposal represents an international consensus on this topic and establishes the first revision of the Banff 2007 working classification of skin-containing vascularized composite allograft pathology.

Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model.

Understanding the host pathways that define susceptibility to Severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in the Syrian hamster model. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.

Low dose interleukin-2 selectively expands circulating regulatory T cells but fails to promote liver allograft tolerance in humans.

BACKGROUND & AIMS: CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential to maintain immunological tolerance and have been shown to promote liver allograft tolerance in both rodents and humans. Low-dose IL-2 (LDIL-2) can expand human endogenous circulating Tregs in vivo, but its role in suppressing antigen-specific responses and promoting Treg trafficking to the sites of inflammation is unknown. Likewise, whether LDIL-2 facilitates the induction of allograft tolerance has not been investigated in humans. METHODS: We conducted a clinical trial in stable liver transplant recipients 2-6 years post-transplant to determine the capacity of LDIL-2 to suppress allospecific immune responses and allow for the complete discontinuation of maintenance immunosuppression (ClinicalTrials.gov NCT02949492). One month after LDIL-2 was initiated, those exhibiting at least a 2-fold increase in circulating Tregs gradually discontinued immunosuppression over a 4-month period while continuing LDIL-2 for a total treatment duration of 6 months. RESULTS: All participants achieved a marked and sustained increase in circulating Tregs. However, this was not associated with the preferential expansion of donor-reactive Tregs and did not promote the accumulation of intrahepatic Tregs. Furthermore, LDIL-2 induced a marked IFNγ-orchestrated transcriptional response in the liver even before immunosuppression weaning was initiated. The trial was terminated after the first 6 participants failed to reach the primary endpoint owing to rejection requiring reinstitution of immunosuppression. CONCLUSIONS: The expansion of circulating Tregs in response to LDIL-2 is not sufficient to control alloimmunity and to promote liver allograft tolerance, due, at least in part, to off-target effects that increase liver immunogenicity. Our trial provides unique insight into the mechanisms of action of immunomodulatory therapies such as LDIL-2 and their limitations in promoting alloantigen-specific effects and immunological tolerance. CLINICAL TRIALS REGISTRATION: The study is registered at ClinicalTrials.gov (NCT02949492). IMPACT AND IMPLICATIONS: The administration of low-dose IL-2 is an effective way of increasing the number of circulating regulatory T cells (Tregs), an immunosuppressive lymphocyte subset that is key for the establishment of immunological tolerance, but its use to promote allograft tolerance in the setting of clinical liver transplantation had not been explored before. In liver transplant recipients on tacrolimus monotherapy, low-dose IL-2 effectively expanded circulating Tregs but did not increase the number of Tregs with donor specificity, nor did it promote their trafficking to the transplanted liver. Low-dose IL-2 did not facilitate the discontinuation of tacrolimus and elicited, as an off-target effect, an IFNγ-orchestrated inflammatory response in the liver that resembled T cell-mediated rejection. These results, supporting an unexpected role for IL-2 in regulating the immunogenicity of the liver, highlight the need to carefully evaluate systemic immunoregulatory strategies with investigations that are not restricted to the blood compartment and involve target tissues such as the liver.

Regulatory T-cell therapy approaches.

Regulatory T cells (Tregs) have enormous therapeutic potential to treat a variety of immunopathologies characterized by aberrant immune activation. Adoptive transfer of ex vivo expanded autologous Tregs continues to progress through mid- to late-phase clinical trials in several disease spaces and has generated promising preliminary safety and efficacy signals to date. However, the practicalities of this strategy outside of the clinical trial setting remain challenging. Here, we review the current landscape of regulatory T-cell therapy, considering emergent approaches and technologies presenting novel ways to engage Tregs, and reflect on the progress necessary to deliver their therapeutic potential to patients.

Threat awareness training for non-governmental organizations deploying humanitarian aid workers into conflict environments.

INTRODUCTION: The current war in Ukraine and the subsequent deployment of Non-Governmental Organizations (NGOs) from around the world has highlighted the many potential dangers faced by humanitarian aid workers operating in conflict zones. Humanitarian aid workers may face both direct and indirect threats and aggression while on deployment, and given the rising number of global conflicts, the authors postulate a need to incorporate threat awareness training as part of pre-deployment training. METHODS: A list of the top 22 rated NGOs providing international aid was obtained from CharityWatch. All 22 were contacted via their public email addresses or website contact pages to find out if they provide any form of security, tactical or threat awareness training. RESULTS: Of the 13 NGOs that responded, 7 did not deploy staff into recent conflict zones or surroundings. All 6 NGOs who deployed staff into Ukraine or surrounding border countries, provided either security, tactical or threat awareness training to their staff. CONCLUSION: With the rising number of conflicts and disasters around the world, humanitarian aid workers are increasingly exposed to hostile environments and there is a compelling need for NGOs to ensure staff are adequately trained and prepared to handle any dangers and threats they may face. In this study, all 6 of the studied NGOs which deployed staff to the conflict zone confirmed some type of security or threat awareness training ranging from in-house security briefs to extensive, multi-day, commercially run courses such as Hostile Environment Awareness Training course.

Chimeric antigen receptor-modified human regulatory T cells that constitutively express IL-10 maintain their phenotype and are potently suppressive.

Clinical trials of Treg therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations that harbor a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CARs), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study, we build on our previous work in the development of HLA-A2 CAR-Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL-10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA-A2, and suppressed alloresponses potently. The addition of IL-10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof-of-principle for this cell engineering approach for next-generation Treg therapy in transplantation.

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes.

Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

Joining Forces in Basic Science: ITS Meeting 2.0.

The second International Transplant Science (ITS) meeting jointly organized by the European Society for Organ Transplantation (ESOT), the American Society of Transplantation (AST), and The Transplantation Society (TTS) took place in May 2022 in one of Europe's most iconic cities: Berlin, Germany. The ITS meeting 2022 was designed to serve as an international platform for scientific discussions on the latest ground-breaking discoveries in the field, while providing an excellent opportunity to present cutting-edge research to the scientific community. We think this is fundamental for the exchange of new ideas and establishment of collaborative work between advanced transplant experts, young professionals and early-stage researchers and students. Scientific sessions tackled hot topics in transplantation such as mechanisms of tolerance, biomarkers, big data and artificial intelligence. Our educational pre-meeting focused on the breakthrough and challenges in single-cell multimodal omics. The program included panel discussions illuminating various topics concerning conflicts and problems related to gender, such as challenges for female scientists. Attendees returned to their institutes with not only profound knowledge of the latest discoveries, technologies, and concepts in basic and translational science, but also inspired and excited after discussions and networking sessions with fellow scientists which have been duly missed during the pandemic.

IL-33 drives the production of mouse regulatory T cells with enhanced in vivo suppressive activity in skin transplantation.

Regulatory T cells (Tregs) are crucial mediators of immune homeostasis with the ability to modulate allogeneic response and control transplant rejection. Although Treg-based cell therapies have shown immense promise, methods to optimize current strategies are critical for successful implementation within the clinic. IL-33 is a cytokine with pleiotropic properties and effects on Treg function and development. In this study, we explored the unique properties of Treg populations activated through the IL-33/ST2 pathway, aiming to exploit their tolerogenic properties for cell therapy. We show that treatment with exogenous IL-33 results in a generalized downregulation of genes critical to T cell biology together with an upregulation of Treg-associated genes. Tregs that develop in response to IL-33 upregulate critical Treg-associated markers, yet without developing enhanced in vitro suppressive capacity. Conversely, these Tregs display potent regulatory activity in vivo, promoting long-term skin allograft survival in a stringent transplantation model. Detailed transcriptomic and immunophenotypic analyses of IL-33-expanded Tregs reveal an enhancement in graft-homing chemokine receptors, which may be partly responsible for their superior in vivo activity that is not reflected in vitro. IL-33 treatment is therefore an attractive adjunctive strategy for patients receiving Treg cell therapeutics.

The Fourth International Workshop on Clinical Transplant Tolerance.

The International Workshop on Clinical Transplant Tolerance is a biennial meeting that aims to provide an update on the progress of studies of immunosuppression minimization or withdrawal in solid organ transplantation. The Fourth International Workshop on Clinical Tolerance was held in Pittsburgh, Pennsylvania, September 5-6, 2019. This report is a summary of presentations on the status of clinical trials designed to minimize or withdraw immunosuppressive drugs in kidney, liver, and lung transplantation without subsequent evidence of rejection. All protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. The workshop also included presentations of mechanistic studies designed to improve understanding of the cellular and molecular basis of tolerance and to identify potential predictors/biomarkers of tolerance. Strategies to enhance the safety of hematopoietic cell transplantation and to improve patient selection/risk stratification for clinical trials were also discussed.

Feasibility, long-term safety, and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients.

Short-term outcomes in kidney transplantation are marred by progressive transplant failure and mortality secondary to immunosuppression toxicity. Immune modulation with autologous polyclonal regulatory T cell (Treg) therapy may facilitate immunosuppression reduction promoting better long-term clinical outcomes. In a Phase I clinical trial, 12 kidney transplant recipients received 1-10 × 106 Treg per kg at Day +5 posttransplantation in lieu of induction immunosuppression (Treg Therapy cohort). Nineteen patients received standard immunosuppression (Reference cohort). Primary outcomes were rejection-free and patient survival. Patient and transplant survival was 100%; acute rejection-free survival was 100% in the Treg Therapy versus 78.9% in the reference cohort at 48 months posttransplant. Treg therapy revealed no excess safety concerns. Four patients in the Treg Therapy cohort had mycophenolate mofetil withdrawn successfully and remain on tacrolimus monotherapy. Treg infusion resulted in a long-lasting dose-dependent increase in peripheral blood Tregs together with an increase in marginal zone B cell numbers. We identified a pretransplantation immune phenotype suggesting a high risk of unsuccessful ex-vivo Treg expansion. Autologous Treg therapy is feasible, safe, and is potentially associated with a lower rejection rate than standard immunosuppression. Treg therapy may provide an exciting opportunity to minimize immunosuppression therapy and improve long-term outcomes.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.

Nuclear androgen and progestin receptors inversely affect aggression and social dominance in male zebrafish (Danio rerio).

Aggression is a fundamental behavior displayed universally among animal species, but hyper- or hypo-aggressiveness can be maladaptive with negative consequences for individuals and group members. While the social and ecological significance of aggression is well understood, the specific neurobiological and hormonal mechanisms responsible for mediating aggression have not been fully elucidated. Previous studies have shown a relationship between aggressive acts and circulating gonadal steroids, but whether classical nuclear steroid receptors regulate aggression in animals is still uncertain. We examined whether the nuclear androgen receptor (Ar) and nuclear progestin receptor (Pgr) were necessary for aggressive behaviors and maintenance of a dominance relationship in male zebrafish (Danio rerio). Dyadic social interactions of Ar knockout (ArKO), Pgr knockout (PgrKO) and wildtype (WT) controls were observed for two weeks (2-weeks). ArKO zebrafish were significantly less aggressive and had a less defined dominance relationship, whereas PgrKO dominant zebrafish were significantly and persistently more aggressive with a robust dominance relationship. Our results demonstrate the importance of nuclear steroid hormone receptors in regulating aggression of adult male zebrafish and provide new models for understanding of the mechanisms of aggression.

Cellular therapies in organ transplantation.

Cellular therapy is a promising tool for improving the outcome of organ transplantation. Various cell types with different immunoregulatory and regenerative properties may find application for specific transplant rejection or injury-related indications. The current era is crucial for the development of cellular therapies. Preclinical models have demonstrated the feasibility of efficacious cell therapy in transplantation, early clinical trials have shown safety of several of these therapies, and the first steps towards efficacy studies in humans have been made. In this review, we address the current state of the art of cellular therapies in clinical transplantation and discuss monitoring tools and endpoints for these studies.

Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation.

This meeting report from the XV Banff conference describes the creation of a multiorgan transplant gene panel by the Banff Molecular Diagnostics Working Group (MDWG). This Banff Human Organ Transplant (B-HOT) panel is the culmination of previous work by the MDWG to identify a broadly useful gene panel based on whole transcriptome technology. A data-driven process distilled a gene list from peer-reviewed comprehensive microarray studies that discovered and validated their use in kidney, liver, heart, and lung transplant biopsies. These were supplemented by genes that define relevant cellular pathways and cell types plus 12 reference genes used for normalization. The 770 gene B-HOT panel includes the most pertinent genes related to rejection, tolerance, viral infections, and innate and adaptive immune responses. This commercially available panel uses the NanoString platform, which can quantitate transcripts from formalin-fixed paraffin-embedded samples. The B-HOT panel will facilitate multicenter collaborative clinical research using archival samples and permit the development of an open source large database of standardized analyses, thereby expediting clinical validation studies. The MDWG believes that a pathogenesis and pathway based molecular approach will be valuable for investigators and promote therapeutic decision-making and clinical trials.

Identification, selection, and expansion of non-gene modified alloantigen-reactive Tregs for clinical therapeutic use.

Transplantation is limited by the need for life-long pharmacological immunosuppression, which carries significant morbidity and mortality. Regulatory T cell (Treg) therapy holds significant promise as a strategy to facilitate immunosuppression minimization. Polyclonal Treg therapy has been assessed in a number of Phase I/II clinical trials in both solid organ and hematopoietic transplantation. Attention is now shifting towards the production of alloantigen-reactive Tregs (arTregs) through co-culture with donor antigen. These allospecific cells harbour potent suppressive function and yet their specificity implies a theoretical reduction in off-target effects. This review will cover the progress in the development of arTregs including their potential application for clinical use in transplantation, the knowledge gained so far from clinical trials of Tregs in transplant patients, and future directions for Treg therapy.

Regulatory B cells in transplantation: roadmaps to clinic.

Over the last two decades, an additional and important role for B cells has been established in immune regulation. Preclinical studies demonstrate that regulatory B cells (Breg) can prolong allograft survival in animal models and induce regulatory T cells. Operationally tolerant human kidney transplant recipients demonstrate B-cell-associated gene signatures of immune tolerance, and novel therapeutic agents can induce Bregs in phase I clinical trials in transplantation. Our rapidly expanding appreciation of this novel B-cell subtype has made the road to clinical application a reality. Here, we outline several translational pathways by which Bregs could soon be introduced to the transplant clinic.

Understanding Stem Cell Immunogenicity in Therapeutic Applications.

Stem cells and their differentiated progeny offer great hope for treating disease by providing an unlimited source of cells for repairing or replacing damaged tissue. Initial studies suggested that, unlike 'normal' transplants, specific characteristics of stem cells enabled them to avoid immune attack. However, recent findings have revealed that the immunogenicity of stem cells may have been underestimated. Here, we review the current understanding of the mechanisms of immune recognition associated with stem cell immunogenicity, and discuss the relevance of reprogramming and differentiation strategies used to generate cells or tissue from stem cells for implantation in eliciting an immune response. We examine the effectiveness of current strategies for minimising immune attack in light of our experience in the transplantation field and, in this context, outline important challenges moving forward.