Dysregulated anti-oxidant signalling and compromised mitochondrial integrity negatively influence regulatory T cell function and viability in liver disease.

BACKGROUND: Dysregulated inflammatory responses and oxidative stress are key pathogenic drivers of chronic inflammatory diseases such as liver cirrhosis (LC). Regulatory T cells (Tregs) are essential to prevent excessive immune activation and maintain tissue homeostasis. While inflammatory cues are well known to modulate the function and stability of Tregs, the extent to which Tregs are influenced by oxidative stress has not been fully explored. METHODS: The phenotypic and functional properties of CD4+CD25+CD127lo/- Tregs isolated from patients with LC were compared to healthy controls (HC). Treg redox state was investigated by characterizing intracellular reactive oxygen species (ROS), NADPH oxidase-2 (Nox2) activity, mitochondrial function, morphology, and nuclear factor-erythroid 2-related factor (Nrf2) antioxidant signalling. The relevance of Nrf2 and its downstream target, Heme-oxygenase-1 (HO-1), in Treg function, stability, and survival, was further assessed using mouse models and CRISPR/Cas9-mediated HO-1 knock-out. FINDINGS: Circulating Tregs from LC patients displayed a reduced suppressive function, correlating with liver disease severity, associated with phenotypic abnormalities and increased apoptosis. Mechanistically, this was linked to a dysregulated Nrf2 signalling with resultant lower levels of HO-1, enhanced Nox2 activation, and impaired mitochondrial respiration and integrity. The functional deficit in LC Tregs could be partially recapitulated by culturing control Tregs in patient sera. INTERPRETATION: Our findings reveal that Tregs rely on functional redox homeostasis for their function, stability, and survival. Targeting Treg specific anti-oxidant pathways may have therapeutic potential to reverse the Treg impairment in conditions of oxidative damage such as advanced liver disease. FUNDING: This study was funded by the Wellcome Trust (211113/A/18/Z).

Advances in Liver Transplantation: where are we in the pursuit of transplantation tolerance?

Liver transplantation is the ultimate treatment option for end-stage liver disease. Breakthroughs in surgical practice and immunosuppression have seen considerable advancements in survival after transplantation. However, the intricate management of immunosuppressive regimens, balancing desired immunological quiescence while minimizing toxicity has proven challenging. Diminishing improvements in long-term morbidity and mortality have been inextricably linked with the protracted use of these medications. As such, there is now enormous interest to devise protocols that will allow us to minimize or completely withdraw immunosuppressants after transplantation. Immunosuppression withdrawal trials have proved the reality of tolerance following liver transplantation, however, without intervention will only occur after several years at the risk of potential cumulative immunosuppression-related morbidity. Focus has now been directed at accelerating this phenomenon through tolerance-inducing strategies. In this regard, efforts have seen the use of regulatory cell immunotherapy. Here we focus particularly on regulatory T cells, discussing preclinical data that propagated several clinical trials of adoptive cell therapy in liver transplantation. Furthermore, we describe efforts to further optimize the specificity and survival of regulatory cell therapy guided by concurrent immunomonitoring studies and the development of novel technologies including chimeric antigen receptors and co-administration of low-dose IL-2.

Extraspinal articular tuberculosis: An 11-year retrospective study of demographic features and clinical outcomes in East London.

OBJECTIVES: To describe demographic features, clinical outcomes and diagnostic delay amongst patients with extra-spinal articular tuberculosis (TB) in a low-incidence setting. METHODS: Cases of TB treated at our institution between 2004 and 2014 were identified via the London TB register (LTBR). Demographic features of extra-spinal articular TB cases were compared to controls with TB at all other sites. For articular cases (excluding individuals <16 years or with spinal TB without peripheral joint involvement) clinical data were retrospectively collected. RESULTS: 6,146 TB patients were identified over the study period; 146 (2.4%) cases had extra-spinal articular infection. There was no difference in median age between extra-spinal articular TB cases and controls with TB at other sites (31 vs 32 years, p = 0.57). Articular cases were more likely to be male (70.6% vs 59.5%, p = 0.007), Bangladeshi (28.7% vs 18.0%) or Pakistani (24.0% vs 16.1%) and were less likely to be Black-African (9.5% vs 19.8%) (p < 0.001). 93 cases were included in the case series; 85 (88.5%) were migrants and 83 (89.2%) were South Asian. Knee and elbow joints were affected in 22 (23.7%) and 18 (19.4%) cases respectively. The median durations of pre-healthcare and healthcare associated delay were 16 and 6 weeks respectively. Where mycobacterial culture was performed, 57/75 (76%) were positive for Mycobacterium tuberculosis. 86 (92.5%) cases received standard quadruple therapy for a median of 6 months (IQR 6-9). Recurrence of TB infection occurred in 4 (4.3%) cases and there were no TB related deaths. Seven (7.6%) cases required surgical intervention. CONCLUSIONS: Extra-spinal articular TB more commonly affected men and people of South Asian ethnicity. Significant diagnostic delays were identified, including avoidable healthcare-associated delays.

Optimizing regulatory T cells for therapeutic application in human organ transplantation.

PURPOSE OF REVIEW: Initial clinical trials of adoptive regulatory T-cell (Treg) therapy in solid organ transplantation have proven to be both feasible and well tolerated. With Phase 2 trials underway, efforts have been focused on the optimization of the Treg product. RECENT FINDINGS: With science and our knowledge on the biology of these cells constantly advancing, we have been able to refine our search for a Treg population that would be ideally suited for therapeutic application. This idealized population must be readily isolated, allow for in-vitro expansion, demonstrate potent and specific suppressor function, maintain lineage stability and demonstrate a relevant homing profile. With the advent of innovative cell analysis/isolation techniques and genetic modifications, we are able to choose and design Tregs to fulfil these criteria. SUMMARY: By utilizing advances in science and technology, we can optimize Treg therapy in human organ transplantation maximizing their prospects in the arena of transplantation tolerance.

Cell Therapy in Organ Transplantation: Our Experience on the Clinical Translation of Regulatory T Cells.

Solid organ transplantation is the treatment of choice for patients with end-stage organ dysfunction. Despite improvements in short-term outcome, long-term outcome is suboptimal due to the increased morbidity and mortality associated with the toxicity of immunosuppressive regimens and chronic rejection (1-5). As such, the attention of the transplant community has focused on the development of novel therapeutic strategies to achieve allograft tolerance, a state whereby the immune system of the recipient can be re-educated to accept the allograft, averting the need for long-term immunosuppression. Indeed, reports of "operational" tolerance, whereby the recipient is off all immunosuppressive drugs and maintaining good graft function, is well documented in the literature for both liver and kidney transplantations (6-8). However, this phenomenon is rare and in the setting of liver transplantation has been shown to occur late after transplantation, with the majority of patients maintained on life-long immunosupression to prevent allograft rejection (9). As such, significant research has focused on immune regulation in the context of organ transplantation with regulatory T cells (Tregs) identified as cells holding considerable promise in this endeavor. This review will provide a brief introduction to human Tregs, their phenotypic and functional characterization and focuses on our experience to date at the clinical translation of Treg immunotherapy in the setting of solid organ transplantation.

Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation.

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial, ThRIL, recently commenced at King's College London, proposes to use Treg cell therapy to induce tolerance in liver transplant recipients, the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads, IL-2 and rapamycin. We report the enrichment of a pure, stable population of Tregs (>95% CD4(+)CD25(+)FOXP3(+)), reaching adequate numbers for their clinical application. Our protocol proved successful in, influencing the expansion of superior functional Tregs, as compared to freshly isolated cells, whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF), a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL, will undoubtedly inform the improved management of the liver transplant recipient.

Regulatory T Cells: Serious Contenders in the Promise for Immunological Tolerance in Transplantation.

Regulatory T cells (Tregs) play an important role in immunoregulation and have been shown in animal models to promote transplantation tolerance and curb autoimmunity following their adoptive transfer. The safety and potential therapeutic efficacy of these cells has already been reported in Phase I trials of bone-marrow transplantation and type I diabetes, the success of which has motivated the broadened application of these cells in solid-organ transplantation. Despite major advances in the clinical translation of these cells, there are still key questions to be addressed to ensure that Tregs attest their reputation as ideal candidates for tolerance induction. In this review, we will discuss the unique traits of Tregs that have attracted such fame in the arena of tolerance induction. We will outline the protocols used for their ex vivo expansion and discuss the future directions of Treg cell therapy. In this regard, we will review the concept of Treg heterogeneity, the desire to isolate and expand a functionally superior Treg population and report on the effect of differing culture conditions. The relevance of Treg migratory capacity will also be discussed together with methods of in vivo visualization of the infused cells. Moreover, we will highlight key advances in the identification and expansion of antigen-specific Tregs and discuss their significance for cell therapy application. We will also summarize the clinical parameters that are of importance, alongside cell manufacture, from the choice of immunosuppression regimens to the number of injections in order to direct the success of future efficacy trials of Treg cell therapy. Years of research in the field of tolerance have seen an accumulation of knowledge and expertise in the field of Treg biology. This perpetual progression has been the driving force behind the many successes to date and has put us now within touching distance of our ultimate success, immunological tolerance.