Tipping the balance in autoimmunity: are regulatory t cells the cause, the cure, or both?

Regulatory T cells (Tregs) are a specialized subgroup of T-cell lymphocytes that is crucial for maintaining immune homeostasis and preventing excessive immune responses. Depending on their differentiation route, Tregs can be subdivided into thymically derived Tregs (tTregs) and peripherally induced Tregs (pTregs), which originate from conventional T cells after extrathymic differentiation at peripheral sites. Although the regulatory attributes of tTregs and pTregs partially overlap, their modes of action, protein expression profiles, and functional stability exhibit specific characteristics unique to each subset. Over the last few years, our knowledge of Treg differentiation, maturation, plasticity, and correlations between their phenotypes and functions has increased. Genetic and functional studies in patients with numeric and functional Treg deficiencies have contributed to our mechanistic understanding of immune dysregulation and autoimmune pathologies. This review provides an overview of our current knowledge of Treg biology, discusses monogenetic Treg pathologies and explores the role of Tregs in various other autoimmune disorders. Additionally, we discuss novel approaches that explore Tregs as targets or agents of innovative treatment options.

Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application.

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Adoptive transfer of ex vivo expanded regulatory T cells improves immune cell engraftment and therapy-refractory chronic GvHD.

Graft-versus-host disease (GvHD) is still the major non-relapse, life-limiting complication after hematopoietic stem cell transplantation. Modern pharmacologic immunosuppression is often insufficient and associated with significant side effects. Novel treatment strategies now include adoptive transfer of ex vivo expanded regulatory T cells (Tregs), but their efficacy in chronic GvHD is unknown. We treated three children suffering from severe, therapy-refractory GvHD with polyclonally expanded Tregs generated from the original stem cell donor. Third-line maintenance immunosuppression was tapered to cyclosporin A and low-dose steroids shortly before cell transfer. Regular follow-up included an assessment of the subjective and objective clinical development, safety parameters, and in-depth immune monitoring. All patients showed marked clinical improvement with substantially decreased GvHD activity. Laboratory follow-up showed a significant enhancement of the immunologic engraftment, including lymphocytes and dendritic cells. Monitoring the fate of Tregs by next-generation sequencing demonstrated clonal expansion. In summary, adoptive transfer of Tregs was well tolerated and able to modulate an established undesired T cell mediated allo-response. Although no signs of overimmunosuppression were detectable, the treatment of patients with invasive opportunistic infections should be undertaken with caution. Further controlled studies are necessary to confirm these encouraging effects and eventually pave the way for adoptive Treg therapy in chronic GvHD.

Cyclosporine A but Not Corticosteroids Support Efficacy of Ex Vivo Expanded, Adoptively Transferred Human Tregs in GvHD.

Reshaping the immune balance by adoptive transfer of regulatory T-cells (Tregs) has emerged as a promising strategy to combat undesired immune reactions, including in Graft-versus-Host Disease (GvHD), which is the most lethal non-relapse complication of allogeneic hematopoietic stem cell transplantation. Currently however, little is known about the potentially inhibitory in vivo effects of conventional immunosuppressive drugs, which are routinely used to treat GvHD, on adoptively transferred Tregs. Here we demonstrate drug-specific effects of the conventional immunosuppressive drugs Cyclosporine A, Mycophenolate mofetil and methylprednisolone on adoptively transferred Tregs in a humanized NOD/SCID/IL2Rgamma-/- GvHD mouse model. The clinical course of GvHD and postmortem organ histology, including cellular organ infiltration, showed that co-administration of Cyclosporine A and Tregs is highly beneficial as it enhanced Treg accumulation at inflammatory sites like lung and liver. Similarly, co-administration of Mycophenolate mofetil and Tregs improved clinical signs of GvHD. In contrast, co-administration of methylprednisolone and Tregs resulted in reduced Treg recruitment to inflammatory sites and the fast deterioration of some animals. Consequently, when clinical trials investigating safety and efficacy of adjunctive Treg therapy in GvHD are designed, we suggest co-administering Cyclosporine A, whereas high doses of glucocorticosteroids should be avoided.

Hematopoietic Stem Cell Transplantation Cures Therapy-refractory Aspergillosis in Chronic Granulomatous Disease.

BACKGROUND: Pulmonary invasive aspergillosis is a frequent and life-threatening complication for patients with chronic granulomatous disease (CGD). Despite combined treatment with several groups of antifungal agents, conservative treatment of invasive aspergillosis often remains refractory. Pulmonary invasive aspergillosis is often treated by surgical resection of consolidated lobes or segments, donor granulocyte transfusions and allogeneic hematopoietic stem cell transplantation (HSCT). These options are not mutually exclusive and often combined. METHODS AND RESULTS: We here describe the treatment of 3 patients with CGD who received HSCT upon active pulmonary invasive aspergillosis: Two of them received HSCT as salvage therapy for refractory aspergillosis, and 1 patient received elective HSCT in infancy but developed pulmonary aspergillosis during secondary graft failure. Based on our experience and available literature, we discuss indication as well as timing of HSCT, granulocyte transfusions and surgery in patients with CGD and pulmonary invasive aspergillosis. CONCLUSIONS: Upon diagnosis with invasive aspergillosis in CGD, we propose to start antifungal treatment and preparation for HSCT at the same time. Remission of pulmonary invasive aspergillosis before HSCT remains preferable but is not mandatory. When pulmonary aspergillosis in patients with CGD remains refractory for longer than 3 months on conservative treatment, HSCT without prior surgery or accompanying granulocyte transfusions is a feasible option.

Regulatory T cells for minimising immune suppression in kidney transplantation: phase I/IIa clinical trial.

OBJECTIVE: To assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model. DESIGN: Investigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13). SETTING: Charité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union). PARTICIPANTS: Recipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9). INTERVENTIONS: CD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×106 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48. MAIN OUTCOME MEASURES: The primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio. RESULTS: For all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire. CONCLUSIONS: The application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies. TRIAL REGISTRATION: NCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

Comprehensive Characterization of a Next-Generation Antiviral T-Cell Product and Feasibility for Application in Immunosuppressed Transplant Patients.

Viral infections have a major impact on morbidity and mortality of immunosuppressed solid organ transplant (SOT) patients because of missing or failure of adequate pharmacologic antiviral treatment. Adoptive antiviral T-cell therapy (AVTT), regenerating disturbed endogenous T-cell immunity, emerged as an attractive alternative approach to combat severe viral complications in immunocompromised patients. AVTT is successful in patients after hematopoietic stem cell transplantation where T-cell products (TCPs) are manufactured from healthy donors. In contrast, in the SOT setting TCPs are derived from/applied back to immunosuppressed patients. We and others demonstrated feasibility of TCP generation from SOT patients and first clinical proof-of-concept trials revealing promising data. However, the initial efficacy is frequently lost long-term, because of limited survival of transferred short-lived T-cells indicating a need for next-generation TCPs. Our recent data suggest that Rapamycin treatment during TCP manufacture, conferring partial inhibition of mTOR, might improve its composition. The aim of this study was to confirm these promising observations in a setting closer to clinical challenges and to deeply characterize the next-generation TCPs. Using cytomegalovirus (CMV) as model, our next-generation Rapamycin-treated (Rapa-)TCP showed consistently increased proportions of CD4+ T-cells as well as CD4+ and CD8+ central-memory T-cells (TCM). In addition, Rapamycin sustained T-cell function despite withdrawal of Rapamycin, showed superior T-cell viability and resistance to apoptosis, stable metabolism upon activation, preferential expansion of TCM, partial conversion of other memory T-cell subsets to TCM and increased clonal diversity. On transcriptome level, we observed a gene expression profile denoting long-lived early memory T-cells with potent effector functions. Furthermore, we successfully applied the novel protocol for the generation of Rapa-TCPs to 19/19 SOT patients in a comparative study, irrespective of their history of CMV reactivation. Moreover, comparison of paired TCPs generated before/after transplantation did not reveal inferiority of the latter despite exposition to maintenance immunosuppression post-SOT. Our data imply that the Rapa-TCPs, exhibiting longevity and sustained T-cell memory, are a reasonable treatment option for SOT patients. Based on our success to manufacture Rapa-TCPs from SOT patients under maintenance immunosuppression, now, we seek ultimate clinical proof of efficacy in a clinical study.

Ex vivo expanded natural regulatory T cells from patients with end-stage renal disease or kidney transplantation are useful for autologous cell therapy.

Novel concepts employing autologous, ex vivo expanded natural regulatory T cells (nTreg) for adoptive transfer has potential to prevent organ rejection after kidney transplantation. However, the impact of dialysis and maintenance immunosuppression on the nTreg phenotype and peripheral survival is not well understood, but essential when assessing patient eligibility. The current study investigates regulatory T-cells in dialysis and kidney transplanted patients and the feasibility of generating a clinically useful nTreg product from these patients. Heparinized blood from 200 individuals including healthy controls, dialysis patients with end stage renal disease and patients 1, 5, 10, 15, 20 years after kidney transplantation were analyzed. Differentiation and maturation of nTregs were studied by flow cytometry in order to compare dialysis patients and kidney transplanted patients under maintenance immunosuppression to healthy controls. CD127 expressing CD4+CD25highFoxP3+ nTregs were detectable at increased frequencies in dialysis patients with no negative impact on the nTreg end product quality and therapeutic usefulness of the ex vivo expanded nTregs. Further, despite that immunosuppression mildly altered nTreg maturation, neither dialysis nor pharmacological immunosuppression or previous acute rejection episodes impeded nTreg survival in vivo. Accordingly, the generation of autologous, highly pure nTreg products is feasible and qualifies patients awaiting or having received allogenic kidney transplantation for adoptive nTreg therapy. Thus, our novel treatment approach may enable us to reduce the incidence of organ rejection and reduce the need of long-term immunosuppression.

Peripheral blood-derived virus-specific memory stem T cells mature to functional effector memory subsets with self-renewal potency.

Memory T cells expressing stem cell-like properties have been described recently. The capacity of self-renewal and differentiation into various memory/effector subsets make them attractive for adoptive T cell therapy to combat severe virus infections and tumors. The very few reports on human memory stem T cells (T(SCM)) are restricted to analyses on polyclonal T cells, but extensive data on Ag-specific T(SCM )are missing. This might be due to their very low frequency limiting their enrichment and characterization. In this article, we provide functional and phenotypic data on human viral-specific T(SCM), defined as CD8(+)CD45RA(+)CCR7(+)CD127(+)CD95(+). Whereas <1% of total T cells express the T(SCM) phenotype, human CMV-specific T(SCM) can be detected at frequencies similar to those seen in other subsets, resulting in ∼ 1 /10,000 human CMV-specific T(SCM). A new virus-specific expansion protocol of sort-purified T(SCM) reveals both upregulation of various T cell subset markers and preservation of their stem cell phenotype in a significant proportion, indicating both self-renewal and differentiation potency of virus-specific T cells sharing their TCR repertoire. Furthermore, we describe a simplified culture protocol that allows fast expansion of virus-specific T(SCM) starting from a mixed naive T/T(SCM) pool of PBLs. Due to the clinical-grade compatibility, this might be the basis for novel cell therapeutic options in life-threatening courses of viral and tumor disease.

Interaction of Streptococcus agalactiae and Cellular Innate Immunity in Colonization and Disease.

Streptococcus agalactiae (Group B streptococcus, GBS) is highly adapted to humans, where it is a normal constituent of the intestinal and vaginal flora. Yet, GBS has highly invasive potential and causes excessive inflammation, sepsis, and death at the beginning of life, in the elderly and in diabetic patients. Thus, GBS is a model pathobiont that thrives in the healthy host, but has not lost its potential virulence during coevolution with mankind. It remains incompletely understood how the innate immune system contains GBS in the natural niches, the intestinal and genital tracts, and which molecular events underlie breakdown of mucocutaneous resistance. Newborn infants between days 7 and 90 of life are at risk of a particularly striking sepsis manifestation (late-onset disease), where the transition from colonization to invasion and dissemination, and thus from health to severe sepsis is typically fulminant and not predictable. The great majority of late-onset sepsis cases are caused by one clone, GBS ST17, which expresses HvgA as a signature virulence factor and adhesin. In mice, HvgA promotes the crossing of both the mucosal and the blood-brain barrier. Expression levels of HvgA and other GBS virulence factors, such as pili and toxins, are regulated by the upstream two-component control system CovR/S. This in turn is modulated by acidic epithelial pH, high glucose levels, and during the passage through the mouse intestine. After invasion, GBS has the ability to subvert innate immunity by mechanisms like glycerinaldehyde-3-phosphate-dehydrogenase-dependent induction of IL-10 and ß-protein binding to the inhibitory phagocyte receptors sialic acid binding immunoglobulin-like lectin 5 and 14. On the host side, sensing of GBS nucleic acids and lipopeptides by both Toll-like receptors and the inflammasome appears to be critical for host resistance against GBS. Yet, comprehensive models on the interplay between GBS and human immune cells at the colonizing site are just emerging.