Recombinant adeno-associated virus (rAAV) vectors stand out as highly promising for in vivo gene transfer, particularly in targeting the skeletal muscle for treating muscular genetic diseases or secreting therapeutic factors. Despite the simplicity and efficacy of the established intramuscular (IM) route, it has been often associated with an immune-induced rapid loss of transgene expression, in particular in large animal models, and generally considered irreversible as a consequence of a cytotoxic elimination of transduced cells. Here, we report in a non-human primate model that transgene expression loss after IM delivery of an rAAV1 expressing an immunogenic protein is only transient, with the re-expression of the transgene lasting up to 5 years post-injection. We show that the recovery of transgene expression is due to persisting viral genomes in the injected muscles despite the detection of peripheral anti-transgene cellular immunity. Persisting genomes were observed in the presence of infiltrated mononuclear CD8 and CD4 T lymphocytes, among which we were able to detect FoxP3+ regulatory cells. This is to our knowledge the first report of a transient immune-mediated loss of gene expression in a large animal model after rAAV delivery that should shed new light on the issue of rAAV vector immunogenicity.

IntroductionCD38 is highly expressed on immune cells. It catabolizes NAD+, which is a critical cofactor for enzymes involved in metabolism and energy production. Methods and resultsWe developed TNB-738, a fully human antibody that potently inhibits human CD38 enzymatic activity on human immune cells, resulting in a dose-dependent increase of intracellular NAD+ levels. TNB-738 does not show immune effector functions, does not induce direct cell killing of CD38 positive cells and is not internalized. In vivo, treatment with TNB-738 following infusion of human PBMCs into NSG mice resulted in significantly lower clinical scores, prolonged overall survival, less expansion of engrafted human CD45+ cells and a significant expansion of Tregs. DiscussionCD38 positive T cells regulate NAD+ metabolism in inflamed tissues and blockade of CD38 enzyme activity by TNB-738 could represent a novel class of therapeutics for the treatment of inflammatory conditions, including GvHD.

Cluster of differentiation 38 (CD38) is an ecto-enzyme expressed primarily on immune cells that metabolizes nicotinamide adenine dinucleotide (NAD+) to adenosine diphosphate ribose or cyclic ADP-ribose and nicotinamide. Other substrates of CD38 include nicotinamide adenine dinucleotide phosphate and nicotinamide mononucleotide, a critical NAD+ precursor in the salvage pathway. NAD+ is an important coenzyme involved in several metabolic pathways and is a required cofactor for the function of sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases. Declines in NAD+ levels are associated with metabolic and inflammatory diseases, aging, and neurodegenerative disorders. To inhibit CD38 enzyme activity and boost NAD+ levels, we developed TNB-738, an anti-CD38 biparatopic antibody that pairs two non-competing heavy chain-only antibodies in a bispecific format. By simultaneously binding two distinct epitopes on CD38, TNB-738 potently inhibited its enzymatic activity, which in turn boosted intracellular NAD+ levels and SIRT activities. Due to its silenced IgG4 Fc, TNB-738 did not deplete CD38-expressing cells, in contrast to the clinically available anti-CD38 antibodies, daratumumab, and isatuximab. TNB-738 offers numerous advantages compared to other NAD-boosting therapeutics, including small molecules, and supplements, due to its long half-life, specificity, safety profile, and activity. Overall, TNB-738 represents a novel treatment with broad therapeutic potential for metabolic and inflammatory diseases associated with NAD+ deficiencies.Abbreviations: 7-AAD: 7-aminoactinomycin D; ADCC: antibody dependent cell-mediated cytotoxicity; ADCP: antibody dependent cell-mediated phagocytosis; ADPR: adenosine diphosphate ribose; APC: allophycocyanin; cADPR: cyclic ADP-ribose; cDNA: complementary DNA; BSA: bovine serum albumin; CD38: cluster of differentiation 38; CDC: complement dependent cytotoxicity; CFA: Freund’s complete adjuvant; CHO: Chinese hamster ovary; CCP4: collaborative computational project, number 4; COOT: crystallographic object-oriented toolkit; DAPI: 4′,6-diamidino-2-phenylindole; DNA: deoxyribonucleic acid; DSC: differential scanning calorimetry; 3D: three dimensional; εNAD+: nicotinamide 1,N6-ethenoadenine dinucleotide; ECD: extracellular domain; EGF: epidermal growth factor; FACS: fluorescence activated cell sorting; FcγR: Fc gamma receptors; FITC: fluorescein isothiocyanate; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: immunoglobulin; IFA: incomplete Freund’s adjuvant; IFNγ: Interferon gamma; KB: kinetic buffer; kDa: kilodalton; KEGG: kyoto encyclopedia of genes and genomes; LDH: lactate dehydrogenase; M: molar; mM: millimolar; MFI: mean fluorescent intensity; NA: nicotinic acid; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NAM: nicotinamide; NGS: next-generation sequencing; NHS/EDC: N-Hydroxysuccinimide/ ethyl (dimethylamino propyl) carbodiimide; Ni-NTA: nickel-nitrilotriacetic acid; nL: nanoliter; NK: natural killer; NMN: nicotinamide mononucleotide; OD: optical density; PARP: poly (adenosine diphosphate-ribose) polymerase; PBS: phosphate-buffered saline; PBMC: peripheral blood mononuclear cell; PDB: protein data bank; PE: phycoerythrin; PISA: protein interfaces, surfaces, and assemblies: PK: pharmacokinetics; mol: picomolar; RNA: ribonucleic acid; RLU: relative luminescence units; rpm: rotations per minute; RU: resonance unit; SEC: size exclusion chromatography; SEM: standard error of the mean; SIRT: sirtuins; SPR: surface plasmon resonance; µg: microgram; µM: micromolar; µL: microliter

Kidney transplant survival is shortened by chronic rejection and side effects of standard immunosuppressive drugs. Cell-based immunotherapy with tolerogenic dendritic cells has long been recognized as a promising approach to reduce general immunosuppression. Published trials report the safety and the absence of therapy-related adverse reactions in patients treated with tolerogenic dendritic cells suffering from several inflammatory diseases. Here, we present the first phase I clinical trial results using human autologous tolerogenic dendritic cells (ATDC) in kidney transplantation. Eight patients received ATDC the day before transplantation in conjunction with standard steroids, mycophenolate mofetil and tacrolimus immunosuppression with an option to taper mycophenolate mofetil. ATDC preparations were manufactured in a Good Manufacturing Practice-compliant facility and fulfilled cell count, viability, purity and identity criteria for release. A control group of nine patients received the same standard immunosuppression, except basiliximab induction replaced ATDC therapy and mycophenolate tapering was not allowed. During the three-year follow-up, no deaths occurred and there was 100% graft survival. No significant increase of adverse events was associated with ATDC infusion. Episodes of rejection were observed in two patients from the ATDC group and one patient from the control group. However, all rejections were successfully treated by glucocorticoids. Mycophenolate was successfully reduced/stopped in five patients from the ATDC group, allowing tacrolimus monotherapy for two of them. Regarding immune monitoring, reduced CD8 T cell activation markers and increased Foxp3 expression were observed in the ATDC group. Thus, our results demonstrate ATDC administration safety in kidney-transplant recipients.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2–related murine β-coronavirus. Tmem176b −/− mice infected with murine β-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical β-coronavirus disease.

The use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαβγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rβγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule's in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

Summary Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. Here we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in RORγt + cells and conventional dendritic cells (cDCs) using germline and conditional double knock-out (DKO) mice. While Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells (ILC3s) in the intestinal mucosa, we found that they were required in cDCs for optimal antigen processing and presentation to CD4 + T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC II compartment (MIIC) of DCs for the fine regulation of antigen presentation and naive CD4 + T cell priming.

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. Copyright (C) 2020 Elsevier Ltd. All rights reserved.

Background. Kidney transplantation is the therapeutic of choice for patients with kidney failure. While immunosuppressive drugs can control graft rejection, their use is associated with increased infections and cancer, and they do not effectively control chronic graft rejection. Cell therapy is an attractive strategy to minimize the use of pharmacological drugs. Methods. We recently developed a protocol to generate human monocyte-derived autologous tolerogenic dendritic cells (ATDCs) from healthy volunteers. Herein, we transferred the ATDC manufacturing protocol to a Good Manufacturing Practice (GMP)-compliant facility. Furthermore, we compared the phenotype and in vitro functions of ATDCs generated from patients with end-stage renal disease to those generated from healthy volunteers. Results. We describe the critical steps for GMP-compliant production of ATDCs and define the quality criteria required to allow release of the cell products. Furthermore, we showed that ATDCs generated from healthy volunteers and patients with kidney failure display the same tolerogenic profile based on their phenotype, resistance to maturation, and ability to modulate T-cell responses. Conclusions. Together, these results allowed us to define the production process and the quality criteria for the release of ATDCs before their administration in patients receiving a kidney transplant.

Cell therapy is a promising strategy for treating patients suffering from autoimmune or inflammatory diseases or receiving a transplant. Based on our preclinical studies, we have generated human autologous tolerogenic dendritic cells (ATDCs), which are being tested in a first-in-man clinical trial in kidney transplant recipients. Here, we report that ATDCs represent a unique subset of monocyte-derived cells based on phenotypic, transcriptomic, and metabolic analyses. ATDCs are characterized by their suppression of T cell proliferation and their expansion of Tregs through secreted factors. ATDCs produce high levels of lactate that shape T cell responses toward tolerance. Indeed, T cells take up ATDC-secreted lactate, leading to a decrease of their glycolysis. In vivo, ATDCs promote elevated levels of circulating lactate and delay graft-versus-host disease by reducing T cell proliferative capacity. The suppression of T cell immunity through lactate production by ATDCs is a novel mechanism that distinguishes ATDCs from other cell-based immunotherapies.

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8+ T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1β activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8+ T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers.

Over the past century, solid organ transplantation has been improved both at a surgical and postoperative level. However, despite the improvement in efficiency, safety, and survival, we are still far from obtaining full acceptance of all kinds of allograft in the absence of concomitant treatments. Today, transplanted patients are treated with immunosuppressive drugs (IS) to minimize immunological response in order to prevent graft rejection. Nevertheless, the lack of specificity of IS leads to an increase in the risk of cancer and infections. At this point, cell therapies have been shown as a novel promising resource to minimize the use of IS in transplantation. The main strength of cell therapy is the opportunity to generate allograft-specific tolerance, promoting in this way long-term allograft survival. Among several other regulatory cell types, tolerogenic monocyte-derived dendritic cells (Tol-MoDCs) appear to be an interesting candidate for cell therapy due to their ability to perform specific antigen presentation and to polarize immune response to immunotolerance. In this review, we describe the characteristics and the mechanisms of action of both human Tol-MoDCs and rodent tolerogenic bone marrow-derived DCs (Tol-BMDCs). Furthermore, studies performed in transplantation models in rodents and non-human primates corroborate the potential of Tol-BMDCs for immunoregulation. In consequence, Tol-MoDCs have been recently evaluated in sundry clinical trials in autoimmune diseases and shown to be safe. In addition to autoimmune diseases clinical trials, Tol-MoDC is currently used in the first phase I/II clinical trials in transplantation. Translation of Tol-MoDCs to clinical application in transplantation will also be discussed in this review.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

Myeloid cells play a pivotal role in regulating innate and adaptive immune responses. In inflammation, autoimmunity, and after transplantation, myeloid cells have contrasting roles: on the one hand they initiate the immune response, promoting activation and expansion of effector T-cells, and on the other, they counter-regulate inflammation, maintain tissue homeostasis, and promote tolerance. The latter activities are mediated by several myeloid cells including polymorphonuclear neutrophils, macrophages, myeloid-derived suppressor cells, and dendritic cells. Since these cells have been associated with immune suppression and tolerance, they will be further referred to as myeloid regulatory cells (MRCs). In recent years, MRCs have emerged as a therapeutic target or have been regarded as a potential cellular therapeutic product for tolerance induction. However, several open questions must be addressed to enable the therapeutic application of MRCs including: how do they function at the site of inflammation, how to best target these cells to modulate their activities, and how to isolate or to generate pure populations for adoptive cell therapies. In this review, we will give an overview of the current knowledge on MRCs in inflammation, autoimmunity, and transplantation. We will discuss current strategies to target MRCs and to exploit their tolerogenic potential as a cell-based therapy.

Dendritic cells (DCs) represent essential antigen-presenting cells that are critical for linking innate and adaptive immunity, and influencing T-cell responses. Among pattern recognition receptors, DCs express C-type lectin receptors triggered by both exogenous and endogenous ligands, therefore dictating pathogen response, and also shaping T-cell immunity. We previously described in rat, the expression of the orphan C-type lectin-like receptor-1 (CLEC-1) by DCs and demonstrated in vitro its inhibitory role in downstream T helper 17 (Th17) activation. In this study, we examined the expression and functionality of CLEC-1 in human DCs, and show a cell-surface expression on the CD16- subpopulation of blood DCs and on monocyte-derived DCs (moDCs). CLEC-1 expression on moDCs is downregulated by inflammatory stimuli and enhanced by transforming growth factor β. Moreover, we demonstrate that CLEC-1 is a functional receptor on human moDCs and that although not modulating the spleen tyrosine kinase-dependent canonical nuclear factor-κB pathway, represses subsequent Th17 responses. Interestingly, a decreased expression of CLEC1A in human lung transplants is predictive of the development of chronic rejection and is associated with a higher level of interleukin 17A (IL17A). Importantly, using CLEC-1-deficient rats, we showed that disruption of CLEC-1 signaling led to an enhanced Il12p40 subunit expression in DCs, and to an exacerbation of downstream in vitro and in vivo CD4+ Th1 and Th17 responses. Collectively, our results establish a role for CLEC-1 as an inhibitory receptor in DCs able to dampen activation and downstream effector Th responses. As a cell-surface receptor, CLEC-1 may represent a useful therapeutic target for modulating T-cell immune responses in a clinical setting.

As pediatric liver transplantation comes of age, experts gathered to discuss current paradigms and define gaps in knowledge warranting research to further improve patient and graft outcomes. Identified areas ripe for collaborative research include understanding the molecular and cellular mechanisms of tolerance and the role of donor-specific antibodies, considering ways to expand donor pool, minimizing long-term side effects of immunosuppression, and fine-tuning surgical techniques to minimize biliary and vascular complications.

Although the occurrence of acute rejection was significantly reduced and the allograft survival at 1 year was massively improved by the development of pharmacological immunosuppressive drugs, little progress has been made regarding long-term graft survival. Cell therapy appears to be an innovative and promising strategy to minimize the use of immunosuppression in transplantation and consequently increases long-term graft survival. The strength of cell therapy is that it will induce graft-specific tolerance and not a general immunosuppression of the patients. Several candidates, such as tolerogenic dendritic cells, have been gaining interest as an efficient means of promoting antigen-specific tolerance over recent years. Studies performed in rodent models have demonstrated the feasibility and efficacy of tolerogenic dendritic cells for the induction of tolerance in transplantation. In parallel, protocols to generate human tolerogenic dendritic cells in vitro have been defined, and some phase I clinical trials in autoimmune diseases have been recently performed to evaluate the safety of tolerogenic dendritic cell therapy. In this review, we will focus on the potential therapeutic interest of these cells in transplantation as well as their generation and characterization in humans. Finally, we will describe our current clinical trial using autologous tolerogenic dendritic cells in transplantation.

Retinoid-related orphan receptor gamma t (RORγt) is a master transcription factor central to type 17 immunity involving cells such as T helper 17, group 3 innate lymphoid cells or IL-17-producing γδ T cells. Here we show that the intracellular ion channel TMEM176B and its homologue TMEM176A are strongly expressed in these RORγt(+) cells. We demonstrate that TMEM176A and B exhibit a similar cation channel activity and mainly colocalise in close proximity to the trans-Golgi network. Strikingly, in the mouse, the loss of Tmem176b is systematically associated with a strong upregulation of Tmem176a. While Tmem176b single-deficiency has no effect on the course of experimental autoimmune encephalomyelitis, T cell or DSS-induced colitis, it significantly reduces imiquimod-induced psoriasis-like skin inflammation. These findings shed light on a potentially novel specific process linked to post-Golgi trafficking for modulating the function of RORγt(+) cells and indicate that both homologues should be simultaneously targeted to clearly elucidate the role of this intracellular ion flow.

Dendritic cells (DC) are the most important subset of antigen presenting cells (APC) that are able to polarize the immune response to pro-inflammatory or anti-inflammatory response. This duality places DC in the axis between tolerance and immunogenicity. The regulation of polarization is the key point in autoimmune diseases and organ transplantation. In order to manipulate this duality, DC have been generated ex vivo with a tolerogenic or immunogenic profile since several years. During the last decade, cell therapy using tolerogenic DC (TolDC) has been shown to be safe and effective both in autoimmune diseases and transplantation models in animals. Since 2011, recipient TolDC has been tested in clinical trials in type 1 diabetes, rheumatoid arthritis and Crohn’s diseases with favourable results in terms of safety. Indeed, other clinical trials are ongoing including a phase I/II clinical assay in kidney transplantation. In this review, we will discuss the potential of TolDC that has been demonstrated in animal models and used in clinical trials.