Circadian clocks orchestrate metabolic processes in the whole body and their response to food. Therefore, not only what and how much, but also when we eat has a significant impact on metabolism. In this context, daytime of carbohydrate and fat intake was recently shown to alter the metabolic state and potentially affect the disease risk. However, molecular mechanisms of this regulation in humans are poorly understood. In this crossover trial, we investigated the effects of two isocaloric 4-week dietary patterns - high carbohydrate in the morning and high fat in the afternoon (HC/HF) and the reverse (HF/HC) - on the subcutaneous adipose tissue (SAT) in overweight non-diabetic men. The SAT transcriptome was assessed using microarrays. Additionally, gene expression in peripheral blood monocytes (PBMCs) was analysed by qPCRs. Analysis of SAT samples collected across the day identified 1386 genes exhibiting diurnal oscillations. In SAT, both oscillatory and non-oscillatory genes related to lipid and glucose metabolism were modulated by the timing of macronutrient intake. Notably, expression of inflammatory response genes in SAT was elevated after HC/HF compared to HF/HC, suggesting that the HC/HF diet might promote an early proinflammatory state in SAT due to higher fat intake in the afternoon. Diet-induced remodelling of the SAT transcriptome was partly reflected in PBMCs. These findings demonstrate that diurnal macronutrient distribution significantly reshapes the SAT transcriptome, underscoring the relevance of eating timing-based (chrononutritional) strategies for prevention of metabolic dysfunction and systemic inflammation associated with obesity and type 2 diabetes.

The cellular composition that defines the tumor ecosystem plays a crucial role in determining the response of tumors to immune checkpoint inhibitors (ICi) and to pharmacological blockade of BRAF (BRAFi). However, the compositional changes that likely shape the ecosystems of primary melanomas for metastasis to the brain on the one hand and the progression of brain metastases on the other remain unclear. In this study, we resolved the spatial landscapes of progressive melanoma brain metastases (MBM) by profiling 13 regions from 8 distinct metastases, together with one matched primary tumor spanning various stages of tumor development and therapeutic intervention. Single cell resolved spatial transcriptomics-based profiling uncovered distinct and common patterns of MBM progression. Our approach revealed remarkable cellular and spatial heterogeneity and identified key tumor subsets of MET or NGFR expressing cell populations. Moreover, spatial profiling of tumors which have progressed under ICi or BRAFi identified BZW2+ tumor cells contributing to an immune-suppressive microenvironment, preventing immune cell infiltration and dispersion within the tumor. Elevated BZW2 expression was associated with resistance to ICi therapy and is inversely correlated with the antigen transporter TAP1. Spatially-aware automated profiling of immune cell-enriched areas revealed ″hot″ niches characterized by tumor cell expression of TAP1, PD-L2, HLA-DRA whereas ″cold″ niches featured high levels of particularly BZW2 and SOX4. The latter suppressor of translation and transcriptional regulator was co-localized with the clinically targetable MET receptor in a subset of tumor cells that exhibit immune evasion features. In summary, we provide for the first time, spatially resolved landscapes of MBM that reveal insights into the cellular heterogeneity of tumors and the mechanisms underlying immune escape. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the German Federal Ministry of Education and Research (BMBF): NATON, No. 01KX2121 to H.R. and Seed funding from University Medicine Greifswald to K.P. and by the European Regional Development Fund (EFRE) (Target-H; FV-2024-0022) to JR. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Charite-Universitaetsmedizin Berlin and of Universitaetsmedizin Greifswald gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data generated in this study will be made publicly available by deposition in GEO (Gene Expression Omnibus) and Zenodo.

Immune evasion by escape mutations subverts immunity against SARS-CoV-2. A role of pan-coronavirus immunity for more durable protection is being discussed, but has remained understudied. We here investigated the effects of age, mutations, and homo-/heterologous vaccination regimens on the dominant pan-coronavirus-specific cellular and humoral epitope iCope after SARS-CoV-2 infection and vaccination in detail. In older individuals, the quantitatively and qualitatively reduced iCope-reactive CD4+ T cell responses with narrow TCR repertoires could not be enhanced by vaccination and were further compromised by emerging spike mutations. In contrast, pan-coronavirus-reactive humoral immunity was affected only by mutations and not by age. Our results reveal a distinct deficiency of the dichotomous layer of pan-coronavirus immunity in the older, critical for long-term protection against SARS-CoV-2 variants.

The development of brain metastases hallmarks disease progression in 20–40% of melanoma patients and is a serious obstacle to therapy. Understanding the processes involved in the development and maintenance of melanoma brain metastases (MBM) is critical for the discovery of novel therapeutic strategies. Here, we generated transcriptome and methylome profiles of MBM showing high or low abundance of infiltrated Iba1 high tumor-associated microglia and macrophages (TAMs). Our survey identified potential prognostic markers of favorable disease course and response to immune checkpoint inhibitor (ICi) therapy, among them APBB1IP and the interferon-responsive gene ITGB7 . In MBM with high ITGB7/APBB1IP levels, the accumulation of TAMs correlated significantly with the immune score. Signature-based deconvolution of MBM via single sample GSEA revealed enrichment of interferon-response and immune signatures and revealed inflammation, stress and MET receptor signaling. MET receptor phosphorylation/activation maybe elicited by inflammatory processes in brain metastatic melanoma cells via stroma cell-released HGF. We found phospho-MET Y1234/1235 in a subset of MBM and observed a marked response of brain metastasis-derived cell lines (BMCs) that lacked druggable BRAF mutations or developed resistance to BRAF inhibitors (BRAFi) in vivo to MET inhibitors PHA-665752 and ARQ197 (tivantinib). In summary, the activation of MET receptor in brain colonizing melanoma cells by stromal cell-released HGF may promote tumor self-maintenance and expansion and might counteract ICi therapy. Therefore, therapeutic targeting of MET possibly serves as a promising strategy to control intracranial progressive disease and improve patient survival.

Cardiovascular diseases are a significant cause of illness and death worldwide, often resulting in myofibroblast differentiation, pathological remodeling, and fibrosis, characterized by excessive extracellular matrix protein deposition. Treatment options for cardiac fibrosis that can effectively target myofibroblast activation and ECM deposition are limited, necessitating an unmet need for new therapeutic approaches. In recent years, microcurrent therapy has demonstrated promising therapeutic effects, showcasing its translational potential in cardiac care. This study therefore sought to investigate the effects of microcurrent therapy on cardiac myofibroblasts, aiming to unravel its potential as a treatment for cardiac fibrosis and heart failure. The experimental design involved the differentiation of primary rat cardiac fibroblasts into myofibroblasts. Subsequently, these cells were subjected to microcurrent (MC) treatment at 1 and 2 µA/cm2 DC with and without polarity reversal. We then investigated the impact of microcurrent treatment on myofibroblast cell behavior, including protein and gene expression, by performing various assays and analyses comparing them to untreated myofibroblasts and cardiac fibroblasts. The application of microcurrents resulted in distinct transcriptional signatures and improved cellular processes. Gene expression analysis showed alterations in myofibroblast markers, extracellular matrix components, and pro-inflammatory cytokines. These observations show signs of microcurrent-mediated reversal of myofibroblast phenotype, possibly reducing cardiac fibrosis, and providing insights for cardiac tissue repair.

Background Exercise exerts many health benefits by directly inducing molecular alterations in physically utilized skeletal muscle. Molecular adaptations of subcutaneous adipose tissue (SCAT) might also contribute to the prevention of metabolic diseases. Aim To characterize the response of human SCAT based on changes in transcripts and mitochondrial respiration to acute and repeated bouts of exercise in comparison to skeletal muscle. Methods Sedentary participants (27 ± 4 yrs) with overweight or obesity underwent 8-week supervised endurance exercise 3×1h/week at 80% VO2peak. Before, 60 min after the first and last exercise bout and 5 days post intervention, biopsies were taken for transcriptomic analyses and high-resolution respirometry ( n = 14, 8 female/6 male). Results In SCAT, we found 37 acutely regulated transcripts (FC > 1.2, FDR < 10%) after the first exercise bout compared to 394, respectively, in skeletal muscle. Regulation of only 5 transcripts overlapped between tissues highlighting their differential response. Upstream and enrichment analyses revealed reduced transcripts of lipid uptake, storage and lipogenesis directly after exercise in SCAT and point to β-adrenergic regulation as potential major driver. The data also suggest an exercise-induced modulation of the circadian clock in SCAT. Neither term was associated with transcriptomic changes in skeletal muscle. No evidence for beigeing/browning was found in SCAT along with unchanged respiration. Conclusions Adipose tissue responds completely distinct from adaptations of skeletal muscle to exercise. The acute and repeated reduction in transcripts of lipid storage and lipogenesis, interconnected with a modulated circadian rhythm, can counteract metabolic syndrome progression toward diabetes.

Background Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. Methods Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. Results We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. Conclusions Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential. Graphical Abstract

Currently available COVID-19 vaccines include inactivated virus, live attenuated virus, mRNA-based, viral vectored and adjuvanted protein-subunit-based vaccines. All of them contain the spike glycoprotein as the main immunogen and result in reduced disease severity upon SARS-CoV-2 infection. While we and others have shown that mRNA-based vaccination reactivates pre-existing, cross-reactive immunity, the effect of vector vaccines in this regard is unknown. Here, we studied cellular and humoral responses in heterologous adenovirus-vector-based ChAdOx1 nCOV-19 (AZ; Vaxzeria, AstraZeneca) and mRNA-based BNT162b2 (BNT; Comirnaty, BioNTech/Pfizer) vaccination and compared it to a homologous BNT vaccination regimen. AZ primary vaccination did not lead to measurable reactivation of cross-reactive cellular and humoral immunity compared to BNT primary vaccination. Moreover, humoral immunity induced by primary vaccination with AZ displayed differences in linear spike peptide epitope coverage and a lack of anti-S2 IgG antibodies. Contrary to primary AZ vaccination, secondary vaccination with BNT reactivated pre-existing, cross-reactive immunity, comparable to homologous primary and secondary mRNA vaccination. While induced anti-S1 IgG antibody titers were higher after heterologous vaccination, induced CD4+ T cell responses were highest in homologous vaccinated. However, the overall TCR repertoire breadth was comparable between heterologous AZ-BNT-vaccinated and homologous BNT-BNT-vaccinated individuals, matching TCR repertoire breadths after SARS-CoV-2 infection, too. The reasons why AZ and BNT primary vaccination elicits different immune response patterns to essentially the same antigen, and the associated benefits and risks, need further investigation to inform vaccine and vaccination schedule development.

The development of brain metastases hallmarks disease progression in 20-40% of melanoma patients and is a serious obstacle to therapy. Understanding the processes involved in the development and maintenance of melanoma brain metastases (MBM) is critical for the discovery of novel therapeutic strategies. Here, we generated transcriptome and methylome profiles of MBM showing high or low abundance of infiltrated Iba1 high tumor-associated microglia and macrophages (TAMs). Our survey identified potential prognostic markers of favorable disease course and response to immune checkpoint inhibitor (ICi) therapy, among them APBB1IP and the interferon-responsive gene ITGB7 . In MBM with high ITGB7/APBB1IP levels, the accumulation of TAMs correlated significantly with the immune score. Signature-based deconvolution of MBM via single sample GSEA revealed enrichment of interferon-response and immune signatures and revealed inflammation, stress and MET receptor signaling. MET receptor phosphorylation/activation maybe elicited by inflammatory processes in brain metastatic melanoma cells via stroma cell-released HGF. We observed phospho-MET Y1234/1235 in a subset of MBM and observed marked response of brain metastasis-derived cell lines (BMCs) that lacked druggable BRAF mutations or developed resistance to BRAF inhibitors (BRAFi) in vivo to MET inhibitors PHA-665752 and ARQ197 (tivantinib). In summary, the activation of MET receptor in brain colonizing melanoma cells by stromal cell-released HGF may promote tumor cells self-maintenance and expansion might counteract ICi therapy. Therefore, therapeutic targeting of MET possibly serves as promising strategy to control intracranial progressive disease and improve patient survival.

BACKGROUND:SARS-CoV-2 mRNA vaccination of healthy individuals is highly immunogenic and protective against severe COVID-19. However, there are limited data on how disease-modifying therapies (DMTs) alter SARS-CoV-2 mRNA vaccine immunogenicity in patients with autoimmune diseases. METHODS:As part of a prospective cohort study, we investigated the induction, stability and boosting of vaccine-specific antibodies, B cells and T cells in patients with multiple sclerosis (MS) on different DMTs after homologous primary, secondary and booster SARS-CoV-2 mRNA vaccinations. Of 126 patients with MS analysed, 105 received either anti-CD20-based B cell depletion (aCD20-BCD), fingolimod, interferon-β, dimethyl fumarate, glatiramer acetate, teriflunomide or natalizumab, and 21 were untreated MS patients for comparison. RESULTS:In contrast to all other MS patients, and even after booster, most aCD20-BCD- and fingolimod-treated patients showed no to markedly reduced anti-S1 IgG, serum neutralising activity and a lack of receptor binding domain-specific and S2-specific B cells. Patients receiving fingolimod additionally lacked spike-reactive CD4+ T cell responses. The duration of fingolimod treatment, rather than peripheral blood B and T cell counts prior to vaccination, determined whether a humoral immune response was elicited. CONCLUSIONS:The lack of immunogenicity under long-term fingolimod treatment demonstrates that functional immune responses require not only immune cells themselves, but also access of these cells to the site of inoculation and their unimpeded movement. The absence of humoral and T cell responses suggests that fingolimod-treated patients with MS are at risk for severe SARS-CoV-2 infections despite booster vaccinations, which is highly relevant for clinical decision-making and adapted protective measures, particularly considering additional recently approved sphingosine-1-phosphate receptor antagonists for MS treatment.

Intestinal parasitic nematodes affect a quarter of the world's population, typically eliciting prominent effector Th2-driven host immune responses. As not all infected hosts develop protection against reinfection, our current understanding of nematode-induced memory Th2 responses remains limited. Here, we investigated the activation of memory Th2 cells and the mechanisms driving early recall responses to the enteric nematode Heligmosomoides polygyrus in mice. We show that nematode-cured mice harbor memory Th2 cells in lymphoid and non-lymphoid organs with distinct transcriptional profiles, expressing recirculation markers like CCR7 and CD62-L in the mesenteric lymph nodes (mLN), and costimulatory markers like Ox40, as well as tissue homing and activation markers like CCR2, CD69 and CD40L in the gut and peritoneal cavity (PEC). While memory Th2 cells persist systemically in both lymphoid and non-lymphoid tissues following cure of infection, peritoneal memory Th2 cells in particular displayed an initial prominent expansion and strong parasite-specific Th2 responses during early recall responses to a challenge nematode infection. This effect was paralleled by a significant influx of dendritic cells (DC) and eosinophils, both also appearing exclusively in the peritoneal cavity of reinfected mice. In addition, we show that within the peritoneal membrane lined by peritoneal mesothelial cells (PeM), the gene expression levels of cell adhesion markers VCAM-1 and ICAM-1 decrease significantly in response to a secondary infection. Overall, our findings indicate that the host peritoneal cavity in particular harbors prominent memory Th2 cells and appears to respond directly to H. polygyrus by an early recall response via differential regulation of cell adhesion markers, marking the peritoneal cavity an important site for host immune responses to an enteric pathogen.

Stromal cells interact with immune cells during initiation and resolution of immune responses, though the precise underlying mechanisms remain to be resolved. Lessons learned from stromal cell-based therapies indicate that environmental signals instruct their immunomodulatory action contributing to immune response control. Here, to the best of our knowledge, we show a novel function for the guanine-exchange factor DOCK2 in regulating immunosuppressive function in three human stromal cell models and by siRNA-mediated DOCK2 knockdown. To identify immune function-related stromal cell molecular signatures, we first reprogrammed mesenchymal stem/progenitor cells (MSPCs) into induced pluripotent stem cells (iPSCs) before differentiating these iPSCs in a back-loop into MSPCs. The iPSCs and immature iPS-MSPCs lacked immunosuppressive potential. Successive maturation facilitated immunomodulation, while maintaining clonogenicity, comparable to their parental MSPCs. Sequential transcriptomics and methylomics displayed time-dependent immune-related gene expression trajectories, including DOCK2, eventually resembling parental MSPCs. Severe combined immunodeficiency (SCID) patient-derived fibroblasts harboring bi-allelic DOCK2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. Conditional DOCK2 siRNA knockdown in iPS-MSPCs and fibroblasts also immediately reduced immunomodulatory capacity. Conclusively, CRISPR/Cas9-mediated DOCK2 knockout in iPS-MSPCs also resulted in significantly reduced immunomodulation, reduced CDC42 Rho family GTPase activation and blunted filopodia formation. These data identify G protein signaling as key element devising stromal cell immunomodulation.

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms.

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient's prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAFmut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients.

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

T cells need to adapt their cellular metabolism for effector cell differentiation. This relies on alterations in mitochondrial physiology. Which signals and molecules regulate those alterations remains unclear. We recently reported, that the mitochondrial protein TCAIM inhibits activation-induced changes in mitochondrial morphology and function and thus, CD4 + effector T cell formation. Using conditional TCAIM knock-in (KI) and knockout (KO) mice, we now show that it also applies to CD8 + T cells and more importantly, delineate the molecular processes in mitochondria by which TCAIM controls effector cell differentiation. TCAIM KI resulted in reduced activation-induced HIF1α protein expression. Metabolomics and transcriptional data in combination with mathematical flux modeling revealed an impaired induction of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis in TCAIM KI CD8 + T cells. Addition of cholesterol completely rescued HIF1α protein expression, activation and proliferation of TCAIM KI CD8 + T cells. At the molecular level, TCAIM delayed activation-induced mitochondria-ER contact (MERC) formation by binding to MERC promoting proteins such as RMD3 and VDAC2. In summary, we demonstrate that TCAIM suppresses effector cell differentiation by inhibiting MERC formation, which induce HIF1α-mediated increase in cellular metabolism and cholesterol biosynthesis.

The prevailing ‘division of labor’ concept in cellular immunity is that CD8 + T cells primarily utilize cytotoxic functions to kill target cells, while CD4 + T cells exert helper/inducer functions. Multiple subsets of CD4 + memory T cells have been characterized by distinct chemokine receptor expression. Here, we demonstrate that analogous CD8 + memory T-cell subsets exist, characterized by identical chemokine receptor expression signatures and controlled by similar generic programs. Among them, Tc2, Tc17 and Tc22 cells, in contrast to Tc1 and Tc17 + 1 cells, express IL-6R but not SLAMF7, completely lack cytotoxicity and instead display helper functions including CD40L expression. CD8 + helper T cells exhibit a unique TCR repertoire, express genes related to skin resident memory T cells (T RM ) and are altered in the inflammatory skin disease psoriasis. Our findings reveal that the conventional view of CD4 + and CD8 + T cell capabilities and functions in human health and disease needs to be revised.

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.

AbstractObjectiveTo 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.DesignInvestigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13).SettingCharité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union).ParticipantsRecipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9).InterventionsCD4+ 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 measuresThe 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.ResultsFor 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.ConclusionsThe 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 registrationNCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).