In Vitro Transcribed mRNA Immunogenicity Induces Chemokine-Mediated Lymphocyte Recruitment and Can Be Gradually Tailored by Uridine Modification.

Beyond SARS-CoV2 vaccines, mRNA drugs are being explored to overcome today's greatest healthcare burdens, including cancer and cardiovascular disease. Synthetic mRNA triggers immune responses in transfected cells, which can be reduced by chemically modified nucleotides. However, the side effects of mRNA-triggered immune activation on cell function and how different nucleotides, such as the N1-methylpseudouridine (m1Ψ) used in SARS-CoV2 vaccines, can modulate cellular responses is not fully understood. Here, cellular responses toward a library of uridine-modified mRNAs are investigated in primary human cells. Targeted proteomics analyses reveal that unmodified mRNA induces a pro-inflammatory paracrine pattern marked by the secretion of chemokines, which recruit T and B lymphocytes toward transfected cells. Importantly, the magnitude of mRNA-induced changes in cell function varies quantitatively between unmodified, Ψ-, m1Ψ-, and 5moU-modified mRNA and can be gradually tailored, with implications for deliberately exploiting this effect in mRNA drug design. Indeed, both the immunosuppressive effect of stromal cells on T-cell proliferation, and the anti-inflammatory effect of IL-10 mRNA are enhanced by appropriate uridine modification. The results provide new insights into the effects of mRNA drugs on cell function and cell-cell communication and open new possibilities to tailor mRNA-triggered immune activation to the desired pro- or anti-inflammatory application.

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T-cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and redirection of various immune cell types, including conventional T-cells, TCRγ/δ T-cells, regulatory T-cells, and NK-cells. In T-cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T-cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T-cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK-cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3 ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 ζ-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant ( TRAC )-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 ζ-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 ζ conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key points: Integration of ζ-deficient CARs into CD3 ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

CAR NK-92 cell-mediated depletion of residual TCR+ cells for ultrapure allogeneic TCR-deleted CAR T-cell products.

Graft-versus-host disease (GVHD) is a major risk of the administration of allogeneic chimeric antigen receptor (CAR)-redirected T cells to patients who are HLA unmatched. Gene editing can be used to disrupt potentially alloreactive T-cell receptors (TCRs) in CAR T cells and reduce the risk of GVHD. Despite the high knockout rates achieved with the optimized methods, a subsequent purification step is necessary to obtain a safe allogeneic product. To date, magnetic cell separation (MACS) has been the gold standard for purifying TCRα/ß- CAR T cells, but product purity can still be insufficient to prevent GVHD. We developed a novel and highly efficient approach to eliminate residual TCR/CD3+ T cells after TCRα constant (TRAC) gene editing by adding a genetically modified CD3-specific CAR NK-92 cell line during ex vivo expansion. Two consecutive cocultures with irradiated, short-lived, CAR NK-92 cells allowed for the production of TCR- CAR T cells with <0.01% TCR+ T cells, marking a 45-fold reduction of TCR+ cells compared with MACS purification. Through an NK-92 cell-mediated feeder effect and circumventing MACS-associated cell loss, our approach increased the total TCR- CAR T-cell yield approximately threefold while retaining cytotoxic activity and a favorable T-cell phenotype. Scaling in a semiclosed G-Rex bioreactor device provides a proof-of-principle for large-batch manufacturing, allowing for an improved cost-per-dose ratio. Overall, this cell-mediated purification method has the potential to advance the production process of safe off-the-shelf CAR T cells for clinical applications.

Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells.

BACKGROUND: Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. RESULTS: Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro. CONCLUSIONS: We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.

In vitro and in vivo evidence that the switch from calcineurin to mTOR inhibitors may be a strategy for immunosuppression in Epstein-Barr virus-associated post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder is a life-threatening complication of immunosuppression following transplantation mediated by failure of T cells to control Epstein-Barr virus (EBV)-infected and transformed B cells. Typically, a modification or reduction of immunosuppression is recommended, but insufficiently defined thus far. In order to help delineate this, we characterized EBV-antigen-specific T cells and lymphoblastoid cell lines from healthy donors and in patients with a kidney transplant in the absence or presence of the standard immunosuppressants tacrolimus, cyclosporin A, prednisolone, rapamycin, and mycophenolic acid. Phenotypes of lymphoblastoid cell-lines and T cells, T cell-receptor-repertoire diversity, and T-cell reactivity upon co-culture with autologous lymphoblastoid cell lines were analyzed. Rapamycin and mycophenolic acid inhibited lymphoblastoid cell-line proliferation. T cells treated with prednisolone and rapamycin showed nearly normal cytokine production. Proliferation and the viability of T cells were decreased by mycophenolic acid, while tacrolimus and cyclosporin A were strong suppressors of T-cell function including their killing activity. Overall, our study provides a basis for the clinical decision for the modification and reduction of immunosuppression and adds information to the complex balance of maintaining anti-viral immunity while preventing acute rejection. Thus, an immunosuppressive regime based on mTOR inhibition and reduced or withdrawn calcineurin inhibitors could be a promising strategy for patients with increased risk of or manifested EBV-associated post-transplant lymphoproliferative disorder.

Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells.

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR+ T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.

Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients.

Solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines. For this reason, SOT patients have a high risk of developing severe coronavirus disease 2019 (COVID-19) and an increased risk of death from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Moreover, the efficiency of immunotherapies and vaccines is reduced due to the constant immunosuppression in this patient group. Here, we propose adoptive transfer of SARS-CoV-2-specific T cells made resistant to a common immunosuppressant, tacrolimus, for optimized performance in the immunosuppressed patient. Using a ribonucleoprotein approach of CRISPR-Cas9 technology, we have generated tacrolimus-resistant SARS-CoV-2-specific T cell products from convalescent donors and demonstrate their specificity and function through characterizations at the single-cell level, including flow cytometry, single-cell RNA (scRNA) Cellular Indexing of Transcriptomes and Epitopes (CITE), and T cell receptor (TCR) sequencing analyses. Based on the promising results, we aim for clinical validation of this approach in transplant recipients. Additionally, we propose a combinatory approach with tacrolimus, to prevent an overshooting immune response manifested as bystander T cell activation in the setting of severe COVID-19 immunopathology, and tacrolimus-resistant SARS-CoV-2-specific T cell products, allowing for efficient clearance of viral infection. Our strategy has the potential to prevent severe COVID-19 courses in SOT or autoimmunity settings and to prevent immunopathology while providing viral clearance in severe non-transplant COVID-19 cases.

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.

Strong Expansion of Human Regulatory T Cells for Adoptive Cell Therapy Results in Epigenetic Changes Which May Impact Their Survival and Function.

Adoptive transfer of regulatory T cells (Treg) is a promising new therapeutic option to treat detrimental inflammatory conditions after transplantation and during autoimmune disease. To reach sufficient cell yield for treatment, ex vivo isolated autologous or allogenic Tregs need to be expanded extensively in vitro during manufacturing of the Treg product. However, repetitive cycles of restimulation and prolonged culture have been shown to impact T cell phenotypes, functionality and fitness. It is therefore critical to scrutinize the molecular changes which occur during T cell product generation, and reexamine current manufacturing practices. We performed genome-wide DNA methylation profiling of cells throughout the manufacturing process of a polyclonal Treg product that has proven safety and hints of therapeutic efficacy in kidney transplant patients. We found progressive DNA methylation changes over the duration of culture, which were donor-independent and reproducible between manufacturing runs. Differentially methylated regions (DMRs) in the final products were significantly enriched at promoters and enhancers of genes implicated in T cell activation. Additionally, significant hypomethylation did also occur in promoters of genes implicated in functional exhaustion in conventional T cells, some of which, however, have been reported to strengthen immunosuppressive effector function in Tregs. At the same time, a set of reported Treg-specific demethylated regions increased methylation levels with culture, indicating a possible destabilization of Treg identity during manufacturing, which was independent of the purity of the starting material. Together, our results indicate that the repetitive TCR-mediated stimulation lead to epigenetic changes that might impact functionality of Treg products in multiple ways, by possibly shifting to an effector Treg phenotype with enhanced functional activity or by risking destabilization of Treg identity and impaired TCR activation. Our analyses also illustrate the value of epigenetic profiling for the evaluation of T cell product manufacturing pipelines, which might open new avenues for the improvement of current adoptive Treg therapies with relevance for conventional effector T cell products.

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.

Functional Surfactants for Molecular Fishing, Capsule Creation, and Single-Cell Gene Expression.

Creating a single surfactant that is open to manipulation, while maintaining its surface activity, robustness, and compatibility, to expand the landscape of surfactant-dependent assays is extremely challenging. We report an oxidation-responsive precursor with thioethers and multiple 1,2-diols for creating a variety of functional surfactants from one parent surfactant. Using these multifunctional surfactants, we stabilize microfluidics-generated aqueous droplets. The droplets encapsulate different components and immerse in a bioinert oil with distinct interfaces where an azide-bearing surfactant allow fishing of biomolecules from the droplets, aldehyde-bearing surfactant allow fabrication of microcapsules, and hydroxyl-bearing surfactants, with/without oxidized thioethers, allow monitoring of single-cell gene expression. Creating multifunctional surfactants poses opportunities for broad applications, including adsorption, bioanalytics, catalysis, formulations, coatings, and programmable subset of emulsions.

Cas9-directed immune tolerance in humans-a model to evaluate regulatory T cells in gene therapy?

The dichotomic nature of the adaptive immune response governs the outcome of clinical gene therapy. On the one hand, neutralizing antibodies and cytotoxic T cells can have a dramatic impact on the efficacy and safety of human gene therapies. On the other hand, regulatory T cells (Treg) can promote tolerance toward transgenes thereby enabling long-term benefits of in vivo gene therapy after a single administration. Pre-existing antibodies and T cell immunity has been a major obstacle for in vivo gene therapies with viral vectors. As CRISPR-Cas9 gene editing advances toward the clinics, the technology's inherent immunogenicity must be addressed in order to guide clinical treatment decisions. This review summarizes the recent evidence on Cas9-specific immunity in humans-including early results from clinical trials-and discusses the risks for in vivo gene therapies. Finally, we focus on solutions and highlight the potential role of Cas9-specific Treg cells to promote immune tolerance. As a "beneficial alliance" beyond Cas9-immunity, antigen-specific Treg cells may serve as a living and targeted immunosuppressant to increase safety and efficacy of gene therapy.

The intratumoral CXCR3 chemokine system is predictive of chemotherapy response in human bladder cancer.

Chemotherapy has direct toxic effects on cancer cells; however, long-term cancer control and complete remission are likely to involve CD8+ T cell immune responses. To study the role of CD8+ T cell infiltration in the success of chemotherapy, we examined patients with muscle invasive bladder cancer (MIBC) who were categorized on the basis of the response to neoadjuvant chemotherapy (NAC). We identified the intratumoral CXCR3 chemokine system (ligands and receptor splice variants) as a critical component for tumor eradication upon NAC in MIBC. Through characterization of CD8+ T cells, we found that stem-like T cell subpopulations with abundant CXCR3alt, a variant form of the CXCL11 receptor, responded to CXCL11 in culture as demonstrated by migration and enhanced effector function. In tumor biopsies of patients with MIBC accessed before treatment, CXCL11 abundance correlated with high numbers of tumor-infiltrating T cells and response to NAC. The presence of CXCR3alt and CXCL11 was associated with improved overall survival in MIBC. Evaluation of both CXCR3alt and CXCL11 enabled discrimination between responder and nonresponder patients with MIBC before treatment. We validated the prognostic role of the CXCR3-CXCL11 chemokine system in an independent cohort of chemotherapy-treated and chemotherapy-naïve patients with MIBC from data in TCGA. In summary, our data revealed stimulatory activity of the CXCR3alt-CXCL11 chemokine system on CD8+ T cells that is predictive of chemotherapy responsiveness in MIBC. This may offer immunotherapeutic options for targeted activation of intratumoral stem-like T cells in solid tumors.

CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.

Viral infections, such as with cytomegalovirus (CMV), remain a major risk factor for mortality and morbidity of transplant recipients because of their requirement for lifelong immunosuppression (IS). Antiviral drugs often cause toxicity and sometimes fail to control disease. Thus, regeneration of the antiviral immune response by adoptive antiviral T cell therapy is an attractive alternative. Our recent data, however, show only short-term efficacy in some solid organ recipients, possibly because of malfunction in transferred T cells caused by ongoing IS. We developed a vector-free clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based good manufacturing practice (GMP)-compliant protocol that efficiently targets and knocks out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), required for the immunosuppressive function of tacrolimus. This was achieved by transient delivery of ribonucleoprotein complexes into CMV-specific T cells by electroporation. We confirmed the tacrolimus resistance of our gene-edited T cell products in vitro and demonstrated performance comparable with non-tacrolimus-treated unmodified T cells. The alternative calcineurin inhibitor cyclosporine A can be administered as a safety switch to shut down tacrolimus-resistant T cell activity in case of adverse effects. Furthermore, we performed safety assessments as a prerequisite for translation to first-in-human applications.

The role of soluble mediators in the clinical course of EBV infection and B cell homeostasis after kidney transplantation.

Epstein-Barr virus (EBV) reactivation can lead to serious complications in kidney transplant patients, including post-transplant lymphoproliferative disorder (PTLD). Here, we have assessed the impact of EBV on B cell homeostasis at cellular and humoral level. In a multicenter study monitoring 540 kidney transplant patients during the first post-transplant year, EBV reactivation was detected in 109 patients. Thirteen soluble factors and B cell counts were analyzed in an EBV+ sub-cohort (N = 54) before, at peak and after EBV clearance and compared to a control group (N = 50). The B cell activating factor (BAFF) was significantly elevated among EBV+ patients. No additional soluble factors were associated with EBV. Importantly, in vitro experiments confirmed the proliferative effect of BAFF on EBV-infected B cells, simultaneously promoting EBV production. In contrast, elevated levels of BAFF in EBV+ patients did not lead to B cell expansion in vivo. Moreover, diminished positive inter-correlations of soluble factors and alterations of the bi-directional interplay between B cell and soluble factors were observed in EBV+ patients at peak and after clearance. Our data suggest that such alterations may counteract the proliferative effect of BAFF, preventing B cell expansion. The role of these alterations in lymphoma development should be analyzed in future studies.

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).

Transient antibody targeting of CD45RC inhibits the development of graft-versus-host disease.

Allogeneic bone marrow transplantation (BMT) is a widely spread treatment of many hematological diseases, but its most important side effect is graft-versus-host disease (GVHD). Despite the development of new therapies, acute GVHD (aGVHD) occurs in 30% to 50% of allogeneic BMT and is characterized by the generation of effector T (Teff) cells with production of inflammatory cytokines. We previously demonstrated that a short anti-CD45RC monoclonal antibody (mAb) treatment in a heart allograft rat model transiently decreased CD45RChigh Teff cells and increased regulatory T cell (Treg) number and function allowing long-term donor-specific tolerance. Here, we demonstrated in rat and mouse allogeneic GVHD, as well as in xenogeneic GVHD mediated by human T cells in NSG mice, that both ex vivo depletion of CD45RChigh T cells and in vivo treatment with short-course anti-CD45RC mAbs inhibited aGVHD. In the rat model, we demonstrated that long surviving animals treated with anti-CD45RC mAbs were fully engrafted with donor cells and developed a donor-specific tolerance. Finally, we validated the rejection of a human tumor in NSG mice infused with human cells and treated with anti-CD45RC mAbs. The anti-human CD45RC mAbs showed a favorable safety profile because it did not abolish human memory antiviral immune responses, nor trigger cytokine release in in vitro assays. Altogether, our results show the potential of a prophylactic treatment with anti-human CD45RC mAbs in combination with rapamycin as a new therapy to treat aGVHD without abolishing the antitumor effect.