B-cell-directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates.

ABSTRACT: Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

Comparing laboratory and industrial yeast platforms for the direct conversion of cellobiose into ethanol under simulated industrial conditions.

An engineered yeast producing all the cellulases needed for cellulose saccharification could produce ethanol from lignocellulose at a lower cost. This study aimed to express fungal ß-glucosidases in Saccharomyces cerevisiae to convert cellobiose into ethanol. Furthermore, two engineering platforms (laboratory vs industrial strain) have been considered towards the successful deployment of the engineered yeast under simulated industrial conditions. The industrial S. cerevisiae M2n strain was engineered through the δ-integration of the ß-glucosidase Pccbgl1 of Phanerochaete chrysosporium. The most efficient recombinant, M2n[pBKD2-Pccbgl1]-C1, was compared to the laboratory S. cerevisiae Y294[Pccbgl1] strain, expressing Pccbgl1 from episomal plasmids, in terms of cellobiose fermentation in a steam exploded sugarcane bagasse pre-hydrolysate. Saccharomyces cerevisiae Y294[Pccbgl1] was severely hampered by the pre-hydrolysate. The industrial M2n[pBKD2-Pccbgl1]-C1 could tolerate high inhibitors-loading in pre-hydrolysate under aerobic conditions. However, in oxygen limited environment, the engineered industrial strain displayed ethanol yield higher than the laboratory Y294[Pccbgl1] only when supplemented with supernatant containing further recombinant ß-glucosidase. This study showed that the choice of the host strain is crucial to ensure bioethanol production from lignocellulose. A novel cellobiose-to-ethanol route has been developed and the recombinant industrial yeast could be a promising platform towards the future consolidated bioprocessing of lignocellulose into ethanol.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases.

The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch-based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon-optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α-amylase (SFA1) genes were δ-integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1-SFA1] and MEL2[TLG1-SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch-to-ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre-treatment or commercial enzyme addition) using industrial yeast strains co-secreting both a glucoamylase and α-amylase.

Human OX40L-CAR-Tregs target activated antigen-presenting cells and control T cell alloreactivity.

Regulatory T cells (Tregs) make major contributions to immune homeostasis. Because Treg dysfunction can lead to both allo- and autoimmunity, there is interest in correcting these disorders through Treg adoptive transfer. Two of the central challenges in clinically deploying Treg cellular therapies are ensuring phenotypic stability and maximizing potency. Here, we describe an approach to address both issues through the creation of OX40 ligand (OX40L)-specific chimeric antigen receptor (CAR)-Tregs under the control of a synthetic forkhead box P3 (FOXP3) promoter. The creation of these CAR-Tregs enabled selective Treg stimulation by engagement of OX40L, a key activation antigen in alloimmunity, including both graft-versus-host disease and solid organ transplant rejection, and autoimmunity, including rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. We demonstrated that OX40L-CAR-Tregs were robustly activated in the presence of OX40L-expressing cells, leading to up-regulation of Treg suppressive proteins without induction of proinflammatory cytokine production. Compared with control Tregs, OX40L-CAR-Tregs more potently suppressed alloreactive T cell proliferation in vitro and were directly inhibitory toward activated monocyte-derived dendritic cells (DCs). We identified trogocytosis as one of the central mechanisms by which these CAR-Tregs effectively decrease extracellular display of OX40L, resulting in decreased DC stimulatory capacity. OX40L-CAR-Tregs demonstrated an enhanced ability to control xenogeneic graft-versus-host disease compared with control Tregs without abolishing the graft-versus-leukemia effect. These results suggest that OX40L-CAR-Tregs may have wide applicability as a potent cellular therapy to control both allo- and autoimmune diseases.

Selection of Superior Yeast Strains for the Fermentation of Lignocellulosic Steam-Exploded Residues.

The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants.

Identification and Tracking of Alloreactive T Cell Clones in Rhesus Macaques Through the RM-scTCR-Seq Platform.

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell's transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, 'RM-scTCR-Seq', which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR's with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.

A yeast metabolome-based model for an ecotoxicological approach in the management of lignocellulosic ethanol stillage.

Lignocellulosic bioethanol production results in huge amounts of stillage, a potentially polluting by-product. Stillage, rich in heavy metals and, mainly, inhibitors, requires specific toxicity studies to be adequately managed. To this purpose, we applied an FTIR ecotoxicological bioassay to evaluate the toxicity of lignocellulosic stillage. Two weak acids and furans, most frequently found in lignocellulosic stillage, have been tested in different mixtures against three Saccharomyces cerevisiae strains. The metabolomic reaction of the test microbes and the mortality induced at various levels of inhibitor concentration showed that the strains are representative of three different types of response. Furthermore, the relationship between concentrations and FTIR synthetic stress indexes has been studied, with the aim of defining a model able to predict the concentrations of inhibitors in stillage, resulting in an optimized predictive model for all the strains. This approach represents a promising tool to support the ecotoxicological management of lignocellulosic stillage.

Metabolomic Alterations Do Not Induce Metabolic Burden in the Industrial Yeast M2n[pBKD2-Pccbgl1]-C1 Engineered by Multiple δ-Integration of a Fungal ß-Glucosidase Gene.

In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple δ-integration of the ß-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the δ-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple δ-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.

Production of bioethanol from multiple waste streams of rice milling.

This work describes the feasibility of using rice milling by-products as feedstock for bioethanol. Starch-rich residues (rice bran, broken, unripe and discolored rice) were individually fermented (20%w/v) through Consolidated Bioprocessing by two industrial engineered yeast secreting fungal amylases. Rice husk (20%w/v), mainly composed by lignocellulose, was pre-treated at 55°C with alkaline peroxide, saccharified through optimized dosages of commercial enzymes (Cellic® CTec2) and fermented by the recombinant strains. Finally, a blend of all the rice by-products, formulated as a mixture (20%w/v) according to their proportions at milling plants, were co-processed to ethanol by optimized pre-treatment, saccharification and fermentation by amylolytic strains. Fermenting efficiency for each by-product was high (above 88% of the theoretical) and further confirmed on the blend of residues (nearly 52g/L ethanol). These results demonstrated for the first time that the co-conversion of multiple waste streams is a promising option for second generation ethanol production.