A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells.

The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.

Machine learning-based detection of adventitious microbes in T-cell therapy cultures using long-read sequencing.

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP <71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated, and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimized experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 colony-forming units (CFU)/mL using metagenomic classification. Machine learning can be coupled with long-read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP <71> organisms to 10 CFU/mL. IMPORTANCE This research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long-read nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

Cell Granularity Reflects Immune Cell Function and Enables Selection of Lymphocytes with Superior Attributes for Immunotherapy.

In keeping with the rule of "form follows function", morphological aspects of a cell can reflect its role. Here, it is shown that the cellular granularity of a lymphocyte, represented by its intrinsic side scatter (SSC), is a potent indicator of its cell state and function. The granularity of a lymphocyte increases from naïve to terminal effector state. High-throughput cell-sorting yields a SSChigh population that can mediate immediate effector functions, and a highly prolific SSClow population that can give rise to the replenishment of the memory pool. CAR-T cells derived from the younger SSClow population possess desirable attributes for immunotherapy, manifested by increased naïve-like cells and stem cell memory (TSCM )-like cells together with a balanced CD4/CD8 ratio, as well as enhanced target-killing in vitro and in vivo. Altogether, lymphocyte segregation based on biophysical properties is an effective approach for label-free selection of cells that share collective functions and can have important applications for cell-based immunotherapies.

IRF-7 Mediates Type I IFN Responses in Endotoxin-Challenged Mice.

IRF-7 mediates robust production of type I IFN via MyD88 of the TLR9 pathway in plasmacytoid dendritic cells (pDCs). Previous in vitro studies using bone marrow-derived dendritic cells lacking either Irf7 or Irf3 have demonstrated that only IRF-3 is required for IFN-ß production in the TLR4 pathway. Here, we show that IRF-7 is essential for both type I IFN induction and IL-1ß responses via TLR4 in mice. Mice lacking Irf7 were defective in production of both IFN-ß and IL-1ß, an IFN-ß-induced pro-inflammatory cytokine, after LPS challenge. IFN-ß production in response to LPS was impaired in IRF-7-deficient macrophages, but not dendritic cells. Unlike pDCs, IRF-7 is activated by the TRIF-, but not MyD88-, dependent pathway via TBK-1 in macrophages after LPS stimulation. Like pDCs, resting macrophages constitutively expressed IRF-7 protein. This basal IRF-7 protein was completely abolished in either Ifnar1-/- or Stat1-/- macrophages, which corresponded with the loss of LPS-stimulated IFN-ß induction in these macrophages. These findings demonstrate that macrophage IRF-7 is critical for LPS-induced type I IFN responses, which in turn facilitate IL-1ß production in mice.

Activation and regulation of interferon-ß in immune responses.

Interferons (IFNs) were discovered more than half a century ago, and extensive research has since identified multifarious roles for type I IFN in human immune responses. Here, we review the functions of IFN-ß in innate and adaptive immunity. We also discuss the activation and influence of IFN-ß on myeloid cell types, including monocytes and dendritic cells, as well as address the effects of IFN-ß on T cells and B cells. Findings from our own laboratory, which explores the molecular mechanisms of IFN-ß activation by LPS and viruses, as well as from other groups investigating the regulation of IFN-ß by viral proteins and endogenous factors are described. The effects of post-translational modifications of the interferon regulatory factor (IRF)-3 on IFN-ß induction are also highlighted. Many unanswered questions remain concerning the regulation of the type I IFN response in inflammation, especially the role of transcription factors in the modulation of inflammatory gene expression, and these questions will form the basis for exciting avenues of future research.

IRF8 and IRF3 cooperatively regulate rapid interferon-ß induction in human blood monocytes.

Robust and rapid induction of interferon-ß (IFN-ß) in monocytes after pathogenic stimulation is a hallmark of innate immune responses. Here, we reveal the molecular mechanism underlying this key property that is exclusive to human blood monocytes. We found that IFN-ß was produced rapidly in primary human monocytes as a result of cooperation between the myeloid-specific transcription factor IRF8 and the ubiquitous transcription factor IRF3. Knockdown of IRF8 in monocytes abrogated IFN-ß transcription, whereas reintroduction of IRF8 into the IRF8(-/-) 32Dcl3 murine myeloid cell line reinstated IFN-ß transcription. Moreover, we provide evidence that IRF8 constitutively binds to the ETS/IRF composite element of the IFN-ß promoter region together with PU.1 in vivo. Furthermore we uncovered a requirement for IRF3, a master regulator of IFN-ß production, as a previously un-indentified interaction partner of IRF8. We mapped the protein-protein interacting regions of IRF3 and IRF8, and found that their interaction was independent of the DNA-binding domain and the IRF association domain of IRF8 and IRF3, respectively. Therefore, we propose a model for the rapid induction of IFN-ß in monocytes, whereby IRF8 and PU.1 form a scaffold complex on the IFN-ß promoter to facilitate the recruitment of IRF3, thus enabling rapid IFN-ß transcription.