Deep mapping of the TCR-antigen interface using pMHC-pseudotyped viruses and yeast display.

T cell receptor (TCR) specificity is central to the efficacy of T cell therapies, yet scalable methods to map how TCR sequences shape antigen recognition remain limited. To address this, we introduce VelociRAPTR, a library-on-library approach that combines yeast-displayed TCR libraries with pMHC-displaying virus-like particles (pMHC-VLPs) to rapidly screen millions of TCR-antigen interactions. We show that pMHC-VLPs efficiently bind TCRs on yeast and generate equivalent data to recombinantly produced pMHC protein.

Automated, aseptic sampling with small-volume capacity from microbioreactors for cell therapy process analysis.

Current workflows in autologous cell therapy manufacturing are reliant on manual processes that are difficult to scale out to meet patient demands. High throughput bioreactor systems that enable multiple cultures to occur in parallel can address this need, but require good bioprocess monitoring workflows to produce good quality cell therapy products. Commercial sampling systems have thus been developed for better feedback control and monitoring capabilities.

High-throughput screening for class I peptide MHC binding via yeast surface display.

T cells rely on short peptides presented by highly polymorphic major histocompatibility complexes (MHCs) to selectively initiate adaptive immune responses. Despite its importance, few techniques can systemically evaluate stable peptide presentation across diverse MHC alleles. Here, we describe a yeast display pipeline that can be deployed to rapidly screen proteomic space to identify class I pMHC binders across many alleles.

Use of cellular FAD autofluorescence as a label-free cellular attribute for the production of chimeric antigen receptor T cells.

Chimeric antigen receptor (CAR) T-cell therapy has become an attractive approach for treating hematologic malignancies. However, the accessibility of this therapy is limited by factors such as complex manufacturing processes, limited capacity of manufacturing facilities, and the requirement of a highly skilled workforce for the manual steps of CAR T-cell production. To minimize manual processes, the CAR T-cell manufacturing field is shifting towards closed and automated systems, including analytical tools that offer intermittent monitoring of cells in production.

Cell trajectory modulation: rapid microfluidic biophysical profiling of CAR T cell functional phenotypes.

Chimeric Antigen Receptor (CAR) T cell therapy is a pivotal treatment for hematological malignancies. However, CAR T cell products exhibit batch-to-batch variability in cell number, quality, and in vivo efficacy due to donor-to-donor heterogeneity, and pre/post-manufacturing processes, and the manufacturing of such products necessitates careful testing, both post-manufacturing and pre-infusion. Here, we introduce the Cell Trajectory Modulation (CTM) assay, a microfluidic, label-free approach for the rapid evaluation of the functional attributes of CAR T cells based on biophysical features (i.

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells.

In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we developed a single-transcript, microRNA-based incoherent feedforward loop called compact microRNA-mediated attenuator of noise and dosage (ComMAND). We experimentally tuned the ComMAND output profile, and we modeled the system to explore additional tuning strategies.

Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence.

The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function-particularly at the molecular level-are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures.

Expanding the CAR toolbox with high throughput screening strategies for CAR domain exploration: a comprehensive review.

Chimeric antigen receptor (CAR)-T-cell therapy has been highly successful in the treatment of B-cell hematological malignancies. CARs are modular synthetic molecules that can redirect immune cells towards target cells with antibody-like specificity. Despite their modularity, CARs used in the clinic are currently composed of a limited set of domains, mostly derived from IgG, CD8α, 4-1BB, CD28 and CD3ζ.

Rapid Universal Detection of High-Risk and Low-Abundance Microbial Contaminations in CAR-T Cell Therapy.

Live microbial contamination poses high risks to cell and gene therapies, threatening manufacturing processes and patient safety. Rapid, sensitive detection of live microbes in complex environments, such as CAR-T cell cultures, remains an urgent need. Here, an innovative sample-to-result workflow is introduced using digital loop-mediated isothermal amplification (dLAMP), enhanced by Electrostatic Microfiltration (EM)-based enrichment, for rapid sterility testing.

Pooled screening for CAR function identifies novel IL-13Rα2-targeted CARs for treatment of glioblastoma.

Background: Chimeric antigen receptor (CAR) therapies have demonstrated potent efficacy in treating B-cell malignancies, but have yet to meaningfully translate to solid tumors. Nonetheless, they are of particular interest for the treatment of glioblastoma, which is an aggressive form of brain cancer with few effective therapeutic options, due to their ability to cross the highly selective blood-brain barrier.

Methods: Here, we use our pooled screening platform, CARPOOL, to expedite the discovery of CARs with antitumor functions necessary for solid tumor efficacy.

Extracellular viral microRNAs as biomarkers of virus infection in human cells.

Nucleic acid amplification tests (NAATs) have enabled fast and sensitive detection of virus infections but are unable to discriminate between live and dead/inert viral fragments or between latent and reactivated virus infections. Here, we show that extracellular viral microRNAs (viral exmiRs) are cell-free candidate biomarkers of live, latent, and reactivated virus infections, achieving fast (under 1 day) and sensitive (30 attomolar [aM]) detection by quantitative real-time reverse transcription PCR (real-time RT-qPCR). We report that spent-media-derived Epstein-Barr virus (EBV) miR-BART10-3p and herpes simplex virus 1 (HSV-1) miR-H5 are biomarkers of live EBV-2 and HSV-1 infection of T cell cultures, respectively.

The T cell receptor sequence influences the likelihood of T cell memory formation.

The amino acid sequence of the T cell receptor (TCR) varies between T cells of an individual's immune system. Particular TCR residues nearly guarantee mucosal-associated invariant T (MAIT) and natural killer T (NKT) cell transcriptional fates. To define how the TCR sequence affects T cell fates, we analyze the paired αβTCR sequence and transcriptome of 961,531 single cells.

iSECRETE: Integrating Microfluidics and DNA Proximity Amplification for Synchronous Single-Cell Activation and IFN-γ Secretion Profiling.

Cytokines, crucial in immune modulation, impact disease progression when their secretion is dysregulated. Existing methods for profiling cytokine secretion suffer from time-consuming and labor-intensive processes and often fail to capture the dynamic nature of immune responses. Here, iSECRETE, an integrated platform that enables synchronous cell activation, wash-free, and target-responsive protein detection for single-cell IFN-γ cytokine secretion analysis within 30 min at room temperature is presented.

SARS-CoV-2 spike does not interact with the T cell receptor or directly activate T cells.

SARS-CoV-2infection can induce multisystem inflammatory syndrome in children, which resembles superantigen-induced toxic shock syndrome. Recent work has suggested that the SARS-CoV-2 spike (S) protein could act as a superantigen by binding T cell receptors (TCRs) and inducing broad antigen-independent T cell responses. Structure-based computational modeling identified potential TCR-binding sites near the S receptor-binding domain, in addition to a site with homology to known neurotoxins.

Separation of Activated T Cells Using Multidimensional Double Spiral (MDDS) Inertial Microfluidics for High-Efficiency CAR T Cell Manufacturing.

This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells.

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.

Pooled screening for CAR function identifies novel IL13Rα2-targeted CARs for treatment of glioblastoma.

Chimeric antigen receptor therapies have demonstrated potent efficacy in treating B cell malignancies, but have yet to meaningfully translate to solid tumors. Here, we utilize our pooled screening platform, CARPOOL, to expedite the discovery of CARs with anti-tumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.

Library-based single-cell analysis of CAR signaling reveals drivers of persistence.

The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function-particularly at the molecular level-are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures.

Polymorphic residues in HLA-B that mediate HIV control distinctly modulate peptide interactions with both TCR and KIR molecules.

Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8 T cell recognition, substitution of Val97 had no significant impact.

A biomaterial platform for T cell-specific gene delivery.

Efficient T cell engineering is central to the success of CAR T cell therapy but involves multiple time-consuming manipulations, including T cell isolation, activation, and transduction. These steps add complexity and delay CAR T cell manufacturing, which takes a mean time of 4 weeks. To streamline T cell engineering, we strategically combine two critical engineering solutions - T cell-specific lentiviral vectors and macroporous scaffolds - that enable T cell activation and transduction in a simple, single step.

Re-centauring T cell antigen discovery around CD4 T cells.

In a recent issue of Cell, Dezfulian et al. develop a genome-scale platform to enable high-throughput identification of CD4 T cell epitopes. This platform enables unbiased screens to discover antigens recognized by CD4 T cells in cancer, infectious diseases, and autoimmunity.