TCR ligand potency differentially impacts PD-1 inhibitory effects on diverse signaling pathways.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs.

Early B cell factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells.

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis.

Tuning T cell receptor sensitivity through catch bond engineering.

Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds.

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation.

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution.

Efficient Immune Cell Genome Engineering with Enhanced CRISPR Editing Tools.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods.

IFN-mediated negative feedback supports bacteria class-specific macrophage inflammatory responses.

Despite existing evidence for tuning of innate immunity to different classes of bacteria, the molecular mechanisms used by macrophages to tailor inflammatory responses to specific pathogens remain incompletely defined. By stimulating mouse macrophages with a titration matrix of TLR ligand pairs, we identified distinct stimulus requirements for activating and inhibitory events that evoked diverse cytokine production dynamics. These regulatory events were linked to patterns of inflammatory responses that distinguished between Gram-positive and Gram-negative bacteria, both in vitro and after in vivo lung infection.

Molecular Determinants of Scaffold-induced Linear Ubiquitinylation of B Cell Lymphoma/Leukemia 10 (Bcl10) during T Cell Receptor and Oncogenic Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling.

The activation of NF-κB downstream of T cell receptor (TCR) engagement is a key signaling step required for normal lymphocyte function during the adaptive immune response. During TCR signaling, the adaptor protein Bcl10 is inducibly recruited to the CARD11 scaffold protein as part of a multicomponent complex that induces IκB kinase (IKK) activity and NF-κB activation. Here, we show that a consequence of this recruitment is the TCR-induced conjugation of Bcl10 with linear-linked polyubiquitin chains to generate the signaling intermediate Lin(Ub)-Bcl10, which is required for the association of Bcl10 with the NEMO subunit of the IKK complex.

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181.

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner.

Microtransplantation of whole ganglionic eminence cells ameliorates motor deficit, enlarges the volume of grafts, and prolongs survival in a rat model of Huntington's disease.

Studies have demonstrated that embryonic cell therapy is a potential approach for the treatment of Huntington's disease (HD). However, because of the limited resource of embryos, greater attention is needed in developing more efficient surgical techniques that not only enhance the therapy outcome but also avoid inefficient therapeutics of transplantation. In this study, we explored the curative effects of two different transplantation methods using a rat model of HD.

A quantitative signaling screen identifies CARD11 mutations in the CARD and LATCH domains that induce Bcl10 ubiquitination and human lymphoma cell survival.

Antigen receptor signaling to NF-κB, essential for normal lymphocyte activation, is dysregulated in several types of lymphoma. During normal signaling, the multidomain adapter CARD11 transitions from a closed, inactive state to an open, active scaffold that assembles a multiprotein complex, leading to NF-κB activation. The regulation of CARD11 scaffold function is bypassed by lymphoma-associated oncogenic CARD11 mutations that induce spontaneous signaling.

Anergic T cells are metabolically anergic.

Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function.