T cells expressing chimeric antigen receptors (CARs) are promising cancer therapeutic agents, with the prospect of becoming the ultimate smart cancer therapeutics. To expand the capability of CAR T cells, here, we present a split, universal, and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical "upgrades," such as the ability to switch targets without re-engineering the T cells, finely tune T cell activation strength, and sense and logically respond to multiple antigens. These features are useful to combat relapse, mitigate over-activation, and enhance specificity. We test our SUPRA system against two different tumor models to demonstrate its broad utility and humanize its components to minimize potential immunogenicity concerns. Furthermore, we extend the orthogonal SUPRA CAR system to regulate different T cell subsets independently, demonstrating a dually inducible CAR system. Together, these SUPRA CARs illustrate that multiple advanced logic and control features can be implemented into a single, integrated system.

Autonomous oscillations found in gene expression and metabolic, cardiac and neuronal systems have attracted significant attention both because of their obvious biological roles and their intriguing dynamics. In addition, de novo designed oscillators have been demonstrated, using components that are not part of the natural oscillators. Such oscillators are useful in testing the design principles and in exploring potential applications not limited by natural cellular behaviour. To achieve transcriptional and metabolic integration characteristic of natural oscillators, here we designed and constructed a synthetic circuit in Escherichia coli K12, using glycolytic flux to generate oscillation through the signalling metabolite acetyl phosphate. If two metabolite pools are interconverted by two enzymes that are placed under the transcriptional control of acetyl phosphate, the system oscillates when the glycolytic rate exceeds a critical value. We used bifurcation analysis to identify the boundaries of oscillation, and verified these experimentally. This work demonstrates the possibility of using metabolic flux as a control factor in system-wide oscillation, as well as the predictability of a de novo gene-metabolic circuit designed using nonlinear dynamic analysis.

A role for caspase-10, previously implicated in the autoimmune lymphoproliferative syndrome, in death receptor signaling has not been directly shown. Here we show that caspase-10 can function independently of caspase-8 in initiating Fas-and tumor necrosis factor-related apoptosis-inducing ligand-receptor-mediated apoptosis. Moreover, Fas crosslinking in primary human T cells leads to the recruitment and activation of caspase-10. Fluorescent resonance energy transfer analysis indicates that the death-effector domains of caspase-8 and -10 both interact with the death-effector domain of FADD. Nonetheless, we find that caspase-8 and -10 may have different apoptosis substrates and therefore potentially distinct roles in death receptor signaling or other cellular processes.D eath receptors (DRs) of the tumor necrosis factor receptor superfamily contain a conserved ''death domain'' in their intracellular region (1). Fas associates with an adaptor molecule, FADD, through homotypic death domain interactions (2, 3). FADD then recruits caspase-8 through homotypic interactions of death-effector domains (DEDs), leading to caspase-8 activation and apoptosis (4, 5). Other DRs may also signal by recruiting caspase-8 through FADD. Among at least 14 mammalian caspases identified so far, only caspase-10 shares homologous DEDs with caspase-8 (6, 7), suggesting that caspase-10 may also function by interacting with DRs. However, recent studies have failed to detect caspase-10 in DR signaling complexes (8, 9).We have studied patients with autoimmune lymphoproliferative syndrome, a genetic disorder of lymphocyte homeostasis and immune tolerance. Some of these patients harbor a dominant interfering mutation in caspase-10 (10). Caspase-10 abnormalities are associated with defective DR-mediated apoptosis in T cells and dendritic cells. Another homozygous-altered allele was found to be associated with autoimmune lymphoproliferative syndrome (10). Recently, this same mutant allele (V367I in caspase-10 S or V410I in caspase-10 L ) has been found to be present at high frequency in heterozygous form in certain populations (11). Given that this allele encodes a protein with reduced caspase activity, the wide occurrence in this allele may suggest a broader role as a susceptibility factor in immune diseases. Here we show that caspase-10 enters DR signaling complexes in primary T cells and that caspase-10 can functionally substitute for caspase-8, providing direct evidence for a physiological role of caspase-10 in DR signal transduction. Experimental ProceduresCell Line, Abs, and Plasmids. Wild-type Jurkat cells and caspase-8-deficient Jurkat mutant, I9.2, were kindly provided by John Blenis (12). Monoclonal anti-caspase-10 Ab specific for caspase-10 DEDs was from the Medical and Biological Laboratories (Nagoya, Japan). MAbs to caspase-3, caspase-8, RIP, and FADD were from PharMingen. Anti-APO1.3 (anti-Fas) was obtained from Kamiya Biomedical (Thousand Oaks, CA). Polyclonal rabbit anti-Fas Ab was from Marcus Peter (University of Chicago). Full-len...

Genetic circuits engineered for mammalian cells often require extensive fine-tuning to perform their intended functions. To overcome this problem, we present a generalizable biocomputing platform that can engineer genetic circuits which function in human cells with minimal optimization. We used our Boolean Logic and Arithmetic through DNA Excision (BLADE) platform to build more than 100 multi-input-multi-output circuits. We devised a quantitative metric to evaluate the performance of the circuits in human embryonic kidney and Jurkat T cells. Of 113 circuits analysed, 109 functioned (96.5%) with the correct specified behavior without any optimization. We used our platform to build a three-input, two-output Full Adder and six-input, one-output Boolean Logic Look Up Table. We also used BLADE to design circuits with temporal small molecule-mediated inducible control and circuits that incorporate CRISPR/Cas9 to regulate endogenous mammalian genes.

Tubulointerstitial fibrosis is a common consequence of a diverse range of kidney diseases that lead to end-stage renal failure. The degree of fibrosis is related to leukocyte infiltration. Here, we determined the role of different T cell populations on renal fibrosis in the well-characterized mouse model of unilateral ureteric obstruction. Depletion of CD4(+) T cells in wild-type mice with a monoclonal antibody significantly reduced the amount of interstitial expansion and collagen deposition after 2 weeks of obstruction. Reconstitution of lymphopenic RAG knockout mice with purified CD4(+) but not CD8(+) T cells, prior to ureteric obstruction, resulted in a significant increase in interstitial expansion and collagen deposition. Wild-type mice had significantly greater interstitial expansion and collagen deposition compared with lymphopenic RAG(-/-) mice, following ureteric obstruction; however, macrophage infiltration was equivalent in all groups. Thus, our results suggest that renal injury with subsequent fibrosis is likely to be a multifactorial process, with different arms of the immune system involved at different stages. In this ureteric obstruction model, we found a critical role for CD4(+) T cells in kidney fibrosis. These cells could be a potential target of therapeutic intervention to prevent excessive fibrosis and loss of function due to renal injury.

Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signaling pathways, provide a mechanism to evade immune responses during infection1,2. Although these effectors are responsible for pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behavior. Here, we have exploited two effector proteins, the Shigella flexneri OspF protein3 and Yersinia pestis YopH protein4, to systematically rewire kinase-mediated responses in both yeast and mammalian immune cells. Bacterial effector proteins can be directed to selectively inhibit specific mitogen activated protein kinase (MAPK) pathways in yeast by artificially targeting them to pathway specific complexes. Moreover, we show that unique properties of the effectors generate novel pathway behaviors: OspF, which irreversibly inactivates MAPKs4, was used to construct a synthetic feedback circuit that displays novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to precisely tune the T cell response amplitude, or as an inducible pause switch that can temporarily disable T cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications.

Homeostatic proliferation is a normal physiological process triggered by lymphopenia to maintain a constant level of T cells. It becomes the predominant source of new T cells in adulthood after thymus regression. T cells that have undergone homeostatic proliferation acquire the memory phenotype, cause autoimmune disease, and are resistant to tolerance induction protocols. Transplantation is a rare example in which lymphopenia is deliberately induced for its immunosuppressive effect. However, it is not known whether the homeostatic proliferation that follows will have the opposite effect and accelerate rejection. We show that T cells that have undergone homeostatic proliferation acquire a memory phenotype, spontaneously skews toward the Th1 phenotype, even in the absence of antigenic stimulus. Interestingly, in contrast, the percentage of Foxp3+ regulatory T cells increased by 28-fold following homeostatic proliferation. Using a mouse life-sustaining kidney transplant model, we showed that T cells that have gone through homeostatic proliferation in lymphopenic hosts transformed chronic rejection to acute rejection of a single MHC class II-mismatched kidney allograft. T cells that have undergone homeostatic proliferation consistently cause reliable rejection even when bona fide memory T cells cannot. These functional changes are long-lasting and not restricted to the acute phase of homeostatic proliferation. Our findings have important implications for tolerance induction or graft-prolonging protocols involving leukocyte depletion such as irradiation bone marrow chimera, T cell-depleting Abs, and lymphopenia induced by infections such as CMV and HIV.

BackgroundEnteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored.ResultsWe utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally.ConclusionsOur data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0376-y) contains supplementary material, which is available to authorized users.