Constitutive Turbodomains enhance expansion and antitumor activity of allogeneic BCMA CAR T cells in preclinical models.

The magnitude of CAR T cell expansion has been associated with clinical efficacy. Although cytokines can augment CAR T cell proliferation, systemically administered cytokines can result in toxicities. To gain the benefits of cytokine signaling while mitigating toxicities, we designed constitutively active synthetic cytokine receptor chimeras (constitutive Turbodomains) that signal in a CAR T cell-specific manner. The modular design of Turbodomains enables diverse cytokine signaling outputs from a single homodimeric receptor chimera and allows multiplexing of different cytokine signals. Turbodomains containing an IL-2/15Rß-derived signaling domain closely mimicked IL-15 signaling and enhanced CAR T cell potency. Allogeneic TurboCAR T cells targeting BCMA showed no evidence of aberrant proliferation yet displayed enhanced expansion and antitumor activity, prolonging survival and preventing extramedullary relapses in mouse models. These results illustrate the potential of constitutive Turbodomains to achieve selective potentiation of CAR T cells and demonstrate the safety and efficacy of allogeneic BCMA TurboCAR T cells, supporting clinical evaluation in multiple myeloma.

Design and Validation of Inducible TurboCARs with Tunable Induction and Combinatorial Cytokine Signaling.

Although cytokine support can enhance CAR T-cell function, coadministering cytokines or engineering CAR T cells to secrete cytokines can result in toxicities. To mitigate these safety risks, we engineered iTurboCAR T cells that coexpress a novel inducible Turbo (iTurbo) cytokine signaling domain. iTurbo domains consist of modular components that are customizable to a variety of activating inputs, as well as cytokine signaling outputs multiplexable for combinatorial signaling outcomes. Unlike most canonical cytokine receptors that are heterodimeric, iTurbo domains leverage a compact, homodimeric design that minimizes viral vector cargo. Using an iTurbo domain activated by the clinically validated dimerizer, AP1903, homodimeric iTurbo domains instigated signaling that mimicked the endogenous heterodimeric cytokine receptor. Different iTurbo domains programmed iTurboCAR T cells toward divergent phenotypes and resulted in improved antitumor efficacy. iTurbo domains, therefore, offer the flexibility for user-programmable signaling outputs, permitting control over cellular phenotype and function while minimizing viral cargo footprint.

Direct control of CAR T cells through small molecule-regulated antibodies.

Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.

Non-invasive treatment of patients with upper extremity spasticity following stroke using paired trans-spinal and peripheral direct current stimulation.

BACKGROUND: Muscle spasticity is a common impediment to motor recovery in patients with chronic stroke. Standard-of-care treatments such as botulinum toxin injections can temporarily relieve muscle stiffness and pain associated with spasticity, but often at the expense of increased muscle weakness. Recent preclinical investigations of a non-invasive treatment that pairs trans-spinal direct current stimulation and peripheral nerve direct current stimulation (tsDCS+pDCS) provided promising data for a novel approach based on bioelectronic medicine for the treatment of patients with post-stroke spasticity. METHODS: Twenty-six patients with upper limb hemiparesis and wrist spasticity at least 6 months after their initial stroke participated in this single-blind crossover design study to test whether tsDCS+pDCS reduces chronic upper-extremity spasticity. Subjects received five consecutive daily sessions (20 min of stimulation or sham) of anodal tsDCS+pDCS, separated by a one-week washout period. The sham condition always preceded the active condition. Clinical and objective measures of spasticity and motor function were collected before and after each condition, and for five weeks after the completion of the active intervention. RESULTS: Subjects treated with active tsDCS+pDCS demonstrated significant reductions in both Modified Tardieu Scale scores (summed across the upper limb, P < 0.05), and in objective torque measures (Nm) of the spastic catch response at the wrist flexor (P < 0.05), compared to the sham condition. Motor function also improved significantly (measured by the Fugl-Meyer and Wolf Motor Function Test; P < 0.05 for both tests) after active treatment. CONCLUSIONS: tsDCS+pDCS intervention alone significantly reduced upper limb spasticity in participants with stroke. Decreased spasticity was persistent for five weeks after treatment, and was accompanied by improved motor function even though patients were unsupervised and there was no prescribed activity or training during that interval. TRIAL REGISTRATION: NCT03080454, March 15, 2017.

Intensive seated robotic training of the ankle in patients with chronic stroke differentially improves gait.

BACKGROUND: Robotic driven treatment plans targeting isolated joints of the upper limb have improved the sensorimotor condition of patients with stroke. Similar intensive efforts to allay lower limb gait impairment have not been so successful. In patients with stroke, targeted robot assisted training of the ankle joint, in a seated position, has demonstrated significant alterations in ankle stiffness and foot-ankle orientation at foot strike which may provide a new treatment option for gait impairment. OBJECTIVE: To determine if isolated robot-assisted training of the ankle joint improves chronic hemiparetic gait in patients with stroke who are categorized according to baseline gait impairment. METHODS: Patients with chronic stroke (>6mo) and hemiparetic gait (N = 29) received 18 sessions of isolated robot-assisted motor training of the ankle (3×/week for 6 weeks). All participants had stable clinical baseline scores across three admission measures, and no participant was receiving simultaneous outpatient rehabilitation. Baseline gait speed determined three impairment groups: high, >0.8 m/s; medium, 0.4-0.8 m/s; low, <0.4 m/s. Outcome measures included the Berg Balance Scale, the 6 Minute Walk Test, and the 10 Meter Walk Test, and were recorded upon admission, discharge, and 3 months following intervention. RESULTS: Three distinct and significant between-group patterns of recovery emerged for gait speed. The within-group analysis showed that the medium and high group exhibited significant improvements in gait speed and endurance upon discharge, that were maintained at 3-months. Gait speed improvements were clinically significant (>0.16 m/s) for the high function group across all gait speed and endurance measures at discharge and at 3 months. The moderate group also exhibited clinically significant improvements at follow-up on the 10 Meter Walk Test, fast pace (0.16 m/sec), and approached clinical significance for the 10 Meter Walk comfortable pace (0.12 m/sec). The low group had small but significant improvements, at discharge on two of the three gait measures, and these improvements were maintained at 3 months. For balance measures, the low and moderate impairment groups had significant improvements at discharge that were robust on follow-up measure. The high function group demonstrated no significant change in balance. CONCLUSIONS: Joint-specific robotic training of the paretic ankle provided the most benefit to individuals with moderate or mild gait speed impairments after stroke. Baseline gait speed function (low, moderate, high) was associated with three distinct recovery profiles. This suggests that severity-specific intervention may be critical to improving efficiency of stroke recovery.

Analysis of the Rab GTPase Interactome in Dendritic Cells Reveals Anti-microbial Functions of the Rab32 Complex in Bacterial Containment.

Dendritic cells (DCs) orchestrate complex membrane trafficking through an interconnected transportation network linked together by Rab GTPases. Through a tandem affinity purification strategy and mass spectrometry, we depicted an interactomic landscape of major members of the mammalian Rab GTPase family. When complemented with imaging tools, this proteomic analysis provided a global view of intracellular membrane organization. Driven by this analysis, we investigated dynamic changes to the Rab32 subnetwork in DCs induced by L. monocytogenes infection and uncovered an essential role of this subnetwork in controlling the intracellular proliferation of L. monocytogenes. Mechanistically, Rab32 formed a persistent complex with two interacting proteins, PHB and PHB2, to encompass bacteria both during early phagosome formation and after L. monocytogenes escaped the original containment vacuole. Collectively, we have provided a functional compartmentalization overview and an organizational framework of intracellular Rab-mediated vesicle trafficking that can serve as a resource for future investigations.

The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis.

One of the most important factors that limit the potency of CD8+ cytotoxic T lymphocyte (CTL) responses is the tumor microenvironment (TME). Here, we provide evidence that miR-26a is a negative regulator of CTL function in the TME. Specifically, we identified miR-26a as a crucial suppressor gene in CTLs from the TME, as we found that, miR-26a expression was elevated in CTLs to respond to TME secretome stimulation. CTLs from miR-26a-transgenic mice showed impaired IFNγ and granzyme B production in response to their cognate antigen. Conversely, we found that miR-26a inhibition in CTLs could effectively increase the cytotoxicity and suppress tumor growth. Mechanically, we identified EZH2 as a direct target of miR-26a. miR-26a and EZH2 expression were found to be inversely correlated in CTLs, and the inhibition of EZH2 in CTLs impairs CTL function. These functional correlations were validated in a cohort of non-small cell lung cancer patients, indicating that the miR-26a-EZH2 axis is clinically relevant. Our findings suggested that miR-26a silencing as a novel strategy to improve the efficacy of CTL-based cancer immunotherapy.

Diversity index of mucosal resident T lymphocyte repertoire predicts clinical prognosis in gastric cancer.

A characteristic immunopathology of human cancers is the induction of tumor antigen-specific T lymphocyte responses within solid tumor tissues. Current strategies for immune monitoring focus on the quantification of the density and differentiation status of tumor-infiltrating T lymphocytes; however, properties of the TCR repertoire ‒ including antigen specificity, clonality, as well as its prognostic significance ‒ remain elusive. In this study, we enrolled 28 gastric cancer patients and collected tumor tissues, adjacent normal mucosal tissues, and peripheral blood samples to study the landscape and compartmentalization of these patients' TCR ß repertoire by deep sequencing analyses. Our results illustrated antigen-driven expansion within the tumor compartment and the contracted size of shared clonotypes in mucosa and peripheral blood. Most importantly, the diversity of mucosal T lymphocytes could independently predict prognosis, which strongly underscores critical roles of resident mucosal T-cells in executing post-surgery immunosurveillance against tumor relapse.

miR-23a blockade enhances adoptive T cell transfer therapy by preserving immune-competence in the tumor microenvironment.

In adoptive T cell transfer therapy (ACT), the antitumor efficacy of cytotoxic CD8+ T lymphocytes (CTLs) has been limited by tumor-induced immunosuppression. We have demonstrated that miR-23a blockade in tumor-specific CTLs conferred resilience to TGFß-mediated immunosuppression, resulting in superior tumor control. Our studies highlight miR-23a in tumor-specific CTLs as a clinically relevant target to enhance ACT.

Association of CD8(+) T lymphocyte repertoire spreading with the severity of DRESS syndrome.

T-cell receptor (TCR)-mediated cross-recognition is a major mechanism in the pathogenesis of drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome. However, the characteristics of the TCR repertoire and the clinical significance of repertoire reformation throughout the course of DRESS are unknown. Here, we isolated CD4(+) and CD8(+) T-cells from peripheral blood of 8 DRESS patients at 10-day intervals and, sequenced CDR3-regions of the TCRB chain by high-throughput sequencing to analyze the dynamic reformation in the T-cell repertoire hierarchy. Compared with healthy donors, T-cell expanded in peripheral repertoires from DRESS patient. The extent of fluctuation of dominant CD8(+) T-cell clones, but not of CD4(+) counterparts, correlated positively with the clinical severity and helped classify the enrolled subjects into "fluctuant" and "flat" repertoire groups. The anti-herpesvirus response, which was measured using anti-EBV/HHV antibodies, and the proportion of the homologous CD8(+) EBV-specific clonotypes, in the "fluctuant" group was substantial higher than that in the "flat" group. Furthermore, autoimmune sequelae were observed in a cured "fluctuant" patient. Collectively, the clinical relevance of the fluctuant CD8(+) T-cell repertoires supports the notion that herpes virus-mediated continuously de novo priming of newly pathogenic CD8(+) T-cell clones is an alternate mechanism responsible for the pathogenicity of DRESS.

Targeting miR-23a in CD8+ cytotoxic T lymphocytes prevents tumor-dependent immunosuppression.

CD8(+) cytotoxic T lymphocytes (CTLs) have potent antitumor activity and therefore are leading candidates for use in tumor immunotherapy. The application of CTLs for clinical use has been limited by the susceptibility of ex vivo-expanded CTLs to become dysfunctional in response to immunosuppressive microenvironments. Here, we developed a microRNA-targeting (miRNA-targeting) approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor BLIMP-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was upregulated in tumor-infiltrating CTLs, and expression correlated with impaired antitumor potential of patient CTLs. We determined that tumor-derived TGF-ß directly suppresses CTL immune function by elevating miR-23a and downregulating BLIMP-1. Functional blocking of miR-23a in human CTLs enhanced granzyme B expression, and in mice with established tumors, immunotherapy with just a small number of tumor-specific CTLs in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CTLs that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF-ß-mediated tumor immune-evasion pathway.

Biological evaluation of subglutinol a as a novel immunosuppressive agent for inflammation intervention.

Subglutinol A (1) is an immunosuppressive natural product isolated from Fusarium subglutinans, an endophytic fungus from the vine Tripterygium wilfordii. We show that 1 exerts multimodal immune-suppressive effects on activated T cells in vitro: subglutinol A (1) effectively blocks T cell proliferation and survival while profoundly inhibiting pro-inflammatory IFNγ and IL-17 production by fully differentiated effector Th1 and Th17 cells. Our data further reveal that 1 may exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells. Additionally, we demonstrate that 1 significantly reduces lymphocytic infiltration into the footpad and ameliorates footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of 1 as a novel therapeutic for inflammatory diseases.

The Parkinson disease-linked LRRK2 protein mutation I2020T stabilizes an active state conformation leading to increased kinase activity.

The effect of leucine-rich repeat kinase 2 (LRRK2) mutation I2020T on its kinase activity has been controversial, with both increased and decreased effects being reported. We conducted steady-state and pre-steady-state kinetic studies on LRRKtide and its analog LRRKtide(S). Their phosphorylation differs by the rate-limiting steps: product release is rate-limiting for LRRKtide and phosphoryl transfer is rate-limiting for LRRKtide(S). As a result, we observed that the I2020T mutant is more active than wild type (WT) LRRK2 for LRRKtide(S) phosphorylation, whereas it is less active than WT for LRRKtide phosphorylation. Our pre-steady-state kinetic data suggest that (i) the I2020T mutant accelerates the rates of phosphoryl transfer of both reactions by 3-7-fold; (ii) this increase is masked by a rate-limiting product release step for LRRKtide phosphorylation; and (iii) the observed lower activity of the mutant for LRRKtide phosphorylation is a consequence of its instability: the concentration of the active form of the mutant is 3-fold lower than WT. The I2020T mutant has a dramatically low KATP and therefore leads to resistance to ATP competitive inhibitors. Two well known DFG-out or type II inhibitors are also weaker toward the mutant because they inhibit the mutant in an unexpected ATP competitive mechanism. The I2020 residue lies next to the DYG motif of the activation loop of the LRRK2 kinase domain. Our modeling and metadynamic simulations suggest that the I2020T mutant stabilizes the DYG-in active conformation and creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion.