Dexamethasone potentiates chimeric antigen receptor T cell persistence and function by enhancing IL-7Rα expression.

Dexamethasone (dex) is a glucocorticoid that is a mainstay for the treatment of inflammatory pathologies, including immunotherapy-associated toxicities, yet the specific impact of dex on the activity of CAR T cells is not fully understood. We assessed whether dex treatment given ex vivo or as an adjuvant in vivo with CAR T cells impacted the phenotype or function of CAR T cells. We demonstrated that CAR T cell expansion and function were not inhibited by dex. We confirmed this observation using multiple CAR constructs and tumor models, suggesting that this is a general phenomenon. Moreover, we determined that dex upregulated interleukin-7 receptor α on CAR T cells and increased the expression of genes involved in activation, migration, and persistence when supplemented ex vivo. Direct delivery of dex and IL-7 into tumor-bearing mice resulted in increased persistence of adoptively transferred CAR T cells and complete tumor regression. Overall, our studies provide insight into the use of dex to enhance CAR T cell therapy and represent potential novel strategies for augmenting CAR T cell function during production as well as following infusion into patients.

The increasing burden of asthma acute care in Singapore: an update on 15-year population-level evidence.

BACKGROUND: In Singapore, there is currently scarce population-based research informing the recent trends of asthma-related healthcare burdens. In this study, we investigated the past 25-year trends of asthma-related hospitalisations, emergency department (ED) visits and deaths in Singapore and projected the future burdens from 2023 to 2040. METHODS: We acquired annually-measured data from the Singapore Ministry of Health Clinical and National Disease Registry, containing 25-year asthma-related hospitalisation and death rates as well as 15-year ED visit rates. We conducted change-point analysis and generalised linear modelling to identify time intervals with stable trends and estimate asthma-related healthcare utilisation and mortality rates. To project future asthma-related burdens, we developed a probabilistic model which combined projections of future population size with the estimated rate outcomes from the last stable period. RESULTS: Our results show that the asthma hospitalisation rate in Singapore had remained at approximately 80 episodes per 100,000 from 2003 to 2019 and are likely to grow by 1.7% each year (95% CI: 0.7, 5.0%), leading to a total of 163,633 episodes from 2023 to 2040 which corresponds to an estimated $103,075,820 based on 2022 USD. Besides, Singapore's asthma-related ED visit rate was 390 per 100,000 in 2019 and is expected to decline by 3.4% each year (95% CI: - 5.8, 0.0%), leading to a total of 208,145 episodes from 2023 to 2040 which corresponds to USD$15,053,795. In contrast, the 2019 asthma-related mortality rate in Singapore was approximately 0.57 per 100,000 and is likely to stay stably low (change per year: -1.3, 95% CI: - 11.0, 4.3%). Between 2023 and 2040, Singapore's estimated total number of asthma-related deaths is 638 episodes. CONCLUSIONS: Currently, the burden of asthma acute care in Singapore is high; Singapore's asthma-related hospitalisation and ED visit rates are relatively higher than those of other developed economies, and its asthma admission rate is expected to increase significantly over time, possibly indicating excess resource use for asthma. The established national asthma programme in Singapore, together with recent efforts in reinforcing primary care at the national level, provides opportunities to reduce avoidable asthma admissions.

Impact of Guideline-Based Asthma Treatment on Health Services Use in Singapore Before and During COVID-19 Outbreak.

Introduction: To date, the role of standard asthma care in reducing asthma-related health services use (HSU) during the COVID-19 pandemic remains unclear. This study examined the impact of guideline-based asthma treatment on the use of asthma-related emergency department (ED) visits, polyclinic visits (total visits and urgent visits characterized by nebuliser use) before and during the pandemic. Methods: Data from April 2017 to October 2020 was obtained from the National University Health System, one of the three healthcare clusters in Singapore. Using generalized linear models, we estimated the joint effects of the ratio of preventer to reliever dispensations (PRR) and COVID-19 on asthma-related ED visits per hospital per month, total asthma-related polyclinic visits and asthma-related urgent polyclinic visits per clinic per month. Results: Findings show that before the onset of COVID-19, for every 0.5 unit increase in PRR, the number of asthma-related ED visits and urgent polyclinic visits decreased by 12.9% (95% CI: -13.0% to -12.9%) and 6.8% (95% CI: -6.9% to -6.7%), respectively, whereas total asthma-related polyclinic visits increased by 1.0% (95% CI: 0.9% to 1.0%). During the pandemic, a 0.5 unit increase of PRR decreased the number of asthma-related ED visits, urgent and total polyclinic visits by 16.9% (95% CI: -17.0% to - 16.9%), 9.3% (95% CI: -9.5% to -9.2%) and 0.7% (95% CI: -0.8% to -0.7%), respectively. Discussion: These findings suggest that regardless of the COVID-19 pandemic, an increase in PRR consistently reduced the frequency of asthma-related urgent and emergent care, although it barely influenced routine asthma follow-up visits.

Mediation analysis with multiple mediators under unmeasured mediator-outcome confounding.

It is often of interest in the health and social sciences to investigate the joint mediation effects of multiple post-exposure mediating variables. Identification of such joint mediation effects generally require no unmeasured confounding of the outcome with respect to the whole set of mediators. As the number of mediators under consideration grows, this key assumption is likely to be violated as it is often infeasible to intervene on any of the mediators. In this article, we develop a simple two-step method of moments estimation procedure to assess mediation with multiple mediators simultaneously in the presence of potential unmeasured mediator-outcome confounding. Our identification result leverages heterogeneity of the population exposure effect on the mediators, which is plausible under a variety of empirical settings. The proposed estimators are illustrated through both simulations and an application to evaluate the mediating effects of post-traumatic stress disorder symptoms in the association between self-efficacy and fatigue among health care workers during the COVID-19 outbreak.

Pre-clinical data supporting immunotherapy for HIV using CMV-HIV-specific CAR T cells with CMV vaccine.

T cells engineered to express HIV-specific chimeric antigen receptors (CARs) represent a promising strategy to clear HIV-infected cells, but to date have not achieved clinical benefits. A likely hurdle is the limited T cell activation and persistence when HIV antigenemia is low, particularly during antiretroviral therapy (ART). To overcome this issue, we propose to use a cytomegalovirus (CMV) vaccine to stimulate CMV-specific T cells that express CARs directed against the HIV-1 envelope protein gp120. In this study, we use a GMP-compliant platform to engineer CMV-specific T cells to express a second-generation CAR derived from the N6 broadly neutralizing antibody, one of the broadest anti-gp120 neutralizing antibodies. These CMV-HIV CAR T cells exhibit dual effector functions upon in vitro stimulation through their endogenous CMV-specific T cell receptors or the introduced CARs. Using a humanized HIV mouse model, we show that CMV vaccination during ART accelerates CMV-HIV CAR T cell expansion in the peripheral blood and that higher numbers of CMV-HIV CAR T cells were associated with a better control of HIV viral load and fewer HIV antigen p24+ cells in the bone marrow upon ART interruption. Collectively, these data support the clinical development of CMV-HIV CAR T cells in combination with a CMV vaccine in HIV-infected individuals.

CD19-directed CAR T-cell therapy for treatment of primary CNS lymphoma.

CD19-directed chimeric antigen receptor (CD19CAR) T-cell therapy has been successful in treating several B-cell lineage malignancies, including B-cell non-Hodgkin lymphoma (NHL). This modality has not yet been extended to NHL manifesting in the central nervous system (CNS), primarily as a result of concerns for potential toxicity. CD19CAR T cells administered IV are detectable in cerebrospinal fluid (CSF), suggesting that chimeric antigen receptor (CAR) T cells can migrate from the periphery into the CNS, where they can potentially mediate antilymphoma activity. Here, we report the outcome of a subset of patients with primary CNS lymphoma (PCNSL; n = 5) who were treated with CD19CAR T cells in our ongoing phase 1 clinical trial. All patients developed grade ≥ 1 cytokine release syndrome and neurotoxicity post-CAR T-cell infusion; toxicities were reversible and tolerable, and there were no treatment-related deaths. At initial disease response, 3 of 5 patients (60%; 90% confidence interval, 19-92%) seemed to achieve complete remission, as indicated by resolution of enhancing brain lesions; the remaining 2 patients had stable disease. Although the study cohort was small, we demonstrate that using CD19CAR T cells to treat PCNSL can be safe and feasible. This trial was registered at www.clinicaltrials.gov as #NCT02153580.

The Cerebroventricular Environment Modifies CAR T Cells for Potent Activity against Both Central Nervous System and Systemic Lymphoma.

Lymphomas with central nervous system (CNS) involvement confer a worse prognosis than those without CNS involvement, and patients currently have limited treatment options. T cells genetically engineered with CD19-targeted chimeric antigen receptors (CAR) are effective against B-cell malignancies and show tremendous potential in the treatment of systemic lymphoma. We aimed to leverage this strategy toward a more effective therapy for patients with lymphoma with CNS disease. NOD-scid IL2Rgammanull (NSG) mice with CNS and/or systemic lymphoma were treated with CD19-CAR T cells via intracerebroventricular (ICV) or intravenous (IV) injection. CAR T cells isolated after treatment were rigorously examined for phenotype, gene expression, and function. We observed that CAR T cells infused ICV, but not IV, completely and durably eradicated both CNS and systemic lymphoma. CAR T cells delivered ICV migrated efficiently to the periphery, homed to systemic tumors, and expanded in vivo, leading to complete elimination of disease and resistance to tumor rechallenge. Mechanistic studies indicated that ICV-delivered CAR T cells are conditioned by exposure to cerebrospinal fluid in the ICV environment for superior antilymphoma activity and memory function compared with IV-delivered CAR T cells. Further analysis suggested that manipulating cellular metabolism or preactivating therapeutic CAR T cells with antigen ex vivo may improve the efficacy of CAR T cells in vivo Our demonstration that ICV-delivered CD19-CAR T cells had activity against CNS and systemic lymphoma could offer a valuable new strategy for treatment of B-cell malignancies with CNS involvement.

Conditionally Replicating Vectors Mobilize Chimeric Antigen Receptors against HIV.

Human immunodeficiency virus (HIV) is an attractive target for chimeric antigen receptor (CAR) therapy. CAR T cells have proved remarkably potent in targeted killing of cancer cells, and we surmised that CAR T cells could prove useful in eradicating HIV-infected cells. Toward this goal, we interrogate several neutralizing single-chain variable fragments (scFvs) that target different regions of the HIV envelope glycoprotein, gp120. We find here that CAR T cells with scFv from NIH45-46 antibody demonstrated the highest cytotoxicity. Although NIH45-46 CAR T cells are capable of eliminating antigen-expressing cells, we wanted to address HIV reactivation from ex vivo culture of HIV patient-derived CAR T cells. In order to capitalize on the HIV reactivation, we developed a conditionally replicating lentiviral vector (crLV). The crLV can hijack HIV machinery, forming a chimeric lentivirus (LV) instead of HIV and delivered to uninfected cells. We find that CAR T cells generated with crLVs have similar CAR-mediated functionality as traditional CARs. We also demonstrate crLVs' capability of expanding CAR percentage and protecting CD4 CAR T cell in HIV donors. Collectively, we demonstrate here that the novel crLV NIH45-46 CAR can serve as a strategy to combat HIV, as well as overcome HIV reactivation in CD4+ CAR T cells.

Craspedotropis gretathunbergae, a new species of Cyclophoridae (Gastropoda: Caenogastropoda), discovered and described on a field course to Kuala Belalong rainforest, Brunei.

BACKGROUND: Terrestrial Caenogastropoda form an important but threatened component of the Borneo tropical rainforest malacofauna, where the group is nearly as rich in species as the Stylommatophora. They are, however, more sensitive to drought, temperature extremes and forest degradation. NEW INFORMATION: On a field course at Kuala Belalong Field Studies Centre in Brunei Darussalam (Borneo), a new caenogastropod species, belonging to the genus Craspedotropis, was discovered by the course participants. The participants decided to name the species Craspedotropis gretathunbergae n. sp., in honour of the climate change activist Greta Thunberg, as caenogastropod land snails, such as this species, are likely to suffer because of climate change.

Lenalidomide Enhances the Function of CS1 Chimeric Antigen Receptor-Redirected T Cells Against Multiple Myeloma.

Purpose: Multiple myeloma remains an incurable malignancy of plasma cells despite considerable advances in treatment. The purpose of the study was to develop novel chimeric antigen receptors (CAR) for the treatment of multiple myeloma and explore combinatorial therapy using CAR T cells and immunomodulatory drugs such as lenalidomide for increasing treatment efficacy.Experimental Design: We redirected central memory T cells to express second-generation CAR-specific for CS1 and adoptively transferred them into multiple myeloma tumor-bearing mice to test their anti-multiple myeloma activity. CS1 CAR T cells were transduced and expanded in the presence of lenalidomide in vitro The phenotype and effector function of CS1 CAR T cells treated with and without lenalidomide were compared. Finally, CS1 CAR T cells and lenalidomide were administered to treat multiple myeloma-bearing mice as combinatorial therapy.Results: CS1 CAR T cells exhibited efficient antitumor activity when adoptively transferred into mice. Mechanistic studies indicated that the addition of lenalidomide during CS1 CAR T-cell expansion in vitro enhanced the immune functions of CS1 CAR T cells, including cytotoxicity, memory maintenance, Th1 cytokine production, and immune synapse formation. Furthermore, lenalidomide enhanced the antitumor activity and persistence of adoptively transferred CS1 CAR T cells in vivoConclusions: The study demonstrates that lenalidomide improves the anti-multiple myeloma properties of CS1-directed CAR T cells and provides a basis for a planned clinical trial using the combination of lenalidomide with engineered T cells against CS1 in relapsed myeloma. Clin Cancer Res; 24(1); 106-19. ©2017 AACR.

Preclinical data support leveraging CS1 chimeric antigen receptor T-cell therapy for systemic light chain amyloidosis.

BACKGROUND AIMS: Light chain amyloidosis (AL) is a protein deposition disorder that is a result of a plasma cell dyscrasia, similar to multiple myeloma (MM). Immunotherapy is an attractive approach because of the low burden of disease, but the optimal target for AL is unclear. CS1 and B-cell maturation antigen (BCMA) are two potential targets because they are expressed on normal plasma cells and MM cells. METHODS: We performed a prospective study evaluating bone marrow specimens of 20 patients with plasma cell diseases, 10 with AL and 10 with MM. We evaluated the clonal population of plasma cells for BCMA and CS1 expression. We designed a second-generation CS1 chimeric antigen receptor (CAR) construct, comprising a CS1 antigen-specific scFv, shortened hinge region and CD28 costimulatory domain. Purified central memory T cells were activated and transduced with a lentiviral vector encoding the CS1 CAR. Cytotoxicity was evaluated using 51Cr release assays. Five days after tumor inoculation, NSG mice were injected intravenously with CS1 CAR T cells. RESULTS: Whereas CS1 is present on the plasma cells of AL patients, we found BCMA expression in AL to be markedly low. CS1 CAR T cells were cytotoxic against CS1 positive tumor cells and induced durable tumor regressions in mice. DISCUSSION: Our work represents a novel application of CS1-directed CAR T cells while revealing that BCMA would not be a suitable target. We expect AL to be particularly susceptible to CAR T-cell therapy because of the low tumor burden in the bone marrow.

Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy.

BACKGROUND: Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model. METHODS: Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 µM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments. RESULTS: We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once adoptively transferred into CD19+ tumor-bearing mice, Akti treated CD19CAR T cells exhibited more antitumor activity than did untreated CD19CAR T cells. CONCLUSIONS: Inhibition of Akt signaling during ex vivo priming and expansion gives rise to CD19CAR T cell populations that display comparatively higher antitumor activity.

The orphan nuclear receptor Nur77 is a determinant of myofiber size and muscle mass in mice.

We previously showed that the orphan nuclear receptor Nur77 (Nr4a1) plays an important role in the regulation of glucose homeostasis and oxidative metabolism in skeletal muscle. Here, we show using both gain- and loss-of-function models that Nur77 is also a regulator of muscle growth in mice. Transgenic expression of Nur77 in skeletal muscle in mice led to increases in myofiber size. Conversely, mice with global or muscle-specific deficiency in Nur77 exhibited reduced muscle mass and myofiber size. In contrast to Nur77 deficiency, deletion of the highly related nuclear receptor NOR1 (Nr4a3) had minimal effect on muscle mass and myofiber size. We further show that Nur77 mediates its effects on muscle size by orchestrating transcriptional programs that favor muscle growth, including the induction of insulin-like growth factor 1 (IGF1), as well as concomitant downregulation of growth-inhibitory genes, including myostatin, Fbxo32 (MAFbx), and Trim63 (MuRF1). Nur77-mediated increase in IGF1 led to activation of the Akt-mTOR-S6K cascade and the inhibition of FoxO3a activity. The dependence of Nur77 on IGF1 was recapitulated in primary myoblasts, establishing this as a cell-autonomous effect. Collectively, our findings identify Nur77 as a novel regulator of myofiber size and a potential transcriptional link between cellular metabolism and muscle growth.

The effect of caffeine ingestion on human neutrophil oxidative burst responses following time-trial cycling.

Following fixed-duration exercise of submaximal intensity, caffeine ingestion is associated with an attenuation of the exercise-induced decline in N-formyl-methionyl-phenyl-alanine (f-MLP) stimulated neutrophil oxidative burst. However, the response following high-intensity exhaustive exercise is unknown. Nine endurance-trained male cyclists ingested 6 mg caffeine or placebo per kilogram of body mass 60 min before cycling for 90 min at 70% of maximal oxygen consumption (VO2max) and then performing a time-trial requiring an energy expenditure equivalent to 30 min cycling at 70% maximum power output. Time-trial performance was 4% faster in the caffeine than in the placebo trial (P = 0.043). Caffeine was associated with an increased plasma adrenaline concentration after 90 min of exercise (P = 0.046) and immediately after the time-trial (P = 0.02). Caffeine was also associated with an increased serum caffeine concentration (P < 0.01) after 90 min of exercise and immediately after the time-trial, as well as 1 h after the time-trial. However, the f-MLP-stimulated neutrophil oxidative burst response fell after exercise in both trials (P = 0.002). There was no effect of caffeine on circulating leukocyte or neutrophil counts, but the lymphocyte count was significantly lower on caffeine (20%) after the time-trial (P = 0.003). Our results suggest that high-intensity exhaustive exercise negates the attenuation of the exercise-induced decrease in neutrophil oxidative burst responses previously observed when caffeine is ingested before exercise of fixed duration and intensity. This may be associated with the greater increase in adrenaline concentration observed in the present study.