Protective effects of low-intensity pulsed ultrasound (LIPUS) against cerebral ischemic stroke in mice by promoting brain vascular remodeling via the inhibition of ROCK1/p-MLC2 signaling pathway.

Vascular remodeling is essential for patients with cerebral ischemic stroke (CIS). Our previous study proved that low-intensity pulsed ultrasound (LIPUS) could increase cortical hemodynamics. However, the effects and mechanisms of LIPUS on cerebral vascular remodeling after CIS are still unknown. In this study, we applied LIPUS to the mouse brain at 0.5 h after distal middle cerebral artery occlusion (dMCAO) and subsequently daily for a stimulation time of 30 min. Results showed that compared with the dMCAO group, LIPUS markedly increased cerebral blood flow (CBF), reduced brain swelling, and improved functional recovery at day 3 after CIS. LIPUS promoted leptomeningeal vasculature remodeling, enlarged vascular diameter, and increased the average vessel length and density at day 3 after CIS. Proteomic analysis highlighted that LIPUS mainly participated in the regulation of actin cytoskeleton pathway. Rho kinase 1 (ROCK1) was downregulated by LIPUS and participated in regulation of actin cytoskeleton. Subsequently, we verified that ROCK1 was mainly expressed in pericytes. Furthermore, we demonstrated that LIPUS inhibited ROCK1/p-MLC2 signaling pathway after CIS, which had positive effects on vascular remodeling and cerebral blood circulation. In conclusion, our preliminary study revealed the vascular remodeling effects and mechanism of LIPUS in CIS, provided evidence for potential clinical application of LIPUS.

Low-intensity pulsed ultrasound triggers a beneficial neuromodulation in dementia mice with chronic cerebral hypoperfusion via activation of hippocampal Fndc5/irisin signaling.

BACKGROUND: Exercise-related signaling Fndc5/irisin expresses in brain and acts as a crucial regulator of cognitive function, but its detailed roles in vascular dementia (VaD) are still unclear. Low intensity pulsed ultrasound (LIPUS), a novel brain stimulation approach, has been suggested as a promising treatment for dementia. Here, we investigated the activity and efficacy of Fndc5/irisin in experimental VaD, further explored whether the potential effects of LIPUS on VaD is related to Fndc5/irisin. METHODS: Mouse model of VaD was established with chronic cerebral hypoperfusion (CCH) using bilateral common carotid arteries stenosis (BCAS). Transcranial LIPUS was applied 24 h after BCAS and subsequently daily with a stimulation time of 5 min at an ultrasound pressure of 0.51 MPa for a period of 28 days. The levels of Fndc5/irisin in different brain regions, the hippocampal long-term potentiation and anti-inflammatory cytokines were investigated at day 28 after cognitive evaluation. Global Fndc5 knock-out (F5KO), forced expression or knockdown of Fndc5, and recombinant irisin application were respectively employed for mechanism exploration. The neuron dendritic spine density and astrocyte phenotype were detected in vitro. RESULTS: Fndc5/irisin was reduced in hippocampus of BCAS mice, forced expression hippocampal Fndc5 or bilateral intrahippocampal injection of recombinant irisin respectively improved hippocampal synaptic plasticity or inflammatory microenvironment, and then alleviated the cognitive impairments. LIPUS existed a positive efficacy in enhancing hippocampal Fndc5/irisin in BCAS mice, thus triggering a beneficial neuromodulation for VaD protection. Importantly, the neurorestorative effects of LIPUS on CCH-induced damages were totally reversed by knockdown the expression of hippocampal Fndc5 in WT mice, or in F5KO mice. Moreover, Fndc5 mediated the upregulated effects of LIPUS on spine density as well as irisin secretion of hippocampal neurons. The neuron-secreted irisin further drove reactive astrocytes to a neuroprotective phenotype. CONCLUSION: LIPUS induced a neurorestorative stimulation against VaD may be through upregulation of the hippocampal Fndc5/irisin levels. Hippocampal Fndc5/irisin signaling might be a promising strategic target for VaD.

Vector flow mapping analysis of left ventricular vortex performance in type 2 diabetic patients with early chronic kidney disease.

BACKGROUND: Diabetes is the leading cause of chronic kidney disease (CKD) and contributes to an elevated incidence of diastolic dysfunction in the early stages of CKD. Intracardiac vortex is a novel hemodynamic index for perceiving cardiac status. Here, we visualized left ventricular (LV) vortex characteristics using vector flow mapping (VFM) in type 2 diabetic patients with early CKD. METHODS: This cross-sectional study included 67 controls and 89 type 2 diabetic patients with stages 2-3a CKD. All subjects underwent transthoracic echocardiographic examination. LV anterior vortex during early diastole (E-vortex), atrial contraction (A-vortex) and systole (S-vortex) were assessed using VFM in the apical long-axis view. Its relation to glycemia or LV filling echocardiographic parameters were further analyzed using correlation analysis. RESULTS: Type 2 diabetic patients with early CKD had a small area (439.94 ± 132.37 mm2 vs. 381.66 ± 136.85 mm2, P = 0.008) and weak circulation (0.0226 ± 0.0079 m2/s vs. 0.0195 ± 0.0070 m2/s, P = 0.013) of E-vortex, but a large area (281.52 ± 137.27 mm2 vs. 514.83 ± 160.33 mm2, P ˂ 0.001) and intense circulation (0.0149 ± 0.0069 m2/s vs. 0.0250 ± 0.0067 m2/s, P < 0.001) of A-vortex compared to controls. CKD patients with poorly controlled hyperglycemia had stronger A-vortex (area: 479.06 ± 146.78 mm2 vs. 559.96 ± 159.27 mm2, P = 0.015; circulation: 0.0221 ± 0.0058 m2/s vs. 0.0275 ± 0.0064 m2/s, P < 0.001) and S-vortex (area: 524.21 ± 165.52 mm2 vs. 607.87 ± 185.33 mm2, P = 0.029; circulation: 0.0174 ± 0.0072 m2/s vs. 0.0213 ± 0.0074 m2/s, P = 0.015), and a longer relative duration of S-vortex (0.7436 ± 0.0772 vs. 0.7845 ± 0.0752, P = 0.013) than those who had well-controlled hyperglycemia. Glycemia, and E/A (a LV filling parameter) were respectively found to had close correlation to the features of A-vortex and S-vortex (all P < 0.05). CONCLUSIONS: Abnormal LV vortices were detected in type 2 diabetic patients with early CKD using VFM, especially in those who neglected hyperglycemic control. LV vortex might be a promising parameter to slow or halt the hyperglycemia-induced diastolic dysfunction in early CKD.

Assessment of left ventricular energy loss using vector flow mapping in patients with stages 1-3 chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) experience abnormality of intracardiac blood flow status during early-stages of disease. Left ventricular energy loss (EL) derived from vector flow mapping (VFM) represents fluid energy lost as heat in left ventricle and had been used to detect intracardiac blood flow efficiency. We aimed to evaluate the left ventricular EL in stage 1-3 CKD patients, and explored whether hypertension, a main cardiovascular risk, deteriorate the abnormality of intracardiac blood flow status. METHODS: Transthoracic echocardiography was performed in 41 controls and 48 patients with stages 1-3 CKD. CKD patients consisted a subgroup with no hypertension, a subgroup with well-controlled hypertension and a subgroup with poorly controlled hypertension. The EL were calculated in the left ventricle using VFM analysis from the apical 3-chamber view. Furthermore, the correlation and stepwise multiple regression analysis were used to explore the potential independent predictors of left ventricular EL. RESULTS: Compared with controls, stage 1-3 CKD patients showed increased left ventricular EL during total diastole, late diastole, total systole, isovolumic contraction and ejection. CKD patients with poorly controlled hypertension had higher left ventricular EL compared to the other CKD subgroups. Additionally, the ratio of mitral early filling wave peak velocity and early mitral annular peak velocity on septal side, mitral early filling wave peak velocity, and left ventricular mass index were independent predictors of the diastolic EL; whereas systolic blood pressure and left ventricular mass index were independent predictors of the systolic EL. CONCLUSIONS: Left ventricular EL was a useful echocardiographic parameter to evaluate the impaired intracardiac blood flow efficiency in patients with stages 1-3 CKD. Hypertension was a crucial contributor for intracardiac blood flow abnormality. This study might provide valuable clinical data to discern cardiac dysfunction and reduce the cardiovascular risk in early-stage CKD.

Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke.

BACKGROUND: Ischemic stroke is a leading cause of disability worldwide and characteristically accompanied by downregulation of the Wnt/ß-catenin signaling. Activation of Wnt/ß-catenin signaling emerges to attenuate neuroinflammation after ischemic stroke; however, its effect on modulating microglial polarization is largely unknown. Here, we explored whether Wnt/ß-catenin pathway activator TWS119 facilitated long-term neurological recovery via modulating microglia polarization after experimental stroke. METHODS: Ischemic stroke mice model was induced by permanent distal middle cerebral artery occlusion plus 1 h hypoxia. TWS119 was administrated from day 1 to 14 after stroke. Neurological deficits were monitored up to 21 days after stroke. Angiogenesis, neural plasticity, microglial polarization, and microglia-associated inflammatory cytokines were detected in the peri-infarct cortex at days 14 and 21 after stroke. Primary microglia and mouse brain microvascular endothelial cell lines were employed to explore the underlying mechanism in vitro. RESULTS: TWS119 mitigated neurological deficits at days 14 and 21 after experimental stroke, paralleled by acceleration on angiogenesis and neural plasticity in the peri-infarct cortex. Mechanistically, cerebral ischemia induced production of microglia-associated proinflammatory cytokines and priming of activated microglia toward pro-inflammatory polarization, whereas TWS119 ameliorated microglia-mediated neuroinflammatory status following ischemic stroke and promoted angiogenesis by modulating microglia to anti-inflammatory phenotype. The beneficial efficacy of TWS119 in microglial polarization was largely reversed by selective Wnt/ß-catenin pathway blockade in vitro, suggesting that TWS119-enabled pro-inflammatory to anti-inflammatory phenotype switch of microglia was possibly mediated by Wnt/ß-catenin signaling. CONCLUSIONS: Wnt/ß-catenin pathway activator TWS119 ameliorated neuroinflammatory microenvironment following chronic cerebral ischemia via modulating microglia towards anti-inflammatory phenotype, and facilitates neurological recovery in an anti-inflammatory phenotype polarization-dependent manner. Activation of Wnt/ß-catenin pathway following ischemic stroke might be a potential restorative strategy targeting microglia-mediated neuroinflammation.

CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo.

The costimulatory effects of CD27 on T lymphocyte effector function and memory formation has been confined to evaluations in mouse models, in vitro human cell culture systems, and clinical observations. Here, we tested whether CD27 costimulation actively enhances human T-cell function, expansion, and survival in vitro and in vivo. Human T cells transduced to express an antigen-specific chimeric antigen receptor (CAR-T) containing an intracellular CD3 zeta (CD3ζ) chain signaling module with the CD27 costimulatory motif in tandem exerted increased antigen-stimulated effector functions in vitro, including cytokine secretion and cytotoxicity, compared with CAR-T with CD3ζ alone. After antigen stimulation in vitro, CD27-bearing CAR-T cells also proliferated, up-regulated Bcl-X(L) protein expression, resisted apoptosis, and underwent increased numerical expansion. The greatest impact of CD27 was noted in vivo, where transferred CAR-T cells with CD27 demonstrated heightened persistence after infusion, facilitating improved regression of human cancer in a xenogeneic allograft model. This tumor regression was similar to that achieved with CD28- or 4-1BB-costimulated CARs, and heightened persistence was similar to 4-1BB but greater than CD28. Thus, CD27 costimulation enhances expansion, effector function, and survival of human CAR-T cells in vitro and augments human T-cell persistence and antitumor activity in vivo.

The genetic polymorphisms of intercellular cell adhesion molecules and breast cancer susceptibility: a meta-analysis.

Intercellular cell adhesion molecules (ICAMs) genetic polymorphisms have been considered to be implicated in the development of breast cancer. However, the previous reports are conflicting. Therefore, we performed a meta-analysis to assess the association between three polymorphisms, including ICAM1 K469E, ICAM5 V301I, ICAM5 rs281439, and breast cancer risk. The meta-analyses are based on a literature search of PubMed, CNKI and VIP database up until August 2011. Pooled odds ratio (OR) with 95 % confidence interval (CI) was calculated using review manager 5.0.25 package. In total, five populations (2,020 cases and 2,012 controls) on ICAM1 K469E polymorphism, four populations (1,797 cases and 2,244 controls) on ICAM5 V301I polymorphism and five populations (2,744 cases and 3,006 controls) on ICAM5 rs281439 variant were included. Overall, the meta-analysis showed no significant association between ICAM1 K469E polymorphism and breast cancer risk. However, a significant association was observed for ICAM5 V301I polymorphism (VV vs. II: OR = 1.48, 95 % CI 1.04-2.13, P = 0.03; VV/VI vs. II: OR = 1.25, 95 % CI 1.05-1.48, P = 0.01). In addition, there was a significant association between ICAM5 rs281439 variant and breast cancer risk (GG vs. CC: OR = 1.31, 95 % CI 1.03-1.65, P = 0.03). Our meta-analysis suggests that the ICAM5 V301I and rs281439 variants but not ICAM1 K469E polymorphism may contribute to the susceptibility of breast cancer. Given the limited sample sizes, further investigation is needed.

Geniposide protected against cerebral ischemic injury through the anti-inflammatory effect via the NF-κB signaling pathway.

Context: Accumulated evidence indicates that geniposide exhibits neuroprotective effects in ischemic stroke. However, the potential targets of geniposide remain unclear. Objective: We explore the potential targets of geniposide in ischemic stroke. Materials and methods: Adult male C57BL/6 mice were subjected to the middle cerebral artery occlusion (MCAO) model. Mice were randomly divided into five groups: Sham, MCAO, and geniposide-treated (i.p. twice daily for 3 days before MCAO) at doses of 25, 75, or 150 mg/kg. We first examined the neuroprotective effects of geniposide. Then, we further explored via biological information analysis and verified the underlying mechanism in vivo and in vitro. Results: In the current study, geniposide had no toxicity at concentrations of up to 150 mg/kg. Compared with the MCAO group, the 150 mg/kg group of geniposide significantly (P < 0.05) improved neurological deficits, brain edema (79.00 ± 0.57% vs 82.28 ± 0.53%), and infarct volume (45.10 ± 0.24% vs 54.73 ± 2.87%) at 24 h after MCAO. Biological information analysis showed that the protective effect was closely related to the inflammatory response. Geniposide suppressed interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) expression in the brain homogenate, as measured by enzyme-linked immunosorbent assay (ELISA). Geniposide upregulated A20 and downregulated TNF receptor-associated factor-6 and nuclear factor kappa-B phosphorylation in the MCAO model and lipopolysaccharide-treated BV2 cells at 100 µM. Conclusions: Geniposide exhibited a neuroprotective effect via attenuating inflammatory response, as indicated by biological information analysis, in vivo and in vitro experiments, which may provide a potential direction for the application of geniposide in the treatment of ischemic stroke.

Astragaloside IV promotes cerebral angiogenesis and neurological recovery after focal ischemic stroke in mice via activating PI3K/Akt/mTOR signaling pathway.

Our previous work has shown that activating PI3K/Akt/mTOR signaling pathway is involved in angiogenesis after ischemic stroke, and recent studies have revealed that astragaloside IV (AS-IV) exerts beneficial effects on cerebral protection after ischemic stroke. However, it is unclear whether the beneficial effects of AS-IV against ischemic stroke is related to angiogenesis and PI3K/Akt/mTOR signaling pathway. The aim of this study was to investigate the effects of AS-IV on angiogenesis and long-term neurological recovery after focal ischemic stroke as well as the underlying mechanisms. After mice model of distal middle cerebral artery occlusion (dMCAO), AS-IV was administered with low dose (10 mg/kg), medium dose (20 mg/kg) or high dose (40 mg/kg) once daily for 14 days. We report herein that AS-IV (20 mg/kg) significantly ameliorated long-term neurological recovery and attenuated histological damage, while promoting cerebral blood flow recovery in ischemic mice. Moreover, AS-IV administration enhanced microvessel density as well as astrocyte and pericyte coverage around microvessels in the peri-infarct cortex. In vitro, AS-IV promoted endothelial cells (ECs) proliferation and tube formation after oxygen-glucose deprivation (OGD), which was partially inhibited by the specific PI3K inhibitor LY294002. Finally, AS-IV increased the expression of vascular endothelial growth factor (VEGF) through activating the PI3K/AKT/mTOR signaling pathway in the process of promoting angiogenesis. These results suggested that AS-IV may promote angiogenesis after ischemic stroke through increasing the expression of VEGF via PI3K/Akt/mTOR pathway, which unveils novel therapeutic effects of AS-IV and suggests promising application of AS-IV in ischemic stroke.

Chrysophanol facilitates long-term neurological recovery through limiting microglia-mediated neuroinflammation after ischemic stroke in mice.

BACKGROUND: Inflammation plays an important role in ischemic brain injury and affects brain recovery and neuroplasticity. Chrysophanol (CHR), has attracted attention for its protective effects through immunomodulatory and anti-inflammatory properties. However, the effect of CHR for brain recovery and neuroplasticity is not clear. The current study aimed to investigate the effect of CHR in the chronic phase of stroke in mice, and to elucidate the underlying mechanisms. METHODS: C57BL/6 mice were subjected to treatment with Vehicle or CHR immediately through intraperitoneal injection daily for 14 d after distal middle cerebral artery occlusion (dMCAO). Neurological deficits were monitored up to 28 days after stroke. Nissl and Golgi stain, neural plasticity, and microglia-associated inflammatory cytokines were detected. Primary cortical neuron and BV2 microglia cell lines were employed to explore the underlying mechanism in vitro. RESULTS: Compared with Vehicle group, CHR mitigated the histological damage, facilitated the neural plasticity and improved the neurological function up to 4 weeks after stroke. In vitro, CHR promoted the complexity of neurons and the spine density by modulating microglial polarization and reducing the expression of microglia-associated inflammatory cytokines, especially IL-6. In vivo, microglia activation and inflammatory cytokines were significantly increased after dMCAO and downregulated by CHR. Further investigation showed STAT3 is the major downstream effector of IL-6 signaling. CONCLUSIONS: CHR ameliorated microenvironment for neural plasticity and exhibited neuroprotection via arresting microglia toward pro-inflammatory phenotype and downregulation of the expressions of pro-inflammatory cytokines, especially of IL-6. IL-6-STAT3 signaling might be CHR's therapeutic target for neuroinflammatory responses after stroke.

Ginsenoside Rg1 promotes cerebral angiogenesis via the PI3K/Akt/mTOR signaling pathway in ischemic mice.

Angiogenesis plays an important role in the remodeling process of the ischemic brain and the recovery of neurological function after ischemic stroke. Ginsenoside Rg1 has been reported to exert neuroprotective effects on the central nervous system. However, the effects of ginsenoside Rg1 on cerebral angiogenesis in cerebral ischemia remained unclear. The current study aimed to investigate the potential protective effects of ginsenoside Rg1 on cerebral angiogenesis as well as its underlying mechanisms. Mice were subjected to treatment with vehicle or ginsenoside Rg1 daily for 14 d beginning at 24 h after distal middle cerebral artery occlusion (dMCAO). Compared with the dMCAO group, ginsenoside Rg1 improved the neurobehavioral outcomes and reduced the brain infarct volume. Ginsenoside Rg1 treatment increased the expression of the cluster of differentiation 31 (CD31), bromodeoxyuridine+/CD31+ microvessels and GFAP-positive vessels in the peri-infarct cortex. The expression of VEGF was significantly enhanced in ginsenoside Rg1 group. In vitro, human brain microvascular endothelial (hCMEC/D3) cells was successfully cultured, and oxygen and glucose deprivation (OGD) model was established. Ginsenoside Rg1 significantly increased proliferation, migration and tube formation of endothelial cells after OGD, as well as upregulated the expressions of VEGF, HIF-1α, PI3K, p-Akt, and p-mTOR. Furthermore, administration of PI3K/Akt/mTOR signaling pathway inhibitor LY294002 abolished the beneficial effects of ginsenoside Rg1. In conclusion, ginsenoside Rg1 promoted cerebral angiogenesis through increasing the expression of VEGF via PI3K/Akt/mTOR signaling pathway after ischemic stroke.

Control of triple-negative breast cancer using ex vivo self-enriched, costimulated NKG2D CAR T cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive disease that currently lacks effective targeted therapy. NKG2D ligands (NKG2DLs) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. METHODS: We applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human TNBCs. Lentiviral vectors were used to express the extracellular domain of human NKG2D that binds various NKG2DLs, fused to signaling domains derived from T cell receptor CD3 zeta alone or with CD27 or 4-1BB (CD137) costimulatory domain. RESULTS: Interleukin-2 (IL-2) promoted the expansion and self-enrichment of NKG2D-redirected CAR T cells in vitro. High CD25 expression on first-generation NKG2D CAR T cells was essential for the self-enrichment effect in the presence of IL-2, but not for CARs containing CD27 or 4-1BB domains. Importantly, self-enriched NKG2D CAR T cells effectively recognized and eliminated TNBC cell lines in vitro, and adoptive transfer of T cells expressing NKG2D CARs with CD27 or 4-1BB specifically enhanced NKG2D CAR surface expression, T cell persistence, and the regression of established MDA-MB-231 TNBC in vivo. NKG2D-z CAR T cells lacking costimulatory domains were less effective, highlighting the need for costimulatory signals. CONCLUSIONS: These results demonstrate that CD27 or 4-1BB costimulated, self-enriched NKG2D CAR-redirected T cells mediate anti-tumor activity against TNBC tumor, which represent a promising immunotherapeutic approach to TNBC treatment.

Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level.

BACKGROUND: The poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC. METHODS: The aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo. RESULTS: We found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα. CONCLUSIONS: Taken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.

Multiparameter comparative analysis reveals differential impacts of various cytokines on CART cell phenotype and function ex vivo and in vivo.

Exogenous cytokines are widely applied to enhance the anti-tumor ability of immune cells. However, systematic comparative studies of their effects on chimeric antigen receptor (CAR)-engineered T (CART) cells are lacking. In this study, CART cells targeting folate receptor-alpha were generated and expanded ex vivo in the presence of different cytokines (IL-2, IL-7, IL-15, IL-18, and IL-21), and their expansion, phenotype and cytotoxic capacity were evaluated, in vitro and in vivo. Moreover, the effect of the administration of these cytokines along with CART cells in vivo was also studied. IL-2, IL-7, and IL-15 favored the ex vivo expansion of CART cells compared to other cytokines or no cytokine treatment. IL-7 induced the highest proportion of memory stem cell-like CART cells in the final product, and IL-21 supported the expansion of CART cells with a younger phenotype, while IL-2 induced more differentiated CART cells. IL-2 and IL-15-exposed CART cells secreted more proinflammatory cytokines and presented stronger tumor-lysis ability in vitro. However, when tested in vivo, CART cells exposed to IL-2 ex vivo showed the least anti-tumor effect. In contrast, the administration of IL-15 and IL-21 in combination with CART cells in vivo increased their tumor killing capacity. According to our results, IL-7 and IL-15 show promise to promote ex vivo expansion of CART cells, while IL-15 and IL-21 seem better suited for in vivo administration after CART cell infusion. Collectively, these results may have a profound impact on the efficacy of CART cells in both hematologic and solid cancers.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity.

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate "on-target" toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and "on-target off-tumor" toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs.

CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor.

PURPOSE: Upregulation of CD137 (4-1BB) on recently activated CD8(+) T cells has been used to identify rare viral or tumor antigen-specific T cells from peripheral blood. Here, we evaluated the immunobiology of CD137 in human cancer and the utility of a CD137-positive separation methodology for the identification and enrichment of fresh tumor-reactive tumor-infiltrating lymphocytes (TIL) or tumor-associated lymphocytes (TAL) from ascites for use in adoptive immunotherapy. EXPERIMENTAL DESIGN: TILs from resected ovarian cancer or melanoma were measured for surface CD137 expression directly or after overnight incubation in the presence of tumor cells and homeostatic cytokines. CD137(pos) TILs were sorted and evaluated for antitumor activity in vitro and in vivo. RESULTS: Fresh ovarian TILs and TALs naturally expressed higher levels of CD137 than circulating T cells. An HLA-dependent increase in CD137 expression was observed following incubation of fresh enzyme-digested tumor or ascites in IL-7 and IL-15 cytokines, but not IL-2. Enriched CD137(pos) TILs, but not PD-1(pos) or PD-1(neg) CD137(neg) cells, possessed autologous tumor reactivity in vitro and in vivo. In melanoma studies, all MART-1-specific CD8(+) TILs upregulated CD137 expression after incubation with HLA-matched, MART-expressing cancer cells and antigen-specific effector function was restricted to the CD137(pos) subset in vitro. CD137(pos) TILs also mediated superior antitumor effects in vivo, compared with CD137(neg) TILs. CONCLUSIONS: Our findings reveal a role for the TNFR-family member CD137 in the immunobiology of human cancer where it is preferentially expressed on tumor-reactive subset of TILs, thus rationalizing its agonistic engagement in vivo and its use in TIL selection for adoptive immunotherapy trials.

Finding a needle in a haystack: Activation-induced CD137 expression accurately identifies naturally occurring tumor-reactive T cells in cancer patients.

We have recently identified tumor necrosis factor receptor superfamily, member 9 (TNFRSF9, best known as CD137 or 4-1BB) as a biomarker of tumor-reactive T cells naturally occurring in cancer patients, and developed a rapid, accurate system to comprehensively isolate lymphocytes with tumor-rejecting properties from human biopsies. Our findings reveal a previously unappreciated role for CD137, a co-stimulatory TNFR family member, in the immunobiology of human cancer.

Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition.

NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4(+) and CD8(+) NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer.