Endogenous bystander killing mechanisms enhance the activity of novel FAP-specific CAR-T cells against glioblastoma.

Objectives: CAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma. Methods: We generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function in vitro (using real-time impedance, flow cytometry, imaging and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma). Results: FAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells. Conclusions: Our findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.

Safety and biological outcomes following a phase 1 trial of GD2-specific CAR-T cells in patients with GD2-positive metastatic melanoma and other solid cancers.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapies specific for the CD19 and B-cell maturation antigen have become an approved standard of care worldwide for relapsed and refractory B-cell malignancies. If CAR-T cell therapy for non-hematological malignancies is to achieve the same stage of clinical development, then iterative early-phase clinical testing can add value to the clinical development process for evaluating CAR-T cell products containing different CAR designs and manufactured under differing conditions. METHODS: We conducted a phase 1 trial of third-generation GD2-specific CAR-T cell therapy, which has previously been tested in neuroblastoma patients. In this study, the GD2-CAR-T therapy was evaluated for the first time in metastatic melanoma patients in combination with BRAF/MEK inhibitor therapy, and as a monotherapy in patients with colorectal cancer and a patient with fibromyxoid sarcoma. Feasibility and safety were determined and persistence studies, multiplex cytokine arrays on sera and detailed immune phenotyping of the original CAR-T products, the circulating CAR-T cells, and, in select patients, the tumor-infiltrating CAR-T cells were performed. RESULTS: We demonstrate the feasibility of manufacturing CAR-T products at point of care for patients with solid cancer and show that a single intravenous infusion was well tolerated with no dose-limiting toxicities or severe adverse events. In addition, we note significant improvements in CAR-T cell immune phenotype, and expansion when a modified manufacturing procedure was adopted for the latter 6 patients recruited to this 12-patient trial. We also show evidence of CAR-T cell-mediated immune activity and in some patients expanded subsets of circulating myeloid cells after CAR-T cell therapy. CONCLUSIONS: This is the first report of third-generation GD2-targeting CAR-T cells in patients with metastatic melanoma and other solid cancers such as colorectal cancer, showing feasibility, safety and immune activity, but limited clinical effect. TRIAL REGISTRATION NUMBER: ACTRN12613000198729.

Depression and associated factors among HIV-positive smokers receiving care at HIV outpatient clinics in Vietnam: a cross-sectional analysis.

OBJECTIVES: To assess the prevalence of depressive symptoms and associated factors among people living with HIV (PLWH) who were current cigarette smokers and receiving treatment at HIV outpatient clinics (OPCs) in Vietnam. DESIGN: A cross-sectional survey of smokers living with HIV. SETTING: The study was carried out in 13 HIV OPCs located in Ha Noi, Vietnam. PARTICIPANTS: The study included 527 PLWH aged 18 and above who were smokers and were receiving treatment at HIV OPCs. OUTCOME MEASURES: The study used the Centre for Epidemiology Scale for Depression to assess depressive symptoms. The associations between depressive symptoms, tobacco dependence and other characteristics were explored using bivariate and Poisson regression analyses. RESULTS: The prevalence of depressive symptoms among smokers living with HIV was 38.3%. HIV-positive smokers who were female (prevalence ratio, PR 1.51, 95% CI 1.02 to 2.22), unmarried (PR 2.06, 95% CI 1.54 to 2.76), had a higher level of tobacco dependence (PR 1.06, 95% CI 1.01 to 1.11) and reported their health as fair or poor (PR 1.66, 95% CI 1.22 to 2.26) were more likely to have depression symptoms compared with HIV-positive smokers who were male, married, had a lower level of tobacco dependence and self-reported their health as good, very good or excellent. CONCLUSION: The prevalence of depressive symptoms among smokers receiving HIV care at HIV OPCs was high. Both depression and tobacco use screening and treatment should be included as part of ongoing care treatment plans at HIV OPCs.

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma.

BACKGROUND: Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited. METHODS: We profiled an extensive biobank of patients' biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. RESULTS: Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. CONCLUSIONS: Targeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.

Management of Frail and Older Homebound Patients With Heart Failure: A Contemporary Virtual Ambulatory Model.

BACKGROUND: Heart failure (HF) affects many patients who are older and frail, presenting multiple physical barriers to accessing specialty care in a traditional ambulatory clinic model. Here, we present an assisted virtual care model in which a home visiting nurse facilitated video visits with a HF cardiologist to follow homebound, frail, and older patients with HF. METHODS: This is a pragmatic, quasi-experimental, pre-post, single-centre study. It included homebound, frail, and older patients with HF from 2015 to 2019 who were followed for 1 year; in-person clinic visits were completely replaced by nurse-facilitated virtual video visits. Outcomes evaluated included annualized hospitalization rate, number of hospitalization days, and number of emergency department visits. RESULTS: A total of 49 patients were included, with a median age of 86 (83-93) years, and were followed for 1 year after enrollment. Among patients enrolled, HF with preserved ejection fraction was the most common subtype (57%). Compared to the year prior to enrollment, patients had a lower mortality-adjusted all-cause annualized hospitalization rate in the year following enrollment (2.57 vs 1.78, P < 0.0001). Compared to the year prior, the number of mortality-adjusted all-cause hospitalization days was significantly lower in the year following enrollment (27.2 vs 21.4, P < 0.0001). There was a reduction in the number of all-cause annualized emergency department visits (3.10 vs 2.27, P = 0.003). CONCLUSIONS: Nurse-assisted virtual visits may be a preferable strategy for homebound, frail, and older patients with HF to receive longitudinal care. This approach may represent a plausible strategy to care for other patients with significant barriers to accessing specialized cardiac care.

CONTEXTE: L'insuffisance cardiaque (IC) touche de nombreux patients âgés et fragiles, et dresse maints obstacles physiques à l'accès aux soins spécialisés au sein d'un modèle classique de soins cliniques ambulatoires. Dans le présent article, nous exposons un modèle de soins virtuels assistés où une infirmière visiteuse assure par vidéoconsultation, avec un cardiologue, le suivi de patients âgés et fragiles atteints d'IC confinés à la maison. MÉTHODOLOGIE: Une étude monocentrique et pragmatique, quasi expérimentale de type « avant-après ¼, a été menée de 2015 à 2019 auprès de patients âgés et fragiles atteints d'IC confinés à la maison. Les patients ont été suivis durant un an; les consultations en personne ont été entièrement remplacées par des vidéoconsultations effectuées par une infirmière. Les paramètres évalués comprenaient le taux annualisé d'hospitalisation, le nombre de jours d'hospitalisation et le nombre de consultations aux urgences. RÉSULTATS: Au total, 49 patients dont l'âge médian était de 86 ans (83-93 ans) ont été suivis durant un an à compter de leur admission à l'étude. L'IC à fraction d'éjection préservée était le sous-type d'IC le plus fréquent (57 %) chez les patients participant à l'étude. Par comparaison à l'année précédente, le taux annualisé d'hospitalisation toutes causes confondues ajusté en fonction de la mortalité a été plus faible chez les patients au cours de l'année où ils ont été suivis dans le cadre de l'étude (2,57 vs 1,78, P < 0,0001). Toujours par comparaison à l'année précédente, le nombre de jours d'hospitalisation toutes causes confondues ajusté en fonction de la mortalité a été significativement inférieur chez les patients au cours de l'année où ils ont été suivis dans le cadre de l'étude (27,2 vs 21,4, P < 0,0001). Le nombre annualisé de consultations aux urgences toutes causes confondues a quant à lui diminué (3,10 vs 2,27, P = 0,003). CONCLUSIONS: Les consultations virtuelles assistées par une infirmière peuvent constituer une stratégie à privilégier dans la prestation de soins longitudinaux à des patients âgés et fragiles atteints d'IC qui sont confinés à la maison. Cette approche pourrait représenter une stratégie plausible pour prodiguer des soins à d'autres patients qui sont confrontés à d'importants obstacles limitant leur accès à des soins spécialisés en cardiologie.

Effects of Chemotherapy Agents on Circulating Leukocyte Populations: Potential Implications for the Success of CAR-T Cell Therapies.

Adoptive T-cell therapy using autologous T cells genetically modified to express cancer-specific chimeric antigen receptors (CAR) has emerged as a novel approach for cancer treatment. CAR-T cell therapy has been approved in several major jurisdictions for treating refractory or relapsed cases of B-cell precursor acute lymphoblastic leukaemia and diffuse large B-cell lymphoma. However, in solid cancer patients, several clinical studies of CAR-T cell therapy have demonstrated minimal therapeutic effects, thus encouraging interest in better integrating CAR-T cells with other treatments such as conventional cytotoxic chemotherapy. Increasing evidence shows that not only do chemotherapy drugs have tumoricidal effects, but also significantly modulate the immune system. Here, we discuss immunomodulatory effects of chemotherapy drugs on circulating leukocyte populations, including their ability to enhance cytotoxic effects and preserve the frequency of CD8+ T cells and to deplete immunosuppressive populations including regulatory T cells and myeloid-derived suppressor cells. By modulating the abundance and phenotype of leukocytes in the blood (the 'raw material' for CAR-T cell manufacturing), we propose that prior chemotherapy could facilitate production of the most effective CAR-T cell products. Further research is required to directly test this concept and identify strategies for the optimal integration of CAR-T cell therapies with cytotoxic chemotherapy for solid cancers.

Optimization of manufacturing conditions for chimeric antigen receptor T cells to favor cells with a central memory phenotype.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells are genetically engineered to recognize tumor-associated antigens and have potent cytolytic activity against tumors. Adoptive therapy with CAR-T cells has been highly successful in B-cell leukemia and lymphoma. However, in solid tumor settings, CAR-T cells face a particularly hostile tumor microenvironment where multiple immune suppressive factors serve to thwart the anti-cancer immune response. Clinical trials of solid tumor antigen-targeted CAR-T cells have shown limited efficacy, and issues for current CAR-T cell therapies include failures of expansion and persistence, tumor entry, deletion and functional exhaustion. METHODS: We compared our standard protocol for CAR-T cell manufacturing, currently used to generate CAR-T cells for a phase 1 clinical trial, with two alternative approaches for T-cell activation and expansion. The resulting cultures were analyzed using multicolor flow cytometry, cytokine bead array and xCELLigence cytotoxicity assays. RESULTS: We have found that by changing the method of activation we can promote generation of CAR-T cells with enhanced CD62L and CCR7 expression, increased interleukin (IL)-2 production and retention of cytolytic activity, albeit with slower kinetics. DISCUSSION: We propose that these phenotypic characteristics are consistent with a central memory phenotype that will better enable CAR-T cell survival and persistence after activation in vivo, and we aim to test this in a continuation of our current phase 1 clinical trial of CAR-T cells in patients with advanced melanoma.

Rapid mitogenic regulation of the mTORC1 inhibitor, DEPTOR, by phosphatidic acid.

The mammalian target of rapamycin complex 1 (mTORC1) is regulated, in part, by the endogenous inhibitor DEPTOR. However, the mechanism of DEPTOR regulation with regard to rapid mTORC1 activation remains unknown. We report that DEPTOR is rapidly and temporarily dissociated from mTORC1 upon mitogenic stimulation, suggesting a mechanism underlying acute mTORC1 activation. This mitogen-stimulated DEPTOR dissociation is blocked by inhibition or depletion of the mTORC1 regulator, phospholipase D (PLD), and recapitulated with the addition of the PLD product phosphatidic acid (PA). Our mass spectrometry analysis has independently identified DEPTOR as an mTOR binding partner dissociated by PA. Interestingly, only PA species with unsaturated fatty acid chains, such as those produced by PLD, are capable of displacing DEPTOR and activating mTORC1, with high affinity for the FRB domain of mTOR. Our findings reveal a mechanism of mTOR regulation and provide a molecular explanation for the exquisite specificity of PA function.

Regulatory T cells dynamically control the primary immune response to foreign antigen.

The population dynamics that enable a small number of regulatory T (T(R)) cells to control the immune responses to foreign Ags by the much larger conventional T cell subset were investigated. During the primary immune response, the expansion and contraction of conventional and T(R) cells occurred in synchrony. Importantly, the relative accumulation of T(R) cells at peak response significantly exceeded that of conventional T cells, reflecting extensive cell division within the T(R) cell pool. Transfer of a polyclonal T(R) cell population before immunization antagonized both polyclonal and TCR transgenic responses, whereas blocking T(R) cell function enhanced those responses. These results define an inverse quantitative relationship between T(R) and conventional T cells that controls the magnitude of the primary immune response. The high frequency of dividing T(R) cells suggests degenerate TCR specificity enabling activation by a broad spectrum of Ags.

Regulatory T cell development in the absence of functional Foxp3.

Although the development of regulatory T cells (T(reg) cells) in the thymus is defined by expression of the lineage marker Foxp3, the precise function of Foxp3 in T(reg) cell lineage commitment is unknown. Here we examined T(reg) cell development and function in mice with a Foxp3 allele that directs expression of a nonfunctional fusion protein of Foxp3 and enhanced green fluorescent protein (Foxp3DeltaEGFP). Thymocyte development in Foxp3DeltaEGFP male mice and Foxp3DeltaEGFP/+ female mice recapitulated that of wild-type mice. Although mature EGFP(+) CD4(+) T cells from Foxp3DeltaEGFP mice lacked suppressor function, they maintained the characteristic T(reg) cell 'genetic signature' and failed to develop from EGFP(-) CD4(+) T cells when transferred into lymphopenic hosts, indicative of their common ontogeny with T(reg) cells. Our results indicate that T(reg) cell effector function but not lineage commitment requires the expression of functional Foxp3 protein.

Long-term memory deficits in Pavlovian fear conditioning in Ca2+/calmodulin kinase kinase alpha-deficient mice.

Signaling by the Ca(2+)/calmodulin kinase (CaMK) cascade has been implicated in neuronal gene transcription, synaptic plasticity, and long-term memory consolidation. The CaM kinase kinase alpha (CaMKKalpha) isoform is an upstream component of the CaMK cascade whose function in different behavioral and learning and memory paradigms was analyzed by targeted gene disruption in mice. CaMKKalpha mutants exhibited normal long-term spatial memory formation and cued fear conditioning but showed deficits in context fear during both conditioning and long-term follow-up testing. They also exhibited impaired activation of the downstream kinase CaMKIV/Gr and its substrate, the transcription factor cyclic AMP-responsive element binding protein (CREB) upon fear conditioning. Unlike CaMKIV/Gr-deficient mice, the CaMKKalpha mutants exhibited normal long-term potentiation and normal levels of anxiety-like behavior. These results demonstrate a selective role for CaMKKalpha in contextual fear memory and suggest that different combinations of upstream and downstream components of the CaMK cascade may serve distinct physiological functions.

Allergic dysregulation and hyperimmunoglobulinemia E in Foxp3 mutant mice.

BACKGROUND: Regulatory T cells have been proposed to play an important role in regulating allergic inflammation. The transcription factor Foxp3 is a master switch gene that controls the development and function of natural and adaptive CD4(+)CD25(+) regulatory T (T(R)) cells. In human subjects loss-of-function Foxp3 mutations trigger lymphoproliferation, autoimmunity, and intense allergic inflammation in a disease termed immune dysregulation polyendocrinopathy enteropathy-X-linked syndrome. OBJECTIVE: We sought to examine the evolution and attributes of allergic inflammation in mice with a targeted loss-of-function mutation in the murine Foxp3 gene that recapitulates a known disease-causing human Foxp3 mutation. METHODS: Foxp3 mutant mice were generated by means of knock-in mutagenesis and were analyzed for histologic, immunologic, and hematologic abnormalities. The role of signal transducer and activator of transcription 6 (Stat6) in disease pathogenesis was analyzed by using Stat6 and Foxp3 double-mutant mice. RESULTS: Foxp3 mutant mice developed an intense multiorgan inflammatory response associated with allergic airway inflammation, a striking hyperimmunoglobulinemia E, eosinophilia, and dysregulated T(H)1 and T(H)2 cytokine production in the absence of overt T(H)2 skewing. Concurrent Stat6 deficiency reversed the hyperimmunoglobulinemia E and eosinophilia and delayed mortality, which is consistent with a pathogenic role for allergic inflammation in Foxp3 deficiency. CONCLUSION: Allergic dysregulation is a common and fundamental consequence of loss of CD4(+)CD25(+) T(R) cells caused by Foxp3 deficiency in different species. Abnormalities affecting T(R) cells might contribute to a variety of allergic diseases.

Critical function of the CD40 pathway in parvovirus B19 infection revealed by a hypomorphic CD40 ligand mutation.

Parvovirus B19-induced chronic anemia has been associated with failure to mount an effective neutralizing antibody response. We describe an adolescent male with a 13-year history of parvovirus B19-induced anemia as the primary manifestation of X-linked hyper IgM immunodeficiency (XHIM). This patient, whose serum IgG concentration was at the low end of the normal range and who mounted IgG antibody responses to T cell-dependent antigens, suffered from a nonsense mutation (R11 --> X) in the CD40 ligand (CD40L) gene. This resulted in low-level expression of a mutant CD40L predicted to lack the cytoplasmic domain. Intravenous immunoglobulin therapy alone or in combination with interferon gamma, given in the context of impaired Th1 cytokine production, suppressed but did not eradicate the infection. These results highlight the critical function of the CD40/CD40L pathway in parvovirus B19 infection and suggest that subtle defects in this pathway may underlie cases of chronic parvovirus B19 infection atypical of XHIM.