Abstract
Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to ...poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (Le
Y
)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, Le
Y
CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although Le
Y
CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.
Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we ...explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.
Autocrine priming of cells by small quantities of constitutively produced type I interferon (IFN) is a well-known phenomenon. In the absence of type I IFN priming, cells display attenuated responses ...to other cytokines, such as anti-viral protection in response to IFNgamma. This phenomenon was proposed to be because IFNalpha/beta receptor1 (IFNAR1) is a component of the IFNgamma receptor (IFNGR), but our new data are more consistent with a previously proposed model indicating that regulated expression of STAT1 may also play a critical role in the priming process. Initially, we noticed that DNA binding activity of STAT1 was attenuated in c-Jun(-/-) fibroblasts because they expressed lower levels of STAT1 than wild-type cells. However, expression of STAT1 was rescued by culturing c-Jun(-/-) fibroblasts in media conditioned by wild-type fibroblasts suggesting they secreted a STAT1-inducing factor. The STAT1-inducing factor in fibroblast-conditioned media was IFNbeta, as it was inhibited by antibodies to IFNAR1, or when IFNbeta expression was knocked down in wild-type cells. IFNAR1(-/-) fibroblasts, which cannot respond to this priming, also expressed reduced levels of STAT1, which correlated with their poor responses to IFNgamma. The lack of priming in IFNAR1(-/-) fibroblasts was compensated by over-expression of STAT1, which rescued molecular responses to IFNgamma and restored the ability of IFNgamma to induce protective anti-viral immunity. This study provides a comprehensive description of the molecular events involved in priming by type I IFN. Adding to the previous working model that proposed an interaction between type I and II IFN receptors, our work and that of others demonstrates that type I IFN primes IFNgamma-mediated immune responses by regulating expression of STAT1. This may also explain how type I IFN can additionally prime cells to respond to a range of other cytokines that use STAT1 (e.g., IL-6, M-CSF, IL-10) and suggests a potential mechanism for the changing levels of STAT1 expression observed during viral infection.
Chimeric antigen receptor (CAR)-T-cell therapy is a promising approach for the treatment of childhood cancers, particularly high-risk tumors that fail to respond to standard therapies. CAR-T cells ...have been highly successful in treating some types of hematological malignancies. However, CAR-T cells targeting solid cancers have had limited success so far for multiple reasons, including their poor long-term persistence and proliferation. Evidence is emerging to show that maintaining CAR-T cells in an early, less-differentiated state in vitro results in superior persistence, proliferation, and antitumor effects in vivo. Children are ideal candidates for receiving less-differentiated CAR-T cells, because their peripheral T-cell pool primarily comprises naïve cells that could readily be harvested in large numbers to generate early-phenotype CAR-T cells. Although several studies have reported different approaches to successfully generate early CAR-T cells, there are only a few clinical trials testing these in adult patients. No trials are currently testing early CAR-T cells in children. Here, we summarize the different strategies used to maintain CAR-T cells in an early phenotypic stage and present evidence suggesting that this approach may be particularly relevant to treating childhood cancers.
•Early-phenotype CAR-T cells in the good manufacturing practice product are associated with improved CAR-T-cell persistence and clinical outcome in acute lymphoblastic leukemia.•Children have a high frequency of naïve T cells, which are an optimal source for generation of early-phenotype CAR-T cells.•Early CAR-T cells can be generated using memory cytokines, inhibitors, or genetic modification of T-cell signaling pathways.•Protocols to prevent CAR tonic signaling are also key to generate early-phenotype CAR-T cells.
Guidelines recommend normocapnia for adults with coma who are resuscitated after out-of-hospital cardiac arrest. However, mild hypercapnia increases cerebral blood flow and may improve neurologic ...outcomes.
We randomly assigned adults with coma who had been resuscitated after out-of-hospital cardiac arrest of presumed cardiac or unknown cause and admitted to the intensive care unit (ICU) in a 1:1 ratio to either 24 hours of mild hypercapnia (target partial pressure of arterial carbon dioxide Paco
, 50 to 55 mm Hg) or normocapnia (target Paco
, 35 to 45 mm Hg). The primary outcome was a favorable neurologic outcome, defined as a score of 5 (indicating lower moderate disability) or higher, as assessed with the use of the Glasgow Outcome Scale-Extended (range, 1 death to 8, with higher scores indicating better neurologic outcome) at 6 months. Secondary outcomes included death within 6 months.
A total of 1700 patients from 63 ICUs in 17 countries were recruited, with 847 patients assigned to targeted mild hypercapnia and 853 to targeted normocapnia. A favorable neurologic outcome at 6 months occurred in 332 of 764 patients (43.5%) in the mild hypercapnia group and in 350 of 784 (44.6%) in the normocapnia group (relative risk, 0.98; 95% confidence interval CI, 0.87 to 1.11; P = 0.76). Death within 6 months after randomization occurred in 393 of 816 patients (48.2%) in the mild hypercapnia group and in 382 of 832 (45.9%) in the normocapnia group (relative risk, 1.05; 95% CI, 0.94 to 1.16). The incidence of adverse events did not differ significantly between groups.
In patients with coma who were resuscitated after out-of-hospital cardiac arrest, targeted mild hypercapnia did not lead to better neurologic outcomes at 6 months than targeted normocapnia. (Funded by the National Health and Medical Research Council of Australia and others; TAME ClinicalTrials.gov number, NCT03114033.).
Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein ...perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.
We established an international consortium to review and discuss relevant clinical evidence in order to develop expert consensus statements related to cancer management during the severe acute ...respiratory syndrome coronavirus 2-related disease (COVID-19) pandemic. The steering committee prepared 10 working packages addressing significant clinical questions from diagnosis to surgery. During a virtual consensus meeting of 62 global experts and one patient advocate, led by the European Society for Medical Oncology, statements were discussed, amended and voted upon. When consensus could not be reached, the panel revised statements until a consensus was reached. Overall, the expert panel agreed on 28 consensus statements that can be used to overcome many of the clinical and technical areas of uncertainty ranging from diagnosis to therapeutic planning and treatment during the COVID-19 pandemic.
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