Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced ...dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory “cytokine-storm”, a cocktail of interferon gamma, interleukin 1β, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.
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•Pro-inflammatory factors drive systolic and diastolic cardiac dysfunction•BRD4 activation drives diastolic dysfunction and is blocked by clinically relevant drugs•BET inhibition decreases ACE2 expression and decreases SARS-CoV2 infection
COVID-19 causes cardiac injury, although mechanisms and effective therapeutics are lacking. In this study, Mills et al., show that cytokines elevated in COVID-19 patients drive cardiac dysfunction. These responses are mapped using phosphoproteomics and single nuclei RNA sequencing, enabling a targeted drug screen to identify therapeutics for rapid repurposing. BET inhibitors were identified as leading candidates to block cardiac dysfunction and decrease SARS-CoV-2 cardiac infection.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the ...exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.
IntroductionNKR-2 are autologous T cells genetically modified to express a chimeric antigen receptor (CAR) comprising a fusion of the natural killer group 2D (NKG2D) receptor with the CD3ζ signalling ...domain, which associates with the adaptor molecule DNAX-activating protein of 10 kDa (DAP10) to provide co-stimulatory signal upon ligand binding. NKG2D binds eight different ligands expressed on the cell surface of many tumour cells and which are normally absent on non-neoplastic cells. In preclinical studies, NKR-2 demonstrated long-term antitumour activity towards a breadth of tumour indications, with maximum efficacy observed after multiple NKR-2 administrations. Importantly, NKR-2 targeted tumour cells and tumour neovasculature and the local tumour immunosuppressive microenvironment and this mechanism of action of NKR-2 was established in the absence of preconditioning.Methods and analysisThis open-label phase I study will assess the safety and clinical activity of NKR-2 treatment administered three times, with a 2-week interval between each administration in different tumour types. The study will contain two consecutive segments: a dose escalation phase followed by an expansion phase. The dose escalation study involves two arms, one in solid tumours (five specific indications) and one in haematological tumours (two specific indications) and will include three dose levels in each arm: 3×108, 1×109 and 3×109 NKR-2 per injection. On the identification of the recommended dose in the first segment, based on dose-limiting toxicity occurrences, the study will expand to seven different cohorts examining the seven different tumour types separately. Clinical responses will be determined according to standard Response Evaluation Criteria In Solid Tumors (RECIST) criteria for solid tumours or international working group response criteria in haematological tumours.Ethics approval and disseminationEthical approval has been obtained at all sites. Written informed consent will be taken from all participants. The results of this study will be disseminated through presentation at international scientific conferences and reported in peer-reviewed scientific journals.Trial registration numberNCT03018405, EudraCT 2016-003312-12; Pre-result.
T cells engrafted with chimeric AgRs (CAR) are showing exciting potential for targeting B cell malignancies in early-phase clinical trials. To determine whether the second-generation CAR was ...essential for optimal antitumor activity, two CD28-based CAR constructs targeting CD19 were tested for their ability to redirect mouse T cell function against established B cell lymphoma in a BALB/c syngeneic model system. T cells armed with either CAR eliminated A20 B cell lymphoma in vivo; however, one construct induced a T cell dose-dependent acute toxicity associated with a raised serum Th1 type cytokine profile on transfer into preconditioned mice. Moreover, a chronic toxicity manifested as granuloma-like formation in spleen, liver, and lymph nodes was observed in animals receiving T cells bearing either CD28 CAR, albeit with different kinetics dependent upon the specific receptor used. This phenotype was associated with an expansion of CD4+ CAR+ T cells and CD11b+ Gr-1(+) myeloid cells and increased serum Th2-type cytokines, including IL-10 and IL-13. Mouse T cells engrafted with a first-generation CAR failed to develop such autotoxicity, whereas toxicity was not apparent when T cells bearing the same receptors were transferred into C57BL/6 or C3H animals. In summary, the adoptive transfer of second-generation CD19-specific CAR T cells can result in a cell dose-dependent acute toxicity, whereas the prolonged secretion of high levels of Th2 cytokines from these CAR T cells in vivo drives a granulomatous reaction resulting in chronic toxicity. Strategies that prevent a prolonged Th2-cytokine biased CAR T cell response are clearly warranted.
Importance of the field:
The active suppression of immune responses against tumor is a major barrier to the likely success of cancer immunotherapy. There is now compelling evidence implicating T ...regulatory cells (Tregs) as being key players driving immune suppression. Elevated frequencies of Tregs within the peripheral circulation and tumor microenvironment of cancer patients correlate with poor prognosis and reduced survival. Understanding the mechanism of Treg elevation is critical for the development of new approaches aiming to modulate the frequency and function of Tregs to enhance the efficacy of cancer immune-based therapies.
Areas covered in this review:
This review focuses on current knowledge concerning Tregs in cancer and discusses putative mechanisms which underlie the expansion of Tregs in cancer patients. Additionally, we review current strategies to deplete/suppress Treg activity, the limitations of these strategies and future perspective for improving their efficacy.
What the reader will gain:
An insight of the current aspects concerning Treg subsets in cancer and an overview of recent advances in the identification of Treg-specific markers, in addition to the potential strategies to target Tregs for enhancing antitumor immunity.
Take home message:
Mechanisms by which Treg functions modulate the immune response to tumors are becoming further understood. However, specific markers to tumor-specific/induced Tregs are yet to be clearly identified, which is a major limitation in optimizing strategies to specifically target Tregs in cancer. Despite this, strategies aimed at modulating Tregs in patients are providing some early encouraging results supporting the overall concept and indicating that further studies are clearly warranted.
Recombinant TCRs confer specificity to T cells and trigger their activation. Receptors with Ab-derived binding domains have the advantages of MHC-independent Ag recognition and of targeting a variety ...of chemically different molecules. We explored the impact of the position of a defined epitope within the target molecule on the efficacy of receptor-mediated T cell activation. T cells were grafted with recombinant immunoreceptors that recognize either the membrane distal N or the proximal A3 domain of carcinoembryonic Ag (CEA). Upon binding to isolated, solid-phase immobilized CEA, receptor-mediated T cell activation correlates with the binding efficiency, irrespectively, of the epitope position. Upon binding to CEA expressed on the cell membrane, in contrast, the A3 epitope mediates more efficiently T cell activation than the N epitope, although the N epitope is bound with higher affinity. The CEA N epitope when expressed in a more membrane proximal position, however, activated receptor grafted T cells with higher efficiency than in the distal position. The position of the targeted epitope within the molecule obviously has major impact on the efficacy of T cell activation independently of the binding efficiency of the immunoreceptor.
T cells gene-modified to express chimeric Ag receptors (CARs) have shown potent antitumor activity in vivo and are in clinical trials at locations worldwide. However, CAR activity has been ...investigated in mouse models in which Ag expression is restricted to the tumor. To explore the impact of normal tissue expression of the target Ag, we developed a mouse CD19-specific CAR to investigate antitumor efficacy against a syngeneic B cell lymphoma cell line within a background of normal CD19(+) host B cells. Mouse T cells engrafted with the amCD19CD3zeta CAR specifically lysed A20 lymphoma targets and B cells in vitro. These T cells also eradicated a 12-d established disseminated A20 lymphoma in mice preconditioned with 6 Gy total body irradiation. In the short-term (7 d after adoptive transfer), amCD19z T cells underwent Ag-dependent proliferation in vivo with a concomitant depletion in host B cell levels. However, the levels of amCD19z CAR(+) T cells decreased significantly at later time points, at which point host B cells returned, eventually reaching normal levels. In contrast, CAR(+) T cells lacking a signaling domain or specificity for mCD19 persisted over extended periods in blood and spleen. Importantly, no overt clinical signs of autotoxicity were observed in tumor-free or tumor-bearing mice treated with amCD19z T cells over an extended period of time. These observations highlight the importance of studying the activity of CAR(+) T cells in autologous models that have the normal range of tissue expression of Ag.
Purpose: To study the production of chemokines by colorectal hepatic metastases.
Experimental Design: Biopsies of resected colorectal hepatic metastases and nonneoplastic adjacent liver tissue were ...screened for chemokines using
protein arrays and results were confirmed by ELISA and immunohistochemistry.
Results: Two chemokines, eotaxin-2 and MCP-1, were found at elevated levels within the tumor biopsy compared with adjacent liver.
The relative increase in expression from tumor was much higher for eotaxin-2 than MCP-1, with 10 of 25 donors having a >100-fold
increase in expression compared with 0 of 24 donors for MCP-1. In a parallel analysis, eotaxin-2 was also found at elevated
levels in the tumor region of primary colorectal cancer biopsies. Immunohistochemical staining indicated that carcinoembryonic
antigen–positive tumor cells stained strongly for eotaxin-2, implicating these cells as the predominant source of the chemokine.
In vitro studies confirmed that several colorectal tumor lines produce eotaxin-2 and that secretion of this chemokine could be depressed
by IFN-γ and enhanced by the Th2-type cytokines interleukin-4 and interleukin-13. Jurkat T cells were engineered to express
the receptor for eotaxin-2 (CCR3). These cells effectively migrated in response to eotaxin-2 protein, suggesting that immune
cells gene modified to express a chemokine receptor may have improved abilities to home to tumor.
Conclusions: Taken together, these observations confirm eotaxin-2 as a chemokine strongly associated with primary and metastatic tumors
of colorectal origin. Furthermore, the importance of this result may be a useful tool in the development of targeted therapeutic
approaches to colorectal tumors.
The central goal of cancer immunotherapy is to control tumors through the mobilization of the patient's immune system. Vaccines targeting the Her2/neu proto-oncogene have been tested with some early ...encouraging responses in breast cancer. However, a more effective set of vaccines targeting specific immune cell subtypes may provide a more potent means to stimulate anti-tumor immunity. Dendritic cell-specific antibodies fused with the Her2/neu protein proved effective at generating immune responses in preclinical models. Importantly, only low amounts of protein vaccine were required to generate this response, which has potentially significant implications for the future clinical development of Her2/neu-targeted vaccines and other vaccine targets.
Abstract
Chimeric Ag receptors (CARs) expressed in T cells permit the redirected lysis of tumor cells in an MHC-unrestricted manner. In the Jurkat T cell model system, expression of a ...carcinoembryonic Ag-specific CD3ζ CAR (MFEζ) resulted in an increased sensitivity of the transduced Jurkat cell to generate cytokines when stimulated through the endogenous TCR complex. This effect was driven through two key characteristics of the MFEζ CAR: 1) receptor dimerization and 2) the interaction of the CAR with the endogenous TCR complex. Mutations of the CAR transmembrane domain that abrogated these interactions resulted in a reduced functional capacity of the MFEζ CAR to respond to carcinoembryonic Ag protein Ag. Taken together, these results indicate that CARs containing the CD3ζ transmembrane domain can form a complex with the endogenous TCR that may be beneficial for optimal T cell activation. This observation has potential implications for the future design of CARs for cancer therapy.
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