Live cell evaluation of granzyme delivery and death receptor signaling in tumor cells targeted by human natural killer cells

Vrazo, Alexandra C.; Hontz, Adrianne E.; Figueira, Sarah K.; Butler, Braeden L.; Ferrell, Julie; Binkowski, Brock F.; Li, Jinzhu; Risma, Kimberly A.

doi:10.1182/blood-2015-03-632273

Cited by 26 publications

(20 citation statements)

References 60 publications

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“…Activation of the sensors occurred within 2 hours, which is compatible with the proposed kinetics of Gr delivery. 1,8 Similar results were obtained in HeLa cervix carcinoma cells (supplemental Figure 1, available on the Blood Web site). Primary NK and LAK cells also activated the GrM sensors ( Figure 1G-H, respectively).…”

supporting

confidence: 79%

“…GrM is expressed by lymphocytes of both the innate and adaptive immune system and plays a role in the control of cancer, viruses, and inflammation. 2 Here, we report the development and comparison of 2 distinct variants of GrM gain-of-function biosensors: the GrM GloSensor, which is similar to the sensors described by Vrazo et al, 1 and the GrM pro-interleukin (IL)-1b-gaussia luciferase (iGLuc) sensor 3 ( Figure 1A). …”

mentioning

confidence: 89%

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Bioluminescent reporters to monitor killer cell–mediated delivery of granzymes inside target cells

Poot

Erp

Meeldijk

et al. 2015

Blood

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supporting

confidence: 79%

mentioning

confidence: 89%

Bioluminescent reporters to monitor killer cell–mediated delivery of granzymes inside target cells

Poot

Erp

Meeldijk

et al. 2015

Blood

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“…31 Our data suggest that moderate, but subnormal levels of perforin in CD8 + T and NK cells are sufficient to provide the feedback to decrease the exaggerated inflammation and immune response; however, complete correction and recovery from HLH, including viral clearance, requires a much higher level of perforin expression (i.e., near normal levels), and was achievable by the MNDU3 promoter/enhancer. It will be of interest to study the expression of this promoter in primary CD8…”

Section: Cd8mentioning

confidence: 72%

“…22,31 The perforin cDNA in LV vectors lacks the 3¢ UTR and is hence unaffected by the shRNA. KHYG1-KD cells were transduced with all three LV vectors at a low MOI and sorted for tNGFR-expressing cells to enrich for The relative perforin mRNA expression in MND4T KHYG1-KD cells was about 2-fold higher than that in PGK4T and PRF0T cells and nearly comparable to wild-type perforin expression in KHYG1 cells (Fig.…”

Section: Regulated Perforin LV Vectors and Their Relative Expression mentioning

confidence: 99%

High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis

et al. 2016

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K.R., M.J., and P.M. share senior authorship.Perforin-1 mutations result in a potentially fatal hemophagocytic lymphohistiocytosis (HLH) with heightened immune activation, hypercytokinemia, pancytopenia, and end-organ damage. At present, hematopoietic stem cell (HSC) transplantation is curative, but limited by donor availability and associated mortality, making gene therapy an attractive alternative approach for HLH. We reported that perforin expression driven by cellular promoters in lentiviral (LV) vectors resulted in significant, albeit partial, correction of the inflammatory features in a murine model of HLH. We hypothesized that the level of perforin expression achieved per cell from ectopic moderate-strength cellular promoters (phosphoglycerate kinase gene/perforin-1 gene) is inadequate and thus engineered an LV vector using a viral promoter (MND; a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer) containing microRNA126 target sequences to restrict perforin expression in HSCs. We show here that the MND-LV vector restored perforin expression to normal levels in a perforin-deficient human natural killer cell line and perforin gene-corrected Perforin1-/-transplant recipients, whereas cellular promoters drove only partial correction. On lymphocytic choriomeningitis virus challenge, the clinical scores and survival improved only with the MND-LV vector, but inflammatory markers and cytotoxicity were improved with all LV vectors. Our studies suggest that although moderate levels of expression can result in partial amelioration of the HLH phenotype, high levels of perforin expression per cell are required for complete correction of HLH.

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“…EGTA, a Ca 2+ -specific chelator that inhibits the release of cytolytic granules (25), was used in the presence of Mg 2+ supplementation. EGTA strongly decreased MAIT cell degranulation as determined by activation was blocked using nafamostat mesylate (NAM) and Ac-IETD-CHO, respectively [26,27]. NAM had no effect on MAIT cell degranulation, caspase 3 activation, and bacterial counts ( Fig.…”

Section: Mait Cells Mediate Antimicrobial Activity Through the Cytolymentioning

confidence: 93%

Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance inEscherichia coli

Boulouis

Sia

Gulam

et al. 2020

Preprint

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One Sentence Summary: Potent antimicrobial activity of human MAIT cells overcomes carbapenem-resistance in control of Escherichia coli Category of manuscript: Research Article. Abstract word count: 220 References: 69 Display items: 6 figures, 7 supplementary figures, 3 supplementary tables. Word count incl. main text, references, and figure legends: 10041. 3 Abstract Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans, and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1restricted antimicrobial activity against E. coli clinical strains in a manner dependent on the activity of cytolytic proteins, but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to TCR-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living E. coli. Furthermore, MAIT cell-mediated bacterial control extend to multidrugresistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity, and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli. 4 [Main Text: ] Introduction Mucosa-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in mucosal tissues, the liver, lungs and gastrotintestinal tract, and in peripheral blood [1]. MAIT cells are mostly CD8α + [2, 3], express a semi-invariant T cell receptor (TCR), and recognize antigens in complex with the MHC-Ib-related protein (MR1) [4]. MR1 displays an extraordinary level of evolutionary conservation among placental and marsupial mammals [5], strongly supporting the notion that MR1 and MAIT cells perform critical functions in the immune system. The MR1presented antigens recognized by MAIT cells are derivatives of intermediates in the microbial synthesis of vitamin B 2 (riboflavin) and are produced by many bacteria [6-8]. Riboflavin is a critical component in a wide variety of bacterial cellular processes [9]. MAIT cells are thus able to recognize and respond to a broad set of bacteria [10]. Following TCR-mediated recognition of MR1-presented bacterial riboflavin metabolite antigens, MAIT cells rapidly media...

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Live cell evaluation of granzyme delivery and death receptor signaling in tumor cells targeted by human natural killer cells

Abstract: Key Points Natural killer cell granzyme B, A, and K delivery and subsequent caspase activation is rapid after conjugation with tumor target cells. Natural killer cells also induce caspase activation through death receptor ligation that can be monitored in real time.

Cited by 26 publications

References 60 publications

Bioluminescent reporters to monitor killer cell–mediated delivery of granzymes inside target cells

Bioluminescent reporters to monitor killer cell–mediated delivery of granzymes inside target cells

High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis

Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance inEscherichia coli

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