Constitutive activation of WASp leads to abnormal cytotoxic cells with increased granzyme B and degranulation response to target cells

ABSTRACT Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.

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Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils

Blood ◽

10.1182/blood-2003-04-1069 ◽

2004 ◽

Vol 103 (3) ◽

pp. 1099-1104 ◽

Cited By ~ 71

Author(s):

Christof Wagner ◽

Christof Iking-Konert ◽

Birgit Denefleh ◽

Sabine Stegmaier ◽

Friederike Hug ◽

...

Keyword(s):

Granzyme B ◽

Constitutive Expression ◽

Jurkat Cells ◽

Polymorphonuclear Neutrophils ◽

Target Cells ◽

Dependent Manner ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Cytotoxic Molecules ◽

Chicken Erythrocytes

AbstractPolymorphonuclear neutrophils (PMNs) produce an abundance of bactericidal and cytotoxic molecules consistent with their role as first-line defense against bacterial infection. PMNs, however, also cause efficient cellular cytotoxicity when targeted through Fc receptors to appropriate antibody-coated target cells. Although this so-called antibody-dependent cellular cytotoxicity (ADCC) was described many years ago, the mechanism of killing is still elusive. We now have found that PMNs contain perforin and granzyme B, the 2 molecules known as the cytotoxic entity of natural killer cells and of cytotoxic T lymphocytes as well. Lysates of PMNs were lytic for chicken erythrocytes in a time-, temperature-, and Ca2+-dependent manner. Moreover, apoptosis of Jurkat cells was induced, consistent with the observation that the PMN lysates contain enzymatically active granzyme B. Taken together, our data provide evidence for the presence of perforin and granzyme B within the cytotoxic arsenal of PMNs. (Blood. 2004;103:1099-1104)

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Granzyme B Is a Biomarker for Suspicion of Malignant Seromas Around Breast Implants

Aesthetic Surgery Journal ◽

10.1093/asj/sjaa302 ◽

2020 ◽

Author(s):

Marshall E Kadin ◽

John Morgan ◽

Haiying Xu ◽

Caroline Glicksman ◽

David Sieber ◽

...

Keyword(s):

Early Detection ◽

Tumor Cells ◽

Breast Implant ◽

Granzyme B ◽

Breast Implants ◽

Large Cell ◽

Target Cells ◽

Cytotoxic Lymphocytes ◽

Tumor Cell Death ◽

Level Of Evidence

Abstract Background Granzyme B (GrB) is a serine protease secreted, along with pore-forming perforin, by cytotoxic lymphocytes to mediate apoptosis in target cells. GrB has been detected in tumor cells associated with systemic and breast implant–associated anaplastic large cell lymphoma (BIA-ALCL) but its potential use for detection of early BIA-ALCL has not been fully investigated. Objectives Prompted by the increased incidence of BIA-ALCL, the aim of this study was to assess GrB as a new biomarker to detect early disease in malignant seromas and to better understand the nature of the neoplastic cell. Methods A Human XL Cytokine Discovery Magnetic Luminex 45-plex Fixed Panel Performance Assay was used to compare cytokine levels in cell culture supernatants of BIA-ALCL and other T-cell lymphomas, as well as malignant and benign seromas surrounding breast implants. Immunohistochemistry was employed to localize GrB to cells in seromas and capsular infiltrates. Results Differences in GrB concentrations between malignant and benign seromas were significant (P < 0.001). GrB was found in and around apoptotic tumor cells, suggesting that the protease may be involved in tumor cell death. Conclusions GrB is a useful marker for early detection of malignant seromas and to identify tumor cells in seromas and capsular infiltrates. Because there is an overlap between the lowest concentrations of soluble GrB in malignant seromas and the highest concentrations of GrB in benign seromas, it is recommended that GrB be used only as part of a panel of biomarkers for the screening and early detection of BIA-ALCL. Level of Evidence: 5

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The differential contribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated apoptosis is determined by the quality of target cells

European Journal of Immunology ◽

10.1002/1521-4141(200207)32:7<1980::aid-immu1980>3.0.co;2-z ◽

2002 ◽

Vol 32 (7) ◽

pp. 1980 ◽

Cited By ~ 40

Author(s):

Julián Pardo ◽

Sandra Balkow ◽

Alberto Anel ◽

Markus M. Simon

Keyword(s):

T Lymphocyte ◽

Cytotoxic T Lymphocyte ◽

Granzyme B ◽

Target Cells ◽

Granzyme A ◽

Differential Contribution

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Granzyme B Short-Circuits the Need for Caspase 8 Activity during Granule-Mediated Cytotoxic T-Lymphocyte Killing by Directly Cleaving Bid

Molecular and Cellular Biology ◽

10.1128/mcb.20.11.3781-3794.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 3781-3794 ◽

Cited By ~ 220

Author(s):

Michele Barry ◽

Jeffrey A. Heibein ◽

Michael J. Pinkoski ◽

Siow-Fong Lee ◽

Richard W. Moyer ◽

...

Keyword(s):

Dna Fragmentation ◽

Caspase 3 ◽

Cytotoxic T Lymphocyte ◽

Granzyme B ◽

Serine Proteinase ◽

Target Cells ◽

Caspase 8 ◽

Intact Cells ◽

Whole Cells

ABSTRACT Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.

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Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing

International Journal of Molecular Sciences ◽

10.3390/ijms20184509 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4509 ◽

Cited By ~ 12

Author(s):

Francisca Hofmann ◽

Mariela Navarrete ◽

Javiera Álvarez ◽

Israel Guerrero ◽

María Alejandra Gleisner ◽

...

Keyword(s):

Tumor Cell ◽

Gap Junctions ◽

Immunological Synapse ◽

Granzyme B ◽

Melanoma Cells ◽

Cell Killing ◽

Target Cells ◽

Cx43 Expression ◽

B16f10 Cells ◽

Immunological Synapses

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell–tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing.

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Sinonasal T-Cell Expression of Cytotoxic Mediators Granzyme B and Perforin is Reduced in Patients with Chronic Rhinosinusitis

American Journal of Rhinology and Allergy ◽

10.2500/ajra.2017.31.4474 ◽

2017 ◽

Vol 31 (6) ◽

pp. 352-356 ◽

Cited By ~ 1

Author(s):

Sarah E. Smith ◽

Rodney J. Schlosser ◽

James R. Yawn ◽

Jose L. Mattos ◽

Zachary M. Soler ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Chronic Rhinosinusitis ◽

Nasal Polyp ◽

Granzyme B ◽

Cytotoxic Cells ◽

Sinonasal Mucosa ◽

Perforin Expression

Background CD8+ T cells and natural killer (NK) cells are cytotoxic cells that use granzyme B (GrB) and perforin. Defective cytotoxic function is known to play a role in dysregulated immune response as seen in chronic sinusitis, also referred to as chronic rhinosinusitis (CRS). However, to our knowledge, in the United States, neither GrB or perforin expression has been reported in patients with CRS. Objective The aim of this study was to investigate sinonasal cytotoxic cells, their mediators, and cell-specific distribution of these mediators in patients with CRS with nasal polyp (CRSwNP) and in patients with CRS without nasal polyp (CRSsNP). Methods Blood and sinus tissue samples were taken from patients with CRSsNP (n = 8) and CRSwNP (n = 8) at the time of surgery. Control subjects (n = 8) underwent surgery for cerebrospinal fluid leak repair or to remove non-hormone-secreting pituitary tumors. The cells were analyzed via flow cytometry by using CD8 expression to identify cytotoxic T cells and CD56 expression to identify NK cells. Intracellular GrB and perforin expression were analyzed with flow cytometry. Results We observed no significant differences in plasma or peripheral blood immune cell numbers or specific levels of GrB or perforin among the groups. In the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP, there was a significant decrease in GrB and perforin levels (p <0.05) despite similar or increased numbers of cytotoxic cells when compared with the controls. The overall decrease in GrB and perforin in the sinonasal mucosa of the patients with CRSsNP and the patients with CRSwNP was due to decreased T cell production. There was no difference in total NK cell count or expression of perforin or GrB among all the groups. Conclusion Total levels of sinonasal GrB and perforin were decreased in the sinonasal mucosa of both the patients with CRSwNP and the patients with CRSsNP compared with the controls, whereas sinonasal CD8+ T cells, (but not NK cells,), intracellular stores of GrB and perforin were reduced in the patients with CRSwNP compared with the controls.

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Granzyme B-induced Cell Death Involves Induction of p53 Tumor Suppressor Gene and Its Activation in Tumor Target Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m705290200 ◽

2007 ◽

Vol 282 (45) ◽

pp. 32991-32999 ◽

Cited By ~ 20

Author(s):

Franck Meslin ◽

Jerome Thiery ◽

Catherine Richon ◽

Abdelali Jalil ◽

Salem Chouaib

Keyword(s):

Cell Death ◽

Tumor Suppressor ◽

Tumor Suppressor Gene ◽

Granzyme B ◽

Suppressor Gene ◽

Target Cells ◽

Tumor Target ◽

P53 Tumor Suppressor ◽

P53 Tumor Suppressor Gene

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IPH2101, a Novel Anti-Inhibitory KIR Monoclonal Antibody, and Lenalidomide Combine to Enhance the Natural Killer (NK) Cell Versus Multiple Myeloma (MM) Effect.

Blood ◽

10.1182/blood.v114.22.3870.3870 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3870-3870 ◽

Cited By ~ 1

Author(s):

Don Benson ◽

Courtney E Bakan ◽

Shuhong Zhang ◽

Lana Alghothani ◽

Jing Liang ◽

...

Keyword(s):

Clinical Trial ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Phase 1 ◽

Granzyme B ◽

Cell Activity ◽

Target Cells ◽

Nk Cell Activity ◽

Immune Complex Formation

Abstract Abstract 3870 Poster Board III-806 Background NK cell activity against tumor cells is regulated by a balance of inhibitory and activating signals mediated by receptors on NK cells that recognize inhibitory and activating ligands expressed by cancer cells. IPH2101 (1-7F9) is a novel monoclonal anti-inhibitor KIR blocking antibody that has been shown to augment NK cell function against MM targets. Moreover, lenalidomide has been shown to expand and activate NK cells in vivo and in vitro. We have previously reported that the combination of IPH2101 and lenalidomide enhances NK cell mediated cytotoxicity against MM cells compared to each agent alone (Zhang et al., AACR 2009). We expand our studies to investigate potential mechanisms for the enhancement of NK cell activity by the combination of IPH2101 and lenalidomide. Methods The effects of IPH2101 and lenalidomide alone and in combination were studied using primary human NK cells from healthy donors as well as from MM patients. The MM cell lines U266 and RPMI 8226 as well as primary tumor cells from marrow aspirates of MM patients served as target cells. The effect of lenalidomide on MM activating and inhibitory ligand expression was studied by flow cytometry. NK cell trafficking was investigated with standard transwell plate migration assay. Immune complex formation between NK cell effectors and MM tumor targets was characterized by flow cytometry in control conditions and with NK cells pre-treated with IPH2101 and lenalidomide. The effects of IPH2101 and lenalidomide were studied regarding interferon-gamma and granzyme B production by ELISPOT and target-specific cytotoxicity studies were conducted to complement effector-based assays. Results IPH2101 (30 ug/ml) significantly enhanced cytotoxicity against U266 cells and primary MM tumor cells by both purified NK cells at effector:target (E:T) ratios of 10:1 or less, and also of freshly isolated peripheral blood mononuclear cells (PBMC) at E:T ratios of 60:1 or less, from more than 10 random donors. In addition, treatment of PBMC with 5-10 μmol/L lenalidomide for 72h without interleukin (IL)-2 increased NK cell lysis of U266. Treatment of PBMC from normal donors did not enhance the expression of the NK receptors KIR, NKG2D, NCR, TRAIL, and DNAM-1. Incubation of U266 cells with lenalidomide (5 uM) for 3-5 days resulted in significant enhancement of cytotoxicity by normal donor NK cells. This was associated with upregulation of the activating ligands, MICA, ULBP-2, DR4, and CD112. Using blocking antibodies to NKG2D, TRAIL, and DNAM-1, lenalidomide enhancement of MM cell killing was abrogated indicating the importance of the modulation of the ligands to the latter receptors by lenalidomide. Although IPH2101 and lenalidomide did not significantly increase NK cell migration into normal media, migration was enhanced 2.98-fold (+/− 0.36, p < 0.05) towards U266 cell targets (n= 3, p < 0.05) and MM patient serum 3.2-fold (+/− 0.4, n=3, p < 0.05). IPH2101 and lenalidomide also led to a 2.3-fold (+/− 0.43, p < 0.05) increase in immune complex formation between NK cells and MM tumor cells. IPH2101 and lenalidomide also augmented NK cell interferon gamma production against MM (control mean 303 spots/well +/− 13 versus 525 +/− 83, n=3, p < 0.05) and granzyme B production (control mean 115 +/− 98 versus 449 +/−72, n=3, p < 0.05). Importantly, in all experiments described herein, the effects of IPH2101 and lenalidomide together were greater than either agent alone. Conclusions Taken together, our data suggest that IPH2101 and lenalidomide may exert complementary mechanisms on both effector and target cells to enhance NK cell mediated killing of MM cells. Moreover, these agents have no predicted clinical cross-toxicities. A single-agent phase 1 clinical trial of IPH2101 has shown the mAb to be safe and well tolerated in MM patients. These findings support a phase 1/2 clinical trial of IPH2101 with lenalidomide as a first dual-innate immunotherapy for patients with MM. Disclosures: Andre: Innate Pharma: Employment. Squiban:Innate pharma: Employment. Romagne:Innate Pharma: Employment.

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Immunopharmacodynamic response to blinatumomab in patients with relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL).

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.7020 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 7020-7020 ◽

Cited By ~ 6

Author(s):

Andrea Schub ◽

Virginie Nägele ◽

Gerhard Zugmaier ◽

Christian Brandl ◽

Youssef Hijazi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Lymphoblastic Leukemia ◽

Granzyme B ◽

Significant Proportion ◽

Target Cells ◽

Cell Counts

7020 Background: Blinatumomab is an anti-CD19/anti-CD3 bispecific T cell engager (BiTE) that induces target cell-dependent, polyclonal T cell activation and proliferation, resulting in redirected lysis of CD19+ target cells. Methods: In a phase 2 study, adult patients (N=36) with relapsed/refractory B-precursor ALL received continuous blinatumomab IV infusion for 28 days in ≤5 treatment/consolidation cycles. Whole blood and serum samples were collected throughout treatment and analyzed for lymphocyte subpopulations, cytokines, granzyme B, and blinatumomab serum concentrations. Results: Lymphocytes in all patients responded in a similar fashion. After infusion start, peripheral B cell counts dropped to ≤1 B cell/μL in <1 week and remained undetectable throughout treatment. Peripheral T cells showed a redistribution characterized by swift disappearance within the first 2-6 hrs and subsequent recovery to baseline within several days. Otherwise, T cell counts remained at least stable in most patients. In some patients even an expansion of the T cell compartments was observed, most likely due to specific proliferation of activated T cells but could not be defined as prerequisite for treatment efficacy. During the first infusion days, a significant proportion of T cells newly expressed the activation marker CD69, and the T cell effector molecule granzyme B was detectable in serum. Additionally, a transient cytokine release dominated by IL-10, IL-6 and IFN-γ was observed in most patients shortly after first infusion start, which was alleviated or absent in subsequent cycles. Blinatumomab serum steady state concentrations (mean±SD) were 198±61 pg/mL and 694±236 pg/mL at doses of 5 and 15 μg/m²/d, respectively, which is comparable to those from previous studies. Conclusions: Immunopharmacodynamic response to blinatumomab was characterized by B cell depletion, T cell activation and redistribution, and release of granzyme B and cytokines, suggesting T cell engagement according to the expected BiTE mode of action. The tested pharmacodynamic markers did not allow for predictive differentiation between patients achieving a hematologic response and those who did not. Clinical trial information: NCT01209286.

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