The Critical Role of the NKG2D Receptor in CD8+CTL and CD8+CD56+ NKT Cell Cytotoxicity.

Abstract We previously demonstrated the ability to grow and ex vivo expand mobilized peripheral blood mononuclear cells (PBMNC) from myeloma patients into aggressive cytotoxic effector cells (Blood102; 422b, 2003). These experiments were designed to test the function and mechanism of tumor cell killing of these cells. Peripheral blood stem cells (PBSC) were collected from myeloma pts after mobilizing with cyclophosphamide and rhG-CSF and cultured in Aim-V serum-free medium at 37 and 5% CO2. After 2 hrs, the non-adherent cells were removed and placed in culture with Aim-V, IL-2 (50 IU/ml) and OKT-3 (50 ng/ml) for 7 days. Cytotoxicity of the expanded cells was tested on Day 0 and Day 7 using a chromium release assay. To identify the cytotoxic potential of cell subsets, cell populations were depleted using the Auto MACS Magnetic Cell Sorter (Miltenyi Biotec Auburn, CA) and cytotoxicity assays were repeated. Since the CD8+ cell(s) were the most cytotoxic, the CD8+ cells were isolated and their mechanisms of tumor cell killing were evaluated by testing killing through MHC Class I, T cell receptor or the NKG2D receptor. The ex vivo expanded population was extremely cytotoxic and killed RPMI 8226 myeloma cells at 60% lysis (+/− 1.6%) (E:T of 100:1) when compared to 3.9 % on day 0 (+/− 0.8%). CD8+ or CD8+CD56+ cell subsets contributed to > 83.3 % (+/− 1.5%) of the killing. Blocking the TCR pathway (Redirected Cytotoxicity Assay) had no effect on killing and blocking the MHC Class I molecules decreased cytotoxicity by only 6%. When the NKG2D receptor was blocked, cytotoxicity by the CD8+ cells decreased by 48% (+/− 2%), demonstrating the critical role of the NKG2D in these CD8+ cell populations. The expanded cytotoxic effector cells aggressively lyse myeloma tumor cells in a MHC and non-MHC restricted fashion. These ex vivo expanded CD8+ cells likely acquire the NKG2D receptor and kill tumor cells in a non-MHC restricted manner. Since MHC expression is often low or absent on myeloma cells and the NKG2D ligands are fairly specific to tumor cells, the infusion of these ex vivo expanded cells following transplant may improve clinical outcomes.

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NLRP7 Promotes Choriocarcinoma Growth and Progression through the Establishment of an Immunosuppressive Microenvironment

Cancers ◽

10.3390/cancers13122999 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2999

Author(s):

Deborah Reynaud ◽

Roland Abi Nahed ◽

Nicolas Lemaitre ◽

Pierre-Adrien Bolze ◽

Wael Traboulsi ◽

...

Keyword(s):

Immune Response ◽

Tumor Cells ◽

Major Gene ◽

Critical Role ◽

Orthotopic Model ◽

Independent Manner ◽

Maternal Immune Response ◽

The Impact

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC; (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization; and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

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The Enigma of Eosinophil Degranulation

International Journal of Molecular Sciences ◽

10.3390/ijms22137091 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7091

Author(s):

Timothée Fettrelet ◽

Lea Gigon ◽

Alexander Karaulov ◽

Shida Yousefi ◽

Hans-Uwe Simon

Keyword(s):

Blood Cells ◽

Inflammatory Diseases ◽

White Blood Cells ◽

Exact Role ◽

Effector Cells ◽

Effector Functions ◽

Cytotoxic Effector ◽

Eosinophil Degranulation

Eosinophils are specialized white blood cells, which are involved in the pathology of diverse allergic and nonallergic inflammatory diseases. Eosinophils are traditionally known as cytotoxic effector cells but have been suggested to additionally play a role in immunomodulation and maintenance of homeostasis. The exact role of these granule-containing leukocytes in health and diseases is still a matter of debate. Degranulation is one of the key effector functions of eosinophils in response to diverse stimuli. The different degranulation patterns occurring in eosinophils (piecemeal degranulation, exocytosis and cytolysis) have been extensively studied in the last few years. However, the exact mechanism of the diverse degranulation types remains unknown and is still under investigation. In this review, we focus on recent findings and highlight the diversity of stimulation and methods used to evaluate eosinophil degranulation.

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Emerging Role of Phospholipase-Derived Cleavage Products in Regulating Eosinophil Activity: Focus on Lysophospholipids, Polyunsaturated Fatty Acids and Eicosanoids

International Journal of Molecular Sciences ◽

10.3390/ijms22094356 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4356

Author(s):

Eva Knuplez ◽

Eva Maria Sturm ◽

Gunther Marsche

Keyword(s):

Fatty Acids ◽

Polyunsaturated Fatty Acids ◽

Critical Role ◽

Allergic Inflammation ◽

Lipid Mediators ◽

Comprehensive Overview ◽

Local Immunity ◽

Effector Cells ◽

Inflammatory Phenotype

Eosinophils are important effector cells involved in allergic inflammation. When stimulated, eosinophils release a variety of mediators initiating, propagating, and maintaining local inflammation. Both, the activity and concentration of secreted and cytosolic phospholipases (PLAs) are increased in allergic inflammation, promoting the cleavage of phospholipids and thus the production of reactive lipid mediators. Eosinophils express high levels of secreted phospholipase A2 compared to other leukocytes, indicating their direct involvement in the production of lipid mediators during allergic inflammation. On the other side, eosinophils have also been recognized as crucial mediators with regulatory and homeostatic roles in local immunity and repair. Thus, targeting the complex network of lipid mediators offer a unique opportunity to target the over-activation and ‘pro-inflammatory’ phenotype of eosinophils without compromising the survival and functions of tissue-resident and homeostatic eosinophils. Here we provide a comprehensive overview of the critical role of phospholipase-derived lipid mediators in modulating eosinophil activity in health and disease. We focus on lysophospholipids, polyunsaturated fatty acids, and eicosanoids with exciting new perspectives for future drug development.

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Cytotoxic effector cells with antitumor activity can be amplified ex vivo from biopsies or blood of patients with renal cell carcinoma for cell therapy use

Cancer Immunology Immunotherapy ◽

10.1007/s00262-003-0412-9 ◽

2003 ◽

Vol 52 (11) ◽

pp. 699-707 ◽

Cited By ~ 5

Author(s):

Françoise Bouet-Toussaint ◽

Jean-Jacques Patard ◽

Alban Gervais ◽

Noëlle Genetet ◽

Cécile Thomas de la Pintière ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Antitumor Activity ◽

Cell Therapy ◽

Cell Carcinoma ◽

Renal Cell ◽

Ex Vivo ◽

Effector Cells ◽

Cytotoxic Effector

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P01.01 The role of exosome miRNA between tumor cells and microglias during the progression of medulloblastoma

Neuro-Oncology ◽

10.1093/neuonc/noz126.079 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii24-iii24

Author(s):

Q Chang ◽

L Zhu ◽

N Li

Keyword(s):

Tumor Cells ◽

Critical Role ◽

Expression Level ◽

Migration And Invasion ◽

Specific Marker ◽

Migration Ability ◽

Daoy Cell ◽

Bv2 Cells ◽

Close Relationship

Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant paediatric brain tumor. Recent studies show that M2 cells were relative more abundant in Shh subtype of MBs compared with other three subtypes. It’s known that M2 cells have close relationship with many tumors’ progression. But if they play any role in the progression of Shh subtype of MB is not yet clear. Many studies demonstrate that exosomes carring miRNAs have close relationship with tumor invasion. The aim of present study is to clarify the role of exosome miRNA between tumor cells and microglias during the progression of Shh subtype of medulloblastoma. MATERIAL AND METHODS Immunofluerescence staining using iNOS and Arg1, which is M1 and M2 specific marker, respectively, was performed in four subtypes of MBs. After coculture of exosomes extracted from Shh subtype of MB cell (DAOY) with microglia cell (BV2), Q-PCR and ELISA assay were done to evaluate the polarization status of the microglia. Transwell and scratch assay were then performed to detect the migration ability of DAOY cell after treatment of exosomes from polirized M2 cells. MiRNA sequencing by Ion Proton technology was then done to analyze the miRNAs expression level between Shh subtype and other subtype of MBs. Transformation assay was used to overexpress and inhibit the expression of these miRNAs respectively to further clarify the role of exosome miRNA in the polarization of BV2 cells. RESULTS M2 cells were observed more abundant than other three subtypes of tumors, supporting that M2 cells play some role in this subtype of MBs. Exosomes of DAOY cells can induce the polarization of M2 cells. The polarized M2 cells can improved the migration and invasion ability of DAOY cell. Dozens of miRNAs were identified with different expression level between Shh subtype of MBs and other subtype of MB cells. Among them, 4 miRNAs were reported to be related with polariztion of M2 in many other lesions. Three of the 4 miRNAs can induce the polarization of M2 in present study. CONCLUSION Our study demonstrated exosome miRNA play a critical role between tumor cells and microglias during the progression of Shh subtype of medulloblastoma.

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Functional and phenotypic characterization of human peripheral blood stem cell harvests: a comparative analysis of cells from consecutive collections

Blood ◽

10.1182/blood.v85.7.1964.bloodjournal8571964 ◽

1995 ◽

Vol 85 (7) ◽

pp. 1964-1970 ◽

Cited By ~ 35

Author(s):

DJ Verbik ◽

JD Jackson ◽

SJ Pirruccello ◽

KD Patil ◽

A Kessinger ◽

...

Keyword(s):

Stem Cell ◽

Cancer Patients ◽

Peripheral Blood Stem Cell ◽

Ex Vivo ◽

High Dose ◽

Hematopoietic Stem ◽

Effector Cells ◽

Antitumor Effector ◽

Cytotoxic Effector

A considerable number of patients with malignancies who are treated with high-dose therapy and hematopoietic stem cell transplantation subsequently relapse. Analyses of peripheral blood stem cell (PBSC) harvests obtained from 49 cancer patients showed that the PBSC harvest contained precursors for antitumor effector cells. Ex vivo manipulation of these harvests to maximize the antitumor effector cell activity may provide a new therapeutic approach to decrease or eliminate any minimal residual disease that remains after high-dose therapy. Characterization of PBSC from consecutive collections determined the collections best suited for ex vivo augmentation of antitumor cytotoxic effector cells. We report the results of a functional and phenotypical characterization of PBSC obtained from six consecutive collections from 18 cancer patients receiving granulocyte-macrophage colony-stimulating factor (GM-CSF) for hematopoietic stem/progenitor cell mobilization. The PBSC were evaluated for their cytotoxicity using the 51Cr-release assay. The frequency and subsets of lymphocytes were determined using flow cytometry with appropriate specific marker antibodies and differential cell counts. The content of hematopoietic progenitor cells in each collection was determined using a colony-forming unit granulocyte-macrophage (CFU-GM) culture assay. The frequency of cytotoxic effector cells including lymphokine-activated killer (LAK) cell precursors and lymphocytes was significantly greater (P < .05) in the early collections, whereas the later collections contained significantly (P < .05) more CFU-GM progenitor cells and fewer cytotoxic effector cells. Thus, our results show that PBSC obtained from advanced cancer patients do contain considerable levels of precursor cells for the generation of LAK cell populations. These results suggest that cells from the earlier collections are best suited for ex vivo manipulation to augment the antitumor effects.

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Critical role of PD-L1 expression on non-tumor cells rather than on tumor cells for effective anti-PD-L1 immunotherapy in a transplantable mouse hematopoietic tumor model

Cancer Immunology Immunotherapy ◽

10.1007/s00262-020-02520-z ◽

2020 ◽

Vol 69 (6) ◽

pp. 1001-1014 ◽

Cited By ~ 1

Author(s):

Jose-Ignacio Rodriguez-Barbosa ◽

Miyuki Azuma ◽

Gennadiy Zelinskyy ◽

Jose-Antonio Perez-Simon ◽

Maria-Luisa del Rio

Keyword(s):

Tumor Cells ◽

Critical Role ◽

Tumor Model ◽

Transplantable Mouse

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Lymphocyte-mediated cytotoxicity against tumor cells. II. Characteristics and tissue distribution of concanavalin A- activated cytotoxic effector cells

European Journal of Immunology ◽

10.1002/eji.1830060502 ◽

1976 ◽

Vol 6 (5) ◽

pp. 309-316 ◽

Cited By ~ 5

Author(s):

J. D. Waterfield ◽

Elizabeth M. Waterfield

Keyword(s):

Tissue Distribution ◽

Tumor Cells ◽

Concanavalin A ◽

Effector Cells ◽

Cytotoxic Effector

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The Role of ERK5 Signaling in Tolerance Induction by Veto CTLs.

Blood ◽

10.1182/blood.v106.11.3302.3302 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3302-3302

Author(s):

Shlomit Reich-Zeliger ◽

Tamar Hanoch ◽

Rony Seger ◽

Yair Reisner

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cellular Systems ◽

Class I ◽

Spleen Cells ◽

Effector Cells ◽

Cd8 Cells ◽

Veto Cells ◽

Specific Inhibitors

Abstract Several bone marrow cells and lymphocyte subpopulations, known as ‘veto cells’, were shown to induce transplantation tolerance across major histocompatibility antigens. Recently, it has been suggested that anti-3rd party CTLs depleted of alloreactivity against the host are endowed with marked veto activity and can facilitate bone marrow allografting. The veto mechanism is still obscure. While early studies emphasized the role of CD8 mediated apoptosis, we showed that the veto activity of anti-3rd party CD8+ CTLs is dependent upon the simultaneous expression of both CD8 and FasL. Thus, it seems that although Fas is upregulated on the effector T cells upon engagement of their TCR by class I of the veto cells, the presence of FasL on the veto cells cannot result in apoptosis of the effector cells unless CD8 on the veto cells is available and can interact with class 1 on the effector cells. Thus, the interaction of CD8 on the veto cells with class I on the effector cells seems to be associated with an increased susceptability of the effector cells to FasL killing. To further delineate the mechanism of the veto effect we have now studied the role of different signaling pathways using specific inhibitors. Spleen CD8 T cells from 2C mice (H2b) bearing TCR transgene directed against (H2d) were used as effector cells and anti FVB (H2q, third party) CTLs generated from Balb/c spleen cells (H2d) were used as veto cells. The addition of the latter cells to MLR of 2C against Balb/c (H2d) simulators, leads to deletion of the 2C effector CD8 cells within 72 hrs. Deletion monitored by FACS analysis of cells stained with anti-TCR transgene antibody (1B2+) revealed reduction from 46%±11% 1B2+CD8+ cells to 6%±3% 1B2+CD8+ cells in 6 different experiments. In contrast, veto CTLs generated from SJL (H2s) spleen cells that do not display the H2 recognized by the 2C effector cells, did not result in a significant deletion of the effector cells (42%±12% 1B2+CD8+ cells in 6 different experiments). The specific deletion exhibited by veto CTLs of Balb/c origin, can be inhibited by MEK1/2/5 inhibitors such as U0126, reducing the veto activity from 85.5%±7% to 16%±14% in 6 different experiments. The effective concentration of U0126 was relatively high (10μM), and lower concentration of this drug (1μM) had no response, indicating a potential involvement of the MEK5/ERK5 cascade rather than the MEK1/2-ERK1/2 cascade, in the veto effect. In addition, no inhibition of veto activity could be found with specific inhibitors of other signaling molecules such as JNK, P38, PI3K or PKC. Considering that Fas expression on the effector cells is critical for the veto activity, it is interesting that the ERK inhibitor didn’t affect the level of Fas on the effectors (93%±3% of 1B2+CD8+ upregulate FAS in the presence of U0126 in 7 different experiments). Also, this inhibition is not likely mediated by affecting the veto CTLs, as pretreatment of the latter cells with ERK inhibitor didn’t diminish the veto effect. The pro-apoptotic effects of MEK5-ERK5 cascade in this system is intriguing because these kinases are usually thought to promote proliferation and survival in most cellular systems. Therefore, the veto cells exhibit a unique signaling system, which utilizes ERK5 cascade to induce apoptosis. Further studies are directed at the potential links between the ERK5 cascade, the Fas system and the rest of the apoptotic machinery.

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