scholarly journals Photochemical Treatment of Drosophila APCs Can Eliminate Associated Viruses and Maintain the APC Function for Generating Antigen-Specific CTLs Ex Vivo

2018 ◽  
Vol 2018 ◽  
pp. 1-13
Author(s):  
Jun Ye ◽  
Chunxia Yang ◽  
Zeling Cai ◽  
Weixing Shi ◽  
Hong Yu
Keyword(s):  
Mhc Class I ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Cytotoxic T Cells ◽  
Class I ◽  
Specific Ctls ◽  
Melanoma Antigens ◽  
Insect Viruses

Drosophila cells transfected with MHC class I and a number of costimulation molecules including B7.1, ICAM, LFA-3, and CD70 are potent antigen-presenting cells (APCs) for the generation of antigen-specific cytotoxic T cells (CTLs) in vitro. Using Drosophila APCs, CTLs specific for melanoma antigens have been generated in vitro and adoptively transferred to melanoma patients. However, the recent discovery that Drosophila cells can carry insect viruses raises the potential risk of Drosophila APCs transmitting xenogenic viruses to patient CTLs. In this study, we have investigated photoreactive methods to inactivate insect viruses in APC. A clinical grade psoralen compound, 8-MOP (UVADEX) in combination with UVA treatment (5 joules/cm2) can be used to inactivate Drosophila cell viruses. UVADEX treatment is sufficient to inactivate insect viruses but does not affect the expression of MHC class I molecules and costimulation molecules on Drosophila APCs. In fact, UVADEX treatment prevents Drosophila APC growth while maintaining APC function. Furthermore, UVADEX-treated Drosophila APCs maintain or have enhanced APC function as determined by enhanced T cell activation, proliferation, and CTL generation. Thus, the use of UVADEX-treated Drosophila APCs may provide a valuable tool for immunotherapy to generate tumor antigen-specific CTLs.

scholarly journals CD14 Expressing Precursors Give Rise to Highly Functional Conventional Dendritic Cells for Use as Dendritic Cell Vaccine

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3818
Author(s):  
Maud Plantinga ◽  
Denise A. M. H. van den Beemt ◽  
Ester Dünnebach ◽  
Stefan Nierkens
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Dendritic Cell Vaccine ◽  
Cell Vaccine ◽  
Activated T Cells

Induction of long-lasting immunity by dendritic cells (DCs) makes them attractive candidates for anti-tumor vaccination. Although DC vaccinations are generally considered safe, clinical responses remain inconsistent in clinical trials. This initiated studies to identify subsets of DCs with superior capabilities to induce effective and memory anti-tumor responses. The use of primary DCs has been suggested to overcome the functional limitations of ex vivo monocyte-derived DCs (moDC). The ontogeny of primary DCs has recently been revised by the introduction of DC3, which phenotypically resembles conventional (c)DC2 as well as moDC. Previously, we developed a protocol to generate cDC2s from cord blood (CB)-derived stem cells via a CD115-expressing precursor. Here, we performed index sorting and single-cell RNA-sequencing to define the heterogeneity of in vitro developed DC precursors and identified CD14+CD115+ expressing cells that develop into CD1c++DCs and the remainder cells brought about CD123+DCs, as well as assessed their potency. The maturation status and T-cell activation potential were assessed using flow cytometry. CD123+DCs were specifically prone to take up antigens but only modestly activated T-cells. In contrast, CD1c++ are highly mature and specialized in both naïve as well as antigen-experienced T-cell activation. These findings show in vitro functional diversity between cord blood stem cell-derived CD123+DC and CD1c++DCs and may advance the efficiency of DC-based vaccines.

scholarly journals In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

2006 ◽  
Vol 74 (7) ◽  
pp. 3817-3824 ◽  
Author(s):  
Karen L. Wozniak ◽  
Jatin M. Vyas ◽  
Stuart M. Levitz
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Mouse Lung

ABSTRACT Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

scholarly journals GABAA receptor α4-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model

2017 ◽  
Vol 313 (2) ◽  
pp. L406-L415 ◽  
Author(s):  
Gene T. Yocum ◽  
Damian L. Turner ◽  
Jennifer Danielsson ◽  
Matthew B. Barajas ◽  
Yi Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Asthma Model

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

Generation of Antigen Specific Cytotoxic T Lymphocytes (CTL) by Dendritic Cells (DCs) Transfected with In Vitro Transcribed (IVT) SART-1 and WT-1 mRNA.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3859-3859
Author(s):  
Anri Saito ◽  
Miwako Narita ◽  
Toshio Yano ◽  
Naoko Sato ◽  
Asuka Sekiguchi ◽  
...  
Keyword(s):  
Mhc Class I ◽  
Tumor Antigen ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Target Cells ◽  
Mhc Restriction ◽  
Specific Ctls ◽  
Specific Antigens

Abstract Transfection with tumor antigen RNA is one of the promising tools not only because of a possible sufficient amplification of tumor antigen RNA but also because of the absence of antigen peptides-associated MHC restriction. Several succeeded experiments about generation of CTLs using DCs transfeced in vitro transcribed (IVT) cancer specific antigen mRNA such as PSA, CEA, hTERT and MUC-1 have been reported in these a few years. In addition, recent reports about the simultaneous presentation of peptides in both MHC class I and class II molecules on DCs after mRNA electroporation show another superiority of mRNA transfection into DCs. In this presentation, we demonstrate successful generation of tumor antigen specific CTLs using with DCs transfected with IVT mRNA such as SART-1 and WT-1 by electroporation. This is the first report about the generation of SART-1 and WT-1 specific CTLs by using mRNA transfected DCs. [Methods] HLA-A24 positive human PB CD14+ cell-derived DCs were transfected with IVT mRNA (SART-1and WT-1) by electroporation. MRNA transfected DCs were co-cultured with autologous lymphocytes. The bulk co-cultures were re-stimulated several times with same DCs. CD8+ cells were separated and CTL activity was evaluated by 51chromium release assay. To determine whether the induced CTL cells could recognize the target cells in an HLA class I restricted manner, anti-HLA class I monoclonal antibodies were utilized to block the cytotoxicity of effectors. [Results] Electroporation of mRNA showed no effect on the surface phenotypes and antigen presenting ability of DCs. In addition to the demonstration of efficient transfection of M1 mRNA into DCs by using RT-PCR, which eliminated the amplification of transfected mRNA by the treatment with RNase before RNA extraction from the transfected cells, we identified the definite expression of WT-1 protein in the cytoplasm of DCs by using immunoblotting. CTL assay indicated that 1) DCs transfected with mRNA stimulated the generation of antigen-specific CTLs which are capable of lysing autologous DCs transfected with the same mRNA. 2) CTLs also demonstrated cytotoxic ability against cell lines such as KE-4 presenting SART-1 peptides on HLA-A24, MEGO1 presenting WT-1 peptides on MHC class I, and HLA-A24 cDNA transfected T2 which were used as target cells after co- incubation with 9 mer SART-1 peptides with strong affinity to HLA-A24. 3) Each cytotoxicities were markedly blocked after co-incubation of target cells with anti-MHC class I antibody and not inhibited with anti-MHC class II antibody. [Conclusion] Our results showed that IVT mRNA-transfected DCs which is constructed non-virally have a highly efficient ability to stimulate specific T-cell immunity against tumor. Unlike peptide- or tumor cells extract-pulsed DCs based vaccines, anti-tumor immunotherapy using the DCs transfected with antigen mRNA could be extended to a wide range of patients who have previously been excluded from clinical trials for the reason of the un-identification of tumor specific antigens, for the reason of the impossibility of obtaining sufficient tumor specimens, or for the reason of MHC restriction of the tumor specific antigens.

T-cell redirected killing and cure of pancreatic and gastric cancer xenografts by targeting Trop-2.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 262-262
Author(s):  
David M. Goldenberg ◽  
Edmund A. Rossi ◽  
Diane L Rossi ◽  
Thomas M. Cardillo ◽  
Chien-Hsing Chang
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Control Group ◽  
Calcium Signal ◽  
Target Cells ◽  
Normal Tissues

262 Background: Trop-2 [also called tumor-associated calcium signal transducer 2 (TACSTD2), EGP-1 (epithelial glycoprotein-1), GA733-1, or M1S1]is a 35 kDa transmembrane glycoprotein that is overexpressed relative to normal tissues in a variety of human cancers, including pancreatic and gastric carcinomas, where increased expression correlates with poor prognosis. Trop-2 appears to be more tumor-specific than the related molecule, EpCAM (Trop-1). MT110, the EpCAM antibody x CD3 bispecific T-cell engager (BiTE), is currently undergoing a Phase I study in various solid tumors, including lung, gastric, colorectal, breast, prostate, and ovarian cancers. We produced a similar T-cell redirecting bispecific tandem scFv, E1-3, using the variable domains of hRS7 (humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). Methods: T-cell activation, cytokine induction and cytotoxicity were evaluated ex vivo using PBMCs or purified T cells with human pancreatic (Capan-1 and BxPC3) and gastric (NCI-N87) cancer cell lines as target cells. In vivo activity was assayed with NCI-N87 xenografts that were inoculated s.c. in a mixture with twice the number of human PBMCs and matrigel. Results: In the presence of target cells and PBMCs, E1-3 potently induced T-cell activation, proliferation, and dose-dependent cytokine production of IL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7 ng/mL), TNF-α (>1 ng/mL) and IFN-γ (>50 ng/mL). In vitro, E1-3 mediated a highly potent T-cell lysis of BxPC3 [IC50=0.09(±0.04) pM], Capan-1 [IC50=1.2(±1.1) pM] and NCI-N87 [IC50=1.2(±1.2) pM] target cells. In vivo, two 50-µg doses of E1-3 given three days apart cured all of the mice (N=8) bearing NCI-N87 xenografts (P=0.0005; Log-Rank). Tumors in the control group (PBMCs only) reached the endpoint (TV>1 cm3) with a median of 39.5 days. All mice remained tumor-free in the E1-3 group at 78 days. Conclusions: Trop-2 is an attractive target for T-cell-mediated killing of pancreatic, gastric and other epithelial cancers.

scholarly journals Targeting HIV-1 Gag into the Defective Ribosomal Product Pathway Enhances MHC Class I Antigen Presentation and CD8+T Cell Activation

2007 ◽  
Vol 180 (1) ◽  
pp. 372-382 ◽  
Author(s):  
Andreas Goldwich ◽  
Sabine S. C. Hahn ◽  
Sandra Schreiber ◽  
Stefanie Meier ◽  
Eckhart Kämpgen ◽  
...  
Keyword(s):  
T Cell ◽  
Antigen Presentation ◽  
Mhc Class I ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
Class I ◽  
Mhc Class I Antigen ◽  
Hiv 1

Expansion of CD8+ Cytotoxic T Cells in vitro and in vivo Using MHC Class I Tetramers

Tumor Biology ◽  
2007 ◽  
Vol 28 (2) ◽  
pp. 70-76 ◽  
Author(s):  
Philip Savage ◽  
Maggie Millrain ◽  
Sofia Dimakou ◽  
Justin Stebbing ◽  
Julian Dyson
Keyword(s):  
T Cells ◽  
Mhc Class I ◽  
Cytotoxic T Cells ◽  
Class I
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