Dendritic Cells Tranfected with CML-28 cDNA Induce CML28-Specific Cytotoxic T Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5248-5248
Author(s):  
Hongsheng Zhou ◽  
Donghua Zhang ◽  
Yaya Wang ◽  
Yi Xiao ◽  
Wei Huang ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Tumor Antigen ◽  
Mononuclear Cells ◽  
Cytotoxic T Cells ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Target Cells ◽  
Peripheral Blood Mononuclear

Abstract Dendritic cells (DC) are the most potent antigen-presenting cells known; the use of DC loaded with tumor antigen is one of the most promising approaches to induce specific immune response. CML28 is an attractive candidate. According to the published article and our following study, CML28 is a broadly immunogenic antigen, which is overexpessed in leukemia cells, including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and chronic myelogenous leukemia (CML); but not in normal hematopoietic tissues or other normal tissue, with the exception of testis. In our study, DCs were generated from peripheral blood mononuclear cells (PBMC) by culture with GM-CSF, IL-4 and TNF-α. The full CML28 cDNA was amplified from K562 cell and then cloned into plasmid pcDNA3.1 HisA. Load DC with the constructed plasmid pcDNA3.1 HisA-CML28 by electroporation. The results of FACS analysis and Western Blot respectively demonstrate that the plasmid transfected DC retain immunophenotypical characteristics and can express the fusion protein CML28-His. Cytotoxic T lymphocytes (CTL) were generated from autologous PBMC stimulated by the transfected DCs. Cytoxicity assay in vitro revealed that CTL can recognize and lyse CML28-expressing target cells, such as the transfected DCs but not normal autologous DCs, which show that the plasmid pcDNA 3.1 HisA-CML28 transfected DC can induce CML28-specific CTL. Our studies demonstrate that CML28 can be a promising target of tumor antigen for leukemia-specific immunotherapy and DC loaded with CML28 tumor antigen can be a promising tool to induce specific anti-leukemia immune response.

Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 571-576 ◽  
Author(s):  
Susanne M. Schmidt ◽  
Kerstin Schag ◽  
Martin R. Müller ◽  
Markus M. Weck ◽  
Silke Appel ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Survivin Expression ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Specific Ctls ◽  
Apoptosis Protein ◽  
Broad Variety

AbstractSurvivin, a member of the inhibitor of apoptosis protein family, is expressed in almost all types of malignancies, making this protein a useful tool for the development of broadly applicable vaccination therapies. We used a recently identified HLA-A2 binding peptide and dendritic cells (DCs) from healthy donors to induce survivin-specific cytotoxic T lymphocytes (CTLs) in vitro. These T cells efficiently lysed target cells pulsed with the cognate peptide. Furthermore, survivin-specific CTLs recognized HLA-A2–matched tumor cell lines and primary malignant cells from patients with leukemia in an antigen-specific and HLA-restricted manner as demonstrated with the use of cold target inhibition assays and blocking antibodies. To validate the immunogenicity of survivin we performed the experiments in an autologous setting and used monocyte-derived DCs as targets. Interestingly, we found that DCs up-regulate survivin expression on stimulation with tumor necrosis factor α (TNF-α). However, these mature DCs were not recognized by survivin-specific CTLs, whereas they lysed autologous mature DCs pulsed with the antigenic peptide or transfected with whole tumor RNA purified from a survivin-expressing cell line. To further analyze the possible use of survivin-specific CTLs in cancer therapies, we induced survivin-specific CTLs using peripheral blood mononuclear cells (PBMNCs) and DCs from a patient with chronic lymphocytic leukemia (CLL). The in vitro–generated T cells efficiently recognized autologous malignant CLL cells, whereas they spared autologous-purified nonmalignant B cells or DCs. Our results demonstrate that survivin epitopes are presented on a broad variety of malignancies and can be applied in vaccination therapies.

Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies

Blood ◽  
2009 ◽  
Vol 113 (20) ◽  
pp. 4875-4884 ◽  
Author(s):  
Julie Gertner-Dardenne ◽  
Cecile Bonnafous ◽  
Christine Bezombes ◽  
Aude-Hélène Capietto ◽  
Virginie Scaglione ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Γδ T Cells ◽  
Lymphocytic Leukemia ◽  
Lymphoid Cells ◽  
Clinical Activity ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Dependent Cell

In human blood, 1% to 5% of lymphocytes are γδ T cells; they mostly express the γδ T-cell receptor (TCR)Vγ9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by γδ T cells is unknown. Here we report that, in association with the CD20+-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVγ9+ cell binding to CD20+ lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVγ9+ cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20+ cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by γδ T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVγ9+ lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.

scholarly journals Early response of monocyte-derived macrophages from vaccinated and non-vaccinated goats against in vitro infection with Mycobacterium avium subsp. paratuberculosis

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Noive Arteche-Villasol ◽  
Daniel Gutiérrez-Expósito ◽  
Raquel Vallejo ◽  
Jose Espinosa ◽  
Natalia Elguezabal ◽  
...  
Keyword(s):  
Immune Response ◽  
Mycobacterium Avium ◽  
Mononuclear Cells ◽  
Control Method ◽  
Quantitative Polymerase Chain Reaction ◽  
Early Response ◽  
Effective Control ◽  
Target Cells ◽  
Peripheral Blood Mononuclear

AbstractParatuberculosis is a disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Vaccination is the most cost-effective control method. However, despite the fact that macrophages are the main target cells for this pathogen, the precise mechanisms behind the response of the macrophage to Map infection and how it is modified by vaccination are yet poorly understood. The aim of this study was to investigate the effect of Silirum® vaccination in the early immune response of caprine monocyte-derived macrophages (CaMØs). Peripheral blood mononuclear cells (PBMCs) were obtained from vaccinated and non-vaccinated goats, cultured in vitro until differentiation to macrophages and infected with Map. After a 24 h incubation, Map viability and DNA were assessed in culture by viable colony count and real time quantitative polymerase chain reaction (qPCR). In addition, Map phagocytosis and expression of IL-10, IL-12, IFN-γ, TNF-α, IL-17A, IL-1β, iNOS, IL-6 and MIP-1β were also evaluated through immunofluorescence labelling and reverse transcriptase qPCR (RT-qPCR), respectively. A significant reduction of Map viability was observed in both supernatants (P < 0.05) and CaMØs (P < 0.001) from the vaccinated group. Similarly, the percentage of infected CaMØs and the number of internalized Map by CaMØs (P < 0.0001) was higher in the vaccinated group. Finally, iNOS (P < 0.01) and IL-10 were significantly up-regulated in CaMØs from vaccinated goats, whereas only MIP-1β was up-regulated in non-vaccinated animals (P < 0.05). These results show that vaccination modifies the immune response of CaMØs, suggesting that the phagocytosis and microbiocidal activity of macrophages against Map is enhanced after vaccination.

BTK Inhibitors, Irrespective of ITK Inhibition, Increase Efficacy of a CD19/CD3 Bispecific Antibody in CLL

Blood ◽  
2021 ◽  
Author(s):  
Maissa Mhibik ◽  
Erika M. Gaglione ◽  
David Eik ◽  
Ellen K Kendall ◽  
Amy Blackburn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic Activity ◽  
Kinase Inhibitors ◽  
Bispecific Antibody ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Peripheral Blood Mononuclear

Bruton Tyrosine Kinase inhibitors (BTKis) are a preferred treatment for patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, while effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3 bispecific antibody (bsAb) that recruits autologous T cell cytotoxicity against CLL cells in vitro. Compared to observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits IL2 inducible T cell Kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive, and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared to that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including CTLA-4 and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

scholarly journals Cytokine influence on killing of fresh chronic lymphocytic leukemia cells by human leukocytes

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2707-2715 ◽  
Author(s):  
D Cemerlic ◽  
B Dadey ◽  
T Han ◽  
L Vaickus
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Mononuclear Cells ◽  
Protein A ◽  
Human Leukocyte ◽  
Lymphocytic Leukemia ◽  
Target Antigen ◽  
Peripheral Blood Mononuclear ◽  
Ifn Gamma ◽  
Hla Dr

Abstract The feasibility of combining the Lym-1 monoclonal antibody (MoAb) with interferon-gamma (IFN-gamma) in the treatment of chronic lymphocytic leukemia (CLL) was evaluated. We used an in vitro tumor lysis model that incorporated fresh CLL cells from 21 different patients as targets for two distinct normal human leukocyte effector subsets, neutrophils, and peripheral blood mononuclear cells (PBMCs). Lym-1 antigen (Lym-1- Ag) expression varied greatly and did not correlate with the expression of other CLL-associated antigens such as CD5, CD19, or HLA-DR. CLL cells were not lysed by neutrophils alone or with IFN-gamma in the absence of Lym-1. Neutrophil Lym-1-dependent cytotoxicity (ADCC) in the absence of IFN-gamma was weak and inconsistent. IFN-gamma exposure induced MoAb-dependent lysis of 80% of 21 CLL targets and resulted in an eightfold augmentation of neutrophil ADCC against the remainder. Cytotoxicity correlated directly and positively with Lym-1-Ag expression. Confirmation of the need for interaction between neutrophil IgG Fc receptors (Fc gamma Rs) and the Fc portion of the Lym-1 MoAb was obtained by demonstrating that purified Staphylococcus aureus Protein A (SpA) inhibited ADCC. IFN-gamma exposure caused no consistent alternations in Lym-1-Ag expression on CLL cells so that target antigen upregulation was unlikely to account for augmentation of neutrophil ADCC. PBMCs alone, exposed to interkeukin-2 (IL-2) or IFN-gamma, or with Lym-1 in the presence or absence of IL-2 or IFN-gamma were unable to lyse CLL targets. PBMCs were able to kill Raji Burkitt lymphoma cells in conjunction with Lym-1, so their ability to interact with Lym- 1-coated targets and their lytic functions appeared intact. These results emphasize the importance of examining fresh tumor cells with different leukocyte effector subsets before designing a clinical trial that combines a therapeutic MoAb with a cytokine.

scholarly journals Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors

2015 ◽  
Vol 90 (5) ◽  
pp. 2316-2331 ◽  
Author(s):  
Nadeene E. Riddick ◽  
Fan Wu ◽  
Kenta Matsuda ◽  
Sonya Whitted ◽  
Ilnour Ourmanov ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Rhesus Macaques ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Sooty Mangabeys ◽  
Natural Hosts

ABSTRACTAfrican green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIVin vivo, while human-derived CXCR6 and GPR15 also appear to be usedin vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptorsin vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4+T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4+T cells and are potential alternative coreceptors for SIVagm usein vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals.IMPORTANCEAfrican green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cellsin vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptorsin vitro. In this study, we investigated whether SIV from vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of the CCR5 antagonist maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm entry. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entryin vitroand may serve as entry coreceptors for SIVagmin vivo, since their mRNAs were detected in AGM memory CD4+T cells, the preferred target cells of SIV.

Monosodium Urate Crystals Bind with Idiotype Protein and Promote Dendritic Cells to Induce Cytotoxic T Cells in Vitro

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4904-4904
Author(s):  
Ippei Sakamaki ◽  
Kunihiro Inai ◽  
Takanori Ueda ◽  
Hiroshi Tsutani
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cytotoxic T Cells ◽  
Target Cells ◽  
Monosodium Urate ◽  
Cd4 Cells ◽  
Antigenic Protein ◽  
Positively Charged ◽  
Msu Crystals

Abstract Monosodium urate (MSU) crystals have been studied to act as a key substance in local immunoreactions. MSU released from damaged cells works as an endogenous danger signal to antigen-presenting cells. MSU crystals evoke specific cell immunity and work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DCs). We focused on the application of MSU crystals as a not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTLs) efficiently in vitro. We confirmed that MSU crystals facilitated human DCs to express the maturation marker, CD83, deliver (Fab′)2 attaching to the crystals. In order to determine whether MSU crystals facilitate the T-cell proliferation activity of DCs, the proliferative effects of DCs on allogeneic CD4+ cells were investigated. DCs pulsed with MSU crystals significantly facilitated the proliferation of allogeneic CD4+ cells when compared to DCs alone. The stimulation index (SI) was 2.5 ± 0.1 and 1.7 ± 0.1, respectively. When using DCs pulsed with the Fab attached to MSU crystals, the proliferation of CD4+ cells was significantly greater than when using DCs pulsed with Fab alone. The SI was 2.6 ± 0.2 and 1.9 ± 0.1, respectively. No significant differences were seen in the proliferation of allogeneic CD4+ cells between DCs pulsed with the Fab attached to MSU and DCs pulsed with MSU alone. We selected the multiple myeloma IM-9 cell line and its product idiotype (Id) protein as an ideal pair of target cells and positively charged tumor-specific antigen, respectively. After sensitizing DCs derived from HLA-A matched volunteers pulsed with tumor-specific monoclonal IgG-Fab fragments (IM-9 Fab) attached to MSU crystals, the CD8+ T cells stimulated by the DCs killed significantly more target cells (38.5 ± 3.5%, n=4) than those stimulated by DCs pulsed with IM-9 Fab alone (3.5 ± 7.5%). These cytotoxic effects of CD8+ cells stimulated by the DCs pulsed with IM-9 Fab attached to MSU crystals were reduced (3.6 ± 1.7%) when MSU crystals were pre-coated with fetal bovine serum to block to bind with IM-9 Fab. For efficient induction of CTLs, it is necessary for Id proteins to attach to MSU crystals. MSU crystals have some advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DCs, as well as an adjuvant promoting DC maturation and inducing CTLs.

scholarly journals Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1963-1969 ◽  
Author(s):  
Daniel G. Kavanagh ◽  
Daniel E. Kaufmann ◽  
Sherzana Sunderji ◽  
Nicole Frahm ◽  
Sylvie Le Gall ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Cytotoxic T Cells ◽  
T Cell Response ◽  
Peripheral Blood Mononuclear ◽  
T Cell Lines

Transfection with synthetic mRNA is a safe and efficient method of delivering antigens to dendritic cells for immunotherapy. Targeting antigens to the lysosome can sometimes enhance the CD4+ T-cell response. We transfected antigen-presenting cells (APCs) with mRNA encoding Gag-p24 and cytoplasmic, lysosomal, and secreted forms of Nef. Antigen-specific cytotoxic T cells were able to lyse the majority of transfected targets, indicating that transfection was efficient. Transfection of APCs with a Nef construct bearing lysosomal targeting signals produced rapid and prolonged antigen presentation to CD4+ and CD8+ T cells. Polyclonal CD4+ and CD8+ T-cell lines recognizing multiple distinct epitopes were expanded by coculture of transfected dendritic cells with peripheral blood mononuclear cells from viremic and aviremic HIV-infected subjects. Importantly, lysosome-targeted antigen drove a significantly greater expansion of Nef-specific CD4+ T cells than cytoplasmic antigen. The frequency of recognition of CD8 but not CD4 epitopes by mRNA-expanded T cells was inversely proportional to sequence entropy and was similar to ex vivo responses from a large chronic cohort. Thus human dendritic cells transfected with mRNA encoding lysosome-targeted HIV antigen can expand a broad, polyclonal repertoire of antiviral T cells, offering a promising approach to HIV immunotherapy.

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