scholarly journals Whole genome and transcriptome analysis of genetic determinants associated with cyclophosphamide in vitro in pediatric acute leukemias

2021 ◽  
Vol 10 (6) ◽  
pp. 229-238
Author(s):  
Joanna Szczepanek ◽  
Sylwia Górka ◽  
Krzysztof Domagalski ◽  
Joanna Dejewska ◽  
Monika Pogorzała ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Cytokine Signaling ◽  
Comparative Genomic ◽  
Genetic Profile ◽  
Acute Leukemias

Introduction: The main objective was to implement the determinations of the ex vivo resistance to cyclophosphamide and to identify the genetic profile for pediatric patients with acute leukemias. Methods: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). We performed also array-based comparative genomic hybridization using a SurePrint G3 Human CGH Microarray. Data was analyzed by bioinformatics tools. Verification of the relative expression level of 20 genes was carried out by qRT- PCR. Results: We observed a multitude of differentially expressed genes, e.g. ANXA1 (FC=3,04), BCL2A1 (FC=2,69), SERPINA1 (FC=2,12), DHRS7 (FC=2,13), PCDH9 (FC=- 4,58), TTC28 (FC=-2,25) and DUSP1 (FC=-2,91). The expression of genes that code for inflammation mediated by chemokine and cytokine signaling, Wnt, angiogenesis and integrin signaling and T cell activation pathways genes affect the sensitivity of leukemic blasts to cyclophosphamide. Transcriptome level changes are associated with chromosomal aberrations, especially located on chromosomes 8, 10, 14, 15, 16 and 22. Conclusion: Our work delineated genes with differentiated expression and recurrent copy number changes, and revealed novel amplified loci and frequent deletions in resistant to cyclophosphamide cells, which may guide future work aimed at identifying the relevant target genes. In particular, deletion seems to be a frequent mechanism of IFIT3 gene inactivation. ANXA1, SERPINA1, TCF7 and BCL2A1 may also be included among the candidate genes of resistance (Ontological analysis).

Phase II trial of a GM-CSF-producing and CD40L-expressing cell line combined with allogeneic tumor antigen as a novel vaccine for metastatic lung adenocarcinoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 2559-2559
Author(s):  
Ben C. Creelan ◽  
Scott Antonia ◽  
David Noyes ◽  
Terri B. Hunter ◽  
George R. Simon ◽  
...  
Keyword(s):  
T Cell ◽  
Lung Adenocarcinoma ◽  
Cell Line ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Clinical Activity ◽  
Metastatic Lung ◽  
Grade 3

2559 Background: We created a vaccine in which irradiated allogeneic lung adenocarcinoma cells are combined with a bystander K562 cell line transfected with hCD40L and hGM-CSF. By recruiting and activating dendritic cells, we hypothesized the vaccine would induce tumor regression in metastatic lung adenocarcinoma. Methods: Intradermal vaccine was given every 14 days x3, followed by monthly x3. Cyclophosphamide (300 mg/m2 IV) was administered before 1st and 4th vaccines to deplete regulatory T-cells. All-trans retinoic acid was given (150/mg/m2/day) after 1st and 4th vaccines to enhance dendritic differentiation. Peripheral blood mononuclear cells (PBMCs) were collected at baseline and after each vaccination. T-cell activation profiles were analyzed by ELISpot assay and tested by generalized Wilcoxon for correlation to survival. Results: 24 participants were accrued at a single center from 10/2006 to 6/2008, with median age 64 and median of 3 previous lines of chemotherapy prior to entry. 20 were former smokers and 4 had brain metastases. A total of 101 vaccines were administered. Common toxicities of any grade were joint pain (79%) and fatigue (75%). Significant adverse events included a grade 3 hypotension and a grade 3 acute respiratory distress. No confirmed complete or partial radiologic responses were observed. Median overall survival (OS) was 8.0 mo (95% CI 3.5 – 12.5) and median time-to-progression was 2.4 mo (95% CI 0.3 – 4.6). Presence of HLA-A2 conferred reduced risk of progression (HR 0.37, 95% CI 0.14 -0.89, p=0.02) and trend to improved OS (HR 0.59, p = 0.06). Of 14 participants with evaluable PBMCs, 5 demonstrated sustained tumor peptide-specific T-cell activation after vaccination. Ex vivo peptide immune response correlated with improved OS compared to non-responders (23 vs. 7 mo, HR 0.48, p = 0.04). Conclusions: Vaccine administration was feasible and tolerable in a heavily pretreated population of metastatic lung cancer. These data suggest the vaccine has clinical activity in the subset with peptide-induced T-cell immune responses and warrants further investigation. A randomized trial of the vaccine is currently in development.

scholarly journals Quantitative and time-resolved miRNA pattern of early human T cell activation

2020 ◽  
Vol 48 (18) ◽  
pp. 10164-10183
Author(s):  
Caroline Diener ◽  
Martin Hart ◽  
Tim Kehl ◽  
Stefanie Rheinheimer ◽  
Nicole Ludwig ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Expression Profiles ◽  
T Cell Response ◽  
Activation Process ◽  
Time Resolved ◽  
Quantitative Expression ◽  
Human T Cell

Abstract T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human T-cell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR-155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR-155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response.

Regulatory T Cell Counts and Development of Malignancy in Patients with HIV Infection

2020 ◽  
Vol 18 (3) ◽  
pp. 201-209
Author(s):  
Marianna Politou ◽  
Sofia Boti ◽  
Theodoros Androutsakos ◽  
Athanasios Kontos ◽  
Abraham Pouliakis ◽  
...  
Keyword(s):  
T Cell ◽  
Hiv Infection ◽  
T Cell Activation ◽  
Negative Correlation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Linear Regression Analysis ◽  
Cell Mediated Immunity ◽  
Cell Counts

Background: T-regulatory cells (Tregs) play an important role in maintaining homeostasis by attenuating the cytokine response to T-cell receptor (TCR) stimulation and by suppressing the functioning of neighboring immune cells. In Human Immunodeficiency Virus (HIV) infection, Tregs can be either beneficial, by suppressing generalized T-cell activation, or detrimental, by suppressing protective anti-HIV cell-mediated immunity. An imbalance of Tregs and effector T-cells can blunt immune responses to malignant cells or facilitate inflammation-mediated pathologies. Objective: The purpose of our study was to explore the possible correlation between Tregs’ concentration and HIV infection’s parameters as well as the development of hematological and solid malignancies. Methods: In a longitudinal prospective study, ex vivo phenotyping of fresh peripheral blood mononuclear cells from patients with primary HIV infection was performed at baseline. All patients were then followed up every 3 months and the development of solid or hematological malignancies was noted. Results: A total of 155 patients were included in the study and the median follow-up period was 64 months. Treg counts were significantly higher among males, patients with high viral load (>350 copies/ml) and patients with virological failure to antiretroviral treatment (ART). Linear regression analysis showed a significant negative correlation between Treg levels and CD4 (+) T-cell counts. Patients with neoplasia had lower levels of Tregs while increasing levels showed a negative correlation with the development of neoplasia. Conclusion: In our population of HIV-infected patients, high levels of Tregs were associated with disease progression, and low baseline levels were associated with a higher probability of developing neoplasia.

scholarly journals CTLA4Ig Gene Transfer Prolongs Survival and Induces Donor-Specific Tolerance in a Rat Renal Allograft

2000 ◽  
Vol 11 (4) ◽  
pp. 747-752 ◽  
Author(s):  
SUSANNA TOMASONI ◽  
NADIA AZZOLLINI ◽  
FEDERICA CASIRAGHI ◽  
MAURIZIO C. CAPOGROSSI ◽  
GIUSEPPE REMUZZI ◽  
...  
Keyword(s):  
Gene Transfer ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Genetic Manipulation ◽  
Ex Vivo ◽  
Large Animal ◽  
Animal Testing ◽  
Major Step ◽  
Th1 And Th2 Cytokines

Abstract. Organ transplantation requires lifelong antirejection therapy, which carries the risk of infection and cancer. A revolutionary approach is to transduce the organ graft with immunomodulatory genes to render them tolerated with no need of systemic immunosuppression. Prolonged allograft survival was achieved by adenovirus-mediated transduction of the cold-preserved kidney with sequences encoding CTLA4Ig, a recombinant fusion protein that blocks T cell activation. Organ expression of the transgene was achieved associated with mild infiltration of mononuclear cells in the transfected kidney. Mixed lymphocyte reaction as well as the production of both Th1 and Th2 cytokines were reduced. Thus, the gene transfer technique to prolong graft survival is indeed effective and safe and can induce donor-specific unresponsiveness. Pending appropriate large animal testing,ex vivogenetic manipulation of the organ before surgery may hopefully represent a major step forward in human transplant medicine.

scholarly journals Oncolytic Reovirus Immune Priming: A Phase 1b Study of Reolysin with Bortezomib and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4507-4507 ◽  
Author(s):  
Kevin R. Kelly ◽  
Kaijin Wu ◽  
Denice Tsao-Wei ◽  
Susan Groshen ◽  
Timothy Junius Triche ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Disease Progression ◽  
T Cell Activation ◽  
Stable Disease ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Phase 1 ◽  
Immune Priming ◽  
Phase 1B

Abstract Background: While novel agents have improved the outcome for multiple myeloma (MM), the disease remains incurable. Our preclinical work has shown that MM cells from relapsed/refractory patients are very sensitive to the combination of Reolysin (a proprietary formulation of an oncolytic reovirus) and bortezomib (BZ), resulting in synergistic levels of endoplasmic reticulum (ER) stress. A pilot phase 1 study showed that Reolysin was well tolerated in relapsed/refractory MM patients and was associated with prolonged stable disease. Methods: Relapsed/refractory MM patients including patients refractory to BZ were included. This is a phase 1b study of 3 escalating doses of Reolysin (cohort 1; 3 x1010 TCID50, cohort 2; 4.5 x1010 TCID50, and cohort 3; 9 x1010 TCID50). Reolysin is given on days 1, 2, 8, 9, 15, and 16. Patients receive 40 mg dexamethasone and 1.5 mg/m2bortezomib on days 1, 8, and 15. Cycles are repeated every 28 days in the absence of disease progression or unacceptable toxicity. Results: Eight patients have been enrolled, seven were male and the median age was 55 (range, 33 - 66). The median number of prior therapies was 4 (range 1 to 6). All patients were previously exposed to BZ, and 6 patients were previously exposed to both an immunomodulatory agent and carfilzomib. Most patients had ISS stage I disease at study entry (n=5), 2 had stage II and 1 had stage III. The combination was well tolerated and most treatment-related toxicities were transient and easily managed with supportive care. The most common treatment-related toxicities were grade 1 diarrhea (n=4), grade 1 fatigue (n=4), grade 1 flu-like symptoms (n=5) and grade 1 headache (n=4). No dose limiting toxicities occurred in cohort 1 or 2. Three patients completed 1 cycle of treatment only, 2 completed 3 cycles, and 1 patient completed 4 cycles. One patient remains on treatment. Reasons for treatment discontinuation included disease progression (n=4), clinical deterioration (n=1) and patient withdrawal (n=2). Six patients were evaluable for response, 3 patients had stable disease lasting at least 3 cycles whereas 3 patients had progressive disease at the end of cycle 1. Ex vivo treatment of primary MM cells and MM cell lines (U266 and RPMI-8226) with Reolysin revealed a dramatic induction of PD-L1 expression as measured by qRT-PCR and flow cytometry. Furthermore, ex vivo treatment of MM patient mononuclear cells with Reolysin resulted in NK and T cell activation. These results suggest that the addition of an anti-PD-1 or anti-PD-L1 agent may augment the anti-MM activity of Reolysin. Conclusions: The combination of Reolysin, BZ and dexamethasone is well tolerated in a heavily pretreated MM patient population. Cohort 3 is currently enrolling at the 9 x1010 TCID50 dose level of Reolysin. The potential anti-MM effects of immune activation following Reolysin infusion may be mitigated by MM expression of PD-L1. Additional cohorts exploring the tolerability, efficacy and pharmacodynamics of the combination of Reolysin, BZ or carfilzomib, dexamethasone and an immune checkpoint inhibitor will be added following completion of cohort 3. Disclosures Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coffey:Oncolytics Biotech: Employment. Gill:Oncolytics Biotech: Employment.

scholarly journals Whole blood vs PBMC: compartmental differences in gene expression profiling exemplified in asthma

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Daniel He ◽  
Chen Xi Yang ◽  
Basak Sahin ◽  
Amrit Singh ◽  
Casey P. Shannon ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
T Cell Activation ◽  
Whole Blood ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Research Subjects ◽  
Peripheral Blood Mononuclear

Abstract Background Blood has proven to be a useful resource for molecular analysis in numerous biomedical studies, with peripheral blood mononuclear cells (PBMCs) and whole blood being the major specimen types. However, comparative analyses between these two major compartments (PBMCs and whole blood) are few and far between. In this study, we compared gene expression profiles of PBMCs and whole blood samples obtained from research subjects with or without mild allergic asthma. Methods Whole blood (PAXgene) and PBMC samples were obtained from 5 mild allergic asthmatics and 5 healthy controls. RNA from both sample types was measured for expression of 730 immune-related genes using the NanoString nCounter platform. Results We identified 64 uniquely expressed transcripts in whole blood that reflected a variety of innate, humoral, and adaptive immune processes, and 13 uniquely expressed transcripts in PBMCs which were representative of T-cell and monocyte-mediated processes. Furthermore, analysis of mild allergic asthmatics versus non-asthmatics revealed 47 differentially expressed transcripts in whole blood compared to 1 differentially expressed transcript in PBMCs (FDR < 0.25). Finally, through simultaneous measurement of PBMC proteins on the nCounter assay, we identified CD28 and OX40 (TNFRSF4), both of which are critical co-stimulatory molecules during T-cell activation, as significantly upregulated in asthmatics. Conclusions Whole blood RNA preserved in PAXgene tubes is excellent for producing gene expression data with minimal variability and good sensitivity, suggesting its utility in multi-centre studies requiring measurement of blood gene expression.

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 Endogenous retrovirus-encoded Syncytin-2 contributes to exosome-mediated immunosuppression of T cells†

2019 ◽  
Author(s):  
Adjimon G Lokossou ◽  
Caroline Toudic ◽  
Phuong Trang Nguyen ◽  
Xavier Elisseeff ◽  
Amandine Vargas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Map Kinases ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Endogenous Retrovirus ◽  
Jurkat Cells ◽  
Peripheral Blood Mononuclear ◽  
Interfering Rna

Abstract Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

Sign in / Sign up

Export Citation Format

Share Document