Quantitation of Zap-70 Expression in B-CLLCells Employing a Novel Flow Cytometry Analysis Method.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4785-4785
Author(s):  
Varda R. Deutsch ◽  
Sigi Kay ◽  
Marjorie Pick ◽  
Yair Herishanu ◽  
Ori Rogowsky ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Pearson Correlation ◽  
Analysis Method ◽  
Flow Cytometry Assay ◽  
Expression Levels ◽  
Mutational Status ◽  
The Mean

Abstract IgVH mutational status and molecular cytogenetics have dramatically improved the ability to predict the prognosis of CLL patients. These tests, however, are highly sophisticated, complex and costly for routine use. ZAP-70, a syk family tyrosine kinase normally expressed in T cells, is a newly described marker which correlates with clinical progression and shorter survival in CLL. A flow cytometry assay to detect ZAP-70 described by Crespo et al (1), appears to be the simplest approach for routine clinical stratification in B-CLL. It is highly informative, and has a strong correlation between the expression of ZAP-70 in CLL cells and clinical outcome. However, in this analysis there are some technical aspects that should be improved to enable it to be standardized as a routine flow cytometry assay. ZAP-70 expression in B-CLL cells is not quantitative but assessed relative to its expression in the T- and NK cells (CD3+, CD56+). This approach can be problematic at times, as ZAP-70 levels in T cells vary in CLL patients as well as in normal controls, probably due to its up regulation following activation. An additional quandary in this assay is that all results are recorded relative to the subjectively delineated T-cell gate. Accordingly, small changes in expression in the T cells can significantly alter the results obtained in some B-CLL samples. In this study we aimed to improve the resolution of the assay by performing a quantitative analysis of ZAP-70 expression within the B-CLL cell population which is uncoupled from T cells. Blood samples were stained by the method described by Crespo et al (1) and ZAP 70 levels in B cell populations in CLL patients (CD19+CD5+) and in healthy volunteers (CD19+) were determined using a standard curve generated by an absolute fluorescent standard of FITC high levels beads with a range of 50–2000 x103 molecules of equivalent soluble fluorochrome (MESF) units per microsphere. Quantitation of expression levels were generated using Quick Cal V2.2 via www.bangslab.com. (Bangs laboratories). Using this analysis system the mean expression levels of ZAP 70 were calculated in healthy B cells (n=11) to be 11,177±1812 MESF units while in CLL (n=36) the mean value was >143,000 MESF units. To determine the reliability of this new method and its clinical relevance we compared our results to data generated using the analysis method of Crespo et al (1). We found a significant correlation between the two methods (r2 = 0.7558). Using ROC curve analysis with maximum sensitivity and specificity, our minimum positive value was found to be 46,700 MESF, with >95% sensitivity at 27,000 MESF and >92% specificity at 67,000 MESF and a Pearson correlation of 0.877 (P<0.0005). We conclude that this assay can provide a more reproducible and reliable analysis of Zap-70 expression in B-CLL, which is easily standardized. This analysis is highly specific as it is quantitative, not subjective and uncoupled from T cell activation in the sample.

Development and characterization of a HCMV multiantigen therapeutic vaccine for GBM using the UNITE platform.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14565-e14565
Author(s):  
Amit Adhikari ◽  
Juliete Macauley ◽  
Yoshimi Johnson ◽  
Mike Connolly ◽  
Tim Coleman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
T Cell Response

e14565 Background: Glioblastoma (GBM) is an aggressive form of brain cancer with a median survival of 15 months which has remained unchanged despite technological advances in the standard of care. GBM cells specifically express human cytomegalovirus (HCMV) proteins providing a unique opportunity for targeted therapy. Methods: We utilized our UNITE (UNiversal Intracellular Targeted Expression) platform to develop a multi-antigen DNA vaccine (ITI-1001) that codes for the HCMV proteins- pp65, gB and IE-1. The UNITE platform involves lysosomal targeting technology, fusing lysosome-associated protein 1 (LAMP1) with target antigens resulting in increased antigen presentation by MHC-I and II. ELISpot, flow cytometry and ELISA techniques were used to evaluate the vaccine immunogenicity and a syngeneic, orthotopic GBM mouse model that expresses HCMV proteins was used for efficacy studies. The tumor microenvironment studies were done using flow cytometry and MSD assay. Results: ITI-1001 vaccination showed a robust antigen-specific CD4 and CD8 T cell response in addition to a strong humoral response. Using GBM mouse model, therapeutic treatment of ITI-1001 vaccine resulted in ̃56% survival with subsequent long-term immunity. Investigating the tumor microenvironment showed significant CD4 T cell infiltration as well as enhanced Th1 and CD8 T cell activation. Regulatory T cells were also upregulated upon ITI-1001 vaccination and would be an attractive target to further improve this therapy. In addition, tumor burden negatively correlated with number of activated CD4 T cells (CD4 IFNγ+) reiterating the importance of CD4 activation in ITI-1001 efficacy and potentially identifying treatment responders and non-responders. Further characterization of these two groups showed high infiltration of CD3+, CD4+ and CD8+ T cells in responders compared with non- responders along with higher CD8 T cell activation. Conclusions: Thus, we show that vaccination with HCMV antigens using the ITI-1001-UNITE platform generates strong cellular and humoral immune responses, triggering significant anti-tumor activity that leads to enhanced survival in mice with GBM.

scholarly journals Rapid Flow Cytometry Method for Quantitation of LFA-1-Adhesive T Cells

2006 ◽  
Vol 13 (3) ◽  
pp. 403-408 ◽  
Author(s):  
Brian Crucian ◽  
Mayra Nelman-Gonzalez ◽  
Clarence Sams
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Subsets ◽  
Activated T Cells ◽  
Peripheral Blood Mononuclear ◽  
Simple Method ◽  
Rapid Flow

ABSTRACT Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an “adhesive” state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-μm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3+ CD4+ or CD8+ CD19− CD16− CD45RO+ CD62L+ CD27+ CD57−. A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.

Significant Alterations in T-Cell TH1 and TH2 Cytokine Gene Profiles Associated with G-CSF Mobilization Do Not Occur in T-Cells Mobilized with AMD3100.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3277-3277 ◽  
Author(s):  
Aleah L. Smith ◽  
Joo Jungnam ◽  
Sheila Rao ◽  
Andreas Lundqvist ◽  
Lisa W. Cook ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
False Discovery Rate ◽  
Heat Map ◽  
Expression Levels ◽  
False Discovery ◽  
Healthy Donors ◽  
Regulated Expression ◽  
The Mean ◽  
Permutation Technique

Abstract Previous studies have shown that granulocyte colony stimulating factor (G-CSF) mobilization skews T-cells toward a type 2 cytokine profile, potentially impacting GVHD and other immune mediated events that occur after allogeneic hematopoietic stem cell transplantation (HCT). AMD3100, a selective antagonist of CXCR4, rapidly mobilizes hematopoietic progenitor cells into the circulation, has a synergistic effect on CD34+ cell mobilization when combined with G-CSF, and is currently being evaluated as a single agent to mobilize allografts. Apheresis collections mobilized with a single injection of AMD3100 contain a similar number of T-cells as those collected following 5 daily doses of G-CSF. We investigated whether T-cells mobilized with AMD3100 undergo changes in cytokine polarization status as described to occur with G-CSF mobilization. Using real time PCR, we investigated the expression of 84 genes associated with TH1, TH2, and TH3 T-cell pathways at baseline and following mobilization with a single injection of AMD3100 (dosed at 240 or 320 mcg/kg; n=12 subjects) or following 5 daily doses of G-CSF(n=5 subjects). RNA was extracted from CD3+ T-cells isolated using immunomagnetic beads (>95% purity) from PBMCs collected immediately before mobilization and 6 hours after AMD3100 administration or 5 days after G-CSF mobilization. The RT2 Profiler ™ PCR Array was used which contains pathway specific cytokine genes associated with TH1, TH2, and TH3 cells. Expression levels of 16 genes changed significantly (false discovery rate=0.10) from baseline following G-CSF mobilization; 9 genes were up-regulated and 7 genes were down-regulated from baseline. Five up-regulated and 4 down-regulated genes had greater than a 2-fold change in expression (Figure). In contrast, none of the 84 genes examined, including the 16 altered with G-CSF, changed significantly following AMD3100 administration. Our results are concordant with current literature that shows the expression of several genes effecting T-cell cytokine polarization are altered in G-CSF mobilized T-cells. It has been suggested that the TH2 polarization in G-CSF mobilized products contributes to the comparable incidence of acute GVHD and the higher incidence of chronic GVHD compared to bone marrow allografts. In contrast, T-cells mobilized with AMD3100 appear similar to non-mobilized T-cells, and do not undergo a change in TH1- and TH2-related gene expression. Whether the differences in cytokine polarization of T-lymphocytes mobilized with AMD3100 compared to G-CSF will impact immune reconstitution or other immune sequela (i.e. GVHD, graft-vs.-tumor) associated with HCT is currently being assessed in a pilot allogeneic transplantation trial in humans using AMD3100 to mobilize donors. Figure: Heat map showing expression levels of 16 genes in CD3+ T-cells that changed significantly from baseline following G-CSF mobilization in 5 healthy donors. Samples were analyzed with a two-sample paired t-test, and the corresponding p-values were evaluated based on the permutation technique at a 10% false discovery rate. All samples were normalized to the center of the mean of the pre G-CSF samples with black denoting up-regulated expression and white denoting down-regulated expression. Figure:. Heat map showing expression levels of 16 genes in CD3+ T-cells that changed significantly from baseline following G-CSF mobilization in 5 healthy donors. Samples were analyzed with a two-sample paired t-test, and the corresponding p-values were evaluated based on the permutation technique at a 10% false discovery rate. All samples were normalized to the center of the mean of the pre G-CSF samples with black denoting up-regulated expression and white denoting down-regulated expression.

Evaluation Of CD33 Expression and Functional Analysis Of The CD33/CD3 Bispecific BiTE® Antibody AMG 330 In Primary AML Samples

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 239-239 ◽  
Author(s):  
Christina Krupka ◽  
Peter Kufer ◽  
Roman Kischel ◽  
Gerhard Zugmaier ◽  
Jan Boegeholz ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Ex Vivo ◽  
Natural Setting ◽  
Single Chain ◽  
Activation Markers ◽  
Expression Levels ◽  
Equity Ownership

Abstract Antibody-based immunotherapy represents a promising strategy to specifically target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). We evaluated a single-chain bispecific CD33/CD3 BiTE® antibody (AMG 330) for its suitability as immunotherapy in AML. A prerequisite for successful immunotherapeutic approaches using this molecule is the expression of CD33 on AML blasts including leukemic stem cells (LSCs). Therefore, we quantified CD33 expression on AML blasts and LSCs by flow cytometry (mean fluorescence intensity, MFI) and correlated expression intensity with cytogenetic and molecular disease characteristics in order to identify patient cohorts possibly most suited for CD33-targeted therapies. CD33 expression was detected in >99% of patient samples (n=621, MFI ratio ≥ 1.5) although highly variable. A strong correlation between high CD33 expression levels and NPM1 mutations (p<0.001) was found. In contrast, low CD33 expression levels were significantly associated with complex karyotypes and t(8,21) translocations (p<0.001). Furthermore, LSCs within the CD34+/CD38- compartment displayed CD33 at higher levels than healthy donor stem cells (p=0.047). Importantly, colony formation of CD34+/Lin-cells from healthy donors was not affected after pre-incubation with AMG 330 and T-cells. A major hurdle for measuring cytotoxic effects on AML blasts has long been that primary AML patient samples show progressive cell death within a few days ex-vivo. To simulate the natural setting of target and T-cells in AML patients, we developed a long-term culture system for AML blasts that allowed us to observe these co-cultures for up to 5 weeks. Thus, we were able to show effective elimination of AML blasts within primary samples by AMG 330-activated and expanded residual CD3+/CD45RA-/CCR7+ memory T-lymphocytes. While the functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT vs. CD33DIM AML cell lines in an in-vitro cytotoxicity assay potent anti-leukemic activity on primary AML blasts was observed irrespective of CD33 expression level. At low effector to target ratios (up to 1:79), the recruited T-cells lysed autologous blasts completely in the majority of samples. Further T-cell analysis showed that naive T-cells (CD45RA+/CCR7+) were not expanded by AMG 330; neither were terminally differentiated T-cells (CD45RA+/CCR7-), probably due to their poor proliferative capacity. We did not observe an increase in percentage of CD3+/CD4+/CD25+/FoxP3+regulatory T-cells in the presence of AMG 330, suggesting that these cells may not have impacted AMG 330-mediated T-cell activity in our experiments. Compared to control cultures, T-cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3 upon response to AMG 330, which was partially reversible after complete target cell elimination. However, PD-1 up-regulation did not correlate with an up-regulation of PD-L1 on AML blasts despite substantial INFγ secretion by activated T-cells. This study provides the first correlation of CD33 expression levels to a comprehensive genotype analysis in adult AML patients. While CD33 expression may vary by AML biologic subgroup, AMG 330 exposure led to lysis of AML blasts even in samples with low levels of expression. Targeting CD33 using AMG 330 in primary AML samples led to efficient T-cell activation and expansion as expected from the mechanism of action of BiTE® antibodies. The remarkable ex-vivo activity of AMG 330 supports further development of AMG 330 as an immunotherapy for patients with AML. Disclosures: Kufer: AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Baeuerle:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership.

Targeting The T Cell Component Of The Tumour Microenvironment In Chronic Lymphocytic Leukaemia; A Potential Therapeutic Strategy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4147-4147
Author(s):  
Kirsty M Cuthill ◽  
Andrea Gail Sherman Buggins ◽  
Pj Chana ◽  
Stephen Devereux
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Lymphocytic Leukaemia ◽  
Nuclear Translocation ◽  
Tumour Microenvironment

Abstract It has recently become clear that B cell receptor (BCR) activation plays an important role in the pathogenesis of chronic lymphocytic leukaemia (CLL); a fact that is underlined by the marked efficacy of drugs that inhibit components of this pathway. Although the underlying mechanisms remain unclear, CLL BCRs have been shown to recognize a variety of autoantigens and there is evidence of ongoing activation of a number of downstream signaling molecules including Syk, Erk, Akt and the NFkB and NFAT family of transcription factors. In addition to BCR activation, it is thought that signals from other cells in the tumour microenvironment such as T cells, the vascular endothelium and other stromal cells may also play a role in promoting the growth of the disease. In the present study we chose to revisit the effects of ciclosporin (CsA), a calcineurin antagonist with effects on antigen receptor signaling, in CLL. When this agent is used to treat the autoimmune complications of CLL, concurrent responses in the underlying disease have been noted in about 20% of patients, although the underlying mechanism has not been thoroughly investigated. Since CsA primarily inhibits T cell activation we hypothesized that its effects in CLL might be due to a reduction in T cell mediated co-stimulation in the lymph nodes. We therefore investigated the effect of CsA on the activation of CLL B and T cells using conventional and multispectral imaging flow cytometry to measure the expression of activation markers and the nuclear translocation of NFAT and NFKB family transcription factors. Cells were collected from eight unselected patients with a confirmed diagnosis of CLL for each study. T and B cells were purified by negative immunomagnetic selection and activated by incubation with phorbol ester and ionomycin (PMA/I) or CD40L transfected fibroblasts in the presence of absence of CsA. The activation of CD4+ T cells and CD19+ CLL cells was assessed by staining for CD69/interferon gamma (IFNΥ) and CD69/CD25 respectively. Nuclear translocation of NFATc2 and NFKB p65 was measured by image flow cytometry (Amnis Imagestream). Leukaemia and Lymphoma Research provided the funding for this study. NFkB(p65) translocation at 30 minutes was inhibited by a mean of 22.5% (p=0.0003) in activated CLL CD4+ T cells treated with CsA compared to those treated with vehicle control (VC). Similarly, in the presence of CsA, NFAT-c2 translocation was inhibited by a mean of 24.3% (p=0.008) at 10 minutes in CLL CD4+ T cells compared to those treated with VC. NFkB(p65) translocation was not inhibited (mean of differences=0.63%, p=0.645) and NFAT-c2 translocation was minimally inhibited (mean of differences = -4%, p = 0.007) in activated CLL B Cells treated with CsA. The proportion of activated CLL CD4+ T cells expressing both CD69 and IFNΥ was reduced by 13.2% (p=0.003) in the presence of CsA whereas there was no inhibition of CD25(-1.5, p=0.16) and CD69(-1.4, p=0.5) expression in activated CLL B cells treated with CsA. In summary, CsA had a profound effect on CD4+ T cell activation in patients with CLL, as demonstrated by the reduction in NFkB (p65), NFAT-c2 nuclear translocation and CD69/IFNΥ expressing cells. In contrast, there was a minimal effect on NFAT-c2 translocation in activated CLL B cells and no impact on NFkB (p65) translocation or the expression of CD25 and CD69. These findings suggest that the previously documented activity of CsA in CLL is not due to a direct effect on the tumour but is instead indirect and mediated through inhibition of other microenvironment derived signals such as those provided by activated CD4+ T cells. Since it is likely that these co-stimulatory effects act in concert other signals, such as those induced by BCR activation, reexamination of CsA and similar agents in CLL would thus seem warranted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals P06.02 Enhancing CAR T cell persistence and memory through modulating mitochondrial function

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A42.1-A42
Author(s):  
A Hosseini Rad ◽  
G Min Yi Tan ◽  
A Poudel ◽  
A McLellan
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Viability ◽  
T Cell Activation ◽  
Mitochondrial Function ◽  
Luciferase Reporter ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundCAR T cell therapy for solid tumours has achieved limited success compared to its application to B cell malignancies. One reason for this failure is the low differentiation rate to memory subsets and low persistence of CAR T cells due to activation-induced cell death (AICD) in lymphoid tissue and the tumour microenvironment. In this study, we have expressed the MCL1 gene within CAR T cells to overcome losses by AICD in adoptively transferred T cells. The MCL1 gene expresses two isoforms; the long isoform localises to the outer membrane of mitochondria and inhibits the CD95 signalling death pathway, while the short isoform localises to the inner membrane of mitochondria to enhance mitochondrial oxidation, phosphorylation and fusion. In addition, we have also utilized a microRNA (miR) 429 to promote memory T cell formation through the suppression of genes such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell activation inhibitor, mitochondrial (TCAIM) and mitochondrial fission factor (MFF).Materials and MethodsOverexpression of MCL1 was confirmed at both mRNA and protein level by real time RT-PCR (qPCR) and western blot. Similarly, overexpression of miR-429 was measured by qPCR and specific binding of miR-429 to the 3′ UTR of target genes was confirmed by luciferase reporter assay. Mitochondrial depolarization and cell viability were assessed by TMRE mitochondrial membrane potential assay (flow-cytometry) and resazurin assay. The effect of MCL1 or miR429 overexpression on HER2-CAR T cells was determined by flow cytometry. Soluble leucine-zipper CD95L (https://www.addgene.org/104349/) was expressed and purified from Expi293 cells.ResultsOverexpression of MCL1 in both Jurkat T cells and primary human T cells protected cells against mitochondria depolarization as well as the loss of cell viability in response to CD95L-triggering. Expression of miR429 downregulated TIA1, TCAIM and MFF. A HER2-CAR construct with either MCL1 or miR429 in a lentiviral system was successfully designed and transduced into primary T cells. Mitochondria in transduced T demonstrated enlarged and fusion morphology - a classic feature of memory T cells.ConclusionsOverexpressing MCL1 or miR429 significantly improves mitochondrial function in T cells. This approach will be used to increase persistence of adoptively transferred CAR T cells.Disclosure InformationA. Hosseini Rad: None. G. Min Yi Tan: None. A. Poudel: None. A. McLellan: None.

scholarly journals EXTH-45. DELTA-24-RGDOX ACTIVATION OF THE IDO-KYN-AHR CASCADE IN GLIOBLASTOMA: OLD TARGETS FOR A NEW THERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii96-ii97
Author(s):  
Teresa Nguyen ◽  
Dong Ho Shin ◽  
Hong Jiang ◽  
Derek Wainwright ◽  
Sagar Sohoni ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Oncolytic Viruses ◽  
Immune Memory ◽  
Tryptophan Depletion

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases proliferation and induces apoptosis of surrounding effector T-cells. Kynurenine, a metabolite of tryptophan, induces T-cell differentiation into immunosuppressive Tregs. Excess kynurenine elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, Delta-24-RGDOX, triggers IFNγ production contributing to a positive IDO-Kynurenine-AhR feedback loop. We hypothesized that combining Delta-24-RGDOX with IDO inhibitors will synergize to effectively treat glioblastoma. We characterized IDO and AhR in Delta-24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo. We also observed induction of AhR in CD4+ and CD8+ T-cells by Delta-24-RGDOX in vivo. Delta-24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. We used a murine glioblastoma model to test the efficacy of combining Delta-24-RGDOX with IDO inhibitor, 1MT/indoximod; the combination produced 30% more long-term survivors compared Delta-24-RGDOX alone (P=0.03), which we showed, through lymphocytic depletion studies, was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and myeloid derived suppressor cells compared to Delta-24-RGDOX single treatment (P≤0.05). This microenvironment remodeling correlated with complete tumor elimination. Altogether, Delta-24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression.

scholarly journals Human Memory CD4+T Cell Immune Responses against Giardia lamblia

2015 ◽  
Vol 23 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Christina Skår Saghaug ◽  
Steinar Sørnes ◽  
Dimitra Peirasmaki ◽  
Staffan Svärd ◽  
Nina Langeland ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Giardia Lamblia ◽  
Mononuclear Cells ◽  
T Cell Response ◽  
Effector Memory ◽  
Content Type ◽  
Tnf Α

ABSTRACTThe intestinal protozoan parasiteGiardia lambliamay cause severe prolonged diarrheal disease or pass unnoticed as an asymptomatic infection. T cells seem to play an important role in the immune response toGiardiainfection, and memory responses may last years. Recently, TH17 responses have been found in three animal studies ofGiardiainfection. The aim of this study was to characterize the human CD4+T cell responses toGiardia. Peripheral blood mononuclear cells (PBMCs) were obtained from 21 returning travelers with recent or ongoing giardiasis and 12 low-risk healthy controls and stimulatedin vitrowithGiardia lambliaproteins. Production of tumor necrosis factor alpha (TNF-α), gamma interferon, interleukin-17A (IL-17A), IL-10, and IL-4 was measured in CD4+effector memory (EM) T cells after 24 h by flow cytometry. After 6 days of culture, activation and proliferation were measured by flow cytometry, while an array of inflammatory cytokine levels in supernatants were measured with multiplex assays. We found the number of IL-17A-producing CD4+EM T cells, as well as that of cells simultaneously producing both IL-17A and TNF-α, to be significantly elevated in theGiardia-exposed individuals after 24 h of antigen stimulation. In supernatants of PBMCs stimulated withGiardiaantigens for 6 days, we found inflammation-associated cytokines, including 1L-17A, as well as CD4+T cell activation and proliferation, to be significantly elevated in theGiardia-exposed individuals. We conclude that symptomaticGiardiainfection in humans induces a CD4+EM T cell response of which IL-17A production seems to be an important component.

scholarly journals Epigenetic mechanisms, T-cell activation, andCCR5genetics interact to regulate T-cell expression of CCR5, the major HIV-1 coreceptor

2015 ◽  
Vol 112 (34) ◽  
pp. E4762-E4771 ◽  
Author(s):  
German G. Gornalusse ◽  
Srinivas Mummidi ◽  
Alvaro A. Gaitan ◽  
Fabio Jimenez ◽  
Veron Ramsuran ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Methylation Status ◽  
T Cell Subsets ◽  
Critical Determinant ◽  
Expression Levels ◽  
Cell Expression ◽  
Hiv Aids

T-cell expression levels of CC chemokine receptor 5 (CCR5) are a critical determinant of HIV/AIDS susceptibility, and manifest wide variations (i) between T-cell subsets and among individuals and (ii) in T-cell activation-induced increases in expression levels. We demonstrate that a unifying mechanism for this variation is differences in constitutive and T-cell activation-induced DNA methylation status ofCCR5 cis-regulatory regions (cis-regions). Commencing at an evolutionarily conserved CpG (CpG −41),CCR5 cis-regions manifest lower vs. higher methylation in T cells with higher vs. lower CCR5 levels (memory vs. naïve T cells) and in memory T cells with higher vs. lower CCR5 levels. HIV-related and in vitro induced T-cell activation is associated with demethylation of thesecis-regions.CCR5haplotypes associated with increased vs. decreased gene/surface expression levels and HIV/AIDS susceptibility magnify vs. dampen T-cell activation-associated demethylation. Methylation status ofCCR5intron 2 explains a larger proportion of the variation in CCR5 levels than genotype or T-cell activation. The ancestral, protectiveCCR5-HHA haplotype bears a polymorphism at CpG −41 that is (i) specific to southern Africa, (ii) abrogates binding of the transcription factor CREB1 to thiscis-region, and (iii) exhibits a trend for overrepresentation in persons with reduced susceptibility to HIV and disease progression. Genotypes lacking theCCR5-Δ32 mutation but with hypermethylatedcis-regions have CCR5 levels similar to genotypes heterozygous forCCR5-Δ32. In HIV-infected individuals,CCR5 cis-regions remain demethylated, despite restoration of CD4+ counts (≥800 cells per mm3) with antiretroviral therapy. Thus, methylation content ofCCR5 cis-regions is a central epigenetic determinant of T-cell CCR5 levels, and possibly HIV-related outcomes.

T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4692-4692
Author(s):  
Mauro Di Ianni ◽  
Lorenzo Moretti ◽  
Beatrice Del Papa ◽  
Maria De Ioanni ◽  
Adelmo Terenzi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Activated T Cells ◽  
Mutational Status ◽  
The Impact

Abstract As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.

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