scholarly journals T Cell Activation By OKT3 Is a Function of the Percent Monocytes in PBMC of Healthy Donors and MDS Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4870-4870
Author(s):  
Alison Tarke ◽  
Valentina Ferrari ◽  
Hannah Fields ◽  
Luca Ferrari ◽  
Franco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Transplant ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Healthy Donors

Background: Myelodysplastic Syndromes (MDS) are a heterogeneous hematologic malignancy characterized by bone marrow failure and cytopenias. The median survival rate for patients with higher-risk MDS who fail standard-of-care chemotherapy with hypomethylating agents (HMAs) is less than 6 months, and the only curative treatment for these patients is hematopoietic stem cell transplant (HSCT). Over the past 10 years, immunotherapy as a cancer treatment has achieved variable levels of success in different tumor types. There are currently 22 active clinical trials of immunotherapies for MDS (www.clinicaltrials.gov; 7/30/19), including our phase I clinical trial with a personalized adoptive cellular therapy targeting MDS patient neoantigens (NCT 03258359). Because MDS patients are frequently monocytopenic and the existing literature is inconsistent regarding the ability of MDS patients' monocytes to support T cell activation, we compared the activation of MDS T cells with those of healthy donors in the presence of autologous monocytes. Methods: Peripheral blood mononuclear cells (PBMC) from 5 healthy donors and 7 higher-risk MDS patients were cryopreserved after Ficoll separation. These PBMC were thawed and aliquoted into 6 replicate wells of 200,000 cells in 96-well u-bottom plates in R-10 culture medium. Half of the wells were treated with 25 ng/mL OKT3 and 200 U/mL IL-2. After 48 hours at 37˚C with 5% CO2, the wells were collected for analysis by flow cytometry. Beads were used to detect T cell activation induced secretion of IFNg, TNFa, IL-4, IL-10, and IL-17 in the supernatant and fluorescent antibodies were used to phenotype viable cells for CD3, CD4, CD8, and the T cell activation markers, CD69, CD25, CTLA-4, PD-1, and HLA-DR. Results: We measured a higher release of IFNg and TNFa in donor PBMC compared to MDS patients after OKT3/IL-2 activation, p < 0.01 and 0.04, respectively by 2-way ANOVA. The expression of CD69, CD25, HLA-DR, and CTLA4 increased variably on activated T cells from donors or MDS patients, but expression of CD4+CD25+ was more frequent on donor T cells after activation (p = 0.03). Activation also resulted in a higher frequency of PD-1 expression on donor CD4+ and CD8+ T cells than on MDS T cells (p < 0.01 and < 0.01, respectively). Interestingly, on both MDS and normal T cells the percentage of CD8+PD1+ activated cells correlated strongly with the percent of CD14+ monocytes present in the PBMC (R2 = 0.92 and 0.60 respectively; Fig 1a and 1b). We designed further experiments to test whether this was a patient intrinsic phenomenon, or if the absolute number of CD14+ monocytes in the PBMC was associated with different levels of PD1 expression upon T cell activation. First, we separated CD14+ cells from the PBMC of a patient with MDS using magnetic beads. Then CD14+ cells were added back to the CD14-depleted PBMC at a final percent of 0.5, 5, 10, 20, 35, 70, or 100% of the original amount. Unmodified PBMC was included as a control and all cells were stimulated with OKT3 and IL-2 or left in R-10 medium without stimulus. After 24, 48, and 70 hours, samples were collected to analyze by flow cytometry for CD3, CD4, CD8, CD14, and PD1 expression. The results show that an increasing percent of monocytes corresponded to the increased expression of PD1 on CD8+ and CD4+ T cells. Conclusion: Our results show that there are variable reductions in markers of T cell activation and cytokine secretion in MDS patients compared to healthy donors. We also observed that the fold increase in activation induced PD-1 expression was well correlated with the percent of CD14+ monocytes in the PBMC of both MDS patients and healthy donors. Direct experimentation revealed that this correlation is a cause-effect relationship. We are continuing to investigate the role of monocytes in T cell activation in MDS patients. Disclosures Bejar: Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Lane:PersImmune, Inc.: Employment.

Demonstration of a Direct Immunomodulatory Role for Vitamin D in Human T Cells Using Flow Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2577-2577
Author(s):  
Richard W. Joseph ◽  
Tae Kon Kim ◽  
Lisa St. John ◽  
Jahan Khalili ◽  
Uday R Popat ◽  
...  
Keyword(s):  
Vitamin D ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
Activation Markers

Abstract Clinical and epidemiological studies have demonstrated an increasingly stronger link between Vitamin D deficiency and a broad array of illnesses characterized by inflammation, including autoimmune diseases, coronary artery disease, and cancers. Vitamin D is a steroid hormone that exerts the majority of its biologic effects via the binding of the intracellular Vitamin D receptor (VDR). While upregulation of VDR has been demonstrated in activated bulk T cells using traditional approaches (e.g., western blotting), such assays cannot precisely define VDR distribution and kinetics. To overcome these limitations, we developed what we believe to be the first flow cytometric assay to quantify VDR expression at a single-cell level. We used a primary antibody against VDR (mouse monoclonal IgG2a to human VDR) in permeabilized T cells followed by a labeled secondary antibody. We detected a positive cell population using flow cytometry that was sharply increased following activation, consistent with upregulation of VDR confirmed by immunoblotting of sorted cells. We then applied this validated assay to define the kinetics of VDR upregulation in activated T cells. We stimulated PBMC with PMA:Ionomycin (P:I) for varying intervals and assessed intracellular VDR using flow cytometry. VDR is significantly upregulated by 15 min after stimulation, reaches a plateau after 6 hr, and may remain elevated for up to 7 d. We compared VDR to classical early and late T cell activation markers (CD69 and CD25, respectively), and we found that VDR was upregulated as consistently as (but even earlier than) CD69, and that VDR and CD25 were both consistently upregulated at later intervals (p&lt;0.0001). To examine the association between VDR expression and proliferation, we stimulated CFSE-labeled T cells with OKT3 (2mg/ml) for 5 d and found that proliferating T cells expressed a significantly higher level of VDR than resting T cells, which maintained baseline VDR expression (p&lt;0.0001). To assess the association between T cell cytokine production and VDR expression, we stimulated T cells with (P:I) for 6 hr in the presence of brefeldin A, and we confirmed that all cytokine-producing cells (TNFα, IL-2, IFNγ) were contained within the VDR-high population. We then assessed whether physiologic concentrations of Vitamin D could inhibit T cell proliferation in vitro. We stimulated CFSE-labeled PBMC with either OKT3 or irradiated allogeneic dendritic cells (DC) in the presence or absence of physiologic concentrations of calcitriol (50 nm) for 5 to 7 d. The presence of calcitriol during OKT3 stimulation resulted in significantly reduced cell division (p=0.004, n=5). Using a previously validated phenotype to demarcate activated alloreactive CD4+ T cells (CD4hiCD38+), we demonstrated that physiologic calcitriol supplementation decreased alloreactive activation following 7 d stimulation with allogeneic DC (p=0.0003, n=10). In conclusion, VDR is a consistent and specific early and late marker of T cell activation, suggesting a direct role for the Vitamin D axis in immunoregulation. Furthermore, physiological concentrations of Vitamin D can inhibit T cell proliferation induced by polyclonal stimuli, including allogeneic DC. These data provide confirmation for a direct immunoregulatory role for Vitamin D and suggest that further mechanistic and clinical studies may yield novel therapeutic strategies for inflammatory conditions, including graft-versus-host disease.

CVID patients with autoimmunity have elevated T cell expression of granzyme B and HLA-DR and reduced levels of Treg cells

2012 ◽  
Vol 66 (2) ◽  
pp. 146-150 ◽  
Author(s):  
Clive R D Carter ◽  
Ganesha Aravind ◽  
Natuley L Smalle ◽  
June Y Cole ◽  
Sinisa Savic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Granzyme B ◽  
Treg Cells ◽  
T Cell Subsets ◽  
Activated T Cells ◽  
Activation Markers ◽  
Hla Dr

AimsCommon variable immunodeficiency (CVID) is a primary antibody immunodeficiency with approximately 20% of patients reporting additional autoimmune symptoms. The primary aim of this study was to compare the levels of activated and regulatory T cells (Treg cells) in CVID patients in an attempt to clarify their possible interactions leading to the generation of autoimmunity.MethodsImmunophenotyping of T cells was performed by flow cytometry using a whole blood approach. Surface expression of human leukocyte antigen HLA class II DR and intracellular levels of granzyme B in T cell subsets were assessed; Treg levels were measured using CD4 CD25, FOXp3 and CTLA-4.ResultsCVID patients had higher levels of granzyme B and HLA-DR on CD8+ T cells compared with control values (mean of 59% vs 30% and 45% vs 21%, respectively). Patients also had reduced levels of Treg cells compared with control values (con mean=3.24% vs pat=2.54%). Patients with autoimmunity (5/23) had a similar level of T cell activation markers to the rest of the patients but with lower Treg cells (mean of 1.1%) and reduced CD25 and CTLA-4 expression. Patients with autoimmunity had a higher ratio of activated to Treg cells compared with patients with no autoimmune symptoms.ConclusionsThese results highlight that reduced levels of Treg cells were associated with elevated levels of activated T cells, suggesting that reduced Treg cells in these patients may have functional consequences in allowing exaggerated T cell responses.

scholarly journals Modulation of T-Cell Activation Markers Expression by the Adipose Tissue–Derived Mesenchymal Stem Cells of Patients with Rheumatic Diseases

2020 ◽  
Vol 29 ◽  
pp. 096368972094568
Author(s):  
Ewa Kuca-Warnawin ◽  
Iwona Janicka ◽  
Piotr Szczęsny ◽  
Marzena Olesińska ◽  
Krzysztof Bonek ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Activation Markers ◽  
Cd25 Expression ◽  
Hla Dr

Background: Activated T lymphocytes play an important role in the pathogenesis of rheumatic diseases (RD). Mesenchymal stem cells (MSCs) possess immunoregulatory activities but such functions of MSCs from bone marrow of systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and ankylosing spondylitis (AS) patients are impaired. Adipose tissue–derived MSCs (ASCs) are an optional pool of therapeutically useful MSCs, but biology of these cells in RD is poorly known. This study aimed at investigating the effect of ASCs from RD patients and healthy donors (HD) on the expression of the key T-cell activation markers. Methods: ASCs were isolated from subcutaneous abdominal fat from SLE ( n = 16), SSc ( n = 18), and AS ( n = 16) patients, while five human ASCs lines from HD were used as a control. Untreated and cytokine (tumor necrosis factor α + interferon γ)-treated ASCs were co-cultured with allogenic, mitogen (phytohemagglutinin)-stimulated peripheral blood mononuclear cells (PBMCs) or purified anti-CD3/CD28-activated CD4+ T lymphocytes. Contacting and noncontacting ASCs-PBMCs co-cultures were performed. RD/ASCs were analyzed in co-cultures with both allogeneic and autologous PBMCs. Flow cytometry analysis was used to evaluate expression of CD25, HLA-DR, and CD69 molecules on CD4+ and CD8+ cells. Results: In co-cultures with allogeneic, activated CD4+ T cells and PBMCs, HD/ASCs and RD/ASCs downregulated CD25 and HLA-DR, while upregulated CD69 molecules expression on both CD4+ and CD8+ cells with comparable potency. This modulatory effect was similar in contacting and noncontacting co-cultures. RD/ASCs exerted weaker inhibitory effect on CD25 expression on autologous than allogeneic CD4+ and CD8+ T cells. Conclusion: RD/ASCs retain normal capability to regulate expression of activation markers on allogeneic T cells. Both HD/ASCs and RD/ASCs exert this effect independently of their activation status, mostly through the indirect pathway and soluble factors. However, autologous CD4+ and CD8+ T cells are partially resistant to RD/ASCs inhibition of CD25 expression, suggesting weaker control of T-cell activation in vivo.

scholarly journals HIV and Tuberculosis co-infection impacts T-cell activation markers but not the numbers subset of regulatory T-cells in HIV-1 infected patients

2013 ◽  
Vol 2 (1) ◽  
Author(s):  
Moustapha Mbow ◽  
Ndèye S.S. Santos ◽  
Makhtar Camara ◽  
Awa Ba ◽  
Aliou Niang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Hiv Infection ◽  
T Cell Activation ◽  
Cell Activation ◽  
Treg Cells ◽  
Activation Markers ◽  
Hla Dr ◽  
Hiv 1

Background: Tuberculosis (TB) has been shown to accelerate the clinical course of HIV infection, but the mechanisms by which this occurs are not well understood. Regulatory T-cells (Tregs)are known to dampen hyperactivation of the immune cells, but it remains unclear whether hyperactivation of T-cells in HIV infection is associated with a decrease of Tregs and what the effect Mycobacterium tuberculosis (MTB) co-infection has on T-cell activation and Tregs.Objectives: In this study, we aim to evaluate whether active TB is associated with the increased expression of T-cell activation markers and reduced number of Treg cells in HIV-1-infected patients.Methods: This study was conducted on 69 subjects consisting of 20 HIV-infected patients,20 HIV and MTB co-infected patients, 19 MTB-infected patients and 10 uninfected control subjects negative for both MTB and HIV. The frequencies of T-cell activation markers (CD38 and HLA-DR) and Treg cells (CD4+CD25+CD127-) were measured by flow cytometry.Results: Significantly higher expression of CD38 and HLA-DR on CD4+ and CD8+ T-cells was found in MTB and HIV co-infected patients compared with HIV-infected patients. However,no significant difference in the percentage of Treg cells was reported between HIV patients with TB and those without. The study also showed a negative correlation between regulatoryT-cells frequency and CD4+ T-cell counts.Conclusion: These results suggest that TB enhances the expression of peripheral T-cell activation markers during HIV infection, whilst having no impact on the percentages of Tregcells.

Flow Cytometry Analysis of Peripheral Blood CD4+/CD25+/FOXP3+ T Regulator Cells from 11 Patients Treated with Ipilimumab Following Relapse of Malignancy after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3240-3240
Author(s):  
Jiehua Zhou ◽  
Ruikun Zhong ◽  
Sue Corringham ◽  
Teresa Sapp ◽  
Robert Soiffer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Single Dose ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Activated T Cells ◽  
Hematopoietic Stem

Abstract We conducted a multi-center dose-escalation trial to assess whether CTLA-4 blockade is safe and efficacious as immunotherapy in patients with relapse of malignancy following allogeneic hematopoietic stem cell transplantation (allo-HCT). We hypothesized that since CTLA-4 is a negative regulator of effector T-cell activation, inhibition of CTLA-4 ligation with a blocking human monoclonal antibody (ipilimumab) will augment graft-versus-malignancy. Here we report on the effects of a single dose of ipilimumab on in vivo T cell subsets from 11 patients treated at the highest dose level (3 mg/kg) and compare these findings with those in normal volunteer donor lymphocytes. We analyzed the expression of intracellular CTLA-4 and FOXP3 on CD4+/CD25+ Treg cells, intracellular cytokines and surface markers for T-cell activation on peripheral T cells from 11 patients and 9 normal donors. Peripheral blood mononuclear cells (PBMC) from patients before (day 0) and after the treatment at day 1, 3, 7, 14 and 30 were stained with a panel of antibodies and analyzed by flow cytometry. Lymphocytes from normal donors at time zero and 3 days after culture in IL-2 (200u/ml) were used as controls. The pre-treatment expression of intracellular CTLA-4 was significantly higher in CD4+ T cells from patients than normal controls and was increased further after antibody treatment from 7.1±3.8% at day 0 to 18.2±7.1% at day 30 (p=0.02). Although the expression of FOXP3 in CD4+/CD25+ T cells was higher in patients than in normal donors (6.3±4.8% compared with 2.0±1.6%, p=0.02), there was no significant change in the levels following ipilimumab infusion. The expression of CD4+/CD25high in the patients was 7.7±2.8%, higher than the normal donors (2.3±1.1%). However, 6/11 cases had increased expression while others had decrease or no change, overall there was no statistically significant change. CD4+/CD25low activated T cells were elevated in 10/11 patients before ipilimumab (42.1±8.5%). Their levels were not affected by CTLA-4 blockade. CD8+/CD69+ activated T-cells were significantly increased in 8/11 patients within the 30 days after ipilimumab treatment but typically returned to baseline values on longer follow-up. CD4+/CD69+ and CD4+/HLA-DR+ T-cells were unchanged following ipilimumab treatment. These data show that a single dose of ipilimumab enhances levels of some subsets of activated T cells without a significant effect on cells with a T-regulatory phenotype.

scholarly journals CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 580-588 ◽  
Author(s):  
Kathrin Gollmer ◽  
François Asperti-Boursin ◽  
Yoshihiko Tanaka ◽  
Klaus Okkenhaug ◽  
Bart Vanhaesebroeck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Early Time ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

scholarly journals TRPM2 Is Not Required for T-Cell Activation and Differentiation

2022 ◽  
Vol 12 ◽  
Author(s):  
Niels C. Lory ◽  
Mikolaj Nawrocki ◽  
Martina Corazza ◽  
Joanna Schmid ◽  
Valéa Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transient Receptor Potential ◽  
Cell Function ◽  
Intestinal Inflammation ◽  
Activation Markers

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

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.

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.

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