Flow cytometric analysis of the stimulatory response of T cell subsets from normal and HIV-1+ individuals to various mitogenic stimuli in vitro

Abstract Background Coronary heart disease (CHD) is causing by the aberrant aggregation of immune cells in plaque. This study aimed to identify abnormal T cell subtypes and inflammatory factors in CHD patients.Methods and results T cell subsets from 187 CHD patients were analyzed using flow cytometry. Plasma concentration of cytokines were analyzed by Luminex. Flow cytometric analysis revealed that the number of ThGM cells was higher in CHD patients. The proportion of Th17 and Th1 cells were also increased in CHD patients. levels of IL-4, IL-5, IL-6, and IL-10 were significantly higher in CHD patients (P<0.05). However, levels of GM-CSF were slightly lower in CHD patients. Conclusions ThGM can be considered as a diagnostic marker of CDH.

Download Full-text

Analysis of In Vitro Effects of Bortezomib (Velcade) on T Cell Prolymphocytic Leukemia (T-PLL).

Blood ◽

10.1182/blood.v108.11.4426.4426 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4426-4426

Author(s):

Fulya Ozpuyan ◽

Paul N. Meyer ◽

Hytham Al-Masri ◽

Hongyu Ni ◽

Serhan Alkan

Keyword(s):

T Cell ◽

Lymphoproliferative Disorder ◽

Flow Cytometric Analysis ◽

Leukemic Cells ◽

Cell Phenotype ◽

Base Line ◽

Prolymphocytic Leukemia ◽

Bortezomib Treatment ◽

Flow Cytometric

Abstract T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoproliferative disorder with postthymic T cell phenotype and prolymphocytic morphology. In the majority of patients, the leukemic process progresses rapidly and patients die shortly after diagnosis (median survival of 7 months). Bortezomib, the first proteasome inhibitor to be approved for use in haematological malignancies such as multiple myeloma, is beginning to be utilized as an effective anti-neoplastic agent in other hematopoietic and non-hematopoietic neoplastic disorders. We report here the in vitro apoptotic effects of bortezomib on leukemic cells isolated from three T-PLL patients. Interestingly, one of the patient’s leukemia developed in the setting of immunosupression due to transplant therapy (post-transplant lymphoproliferative disorder). Flow cytometric analysis of leukemic cells of the three patients showed CD8, double CD4+CD8+ and double CD4−CD8− immunophenotypic features. All cases showed monoclonal band pattern by T-cell receptor (TCR) gene rearrangement as analyzed by the PCR amplification of the TCR gamma heavy chain gene. Freshly isolated leukemic cells with the CD8 phenotype T-PLL analyzed for apoptosis after ficoll hypaque separation and cultured in the presence of various concentration of Bortezomib (0.001, 0.01, 0.1, 1, and 10 uM) for dose curve analysis. Apoptosis of the leukemic cells was determined by Annexin-V and 7-AAD staining and flow cytometric analysis after incubation at 24 and 72 hours, respectively. Samples treated for 72 hours showed higher rate of apoptosis compared to 24 hours: 10 uM (62% increase above the base line of control cells), 1 uM (58%), 0.1 uM (55%), 0.01 uM (40%) and 0.001 uM (0%) concentrations while samples treated for 24 hours with 10 uM showed (42% increase above the base line of control cells) and 1 uM (33% increase above the base line). Light microscopic analysis of leukemic cells treated with Bortezomib confirmed that the majority of cells undergo apoptosis with Bortezomib treatment as it revealed nuclear fragmentation and apoptotic bodies. Leukemic cells recovered from cryopreservation from the second and third T-PLL patient samples analyzed also showed significant increase in early and late apoptosis at 24 hours with Bortezomib treatment (10nm). These results suggest that Bortezomib may provide an alternate therapy in the treatment of T-PLL. Future collaborative efforts investigating efficacy with Bortezomib as a single agent or in combination with other therapeutic agents will be crucial to improving survival for patients with this disease.

Download Full-text

Flow Cytometric Analysis Revealed a Significant Accumulation of Terminally Differentiated T cell Subtypes in the Circulating Lymphocytes of Cytomegalovirus (CMV) Positive Follicular Lymphoma Patients

10.47363/jcbr/2021(3)132 ◽

2021 ◽

pp. 1-9

Author(s):

Lugos MD ◽

◽

Dangana A ◽

Ntuhun BD ◽

Oluwatayo BO ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Follicular Lymphoma ◽

B Cell ◽

Flow Cytometric Analysis ◽

B Cell Lymphoma ◽

T Cell Subsets ◽

Cmv Infection ◽

Flow Cytometric ◽

The Impact

Follicular lymphoma (FL), a non-Hodgkin lymphoma, is an indolent cancer of the B cell lineage that runs a chronic deterioration course that can result in multiple treatment episodes leading to resistance and possible transformation to diffuse large B cell lymphoma. Cytomegalovirus (CMV) reactivation during chemotherapy or after an organ or hematopoietic stem cell transplantation is a major cause of morbidity and mortality. This study tests the hypothesis that some of the heterogeneity of FL might result from chronic infection with Cytomegalovirus (CMV). This research was intended to appraise the impact of CMV infection on the subtypes of T cells in follicular lymphoma patients. We accessed stored peripheral blood mononuclear cells (PMBCs) from patients of known CMV serostatus recruited into an FL clinical trial. We undertook a multicolour flow cytometric analysis of the PBMCs and compared the number of lymphocyte subtypes of CMV-positive and CMV-negative FL patients. Data showed a significant increase in the quantity of terminally differentiated (TEMRA) T cell subsets, including EM3-CD8 (P=0.005), EM3-CD4 (P=0.018), E-CD4 (P=0.029), E-CD8 (P=0.033) and pE2-CD4 (P=0.046) phenotypes, as well as increased NKT cells (P=0.031) among CMV-positive patients compared to the negative group. Our findings support the hypothesis that recurrent infections characterise CMV infection in FL due to accelerated immune senescence and the accumulation of exhausted T cells. Based on the data, a case could be argued for the routine application of CMV screening in FL before treatment with chemo-immunotherapy to implement enhanced infection surveillance in CMV-positive patients. These discoveries can eventually help improve the treatment approaches in the management of FL toward a combinatorial viewpoint for direct cytotoxic and indirect immunomodulatory outlook

Download Full-text

HIV-1-Specific CD11c+ CD8+ T Cells Display Low PD-1 Expression and Strong Anti-HIV-1 Activity

Frontiers in Immunology ◽

10.3389/fimmu.2021.757457 ◽

2021 ◽

Vol 12 ◽

Author(s):

An-Liang Guo ◽

Jin-Fang Zhao ◽

Lin Gao ◽

Hui-Huang Huang ◽

Ji-Yuan Zhang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Subset ◽

T Cell Subsets ◽

Hla Dr ◽

Tnf Α ◽

Treatment Naïve ◽

Vitro Analysis ◽

Hiv 1

Exhaustion of HIV-1-specific CD8+ T cells prevents optimal control of HIV-1 infection. Identifying unconventional CD8+ T cell subsets to effectively control HIV-1 replication is vital. In this study, the role of CD11c+ CD8+ T cells during HIV-1 infection was evaluated. The frequencies of CD11c+ CD8+ T cells significantly increased and were negatively correlated with viral load in HIV-1-infected treatment-naïve patients. HIV-1-specific cells were enriched more in CD11c+ CD8+ T cells than in CD11c- CD8+ T cells, which could be induced by HIV-1-derived overlapping peptides, marking an HIV-1-specific CD8+ T cell population. This subset expressed higher levels of activating markers (CD38 and HLA-DR), cytotoxic markers (granzyme B, perforin, and CD107a), and cytokines (IL-2 and TNF-α), with lower levels of PD-1 compared to the CD11c- CD8+ T cell subset. In vitro analysis verified that CD11c+ CD8+ T cells displayed a stronger HIV-1-specific killing capacity than the CD11c- counterparts. These findings indicate that CD11c+ CD8+ T cells have potent immunotherapeutic efficacy in controlling HIV-1 infection.

Download Full-text

Rapid and Semi-Automated Screening of Multiple Samples for Antigen-Specific T Lymphocytes

Blood ◽

10.1182/blood.v120.21.4839.4839 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4839-4839

Author(s):

Anne Richter ◽

Michaela Niemöller ◽

Inken Verwohl ◽

Katrin Lange ◽

Anna Foerster-Marniok ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Flow Cytometric Analysis ◽

T Cell Response ◽

Activation Markers ◽

Cell Stimulation ◽

Flow Cytometric ◽

T Cell Stimulation ◽

Multiple Samples

Abstract Abstract 4839 Functional characterizations of T lymphocytes are performed to gain understanding on their contribution in certain immunological situations, to monitor the course of diseases, and to track therapeutic interventions. Meanwhile the flow cytometric analysis of antigen-specific T cells examined for intracellular cytokine production and expression of activation markers after a short-term in vitro antigenic challenge is a well-established method for research applications. Despite the advantages of this approach to qualify samples on a single cell level and by multiple parameters, the broad use of this analysis for immune monitoring purposes is hampered. Screening of a lot of samples is time-consuming, requires many manual handling steps, and operator experience in flow cytometric analysis of stimulated T cell samples. To overcome these hurdles, we worked out a complete strategy to rapidly study cytokine and activation marker profiles in antigen-specific T cells of multiple samples by a semi-automated process. For the simultaneous analysis of multiple samples we examined for the T cell stimulation an antigen pre-coated 96-strip-well culture system. This flexible and ready-to-use format provides the opportunity to screen either for a single antigen or in parallel for up to twelve antigen specificities by combining 8-well-strips possessing different antigens. The coated antigens consist of pools of overlapping 15-mer peptides derived from a single viral protein of cytomegalovirus, Epstein-Barr-Virus, or adenovirus. The peptide pools have been designed for activation of the specific CD4+ as well as CD8+ T cells. They are solubilized and thereby accessible for T cell stimulation after addition of a cell sample, e.g. peripheral blood mononuclear cells, suspended in culture medium into the antigen-coated well. After a stimulation period of six hours the induced T cell response is comparable to the activation with a conventional lyophilized and reconstituted peptide pool. To reduce the time and work load for cell harvesting, fixation, permeabilization, and staining, we developed a protocol and reagents to allow a rapid and easy-to-handle intracellular staining procedure. Compared to conventional staining protocols, all steps are executed in the 96-strip-well culture plate, i.e. cell harvesting is dispensable. Without any washing step, cells are fixed and stained with defined reagent cocktails containing antibodies to identify virus-specific CD3+ CD4+ CD154+ and/or CD3+ CD8+ T cells and various Anti-cytokine-fluorochrome conjugates to evaluate the cytokine pattern. With these modifications, we drastically diminished the overall processing time for the staining of up to 96 samples to only 50 minutes. Furthermore, we integrated an automated flow cytometric analysis process. This includes the possibility to measure the samples in the 96-strip-well plates hands-free using pre-defined experiment settings and acquisition templates. We also applied an automated gating strategy for the data analysis. Finally, a report summarizes the results of the T cell response against several viral proteins for all samples tested, e.g. frequencies of cytokine+ CD154+ CD4+ and cytokine+ CD8+ T cell subsets are indicated. Hands-on time for the multi-sample acquisition and analysis is only minimal and the standardized reagents/protocol and sample analysis process decrease inter- and intra-assay variations. In summary, with our newly developed tools and protocols for in vitro T cell stimulation, staining of activation markers as well as intracellular cytokines, and automated flow cytometric analysis we have set up a fast and convenient procedure to routinely monitor antigen-specific T cell responses. Disclosures: Richter: Miltenyi Biotec GmbH: Employment. Niemöller:Miltenyi Biotec GmbH: Employment. Verwohl:Miltenyi Biotec GmbH: Employment. Lange:Miltenyi Biotec GmbH: Employment. Foerster-Marniok:Miltenyi Biotec GmbH: Employment. Brauns:Miltenyi Biotec GmbH: Employment. Kramer:Miltenyi Biotec GmbH: Employment. Höher-Peters:Miltenyi Biotec GmbH: Employment. Büscher:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment.

Download Full-text

Flow cytometric analysis of T cell subsets in paired samples of cerebrospinal fluid and peripheral blood from patients with neurological and psychiatric disorders

Brain Behavior and Immunity ◽

10.1016/j.bbi.2008.08.003 ◽

2009 ◽

Vol 23 (1) ◽

pp. 134-142 ◽

Cited By ~ 32

Author(s):

Horst-G. Maxeiner ◽

Markus Thomas Rojewski ◽

Anita Schmitt ◽

Hayrettin Tumani ◽

Karl Bechter ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

T Cell ◽

Psychiatric Disorders ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

T Cell Subsets ◽

Paired Samples ◽

Flow Cytometric

Download Full-text

Cytotoxic CD8+ T Cells Expressing CXCR5 Are Detectable in HIV-1 Elite Controllers After Prolonged In Vitro Peptide Stimulation

Frontiers in Immunology ◽

10.3389/fimmu.2020.622343 ◽

2021 ◽

Vol 11 ◽

Author(s):

Philipp Adams ◽

Gilles Iserentant ◽

Jean-Yves Servais ◽

Linos Vandekerckhove ◽

Guido Vanham ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Subsets ◽

Color Flow ◽

Cell Immunity ◽

Ifn Γ ◽

Peptide Stimulation ◽

Hiv Controllers ◽

Hiv 1

Antiretroviral therapy (ART) is not curative as HIV-1 persists in long-lived viral reservoirs. Consequently, patients are dependent on life-long drug adherence with possible side effects. To overcome these limitations strategies of a functional cure aim at ART free viral remission. In this study, we sought to identify detailed subsets of anti-viral CD8+ T cell immunity linked to natural long-term control of HIV-1 infection. Here, we analyzed HIV controllers and ART suppressed progressors for in vitro viral suppressive capacity (VSC) at baseline and after peptide stimulation. Functional properties and phenotypes of CD8+ T cells were assessed by IFN-γ ELISPOT and 18 color flow cytometry. HIV controllers showed significantly increased suppression at baseline as well as after peptide stimulation. IFN-γ secretion and the proliferation marker Ki67 positively correlated with VSC. Moreover, the detailed phenotype of three distinct multifunctional memory CD8+ T cell subsets were specific traits of HIV controllers of which two correlated convincingly with VSC. Our results underline the importance of multifunctional CD8+ T cell responses during natural control. Especially the role of CXCR5 expressing cytotoxic subsets emphasizes potential surveillance in sites of reservoir persistence and demand further study.

Download Full-text

Antiapoptotic Clone 11-Derived Peptides Induce In Vitro Death of CD4+ T Cells Susceptible to HIV-1 Infection

Journal of Virology ◽

10.1128/jvi.00611-20 ◽

2020 ◽

Vol 94 (14) ◽

Cited By ~ 1

Author(s):

Anastassia Mikhailova ◽

José Carlos Valle-Casuso ◽

Annie David ◽

Valérie Monceaux ◽

Stevenn Volant ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Subsets ◽

T Cell Memory ◽

Target Cells ◽

Cytotoxic Action ◽

Cell Memory ◽

Infected Cells ◽

Hiv 1

ABSTRACT HIV-1 successfully establishes long-term infection in its target cells despite viral cytotoxic effects. We have recently shown that cell metabolism is an important factor driving CD4+ T cell susceptibility to HIV-1 and the survival of infected cells. We show here that expression of antiapoptotic clone 11 (AAC-11), an antiapoptotic factor upregulated in many cancers, increased with progressive CD4+ T cell memory differentiation in association with the expression of cell cycle, activation, and metabolism genes and was correlated with susceptibility to HIV-1 infection. Synthetic peptides based on the LZ domain sequence of AAC-11, responsible for its interaction with molecular partners, were previously shown to be cytotoxic to cancer cells. Here, we observed that these peptides also blocked HIV-1 infection by inducing the death of HIV-1-susceptible primary CD4+ T cells across all T cell subsets. The peptides targeted metabolically active cells and had the greatest effect on effector and transitional CD4+ T cell memory subsets. Our results suggest that the AAC-11 survival pathway is potentially involved in the survival of HIV-1-infectible cells and provide proof of principle that some cellular characteristics can be targeted to eliminate the cells offering the best conditions to sustain HIV-1 replication. IMPORTANCE Although antiretroviral treatment efficiently blocks HIV multiplication, it cannot eliminate cells already carrying integrated proviruses. In the search for an HIV cure, the identification of new potential targets to selectively eliminate infected cells is of the outmost importance. We show here that peptides derived from antiapoptotic clone 11 (AAC-11), whose expression levels correlated with susceptibility to HIV-1 infection of CD4+ T cells, induced cytotoxicity in CD4+ T cells showing the highest levels of activation and metabolic activity, conditions known to favor HIV-1 infection. Accordingly, CD4+ T cells that survived the cytotoxic action of the AAC-11 peptides were resistant to HIV-1 replication. Our results identify a new potential molecular pathway to target HIV-1 infection.

Download Full-text

Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4+ T Cells

Journal of Virology ◽

10.1128/jvi.00969-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 17

Author(s):

Deanna A. Kulpa ◽

Aarthi Talla ◽

Jessica H. Brehm ◽

Susan Pereira Ribeiro ◽

Sally Yuan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

T Cell Subsets ◽

Effector Memory ◽

Cell Surface Markers ◽

Cell Cycle Entry ◽

Latent Hiv ◽

Hiv 1

ABSTRACT During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal. IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

Download Full-text