024 Comparative analysis of ex vivo assays aimed at identifying Desmoglein 3 reactive CD4+ T cells in pemphigus vulgaris

Abstract Pemphigus vulgaris (PV) is a chronic and potentially life-threatening autoimmune blistering disease. Aberrant mTOR pathway activity is involved in many autoimmune diseases. This study investigated the correlation of mTOR pathway (PI3K/AKT/mTOR/p70S6K) activity with the loss of balance in T helper 2/regulatory T (Th2/Treg) cells in the peripheral blood of PV patients. CD4+ T cells were isolated from 15 PV patients and 15 healthy controls (HCs), the ratios of Th2/CD4+ T cells and Treg/CD4+ T cells, the activity of the mTOR pathway (PI3K/AKT/mTOR/p70S6K), the transcription factors and cytokines of Th2 and Treg cells were detected. Primary CD4+ T cells from PV patients were cultured under Th2- or Treg-polarizing conditions with or without rapamycin in vitro. We found that PV patients showed significantly elevated serum IL-4 when compared with HCs, and serum IL-4 level was positively correlated with the titer of anti-Dsg1/3 antibody and disease severity, while the serum TGF-β level was negatively correlated with the titer of anti-Dsg3 antibody and disease severity. Meanwhile, PV patients showed increased Th2/CD4+ T cell ratio; decreased Treg/CD4+ T cell ratio; elevated mRNA of PI3K, AKT, mTOR and protein of PI3K (P85), AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), p-p70S6K (Thr389), GATA3; reduced protein of forkhead box protein 3. Rapamycin inhibited Th2 cell differentiation and promoted Treg cell differentiation in vitro. These data suggest a close association between mTOR pathway activation and the loss of balance in Th2/Treg cells in peripheral blood of PV patients. Inhibiting mTORC1 can help restore the Th2/Treg balance.

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Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

Acta Marisiensis - Seria Medica ◽

10.2478/amma-2021-0012 ◽

2021 ◽

Vol 67 (2) ◽

pp. 95-101

Author(s):

Monica Vuță ◽

Ionela-Maria Cotoi ◽

Ion Bogdan Mănescu ◽

Doina Ramona Manu ◽

Minodora Dobreanu

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Cytokine Production ◽

Ex Vivo ◽

Gradient Centrifugation ◽

Intracellular Cytokine Staining ◽

Interleukin 2 ◽

Middle Aged

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

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Inconsistent Reversal of HIV-1 Latency Ex Vivo by Antigens of HIV-1, CMV, and Other Infectious Agents

10.21203/rs.3.rs-35386/v1 ◽

2020 ◽

Author(s):

Thomas Vollbrecht ◽

Aaron O. Angerstein ◽

Bryson Menke ◽

Nikesh M. Kumar ◽

Michelli Faria Oliveira ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Ex Vivo ◽

Latent Reservoir ◽

Antigen Specificity ◽

Pcr Assay ◽

Hiv 1

Abstract BackgroundA reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV and other common pathogens to reverse latency. ResultsWe obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. ConclusionsIn this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

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Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

Journal of Experimental Medicine ◽

10.1084/jem.20160576 ◽

2016 ◽

Vol 213 (11) ◽

pp. 2413-2435 ◽

Cited By ~ 53

Author(s):

Yi Wang ◽

Cindy S. Ma ◽

Yun Ling ◽

Aziz Bousfiha ◽

Yildiz Camcioglu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Ex Vivo ◽

Cell Phenotype ◽

Loss Of Function ◽

Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

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Ex Vivo Phenotype and Frequency of Influenza Virus-Specific CD4 Memory T Cells

Journal of Virology ◽

10.1128/jvi.78.13.7284-7287.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 7284-7287 ◽

Cited By ~ 54

Author(s):

Michaela Lucas ◽

Cheryl L. Day ◽

Jessica R. Wyer ◽

Sharon L. Cunliffe ◽

Andrew Loughry ◽

...

Keyword(s):

T Cells ◽

Influenza Virus ◽

Cd4 T Cells ◽

Memory T Cells ◽

Ex Vivo ◽

Class Ii ◽

Low Frequencies ◽

Tetramer Staining ◽

Specific Memory ◽

Memory Cd4 T Cells

ABSTRACT Recent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonpersistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct “central memory” CD62L+ phenotype.

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