Article

A sensitive method for detecting proliferation of rare autoantigen-specific human T cells

Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.
Journal of Immunological Methods (Impact Factor: 1.82). 01/2004; 283(1-2):173-83. DOI: 10.1016/j.jim.2003.09.004
Source: PubMed

ABSTRACT

The ability to measure proliferation of rare antigen-specific T cells among many bystanders is critical for the evaluation of cellular immune function in health and disease. T-cell proliferation in response to antigen has been measured almost exclusively by 3H-thymidine incorporation. This method does not directly identify the phenotype of the proliferating cells and is frequently not sufficiently sensitive to detect rare autoantigen-specific T cells. To overcome these problems, we developed a novel assay for antigen-specific human T-cell proliferation. Peripheral blood mononuclear cells (PBMC) were labelled with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) and cells that proliferated in response to antigen, with resultant reduction in CFSE intensity, were measured directly by flow cytometry. This assay was more sensitive than 3H-thymidine incorporation and detected the proliferation of rare antigen-specific CD4(+) T cells at 10-fold lower antigen concentrations. It also allowed the phenotype of the proliferating cells to be directly determined. Using the CFSE assay we were able to measure directly the proliferation of human CD4(+) T cells from healthy donors in response to the type 1 diabetes autoantigens glutamic acid decarboxylase (GAD) and proinsulin (PI).

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Available from: Stuart I Mannering, Jul 09, 2014
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    • "For CB T-cell responses, T cells (CD4+ or CD4+CD25−) were labeled with CFSE (Invitrogen, Carlsbad, CA) as previously described (9). CFSE-labeled T cells (1.5 × 105) were added to autologous mature DCs loaded with or without antigen at a ratio of 1 DC:40 T cells in round-bottom 96-well plates. "
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    ABSTRACT: Islet autoimmunity precedes type 1 diabetes onset. We previously found that islet autoimmunity rarely starts before age six months but reaches its highest incidence already at around 1 year of age. We now examine whether homeostatic expansion and immune competence changes seen in a maturating immune system may account for this marked variation in islet autoimmunity risk in the first year of life. We found naïve proinsulin- and GAD65-responsive T cells in cord blood of healthy newborns, with highest responses observed in children with type 1 diabetes susceptible HLA-DRB1/DQB1 genotypes. Homeostatic expansion characteristics with increased IL-7 concentrations and enhanced T cell responsiveness to IL-7 were observed throughout the first year of life. However, the ability of antigen-presenting cells to activate naïve T cells was compromised at birth, and cord blood monocytes had low surface expression of CD40 and HLA class II. In contrast, antigen presentation and expression of these molecules had reached competent adult levels by the high incidence age of 8 months. We propose that temporal changes in islet autoimmunity seroconversion in infants are a consequence of the changing balance between homeostatic drive and antigen presentation competence. These findings are relevant for early prevention of type 1 diabetes.
    Full-text · Article · Jan 2013 · Diabetes
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    • "+ cells that have proliferated in the presence of antigen: without antigen (Mannering et al., 2003a). Fig. 2. "
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    ABSTRACT: Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined (1). This is particularly true in autoimmune diseases and cancer(2). Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes (1,3,4,5). To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays (6). Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.
    Full-text · Article · Sep 2012 · Journal of Visualized Experiments
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    • "A proliferation assay based on cell staining with 5,6- carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry analysis has been described as an alternative approach [7] [17]. This method has been employed in a wide range of applications, including the detection of T-cell responses against autoantigens and the activity of regulatory T cells [18] [19]. However, the applicability of this assay in HTLV-1-infected patients has not been established. "
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    ABSTRACT: The spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) is a hallmark of the human T-lymphotropic virus (HTLV) type-1. Cell proliferation is usually measured using a [ 3 H]thymidine incorporation assay. This study aims to quantify the spontaneous proliferation of PBMCs using flow cytometry. PBMCs were cultured for 24 to 120 hours in the presence of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). For comparison, PBMCs were also cultured with [ 3 H]thymidine. The cutoff values for spontaneous proliferation were >0.06 for the division index and >5.8% for the percentage of divided cells. Sixty-two percent of HTLV-1-infected individuals presented spontaneous proliferation of PBMCs, which was detected in the first 24 hours. Moreover, proliferation was detected in CD4 + and CD8 + T-lymphocyte subsets. A positive correlation was found between the division index and [ 3 H]thymidine incorporation. This method may prove useful to better understand the phenomenon of spontaneous proliferation of PBMC of patients infected with HTLV-1.
    Full-text · Article · Dec 2011
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Questions & Answers about this publication

  • Stuart I Mannering added an answer in T Cell Stimulation:
    Is mixed lymphocyte reaction(MLR)or ELISPOT more sensitive to T cell activation?

    MLR and ELISPOT----- mix the T cells with the  stimulator(cells),which one is more sensitive to the stimulator? 

    Stuart I Mannering

    If you're measuring proliferation in your MLR by 3H-thymidine incorporation an IFNg ELISpot will be more sensitive. It might be worth using a CFSE-based proliferation assays are equally sensitive as ELISpot an allow you to analyze the responding cells (see attached papers).

    Serum free media do work, but in our experience the cells function better in the presence of 5% serum. We routinely use pooled human male serum (see Optimisation of conditions... paper). This is fairly old now, but I hope still useful.

    Good luck!

    Stuart

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      [Show abstract] [Hide abstract]
      ABSTRACT: The ability to measure proliferation of rare antigen-specific T cells among many bystanders is critical for the evaluation of cellular immune function in health and disease. T-cell proliferation in response to antigen has been measured almost exclusively by 3H-thymidine incorporation. This method does not directly identify the phenotype of the proliferating cells and is frequently not sufficiently sensitive to detect rare autoantigen-specific T cells. To overcome these problems, we developed a novel assay for antigen-specific human T-cell proliferation. Peripheral blood mononuclear cells (PBMC) were labelled with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) and cells that proliferated in response to antigen, with resultant reduction in CFSE intensity, were measured directly by flow cytometry. This assay was more sensitive than 3H-thymidine incorporation and detected the proliferation of rare antigen-specific CD4(+) T cells at 10-fold lower antigen concentrations. It also allowed the phenotype of the proliferating cells to be directly determined. Using the CFSE assay we were able to measure directly the proliferation of human CD4(+) T cells from healthy donors in response to the type 1 diabetes autoantigens glutamic acid decarboxylase (GAD) and proinsulin (PI).
      Full-text · Article · Jan 2004 · Journal of Immunological Methods

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