A high-throughput single-cell analysis of human CD8(+) T cell functions reveals discordance for cytokine secretion and cytolysis

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
The Journal of clinical investigation (Impact Factor: 13.22). 10/2011; 121(11):4322-31. DOI: 10.1172/JCI58653
Source: PubMed


CD8+ T cells are a key component of the adaptive immune response to viral infection. An inadequate CD8+ T cell response is thought to be partly responsible for the persistent chronic infection that arises following infection with HIV. It is therefore critical to identify ways to define what constitutes an adequate or inadequate response. IFN-γ production has been used as a measure of T cell function, but the relationship between cytokine production and the ability of a cell to lyse virus-infected cells is not clear. Moreover, the ability to assess multiple CD8+ T cell functions with single-cell resolution using freshly isolated blood samples, and subsequently to recover these cells for further functional analyses, has not been achieved. As described here, to address this need, we have developed a high-throughput, automated assay in 125-pl microwells to simultaneously evaluate the ability of thousands of individual CD8+ T cells from HIV-infected patients to mediate lysis and to produce cytokines. This concurrent, direct analysis enabled us to investigate the correlation between immediate cytotoxic activity and short-term cytokine secretion. The majority of in vivo primed, circulating HIV-specific CD8+ T cells were discordant for cytolysis and cytokine secretion, notably IFN-γ, when encountering cognate antigen presented on defined numbers of cells. Our approach should facilitate determination of signatures of functional variance among individual effector CD8+ T cells, including those from mucosal samples and those induced by vaccines.

Download full-text


Available from: Navin Varadarajan
  • Source
    • "First of all, ICS does not retain cell viability, and thus, recovery of cells for clonal expansion is difficult. In addition, since a protein transport inhibitor is added to retain the cytokines within the cell, it is difficult to determine whether the detected cytokines are intracellular, secreted or membrane-bound [72]. Moreover, dynamic monitoring of an individual cell is difficult since the secretions of the cells upstream may affect others. "
    [Show abstract] [Hide abstract]
    ABSTRACT: More than 60 million people in the world have been diagnosed with human immunodeficiency virus (HIV) infections since the virus was recognized as the causative agent of acquired immunodeficiency syndrome (AIDS) in the 1980s. Even though more than half of the infected patients have died, effective disease treatment and prevention measures have not been established. Antiretroviral therapy is the only proven HIV treatment which sustains suppression of patient viremia. Current routine approaches to treat HIV infections are targeted at developing vaccines that will induce humoral or cell memory immune responses. However, developing an effective vaccine has been challenging because the HIV mutates rapidly, which allows the virus to evade immune surveillances established against the previous strain. In addition, the virus is able to quickly establish a reservoir and treatment is difficult because of the general lack of knowledge about HIV immune response mechanisms. This review introduces common disease symptoms and the progression of HIV infection with a brief summary of the current treatment approaches. Different cellular immune responses against HIV are also discussed, with emphasis on a nanotechnology research that has focused on probing T cell response to HIV infection. Furthermore, we discuss recent noteworthy nanotechnology updates on T cell response screening that are focused on HIV infection. Finally, we review potential future treatment strategies based on the correlations between T cell response and HIV infection.
    Full-text · Article · Jul 2014 · Bioscience Reports
  • Source
    • "They also emphasize that even with optimal sampling and specimen processing procedures studies of genital immunity are hampered by the relatively low cell numbers available. Single cell analysis technologies, which are currently being developed with promising speed, offer the best prospect of overcoming this basic limitation [36], [37]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. WE ENROLLED WOMEN FROM FOUR SITES IN AFRICA AND THE US TO COMPARE THREE GENITAL LEUKOCYTE SAMPLING METHODS: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4(+) T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor. CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.
    Full-text · Article · Jan 2014 · PLoS ONE
  • Source
    • "While flow cytometry has traditionally been used to determine single cell phenotypes, it cannot provide continuous measurements of proteins in the same individual cells over time7. Recently, the development of microfluidic technology enabled analysis of cell phenomena on the single cell level8910. The reported platforms, mostly based on micro well technologies, do not provide a controllable microenvironment for cell-cell interaction, however, as no reagent mixing is provided. "
    [Show abstract] [Hide abstract]
    ABSTRACT: While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.
    Full-text · Article · Nov 2013 · Scientific Reports
Show more