Fluorospot assay

Eric Tartour, Universite Pierre et Marie Currie, Paris, France

Elispot is one of the most sensitive technique to detect cytokine producing T cells (1, 2). However, in most cases, it does not allow detection of single T lymphocytes simultaneous producing multiple cytokines. Yet, in different clinical situations, the profile of cytokine of T cells may help to characterize them and to determine their function. The only detection of IFNƒ× could not discriminate between type 1 T cells identified by their expression of IFN-gamma, IL-2 and TNF-alpha, and Tr1 cells which produce high levels of IL-10 and moderate amount of IFN-gamma; but no IL-2 or IL-4 (3, 4). Type 1 T cells are considered to be major effector against viral infection, intracellular pathogens and cancers, whereas Tr1 cells are involved in downregulation of immune responses. Similarly, IL-10 could be produced by both Tr1 cells and type 2 T cells which also secrete IL-4 and provide helper activity for B cell development. Recent studies indicate that detection of double IFNgamma-IL-2 producing T-cells provides additional clinical information regarding the prognosis of patients with human immunodeficiency virus that enumeration of IFNgamma or IL-2 secreting T cells alone (5-7). All these points strongly encourage us and other groups to develop techniques to detect multiple cytokine produced by single T cells.

Attempts to develop an immunoenzymatic dual-color Elispot failed because of difficulties in the interpretation of mixed color spots as already reported by other groups (8, 9). With the help of the Diaclone company, we therefore moved to the fluorospot assay based on the use of multiple fluorescent labeled anti-cytokine detection antibodies(10, 11). This assay clearly provides better discrimination and characterization of double cytokine-producing cells than does an enzymatic reaction (Fig 1).

The sensitivity of this technique is in the same range than the conventional immuno-enzymatic Elispot (11). Using this technique we could detect Tr1 T cells in pleural effusion, polarized type 1 and type 2 specific tetanus toxoid T cells (11) and IL-2 and/or IFNgamma-secreting anti-HIV specific CD8+T cells (Fig 2). Dual color for IL-2-IFNgamma, IFNgamma-IL-5, IFNgamma-IL-10 are already available and we plan to develop an IFNgamma-perforin assay. In collaboration with the Zeiss company, a special automated Elispot reader consisting of a light microscope with incident fluorescence illumination and an integrated digital color camera has been adapted for this technique. Notes for optimization - The PVDF flat bottom plates gave the best results when compared to plastic plates. This technique did not require the use of black plates. - During dual color fluorospot, one must be aware of cross-reactivity between secondary and/or primary antibodies. Therefore, we recommend that one use species depleted antibodies and Fab¡¦2 antibodies. - When dual-color fluorospot is performed, competition between capture antibodies may occur and this will introduce bias in the detection spots. When antibodies are used for the first time, to ensure that the same concentrations of different anti-cytokine were bound, we simultaneously tested the frequency of the cells producing the different cytokines with single color or double color fluorospot. - Because the fluorescence illumination does not cross the PVDF membranes, the light from illuminator has to come from the top of the plates.

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