title: Detection of low-frequency antigen-specific IL-10-producing CD4(+) T cells via ELISPOT in PBMC: cognate vs. nonspecific production of the cytokine.
abstract: Single-cell resolution cytokine ELISPOT assays are increasingly used to gain insights into clonal sizes of type 1 and type 2 effector T cell populations in vivo. However, ELISPOT assays permitting monitoring of regulatory IL-10-producing T cells have so far not been established. Unlike IFN-gamma, IL-2, IL-4, and IL-5 assays performed on PBMC in which the recall antigen-induced cytokine spots are T cell-derived, we show here that in such assays IL-10 is primarily monocyte-derived. T cell-derived IL-10 spots were 80 x 10(3) microm(2) in size, seven times larger than spots produced by monocytes, and B cells produced even smaller spots. Based on spot size gating and the use of B cells as APC, we have established test conditions that permit measurement of cognate IL-10 production by low-frequency antigen-specific T cells. IL-10-producing PPD-specific CD4(+) T cells were detected in frequencies comparable to IFN-gamma-secreting CD4(+) T cells in tuberculosis patients, but not in uninfected healthy control individuals. In contrast, IL-10-secreting CD4(+) T cells specific for a panel of recall antigens could not be detected in frequencies >1/100,000 in healthy individuals whose CD4(+) cells responded to these antigens with type 1 or type 2 cytokine production in the 1:100,000-1:1000 frequency range. Therefore, the induction of IL-10-producing T cells seems to be under tighter control than that of Th1/Th2 cells, apparently confined to states of chronic immune stimulation. Access to low-frequency immune monitoring of IL-10-producing T cells will provide new insights into the role of regulatory T cells in health and disease.
authors.affiliation: Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4943, USA.
authors.forename: Robert E M
authors.initials: RE
authors.lastname: Guerkov
authors.forename: Oleg S
authors.initials: OS
authors.lastname: Targoni
authors.forename: Christian R
authors.initials: CR
authors.lastname: Kreher
authors.forename: Bernhard O
authors.initials: BO
authors.lastname: Boehm
authors.forename: Maria T
authors.initials: MT
authors.lastname: Herrera
authors.forename: Magdalena
authors.initials: M
authors.lastname: Tary-Lehmann
authors.forename: Paul V
authors.initials: PV
authors.lastname: Lehmann
authors.forename: Stephan K
authors.initials: SK
authors.lastname: Schwander
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000378:metabolism*
chemical_list: D016753:Interleukin-10