Abstract
Experimental allergic encephalomyelitis (EAE) is mediated by neuroantigen-specific pro-inflammatory T cells of the Th1 and Th17 effector class. Th-17 cells can be clearly defined by expression of IL-17, but not IFN-γ, IL-2 or IL-3. Th1 cells do not express IL-17, but it is unclear presently to what extent they co-express the cytokines canonically assigned to Th1 immunity (i.e., IFN-γ, IL-2 and IL-3) and whether CD4 cells producing these cytokines indeed belong to a single Th1 lineage. It is also unclear to what extent the Th1 response in EAE entails polyfunctional T cells that co-express IFN-γ and IL-2. Therefore, we dissected the Th1 cytokine signature of neuroantigen-specific CD4 cells studying at single cell resolution co-expression of IFN-γ, IL-2 and IL-3 using dual color cytokine ELISPOT analysis. Shortly after immunization, in the draining lymph nodes (dLN), the overall cytokine signature of the neuroantigen-specific CD4 cells was highly type 1-polarized, but IFN-γ, IL-2, and IL-3 were each secreted by different CD4 cells in a mutually exclusive manner. This single cell – single cytokine profile was stable through the course of chronic EAE–polyfunctional CD4 cells co-expressing IL-2 and IFN-γ presented less than 5% of the neuroantigen-specific T cells, even in the inflamed CNS itself. The neuroantigen-specific CD4 cells that expressed IFN-γ, IL-2 and IL-3 in a mutually exclusive manner exhibited similar functional avidities and kinetics of cytokine production, but showed different tissue distributions. These data suggest that Th1 cells do not belong to a single lineage, but different Th1 subpopulations jointly mediate Th1 immunity.
Highlights
While considerable progress has been made in understanding the differentiation of CD4 cells into effector cell lineages that express IFN-Ȗ 7K,/-4 (Th2) [1] and IL-17 (Th17) [2,3], at present it is unclear to what extent individual T cells within these subsets co-express other cytokines
We studied whether the autoimmune CD4 cells in the target organ, the central nervous system (CNS), would differ from those residing in the immune periphery in their cytokine signatures, functional avidities, and the kinetics of cytokine production, and whether these parameters would change over the course of chronic-relapsing immune pathology
CD4 cells were purified from the draining lymph nodes (dLN) of the immunized mice and were tested on naive LN cells functioning as APC
Summary
While considerable progress has been made in understanding the differentiation of CD4 cells into effector cell lineages that express IFN-Ȗ 7K ,/-4 (Th2) [1] and IL-17 (Th17) [2,3], at present it is unclear to what extent individual T cells within these subsets co-express other cytokines. Restimulated T cells, including T cell lines and clones, are prone to lose differentiation in cell culture, to undergo replicative senescence after about 15 cycles of proliferation in vitro [8] and to develop chromosomal aberrations [9], all of which can affect cytokine expression/co-expression. While such senescent cells can be sustained in culture, it is unclear whether they can survive in vivo. It is still to be determined to what extent the cytokine expression patterns established for T cell clones, T cell lines and in vitro-propagated TCR-transgenic cells reflect T cell biology in vivo and whether T cells progress towards cytokine co-expressing phenotypes upon continuous stimulation in vivo, for example, when chronically stimulated in an autoimmune setting
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