Abstract
BackgroundHere, we evaluated the hypothesis that CD8+ T cell responses to caspase-cleaved antigens derived from effector T cells undergoing apoptosis, may contribute to multiple sclerosis (MS) immunopathology.MethodsThe percentage of autoreactive CD8+ T effector cells specific for various apoptotic T cell-associated self-epitopes (apoptotic epitopes) were detected in the peripheral blood and cerebrospinal fluid (CSF) by both enzyme-linked immunospot and dextramers of class I molecules complexed with relevant apoptotic epitopes. Moreover, the capacity of dextramer+ CD8+ T cells to produce interferon (IFN)-γ and/or interleukin (IL)-17 in response to the relevant apoptotic epitopes was evaluated by the intracellular cytokine staining. Cross-presentation assay of apoptotic T cells by dendritic cells was also evaluated ex vivo.ResultsWe found that polyfunctional (IFN-γ and/or IL-17 producing) autoreactive CD8+ T cells specific for apoptotic epitopes were represented in MS patients with frequencies significantly higher than in healthy donors. These autoreactive CD8+ T cells with a strong potential to produce IFN-γ or IL-17 in response to the relevant apoptotic epitopes were significantly accumulated in the CSF from the same patients. In addition, the frequencies of these autoreactive CD8+ T cells correlated with the disease disability. Cross-presentation assay revealed that caspase-cleaved cellular proteins are required to activate apoptotic epitope-specific CD8+ T cells ex vivo.ConclusionTaken together, these data indicate that apoptotic epitope-specific CD8+ T cells with strong inflammatory potential are recruited at the level of the inflammatory site, where they may be involved in MS immunopathology through the production of high levels of inflammatory cytokines.
Highlights
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease representing a major cause of neurological disability in the Western world [1]
We demonstrated that the proteome of apoptotic T cells include prominent caspase-cleaved cellular proteins (namely, fragments of actin cytoplasmic 1 (ACTB) [Swissprot: P60709], heterogeneous nuclear ribonucleoprotein (ROK) [Swissprot: P61978], lamin B1 (LAM1) [Swissprot: P20700], non-muscle myosin heavy chain 9 (MYH9) [Swissprot: P35579], vimentin (VIME) [Swissprot: P08670], proteasome component C2 (PSA1) [Swissprot: P25786], rho GDP dissociation inhibitor 2 (GDIS) [Swissprot: P50395], and 60S acidic ribosomal protein P2 (RLA) [Swissprot: P05387]), and a high proportion of distinct epitopes in these fragments can be cross-presented by Dendritic cell (DC) via the classical major histocompatibility complex (MHC) class I pathway to a wide repertoire of autoreactive CD8+ T cells [25,29]
Multispecific CD8+ T effector memory (TEM) cell responses to apoptotic epitopes Freshly isolated CD8+ T cells from consecutive HLAA2+ patients with multiple sclerosis (MS) (Table 1) and healthy donors (HD) were tested for the capacity to form IFN-γ spots in an enzyme-linked immunospot (ELISPOT) assay within 4 to 6 h of contact either with 12 pools containing a total of 90 synthetic HLA-A2-binding apoptotic peptides (Additional file 1: Table S1) [25,29,35], or with overlapping peptides spanning the entire sequence of the myelin basic protein (MBP) (Additional file 1: Table S2) [28]
Summary
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease representing a major cause of neurological disability in the Western world [1]. IL-17 produced by CD4+ or CD8+ T cells specific to myelin proteins (likely in response to IL-23 and granulocyte monocyte-colony stimulating factor, GM-CSF) plays a preponderant role in the autoimmune demyelinating disease found in humans and mice [8,9,10,11,12,13,14,15]. We have previously shown that myelin basic protein (MBP)-specific T cells produce IFN-γ in response to autologous DCs loaded with apoptotic oligodendrocytes in vitro [28]. This result indicated that myelinspecific antigens presented within the apoptotic glial cells may be involved in the first relevant MS pathogenetic step. We evaluated the hypothesis that CD8+ T cell responses to caspase-cleaved antigens derived from effector T cells undergoing apoptosis, may contribute to multiple sclerosis (MS) immunopathology
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