[Show abstract][Hide abstract] ABSTRACT: Objective: Studies evaluating T-cell recognition of myelin oligodendrocyte glycoprotein (MOG) in
multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), have
focused mostly on its 117 amino acid (aa) extracellular domain, especially peptide (p) 35-55.
We characterized T-cell responses to the entire 218 aa MOG sequence, including its transmembrane
and cytoplasmic domains.
Methods: T-cell recognition in mice was examined using overlapping peptides and intact fulllength
mouse MOG. EAE was evaluated by peptide immunization and by adoptive transfer of
MOG epitope-specific T cells. Frequency of epitope-specific T cells was examined by ELISPOT.
Results: Three T-cell determinants of MOG were discovered in its transmembrane and cytoplasmic
domains, p119–132, p181–195, and p186–200. Transmembrane MOG p119-132 induced clinical
EAE, CNS inflammation, and demyelination as potently as p35-55 in C57BL/6 mice and other
H-2b strains. p119-128 contained its minimal encephalitogenic epitope. p119-132 did not cause
disease in EAE-susceptible non-H-2b strains, including Biozzi, NOD, and PL/J. MOG p119-132–
specific T cells produced Th1 and Th17 cytokines and transferred EAE to wild-type recipient mice.
After immunization with full-length MOG, a significantly higher frequency of MOG-reactive T cells
responded to p119-132 than to p35-55, demonstrating that p119-132 is an immunodominant
encephalitogenic epitope.MOG p181-195 did not cause EAE, andMOG p181-195–specific T cells
could not transfer EAE into wild-type or highly susceptible T- and B-cell–deficient mice.
Conclusions: Transmembrane and cytoplasmic domains of MOG contain immunodominant T-cell
epitopes in EAE. A CNS autoantigen can also contain nonpathogenic stimulatory T-cell epitopes.
Recognition that a myelin antigen contains multiple encephalitogenic and nonencephalitogenic
determinants may have implications for therapeutic development in MS.
[Show abstract][Hide abstract] ABSTRACT: The enzyme-linked immunospot (ELISPOT) assay is a widely used method for enumerating antigen-specific cytokine-producing or antibody-secreting immune cells. It is one of the most effective immunological and diagnostic approaches to detect and quantify low-frequency cytokine- or antibody-producing cells in human and animal tissues, such as peripheral blood, lymph nodes, and spleen. Detection and quantification of specific cytokine-producing cells by the ELISPOT assay is based on the formation of visible spots at the site of cytokine release by the cells under investigation (e.g., T cells) using pairs of different capture and detection antibodies under optimized conditions.Here we focus mainly on practical, optimized protocols for cytokine ELISPOT assays for detection of mouse and human cytokine-producing immune cells (e.g., peripheral blood mononuclear cells, PBMC), including suggestions for trouble-shooting and optimizing steps for problematic tissue samples.
Methods in Molecular Biology 08/2014; DOI:10.1007/7651_2014_111 · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induction of experimental autoimmune encephalomyelitis (EAE) in susceptible animals requires reactivation of encephalitogenic CD4(+) T cells by APCs in the CNS. However, it has remained unresolved from where APCs in the CNS acquire myelin Ag for T cell activation and under which conditions, that is, whether only during EAE or also in the naive CNS. In this study, we investigated the kinetics of myelin Ag uptake by CNS APCs during EAE and in the naive CNS. Our results show that during EAE CX3CR1(+)CD11b(+) microglia were the first APCs in the CNS to contain myelin Ag upon induction of disease, albeit in very small numbers. Dendritic cells (DCs) arrived in the CNS in sizable numbers significantly later (day 5 postimmunization), without detectable myelin Ag, but acquired it by day 7 postimmunization. Furthermore, a sharp increase in neuroantigen-containing DCs coincided with the onset of EAE symptoms. Importantly, in naive mice a low but consistent number of microglia contained myelin Ag, suggesting release by oligodendrocytes under steady state conditions. Although microglia isolated from naive brain and spinal cord did not elicit a strong CD4(+) T cell response in vitro, myelin Ag-containing microglia may still play a local role in modulating encephalitogenic CD4(+) T cell responses in early EAE prior to the arrival of other professional APCs, such as DCs. Finally, newly arriving DCs in the CNS not yet loaded with myelin Ag before the onset of EAE may be a potential therapeutic target.
The Journal of Immunology 11/2013; 191(12). DOI:10.4049/jimmunol.1300771 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The strong association of HLA-DR2b (DRB1*1501) with multiple sclerosis (MS) suggests this molecule as prime target for specific immunotherapy. Inhibition of HLA-DR2b-restricted myelin-specific T cells has the potential to selectively prevent CNS pathology mediated by these MHC molecules without undesired global immunosuppression. In this study, we report development of a highly selective small molecule inhibitor of peptide binding and presentation by HLA-DR2b. PV-267, the candidate molecule used in these studies, inhibited cytokine production and proliferation of myelin-specific HLA-DR2b-restricted T cells. PV-267 had no significant effect on T cell responses mediated by other MHC class II molecules, including HLA-DR1, -DR4, or -DR9. Importantly, PV-267 did not induce nonspecific immune activation of human PBMC. Lastly, PV-267 showed treatment efficacy both in preventing experimental autoimmune encephalomyelitis and in treating established disease. The results suggest that blocking the MS-associated HLA-DR2b allele with small molecule inhibitors may be a promising therapeutic strategy for the treatment of MS.
The Journal of Immunology 10/2013; 191(10). DOI:10.4049/jimmunol.1300407 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T-bet was initially described as a T-box transcription factor with an essential role in orchestrating Th1 cell differentiation. Subsequently, it was determined that T-bet controls the expression of numerous cytokines and their receptors, adhesion molecules and chemokine receptors, and therefore determines the differentiation and development status of many types of immune cells. The critical role of T-bet in autoimmune diseases, particularly multiple sclerosis and its animal model experimental autoimmune encephalomyelitis, implicates it as a potential biomarker for pathogenic T cells as well as a therapeutic drug target.
[Show abstract][Hide abstract] ABSTRACT: Ab-mediated blockade of the adhesion molecule VLA-4 has been shown to ameliorate disease in human multiple sclerosis patients and experimental autoimmune encephalomyelitis (EAE) animal models. We wanted to determine whether anti-VLA-4 Ab treatment affected the function and persistence of autoreactive T cells in mice with EAE. Unexpectedly, we observed a high level of mortality in anti-VLA-4 mAb (PS/2)-treated mice with actively induced EAE despite decreased disease severity. Investigation of the underlying mechanism showed that injection of PS/2 mAb in combination with pertussis toxin resulted in anaphylaxis and mortality. Furthermore, the data showed that CD4(+) T cells were required for this effect and suggested a role for IL-1β and TNF-α in the underlying pathology. The results reveal a previously not appreciated deleterious effect of anti-VLA-4 Ab treatment in combination with exposure to pertussis toxin.
The Journal of Immunology 03/2011; 186(5):2750-6. DOI:10.4049/jimmunol.1000907 · 5.36 Impact Factor