[Show abstract][Hide abstract] ABSTRACT: Glucocorticoids (GCs) are used as standard treatment for acute attacks of multiple sclerosis (MS). However, GCs eventually lose efficacy and do not prevent disease progression. Macrophage migration inhibitory factor (MIF) is the only known proinflammatory cytokine induced by GCs that inhibits their anti-inflammatory effects. Therefore, we investigated whether MIF plays a role in resistance to GC treatment in experimental autoimmune encephalomyelitis (EAE), an animal model of MS.
EAE was induced in wild-type (Wt) and MIF knockout (MIF(-/-)) mice followed by treatment with dexamethasone (Dex) before or upon disease onset. Splenocytes and brain mononuclear cells were harvested for cytokine ELISPOT assay and flow cytometry analysis.
Treatment of EAE with Dex was substantially more efficacious in MIF(-/-) mice than Wt mice. Dex treatment decreased MOG35-55-induced cytokine production by Wt or MIF(-/-) CD4(+) T cells only at the onset of EAE but inhibited upregulation of T-bet during acute and chronic phases of disease, particularly in MIF(-/-) mice. Furthermore, passive EAE induced by adoptive transfer of T cells showed that Dex was highly effective in ameliorating disease induced by MIF(-/-) CD4(+) T cells but not by Wt CD4(+) T cells. The expression of T-bet and VLA-4 was decreased in CD4(+) T cells in MIF(-/-) mice compared with Wt mice.
Our data establish MIF as a key molecule in resistance of pathogenic CD4(+) T cells to GC treatment in EAE and as a potential target to enhance the effectiveness of steroid treatment in neuroinflammatory disorders.
[Show abstract][Hide abstract] ABSTRACT: Objective
The objective of this study was to determine whether reactivation of Epstein-Barr (EBV) or activation of the anti-EBV immune response correlates with MS disease activity on MR imaging.Methods
Subjects with early, active relapsing-remitting MS were studied for 16 weeks with blood and saliva samples collected every 2 weeks and brain MRI performed every 4 weeks. We isolated peripheral blood mononuclear cells from each blood sample and tested the immune response to EBV, autologous EBV-infected lymphoblastoid cell lines (LCL), human herpesvirus 6 (HHV6), varicella zoster virus (VZV), tetanus, and mitogens. We measured the proliferative response and the number of interferon-γ secreting cells with ELISPOT. We measured the amounts of EBV, HHV6, and VZV DNA in blood and saliva with quantitative PCR. On MRI, we measured number and volume of contrast enhancing and T2 lesions. We tested for correlation between the immunologic assays and the MRI results, assessing different time intervals between the MRI and immunologic assays.ResultsWe studied 20 subjects. Ten had enhancing lesions on one or more MRI scans and one had new T2 lesions without enhancement. The most significant correlation was between proliferation to autologous LCL and the number of combined unique active lesions on MRI 4 weeks later. Both proliferation and number of cells secreting interferon-γ in response to LCL correlated with the number of enhancing lesions 8 weeks later.Conclusions
We find evidence for correlation of antiviral immune responses in the blood with subsequent disease activity on MRI scans.
[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.
T-cell recognition in mice was examined using overlapping peptides and intact full-length 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.
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-2(b) strains. p119-128 contained its minimal encephalitogenic epitope. p119-132 did not cause disease in EAE-susceptible non-H-2(b) 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, and MOG p181-195-specific T cells could not transfer EAE into wild-type or highly susceptible T- and B-cell-deficient mice.
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.
[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 · 4.92 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 · 4.92 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 · 4.92 Impact Factor