Lymph node-derived donor encephalitogenic CD4+ T cells in C57BL/6 mice adoptive transfer experimental autoimmune encephalomyelitis highly express GM-CSF and T-bet

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA.
Journal of Neuroinflammation (Impact Factor: 5.41). 06/2011; 8(1):73. DOI: 10.1186/1742-2094-8-73
Source: PubMed


Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 μg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.

Download full-text


Available from: Scott S Zamvil,
  • Source
    • "Brains were pressed through a 70-μm nylon mesh cell strainer. Brain cells from all mice in each experimental group were pooled and processed as previously described (Cravens et al., 2011, 2013). In brief, brain cells were washed twice in 37% Percoll and CNS mononuclear cells were isolated by centrifugation at 2118 ×g for 15 min at 22 °C over a 30/70% Percoll gradient. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Immune surveillance of the CNS is critical for preventing infections, however there is no accepted experimental model to assess the risk of infection when utilizing disease-modifying agents. We tested two approved agents for patients with multiple sclerosis (MS), glatiramer acetate and fingolimod, in an experimental model of CNS immune surveillance. C57BL/6 mice were infected with the ME49 strain of the neuroinvasive parasite Toxoplasma gondii (T. gondii) and then treated with GA and fingolimod. Neither treatment affected host survival, however differences were observed in parasite load and in leukocyte numbers in the brains of infected animals. Here we demonstrate that this model could be a useful tool for analyzing immune surveillance.
    Journal of Neuroimmunology 09/2014; 276(1-2). DOI:10.1016/j.jneuroim.2014.08.624 · 2.47 Impact Factor
  • Source
    • "Earlier, Huang et al. [9] had described diminished production of IFN-γ in LNCs from CCL2-/- mice, which might reflect the effects of having depleted a peripheral CCL2 pool distinct from that of endothelial cells. Figure 4c further reveals that, following MOG immunization, the percentage of MOG38-49 MHC class II tetramer positive CD4 T cells was in line with another report [29] and similar in LNCs from Astro KO, Endo KO, or WT mice, indicating that the frequency of MOG-specific T cells was comparable among these groups. Hence, our results argue against either astrocyte- or endothelial cell-targeted CCL2 gene deletion having significantly impaired peripheral T cell behavior and immune responsiveness to MOG peptide. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood-brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-gamma production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complexclass II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease.
    Journal of Neuroinflammation 01/2014; 11(1):10. DOI:10.1186/1742-2094-11-10 · 5.41 Impact Factor
  • Source
    • "Supernatants were collected at 48 hrs and 72 hrs and the levels IFNγ and IL-17 were determined by ELISA. (E) To quantify IFNγ expression in neonatal and mature encephalitogenic CD4+ T cells, lymph node cells were prepared for adoptive transfer as described above [22]. IFNγ expression was determined by ELISA after 24, 48, and 72 hrs in culture. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Multiple sclerosis (MS) is thought to be a CD4+ T cell mediated autoimmune demyelinating disease of the central nervous system (CNS) that is rarely diagnosed during infancy. Cellular and molecular mechanisms that confer disease resistance in this age group are unknown. We tested the hypothesis that a differential composition of immune cells within the CNS modulates age-associated susceptibility to CNS autoimmune disease. C57BL/6 mice younger than eight weeks were resistant to experimental autoimmune encephalomyelitis (EAE) following active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35--55. Neonates also developed milder EAE after transfer of adult encephalitogenic T cells primed by adult or neonate antigen presenting cells (APC). There was a significant increase in CD45+ hematopoietic immune cells and CD45+ high side scatter granulocytes in the CNS of adults, but not in neonates. Within the CD45+ immune cell compartment of adults, the accumulation of CD4+ T cells, Gr-1+ and Gr-1- monocytes and CD11c+ dendritic cells (DC) was identified. A significantly greater percentage of CD19+ B cells in the adult CNS expressed MHC II than neonate CNS B cells. Only in the adult CNS could IFNgamma transcripts be detected 10 days post immunization for EAE. IFNgamma is highly expressed by adult donor CD4+ T cells that are adoptively transferred but not by transferred neonate donor cells. In contrast, IL-17 transcripts could not be detected in adult or neonate CNS in this EAE model, and neither adult nor neonate donor CD4+ T cells expressed IL-17 at the time of adoptive transfer.
    Journal of Neuroinflammation 05/2013; 10(1):67. DOI:10.1186/1742-2094-10-67 · 5.41 Impact Factor
Show more