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Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Johnson HL, Willenbring RC, Jin F, Manhart WA, LaFrance SJ, Pirko I, Johnson AJ - PLoS ONE (2014)

Bottom Line: However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood.To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques.Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, Rochester, Minnesota, United States of America; Department of Immunology, Mayo Clinic, Rochester, Minnesota, United States of America; Neurobiology of Disease Graduate Program, Mayo Graduate School, Rochester, Minnesota, United States of America.

ABSTRACT
Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

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Adoptive transfer of CD8+ cells successfully migrate to brain post induction of PIFS.(A) Schematic illustrating the adoptive transfer of sorted perforin competent CD8+ cells into perforin−/− mice followed by induction of PIFS. C57BL/6 perforin−/− mice were irradiated with 400 rads of irradiation and then intravenously injected with 107 GFP+ CD8+ splenocytes (n = 10) or 107 Ly5.1+ CD8+ splenocytes (n = 10). Mice were intracranially infected with TMEV on the following day. PIFS-inducing VP2121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion. MRI analysis was performed on the following day to visualize the extent of CNS vascular permeability and then the CNS was harvested for additional assays. Both negative (Experiment I) and positive (Experiment II) sort experiments yielded high purity of CD8+ T cell transfer. (B) Representative confocal microscopic images illustrating co-localization of Ly5.1 and CD8 protein (Experiment I). CNS-infiltrating Ly5.1+ cells colocalized with CD8+ cells 85.7% of the time as measured by confocal analysis. (C) Confocal microscopy showing of representative brain tissue slice showing successful transfer of GFP+ CD8+ T cells (Experiment II). Purity of transfer was analyzed via flow cytometry, 98.0%+− 0.5% of cells that were CD8 positive and GFP positive.
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pone-0111401-g001: Adoptive transfer of CD8+ cells successfully migrate to brain post induction of PIFS.(A) Schematic illustrating the adoptive transfer of sorted perforin competent CD8+ cells into perforin−/− mice followed by induction of PIFS. C57BL/6 perforin−/− mice were irradiated with 400 rads of irradiation and then intravenously injected with 107 GFP+ CD8+ splenocytes (n = 10) or 107 Ly5.1+ CD8+ splenocytes (n = 10). Mice were intracranially infected with TMEV on the following day. PIFS-inducing VP2121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion. MRI analysis was performed on the following day to visualize the extent of CNS vascular permeability and then the CNS was harvested for additional assays. Both negative (Experiment I) and positive (Experiment II) sort experiments yielded high purity of CD8+ T cell transfer. (B) Representative confocal microscopic images illustrating co-localization of Ly5.1 and CD8 protein (Experiment I). CNS-infiltrating Ly5.1+ cells colocalized with CD8+ cells 85.7% of the time as measured by confocal analysis. (C) Confocal microscopy showing of representative brain tissue slice showing successful transfer of GFP+ CD8+ T cells (Experiment II). Purity of transfer was analyzed via flow cytometry, 98.0%+− 0.5% of cells that were CD8 positive and GFP positive.

Mentions: Spleens of GFP+ or Ly5.1+ mice were removed and strained through a nylon mesh 100 µm filter. CD8+ cells were purified from the resulting lymphocyte population using MACS LS cell purification columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Positive cell sorting was used to obtain GFP+ CD8+ cells and negative cell sorting was used to obtain Ly5.1+ CD8+ cells. Both methods resulted in ∼90% purity as determined by flow cytometric analysis (data not shown). C57BL/6 perforin−/− mice were irradiated with 400 rads of irradiation, given one day to recover, and then intravenously injected with 107 Ly5.1+ CD8+ or GFP+ CD8+ splenocytes (Figure 1A).


Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Johnson HL, Willenbring RC, Jin F, Manhart WA, LaFrance SJ, Pirko I, Johnson AJ - PLoS ONE (2014)

Adoptive transfer of CD8+ cells successfully migrate to brain post induction of PIFS.(A) Schematic illustrating the adoptive transfer of sorted perforin competent CD8+ cells into perforin−/− mice followed by induction of PIFS. C57BL/6 perforin−/− mice were irradiated with 400 rads of irradiation and then intravenously injected with 107 GFP+ CD8+ splenocytes (n = 10) or 107 Ly5.1+ CD8+ splenocytes (n = 10). Mice were intracranially infected with TMEV on the following day. PIFS-inducing VP2121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion. MRI analysis was performed on the following day to visualize the extent of CNS vascular permeability and then the CNS was harvested for additional assays. Both negative (Experiment I) and positive (Experiment II) sort experiments yielded high purity of CD8+ T cell transfer. (B) Representative confocal microscopic images illustrating co-localization of Ly5.1 and CD8 protein (Experiment I). CNS-infiltrating Ly5.1+ cells colocalized with CD8+ cells 85.7% of the time as measured by confocal analysis. (C) Confocal microscopy showing of representative brain tissue slice showing successful transfer of GFP+ CD8+ T cells (Experiment II). Purity of transfer was analyzed via flow cytometry, 98.0%+− 0.5% of cells that were CD8 positive and GFP positive.
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Related In: Results  -  Collection

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pone-0111401-g001: Adoptive transfer of CD8+ cells successfully migrate to brain post induction of PIFS.(A) Schematic illustrating the adoptive transfer of sorted perforin competent CD8+ cells into perforin−/− mice followed by induction of PIFS. C57BL/6 perforin−/− mice were irradiated with 400 rads of irradiation and then intravenously injected with 107 GFP+ CD8+ splenocytes (n = 10) or 107 Ly5.1+ CD8+ splenocytes (n = 10). Mice were intracranially infected with TMEV on the following day. PIFS-inducing VP2121–130 peptide or mock E7 control peptide were intravenously administered on day 7, during the peak of CD8 T cell expansion. MRI analysis was performed on the following day to visualize the extent of CNS vascular permeability and then the CNS was harvested for additional assays. Both negative (Experiment I) and positive (Experiment II) sort experiments yielded high purity of CD8+ T cell transfer. (B) Representative confocal microscopic images illustrating co-localization of Ly5.1 and CD8 protein (Experiment I). CNS-infiltrating Ly5.1+ cells colocalized with CD8+ cells 85.7% of the time as measured by confocal analysis. (C) Confocal microscopy showing of representative brain tissue slice showing successful transfer of GFP+ CD8+ T cells (Experiment II). Purity of transfer was analyzed via flow cytometry, 98.0%+− 0.5% of cells that were CD8 positive and GFP positive.
Mentions: Spleens of GFP+ or Ly5.1+ mice were removed and strained through a nylon mesh 100 µm filter. CD8+ cells were purified from the resulting lymphocyte population using MACS LS cell purification columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Positive cell sorting was used to obtain GFP+ CD8+ cells and negative cell sorting was used to obtain Ly5.1+ CD8+ cells. Both methods resulted in ∼90% purity as determined by flow cytometric analysis (data not shown). C57BL/6 perforin−/− mice were irradiated with 400 rads of irradiation, given one day to recover, and then intravenously injected with 107 Ly5.1+ CD8+ or GFP+ CD8+ splenocytes (Figure 1A).

Bottom Line: However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood.To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques.Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, Rochester, Minnesota, United States of America; Department of Immunology, Mayo Clinic, Rochester, Minnesota, United States of America; Neurobiology of Disease Graduate Program, Mayo Graduate School, Rochester, Minnesota, United States of America.

ABSTRACT
Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.

Show MeSH
Related in: MedlinePlus