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. 2008 Jun 15;180(12):8393-9.
doi: 10.4049/jimmunol.180.12.8393.

In situ activation of antigen-specific CD8+ T cells in the presence of antigen in organotypic brain slices

Changying Ling  1 , Yakov I Verbny, Matthew I Banks, Matyas Sandor, Zsuzsanna Fabry
Affiliations

In situ activation of antigen-specific CD8+ T cells in the presence of antigen in organotypic brain slices

Changying Ling et al. J Immunol. .

Abstract

The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8(+) T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c(+) cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c(+) cells stimulate Ag-specific naive CD8(+) T cells locally in the CNS and may contribute to immune responses in the brain.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
Ag-specific CD8+ T cells migrate and colocalize with CD11c+ cells harboring their cognate Ag (OVA) in organotypic brain slices. A, Diagram illustrates experimental outline. Brain hemi-slices were prepared from B6 mice 1 h after an IC-OVA injection and cocultured with OVA-specific CFSE-labeled CD8+ T cells. Three days after culture, lymph cells were isolated from cocultures and analyzed. B, Confocal images in top panels show the accumulation of OVA-specific CD8+ T cells (green) along the IC-OVA injection track in the brain (right panel). Brain sections prepared from the hemi-slice 3 days after coculture were stained with anti-CD11c Abs (insets only). The inset in each image shows CD11c+ cells (red) and OVA-specific CD8+ T cells (green) in the center of brain slices. A diagram at the bottom shows the anatomic location of images taken and depicts the distribution of OVA-specific CD8+ T cells (green dots) in the brain slices. Data represent analyses from two independent experiments. CC, Corpus callosum; AC, anterior commissure; LV, lateral ventricle; Scale, 250 μm (25 μm for insets).
FIGURE 2
FIGURE 2
The number of OVA-specific (CFSE+Vα2+CD8+) T cells significantly increases in the culture containing brain slices prepared from the IC-OVA hemisphere. Brain hemi-slices with or without (control) an IC-OVA injection site were prepared from B6 mice 1 h after injection and cocultured with CFSE-labeled Vα2+CD8+ T cells. After three days, lymphocytes were isolated from cocultures and analyzed by cytofluorometry. A, Dot plots (gated on CFSE+ lymphocytes) show increased OVA-specific CD8+ T cell frequency (dotted circle) in the cultures with the IC-OVA injection site. B, Histograms show the proliferation of OVA-specific CD8+ T cells in the cultures as detected by cell cycle-dependent decrease of CFSE intensity. C, Quantitative analysis of OVA-specific CD8+ T cells in brain slice cultures. Bars represent the mean ± SE number of OVA-specific CD8+ T cells. *, p < 0.05, paired Student’s t test. Data are representative and were derived from seven independent experiments.
FIGURE 3
FIGURE 3
Ag-specific CD8+ T cell accumulation in organotypic brain slices. Brain hemi-slices containing the IC injection site were prepared from B6 mice 1 h after IC-OVA and IC-PCC immunizations and cocultured with OVA-specific T cells. Three days after culture, the hemi-slices were separated from the culture and lymphocytes were isolated from each hemi-slice and analyzed by cytofluorometry. A, Dot plots (gated on lymphocytes) show the percentage of OVA-specific CD8+ T cells (Vα2+CD8+; dotted circle) per slice (percentage given in the upper right corner of each plot). B, Quantitative analysis of OVA-specific CD8+ T cells in each slice. Bars represent the mean ± SE number of CD8+ T cells. *, p < 0.05, paired Student’s t test. Data are representative and were derived from three independent experiments.
FIGURE 4
FIGURE 4
Brain-resident CD11c+ cells accumulate in the culture supernatant of organotypic brain slices. Brain hemi-slices containing the IC injection sites were prepared from B6 mice 1 h after IC-OVA or IC-PBS injection and cocultured with OVA-specific CD8+ T cells. Three days after coculture, free-floating cells were harvested from the supernatant of each culture and processed for confocal images and cytofluorimetric analysis. A, Representative confocal image shows CFSE−CD11c+ (blue) and OVA-specific CD8+ T cells (CFSE+; green) in the culture supernatant of IC-OVA-injected hemi-slices. Scale bar, 20 μm. B, Histograms (gated on CFSE− cells) show an increased number of CD11c+ cells in the culture supernatant with brain slices containing IC-OVA injection sites. Data are representative of three independent experiments.
FIGURE 5
FIGURE 5
MIP-3β chemokine enhances the migration of brain-resident CD11c+ cells from organotypic brain slices. Brain hemi-slices were prepared from B6 mice 1 h after bilateral IC-OVA immunizations and cocultured with OVA-specific CD8+ T cells with or without MIP-3β (200 ng/ml). Two days after culture, cells were harvested from the supernatant of each culture and processed for cytofluorimetric analysis. A, Histograms (gated on CFSE− cells) show an increased number of CD11c+ cells in the culture with MIP-3β. B, Quantitative analysis of CFSE−CD11c+ cells in culture with or without MIP-3β. Bars represent the mean ± SE number of CD11c+ cells. *, p < 0.05, paired Student’s t test. Data are representative and were derived from three independent experiments.
FIGURE 6
FIGURE 6
The clonal expansion of OVA-specific CD8+ T cells is abolished by depletion of CD11c+ cells from DTR mice. A, Diagram illustrates experimental outline. Brain hemi-slices were prepared from DTR mice 1 h after IC-OVA immunizations with or without a primary DT injection and cocultured with OVA-specific CD8+ T cells from OTI-RAG mice with or without secondary DT treatment. Three days after co-culture, lymphocytes were isolated and processed for cytofluorimetric analysis. B, Histograms (gated on lymphocytes) show the depletion of CD11c+ cells from culture after DT treatments. C, Histograms (gated on CD8+ cells) show that the proliferation of OVA-specific CD8+ T cells in culture is abolished after DT treatments. Data are representative of three independent experiments.
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