Research ArticleNEUROIMMUNOLOGY

Conventional DCs sample and present myelin antigens in the healthy CNS and allow parenchymal T cell entry to initiate neuroinflammation

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Science Immunology  25 Jan 2019:
Vol. 4, Issue 31, eaau8380
DOI: 10.1126/sciimmunol.aau8380
  • Fig. 1 Identification of MHCII+ cells in the steady-state CNS.

    (A) Immune cell populations from the whole steady-state brain (i.e., parenchyma, parenchymal vessels, intact choroid plexus, and the adhering leptomeninges) including the dura mater were analyzed by CyTOF. After gating on live singlets, MHCII+ CD45+ cells were exported as an FCS file using the FlowJo software. (B) The MHCII+ fraction of CNS immune cells was visualized using t-SNE and clustered using the FlowSOM algorithm in R (6907 total cells: max iterations = 5000, perplexity = 50, θ = 0.5). (C) Median relative expression of all panel markers. (D) Frequency of CD45+ (left graph) and MHCII+ cells (right graph) of FlowSOM clusters. (E) FlowSOM cell clusters were analyzed for MHCII, CD86, and PD-L1 expression intensity (after percentile normalization) and plotted as histograms for each cluster using R. Shown are pooled data from n = 3 mice.

  • Fig. 2 Microglial MHCII is dispensable for AT EAE.

    (A) Sall1CreERT2 mice were crossed to Iabfl mice carrying floxed MHCII alleles. (B) Graphical abstract to illustrate the tamoxifen treatment strategy of Sall1Cre/+ Iabfl/fl mice. Cre+ animals and Cre littermates were administered tamoxifen via oral gavage (three times on alternate days) to induce Cre recombinase expression in microglia. (C) MHCII targeting profile of whole steady-state brain APCs in Sall1CreERT2/+ Iabfl/fl and Sall1+/+ Iabfl/fl mice 1 week after tamoxifen treatment (for gating strategy, see fig. S2, C and D). The relative median fluorescence intensity (MFI) has been calculated by normalizing absolute MFIs of each cell population to the MFI of cDC2s (which do express the highest MHCII levels). The MFI of cDC2s of one mouse was set to 100%. Data are representative of one of two independent experiments (n > 7 mice per group). (D) EAE was induced by AT of encephalitogenic 2D2 cells into recipient mice. Data show individual maximal EAE score and number of mice with clinical EAE symptoms. (E to G) At the peak of disease, the CNS (whole brain and spinal cord pooled) was analyzed for inflammatory infiltrates (E) and MHCII targeting of APCs (F and G) by flow cytometry. (E) Infiltrating monocytes (Ly6C+, Ly6G, CD11b+), neutrophils (Ly6Cint, Ly6G+, CD11b+), and CD4+ T cells (CD4+, CD11b, CD11c) were manually gated in FlowJo; absolute numbers (log10) per brain ± SEM are shown. (F) MHCII targeting profile (log10 MFI MHCII) in microglia and (G) other CNS-infiltrating and CNS-resident APCs at peak EAE (relative MFI) (manually gated in FlowJo without using MHCII for the gating). (D to G) Data are pooled from three independent experiments (n > 9 mice per group).

  • Fig. 3 cDCs, but not BAMs or microglia, are required for the reactivation of 2D2 CD4+ T cells in the CNS.

    (A) Cx3cr1CreERT mice were crossed to Iabfl mice. (B) Cx3cr1CreERT2/+ Iabfl/fl (early) mice and Cx3cr1+/+ Iabfl/fl littermates received a short, early tamoxifen treatment via oral gavage (three times on alternate days) 4 weeks before AT EAE or steady-state CNS analysis. Tamoxifen treatment of Cx3cr1CreERT2/+ Iabfl/fl (cont.) and Cx3cr1+/+ Iabfl/fl littermates was initiated 1 week before induction of AT EAE or steady-state brain analysis and continued until the end of the experiment. (C) MHCII targeting profile in APCs of naïve Cx3cr1+/+ Iabfl/fl (white circles) and Cx3cr1CreERT2/+ Iabfl/fl mice (early: gray circles; cont.: red circles) from the whole steady-state brain. Data represent the MFI (±SEM) of MHCII of each manually gated (for gating strategy, see fig. S2, C and D) population and are representative of one of more than three independent experiments (total of n > 12 mice per group). (D) Encephalitogenic 2D2 cells were adoptively transferred, and the clinical outcome was compared between Cx3cr1CreERT2/+ Iabfl/fl mice (early, gray circles; cont., red circles) and Cx3cr1+/+ Iabfl/fl littermates (white circles) over time. Data represent the individual maximal EAE score and the number of mice with clinical EAE (pooled from three independent experiments with n > 14 mice per group); P < 0.0001, χ2 analysis. (E) At the peak of disease (days 12 to 14), the CNS (whole brain and spinal cord pooled) was analyzed for inflammatory infiltrates by flow cytometry. Infiltrating monocytes (Ly6C+, Ly6G, CD11b+), neutrophils (Ly6Cint, Ly6G+, CD11b+), and CD4+ T cells (CD4+, CD11b, CD11c) were manually gated in FlowJo, and absolute numbers (log10) per brain ± SEM are shown. Data are representative of one of three independent experiments (total of n > 10 mice per group).

  • Fig. 4 CNS-associated cDC2s reside in the steady-state brain leptomeninges and reactivate 2D2 T cells in vitro.

    (A) In-depth CyTOF analysis of DC subsets from pre-enriched whole-brain leukocytes (n = 20 naïve brains): DCs were identified and subset from the initial data, subjected to t-SNE dimensionality reduction (3509 total cells; max iterations = 750, perplexity = 200, θ = 0.5), and clustered into three main subsets using FlowSOM-guided clustering according to their marker expression. (B) Relative abundance of the DC subsets within total CNS DCs. (C) Representative immunofluorescence images of CD11c+ MHCII+ CD11b cDC1s and CD11b+ cDC2s in the dura mater, leptomeninges, and choroid plexus of the steady-state brain (n ≥ 2 mice, ≥4 sections per mouse). Scale bar, 50 μm. A zoomed-in view is shown. (D) Brain compartments [leptomeninges, dura mater, choroid plexus, and parenchyma (hippocampus)] were dissected from n = 10 brains. Individual compartments were pooled and single cells were analyzed by flow cytometry followed by computational high-dimensional data analysis. After gating on live singlets, CD45+ cells were exported as an FCS file using the FlowJo software. Shown are pooled data from n = 10 mice, visualized using t-SNE and clustered using the FlowSOM algorithm in R (142,655 total cells; max iterations = 10000, perplexity = 50, θ = 0.5). Plots show FlowSOM clusters of leukocytes overlaid onto a t-SNE map of the combined dataset (left) or shown separately for each compartment as indicated. (E) Data show frequencies of cDC1s, cDC2s, and pDCs of total DCs (left graph) or CD45+ cells (right graph) as determined by FlowSOM clustering. (F) DCs (CD11chi, MHCII+, B220, MerTK), BAMs (MerTK+, CD11b+, CD45hi, MHCII+), microglia (MerTK+, CD45lo, CD11b+), and B cells (CD11b, B220+, CD11c, MHCII+) were isolated from pooled whole steady-state brains of 20 to 25 C57BL/6 mice and incubated with in vivo activated encephalitogenic 2D2 cells in the presence [2000:55,000 (APC:T cell); upper graph] or absence [11,000:55,000 (APC:T cell); lower graph] of MOG35–55 peptide for 72 hours. IFN-γ production was measured by ELISPOT assay. Data represent the mean ± SD cytokine activity of IFN-γ from technical duplicates and are representative of one of two independent experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/31/eaau8380/DC1

    Fig. S1. Identification of MHCII+ cells in the steady-state CNS.

    Fig. S2. Microglial MHCII is dispensable for AT EAE.

    Fig. S3. cDCs, but not BAMs or microglia, are required for the reactivation of 2D2 CD4+ T cells in the CNS.

    Fig. S4. Different DC subsets reside at the steady-state brain interfaces and present myelin Ag to CD4+ 2D2 T cells.

    Table S1. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Identification of MHCII+ cells in the steady-state CNS.
    • Fig. S2. Microglial MHCII is dispensable for AT EAE.
    • Fig. S3. cDCs, but not BAMs or microglia, are required for the reactivation of 2D2 CD4+ T cells in the CNS.
    • Fig. S4. Different DC subsets reside at the steady-state brain interfaces and present myelin Ag to CD4+ 2D2 T cells.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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