Extended Data Fig. 8: Involvement of peri-sinus dural areas in CNS autoimmunity.
From: Distinct roles of the meningeal layers in CNS autoimmunity
CNS-reactive T cells accumulate in higher numbers in the vicinity of the dural sinuses than in the dural hemispheres. a, Quantification of the CNS reactive T cells (T cell/g and absolute number) in the indicated compartments at the peak of T cell infiltration in the indicated transfer and active rat EAE models. Flow cytometry. Mean + s.e.m. Two-tailed t-test was applied for comparison within the dural compartments, one-way ANOVA with post-hoc correction for multiple comparisons for comparison between compartments. Representative data of 2 (bSYN-tEAE, n = 4) experiments and cumulative data of 5 (bSYN-aEAE, n = 13) and 2 (MBP-aEAE, n = 6) independent experiments. b, Top: distribution of TbSYN cells (red) at the COS, in the hemispheric dura and in the leptomeninges. Overview images (fluorescence microscopy) and magnified areas (confocal microscopy) of dural whole-mount and leptomeninges from the same animal. TS: transverse sinus. SSS: superior sagittal sinus. Blue: DAPI. Bottom: distribution of TbSYN cells in the area around the SSS. MMA: middle meningeal artery. Overviews and magnified areas. Fluorescence microscopy. c,d, Intravascular and extravascular T cell motility behavior in sinus and hemispheric dural vessels is comparable. c, Representative TPLSM 15-minute time-projection depicting the intravascular behavior of TOVA cells in the SSS on day 3 after T cell transfer. Note that the attenuation of laser intensity by erythrocytes limits the detection of T cells in the central part of the sinus. Turquoise: TOVA cells; red: 70-kDa dextran labelled vessels. Graph: corresponding quantification of the percentage of TOVA or TbSYN cells crawling, rolling or fast rolling within a 30-minute recording time in the indicated compartments on day 3 p.t. Mean + s.e.m. One-way ANOVA with post-hoc correction for multiple comparisons. Each dot represents one recording. TOVA cells: cumulative data from 28 (leptomeninges), 45 (dura hemisphere) and 6 (dura sinus) recordings. TbSYN cells: cumulative data from 8 (leptomeninges and dura hemisphere) and 6 (dura sinus) recordings. d. Mean velocity (line at mean), straightness (line at mean) and mean square displacement (± s.e.m.) of TbSYN cells recorded by TPLSM on day 3.5 p.t. in leptomeninges, peri-sinus areas and in hemispheric dura. Each dot represents one cell. Kruskall Wallis test with post-hoc correction for multiple comparisons. Cumulative data from 9 (leptomeninges), 14 (dura hemispheres) and 6 (dura sinus) recordings. e, Brain autoantigens are not spontaneously presented in the sinus adjacent areas. e, Experimental set-up as in Fig. 4a. MFI of CD134 and IL2R in TbSYN cells cultured for 48 h with cell suspension from the indicated compartments of naive animals in presence of the cognate antigen. As control (Ct), an irrelevant antigen (OVA) or no antigen were added to the culture (values pooled together). Flow cytometry. Mean + s.e.m. Statistical evaluation as in Fig. 4a. Representative data of 2 independent experiments. The number of analyzed samples is indicated. f-h, CNS-reactive T cells in peri-sinus areas are not activated. f, Expression of pro-inflammatory cytokines in TbSYN cells isolated from the indicated compartments on day 3.5 p.t. Quantitative PCR. House-keeping gene, β-actin. Mean + s.e.m. Representative data of 2 independent experiments. n = 4 (brLepto, brDura sinuses), n = 3 (brDura hemispheres) g, Animals were co-injected with TbSYN and TOVA cells. Relative expression of Il17a in TbSYN cells vs TOVA cells on day 3.5 p.t (ΔΔCt). Quantitative PCR. House-keeping gene, β-actin. Mean + SEM. Representative data of 2 independent experiments. n = 5 (brLepto, brDura hemispheres), n = 4 (brDura sinuses). h, Fluorescent intensity (FI) of CD134 in TbSYN or T MBP cells from the indicated compartments in actively immunized rats at the peak of T cell infiltration. Flow cytometry. Cumulative data of 5 (bSYN-aEAE) and 2 (MBP-aEAE) independent experiments. Medians (continuous line), quartiles (dotted lines) and number of analyzed T cells. e-g, One way-ANOVA with post-hoc correction for multiple comparisons. h, Kruskall Wallis test with post-hoc correction for multiple comparisons. a, c-h: *P < 0.05, **P < 0.01, ***P < 0.01 and ****P < 0.0001. MFI, median fluorescence intensity.
