Immune privilege of the CNS is not the consequence of limited antigen sampling

Abstract

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Figure 1. EGFP-fused neoepitopes are expressed in CNS of PLP-OP and CNP-OP mice.

(A) Map of pZ/EG plasmid. Neomycin resistance, Neo^R; Ampicillin resistance, Amp^R. Arrow, 5′ → 3′(drawn by JK). (B) Mouse breeding strategy (drawn by MGH). (C) Histograms show % EGFP+/− cells. WT (black line); transgenic (green line). Column graphs show average fold-change in mean fluorescent intensity (MFI) over WT. Error bars, SEM. (D) Spinal cord; 1000×. DAPI (blue); α-GFP-FITC (green). Arrows, EGFP⁺ oligodendrocytes.

Figure 2. CNS neoepitopes are immunologically surveyed under normal conditions in the periphery.

Mice received 5 × 10⁵ neoepitope-specific T cells. FACS staining was performed on lymphocytes 6 days post-adoptive transfer. Histograms show CFSE dilution of CD8⁺ Thy1.1⁺ cells from CLNs (WT, PLP-OP, and CNP-OP mice) and MLNs (Vil-OP mouse) and spleen. Numbers indicate frequency of CFSE^high vs. CFSE^low/int cells. N = 8 littermates, n = 7 PLP-OP, n = 7 CNP-OP, and n = 4 Vil-OP mice (2 independent experiments).

Figure 3. Antigen-specific CD8⁺ T cells accumulate in peripheral tissues of mice with high levels of CNS neoepitope expression.

(A) Column graphs indicate mean # of CFSE^low/int LFA-1^high CD8⁺/CD4⁺ Thy1.1⁺ cells/gram tissue. (B) Column graphs indicate mean # of CD8⁺ Thy1.1⁺ IFN-γ⁺ LFA-1^high cells/gram tissue. Error bars, SEM. *, p < 0.05; **, p < 0.01. Data were generated from n = 8 littermates, n = 7 PLP-OP, and n = 7 CNP-OP mice (2–4 experiments).

Figure 4. There is increased peripheral immunosurveillance of CNS neoepitopes under neuroinflammatory conditions.

Nine days post-EAE induction, mice received 5 × 10⁵ neoepitope-specific T cells. FACS was performed within 1–4 days of disease onset. (A) Histograms show percentage of CFSE^low/int/CFSE^high cells. (B) Column graphs indicate mean # of CFSE^low/int LFA-1^high CD8⁺/CD4⁺ Thy1.1⁺ cells/gram tissue. Error bars, SEM. *, p < 0.05; **, p < 0.01. N = 7 littermates, n = 5 PLP-OP and n = 11 CNP-OP mice (3 experiments).

Figure 5. Neoepitope-specific CD8⁺ T cells accumulate in CNS of PLP-OP and CNP-OP mice with EAE.

(A) Column graphs–mean # transgenic cells/gram tissue. N = 7 littermates, n = 5 PLP-OP, n = 11 CNP-OP mice (3 EAE experiments). Error bars, SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.0001 (B) CNP-OP CNS (EAE-D15); 100×. Choroid plexus (CP). White boxes, Thy1.1⁺(red)/CD11c⁺(green) cell interactions. Column graphs–11 brain & 20 spinal cord 400× fields (n = 1 CNP-OP EAE mouse).