Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain

Abstract

Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, has been associated with multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), but direct proof of its involvement in the disease is still missing. To test the idea that MS might result from perturbed EBV infection in the CNS, we investigated expression of EBV markers in postmortem brain tissue from MS cases with different clinical courses. Contrary to previous studies, we found evidence of EBV infection in a substantial proportion of brain-infiltrating B cells and plasma cells in nearly 100% of the MS cases examined (21 of 22), but not in other inflammatory neurological diseases. Ectopic B cell follicles forming in the cerebral meninges of some cases with secondary progressive MS were identified as major sites of EBV persistence. Expression of viral latent proteins was regularly observed in MS brains, whereas viral reactivation appeared restricted to ectopic B cell follicles and acute lesions. Activation of CD8+ T cells with signs of cytotoxicity toward plasma cells was also noted at sites of major accumulations of EBV-infected cells. Whether homing of EBV-infected B cells to the CNS is a primary event in MS development or the consequence of a still unknown disease-related process, we interpret these findings as evidence that EBV persistence and reactivation in the CNS play an important role in MS immunopathology.

Figures

Figure 1.

Immunohistochemical characterization of ectopic B cell follicles with germinal center–like features in the cerebral meninges of a subset of MS cases with secondary progressive disease. (A) Localization of an ectopic B cell follicle (asterisk) and a sparse inflammatory infiltrate in the subarachnoid space lined by the pial membrane (arrows) at the entrance of a cerebral sulcus (hematoxylin counterstaining; CD20 immunostaining in the inset). The adjacent cortical gray matter is extensively demyelinated, as indicated by loss of MOG immunoreactivity. (B–D) An ectopic B cell follicle located in the depth of a cerebral sulcus is shown that comprises the following: aggregated CD20+ B cells and a network of stromal/follicular dendritic cells expressing CXCL13 (B, double immunofluorescence staining for CD20 and CXCL13); proliferating B cells (C, double immunofluorescence staining for CD20 and the proliferation marker Ki67; the inset shows three double labeled CD20+/Ki67+ cells at higher magnification); and peripherally located Ig+ plasma cells (D). Immunostainings shown in B–D were performed in serial brain sections. (E–G) Different ectopic follicles are shown that contain the following: numerous cells expressing AID (E); fewer apoptotic cells expressing activated caspase-3 (F); and numerous cells expressing the antiapoptotic molecule bcl-2 (G). Perivascular bcl2+ cells in a white matter lesion (inset in G) and proliferating CD20+ B cells (arrows) in a large perivascular cuff of an acute lesion (H, double immunofluorescence staining for CD20 and Ki67) are shown. Bars: A, B, D–F, and H, 50 μm; C, G, and inset in G, 20 μm; inset in C, 10 μm.

Figure 2.

Detection of EBER+ cells in the MS brain by in situ hybridization. (A–E) EBV-high MS cases. In situ hybridization for EBER shows enrichment of EBER+ cells (blue-black nuclei) in ectopic B cell follicles located in the meninges of two different MS cases (A and B). Combined in situ hybridization for EBER and immunostaining for CD20 (red surface staining) reveals a high frequency of EBER+ B cells in an ectopic B cell follicle (C) and a lower percentage of B cells expressing EBER in the large perivascular cuff of an acute white matter lesion (D; the same lesion stained for CD20 and Ki67 in an adjacent section is shown in Fig. 1 H). In the insets in C and D, an EBER+/CD138+ plasma cell and an EBER+/CD20+ B cell are shown, respectively, at higher magnification. Perivascular EBER+ (E), CD20+ B cells (inset in E), and CD138+ plasma cells (F) in a large periventricular white matter lesion (E), and some intraparenchymal infiltration by EBER+ cells (E) and plasma cells (F) in the same region. The inset in F shows CD138+ plasma cells positive for EBER inside the parenchyma. (G–I) EBV-low MS cases. EBER+ cells in the meninges (G) and around scarcely infiltrated blood vessels (H and I) in chronic active white matter lesions. The insets in G and I show CD20 immunostaining of the corresponding areas in adjacent sections. In situ hybridization for EBER in a control, EBV-associated B cell lymphoma (J). Bars: A–C, E, G, J, and insets in E, G, and I, 50 μm; D and F, 20 μm; insets in C, D, and F, 10 μm. (K) Statistically significant correlation between the number of CD20+ and EBER+ cells in the white matter (left) and meninges (right) of EBV-low (n = 12) and EBV-high (n = 8) MS cases. EBER+ and CD20+ cells were counted as described in Materials and methods.

Figure 3.

Quantification of CD20+, EBER+, and CD8+ cells in MS brain sections. MS cases with high and low brain inflammation were a posteriori classified as EBV-high and EBV-low, respectively. The graphs show significant differences between the two groups in the number of CD20+, EBER+, and CD8+ cells counted in the white matter (A) and meninges (B). Dot points represent values for each MS case. Each value represents the mean of cell counts performed as described in Materials and methods. The bars represent median values for each group (n = 12 for EBV-low MS cases; n = 8 for EBV-high MS cases).

Figure 4.

Detection of EBV latent and early lytic proteins in the MS brain. In brain sections from EBV-high MS cases, cells expressing EBNA-2 (nuclear staining) are present in the perivascular inflammatory cell infiltrates in acute (A) and chronic active (B) white matter lesions. LMP1+ cells (membrane staining) in a sparse meningeal infiltrate (C) and in an intrameningeal ectopic B cell follicle (D; the inset shows the same follicle stained for EBER in an adjacent section) from EBV-high MS cases. Perivascular EBNA2+ and LMP1+ cells in chronic active lesions from an EBV-low MS case are shown in E and F, respectively. High frequency of BFRF1+ cells in an ectopic intrameningeal B cell follicle from an EBV-high MS case (G). In two different ectopic B cell follicles (H and I), double immunofluorescence for CD20 and BFRF1 shows that BFRF1 is expressed in a substantial proportion of CD20+ B cells, but its intensity is much stronger in CD20− cells (arrows). The insets in G and H highlight the typical perinuclear localization of BFRF1. Double immunofluorescence for Ig and BFRF1 shows that the lytic cycle–associated protein is strongly expressed in a proportion of Ig+ plasma cells in the same ectopic follicle shown in I (J) and in a large perivascular cuff in an acute lesion of an EBV-high MS case (K). Two double-labeled Ig+/BFRF1+ plasma cells are shown at high power magnification (L and M). Bars: K and inset in D, 50 μm; A–J and inset in B, 20 μm; L, M, and insets in G and H, 5 μm.

Figure 5.

Diffuse infiltration of latently and lytically EBV-infected cells in the brain of acute MS cases. (A and B) Presence of LMP1+ cells around scarcely inflamed blood vessels throughout an active lesion of one acute MS case (MSG9; disease duration 23 d). In the same area, perivascular accumulation of CD8+ T cells is observed (C). A highly infiltrated lesion in the white matter of the second acute MS case examined (MSH6; disease duration 10 mo) contains large B cell–enriched perivascular cuffs (D). Immunostainings for BFRF1 and CD8 in adjacent sections show the presence of numerous perivascular BFRF1+ cells (E and F) and CD8+ T cells (G) in the same area. The blood vessels labeled with asterisks in D are shown at high power magnification in E, F, and G. The inset in E shows the typical perinuclear localization of BFRF1. Sparse infiltration of BFRF1+ cells in the white matter of case MSH6 is shown (H). Perivascular (I) and intraparenchymal (J) BFRF1+ cells are shown at high power magnification. Staining of an adjacent section with anti-CD8 mAb shows diffuse infiltration of CD8+ T cells in the same area (K). Presence of isolated cells expressing the structural viral proteins gp350/220 (L and M; membrane staining) and p160 (N; cytoplasmic staining) in the meninges of MSH6 (L and N) and in the white matter of MSG9 (M). Bars: D, 200 μm; A, F, G, K, and L, 50 μm; B, C, E, H, I, M, N, and inset in E, 20 μm; J, 10 μm.

Figure 6.

Analysis of total and EBV-specific OCBs in postmortem CSF from MS cases. The graphs show (A) significantly higher numbers of OCBs in the CSF of EBV-high MS cases (n = 7) versus EBV-low MS cases (n = 9) and (B) no difference in the number of EBV-specific OCBs between EBV-high and EBV-low MS cases. Dot points represent values for each MS case, and the bars represent median values for each group. p-values, calculated by the Mann-Whitney U test, are indicated where statistically significant. (C) Affinity-mediated immunoblotting on EBV antigen-coated nitrocellulose paper of isoelectrofocused CSF from two EBV-low MS cases (lanes 1 and 2 correspond to MS154 and MS102 in Table S1, respectively), anti-EBV EA-D monoclonal (mo) antibody used as positive control (lane 3), and serum (ser) from a patient with monoclonal IgG used as control for binding specificity (lane 4). An additional control for binding specificity included CSF from the MS102 case that was blotted onto casein-coated nitrocellulose paper (absence of reactivity; lane 5). Faint (lane 1) and both faint and strong (lane 2) EBV-specific OCBs are present in MS154 and MS102 cases, respectively (arrows indicate OCBs). Of note, MS102 was also positive for EBV DNA and had the highest frequency of EBER+ and CD8+ cells in white matter lesions and meninges among the EBV-low MS cases.

Figure 7.

CD8+ T cell activation and interaction with EBV-infected cells in the brain of EBV-high MS cases. Double immunofluorescence for CD8 and CD20 (A) and for CD8 and Ig (B) shows accumulation of CD8+ T cells in an intrameningeal ectopic B cell follicle (A) and in the perivascular cuff of an acute white matter lesion (B; same infiltrated blood vessel stained for BFRF1 in an adjacent section is shown in Fig. 4 K). The perivascular infiltrate shown in B contains numerous Ki67+ proliferating cells, of which some are CD8+ T cells (arrows) (C). Note the small cluster of three Ki67+/CD8+ cells shown at higher magnification in the inset in C. Immunohistochemistry with anti–IFN-γ antibody (D) reveals strong immunoreactivity (brown staining) in the cytoplasm of numerous cells in an intrameningeal B cell follicle (same follicle double stained for EBER and CD20 in Fig. 2 C). The insets in D show double stainings for CD8 (brown) and IFN-γ (purple). IFN-γ+/CD8+ cells (black arrows), as well as cells single stained for either CD8 (white arrow) or IFN-γ (arrowheads), are identified in the same follicle (lower inset). Two IFN-γ+ cells, one positive (arrow) and one negative (arrowhead) for CD8, are shown in the upper inset. Double immunofluorescence stainings for CD8 and CD20 (E), CD8 and Ig (F), and CD8 and BFRF1 (G) show that CD8+ T cells with an activated, lymphoblastoid morphology cluster around and extend cytoplasmic processes that contact CD20+ B cells, Ig+ plasma cells, and BFRF1+ cells, respectively. Double immunostaining for perforin and CD8 shows the presence of perforin granules in two out of several CD8+ T cells accumulating in the perivascular cuff of a chronic active lesion (H). Polarization of perforin granules is evident in one CD8+ T cell (arrow), but not in the other (arrowhead). Double immunostaining for perforin and Ig (I and J; inflamed meninges) or CD20 (K; intralesional perivascular cuff) shows polarization of perforin granules toward two Ig+ plasma cells, but not toward CD20+ B cells. Double immunostaining for CD8 and CD107a (L and M) reveals surface distribution of CD107a in some CD8+ T cells (arrows) accumulating in the meninges (L) and in an intraparenchymal perivascular cuff (M). Note the presence in the same microscopic fields of CD8+ T cells that do not express CD107a (arrowheads in L and M) and of a CD8− cell with a cytoplasmic distribution of CD107a (asterisk in M) . Bars: A–C, 50 μm; D, G, M, and inset in C, 20 μm; E, F, H–L, and lower inset in D, 10 μm; upper inset in D, 5 μm. (N) Statistically significant correlation between the number of CD8+ and EBER+ cells in the white matter (left) and meninges (right) of EBV-low (n = 12) and EBV-high (n = 8) MS cases. EBER+ and CD8+ cells were counted as described in Materials and methods.