Characterization of and functional antigen presentation by central nervous system mononuclear cells from mice infected with Theiler's murine encephalomyelitis virus

Abstract

We examined the phenotype and function of cells infiltrating the central nervous system (CNS) of mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV) for evidence that viral antigens are presented to T cells within the CNS. Expression of major histocompatibility complex (MHC) class II in the spinal cords of mice infected with TMEV was found predominantly on macrophages in demyelinating lesions. The distribution of I-As staining overlapped that of the macrophage marker sialoadhesin in frozen sections and coincided with that of another macrophage/microglial cell marker, F4/80, by flow cytometry. In contrast, astrocytes, identified by staining with glial fibrillary acidic protein, rarely expressed detectable MHC class II, although fibrillary gliosis associated with the CNS damage was clearly seen. The costimulatory molecules B7-1 and B7-2 were expressed on the surface of most MHC class II-positive cells in the CNS, at levels exceeding those found in the spleens of the infected mice. Immunohistochemistry revealed that B7-1 and B7-2 colocalized on large F4/80(+) macrophages/microglia in the spinal cord lesions. In contrast, CD4(+) T cells in the lesions expressed mainly B7-2, which was found primarily on blastoid CD4(+) T cells located toward the periphery of the lesions. Most interestingly, plastic-adherent cells freshly isolated from the spinal cords of TMEV-infected mice were able to process and present TMEV and horse myoglobin to antigen-specific T-cell lines. Furthermore, these cells were able to activate a TMEV epitope-specific T-cell line in the absence of added antigen, providing conclusive evidence for the endogenous processing and presentation of virus epitopes within the CNS of persistently infected SJL/J mice.

FIG. 1

Expression of MHC class II in normal and TMEV-infected SJL/J spinal cords. Frozen tissue sections (5 μm) were prepared from spinal cords of TMEV-infected SJL/J mice (A and B). (A) Sections were labeled with MAbs specific for the macrophage marker sialoadhesin (green) and I-A^s (red). Expression of MHC class II largely overlapped that of sialoadhesin (large arrows), but a few singly labeled MHC class II-positive cells were found at the margins of the lesion (open arrowheads). (B) Sections were labeled with the astrocyte marker GFAP (red) and I-A^s (green). Overlap of labeling was observed only rarely on large ramified cells (arrow). Frozen sections (6 μm) from normal spinal cords were also examined (C to E). In the spinal cords of uninfected mice, sialoadhesin (C) and MHC class II (E) are negative except for a few cells in the meninges and vasculature, while GFAP (D) shows punctate labeling of astrocyte processes. A mouse IgG2b isotype control for I-A^s (F) shows hazy background over infiltrating cells and a few bright cells in the meninges of a spinal cord section from a TMEV-infected mouse. Magnification, ×200.

FIG. 2

Flow cytometric analysis of MHC class II, B7-1, and B7-2 expression by macrophages and microglia in the spinal cords of TMEV-infected mice. Splenic and CNS mononuclear cells were isolated from TMEV-infected mice 40 days p.i. and stained with F4/80-PE, anti-B7-1-biotin, or anti-B7-2-biotin, followed by anti-I-A^s-FITC or control antibody, A-PE, and propidium iodide. Most MHC class II⁺ cells in the infected CNS were large F4/80⁺ macrophages and microglia (group D). In the spleen, MHC class II was expressed predominantly on F4/80⁻ cells, most of which were probably B cells (group B). Essentially all of the MHC class II⁺ cells in the infected CNS expressed B7-1 and/or B7-2 (groups G and H). Most of the B7-1 and B7-2 expressing cells were also MHC class II⁺. The splenic MHC class II⁺ cells expressed B7-1 and B7-2 weakly (groups E and F). Identical results were obtained at day 23 p.i. FACS data for viable cells were collected by exclusion of propidium iodide. During analysis, lymphocytes and myelin debris were excluded by electronic gating. Numbers are the percentages of gated events in each quadrant.

FIG. 3

Demonstration of B7-1 and B7-2 in the spinal cords of TMEV-infected mice by immunohistochemistry. Frozen sections (5 to 6 μm) of SJL/J spinal cord were prepared 45 days p.i. and stained sequentially for F4/80 or CD4 (green), followed by B7-1 or B7-2 (red). B7-1 (A) and B7-2 (C) colocalize on large F4/80⁺ macrophages and microglia with the exception of a few F4/80⁺ microglia at the edges of the lesions (arrows). B7-1 and B7-2 do not colocalize to small CD4⁺ T cells in the central part of the lesions (B and D, filled arrows), but a subpopulation of large, blast-like CD4⁺ T cells at the lesion margins express B7-2 (D, open arrows).

FIG. 4

CNS-derived mononuclear cells from TMEV-infected mice endogenously present viral epitopes to the VP2_74–86-specific sTV1 T-cell line. CNS-derived, plastic-adherent mononuclear cells were prepared from symptomatic TMEV-infected SJL/J mice (n = 23, mean clinical score = 1.7) or from mice with R-EAE (n = 24, mean clinical score = 1.7). Various numbers (4 × 10³ to 2.5 × 10⁵) of the CNS APC populations were cultured with 2 × 10⁴ sTV1 cells in the presence and absence of UV-inactivated TMEV virions (5 μg/ml) or VP2_74–86 (0.5 μM). The cultures were pulsed with [³H]TdR at 42 h and harvested 24 h thereafter. For CNS-derived APCs, the background subtracted was the counts per minute obtained from the culture of the same number of R-EAE-derived CNS APC in the absence of antigen.

FIG. 5

Specificity of endogenous antigen presentation by CNS-infiltrating mononuclear cells to TMEV-specific (sTV1) and hMyo-specific T-cell lines. Plastic-adherent, CNS-infiltrating mononuclear cells prepared from mice with TMEV (n = 30, mean score = 1.9) and R-EAE (n = 29, mean score = 1.2) were cultured with sTV1 or hMyo-specific Th1 cells (2 × 10⁴) with UV-inactivated TMEV (BeAn; 5 μg/ml) or hMyo (10 μM), respectively. The results are expressed as the counts per minute in the absence (open bars) or presence (filled bars) of added antigen. Responses of cultures of CNS-derived APC from TMEV-infected mice should be compared to those obtained from the culture of R-EAE-derived CNS APC cultured in the absence of TMEV (group G).