Fatal PML associated with efalizumab therapy: insights into integrin αLβ2 in JC virus control

Abstract

Objectives: Progressive multifocal leukoencephalopathy (PML) has become much more common with monoclonal antibody treatment for multiple sclerosis and other immune-mediated disorders.

Methods: We report 2 patients with severe psoriasis and fatal PML treated for ≥3 years with efalizumab, a neutralizing antibody to αLβ2-leukointegrin (LFA-1). In one patient, we conducted serial studies of peripheral blood and CSF including analyses of leukocyte phenotypes, migration ex vivo, and CDR3 spectratypes with controls coming from HIV-infected patients with PML. Extensive pathologic and histologic analysis was done on autopsy CNS tissue of both patients.

Results: Both patients developed progressive cognitive and motor deficits, and JC virus was identified in CSF. Despite treatment including plasma exchange (PE) and signs of immune reconstitution, both died of PML 2 and 6 months after disease onset. Neuropathologic examination confirmed PML. Efalizumab treatment was associated with reduced transendothelial migration by peripheral T cells in vitro. As expression levels of LFA-1 on peripheral T cells gradually rose after PE, in vitro migration increased. Peripheral and CSF T-cell spectratyping showed CD8+ T-cell clonal expansion but blunted activation, which was restored after PE.

Conclusions: From these data we propose that inhibition of peripheral and intrathecal T-cell activation and suppression of CNS effector-phase migration both characterize efalizumab-associated PML. LFA-1 may be a crucial factor in homeostatic JC virus control.

Figure 1. CSF laboratory findings in patient 1

Abbreviations: β₂-MG = β₂-microglobulin; IgG = immunoglobulin G; IV Ig = IV immunoglobulin; JCV = JC virus; PE = plasma exchange.

Figure 2. Neuropathologic investigation of autopsy CNS tissue from the 2 patients with progressive multifocal leukoencephalopathy

Representative examples of postmortem pathology are shown. The origin of the samples is indicated by patient number in parentheses. (A) Macroscopic image of the occipital lobe (2). The arrowheads show areas of softening and the arrow highlights an area with pronounced cavitation. (B) Scan of a Luxol fast blue (LFB)–hematoxylin & eosin (H&E)–stained section showing a large confluent area of demyelination (2). (C) Microscopic image showing patchy foci of demyelination (2). (D) Macrophages containing LFB-positive myelin debris (arrowheads) and an oligodendrocyte with an intranuclear viral inclusion (arrow) (2). (E) JC virus demonstrated by JC virus–specific immunoreactivity (arrowhead) (1). (F) Ultrastructural demonstration of JC virus in an oligodendrocyte nucleus (2). (G) Perivascular inflammatory infiltrate (H&E) (1). (H–K) Immunohistochemistry for CD3 (H), CD8 (I), CD4 (J), and CD20 (K) (1). (L, M) Plasma cells as demonstrated by H&E (L) and CD138 immunostaining (M) (1). Scale bar = 2 mm (B), 200 μm (C), 20 μm (D, E, and L), 50 μm (H, I, J, K, and M), 100 μm (G), and 50 nm (F).

Figure 3. Immunologic features of patient 1 during progressive multifocal leukoencephalopathy (PML)

(A–F) x-axis shows days in hospital, and time of plasma exchange (PE) treatment is indicated. (A) Binding of efalizumab to CD4+ and CD8+ T cells. MFI = mean fluorescence intensity. (B) T-cell migration over an in vitro blood-brain barrier. (C) LFA-1 expression on CD8+ T cells. GeoMean = geometric mean. (D) Number of migrated cells 2 and 6 weeks after PE. HIV-associated PML (PML (HIV); n = 2), healthy donors (HD; n = 2). (E) T-cell receptor repertoire of CSF cells. GD = Gaussian distributions. (F) Total cell count in the CSF. (G) Phenotypic analysis of T-cell subsets (naïve [Tnaïve], central memory [Tcm], and effector memory [Tem]) as analyzed by the staining for CD4, CD8, CD45RA, and CCR7. T-cell compartments of CD4+ (left row) and CD8+ T cells (right row) are shown for different time points and in different compartments (CSF). Shown are plots of CCR7 (x-axis) and CD45RA (y-axis). Naïve T cells are CCR7+CD45RA+, central memory T cells are CCR7+CD45RA−, and effector memory T cells are CCR7−CD45RA−. Distributions for CD4 and CD8 T cells are shown from CSF (hospital day 42, CSF). Representative examples of patients with HIV-associated PML (PML (HIV)) and healthy donors (HD) are shown. Ef = efalizumab. (H) JC virus (JCV) VP1_p36-specific CD8+ T cells in patient 1: peripheral blood mononuclear cell (PBMC) samples obtained before PE and 1 week after the end of the PE were cultured as described in Methods, and staining with HLA-A*0201/JCV VP1_p36 tetramer was performed at day 14. (I) Cytokine secretion of CD8+ T cells after stimulation with JCV VP1_p36 in patient 1. PBMC samples obtained before PE (left row, hospital day 10) and 1 week after the end of the PE (right row, hospital day 22) were cultured as described in Methods and an intracellular cytokine staining consensus statement with HLA-A*0201/JCV VP1_p36 peptide was performed at day 14. Numbers in the plots represent percentage of cytokine-secreting CD8+ T cells. IFN = interferon; SEB = staphylococcal enterotoxin B; TNF = tumor necrosis factor.

Figure 4. MRI scans and schematic distribution of CNS tissue samples of patient 1 with corresponding virus load and presence of clonally expanded T cells

(A) Initial MRI scan of patient 1 (hospital day 1, left panels) and last MRI scan on day 73 (right panels). (B) Schematic drawing of the sites of assessed samples from autopsy brain tissue. Depicted are findings from the 2 cut brain slices analyzed sagittally (frontal and parietal, each left and right hemisphere, left panel), the distribution of the samples (numbering) in the brain slices (middle panel; samples are always in the sequence of cortex [top], white matter [top], white matter [basal], cortex [basal]). The samples are correlated with the number of dominant clonal expansions in each sample (S), in the white matter of this hemispheric brain slice (WM), the gray matter (GM), the complete hemispheric brain slice (HS), the complete brain slice (BS), and the brain hemispheres (BH). The gray matter/cortex is always highlighted with gray, the right hemisphere with red, and the left hemisphere with green. The virus load in these samples is given in copies (C) per mL. In addition, kidney (K) was analyzed.